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Insulin-like growth factor-I and the endocrine pancreas McClean, Carole Anne 1995

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INSULIN-LIKE GROWTH FACTOR-I AND THE ENDOCRINE PANCREAS by  CAROLE ANNE McCLEAN B . S c , The University of British Columbia, 1986 M.Sc., The University of Saskatchewan, 1989 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E O F DOCTOR O F PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Physiology We accept this thesis as conforming to the required standard  UNIVERSITY OF BRITISH C O L U M B I A December 1995 © C a r o l e Anne McClean , 1995  In presenting  this  degree at the  thesis  in partial  University of  freely available for reference copying  of  department  this or  publication of  British Columbia, and study.  by  his  or  her  the  The University of British ^Columbia Vancouver, Canada  Ipfifl  fag*  requirements  I agree  that the  I further agree  representatives.  may be It  this thesis for financial gain shall not  Department  DE-6 (2/88)  of  thesis for scholarly purposes  permission.  Date  fulfilment  that  advanced  Library shall make it  by the  understood be  an  permission for extensive  granted  is  for  allowed  that without  head  of  my  copying  or  my written  1 1  ABSTRACT Insulin-like growth factor-I (IGF-I) has been determined to have both mitogenic and insulin-like metabolic effects on many tissue types. IGF-I has previously been shown to be secreted from, and to stimulate fi-cell activity in fetal pancreas. The present studies were performed in order to determine if IGF-I was present in adult endocrine pancreatic tissue, and if local IGF-I influenced insulin secretion in the adult islet. IGF-I immunoreactivity was present only in endocrine tissue and double staining techniques revealed that IGF-I immunoreactivity was colocalized only with insulin immunoreactivity. These results were confirmed at the electron microscope level, where IGF-I immunoreactivity was localized to B-cells, and furthermore, only to mature B-cell granules. In order to study pancreatic IGF-I concentrations and secretion, an IGF-I RIA was established, and a method of separating IGF-I from its binding proteins was validated. G H , the primary stimulator of IGF-I secretion from the liver and many other tissues, did not influence IGF-I secretion from either perfused pancreas, or isolated islet preparations. Nor did the insulin secretagogues GIP and glucose. Islet extracts subjected to high performance liquid chromatography (HPLC) revealed an elution peak, corresponding to that of the IGF-I standard, which indicated a low level of IGF-I in adult rat islets (approximately 1 pg/islet). IGF-I receptors were found to be present in islet tissue, as indicated by autoradiographic as well as by  125  I-IGF-I binding studies. IGF-I stimulated insulin secretion  from isolated adult rat islets, particularly at the highest level tested (170.0 nM). This effect was dependent on a stimulatory levels of glucose (8.9, 17.8 mM). Furthermore, low concentrations of IGF-I (1.7 nM, 17.0 nM) potentiated maximal GIP stimulated (10 nM) insulin secretion. Given findings by others that IGF-I can inhibit insulin secretion from the adult islet, it is proposed that in vitro study conditions influence the physiology of pancreatic IGF-I. The stimulatory effect of IGF-I on insulin secretion found in the present study suggests a fetal-like response of fl-cells to IGF-I and it is proposed that the inclusion of fetal serum in islet culture medium may result in adult islets showing fetal characteristics.  Ill  T A B L E OF CONTENTS Page ABSTRACT  ii  T A B L E O F CONTENTS  iii  LIST O F TABLES  viii  LIST O F FIGURES  ix  ACKNOWLEDGMENTS  xii  1. INTRODUCTION  1  1.1. INSULIN-LIKE GROWTH FACTOR-I (IGF-I)  1  1.1.1. HISTORY A N D S T R U C T U R E O F IGF-I  1  1.1.2. SYNTHESIS A N D SECRETION OF IGF-I  4  a) IGF-I gene expression b) Hormonal regulation of IGF-I synthesis and secretion  4 7  c) Physiological and pathophysiological regulation of IGF-I  10  1.1.3. IGF-I BINDING PROTEINS  11  a) IGF-I binding protein-1 (IGFBP-1) b) IGF-I binding protein-2 (IGFBP-2) c) IGF-I binding protein-3 (IGFBP-3) d) IGF-I binding protein 4-6 (IGFBP-4, -5, -6) e) Influence of binding proteins on IGF-I detection in tissues and fluids 1.1.4. IGF-I RECEPTOR BINDING  12 13 13 15  a) Type 1, Type 2 and insulin receptors b) Intracellular signalling via the Type 1 IGF receptor c) Comparison between the IGF-I and insulin receptors d) Regulation of the IGF-I receptor 1.1.5. ACTIONS OF IGF-I a) Endocrine, autocrine and paracrine routes of action b) Metabolic activity of IGF-I c) Mitogenic activity of IGF-I d) Effects of IGF-I on tissue damage e) IGF-I and diabetes  16 18 18 18 20 21 23 23 24 25 28 30  iv  1.2. INSULIN AND THE ENDOCRINE PANCREAS 1.2.1. A N A T O M Y  OF T H E ENDOCRINE PANCREAS  32 32  a) Endocrine cell types  33  b) Anatomy of pancreatic islets  34  1.2.2. G R O W T H A N D  D E V E L O P M E N T OF T H E A D U L T E N D O C R I N E PANCREAS  1.2.3.  R E G U L A T I O N OF INSULIN SYNTHESIS A N D S E C R E T I O N  a) Synthesis of insulin b) Regulation of insulin synthesis and secretion  1.3. IGF-I AND THE ENDOCRINE PANCREAS 1.3.1. IGF-I  A N D ISLET C E L L DIFFERENTIATION A N D PROLIFERATION a) IGF-I and differentiation and proliferation of fetal and neonatal islets b) IGF-I and differentiation and proliferation of adult islet tissue  1.3.2. I N F L U E N C E  37 38 38 41  47 47 47 49  OF IGF-I O N INSULIN SECRETION IN  THE ADULT  50  a) IGF-I receptors in the endocrine pancreas b) Effects of IGF-I in vitro c) Effects of IGF-I in vivo  50 50 51  1.3.3. SYNTHESIS  A N D PRESENCE OF IGF-I IN T H E E N D O C R I N E PANCREAS  52  a) Synthesis of IGF-I in the endocrine pancreas b) Presence of IGF-I in the endocrine pancreas  52 52  1.4. HYPOTHESIS AND OBJECTIVES  2. MATERIALS AND METHODS 2.1. MATERIALS  55 55  2.1.1. ANIMALS 2.1.2. C H E M I C A L S ,  54  55 SUPPLIES A N D I M M U N O R E A G E N T S  55  V  2.2. IGF-I R A D I O I M M U N O A S S A Y A N D BINDING P R O T E I N EXTRACTION  55  2.2.1. IGF-I RADIOIMMUNOASSAY  55  a) Iodination of I G E J b) Purification of I - I G F - I c) Primary antisera dilution for RIA d) Secondary antisera dilution for RIA e) IGF-I standard curve f) IGF-I radioimmunoassay 2.2.2. IGF-I BINDING PROTEIN E X T R A C T I O N  55 56 58 58 59 59 60  a) Use of C-18 cartridges b) Formic acid and acetone extraction c) Sephacryl column chromatography of IGF-I  60 61 62  1 2 5  2.3. E N D O C R I N E P A N C R E A T I C IGF-I  2.3.1. STUDIES O N T H E SECRETION OF P A N C R E A T I C IGF-I a) In situ perfusion of rat pancreas b) Isolation and culture of rat islets c) Characterization of rat islet extract by high performance liquid chromatography (HPLC)  64 64 64 65 67  2.3.2. I M M U N O C Y T O C H E M I C A L D E T E C T I O N O F P A N C R E A T I C IGF-I  68  a) Tissue preparation for light microscopy b) Tissue preparation for electron microscopy c) Immunostaining for light microscopy d) Immunostaining for electron microscopy e) Specificity testing of the IGF-I antibody  68 69 69 70 71  2.4. E F F E C T O F IGF-I O N T H E R A T E N D O C R I N E P A N C R E A S  2.4.1. IGF-I RECEPTOR BINDING  72 72  a) Autoradiographic detection of IGF-I receptors  72  b) Receptor binding with isolated rat islets  72  2.4.2. Conditions for the study of insulin secretion from isolated rat islets  73  a) Islet insulin secretion studies b) Measurement of insulin in medium and cell extracts c) Statistical analysis  73 74 76  vi 3. R E S U L T S 3.1. M E A S U R E M E N T O F IGF-I IN R A T TISSUES A N D F L U I D S  77 77  3.1.1. T H E R A T IGF-I RIA  77  3.1.2. SEPARATION O F IGF-I BINDING PROTEINS  82  A . Reverse phase C\8 Sep-Pak extraction B . Formic acid/acetone extraction  82 84  a) Recovery and measurement of rat IGF-I b) Gel filtration chromatography of tissues and serum  84 85  3.1.3. R E L E A S E OF IGF-I F R O M R A T E N D O C R I N E P A N C R E A S  93  A . In situ perfusion of rat pancreas B . Isolated rat islet secretion studies C . IGF-I in islet and 6-cell extracts  93 93 94  a) Immunoreactive IGF-I determined by RIA b) Immunoreactive IGF-I identified by slot blot  94 94  3.1.4. H P L C C H A R A C T E R I Z A T I O N OF R A T ISLET IGF-I  96  A . Peptide standard elution B . H P L C of rat islets C . H P L C of rat serum  96 96 97  3.2. L O C A L I Z A T I O N O F IGF-I IN T H E P A N C R E A S  99  3.2.1. SPECIFICITY OF T H E IGF-I ANTIBODY  99  A . Displacement of tissue IGF-I immunoreactivity  99  B . Immunoblotting studies  104  3.2.2. L O C A L I Z A T I O N O F IGF-I IN T H E R A T P A N C R E A S  106  A . Immunolocalization of IGF-I at the light microscope level B . Immunolocalization of IGF-I at the electron microscope level 3.2.3. L O C A L I Z A T I O N O F IGF-I IN T H E P A N C R E A S O F O T H E R SPECIES 3.3. IGF-I R E C E P T O R S IN R A T P A N C R E A S  106 110 116 118  3.3.1. P R E S E N C E O F IGF-I RECEPTORS  118  3.3.2. L O C A L I Z A T I O N OF IGF-I RECEPTORS  118  vii  3.4. EFFECT OF IGF-I ON INSULIN SECRETION F R O M ISOLATED RAT ISLETS  121  3.4.1. E F F E C T OF IGF-I O N INSULIN SECRETION WITH INCREASING G L U C O S E CONCENTRATIONS  121  3.4.2. E F F E C T OF INCREASING CONCENTRATIONS O F IGF-I O N INSULIN SECRETION IN T H E PRESENCE O F G L U C O S E A N D GIP STIMULATION  121  3.4.3. E F F E C T O N A N IGF-I M O N O C L O N A L ANTIBODY O N INSULIN SECRETION IN T H E PRESENCE O F G L U C O S E A N D IGF-I STIMULATION  123  A. Effect of an IGF-I monoclonal antibody on glucose stimulated insulin secretion B. Effect of an IGF-I monoclonal antibody on IGF-I stimulated insulin secretion  123  3.4.4. E F F E C T OF IGF-I O N INSULIN SECRETION F R O M ISOLATED ISLETS OVER A 24 HOUR INCUBATION PERIOD  127  3.4.5. E F F E C T OF A N IGF-I VARIANT (DES-AMIDO IGF-I) O N INSULIN SECRETION F R O M ISOLATED ISLETS IN T H E P R E S E N C E O F G L U C O S E STIMULATION  127  3.4.6. E F F E C T OF A P O L Y C L O N A L G L U C A G O N A N T I B O D Y O N INSULIN SECRETION F R O M ISOLATED ISLETS IN T H E P R E S E N C E OF IGF-I A N D G L U C O S E STIMULATION  130  123  4.0 DISCUSSION  132  BIBLIOGRAPHY  152  APPENDIX I  C H E M I C A L SOURCES  188  H  L A B W A R E SOURCES  190  HI I M M U N O R E A G E N T S  191  viii LIST O F T A B L E S NUMBER  TITLE  PAGE  1  Expression of IGF-I mRNA in adult rat tissues  6  2  Islet endocrine cell types  33  3  IGF-I RIA method (summary)  60  4  Recovery of I-IGF-I from formic acid/acetone extracted perfusate and rat serum  84  Recovery of IGF-I from formic acid/acetone extracted serum and perfused pancreas  84  Islet incubation conditions for the detection of secreted IGF-I  93  Islet incubation conditions for the detection of secreted IGF-I and insulin  94  5 6 7  125  ix LIST O F F I G U R E S  NUMBER  TITLE  PAGE  1  Amino acid sequence of rat IGF-I  3  2  Self displacement of  57  3  Calibration of a Sephacryl S-200 (1 X 30 cm) column with standards  63  Purification of I-IGF-I by column chromatography (G-25 Sephadex)  78  Purification of I-IGF-I by column chromatography (G-50 Sephadex)  79  IGF-I antibody binding of I-IGF-I with increasing primary antibody dilutions  80  4 5 6 7  125  I-IGF-I  125  125  125  Effect of second antibody dilution on precipitation of antibody bound  125  I-IGF-I  81  8  The IGF-I RIA standard curve  9  IGF-I RIA of serial dilutions of formic acid/acetone extracted rat serum Elution profile of rat pancreatic extracts incubated with I-IGF-I separated by column chromatography (Sephacryl S-200 HR)  88  Elution profiles of rat serum incubated with I-IGF-I separated by column chromatography (Sephacryl S-200 HR)  89  Elution profile of rat hepatic extract incubated with I-IGF-I separated by column chromatography (Sephacryl S-200 HR)  91  Immunoreactive IGF-I in rat serum following column chromatography (Sephacryl S-200 HR)  92  Detection of IGF-I in an immunoblot of 6-cell extracts  95  H P L C elution profile (30 - 45 % acetonitrile gradient) of IGF-I immunoreactivity detected by RIA in rat islet extracts and rat serum  98  Immunoreactive IGF-I in a section of Wistar rat pancreas  100  10  83  86  125  11  125  12  125  13  14 15  16  X  17  18  19  20  21  22 23 24 25 26  27  28 29 30  31  Absence of IGF-I immunoreactivity in rat pancreas sections following the incubation of the IGF-I antibody with IGF-I  101  Presence of IGF-I immunoreactivity in rat pancreas sections following the incubation of the antibody with insulin  102  Presence of IGF-I immunoreactivity in human pancreas sections, with and without incubation of the IGF-I antibody with insulin  103  Specific detection of IGF-I by the IGF-I monoclonal antibody indicated by an immunoblotting study with homologous peptides  105  Pancreatic localization of IGF-I and colocalization with other hormones determined using double staining techniques with fluorescent detection in serial sections of rat pancreas  107  Immunoreactive IGF-I in the rat pancreas detected using two different polyclonal IGF-I antibodies  108  Immunoreactive IGF-I in pancreatic sections from neonatal (day 1) and 7 day old rats  109  Thin sections of pancreas indicating IGF-I immunoreactivity in rat beta-cells  111  Absence of IGF-I immunoreactivity in microthin sections of non-J3-cells and exocrine rat pancreas  112  Specificity of the IGF-I monoclonal antibody indicated by preincubation with IGF-I and insulin prior to application to microthin rat pancreatic section s  113  Thin section of a rat fi-cell exhibiting insulin immunoreactivity  114  Attempt to colocalize IGF-I and insulin immunoreactivity in the rat 6-cell  115  Presence of IGF-I immunoreactivity in sections of porcine, but not ovine pancreas  117  Localization of IGF-I receptors rat pancreas demonstrated using autoradiographic techniques  120  Localization of IGF-I receptors in sections of rat ovary demonstrated using autoradiographic techniques  120  xi  32  33 34  35 36 37  38  Effect of IGF-I (170 nM) on insulin secretion from isolated rat islets in the presence of increasing glucose stimulation  122  Effect of increasing concentrations of IGF-I on GIP stimulated insulin secretion from isolated rat islets  124  Effect of increasing concentrations of an IGF-I monoclonal antibody on glucose stimulated insulin secretion from isolated rat islets  125  Effect of an IGF-I monoclonal antibody on IGF-I stimulated insulin secretion from isolated rat islets  126  Effect of long term IGF-I incubation (24 h) on insulin secretion from isolated rat islets  128  Effect of an IGF-I variant (des-amido IGF-I) on glucose stimulated insulin secretion from isolated rat islets  129  Effect of a polyclonal glucagon antibody on IGF-I stimulated insulin secretion from isolated rat islets  131  xii ACKNOWLEDGEMENTS  I would like to sincerely thank my supervisor, Dr. C . H . S . Mcintosh for his assistance and support throughout the course of my studies and the preparation of this manuscript. I would also like to thank Dr. A . M . J. Buchan for her time, suggestions and interest in my studies and extend thanks to those in her lab, Narinder Dhatt and Kevin Gibbon for sharing their technical expertise. The guidance of Dr. R . A . Pederson, and the technical assistance from his lab is also much appreciated. I am also grateful to Dr. J.C. Brown for his advice and for extending his unique form of encouragement, and to Dr. K . Kwok for his assistance and suggestions. Many thanks to previously graduated students, Eric Accili, Sue Curtis, Christine Scott, Bruce Verchere as well as to C. A. Courneya for sharing their prolific scientific and conversational abilities, thereby making work into play. I sincerely thank my parents for their never-failing support and encouragement, without which I could never have accomplished so many things in so little time. Special thanks to my mother for her prodding, her apparently boundless energy in the re-assumption of her child care abilities, and for sharing her priceless experience with me. Finally, thanks to my family, Travis and Mitchell who put the world in perspective, and to Chris, who unfailingly helps with everything, and is the crazy glue that holds the whole thing together.  1  1. INTRODUCTION  1.1. INSULIN-LIKE GROWTH FACTOR-I (IGF-I) 1.1.1. HISTORY AND STRUCTURE OF IGF-I IGF-I is a member of the insulin family of peptides which includes insulin, IGF-I, IGFII, proinsulin and relaxin. These peptides show a high degree of structural homology and share some functional similarities (Blundell and Humbel, 1980). The family originated from gene duplication from a common ancestral gene, given that each member of the family is encoded for by a single separate gene, whose internal organization is similar (Haley et al, 1987; Bell et al, 1986; Rotwein et al, 1986; Soares et al, 1986). Insulin-like growth factors were first identified as a result of three biological activities discovered in serum. Salmon and Daughaday gave the term sulphation factor to a serum factor which stimulated the uptake of sulphate into cartilage (Salmon and Daughaday 1957). This factor was present in the serum of normal but not of hypophysectomized rats, and sulphate uptake was not induced by growth hormone (GH) alone. Another set of experiments indicated that serum exerted far more insulin-like activity on adipose and muscle tissue than could be accounted for by insulin itself (Froesch et al, 1963), as measured in the recently developed radioimmunoassay (RIA). This excess activity was named NSILA (non-suppressible insulinlike activity). The third growth promoting activity in serum was termed M S A (multiplication stimulating activity), based on the ability of a serum factor to stimulate cell proliferation in certain cell lines, (Dulak and Temin 1973). These growth promoting and insulin-like properties of serum were collectively termed somatomedin (Daughaday et al, 1972) until 1978 when IGF-I and IGF-II (MSA) were isolated from a Cohn fraction of human plasma (Rinderknecht and Humbel 1978a, 1978b). Since its isolation from serum, and with newly available techniques, the amino acid and gene sequences of IGF-I have been determined. Human IGF-I, isolated from plasma, is a single chain, 70 amino acid peptide (mw 7649) of a basic nature containing three disulphide bonds. IGF-I has 61 % amino acid sequence homology with IGF-II. Like proinsulin, IGF-I retains the connecting C-peptide sequence  2 (cleaved in insulin processing) but, unlike proinsulin, it has an eight amino acid C-terminal extension called the D region. There exists 50% amino acid sequence homology with insulin in the A and B domains, a similarity which results in low affinity receptor cross reactivity, with the binding regions being in the B domain (Humbel 1984). Rat IGF-I was isolated from serum (Rubin et al, 1982) but the full amino acid sequence only determined in 1987, using a complementary D N A (cDNA) sequence (Murphy et al 1987a; Shimatsu and Rotwein 1987) (Figure 1). Three amino acid differences exist between human and rat IGF-I. The primary structure of IGF-I has also been determined from the respective cDNA sequences in the pig (Tavakkol et al, 1988), cow (Honegger and Humbel 1986), mouse (Bell et al, 1986; Stempein et al, 1986) and chicken (Dawe et al, 1988). There exists a high degree of sequence conservation between species, with the human, cow and pig showing identical structures. Chicken and mouse sequences differ from that of the human in 1 and 4 positions respectively. An important role for the hormone is suggested by these species similarities in IGF-I sequence, and by the evolutionary conservation of IGF-I immunoreactivity and biological activity in some birds and reptiles, the Atiantic Hagfish (a primitive cyclostome) and protochordates (Reinecke et al, 1991; 1993b). The insulin-like tertiary structure of the A - and B-domains has been highly conserved over at least 550 million years of vertebrate evolution (Nagamatsu et al, 1991). Several IGF-I variants have been isolated from human Cohn fractions (Van den Brande et al, 1990b). These forms arise either from allelic variation or from incomplete posttranscriptional processing. Such forms may exhibit increased or decreased IGF-I activity. In particular, a truncated IGF-I variant with a three amino acid N-terminal cleavage (des-amido IGF-I) was more potent than IGF-I in stimulating protein and D N A synthesis in cell cultures (Francis et al. 1988) and protein synthesis in vivo in rats (Tomas et al, 1992; 1991; Ballard et al, 1987). Activity of IGF-I variants is dependent on their affinities for the IGF-I binding proteins and for the IGF-I receptor. Peptides with specific amino acid substitutions in the  3  B-Domain  C-Domain  Figure 1. Amino acid sequence of rat IGF-I (from Clemmons 1989). Boxes indicate areas of amino acid sequence homology with insulin. Dashed lines separate the peptide domains.  4  B-domain (Oh et al, 1993; Bayne et al, 1988) and des-amido IGF-I exhibit reduced affinity for IGF-I binding proteins. Amino acid substitutions in the B-domain (position 24) (Cascieri et al, 1988) or deletions in the C-domain (Bayne et al, 1988) reduce IGF-I receptor binding, indicating the importance of these regions. In addition, the substitution of glycine for isoleucine in position 2 of the A-domain in both insulin and IGF-I was found to reduce both insulin receptor binding, and insulin-like effects (stimulation of lipogenesis), as well as the growth promoting effects (stimulation of thymidine incorporation) of insulin and of a synthetic IGF-I/insulin hybrid (Zong et al, 1990).  1.1.2. S Y N T H E S I S A N D S E C R E T I O N O F IGF-I a. IGF-I gene expression The IGF-I gene is located on human chromosome 12, and appears to be a single copy gene in both the rat (Shimatsu and Rotwein 1987) and the human (Rotwein et al, 1986). Both human (Ullrich et al, 1984) and rat (Shimatsu and Rotwein 1987) genes consist of 6 exons (designated l a - 5) and 4 introns. Exon 2 codes for the signal sequence and the B-domain, and exon 3 for the C , A , and D , and part of the B and E domains. Exons 4 and 5 code for an untranslated 3' region and for alternately spliced regions of the E domain. Alternative R N A splicing in both the human and the rat results in two IGF-I precursor proteins, designated IGFIA and IGF-IB, which exhibit differences in the untranslated E-domain. IGF-IA is identical in both species, wheras IGF-IB differs in the human and rat as a result of different splicing. The IGF-IA precursor has been found to be the predominant prohormone in all rat tissues studied (Loweetal. 1988). Studies in the rat suggest that the IGF-IA prohormone contains two potential N glycosylation sites which are absent in the IGF-IB prohormone (Bach et al, 1990). Although it is uncertain whether IGF-IA is glycosylated in vivo, glycosylation of other hormones is associated with increased cellular stability, and with intracellular transport systems. It has also been shown that the E-peptide domain of the IGF-IB prohormone contains an amide sequence which is capable of promoting growth in cell lines, via its own receptor (Seigfried et al, 1992).  5  Multiple size IGF-I mRNA transcripts exist in both human and rat tissues. These consist of two major class sizes ranging from 0.8 - 1.2 and 7.0 - 7.5 kb, with heterogeneity occurring primarily in the 3' untranslated region, representing different polyadenylation sites (Hepler et al, 1990), as well as in the 5' untranslated region. The significance of such heterogeneity may be due to the varying stability of different transcripts. It has been proposed that the half life of 7.0 - 7.5 kb transcripts is much shorter than that of smaller transcripts (Hepler et al, 1990). It has also been suggested that 5* heterogeneity may result in altered translation efficiency (Foyt et al, 1992; Adamo et al, 1991). Although less is known about IGF-I mRNA processing in non-mammalian species, IGF-I cDNAs from chickens (Kajimoto et al, 1989), frogs (Kajimoto and Rotwein 1990; Shuldiner et al, 1990), salmon (Cao et al, 1989) and hagfish (Nagamatsu et al, 1991) have been sequenced. In all species except the hagfish, IGF-I mRNA is expressed in many tissues, with the IGF-IA sequence showing high regional homology to the human sequence. The IGFIB prohormone is absent in lower vertebrates. Varying levels of IGF-I mRNA transcripts, of different sizes, have been found in all rat tissues tested, as reviewed by Rechler and Nissley (1992) (Table 1). The highest abundance of IGF-I mRNA is present in rat liver consistent with the hypothesis that this organ is the primary contributor to circulating IGF-I. Tissue extracts, however, reveal that the concentrations of IGF-I are similar in liver, lung, kidney and testes (D'Ercole et al, 1984). Studies in adult male little mice (lit/lit) also exhibit widespread tissue IGF-I mRNA expression, with the highest levels detectable in the liver, and the pancreas exhibiting the highest non-hepatic expression (Mathews et al, 1986). Very little is known regarding the intracellular processing of IGF-I, or of its storage and secretion from any cell type. A study in Sertoli cells isolated from immature pig testes revealed a vectoral secretion of IGF-I (Skalli et al, 1992). F S H was found to stimulate IGF-I secretion from the basolateral compartment, while stimulating the secretion of IGF-I binding protein-3 (IGFBP-3) from the apical compartment, although the significance of these observations was not clear.  Tissue Liver Uterus Lung Ovary Kidney Heart Testes Pancreas Stomach/intestine S k e l e t a l muscle Mammary g l a n d Brain Placenta Cartilage Pituitary  Species a, b, c a a, c a a,b, c a, c a, c a, c a a a a, c b a a  a - rat b - human c - mouse Table 1. Tissue expression of IGF-I mRNA  M o d i f i e d from Rechler and N i s s l e y  (1992)  7  b. Hormonal regulation of IGF-I synthesis and secretion Growth hormone (GH) is the primary regulator of IGF-I synthesis and secretion by the liver, as well as by other tissues. Regulation Of IGF-I by G H has been studied most intensively, in vivo and in vitro in the rat, with particular attention given to hepatic regulation. G H increased hepatic IGF-I mRNA transcripts of all sizes when administered to rats in vivo, and increases were greater in hypophysectomized, than in normal, rats (Hynes et al, 1987). In vitro, the incubation of G H with cultured rat hepatocytes reveals a dose dependent effect on IGF-I mRNA levels (Kachra et al, 1991; Norstedt and Moller 1987). G H also influences multiple sizes of IGF-I mRNA in cultured hepatocytes. The increase in IGF-I mRNA, which occurs in response to a bolus G H injection, is rapid (3-9 hours) and has been found in normal and hypophysectomized animals, increases in the latter being greater (Hynes et al, 1987; Murphy et al, 1987a; Mathews et al, 1986). G H also increases IGF-I mRNA in primary neural cell cultures (Rotwein et al, 1988) and in a preadipocyte cell line (Doglio et al, 1987). Due to the rapid effect of G H it has been suggested that the hormone acts via transcriptional mechanisms, with a direct link between the G H signal transduction pathway and nuclear events (Bichell et al, 1992). Whereas a single dose of G H is found to result in a rise in all IGF-I mRNA transcripts (Bichell et al, 1992), long term (4 day) exposure to G H results in a greater level of IGF-I mRNA with alternatively spliced exon 5. This is perhaps a result of the increased stability of this mRNA form (Lowe et al, 1988). G H also induced a size specific increase in IGF-I transcripts when administered to lit/lit (GH deficient) mice (Norstedt and Moller 1987). Consistent with the somatomedin hypothesis, and the above observations, perfusion of isolated rat liver with G H stimulates secretion of IGF-I (Schwander et al, 1983). G H administration has been found to increase serum IGF-I levels within 3 hours of administration in hypophysectomized rats (Murphy et al, 1987a). The pattern and level of G H is important to IGF-I synthesis and secretion. Administration of G H to hypophysectomized rats has been found to produce maximal IGF-I secretion with multiple daily doses, whereas continuous infusion of G H is thought to be necessary for optimal up-regulation of hepatic G H receptors  8  (Maiter et al, 1988). Pulsatile G H infusion in male rats was found to be more effective than a continuous G H infusion in stimulating IGF-I mRNA levels in skeletal muscle and rib growth plate, resulting in greater longitudinal growth (Isgaard et al, 1988). Different secretion profiles of G H are thought to influence sexual characteristics in rats, in which the male exhibits a pulsatile secretion, and the female a more continuous level with a higher baseline (Maiter et al, 1988). G H administration results in elevated IGF-I levels in humans (Bengtsson et al, 1993; Jorgensen et al, 1991; Rudman et al, 1990) and G H treatment of G H deficient humans can be used to normalize circulating IGF-I levels (LeRoith et al, 1992). G H administration to stimulate growth is widespread for the treatment of children of short stature due to a variety of reasons including panhypopituitarism, postcranial irradiation, Turner's syndrome, Down's syndrome, intrauterine growth retardation, chronic renal failure, achondroplasia and idiopathic growth deficiency (Strobel and Thomas 1994). Such treatment is effective, and is dependent upon the integrity of the GH/IGF-I axis, with both subcutaneous and intramuscular injections being equally effective in the stimulation of IGF-I levels (Beshyah et al, 1991). G H also stimulates IGF-I gene transcription in rat extra-hepatic tissues including muscle (Turner et al, 1988), heart, lung, kidney (Roberts et al, 1987; Murphy et al, 1987b), pancreas (Hynes et al, 1987), uterus (Murphy and Friesen, 1988), costal cartilage (Isgaard et al, 1988), pituitary (Fagin et al, 1989b), white adipose tissue (Peter et al, 1993) and preadipocytes (Gaskins et al, 1990). The relative levels of different IGF-I mRNA transcripts produced in response to G H is tissue specific. This likely influences individual tissue synthesis of IGF-I due to differing stability of the various mRNA forms (Roberts et al, 1987; Lowe et al, 1988; 1987). IGF-I content in tissues, and levels in the circulation, are sensitive to several other hormones in addition to G H , and are also strongly correlated with general metabolic status. Insulin is thought to be an important regulator of IGF-I secretion from the liver, as shown in vitro in the rat (Scott and Baxter 1986; Maes et al, 1986; Scott et al, 1985; Atkinson et al, 1984; Kogawa et al, 1983; Binoux et al, 1980; Kogawa et al, 1982; Daughaday et al, 1976).  9  Studies of cultured chicken hepatocytes indicate that G H and insulin may act synergistically to stimulate IGF-I production (Houston and O'Neill 1990). In vivo studies in diabetic rats given hepatic portal vein infusions of insulin, showed increased IGF-I secretion and increased IGF-I sensitivity. This was thought to be, in part, a result of altered binding protein regulation and it was proposed that the direct delivery of insulin from the B-cells was as important for growth as for glycemic regulation (Griffen et al, 1987). Humans exhibiting insulin dependent hypoglycemia have been shown to have elevated circulating IGF-I, which is thought to be a result of increased hepatic stimulation (Labib et al, 1990). Insulin has also been found to stimulate IGF-I synthesis in rat aorta (Murphy et al, 1990). Other trophic hormones which stimulate IGF-I synthesis in various tissues include platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) in fibroblasts (Clemmons and Shaw 1986), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa and Sertoli cells (Maruo et al, 1988; Chatelain et al, 1987a, 1987b; Tres et al, 1986; Hammond et al, 1985), thyroid hormones in hepatocytes (Harakawa et al, 1990) and pituitary cells (Fagin et al, 1989a), and glucagon in hepatocytes (Kachra et al, 1991). Dexamethazone, on the other hand, decreases IGF-I mRNA levels in rat neuronal and glial cells (Adamo et al, 1988). In addition, IGF-I synthesis has been found to be regulated by the protonocogene cmyb (Reiss et al, 1991). In two cell lines (Balb/c3T3, Tk-tsl3), the constitutive expression of c-myb R N A increased the expression of both IGF-I and of its receptor, suggesting a potential mechanism for oncogene regulation of cell growth. The intracellular mechanisms by which the above factors might regulate IGF-I mRNA levels have been studied in cultured fibroblasts from the skin of fetal rats (Hovis et al, 1993). The incubation of phorbol esters and plasma peptides in such cultures, demonstrate that the activation of protein kinase-C and the elevation of intracellular C a  2 +  result in decreased IGF-I  mRNA transcript levels (Lowe et al, 1992). The inhibitory effect of elevated intracellular Ca  2 +  on IGF-I mRNA transcript levels in cultured fibroblasts has also been demonstrated  using the C a  2 +  ionophore A23187, and thapsigargin (Hovis et al, 1993). In osteoblast  10  cultures, increased intracellular cAMP stimulated IGF-I synthesis, whereas elevated C a  2 +  had  little effect on IGF-I synthesis, (McCarthy et al, 1990) indicating that intracellular pathways regulating IGF-I synthesis may be tissue dependent.  c. Physiological and pathophysiological regulation of IGF-I Factors such as age, nutritional status, and tissue injury, as well as pathophysiological states such as obesity and diabetes, influence circulating, and perhaps tissue, IGF-I levels by complex and as yet unclear mechanisms. Age related changes in circulating IGF-I in the human are characterized by low levels at birth (70 ng/ml, Hall et al, 1988) with a steady increase to a peak at puberty (300 ng/ml, Merimee et al, 1991), whereupon levels decrease 2.5 times by the third decade and continue to decrease slowly throughout life (LeRoith et al, 1992). Protein and calorie deficiencies alone have both been found to decrease circulating IGF-I levels (Isley et al, 1983), and low fasting levels are rapidly returned to normal upon refeeding (Phillips et al, 1988b). Decreased IGF-I activity is evident in liver perfusate following in situ hepatic perfusion (with Krebs-Ringer bicarbonate buffer), of previously fasted rats (3 d) (Vassilopoulou-Sellin et al, 1980). Hepatic IGF-I secretion has been found to be associated with, and followed by, comparable changes in IGF-I tissue content and circulating levels of IGF-I (Goldstein and Phillips 1989). This indicates the influence of metabolic status on, and the importance of the liver in, the maintenance of peripheral IGF-I levels. These studies also indicated that IGF-I binding proteins from the liver were influenced by fasting and this will be discussed later. Uncontrolled insulin dependant diabetes mellitus (IDDM), in which insulin levels are not maintained, resulting in high or intermittently high glucose levels, has long been known to result in growth retardation in both humans and rats (Phillips and Orawski 1977). In this condition, both peripheral G H and IGF-I levels are reduced in rats. Studies involving the in situ perfusion of rat liver indicated reduced IGF-I activity was released in streptozotocin induced diabetic animals (Vassilopoulou-Sellin et al, 1980). IGF-I is also reduced in poorly  controlled diabetic humans, despite elevated circulating G H (Mauras et al, 1991). In the streptozotocin-induced diabetic rat model, both circulating IGF-I and IGF-I mRNA content in liver, kidney and lung have been found to be reduced (Fagin et al, 1989a). Limited studies of obese humans have revealed normal to elevated circulating IGF-I levels despite reduced G H levels, as well as reduced levels of IGF binding protein-1 (IGFBP1) (Weaver et al, 1990). More recent observations indicate that the increased levels of IGF-I observed in obese humans are likely related to intra-abdominal fat rather than to overall obesity, and are therefore linked to hyperinsulinemia and insulin resistance associated with such fat distribution (Rasmussen et al, 1994). Obese Zucker rats also exhibit reduced G H levels, and normal IGF-I levels with associated hyperinsulinemia (Leidy et al, 1993). Obesity in fat Zucker rats is associated with resistance to IGF-I, as well as to insulin (Jacob et al, 1992), however the situation is unclear in humans as glucose levels have been found to be inversely related to IGF-I levels in obesity (Rasmussen et al, 1994). Elevated IGF-I levels are indicative of, and can also be used to diagnose, acromegaly (Clemmons et al, 1979). In contrast, G H deficiency in children has a high correlation with reduced peripheral IGF-I (Ranke et al, 1988; Lee and Rosenfeld 1987). Localized IGF-I levels are also highly responsive to areas of specific tissue damage. An interaction of the many factors mentioned previously, in addition to other growth factors, appear important in the increased IGF-I mRNA levels which occur in response to local tissue damage as indicated in rat aorta following removal of the endothelial layer (Cercek et al, 1990; Murphy et al, 1990). Local increases in IGF-I mRNA in response to injury have also been found in peripheral nerves (Hansson et al, 1986a) and skeletal muscle (Jennische et al, 1987).  1.1.3. IGF-I BINDING PROTEINS Another important factor which influences IGF-I status is the presence of IGF-I binding proteins. Only an estimated 2-4 % of IGF-I is thought to exist unbound in human plasma (Mukku 1991; Guler et al, 1987; Zapf et al, 1986). IGF-I was initially isolated from a human Cohn fraction as a larger molecular weight molecule, a phenomenon which was later found to  12 be due to the presence of binding proteins (Baumann et al, 1986; Hintz and Liu 1979; Hintz et al, 1974). Similar binding proteins were also found to be present in rat serum (Moses et al, 1976). To date, six structurally homologous IGF-I binding proteins have been identified, and their primary structures determined in both the human and rat as described by Shimasaki and colleagues (1991a; 1991b). These proteins have been designated IGFBP-1 - IGFBP-6. Their synthesis is tissue specific, dependent on physiological status and, when complexed to IGF-I, they can either enhance or inhibit the hormone's actions on target cells (Elgin et al, 1987; Knauer and Smith 1980). IGF-I binding proteins exert influences on IGF-I activity by prolonging its half life, altering distribution of the hormone between body fluids and cell surfaces, and modulating transport and exposure of IGF-I to various tissues. Most studies on IGF-I binding proteins have been performed in the rat, however binding proteins are present in all mammalian, but not non-mammalian, sera (Daughaday et al, 1985).  a. IGF-I binding protein-1 (IGFBP-1) IGFBP-1 was isolated by different groups, from many different tissues and fluids, and later found to be the same protein, as reviewed by Lee and colleagues (1993). This binding protein is largely unsaturated in serum (Lee et al, 1989). The free binding protein, as well as the IGF-I/IGFBP-1 complex is thought to be cleared rapidly either by tissues, or by serum proteases (Lee et al, 1993), exhibiting a half life of 30 - 50 min in rats. The highest levels of IGFBP-1 mRNA have been found in the liver (Julkunen et al. 1988). IGFBP-1 is present in ng/ml levels in human serum, and unlike all other IGF-I binding proteins, levels are rapidly responsive to metabolic changes. Unlike IGFBP-3, synthesis of IGFBP-1 is G H independant and exhibits a diurnal rhythm (Drop et al, 1984). As with IGFBP-3, evidence exists for both enhancement and inhibition of IGF-I activity by IGFBP-1. The primary function of IGFBP-1, appears to be in the inhibition of IGF-I receptor binding. Circulating levels are stimulated by hypoglycemia (Cotterill et al, 1988; Yeoh and Baxter, 1988), and fasting (Busby et al, 1988; Hall et al, 1988) and are inhibited by insulin (Brismar et al, 1988; Suikkari et al, 1988). It has been proposed that elevations in IGFBP-1 levels could prevent the potentially strong  13  hypoglycemic effect of the 2-4 % free IGF-I, in the non-fed state (Lee et al, 1993). In addition, both IGFBP-1 and -2 have been found to equilibrate in extravascular spaces, and may thereby influence IGF-I translocation (Young et al, 1992). Serum IGFBP-1 levels are elevated by poor nutritional status and in diabetes (Brismar et al, 1988; Suikkari et al, 1988). Levels rise during pregnancy in humans, reaching a peak at the end of the first trimester and remaining at this level until parturition (Wang et al, 1991).  b. IGF-I binding protein-2 (IGFBP-2) IGFBP-2 is the predominant binding protein in the fetal circulation, declines at birth, but is still detectable in both adult rat (Gargosky et al, 1990) and human (Clemmons et al, 1991) serum. IGFBP-2 mRNA has been detected in human liver, kidney and brain (Binkert et al, 1989). Concentrations of IGFBP-2 are highest in the central nervous system in adult humans, where IGF-II is the predominant growth factor (Rosenfeld et al, 1989; Hossenlopp et al, 1986). This binding protein has a higher affinity for IGF-II, than it does for IGF-I (Rutanen and Pekonen, 1990). It is therefore likely that IGFBP-2 is most important in fetal IGF regulation (Wang and Chard 1992) and in the central nervous system in the adult. Like IGFBP-1, serum IGFBP-2 is under the acute influence of hormones and metabolites (Ooi et al, 1990; Orlowski et al, 1990).  c. IGF-I binding protein-3 (IGFBP-3) Very little circulating IGF-I exists unbound, and almost all is complexed in a 150 kDa form with IGFBP-3 (Hardouin et al, 1987). IGFBP-3 migrates as a 42/38 kD doublet upon SDS-PAGE electrophoresis and associates with an acid labile subunit of 85 kD. This binding protein is present in mg/1 levels in human serum and has a high affinity for IGF-I. IGF-I and IGFBP-3 form a stable complex increasing the half life of IGF-I from that of its free state (15 min) to approximately 15 hours in the rat (Zapf et al, 1986). This binding protein complex is thought to inhibit the insulin-like effects of high levels of IGF-I which has been estimated to possess a hypoglycemic potential 50 times that of circulating insulin (Yeoh and Baxter, 1988;  14  Froesch et al, 1985). The primary source of IGFBP-3 is thought to be the liver, due to the high level of mRNA found in this tissue, although IGFBP-3 mRNA has also been found in most adult rat tissues (Schwander et al, 1991; Wood et al, 1988). As mentioned earlier, the extent of IGF-I binding to this protein suggests an inhibitory role, but it has also been demonstrated that membrane associated IGFBP-3 exists which potentiates the mitogenic effects of IGF-I (Conover et al, 1990). Such an effect has been found following preincubation of IGFBP-3 with cultured fibroblasts, followed by incubation with IGF-I (Conover 1992; DeMellow and Baxter 1988). IGFBP-3 is thought to associate closely with cells and potentiate type I IGF receptor responsiveness, not via an increase in receptor number, but perhaps by optimizing the presentation of IGF-I to its receptor. Such enhancement of IGF-I activity by IGFBP-3 has been found in a breast cancer carcinoma cell line (Chen et al, 1994). Binding proteins 2 and 3 enhanced IGF-I stimulation of D N A synthesis in M C F - 7 cancer cells. In addition, IGFBP-3 prevented IGF-I receptor down-regulation in response to IGF-I, and is therefore thought be an important mediator of IGF-I activity in breast carcinoma proliferation. Like IGF-I, the synthesis of hepatic IGFBP-3 is stimulated by G H (Blum and Ranke 1990). IGFBP-3 responds to metabolic status in a similar manner to IGF-I , with levels decreasing during states of poor nutrition in the rat (Umezawa et al, 1991) and in poorly controlled I D D M in rats and humans (Batch et al, 1991; Baxter and Martin 1986). In addition, IGFBP-3 specific protease activity has been identified in pregnancy and in severely ill patients. This enzyme activity cleaves IGFBP-3, reducing its affinity for IGF-I, thereby increasing the bioavailability of circulating IGF-I to tissues (Davies et al, 1991). In pregnant rats, IGFBP-3 levels are reduced by over 50 % in late pregnancy (Gargosky et al, 1993). Human studies, however, indicate that IGFBP-3 is still the primary IGF-I reservoir in the pregnant state (Wang and Chard 1992).  15  d. IGF-I binding proteins 4-6 (IGFBP-4, - 5 , -6) Little is known regarding IGFBP4-6 in comparison to the previously discussed proteins. IGFBP-4 has been isolated from adult rat serum and migrates as a doublet of 36/32 kD when reduced and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Shimonaka et al, 1989). Suggested physiological roles for this binding protein include IGF-I regulation in the central nervous system (Cheung et al, 1991) and paracrine or autocrine IGF-I regulation related to bone growth (LaTour et al, 1990). The highest levels of IGFBP-4 mRNA are present in the liver, followed by the brain (Shimasaki et al, 1989). In addition, IGFBP-4, and -5 are the only two IGF binding proteins to be synthesized in rat granulosa cells, where they may play a role in follicular development (Liu et al, 1993). IGFBP-5 migrates as a doublet (31 kD) on non-reducing SDS gels. The proteins primary structure has been determined for both the human and the rat showing a high degree of conservation between the species (97 %) (Kiefer et al, 1991a; 1991b; Shimasaki et al, 1991c). IGFBP-5 mRNA is present in all rat tissues studied, with several exhibiting a higher level of expression than the liver, and the highest level of expression present in the kidney (Shimasaki et al, 1991c). Very little of the protein is present in the circulation. IGF-I has been found to stimulate the secretion of this binding protein from rat fibroblasts (Camacho-Hubner et al, 1991). The affinity of IGFBP-5 for IGF-I is higher than that of any other binding protein, and is approximately 50 X that of the IGF-I receptor. IGFBP-5 has been shown to potentiate the proliferative actions of IGF-II in osteoblasts (Bautista et al, 1991), and a mixture of IGFBP-5 and -6 have been found to enhance the stimulation of mitogenesis in human osteosarcoma cells (Andress and Birnbaum 1991). IGFBP-6 (26 kD) has been found in human cerebrospinal fluid (Roghani et al, 1989) and serum (Shimasaki et al, 1991b; Zapf et al, 1990), and its mRNA has been found in all tissues studied including testis, intestine, adrenal, kidney, stomach, spleen, heart, lung, brain and liver, leading to the conclusion that this protein is ubiquitous (Shimasaki et al, 1991a). The highest level of IGFBP-6 mRNA is present in neural tissue. This, and the high  16  concentrations of IGFBP-6 in cerebrospinal fluid suggests a role in the regulation of IGF activity in the brain. This binding protein has a higher affinity for IGF-II than for IGF-I, further supporting such a role (Shimasaki et al, 1991a).  e. Influence of binding proteins on IGF-I detection in tissues and fluids Another important influence of IGF-I binding proteins is the fact that, as with other hormone associated proteins, they markedly influence the detection and measurement of IGF-I if not removed prior to assay (Breier et al, 1991). With regard to attempts to measure IGF-I it has been stated: "For a while I thought that God had invented BPs to confuse IGF-I determinations. The presence in plasma of free BPs makes it difficult to measure IGF in whole serum and one has to get rid of the free BPs. In addition, if one wishes to measure the total amount of IGF that is present in plasma, you must break the complex apart by acid or some other means." (Hintz 1990).  Early attempts to measure serum IGF-I by RIA indicated levels two to three times lower than those detected following acid/ethanol extraction of serum, as binding proteins compete with antisera for IGF-I (Daughaday et al, 1980). It has been determined that IGFBP3, and aggregates formed following acidification of samples, produce the greatest interference in RIA, whereas IGFBP-1, at the levels found in plasma, does not appear to influence IGF-I RIA results when the IGF-I polyclonal rabbit antiserum (UBK-487) provided by NIFf is used (Breier et al, 1991). Various methods for the removal of IGF-I binding proteins have been developed but, unfortunately, many have been found to produce inconsistent results when used with different tissues, fluids and species, thus making it difficult to compare different experiments. The most widely accepted method involves the acidification of the samples, followed by column chromatography under acidic conditions (Daughaday and Rotwein, 1989). This method was shown to be reliable for the detection of IGF-I in both tissues and fluids in a wide range of species, but is expensive and time consuming, and therefore unsuitable for a large number of samples.  17  Another widely used method involves acidification of samples followed by separation over reverse phase cartridges. The use of cartridges is less time consuming than conventional chromatography, but provides inconsistant removal of binding protein interference from serum of several species, including the human, rat and sheep (Breier et al, 1991; Mesiano et al, 1988; Daughaday et al, 1987). Even less time consuming and less expensive is a widely used method originally developed for human plasma which involves acidification of samples in an acid/ethanol mixture, followed by the separation of precipitated binding proteins by centrifugation (Daughaday et al, 1980). This method, however, was not found to be reproducible for measurements in several pathophysiological conditions, nor in other species including the rat (Mesiano et al, 1988; Daughaday et al, 1987). Recently proposed modifications of the above method have included cryoprecipitation of binding proteins. One such method involves incubation of plasma with HCl/ethanol, followed by precipitation of binding proteins (Breier et al, 1991). This method was found to produce results consistent with acid chromatography extractions, with high recoveries for plasma from sheep, rat, mouse and man. Another more rapid modification, verified for the separation of binding proteins from IGF-II, involves mixing serum samples with formic acid then with acetone. Binding proteins are precipitated by centrifugation at 4 ° C (20 min, 3500 X g) (Bowsher et al, 1991). This method was found to result in high recovery of standard, with low levels of residual binding proteins [determined by F P L C (fast protein liquid chromatography) analysis]. The method was effective with human serum, urine and rat serum. It has become apparent that the method of IGF binding protein extraction must be verified by the investigator when used in different tissues and fluids for the species to be studied, as variation between laboratories using the same procedures can be significant (Bang 1995).  18  1.1.4. IGF-I RECEPTOR BINDING a. Type 1, Type 2 and insulin receptors In addition to high affinity binding to the Type 1 IGF receptor, IGF-I exhibits low affinity binding to the insulin and Type 2 IGF receptors. Cross reactivity of IGF-I and insulin, for each other's receptor, is approximately 100-1000 X lower than for the hormone specific receptor (Massague and Czech, 1982; Redder et al, 1980). IGF-I also exhibits binding to the Type 2 receptor, which is distinct from either the Type 1 or insulin receptors, and is identical to the mannose 6-phosphate receptor. IGF-II shows high affinity binding to both the Type 1 and 2 receptors, and it has been proposed that the cellular effects of both IGF-I and II are mediated by the Type 1 receptor, and the Type 2 receptor serves to internalize and degrade IGF-II (Czech 1989). Like IGF-I, the Type 1 IGF receptor appears well conserved throughout evolution, and has been found in primitive vertebrates (Drakenberg et al, 1993). Molecular cloning revealed high sequence homology (95 %) between the rat and human IGF-I receptors (Ullrich et al, 1986).  b. Intracellular signalling via the Type 1 IGF receptor The IGF-I receptor (Type 1) exhibits both functional and structural homology with the insulin receptor. Both are transmembrane glycoproteins containing two ligand binding extracellular alpha-subunits, each linked by disulphide bonds to a transmembrane 6-subunit (Ullrich et al, 1986). Each U-subunit undergoes tyrosine autophosphorylation upon ligand binding to the alpha-subunit, which is followed by further activation of receptor kinase activity (Zhang and Roth 1992) and tyrosine phosphorylation of a cytosolic protein, initially termed ppl85 (based on migration on SDS-PAGE). This protein, now called insulin receptor substrate-1 (IRS-1), has been identified as a receptor kinase substrate for insulin, IGF-I and interleukin-4 receptor signalling (Prager et al, 1992; Treadway et al, 1991; Soos et al, 1990). IRS-1 has been sequenced and cloned, showing 90 % homology between the rat and human amino acid sequences (Sun et al, 1991). The actions of IRS-1 have been studied most widely  19  with regard to insulin receptor signalling. Although some of insulins biological responses are elicited directly from B-subunit phosphorylation, most cellular actions are thought to be mediated following activation of IRS-1 via the activated insulin receptor (White and Kahn 1994) . As reviewed by White and Kahn (1994), upon insulin receptor binding, IRS-1 recognizes and binds proteins containing src homology 2 domains (SH2), including phosphoinositol-3 (PI-3) kinase (Backer et al, 1992), src homology phosphotyrosine-2 (SHPTP-2, or Syp) (Kuhne et al, 1993), growth factor receptor-bound protein-2 (Grb-2) (Skolnik et al, 1993) and an adaptor protein, Nek (Lee et al, 1994). Activation of these proteins leads to the activation of other cytosolic proteins, in many signalling pathways, resulting in the pleiotropic biological effects of insulin and IGF-I binding. At least two IRS-1 dependent signalling cascades have been determined for both insulin and IGF-I (Jones and Clemmons 1995) . One follows binding of IRS-1 to the p85 regulatory subunits of PI-3 kinase resulting in the formation of phosphatidylinositol-3 phophate (PIP3), which can serve as a signal for cell growth. Like platelet derived growth factor (PDGF), another mitogenic hormone, IGF-I receptor binding has been found to increase the association of PIP3 to its receptor (Yamamoto et al, 1992) as mentioned previously. Although the role of the kinase is unclear for IGF-I, PI-3 kinase has been shown to phosphorylate phosphatidylinositol (PI) to generate a signal for cell growth (Cantley et al, 1991; Carpenter and Cantley 1990; Morrison et al, 1990; 1989; Whitman et al, 1988). Consistent with this, the stimulation of PIP3 activity in bovine luteal cells by both IGF-I and insulin was associated with increased protein and D N A synthesis (Chakravorty et al, 1993). Another cascade involves the binding of IRS-1 to Grb2 resulting in phosphorylation of extracellular signal-related kinases ERK-1 and ERK-2 [MAP (mitogen activating protein) kinases] via activation of the GTP binding protein Ras, which can transmit signals to the nucleus. IGF-I as well as insulin has also been shown to stimulate tyrosine phosphorylation of endogenous c-CRK, a SH2 domain containing-protein which may also be involved in Ras activation (Beitner-John son and LeRoith 1995).  20  Protein Kinase-C (PK-C) has been implicated in the regulation of cell growth, differentiation and secretion (Nishizuka 1986), and has also been found to be activated by IGFI in cultured fibroblasts (Martelli et al, 1991), chondrocytes (Taylor et al, 1988), myocytes (Farese et al, 1989) and neuronal cells (Cortizo et al, 1991). In contrast, IGF-I was found to act independently, but synergistically with PK-C to stimulate cell proliferation in cultured neonatal pulmonary artery smooth muscle (Dempsey et al, 1990), on survival of cultured embryonic chicken ganglia (Crouch and Hendry 1991), and on mitogenesis in cultured rat astrocytes (Tranque et al, 1992) further indicating that IGF-I receptor binding can activate more than one intracellular pathway, at least in these cell types. Like many other hormones, neurotransmitters and growth factors, IGF-I binding to receptors has been found to influence intracellular calcium ( C a ) levels. IGF-I induced 2+  oscillations of intracellular C a  2 +  in cultured BALB/c 3T3 cells as monitored by fura-2  measurements (Kojima et al, 1992). Increases in intracellular C a  2 +  in a cultured rat thyroid  cell line was found to result from the interaction of the IGF-I receptor with a cation channel, via a pertussis toxin sensitive G-protein (Takada et al, 1993). IGF-I, as well as other growth factors, also influences protooncogene levels in several cell types. The synthesis of protooncogenes is thought to be important in cellular regeneration (Makino et al, 1984) and differentiation (Lachman and Skoultchi, 1984).  c. Comparison between IGF-I and insulin receptors Despite the fact that the IGF-I and insulin receptors have much in common, including 50 % amino acid sequence homology, intrinsic 6-subunit tyrosine kinase activity and IRS-1 mediated signalling pathways, ligand binding to the IGF-I and insulin receptors result in distinct biological effects (Prager and Melmed 1993). These differences may be explained by differing, as yet unidentified, intracellular pathways (Jones and Clemmons 1995). Signal divergence is possible both before and after IRS-1 activation, and may be dependent on the relative cellular concentrations of SH2-containing proteins, the relative tissue concentrations of each receptor and the low cross reactivity of IGF-I and insulin for each others receptor.  21  Both the IGF-I and insulin receptors are ubiquitously expressed to a varying extent in different tissue types, and both have isoforms, with differing alpha- and beta-subunits (LeRoith et al, 1994). The two predominant IGF-I receptor isoforms are ubiquitously expressed, exhibit equal affinity for IGF-I, yet have a two fold variation in receptor signalling as indicated by autophosphorylation, substrate phosphorylation and thymidine uptake (Condorelli and Smith 1992). One insulin receptor isoform is expressed exclusively in liver, kidney, muscle fat and fibroblast tissue, and has a reduced affinity for insulin compared to the other isoform (McClain 1991; Yamaguchi et al, 1991). In addition, IGF-I/insulin receptor hybrids have been shown to exist in cultured cells, as well as in placental membranes and fetal skeletal muscle (Jones and Clemmons 1995). These receptors exhibit characteristics of the Type I IGF receptor showing a greater affinity for IGF-I than for insulin. The physiological significance of such receptors, however, is unclear.  d. Regulation of the IGF-I receptor In general, Type 1 tissue IGF receptor numbers are increased in response to decreased peripheral IGF-I levels, and decreased in response to elevated peripheral IGF-I levels (up- and down-regulation respectively) (Rechler and Nissley 1985). The extent to which tissue IGF-I receptor levels are sensitive to G H is unclear, but IGF-I receptor binding is decreased by G H excess (Venkatesan and Davidson, 1990). In vivo, fasted rats exhibit low peripheral IGF-I levels, and increased IGF-I binding is seen in many tissues including the kidney, stomach, testes, lung and heart (Lowe et al, 1989). Increased IGF-I binding to skeletal muscle is also seen in protein deficient rats (Dardevet et al, 1991). The extent of IGF-I receptor down regulation is tissue dependent, with the brain seemingly unresponsive to peripheral IGF-I (Lowe et al, 1989). It has been suggested that such increases or decreases in IGF-I binding may also be a result of altered receptor affinity in the presence of high or low peripheral IGF-I (Lowe et al, 1989). In vitro, cultured human skin  22  fibroblasts and lymphocytes (IM-9) (Rosenfeld 1982), exhibit Type 1 receptor down-regulation when preincubated with IGF-I. Type 1 receptor regulation is also evident in pathophysiological states. The decreased peripheral IGF-I levels seen in streptozotocin-induced diabetic rats is thought to contribute to the increased Type 1 receptor concentration and increased gene expression typical in this model (Werner at al, 1990). In addition, Type 1 receptor expression in isolated mesangial cells of diabetic mice (db/db) was found to be increased under elevated glucose concentrations (Oemar et al, 1991). Both studies implicate IGF-I receptor upregulation in the kidney with the development of diabetic nephropathy. The fate of IGF-I following receptor binding has been studied in chicken embryonic lens cells (Soler et al, 1990). It was determined, using electron microscopy to study the internalization and sub-cellular distribution of IGF-I, that there existed cell specific differences in the cellular localization of IGF-I, which included cytoplasmic and nuclear locations. IGF-I was internalized by epithelial cells following receptor binding and localized to endosomes, Golgi apparatus, lysosomes, and nucleus. It was unclear, in the study, whether the uptake was associated with binding proteins, or whether endocytosis occurred via coated or uncoated invaginations of the cell membrane. Many hormones including G H , prolactin, insulin and somatostatin-28 have been shown to translocate to the nucleus following receptor binding and internalization in various cell types, and these peptides may directly influence transcriptional regulation (Lobie et al, 1994). The biological consequences of IGF-I internalization are as yet unclear. It has, however, been proposed that IGF-I and IGF-II may exert effects through an association with a nuclear anti-oncogene product, retinoblastoma protein (RB) (Radulescu 1994), thereby inhibiting its activity. Furthermore, expression of IGFBP-3 cDNA in a murine fibroblast cell line has been shown to inhibit cell growth, and it is proposed that IGFBP-3 may protect RB by binding nuclear IGF-I. Indeed RB has been shown to have a bipartite nuclear localization signal (NLS) which is essential for its activity, and is also present in the amino acid sequences of human and porcine IGFBP-3.  23  IGF-I uptake and internalization have also been studied in a kidney cell line (opposum kidney; OK) since the proximal cells are a site of peptide hormone metabolism, including that of IGF-I (Fawcett and Rabkin 1995). Following receptor binding, IGF-I was internalized into endosomes, then lysosomes in which degradation took place. Some large molecular weight products were present following degradation, and it was uncertain as to whether these were biologically active within the cell. It was also found that the pathways of IGF-I and insulin degradation appeared to be separate, and that IGF-I was relatively resistant to the processing enzymes. Furthermore, it was found that although IGFBP-3 inhibited binding of IGF-I to the cell receptors, once in the cell the presence of the binding protein enhances its degradation. Evidence therefore exists for the uptake of IGF-I, in several cell types, although the activity of the intact hormone, or of its degradation products following internalization, remain unclear.  1.1.5. A C T I O N S O F IGF-I  a. Endocrine, autocrine and paracrine routes of action The effects of IGF-I can be mediated as a classic hormone (endocrine), being secreted into the circulation, primarily from the liver, and carried to other tissues. The uptake of intravenously infused IGF-I in sheep revealed tissue differences indicating that the endocrine activity of IGF-I may be tissue specific (Hodgkinson et al, 1991). IGF-I also exerts effects on tissues when secreted locally. Given the large number of tissues and cell types found to contain IGF-I mRNA, and the IGF-I and insulin receptors, autocrine and paracrine routes of action are clearly important (Phillips and Vassilopoulou-Sellin 1986; Underwood et al, 1986). The trophic effects of systemically administered IGF-I, in hypophysectomized rats, is weak, suggesting that such effects are primarily locally mediated (Skottner et al, 1987), and circulating IGF-I may, in fact, play a more important role in the maintenance of metabolic status, acting in an insulin-like manner (Sara and Hall 1990). The potential for intracellular autocrine actions of IGF-I have also been investigated, since other peptide hormones, including interleukin-3 and platelet derived growth factor (PDGF), have been found to exert effects without interacting with a cell surface receptor  24 (Bejcek et al, 1989; Dunbar et al, 1989; Keating et al, 1988). Studies of a non-transformed rat follicular cell line suggest, however, that interaction of IGF-I with its cell surface receptor is required for D N A synthesis, at least in this model (Dai et al, 1992a; 1992b). Evidence included the inhibition of IGF-I activity by an IGF-I monoclonal antibody, and the lack of D N A synthesis with the expression of a non-secreted form of IGF-I. As discussed previously, the IGF-I binding proteins influence the activity of IGF-I, and this affects both the endocrine and the locally mediated actions. Although IGFBP-3 is the predominant form in the circulation, the different binding proteins show tissue specific expression (Shimasaki et al, 1991b; Orlowski et al, 1990), and therefore also exhibit peripheral and local activities. Given that the actions of IGF-I on target tissues are thought to be dependent on the relative levels and affinities of binding proteins and the receptor for IGF-I (Sara and Hall 1990), both endocrine and locally mediated actions of IGF-I are dependent on its association with a particular binding protein.  b. Metabolic activity of IGF-I IGF-I can exert metabolic and/or mitogenic effects, on many tissues, the extent of which is dependent on physiological status, as well as on the predominance of insulin or IGF-I receptors in a given tissue, as noted in the discussion of receptors (section 1.1.4.). It is thought that the potentially large hypoglycemic effect of IGF-I is buffered by its association with IGFBP-3 (Yeoh and Baxter 1988; Froesch et al, 1985). The overall effect of an intravenous administration of IGF-I is, however, an acute decrease in plasma glucose in rats (Jacob et al, 1989; Zapf et al, 1986), dogs (Giacca et al, 1990) and humans (Guler et al, 1988). In rats, IGF-I has approximately 50 % of the potency of insulin in mediating this effect (Schmitz et al, 1991; Jacob et al, 1989). Infusions of high concentrations of IGF-I are thought to saturate the binding protein capacity in the short term, with the increased free hormone exerting the hypoglycemic effect (Zapf et al, 1986). It has been shown, however, that even a bolus injection of IGF-I results in very little free hormone after 15 min (Ballard et al, 1991) and that chronic IGF-I administration still has a hypoglycemic effect, even though bound to  IGFBP-3 (Schoenle et al, 1982). This indicates that the binding protein does not completely inhibit the acute metabolic effects of IGF-I, and may act by prolonging the hormones' half life (Schoenle et al, 1982). IGF-I has been found to stimulate glucose uptake and metabolism in many tissues including rat and human muscle (Dimitriadis et al, 1992; Dohm et al, 1990; Zapf et al, 1979), rat and human adipocytes (Kern et al, 1989; Bolinder et al, 1987; Zapf et al, 1986) and rat liver (Jacob et al, 1989). Other insulin-like effects of IGF-I include increases in lipoprotein lipase levels in cultured adipocytes (Kern et al, 1989), expression of GLUT-1 and GLUT-4 glucose transporters in muscle (Duclos et al, 1993), rat hepatic glucose uptake with suppressed hepatic glucose synthesis, and decreased tissue proteolysis, the latter resulting in decreased circulating amino acid levels (Jacob et al, 1989). The influence of IGF-I on glucose metabolism may be mediated predominantly by either the insulin or the Type 1 IGF receptor depending on the tissue and the species. The regulation of glucose uptake and metabolism is mediated primarily by the IGF-I receptor in muscle tissue (Yu and Czech 1984; Pogi et al, 1979). In adipose tissue IGF-I activity is primarily insulin receptor mediated in both humans and rats (Venkatesan and Davidson 1990; Sinha et al, 1989; Caro et al, 1988; Bolinder et al, 1987) where IGF-I receptors have been identified in humans, but not rats (Van Wyk et al, 1985; Massague and Czech 1982; Zapf et al, 1981). IGF-I receptors have been identified in both human (Burguera et al, 1991; Lamas et al, 1991) and rat (Lamas et al, 1991; Venkatesan and Davidson 1990; Caro et al, 1988) hepatic tissue, but the predominant mediator of the hormones' actions is unclear (Kolaczynski and Caro 1994). This may prove to be of importance in the glucose metabolism in pathophysiological states with insulin receptor or post-receptor deficiencies leading to the possibility of IGF-I therapy (Froesch and Hussain 1993).  c) Mitogenic activity of IGF-I The mitogenic activity of IGF-I results in overall growth both pre- and post-natally. Both IGF-I and insulin, and their receptors have been identified at very early embryological  26 stages in amphibians, avians and mammals, as reviewed by de Pablo and coworkers (1990). Both IGF-I and insulin are active in growth and differentiation of embryonic cells, and antibodies to insulin and its receptor have been found to have a deleterious effect on embryonic growth and survival (Liu et al, 1989). Post-natal mitogenic IGF-I activity has been highly investigated due to its importance in overall growth. IGF-I stimulates both somatic and linear growth in hypophysectomized rats (Guler et al, 1988; Skottner et al, 1987; Schoenle et al, 1985; 1982), mice (van Buul-Offers et al, 1986; Smeets and van Buul-Offers 1983) and in intact rats (Philipps et al, 1988a; Hizuki et al, 1986) over a period of several days. Such increases are less, however, than those induced by equimolar doses of G H (Skottner et al, 1987; Schoenle et al, 1985; 1982), and the relative importance of IGF-I and G H in the attainment of linear and somatic growth remains under investigation. The effect of IGF-I on long bone growth has been widely studied in an attempt to determine the relative contributions of IGF-I and G H . Both IGF-I and G H have been found to stimulate [^H]-thymidine incorporation in monolayer cultures of epiphyseal chondrocytes (Ohlsson et al, 1992; Madsen et al, 1983), but appear to act at different stages of cellular maturation. It has been shown that each hormone influences a different cell type in such cultures (Bentham et al, 1993). The effects of IGF-I on cartilage growth may be locally mediated, since an antibody to IGF-I has been shown to block the effects of G H in rat chondrocyte cultures (Scheven and Hamilton, 1991), and on in vitro G H infusion to the arterial supply of a rat hindlimb (Schlechter et al, 1986). Studies in transgenic mice, overexpressing IGF-I, either in the presence or absence of G H , revealed that IGF-I normalized body weight and linear growth in the absence of G H , and increased these parameters in the presence of G H , in comparison to normal littermates (Behringer et al, 1990). Organ weights (duodenum, pancreas, lung) were in proportion to body weight. Brain weight was proportionally higher in all animals over-expressing IGF-I, and liver weight lower in animals not expressing G H , indicating that both G H and IGF-I have specific growth promoting actions.  27  The growth promoting effects of IGF-I and G H appear to be dependent on several factors including binding proteins and other growth factors. Studies of protein deficient rats revealed that neither G H (Thissen et al, 1990), nor IGF-I alone, were able to restore growth parameters including organ and body weights, tail length, and widening of the tail epiphysis (Thissen et al, 1991). These observations are thought to result from G H and IGF-I resistance, as well as to decreased level of IGFBP-3 and the associated reduction in the half life of IGF-I. This is likely a result of nutritional status as studies in 4 week old protein deficient rats indicated that the normalization of circulating IGF-I was not sufficient to stimulate growth of certain organs, wheras growth of the spleen and kidney were enhanced (Thissen et al, 1991). Similarly, these effects were thought to be partially attributable to decreased IGFBP-3 levels, and a partial IGF-I resistance. The effects of IGF-I on cultured chondrocytes appear to be dependent on other growth factors, including fibroblast growth factor (FGF) (Demarquay et al, 1990). The mitogenic effects of IGF-I were found to be relatively weak in comparison to those of F G F , whereas IGF-I was found to stimulate chondrocyte differentiation, promoting the secretion of Type II collagen, while inhibiting Type I collagen secretion which occurs during the maturation of prechondroblasts to chondrocytes (Demarquay et al, 1990). IGF-I exerts widespread influences on many tissues and cell types in addition to its influences on bone, cartilage and overall linear growth. Much evidence exists for the stimulation of brain development (Ballotti et al, 1987; Shemer et al, 1987; Han et al, 1987a; Sara et al, 1983) and growth and differentiation of the the peripheral nervous system ((Nielsen et al, 1991; McMorris et al, 1990; Torres-Aleman et al, 1990; Shemer et al, 1987). IGF-I has also been shown to have trophic effects, stimulating mitosis or cellular differentiation, in many other tissue and cell types including cultured human placental cells (Bhaumick et al, 1992), 3T3 preadipocyte cells (Schmidt et al, 1990), adipocytes (Smith et al, 1988), myoblasts (Florini et al, 1991; Schmid et al, 1983) and osteoblasts (Schmid et al, 1984). IGF-I also stimulates mitogenesis and protein synthesis in cultured mesenchymal cells (Balk et al, 1984; Stiles et al, 1979), and proliferation of canine fundic epithelial cells (Chen et  28  al, 1991). Increased IGF-I mRNA has also been associated with intestinal mucosal hyperplasia seen in G H transgenic mice. In rats implanted with G H secreting tumours, both skeletal and cardiac muscle exhibited hypertrophy in the presence of elevated IGF-I mRNA levels (Turner et al, 1988). As noted previously, many of these actions are not mediated by IGF-I alone, but occur in the presence of other growth factors or hormones. IGF-I, for example, has been found to regulate thyroid function and growth, but is ineffective in the absence of T S H (Eggo et al, 1990; Tode et al, 1989). A role for IGF-I is also suggested in uncontrolled tissue growth. IGF-I receptors have been identified in several malignant cell types including Wilms' tumour, neuroblastoma, phaeochromocytoma, breast cancer and thryoid adenoma (Daughaday 1990; Vanelli et al, 1990). The mechanism by which IGF-I may act is seen in its effects on the cell cycle of various cell lines. Studies in human fibroblasts show that IGF-I increases the transition of cells from the Gj (initial growth) phase to the S phase (DNA synthesis). IGF-I has also been found to be an important factor in the survival of density-inhibited quiescent Balb/c-3T3 murine fibroblast cells (Tarn and Kikuchi 1990). In these cells, IGF-I also acts in the G phase, and x  progression to the S phase is dependent on the ras proteins (Lu and Campisi 1992). Studies in these cells indicate that nuclear pore size is decreased when cells are in quiescence, in comparison to the proliferative stage, thereby reducing the transport of macromolecules from the nucleus to the cytoplasm (Feldherr and Akin 1993). IGF-I was found to stimulate quiescent cells and increase nuclear pore size by acting as a progression factor as above, with epidermal growth factor (EGF), while PDGF was found to be a competence factor, stimulating the progression from the resting phase (G ) to G ^ 0  d. Effects of IGF-I on tissue damage IGF-I mRNA has been found to be increased in several tissues in response to injury, preceding repair. In response to a pancreatectomy, IGF-I mRNA is increased 4 fold in capillary endothelial cells after 3 days, coinciding with the peak mitotic indices of both 6- and  exocrine cells (Brockenbrough et al, 1988) and resulting in an abundance of proliferating ductules, from which pancreatic acinar and islet tissue develop. A similar pattern of IGF-I mRNA expression has been observed in regenerating muscle tissue (Edwall et al, 1989; Jennische et al, 1987), and rat kidney during compensatory renal hypertrophy (Fagin and Melmed 1987). Other tissues exhibiting increased IGF-I mRNA expression following injury include rat sciatic nerve following lesion (Hansson et al, 1986), rat ear epithelium following freezing (Lynch et al, 1987), post-ischemic rat kidney (Matejka and Jennische 1992) and disruption of rat aortic epithelium (Cercek et al, 1990). Locally elevated IGF-I, and treatment of injured tissues with exogenous IGF-I, stimulate tissue regeneration (Skottner et al, 1990), and differentiation (Matejka and Jennische 1992). IGF-I does not act alone in such actions, as many other growth factors have been studied including PDGF-2, transforming growth factor (TGF-alpha), E G F and fibroblast growth factor (FGF), and these show that combinations of growth factors have optimal effects on regeneration. A combination of PDGF-2 and IGF-I, or PDGF-2 and TGF-alpha were found to be the most potent stimulators in healing surgically induced porcine skin wounds (Lynch et al, 1989). As reviewed by Skottner and colleagues (1990), dependent on the tissue, growth factors have complementary roles in tissue repair, which may include mitogenic and chemotactic action of PDGF in the inflammation phase, stimulation of granulation tissue by F G F , increased proliferation of epidermal cells by E G F and stimulation of matrix synthesis by IGF-I. The mechanism by which IGF-I may influence tissue repair has been studied in rat vascular smooth muscle cells (Schini et al, 1994). Under normal physiological conditions, nitric oxide is produced constitutively, primarily by endothelial cells in blood vessels, and serves to inhibit vascular tone and growth while preventing platelet activation (Nathan 1992; Moncada et al, 1992). Following injury, nitric oxide synthase is increased under the influence of locally released interleukin-113 and tumor necrosis factor-alpha (Joly et al, 1992; Nathan 1992). IGF-I has been found to inhibit cytokine induced nitric oxide synthase, which is thought to allow tissue repair functions (Schini et al, 1994).  30  d. IGF-I and diabetes Decreased G H activity is associated with decreased growth potential seen in children with insulin-dependent diabetes mellitus (IDDM). Poorly controlled diabetic humans exhibit normal or high circulating G H levels, and reduced IGF-I levels (Lanes et al, 1985; Amiel et al, 1984; Horner et al, 1981; Merimee et al, 1979; Williams and Savage 1979; Birkbeck 1972; Hansen and Johansen 1970). As noted previously, circulating and tissue IGFBP's and IGF-I receptors are also influenced in the diabetic state. Insulin induced glycemic control partially restores IGF-I levels with little effect on G H (Asplin et al, 1989; Rieu and Binoux 1985; Amiel et al, 1984; Horner et al, 1981; Tamborlane et al, 1981; Kjeldsen et al, 1975) and results in near normal growth. In comparison to normoglycemic boys, those with I D D M treated with insulin, have been found to have normal G H levels, exhibiting altered amplitude and peak secretion and yielding decreased IGF-I levels (Nieves-Rivera et al, 1993). Both G H secretion profiles and IGF-I levels are normalized during puberty, yet G H clearance is decreased in late puberty. In rats, streptozotocin-induced diabetes results in the ablation of G H pulsatility (Robinson et al, 1987) and decreased circulating G H and IGF-I, yielding impaired growth (Yang et al, 1990). Insulin therapy restored hepatic IGF-I mRNA and circulating IGF-I and G H to near normal levels, yielding a positive correlation with increased growth rates (Yang et al, 1990). G H treatment, in contrast, does not increase IGF-I secretion (Scheiwiller et al, 1986). Alterations in IGF-I levels in streptozotocin diabetic rats are tissue specific, as indicated by decreased levels in liver and testes, with increased levels in the kidney and pituitary (Olchovsky et al, 1991). As a result, associated autocrine or paracrine effects include renal hypertrophy (Bach and Jerums 1990; Flyvjerg et al, 1990), also seen in humans (Morgensen et al, 1988), and suppression of G H synthesis and secretion from the pituitary (Olchovsky et al, 1991). Another complication of diabetes associated with IGF-I is retinopathy, as reviewed by Bach and Rechler (1992).  31  Attempts to treat diabetic humans exhibiting severe insulin resistance, (due to defective or severely reduced receptor numbers), with IGF-I, reveal that in such cases IGF-I has a glucose disposal rate of only 36 % that seen in normal subjects (Schoenle et al, 1991). In the short term, IGF-I treatment may be effective in normalizing glycemic control in non-insulin dependent diabetes, but the safety of long term treatment, given the potential for acromegaly and the above-mentioned diabetic complications is of concern (Bach and Rechler 1992).  32 1.2. INSULIN AND T H E ENDOCRINE PANCREAS Insulin, the primary glycemic regulator, is synthesized and released from the endocrine pancreas. One of the first findings (1889) that a substance in the pancreas was important in the regulation of diabetes was the demonstration, by German physiologists, Joseph von Mering and Oscar Minkowski, that the removal of the pancreas resulted in symptoms typical of Type I (insulin dependent) diabetes. It was later shown that damage to the pancreas did not always lead to diabetes, but the preservation of tiny islands of distinctive cells within the exocrine pancreas, were necessary to prevent it. These islands of tissue were discovered in 1869 by Paul Langerhans and termed islets of Langerhans. It was not Until 1921 that insulin was isolated from the pancreas by Frederick G . Banting, Charles H . Best, James J.R. Macleod and James B. Collip at the University of Toronto. Since 1922, insulin therapy has been used in the treatment of diabetes.  1.2.1. ANATOMY OF T H E ENDOCRINE PANCREAS Human and rat endocrine pancreases have been most thoroughly studied (Bonner-Weir 1991; Orci and Perrelet 1985), and species differences exist in the distribution of pancreatic endocrine and exocrine tissue, as well as within the endocrine tissue (islets), and the hormone distribution within the islets (Bonner-Weir 1991). In fetal and neonatal rat, pancreatic endocrine tissue comprises about 20-30 % of the total pancreatic mass. The percent of endocrine tissue increases about five times by adulthood, yet comprises only 1-2 % of the total pancreatic mass, due to a great proliferation of exocrine tissue (McEvoy and Madson 1980). Islet size varies greatly from a few to 5,000 cells in the rat (Bonner-Weir 1991), and up to 12,000 cells in the human (Weir and Bonner-Weir 1990). The majority of islets are small (< 160 /-im, 75 % total islet number, 15 % total islet volume), with the remainder being larger (> 250 itm) and comprising the greater islet volume (Bonner-Weir 1991).  33 a. Endocrine cell types Mammalian islets are made up of four major endocrine cell types:  Cell  type  Size  (nm)  Frequency (% i s l e t s )  Hormone  content  B(13)  250-350  60-80  I n s u l i n (TRH, CGRP p a n c r e a s t a t i n , 7B2, p r o l a c t i n )  A (a)  200-250  15-20  Glucagon, g l i c e n t i n (TRH, CCK, endorphin, GLP-1, PYY, h i s pro-DKP, 7B2, p a n c r e a s t a t i n )  D(S)  200-350  5-10  PP  120-160  15-20  2  Somatostatin (met-enkephalin, CGRP, p a n c r e a s t a t i n ) P a n c r e a t i c p o l y p e p t i d e (mete n k e p h a l i n , PYY, 7B2)  Table 2 . Islet endocrine cell types Modified from Bonner-Weir 1991 Hormones present determined by immunocytochemical techniques. Those in parentheses may be species and age dependent. Glucagon producing A-cells and PP cells are usually mutually exclusive, and dependent on the region of the pancreas. 1  2  With the advent of the electron microscope in the 1950's, the cellular morphology of the pancreatic islets has been clarified. Cell shapes and secretory granules are distinctive and have been reviewed by Bonner-Weir (1991). The 6-cells are polyhedral, and exhibit two types of granules. The mature secretory granules (containing crystalline insulin formations, with low proinsulin content) have an electron dense core surrounded by a light halo, and are encompassed by a thin membrane. The immature granules (containing primarily proinsulin) are moderately electron dense with the absence of a halo, and have a clathrin coat. A-cells are smaller than ft-cells, more columnar in shape and the secretory granules are homogeneous, exhibiting little species variation. Granules have a dense core, a moderately less dense halo and a tightly fitting membrane. D-cells are smaller than A-cells and have a more elongated dendritic shape. Secretory granules are uniformly dense within the granule limiting  34  membrane. PP granules show wide species variation, being elongated and electron dense in the human, and larger, spherical and variably electron dense in the dog and cat.  b. Anatomy of pancreatic islets The cellular composition of islets is dependent on the area within the pancreas. During embryological development the pancreas arises from two or perhaps three loci which merge, yielding a splenic or dorsal area (tail, body and part of head), and a duodenal or ventral area (most of the head). The dorsal area is glucagon (A-cell) rich and PP poor, and is perfused by the coeliac trunk via the gastroduodenal and splenic arteries, then drained by the main dorsal exocrine duct (Baetens et al, 1979; Orci and Perrelet 1985). The ventral area is PP rich and glucagon poor and is served by the superior mesenteric artery via the inferior pancreaticoduodenal artery, then drained by the main ventral exocrine duct. It has been suggested, in studies of isolated islets and of perfused regions, that islets in these different regions respond differently to various stimuli. Splenic islets, for example, have been found to release more insulin in response to glucose than do duodenal islets (Samols 1983; Trimble et al, 1982). In both the human and rat, as well as in other species, there exists a non-random distribution of islet cell types (Bonner-Weir 1991; Orci 1976; Orci and Unger 1975). Typically there is a core of fi-ceils, surrounded by a layer (2-3 cells thick) of non-B-cells. Reaggregation studies of dispersed rat islet cells reveals a return to this distribution, even when the cell type proportions are altered (Halban et al, 1987). Exceptions to this arrangement are found in the monkey and the horse, where there is a core of A - and D-cells surrounded by the fi-cells (Fujita 1973; Fujita and Murakami 1973). In the human, there appears to be a further subdivision, particularly within larger islets, where the islet itself is divided into regions by the vasculature and by connective tissue (Baetens et al, 1979; Orci and Perrelet 1985). Within these regions there exists a core of fi-cells surrounded by non-fi-cells. The islet is delineated from exocrine tissue by a capsule which consists of a single layer of fibroblasts and secreted collagen (Bonner-Weir 1991). This capsule is not continuous  35  between the exocrine and endocrine cells, but does overlay the efferent vasculature exiting the islet. Arterial circulation to the islet involves highly fenestrated capillaries in comparison to less fenestrated capillaries serving the exocrine tissue (Like 1970). The blood flow to the islets is high (10-20 % of the total to the pancreas) in proportion to the islet volume. The pattern of flow within islets is unclear. Studies using static casts and antibody infusions have indicated that arterioles penetrate to the B-cell core, and branch to capillaries which perfuse the core prior to perfusing the non-B-cells (Samols et al, 1988; Bonner-Weir and Orci 1982). Studies on the capillary system of the islet using injected dye in the rabbit (Fraser and Henderson 1980), by measuring flow in the mouse pancreas (Rooth et al, 1985) and by alternating anterograde and retrograde perfusions in the dog and rat (Stagner et al, 1988; Stagner and Samols 1986), indicated that there exists directional flow from the B-cells to the A and D cells. This suggests a primary influence of B-cell products on the A - and D-cells, with little influence in the other direction. These studies indicated that in larger islets, the capillaries coalesce to form collecting venules within the periphery of the islet. In smaller islets, the arterioles perfuse exocrine tissue prior to coalescing into collecting venules. These observations suggest that exocrine secretions have little effect on the islet, that large islets may have litte effect on the exocrine tissue, and that small islets may serve an insulo-acinar portal role (Bonner-Weir 1991). Other studies have revealed that the islet cell mantle is perfused prior to the B-cell core, permitting the perfusion of the B-cells with non-B-cell products (Ohtani et al, 1986; Nishino et al, 1985). Further observations of islet vasculature using fluorescent microspheres indicated that there exist numerous pathways of flow within islets, with the mantle often perfused prior to the B-cell core, suggesting that 6-cells could in fact be regulated by A - and D-cell products, in contrast to previous observations (Liu et al, 1993). This study also revealed a regulation of microsphere flow to, and within, the islet, suggesting that sphincters and vascular contractility influence the blood flow to areas within the islet and are potentially important regulators of glucose homeostasis.  36  fi-cells have been found to form a tube around a central arterial capillary, with each ficell exposing another face to a venous capillary (Bonner-Weir 1988). Like other epithelial cells, B-cells are thought to have an apical and a basolateral membrane, exhibiting efficient secretory response capabilities (Weir and Bonner-Weir 1990). Evidence exists for different subpopulations of 6-cells, some of which are more responsive to stimuli than others, and may be dependent on the location of the islet, or their location within the islet. As noted previously, islets located in areas of different embryonic origin respond differently to stimuli. In addition, it has been shown that the insulin secretory response to glucose varies between cells in cultures of individual fi-cells (Hiriart and Ramirez-Medeles 1991). In-vivo studies also revealed 6-cell heterogeneity based on responses of cells at the core of the islet, and those closer to the periphery (Stefan et al, 1987). Islets are innervated by sympathetic, parasympathetic, and intrinsic neurons (Ahren et al, 1986). Species variation in innervation have made the elucidation of neural effects on islet function difficult (Weir and Bonner-Weir 1990). It has been proposed that islet innervation may be involved in coordinated responses and communication seen by islets throughout the pancreas (Weir and Bonner-Weir 1990). Within each islet, cells can influence each other via blood borne (noted above), paracrine or junctional interactions. The possible influences of cellular interactions include insulin inhibition of A - and D-cells, glucagon stimulation of 6- and D-cells and somatostatin inhibition of A - and fi-cells. Little evidence exists for paracrine interactions in the islet, but it has been suggested that the potential exists for the diffusion of cell products through the interstitium (Bonner-Weir 1993). Non-fi-cells may, for example, exert influences on fi-cells at the periphery of the islet in large islets, and may have a greater influence in smaller islets where the core is smaller. Studies indicate synchronous electrical activity both within and between islets (Scott et al, 1981). Gap junctions between fi-cells, and between fi-cells and non-fi-cells have been identified which allow passage of small molecules (< 900 D) and these may also provide passage of electrical current allowing coordination of cellular activity (Meda et al, 1984).  37 1.2.2. G R O W T H A N D D E V E L O P M E N T O F T H E A D U L T E N D O C R I N E  PANCREAS  The potential for growth and development of the adult endocrine pancreas has been reviewed recently (Bonner-Weir and Smith 1994). Due to the importance of glucose regulation and insufficient insulin activity resulting in diabetes, the regulation of the B-cell population has been under the greatest study. During fetal growth, the B-cell population expands primarily from differentiation of progenitor cells. In the late fetal, neonatal and adult stages, however, the primary means of expansion is via replication of existing B-cells, a capacity which decreases with age (Swenne 1992; Hellerstrom et al, 1988). In fact almost all the adult B-cell population is established by the neonatal period (Vinik et al, 1992). It has been proposed that there is no evidence for proliferating activity of adult rat islet B-cells and that tritiated thymidine incorporation is due to non-islet cell proliferation (De Vroede et al, 1990). Other studies indicate that adult rodent B-cells do have a low level of replicating activity, with approximately 3 % of B-cells arising from preexisting B-cells in 24 hours (Bonner-Weir and Smith 1994; Hellerstrom et al, 1988). This level of replication would be able to double the number of B-cells in 30 days in the (unlikely) total absence of cell death, and is consistent with the observation that the rat B-cell mass doubles between 6 and 10 weeks (Bonner-Weir and Smith 1994). It is thought that the limitation on islet proliferation in later life is dependent on the islet cell population capable of regeneration. Glucose has been found to be able to stimulate proliferation only in cells in the 'proliferative compartment' of the cell cycle (Swenne 1982), which is about 10 % of the B-cell population in fetal rat islets and reduced to less than 3 % in young adult rats (Swenne and Andersson 1984). The remainder of the cells are in an irreversible G phase. The islet cell population is, however, flexible in adulthood and Q  dependent on insulin demands as seen in conditions such as pregnancy, obesity, aging and diabetes (Bonner-Weir and Smith 1994). Factors which stimulate 6-cell proliferation in neonatal and adult islets include glucose and its metabolites (Schuppin et al, 1992; Swenne 1992) and members of the G H family including placental lactogen (PL), prolactin (Prl) and G H (Brelje et al, 1993). In addition,  38  bombesin (Lehy and Puccio 1990), essential amino acids and fetal bovine serum (Swenne 1992) have been shown to stimulate /J-cell replication. It has been noted that the relative potential of the various factors shown to stimulate fi-cell proliferation is unclear, given all the different conditions used for various studies (Bonner-Weir and Smith 1994). It has been difficult to make comparisons as different studies have looked at islets at different stages of maturation, as whole islets, cells, clusters, or monolayers. In addition, until recently, B-cell proliferation was measured as the total tritiated thymidine in T C A precipitates of isolated islet homogenates (thymidine uptake), whereas a more clear indication is given by autoradiography, or with 5-bromodeoxyuridine (BrdU) immunostaining which clarifies the extent of ii-cell proliferation, with regard to that of other islet cell types. A further mechanism of increasing islet growth, is the hypertrophy of pre-existing ficells (Bonner-Weir and Smith 1994). Hypertrophy of cells is thought to be an adaptation to increased demands on insulin production, and can be stimulated to a certain extent by glucose with limited potential to increase insulin production (Borg and Andersson 1981).  1.2.3. R E G U L A T I O N O F INSULIN SYNTHESIS A N D S E C R E T I O N The pathway of insulin synthesis and secretion has been extensively studied and, in comparison to many other hormones, much is known regarding intracellular processing and regulation of secretion.  a. Synthesis of insulin Insulin is encoded by a 600 nucleotide mRNA transcript, which is translated into preproinsulin, a peptide of 109 amino acids. This peptide contains a 23 amino acid hydrophobic region which is thought to promote the association with the endoplasmic reticulum, aiding in the transport of the protein into the cisternal space of the rough endoplasmic reticulum and in vectoral transport for secretion. The mechanism of granular targetting for insulin is, however, unclear. It is possible that an amino acid sorting sequence is  present, or that there exists a spontaneous aggregation of proinsulin based on changes in the trans-golgi network, particularly pH, and C a  2 +  concentrations (Hutton 1994).  The hydrophobic sequence is rapidly cleaved (30 seconds) within the endoplasmic reticulum, to yield proinsulin, an 86 amino acid peptide having a 30-35 amino acid C-chain connecting the A and B chains of insulin. It has been suggested that this C-chain ensures alignment for the formation of disulphide bridges and the final tertiary structure of insulin (Steiner et al, 1972). Proinsulin is then transferred via an energy dependent process to the golgi apparatus where it is packaged, in an environment of neutral pH, into the trans-golgi membranes which contain an ATP-dependent proton pump. The characterization of a family of pro-hormone and pro-protein convertases has clarified the cleavage process of proinsulin to form insulin (Seidah et al, 1991). To date, 7 such proteinases which cleave at pairs of basic residues (pro-protein convertases) have been sequenced (PCI or PC3, PC2, furin, PACE4, PC4, PC5 and PC6), and each show differential tissue distribution and functional specificity. PCI and PC2 are present only in endocrine and neuroendocrine tissues (Day et al, 1992; Kiefer et al, 1991c) and like other members of this family of endopeptidases, are C a  2 +  dependent. Evidence suggests  that PC2 cleaves at L y s - A r g , termed Type-II proinsulin processing (Bennet et al, 1992) and 64  PCI at A r g - A r g 31  32  65  termed Type-I proinsulin processing (Bailyes et al, 1992). The basic  amino acids, exposed by either form of cleavage, are then removed by an exopeptidase, carboxypeptidase H (Davidson and Hutton, 1987), to yield insulin and C-peptide. The finding that insulin and C-peptide are not released in equimolar amounts has led to theories that maturing B-cell granules undergo fusion with other cellular granules through a clathrinmediated process (Hutton 1994). Glucose regulation is seen at this level of insulin synthesis, as both PC3 as well as proinsulin biosynthesis have been shown to be stimulated by elevated glucose (Alarcon et al, 1993). The resulting insulin molecule is less soluble than proinsulin, and precipitates with Zn  2 +  within the granules forming crystals with a ratio of 2 Z n  2 +  to 6 insulin. Under normal  physiological conditions, secretory granules release approximately 95 % insulin and 5 %  40 proinsulin. Transport of mature granules to the cell membrane is thought to be energy dependent and associated with microtubules and microfilaments. Insulin is secreted from Bcells via exocytosis in a regulated manner, with less than one percent of granules releasing insulin constitutively (Rhodes and Halban 1987). This will be discussed further in section 1.2.3.b.(iv). Insulin release has been found to be biphasic in vitro (Grodsky et al, 1963). The biphasic insulin release, seen in perfused pancreatic preparations and perifused islet, is characterized by an insulin peak after approximately 2 minutes following glucose infusion and returning to basal levels after 5-10 minutes. This is followed by a further prolonged rise in secretion for as long as the stimulus persists and is termed the second phase of insulin secretion. Several possible explanations have been suggested for the biphasic release, the most widely accepted being the differential release of insulin from mature B-cell granules close to the cell membrane (first phase release) and from newly synthesized insulin and maturing granules (second phase release) (Grodsky et al, 1973). Other proposed mechanisms for biphasic insulin secretion include differential alignment of insulin granules on microfilaments (Malaisse et al, 1974; Lacy 1970), biphasic electrical activity due to biphasic intracellular Ca  2 +  rises (Meissner 1976), and activation of different second messenger systems (Zawalich et  al, 1983). In vivo, an increase in insulin (5X) is seen with a 50 g oral glucose load humans, with basal levels returning to pre-glucose load levels after 2 hours. In addition, it has been found that circulating levels of insulin exhibit cyclic variations, which reflect a pulsatile release of the hormone (Lefebvre et al, 1987; Stagner et al, 1980; Goodner et al, 1977), and early signs of diabetes are often indicated by the disappearence of such cycles (O'Rahilly et al, 1988).  41  b. Regulation of insulin synthesis and secretion (i) Stimuli of insulin secretion Glucose is the primary modulator of insulin synthesis and secretion, and B-cell function is optimal within a glucose concentration of approximately 4-7 mM, a fluctuation associated with the ingestion of a meal. Glucose, when given intravenously, or when administered in vitro, at levels seen following a meal (peaking at 6 - 7 mM), is a weak stimulus for insulin secretion. The ingestion of a meal or of glucose, however, elicits a much greater insulin response. A complex, highly refined interplay of factors, with their associated second messenger systems has been proposed to describe control of insulin release in response to a meal (Rasmussen et al, 1990). Amino acids, acetylcholine and neuropeptides from intrinsic neurons and hormones, including glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) and the truncated forms of glucagon-like peptide ( G L P - 1 . (7  36)  ,  G L P - 1 _ ) , from the intestine all influence insulin secretion. These factors elicit an increased (7  37)  insulin response with increasing glucose concentrations, and are said to potentiate glucose stimulation (Malaisse et al, 1974), with the overall physiological significance being an absence of stimulated insulin secretion in the absence of elevated glucose levels, and protection from hypoglycemia. The term 'entero-insular axis' refers to the overall influence of the gut on the endocrine pancreas following the ingestion of a meal (Unger and Eisenhaur, 1969). The relationship was first described with the finding that the oral ingestion of glucose resulted in a greater insulin response than did an equal amount of glucose administered intravenously (Mclntyre et al, 1964). Although hormones were initially thought to be the only mediators of this response, there is also evidence for an intestino-pancreatic neural pathway via the coeliac ganglion and a direct innervation of the pancreas from the duodenum, but their significance in the overall pancreatic hormone response to a meal is as yet unclear (Kirchgessner et al, 1989). Although several hormones have been found to potentiate glucose stimulated insulin secretion, the 'incretins' (Labarre 1932) with the greatest influence are GIP and truncated forms of GLP-1 (tGLP-1).  42  GIP, a 42 amino acid peptide hormone, is secreted from cells within the mucosa of the duodenum and jejenum (Polak et al, 1973), primarily in response to the presence of fats (long chain fatty acids; Cleator and Gourlay 1975; Falko et al, 1975; Krarup et al, 1975; Brown et al, 1975; Pederson et al, 1975) and carbohydrates (glucose; Cataland et al, 1974) in the lumen of the small intestine. In addition to its role in the inhibition of gastric acid secretion, for which GIP was originally isolated from impure gastrointestinal C C K preparations (Brown et al, 1969), the hormone was also found to stimulate insulin secretion (Dupre et al, 1973). Following ingestion of a meal, GIP levels increase 5-6 X ih the plasma (to approximately 1.2 ng/ml) and remain elevated for several hours (Kuzio et al; 1974), but stimulate insulin secretion only in the presence of elevated plasma glucose (Dupre et al, 1973), as the name 'glucose-dependent insulinotropic polypeptide' indicates. In addition, GIP has been found to influence other islet hormones in vitro, stimulating glucagon secretion in the absence of hyperglycemia, as well as somatostatin (Verchere 1991). tGLP-1, secreted from the distal small intestine (Lauritsen et al, 1980), has similar insulinotropic actions as GIP, and is also important (Hoist 1994; Nauck et al, 1993). The above factors are thought to interact to produce the characteristic insulin response to the ingestion of oral glucose, or to a meal, which can be divided into a cephalic, an early enteric and a late enteric stage (Jacot et al, 1982; Louis-Sylvestre 1978). The cephalic phase of insulin secretion is mediated mainly by the release of acetylcholine from cholinergic neurons abutting on the B-cell. In some species, C C K or other stimulatory neuropeptides are also released from neurons of the intrinsic nervous system when nutrients enter the intestine during the early enteric phase.  (ii) Intracellular pathways involved in insulin secretion The major pathways involved in insulin secretion are those resulting from the metabolism of glucose, and those mediated by cAMP, calcium, and products of phospholipaseC mediated hydrolysis of phosphoinositides [inositol triphosphate (IP ) /diacylglycerol 3  (DAG)]. Stimulation of these pathways in the B-cell is elicited by the binding of the effectors  43  mentioned above and the subsequent activation of membrane associated G-proteins. In addition to acting individually, these pathways also interact. cAMP and P K - C can increase intracellular Ca  2 +  (Wang et al, 1993; Rajan et al, 1990) and C a  2 +  can regulate enzymes of the c A M P and  P K - C pathways (adenylate cyclase, phospholipase-C, and protein kinase C). Glucagon, GIP and the truncated forms of GLP-1 elicit an insulin response by stimulating B-cell adenylate cyclase, thereby increasing the intracellular c A M P concentration stimulating the uptake of C a  2 +  by voltage dependent (L-type) C a  other pathways. It is unclear whether cAMP increases C a  2 +  2 +  channels, and possibly by  uptake via phosphorylation of the  L-type channels. Acetylcholine and C C K act by stimulating phosphoinositide(PIP )-specific 2  phospholipase C (PI-PLC) resulting in hydrolysis of PIP to IP and diacylglycerol (DAG). 2  IP3 acts as a messenger to release C a  2 +  3  from intracellular pools. The binding of C a  2 +  to  calmodulin stimulates calmodulin-dependent protein kinases which phosphorylates a number of proteins (Wenham et al, 1994; Dunlop and Larkins, 1986; Thams et al, 1984). The metabolism of glucose also results in the formation of D A G which promotes the association of P K - C with the plasma membrane (Persaud et al, 1989; Yamatani et al, 1988) and subsequently phosphorylates a set of cellular proteins important in exocytosis (Alkon and Rasmussen 1988). Overall, the effects of C a  2 +  and cAMP are thought to act synergistically to bring about the  insulin response to a meal (Rasmussen et al, 1990). The mechanism by which glucose elicits an insulin response remains unclear. Glucose is transported into the B-cell via a high K  m  facilitative glucose transporter (Glut-2) (Johnson et  al, 1990; Fukumoto et al, 1988; Thorens et al, 1988). The effects of glucose on insulin secretion are mediated via its metabolism, by glucokinase in the B-cell. Glucokinase appears to control the glucose sensitivity of the cell as the enzyme catalyzes the rate limiting step in the utilization of glucose (German 1993; Liang et al, 1992). B-cells maintain a resting membrane potential of -70 mV by regulating permeability to ions, primarily K stimulated B-cells there exists a large flux of K and C a  2 +  +  +  and C a  2 +  . In non-  out of the B-cell via ATP-dependent channels,  is extruded via ion pumps in the plasma membrane and sequestered in organelles  44 such as the ER. Metabolism of glucose by the 6-cell increases the production of A T P (Ashcroft et al, 1973), which binds and closes ATP-dependent K+ channels (Rajan et al, 1990). This causes a depolarization of the li-cell (Cook et al, 1988; Arkhammar et al, 1987) which activates voltage dependent (L-type) C a  2 +  channels resulting in insulin secretion. It remains  unclear whether it is the increase in A T P itself, or the ratio of A T P / A D P which regulates K+ channels, as difficulties exist in measuring intracellular levels of A T P / A D P . It has been shown that the A T P levels in resting fi-cells are high enough to close all K+ channels, and that perhaps a competition by ADP for the same channel binding site can explain the influence of A T P (Meglasson et al, 1989; Malaisse and Sener, 1987; Ashcroft et al, 1984). Despite findings that A D P levels are several fold lower than those of A T P , it has been proposed that essentially all K  +  channels must be closed for normal physiological function, and there  therefore exists a high sensitivity for any small changes in the A T P / A D P ratio, particularly if A D P influences A T P binding affinity (Cook et al, 1988). In addition, glucose has been shown to stimulate the phosholipase-C (Mathias et al, 1985) and c A M P (Malaisse et al, 1984) pathways, although these effects may be secondary to increases in intracellular C a , as glucose stimulated insulin secretion is not prevented when 2 +  P K - C (Howell et al, 1990; Hughes et al, 1990) or PK-A activity are inhibited. It has also been proposed that glucose stimulates a rise in the intracellular concentration of cyclic A D P ribose (cADP-ribose), which may mediate the insulin secretory response by increasing release of Ca  2 +  from intracellular stores (Takasawa et al, 1993). Overall, glucose stimulates large  amplitude oscillations of intracellular C a , which are thought to result in the cyclic nature of 2 +  insulin release, and which can be abolished by diabetogenic agents such as streptozotocin and alloxan (Grapengiesser et al, 1990). A further level of complexity exists, in that the fi-cell heterogeneity plays a role in insulin responsiveness to glucose. There exist sub-populations of fi-cells which are responsive to lower levels of glucose than are others, and which are characterized by a higher density of pale proinsulin rich secretory granules (Pipeleers 1992). These cells apparently have higher  45 rates of glucose metabolism attributable to higher glucokinase activity as well as glucokinase mRNA, but not to increased glucose transport.  (iii) Inhibition of insulin secretion In addition to stimulatory regulation, insulin secretion is inhibited by hormones including SS and epinephrine, as well as by neurotransmitters, including, norepinephrine and galanin. Multiple mechanisms exist for the inhibition of insulin secretion. Many appear to be mediated following receptor binding and activation of inhibitory G-proteins (G[) as reviewed by McDermott and Sharp (1993). Association of galanin or somatostatin with their respective receptors results in an inhibition of adenylyl-cyclase activity thereby reducing c A M P levels and inhibiting C a  2 +  channel activity via a cAMP independent pathway (Malm et al, 1991). In  addition, both peptides activate A T P sensitive K  +  channels and inhibit voltage dependent C a  2 +  channels directly. This hyperpolarizes the cell, and inhibits the activity of P L C , thereby reducing levels of D A G and reducing PK-C activity. It has also been proposed that there exists a G-protein mediated inhibition late in stimulus-secretion coupling, given findings that galanin inhibits stimulated insulin secretion in the absence of extracellular C a 8Br-cAMP, indicating that inhibition is independent of C a  2 +  2 +  and in the presence of  entry or adenylyl cyclase activity  (McDermott and Sharp 1993).  (iv) Mechanism of exocytosis Insulin is secreted following the fusion of 8-cell dense core granules to the plasma membrane, upon stimulation of the cell by insulin secretagogues. The mechanism by which the 6-cell granules fuse and release their contents has recently been shown to be similar to that of neuronal cells (Sadoul et al, 1995), about which much more is known (Sollner et al, 1993a;b). The mechanism of neurotransmitter release from secretory granules involves a protein 'scaffold' linking the secretory vesicle to the plasma membrane (Monck and Fernandez 1994). The activation of this scaffold may be clathrin mediated, requiring ADP-ribosylation factors (ARFs; Stamnes and Rothman 1993), a family of Ras-related GTP-binding proteins. Non-  46  clathrin mediated vesiculation requires coat proteins (COPs) forming a 'coatomer' (Orci et al, 1993) under A R F regulation. Cytosolic proteins involved in the scaffold structure include annexins, which are Ca^~*~ sensitive and have been found to form filamentous structures important in exocytosis of chromaffin granules (Creutz 1992; Ali et al, 1989). Several proteins in the synaptic vesicle membrane form the scaffold including Rab3a, synaptobrevin, synaptophysin, SV2 and H^ATPases, in association with presynaptic membrane proteins which include syntaxins and C a  2 +  channels (Bennett and Scheller 1993; David et al, 1993;  Horikawa et al, 1993; O'Connor et al, 1993; Bennett et al, 1992). Furthermore, several soluble proteins, previously found to be important in intracellular traffic of transport vesicles, N-ethylmaleimide-sensitive factor (NSF) and related NSF attachment proteins (alpha, beta and gamma SNAPS; Sollner et al, 1993b) may also be important in regulated membrane fusion. Recently, SNAP-25 has been shown to be expressed in rat islets and the use of botulinum toxin, which inhibits neurotransmitter secretion by cleaving SNAP-25, was found to inhibit C a - i n d u c e d insulin secretion (Sadoul et al, 1995) suggesting that a similar protein 2+  scaffolding mediates exocytosis in B-cells.  47  1.3. IGF-I AND T H E ENDOCRINE PANCREAS  1.3.1. IGF-I AND ISLET C E L L DIFFERENTIATION AND PROLIFERATION a. IGF-I and differentiation and proliferation of fetal and neonatal islets Studies of islet neogenesis in rats indicate that IGF-I may play an important role in growth and differentiation of pancreatic tissue. Several studies have implicated IGF-I in fetal endocrine pancreas development and it has been suggested that IGF-I may be the primary mediator of B-cell replication in fetal and neonatal rat islets (Swenne et al, 1988; Billestrup and Martin 1985; Swenne 1985; Rabinovitch et al, 1983; Whittaker and Taylor 1980). Cultures of fetal rat tissue exhibit IGF-I secretion and regulation by G H (Swenne et al, 1987b), and the ability of both IGF-I and IGF-II to stimulate the proliferation of fetal rat 8cells has been documented (Romanus et al, 1985; Rabinovitch et al, 1982). Characterization of fetal rat islet IGF-I, using H P L C , reveals the presence of a peptide of the molecular weight of IGF-I (7000 kD), which is immunoprecipitable with an IGF-I antibody following the removal of high molecular weight binding proteins (Scharfmann et al, 1989). This study did not find a peptide with the characteristics of IGF-II in fetal pancreas. More recent studies of cultured fetal rat islets, however, revealed that islets release more IGF-II than IGF-I, neither of which are affected by glucose stimulation (Hogg et al, 1993). These studies, however, showed that glucose and amino acids did stimulate the release of 4 binding proteins (thought to be IGFBP1,-2,-3 and -4 based on molecular weights). These binding proteins had no effect alone, but synergized with sub-maximal exogenous IGF-I to stimulate D N A synthesis, with most activity seen in the B-cells. Exogenous IGF-I was found to be five times more active in stimulating D N A synthesis in cultured islets than was IGF-II. IGF-I has been found to be present and released in cultures of human fetal (14-21 weeks gestation) pancreatic explants (Hill et al, 1987; Swenne et al, 1987a) and release is associated with binding proteins (Hill et al, 1987). In both studies, glucose concentration was found to have little effect on IGF-I release, but G H (45.5 nM) in the presence of a high concentration of glucose (16.7 mM) increased both pancreatic IGF-I content (114%) and  48  release (117 %) (Swenne et al, 1987a). IGF-I was localized to the fi-cells of the human fetal pancreas (Hill et al, 1987). IGF-I is thought to be a growth factor involved in the maturation of the human pancreas, stimulating cell proliferation and B-cell responsiveness. Long term studies have been done in human fetal islet cell clusters. Over a period of 7 days, IGF-I doubled islet D N A content while suppressing insulin release, whereas after 25-31 days, IGF-I stimulated mean insulin secretion (49 %) (Otonkoski et al, 1988). The extent to which G H or the members of its family regulate B-cell replication via IGF-I is as yet unclear as conflicting results exist (Bonner-Weir and Smith 1994). The effectiveness of the G H family on the stimulation of insulin secretion and B-cell replication was reported to be in the order P L > P r l > G H (Brelje et al, 1993). It has been proposed that, given the effectiveness of PL and Prl, G H acts primarily via the lactogenic receptor, thereby not influencing IGF-I. Indeed, studies in dissociated islet cells from newborn rats revealed that the influence of G H was likely a direct one since IGF-I had only 1/10 the activity of G H in the stimulation of B-cell proliferation and insulin secretion over a 2-3 month culture period (Neilsen, 1982). Further studies with fetal rat pancreatic islets, however, revealed increased Bcell replication upon incubation with glucose, G H , PDGF as well as with IGF-I (Sjoholm et al, 1990). Growth of islets in this study paralleled that of B-cell polyamine content, an amine which has been implicated in the control of cell proliferation in several cell types (Pegg et al, 1989; Pegg 1986; Tabor and Tabor 1984). The influence of IGF-I has also been investigated in newborn rat islets. Islet cells from 3-5 day old rats were found to show a dose dependent increase in B-cell proliferation in the presence of G H , Prl and PL, without changing A - or D-cell proliferation (Billestrup and Neilsen 1991). In this study, IGF-I had no effect on B-cell proliferation, based on incubations done in the presence of G H and an IGF-I monoclonal antibody. These results are in contrast to those of Swenne and colleagues (1987b) in which a polyclonal IGF-I antibody decreased G H stimulated islet cell proliferation.  49  b. IGF-I and differentiation and proliferation of adult islet tissue IGF-I may also be involved in the growth of adult pancreatic tissue. Pancreatectomy (90 %) in rats has been shown to result in regeneration of tissue via an increase in neogenesis from proliferating ductules as well as by replication of existing cells (Brockenbrough et al, 1988). IGF-I expression, localized primarily to proliferating ductular epithelial cells and surrounding connective tissue, was shown to peak three days following pancreatectomy, and decreased to control levels after two weeks (Smith et al, 1991). Peak levels of expression were found to coincide with the peak mitotic index of fi- and exocrine cells, and the decline in expression was consistent with a decrease in proliferating ductules. IGF-I was therefore proposed to have a role in both proliferation of existing cells and in differentiation of ductular epithelial cells (Smith et al, 1991). Evidence also exists for the IGF-I induced stimulation of B-cell replication in adult rat islets (Swenne and Hill 1989; Sieradzki et al, 1987). IGF-I was found to stimulate tritiated thymidine incorporation, insulin biosynthesis and secretion, indicating that IGF-I has a role in regulating the growth and function of adult islets (Sieradzki et al, 1987). Further studies indicated that G H stimulated tritiated-thymidine incorporation, insulin biosynthesis and glucose oxidation, of which only the stimulation of D N A synthesis was partially inhibited by an IGF-I monoclonal antibody (Swenne and Hill 1989). Adult rat islets have also been found to exhibit G H binding sites on the B-cells, and these were primarily lactogenic (Polak et al, 1990). The observation that increased D N A synthesis and insulin secretion were stimulated to a greater degree by Prl than by G H suggests that lactogenic hormones (Prl and PL) are more important regulators of islet growth in the adult rat than is G H , and may influence pancreatic growth in pregnancy (Brelje and Sorenson 1991), although the potential role of IGF-I was not investigated.  50  1.3.2. INFLUENCE OF IGF-I ON INSULIN SECRETION IN T H E ADULT a. IGF-I receptors in the endocrine pancreas IGF-I receptors have been identified on B- and A-cells (Van Schravendijk et al, 1987). Binding studies on purified islet cells from adult male rats revealed the presence of high affinity IGF-I receptor binding, with a lower affinity binding detected for insulin. This finding has prompted the suggestion that insulin, at the high levels encountered in the islet, might act to regulate its own secretion via the IGF-I receptor, given that insulin receptors have not been detected on fi-cells (Leahy and Vandekerkhove 1990; Van Schravendijk et al, 1990; 1987). There was determined to be a high number of Type I IGF-I receptors per B-cell (approximately 12,000), with a lower density per A-cell (approximately 5,000). The low numbers of other islet endocrine cells makes studies of these cells difficult, and the presence of IGF-I receptors on D- or PP- cells is uncertain.  b. Effects of IGF-I in vitro Conflicting results exist regarding the effects of IGF-I on insulin secretion both in vitro and in vivo. Studies in adult isolated rat islets, as indicated previously, have shown a stimulation of DNA synthesis, proinsulin synthesis and insulin secretion following 3 days of culture in the presence of IGF-I, (1.8 itg/ml) particularly in the presence of a stimulatory level of glucose (16.7 mM) (Sieradzki et al, 1987). In other studies on similar long term cultures, however, it was found that although G H (1 ttg/ml) stimulated insulin biosynthesis and secretion, IGF-I (100 ng/ml) had no effect on either parameter (Swenne and Hill 1989). IGF-I has, in fact, been reported to inhibit insulin secretion. In situ perfusion of adult rat pancreas with IGF-I (200 ng/ml) over a 10 minute period was found to inhibit insulin secretion, particularily in the presence of a low stimulatory (7.8 mM) level of glucose, in comparison to a high level of glucose (16.7 mM) (Leahy and Vandekerkhove 1990). In this study, IGF-I levels as low as 2 ng/ml were found to inhibit insulin secretion over a 10 minute period and IGF-I was also able to inhibit arginine-induced insulin secretion, without influencing glucagon release. In addition, B-cell aggregates, perifused over a 15 minute period  51  with IGF-I (5 nM) in the presence of a high level of glucose (20 mM) and glucagon (1 nM), have been shown to exhibit reduced insulin secretion as well as reduced proinsulin synthesis (Van Schravendijk et al, 1990).  c. Effects of IGF-I in vivo The effects of IGF-I have also been investigated in vivo in the human, where conflicting results also exist regarding influences on insulin secretion. Continuous infusion of IGF-I (10 iig/kg/hr; 28 hr) resulted in a 300 % increase in IGF-I levels, and a decrease in plasma insulin, C-peptide and glucagon (Mauras et al, 1992). In this study, IGFBP-3 levels were not affected by IGF-I infusion, nor was protein metabolism. Another study examined the effect of an IGF-I infusion (20 /ig/kg primer then 24 itg/kg/hr; 2 hr) on glucose (2.8 m M , 7.0 mM) stimulated insulin secretion, and found that insulin secretion was suppressed, particularly in the presence of the lower level of glucose, and that C-peptide levels were decreased to a greater extent in all cases (Rennert et al, 1993). The study also showed that glucose metabolism was higher in all cases in the presence of IGF-I infusion, and attributed this to the insulin-like influence of IGF-I. Other studies have also shown that IGF-I infusion in humans lowers basal C-peptide and insulin levels (Boulware et al, 1992; Moxley et al, 1990; Jacob et al, 1989) and lowers the insulin response to a glucose meal (Zenobi et al, 1992a). In contrast, a study of acromegalics, who exhibited elevated serum G H and IGF-I levels and hyperinsulinemia (Hopkins and Holdaway 1992), serum IGF-I, but not G H levels, were found to be significantly correlated with elevated fasting insulin levels. IGF-I was also inversely correlated with the decreased insulin sensitivity and positively correlated with the increased B-cell function that was observed following a glucose infusion. The elevated fasting insulin and increased B-cell function of acromegalics was postulated to result from either an enhanced production of IGF-I, a stimulation of insulin synthesis and release by IGF-I, or from an increase in islet cell mass.  1.3.3. SYNTHESIS AND PRESENCE OF IGF-I IN T H E ENDOCRINE PANCREAS a. Synthesis of IGF-I in the endocrine pancreas Evidence exists for the synthesis of IGF-I by fetal endocrine pancreatic tissue. Fetal islets released IGF-I into culture medium (Hogg et al, 1993; Scharfmann et al, 1989; Hill et al, 1987; Romanus et al, 1985), IGF-I mRNA transcripts were detected in fetal human pancreas (Han et al, 1988; 1987b), and IGF-I synthesis has been measured in isolated rat islets (Scharfmann et al, 1989; Romanus et al, 1985). To date, there is no definitive evidence for the synthesis of IGF-I in the adult endocrine pancreas. Several studies have detected IGF-I mRNA in adult rat (Hansson et al, 1988b) and mouse (Hansson et al, 1989) pancreases, by hybridization studies using R N A extracted from both exocrine and endocrine pancreas, but no evidence exists for the synthesis of IGF-I within the endocrine pancreatic cells. Northern blot analysis of polyadenylated R N A , extracted from G H stimulated adult rat islets, revealed an absence of IGF-I transcripts, whereas transcripts were readily detected in liver RNA extracts (Billestrup and Nielsen 1991). In situ hybridization studies of rat pancreas revealed that IGF-I mRNA was localized to capillary endothelial cells, as well as to regenerative areas including epithelial cells of proliferating ductules and individual connective tissue cells following 90 % pancreatectomy (Smith et al, 1991).  b. Presence of IGF-I in the endocrine pancreas In the fetal rat, immunoreactive IGF-I has been localized to the B-cells of the endocrine pancreas using a monoclonal IGF-I antibody (Hill et al, 1987). In the adult pancreas, several studies reported the presence of immunoreactive IGF-I, albeit in conflicting locations. Immunoreactive IGF-I was first detected in both adult human and mouse endocrine pancreas, without the indication of a specific cell type (Bennington and Spencer 1983). IGF-I was subsequently localized, using a polyclonal rabbit IGF-I antibody (K37), to the f>-, A - and D-cells of rat pancreas (Hansson et al, 1988a). The most prominant staining was said to be in the D-cells and it was concluded from this study that this was the site of IGF-I synthesis and secretion, and that f>- and A-cell immunoreactivity represented receptor  53 binding. It was further proposed that a developmental change in the location of IGF-I existed, with the B-cell being the primary source of IGF-I in the fetal and neonatal rat, and the D-cell the source in the adult. Strong D-cell IGF-I immunoreactivity was also detected in a second study, using the same antibody (K-37) (Hansson et al, 1988b). This study unexpectedly revealed that the liver exhibited low or no IGF-I immunoreactivity, whereas high levels of extractable IGF-I and of IGF-I mRNA was detectable in this tissue. It was suggested that either IGF-I was secreted from the liver immediately following synthesis, and not detectable by the IGF-I antibody, or that the association with binding proteins rendered it undetectable using the antibody. A further study, using 3 different rabbit polyclonal IGF-I antibodies (including K-37 indicated above) revealed that in the rat, dog and human, IGF-I immunoreactivity coexisted with glucagon immunoreactivity, and in man, some, but not all somatostatin immunoreactive cells also exhibited IGF-I immunoreactivity (Maake and Reinecke 1993). It was also suggested in this study, that the use of the K-37 antibody resulted in non-specific binding, when used at high concentrations. It was also found that immunoreactive IGF-II was localized to B-cells. The same three antibodies used in the previous study were also used to detect IGF-I immunoreactivity in the endocrine pancreatic cells of cyclostomes, cartilagenous fish and bony fish (Reinecke et al, 1993a). The study was performed in an attempt to determine the evolutionary divergence of IGF-I and insulin, based on the previous results seen in mammals, indicating that IGF-I and insulin immunoreactivity do not coexist (Maake and Reinecke 1993). In both cyclostomes and cartilagenous fish, IGF-I and insulin immunoreactivity coexisted, whereas in bony fish, there was no colocalization, leading the authors to conclude that evolutionary divergence occurred at the phylogenetic level of bony fish.  54  1.4. HYPOTHESIS A N D OBJECTIVES  Based on findings, in fetal islets, that IGF-I was present in the fi-cells and stimulated the secretion and synthesis of insulin, the present studies were undertaken in order to determine if a similar relationship existed in adult islet tissue. At the commencement of the present studies, limited evidence suggested that IGF-I was present in adult islet tissue, however the localization of the hormone was unclear. Two studies, using the same antibody, indicated immunoreactive IGF-I associated with A - , D- and fi-cells, or with D-cells alone, in contrast to exclusive fi-cell localization in fetal islets. Studies were therefore undertaken to localize IGF-I in the adult endocrine pancreas. In addition, studies were done in order to quantify islet IGF-I, and to determine potential secretagogues of the hormone in this tissue. The influence of IGF-I on insulin secretion was also uncertain. The two studies performed using adult islets yielded contrasting results. One study indicated that IGF-I had no effect on insulin synthesis or secretion but did stimulate islet D N A replication, and the other that IGF-I stimulated insulin synthesis and secretion in addition to D N A proliferation. Studies were therefore performed in order to clarify the potential stimulatory effects of IGF-I on insulin secretion from adult fi-cells. It was therefore proposed that IGF-I was present in the adult endocrine pancreas, and stimulated fi-cell secretion via an autocrine route of action. If present, such stimulatory effects could have a significant effect on the regulation of physiological insulin secretion, particularly with the potentially high local IGF-I levels due to the proposed autocrine or paracrine secretion in addition to those of circulating IGF-I. The present studies were therefore performed to clarify the localization of endocrine pancreatic IGF-I, to quantify levels within adult islets and to determine potential stimulators of islet IGF-I secretion. Studies were also performed to determine the influence of IGF-I on insulin secretion, with the purpose of elucidating possible autocrine effects.  55  2. MATERIALS AND METHODS  2.1 MATERIALS 2.1.1. ANTMALS Wistar Furth rats (250 - 300g) were obtained from the University of British Columbia Animal Care facility, or from Charles River Laboratories, Quebec. Rats were kept up to one week in the Department of Physiology animal facility where they were exposed to a fixed light/dark cycle and fed commercial rat food and water ad libitum.  2.1.2. CHEMICALS, SUPPLIES AND IMMUNOREAGENTS All chemicals, supplies and immunoreagents are listed in the appendices at the end of the thesis (Appendices I, II, and III respectively).  2.2. IGF-I RADIOIMMUNOASSAY AND BINDING PROTEIN EXTRACTION 2.2.1. IGF-I RADIOIMMUNOASSAY a. Iodination of IGF-I A lyophilized aliquot of IGF-I (10 tig) was reconstituted in acetic acid (20 id, 0.1 M) and phosphate buffer (55 pi, 0.05 M , pH 7.4). Three aliquots (25 id) were frozen (-70 ° C ) in polystyrene tubes until immediately prior to iodination when one aliquot was thawed with the addition of phosphate buffer (5 /xl). Iodination followed the method of Hunter and Greenwood (1962) modified as follows. A l l reagents were prepared on the same day as the iodination. Na  125  I (10 til, 0.5 mCi) was added to the IGF-I aliquot and the oxidation reaction was started  with the addition of chloramine-T (20 pi, 1.5 mg/ml). The reaction was allowed to proceed for 30 seconds with light vortexing followed by the addition of sodium metabisulphate (100 pi, 2.0 mg/ml) to reduce and stop the reaction. The reaction mixture was then immediately transferred to a PD-10 column previously equilibrated with BSA (4.0 %). The reaction tube was washed with potassium iodide (300 pi, 10 mg/ml) and the contents transferred to the column. After the reaction mixture had run into the column, the peptide was eluted with  56 phosphate buffer. Fractions (12 x 2.0 ml total volume) were collected in polystyrene tubes, with tubes 2-7 containing BSA (iodination grade, 1.0 ml, 4.0 %). An aliquot (10.0 id) from each fraction was counted on a gamma spectrometer. The iodinated IGF-I peak was diluted with BSA (4.0 % in 0.05 M phosphate buffer to 4.0 ml) and stored in polypropylene snap cap vials (-20 ° C ) until purification. The specific activity of the resulting  125  I-IGF-I was determined by a displacement assay  involving serial dilutions (30,000 cpm - 1,000 cpm) of  125  I-IGF-I. This displacement curve  was plotted with a standard curve using unlabelled IGF-I and the specific activity of the iodinated IGF-I was determined from a plot of cold IGF-I (pg/ml) vs cpm (Figure 2). After each iodination a crude determination of the specific activity of the iodinated hormone was performed using the following calculation: peak 1 (  1 2 5  I-IGF-I,  peak 1 + peak 2  (  1 2 5  amount o f hormone  I,  //Ci) ixCi) (/xg)  This calculation was used to determine consistency between iodinations.  b. Purification of I-IGF-I 12S  Iodinated IGF-I was purified by chromatography on a G-50 Sephadex column within 12 hours prior to radioimmunoassay or binding study. The column (0.9 x 60 cm) was equilibrated with three bed volumes of phosphate buffer (0.05 M , 0.02 % sodium azide, pH 7.4). Bovine serum albumen (1.0 ml, 0.05%) was applied to the column prior to the addition of an aliquot (400 /cl) of  125  I-IGF-I. The iodinated hormone was eluted with phosphate buffer. Fractions (45  x 1.0 ml each) were collected in polystyrene tubes with tubes 11-30 containing BSA (0.5 ml). Aliquots (100 ul) from each fraction were counted on a gamma spectrometer. Two to three fractions containing the iodinated hormone were pooled and stored in a plastic container at 4 ° C until use.  57  oH 0  •  1  10  •  1  20  '  1  30  iodinated IGF-I (cpm x 1000)  Figure 2: Self displacement of I-IGF-I. Specific activity of I-IGF-I was determined by adding increasing concentrations (1700 - 79000 cpm/100 id) of I-IGF-I in the RIA and comparing displacement to that with the unlabelled IGF-I standard curve. The % bound/free (%B/F) were determined for unlabelled and labelled IGF-I. The relationship yielded a slope of 31 pg/16000 cpm and a specific activity of 291 /xCi/itg (2.2 X 10 dpm = 1 uCi, 80 % counting efficiency). 125  125  125  58  c. Primary antisera dilution for RIA The optimal primary antibody titre for the rat IGF-I RIA was determined using a modification of conditions supplied with the antiserum (Lot UBK487 from NIH) (Furlanetto et al, 1977; Copeland et al, 1980; Chatelain et al, 1982). Assay buffer was 0.05 M phosphate (0.02 % sodium azide, 0.05 % Tween 20, p H 7.4). All tubes contained:  300 ill buffer 100 ill antiserum 100 l 125T.TGF-I M  and were precipitated with: 100 fil second antibody 100 /xi normal rabbit serum  Assays were performed in 12 x 75 polystyrene tubes. Dilutions of anti-human IGF-I rabbit serum (1/500, 1/1000, 1/5000, 1/10,000, 1/18,000, final concentrations in assay volume of 500 /xi) were incubated (24 hr, 4 ° C ) with  125  I-IGF-I (7,000 or 10,000 cpm), then goat anti-  rabbit antisera (1/50) and normal rabbit serum (0.4 % or 0.7 %) were added for a further 24 hours (4 ° C ) . Separation of bound and free hormone was by centrifugation at 3,000 rpm (30 min). The supernatant was decanted and the radioactivity in the air dried pellet was counted on a gamma spectrometer.  d. Secondary antisera dilution for RIA The optimal dilution of goat anti-rabbit antibody and of carrier serum (normal rabbit) were determined under the conditions described in the previous section. Goat antibody (1/10, 1/25, 1/50, 1/75, 1/100 initial dilutions) was added with various levels of normal rabbit serum (0.2 %, 0.4 %, 0.6 %, 0.8 %) to  125  I-IGF-I preincubated with primary antibody (1/18,000,  24 hr, 4 ° C ) , and incubated for a further 24 hours (4 ° C ) . Precipitation and determination  59 of radioactivity were as described in section 2.2. l.c Binding was determined according to the following equation:  SAMPLE (cpm)  -  TOTAL (cpm)  NSB (cpm) TOTAL (cpm)  X 100 = %BOUND  where NSB = non-specific binding (determined by the inclusion of a set of assay tubes containing an extra 100 til assay buffer in the absence of primary antisera).  e. I G F - I standard curve IGF-I, from the same source as that used for iodination, was initially included in the assay as in section 2.2.l.d at levels of 10, 100, 1000, 10,000 pg/ml (final concentration in assay). The-standard curve consisted of IGF-I concentrations of 2.5, 5, 10, 25, 50, 100, 250 and 500 pg/ml. Aliquots of these dilutions were made from 10 ng/ml (20 ml) stocks and kept at -70 ° C until use.  f. I G F - I radioimmunoassay The IGF-I RIA was a disequilibrium assay incubated over a period of 72 hours with the dilutions determined previously. All steps were performed over ice or on a refrigerated table. Assay buffer, sample or standard, and rabbit anti-human antiserum were vortex mixed and incubated for a 24 h period (4 ° C ) to allow equilibrium binding of sample (cold) IGF-I by the antiserum. Purified  125  I-IGF-I (7000 cpm) was added, vortexed and incubated for a further 24  h (4 ° C ) to achieve an equilibrium binding of unlabelled and iodinated IGF-I, with the antiserum. Goat anti-rabbit gamma globulin (100 txl) and normal rabbit serum (100 /xl) were added and samples were vortex mixed and incubated for a further 24 h (4 ° C ) to allow binding of first and second antibodies. Antiserum bound IGF-I was precipitated by centrifugation (3,000 X g, 30 min, 4 ° C ) . Samples were decanted and pellets were air dried then counted on a gamma spectrometer.  60 Each assay consisted of 144 tubes which included the following:  Buffer Total  -  count  1 2 5  I - I G F - I Antisera  Replicate Standard or Sample  100 Ml  -  -  3  -  -  3  -  3  Non s p e c i f i c b i n d i n g (NSB)  400 / i i  100 Ml  Total  300 jul  100 Ml  100 Ml  Standards  200 Ml  100 Ml  100 Ml  100 Ml  3  Samples  200 Ml  100 Ml  100 Ml  100 Ml  2  binding  Table 3. IGF-I RIA method (summary)  Each assay also contained a rat serum control sample from which the IGF-I binding proteins had been extracted (see section 2.2.2. b.), diluted by a factor of 5000, which gave a value of approximately 300 ng/ml serum after taking the dilution into account. Determination of the level of IGF-I in each assay sample was performed with a microcomputer programme (RIAPC, Rieger 1988). Calculations followed the formula:  %BOUND = TOTAL(cpm) - SAMPLE (cpm) - TOTAL(cpm) - NSB(cpm) x 100 TOTAL (cpm) TOTAL (cpm) A standard curve was constructed by plotting % BOUND vs IGF-I standard concentration on a semi-logarithmic scale.  2.2.2. IGF-I BINDING PROTEIN EXTRACTION a. Use of C18 cartridges The method of Davenport et al, (1988) was followed. Sep-Pak C18 cartridges were conditioned with isopropyl alcohol (5.0 ml), methanol (5.0 ml), followed by acetic acid (10.0 ml, 4.0 %). Samples (150 M!) were applied to the cartridge following an incubation with hydrochloric acid ( H O , 150 /d, 0.5 M) for 1 h (RT). Samples included phosphate buffer and pancreas perfusion medium (as in section 2.3.1.a) incubated (12 h, 4 ° C ) with either IGF-I  61  standards or with  125  I-IGF-I. Samples were slowly drawn through the column and binding  proteins eluted with acetic acid (10.0 ml, 4.0%). Methanol (0.5 ml) was applied to the column (2.0 min) followed by a further 2.0 ml to elute the free IGF-I. The methanol fraction was then dried under nitrogen gas, lyophilized, reconstituted in distilled water (50.0 /xl) and diluted in phosphate buffer (0.05 M). If neccessary the sample p H was adjusted to 7.4 with sodium hydroxide (1.0 M). Given the poor recovery of IGF-I using the previous method, a modification was also performed. Cartridges were conditioned with distilled water (5.0 ml), followed by acetonitrile (10.0 ml, 60.0 %, 0.1 % trifluoroacetic acid, TFA) then water containing T F A (10 ml, 0.1 % T F A ) . Samples (150 - 600 ul) were applied to the cartridge following incubation with HC1 (0.5 M , 1:1, 1.5 hr), and allowed to remain on the cartridge (5.0 min) which was then washed with water (10.0 ml, 1.0 % TFA) to elute the binding proteins. IGF-I was eluted with acetonitrile (2.5 ml, 60.0 %, 0.1 % TFA). Samples were dried under nitrogen gas, lyophilized and reconstituted in water and phosphate buffer.  b. Formic acid and acetone extraction The method of Bowsher et al, (1991) was used. Samples (100 ul) were vortex mixed with formic acid (50.0 ul, 8.0 M , 0.5 % Tween-20) then acetone (350  in 1.5 ml  polypropylene snap cap tubes (4 ° C ) . Binding proteins were pelleted (3,500 x g , 4 ° C ) and the supernatant either dried under refrigerated centrifugation using a Speed Vac (Savant Instruments, Farmingdale, NY) and reconstituted in water and phosphate buffer, or diluted at least 1:20 in the RIA. Extraction of binding proteins from tissues followed the method of Lee et al, (1991). Tissues were excised from rats, weighed on ice and homogenized in polypropylene tubes using a polytron in 4:1, volume:weight formic acid (3.3 M , 0.5 % Tween-20). Homogenates were centrifuged (40,000 x g, 10 min, 4 ° C ) and aliquots (150 ul) incubated (30 min, 90 ° C ) in capped polypropylene tubes. Acetone (350 itl) was added and samples were  62 vortexed then centrifuged (3,000 x g, 4 ° C , 15 min) to pellet the binding proteins. Supernatants were diluted at least 1:20 in the RIA.  c. Sephacryl column chromatography of  IGF-I  A siliconized glass column (1 x 30 cm) was packed with Sephacryl S-200 HR in phosphate buffer (0.05 M , 0.02 % sodium azide, 150 mM NaCl). The void volume (V ) was 7 ml and G  the total volume (Vj) was 30 ml with a flow rate of 0.3 ml/min under gravity elution. The column was calibrated with standards (high and low molecular weight) which yielded a straight line profile when flow rate was plotted against molecular weight (Figure 3). Prior to the application of samples (buffer, serum and tissue extracts), the column was conditioned with buffer and BSA (1.0 ml, 2.0 %). Rat tissue extracts were obtained either as in section 2.1.2.B.b., or without removal of binding proteins as follows. Fresh rat tissues were homogenized in phosphate buffer (0.05 M) containing E D T A (1.0 mM) and Trasylol (200 K.I.U./ml), and centrifuged in polypropylene tubes (40,000 x g). Aliquots of the supernatant (200 ul) in polypropylene snap cap tubes were again centrifuged (3,500 x g) and the supernatant saved for RIA. Rat blood obtained from the vena cava was allowed to clot (12 hr, 4 ° C ) , centrifuged (3,000 rpm) and the serum was pipetted into polypropylene tubes. Tissue extracts or serum (200 p\, with or without extraction of protein) were either applied directly to the column or preincubated (12 hr, 4 ° C ) with  125  I-IGF-I (100,000 cpm).  Fractions (2.0 min, 590 /xl) were collected in polystyrene tubes. Dependent on the sample, elution profiles were determined by spectrophotometry (absorbance at 280 nm), as well as by IGF-I RIA or by determining the radioactivity in each fraction.  63  1000000 i  Fraction (0.6 ml)  Figure 3: Calibration of a Sephacryl S-200 (1 X 30) column with standards. The void volume (Vo, fraction 13) was 7.0 ml and the total volume (Vt, fraction 50) 30 ml under a gravitational flow rate of 300 /xl/min. Molecular weight standards are indicated: #1 bovine gamma globulin, 158 kD; #2 ovalbumin, 44 kD; #3 myoglobin, 17 kD and #4 cyanocobalamin, 1.35 kD. The best fit line was used to determine the molecular weights of elution peaks.  64  2.3. ENDOCRINE PANCREATIC IGF-I 2.3.1. STUDIES ON T H E SECRETION OF PANCREATIC IGF-I a. In situ perfusion of rat pancreas (i) Surgical procedure Male wistar rats (250 - 275 g) were anaesthetized with sodium pentobarbital (60 mg/kg, i.p.). Paired lateral incisions was made from the sternum to the pubis to expose the abdominal cavity. A double ligature was made around the left renal artery and the left phrenico-abdominal artery, which were subsequently sectioned. Fat and tissue were cleared from the aorta caudal to the mesenteric artery and a loose double ligature was inserted in preparation for catheterization. Fat and connective tissue were cleared from the aorta proximal to the coeliac artery to allow placement of a single loose ligature. The descending colon was then sectioned following the placement of a double ligature. The portal vein was prepared for cannulation by inserting two single loose ligatures, one around the portal vein proximal to the liver and another proximal to the pancreas. A single loose ligature was placed around the inferior vena cava near the right adrenal gland. A duodenal drainage tube was inserted caudal to the pancreas and held in place with a single ligature. The mesenteric vasculature from the drainage tube to the cecum was then laid out, tied off with double ligatures, cut and removed. The spleen was tied off from the pancreas with double ligatures, cut and removed. The stomach was excised after ligatures were placed to tie off the pylorus and to separate the stomach and pancreas. The oesophagus was then cut between a double ligature. Ligatures at the kidney and vena cava were then tightened and the aorta was cannulated caudally and secured with the single ligatures. The aortic suture proximal to the coeliac artery was tightened to ensure flow to the pancreas, which was confirmed by the infusion of heparinized saline (2 ml) through the cannula. The rat was guillotined below the diaphragm, the portal vein was cannulated and the hepatic vessels tied off. The preparation was then put on a heating block and the aortic cannula was attached to perfusion tubing.  65 (ii) Perfusate solutions and  administration  Perfusate was prepared from a concentrated Krebs solution containing potassium 1  chloride (KC1), calcium chloride (CaCl2), magnesium sulfate (MgSCty) and potassium phosphate (KH2PO4). On the day of perfusion this solution was diluted with saline, and sodium bicarbonate (NaHC03), dextran (3 % clinical grade), BSA (0.2 % RIA grade) and glucose were added to yield a final ionic composition of NaHC03 (25 mM), KH2PO4 (1.5 mM),  M g S 0 . 7 H 2 0 (1.2 mM), CaCl2 (2.5 mM), KC1 (4.4 mM) and NaCl (120 mM). 4  Perfusate was introduced to the preparation via a cannula which passed around a heating coil (37.5 ° C ) and through a pump to give a constant flow rate (2 ml/min). The preparation was initially perfused (30 min) with a low glucose (4.4 mM) perfusate, in order to minimize hormonal perturbations from surgery, followed by perfusion under the conditions of study (40 min).  Fractions (1 min) were collected in siliconized glass tubes (12 x 75) over ice, divided  into aliquots ( 2 x 1 ml) and stored (-20 °C) until RIA.  Prior to the IGF-I RIA,  samples were  treated with the formic acid/acetone binding protein extraction protocol described in section 2.2.2.b. Insulin assays were performed as in section 2.4.2.a The G H and GIP gradients were developed by perfusing from a cannula which was connected to a perfusate solution (kept stirring) with a zero concentration of hormone connected to an identical receptacle containing the hormone at the highest concentration to be tested. Hormones were reconstituted immediately prior to perfusion in siliconized tubes with 0.01 M acetic acid and perfusate.  b. Isolation a n d culture of rat islets Islets were isolated from rat pancreas by collagenase digestion followed by a dextran gradient separation technique modified from that of Lacy and Kostianovsky (1967). A concentrated (5X, 2 1) Hanks Buffered Saline Solution (HBSS) was prepared from powder with the addition of sodium bicarbonate and Hepes, and stored (4 ° C ) until use. On the day of islet isolation the solution was diluted, and BSA (1 %, fraction V) was added to prevent the islets from adhering to the various receptacles.  66 Male Wistar rats (250 - 275 g) were anaesthetized with sodium pentobarbital (60 mg/kg i.p.). A midline incision was made from sternum to pubis to expose the abdomen, and the diaphragm was cut to sacrifice the animal. The common bile duct was occluded with a haemostat at the entry to the duodenum. A cannula was placed in the duct, just distal to its bifurcation at the liver, and collagenase (25 mg/ml in HBSS, 10 ml, ice cold) was slowly infused anterogradely. The distended pancreas was excised, with care taken not to puncture the pancreas or any other tissues, rinsed with HBSS and put in a polypropylene tube (50 ml) on ice. The pancreas was kept on ice for a maximum of 10 min before digestion (37 ° C , 10 min). It was then mechanically disrupted by vigorous suspension with a plastic pipette (10 ml). Ice cold HBSS (20 ml) was added, the digest centrifuged (50 x g, 3 min), and the supernatant decanted. The digest was washed a further three times by resuspension in HBSS. Prior to the final wash the digest was passed through a fine plastic mesh (approximately 0.5 mm) to remove large pieces of non-islet tissue. The digest was suspended in dextran (clinical or industrial grade, 29 % in HBSS) in a polypropylene tube (50 ml). One layer of dextran (29 %, 4 ml) was placed under the suspension and lower concentrations (23 %, 7 ml; 11 %, 4 ml) were carefully layered over the suspension. The gradient was then centrifuged (5 min, 50 x g, then 15 min, 500 x g). The islets were present predominantly at the interface of the 29 % and 23 % dextran layers, and were selected by carefully drawing up through the latter layer with a siliconized glass Pasteur pipette. Islets were put in a petri dish (35 mm, coated with black paint for contrast) and non islet tissue was removed by pipette while dispersing the islets with HBSS (ice-cold) under a dissecting microscope. Fragmented islets and islets attached to exocrine or other tissues were discarded. The remaining layers of the gradient were also observed under the microscope and if islets were present these layers were cleared of non islet tissue as above. Islets from one gradient were pooled in a polystyrene culture tube (15 ml). The medium used for the islet culture was Dulbecco's Modified Eagle's Medium ( D M E M , 4.4 mM glucose). Immediately prior to use, heat inactivated calf serum (10 %), penicillin (100 U/ml), streptomycin (100 ttg/ml), and L-glutamine (2 mM) were added and the medium was filter sterilized. Islets were washed by suspending vigorously in culture medium  67  then centrifuging (3 x 10 min, 500 x g) and decanting the supernatant. A l l subsequent procedures were done in a laminar flow hood under sterile conditions. Islets were transferred to a sterile petri dish (35 mm) in culture medium (8 ml) and cultured (95 % air, 5 % C O 2 , 37 ° C , 48 hr).  c. Characterization of rat islet extract by high performance liquid chromatography (HPLC) (i) Preparation of samples Islets (approximately 300), isolated and cultured (12 h) in a petri dish as described above, were resuspended in D M E M (in the absence of fetal calf serum) and centrifuged (2 x 10 min, 500 x g). After the second wash, islets were resuspended in acetic acid (400 /ul, 2 N), boiled (10 min), and centrifuged (10 min, 500 x g). The supernatant was dried in a Speed Vac, resuspended in distilled water (100 ul) and treated with the formic acid/acetone IGF-I binding protein extraction method described in section 2.1.2.B.b. The resulting lyophilized pellet was reconstituted in H P L C grade water (200 ul, 0.1 % TFA) for chromatography. Rat serum (2 ml) was prepared for H P L C by removing IGF-I binding proteins as in section 2.1.2.B.C. Samples were reconstituted in H P L C grade water containing T F A (2 ml, 0.1 % T F A ) and fractionated on a reverse phase C  1 8  cartridge with sample elution in  acetonitrile (60 %, 6 ml). Samples were lyophilized once more and reconstituted in H P L C water (0.1 % T F A ) . Pancreatic extracts were prepared for H P L C by removal of binding proteins as in section 2.1.2.B.b. then fractionated on a C  1 8  cartridges, as for serum.  (ii) HPLC of samples Samples were eluted over a reverse phase C\g column (3.9 x 300 mm) in a Waters H P L C system which consisted of two Model 510 pumps, a Model 712 WISP (Waters Intelligent Sample Processor) controller and a Model 441 absorbance detector. An integrated chromatography sofware programme (Maxima 820, Waters) was used to monitor the system, and to assess the results. The column was calibrated with proinsulin (human, 5 ul, 1.0 uglul),  68  insulin (human, 5 /xl, 1.0 /xg//xl) and IGF-I (recombinant human, 5 /xl, 0.5 /xg//xl) at a flow rate of 1 ml/min. Elution of standards was found to be optimal with a gradient of 33 - 45 % acetonitrile containing 0.1 % T F A run over a 10 minute period (1 ml/min). The column was again equilibrated (70 % acetonitrile, 0.1 % T F A , 15 min, then 30 % acetonitrile, 0.1 % T F A , 10 min) prior to injection of samples. H P L C grade water was injected onto the column and eluted as above between samples in order to ensure that no sample remained on the column. Fractions (35 x 0.5 min were collected in siliconized glass tubes over ice, lyophilized, reconstituted (400 /xl, 0.05 M phosphate buffer) and frozen (-20 ° C ) until RIA.  2.3.2. I M M U N O C Y T O C H E M I C A L D E T E C T I O N O F P A N C R E A T I C IGF-I a. Tissue preparation for light microscopy Rats were anaesthetized with sodium pentobarbital (60 mg/100 g bw i.p.), tissues excised and immediately immersed (24 hr, RT) in Bouin's fixative (71 % saturated picric acid, 24 % formaldehyde, 5.0 % acetic acid), then washed (3 x 20 min) and stored in ethanol (70 %, RT) until processing. Tissue samples were loaded into cassettes and passed through an automatic processing cycle (Histomatic tissue processor, Model 166, Fisher Scientific Co., Fairlawn, N J . ) which consisted of dehydration in an ethanol gradient (80 %, 1 x 30 min; 90 %, 1 x 30 min; 100 %, 3 x 30 min), xylene treatment (2 x 30 min) and infiltration with molten paraffin wax (2 x 1 hr, 60 ° C ) under vacuum. Tissues were embedded and mounted in paraffin wax (60 ° C ) using a tissue embedding center (Model 166, Sybron Canada, St. Laurent, Que.). The paraffin tissue blocks were cooled (-20 ° C ) and sectioned (5.0 - 7.0 /xm) on a rotary microtome (Model 133, Reicher-Jung). Sections were mounted on glycine coated glass slides, dried (12 hr, 37 ° C ) then stored (RT) under dust free conditions. Immediately before use, sections were dewaxed by immersion in xylene ( 2 x 5 min), rinsed in petroleum ether (1 min) and air dried (1 min, RT). Human, ovine and porcine tissues were fixed and treated as above.  69  b. Tissue preparation for electron microscopy Rat pancreas was excised and immediately immersed in gluteraldehyde (2.5 %), cut into small pieces (approximately 1 mm square), fixed (2 hr, RT), then washed in phosphate buffer (0.1 M , 0.1 M sucrose, 3 x 10 min, RT). Tissue was post-fixed in osmium (1 hr, RT) with slow mixing and washed again in phosphate buffer (0.1 M , 3 x 10 min). Fixed pancreas was dehydrated in a gradation of ethanol (70 %, 80 %, 90 %, 1 x 30 min each; 100 %, 3 x 30 min) then infiltrated with propylene oxide (2 x 10 min), propylene oxide/epon (1:1, 2 hr, 1:3, 4 hr) then epon (100 %, 12 hr). Tissues were gravity pelleted in polypropylene capsules in epon, and baked (70 ° C ) overnight. Preparatory thick sections (0.5 urn) were cut and examined under the electron microscope to select blocks containing islets. Thin (80 nm) sections were cut from blocks containing islets and two to three were mounted per grid and baked (60 ° C ) .  c. Immunostaining for light microscopy Tissue sections were incubated in hydrogen peroxide (0.2 % in methanol, 30 min) to diminish endogenous peroxidase activity, then washed in water ( 2 x 5 min) then PBS ( 2 x 5 min). The latter buffer was used throughout for all washes and dilutions. Sections were incubated in BSA (5 % in PBS, 1 h) to reduce non-specific binding of antisera, prior to the application of primary antibodies. IGF-I was detected by exposing sections (24 hr, 4 ° C ) to a monoclonal mouse antihuman IGF-I antibody (dilutions 1 - 100 itg/ml ascites). Immunolocalization of IGF-I involved incubation (24 hr, 4 ° C ) with a biotinylated anti-mouse antibody (1/200, 12 h, 4 ° C ) then with an avidin-biotin complex conjugated to horseradish peroxidase (1 hr, RT). Washes between consecutive steps were in PBS (2 x 10 min). Incubation (5 min) with filtered diaminobenzoic acid (0.3 mg/ml in TBS, 100 ul hydrogen peroxide) was used to demonstrate peroxidase activity. Sections were then lighly counterstained with Mayers haematoxylin and mounted in xylene.  70  Double staining of IGF-I for the other islet hormones involved the above exposure to BSA followed by incubation (48 h, 4 ° C ) of the IGF-I antibody combined with either rabbit anti-glucagon, anti-pancreatic polypeptide, or anti- somatostatin, or with guinea-pig antiinsulin. Insulin, glucagon, somatostatin and pancreatic polypeptide were detected by incubation (2 h, RT) with tetramethylrhodamine isothiocyanate conjugated anti-guinea pig or anti rabbit antibodies. Demonstration of IGF-I included incubation with biotinylated anti-mouse antibodies (2 h, RT) then with fluorescein isothiocyanate conjugated avidin (1.5 h, RT). After each antibody incubation, sections were washed (2 x 10 min) in PBS and mounted in PBS glycerine. Photographs were either taken directly under the microscope or microscopic images were transferred to a VIDAS imaging computer and photographed from its monitor.  d. Immunostaining for electron microscopy Grids containing endocrine pancreas sections were incubated (30 min) in hydrogen peroxide (3 %) then washed by repeatedly dipping in distilled water (15 min). Sections for IGF-I detection were incubated in BSA (5 %, 30 min) then with either the IGF-I monoclonal antibody (8 ug/ml) or the guinea pig anti-insulin antibody (1/1000) (48 h, 4 ° C ) . Grids were then washed in PBS (15 min), PBS containing 0.1 % Tween-20 (15 min), then PBS containing 0.1 % Tween-20 and 0.5 % BSA (15 min). Detection of IGF-I and insulin involved incubation (90 min, RT) with rabbit anti-mouse (1/10) or protein A gold conjugated antibodies (1/10) in PBS containing 0.1 % Tween-20. Sections were counterstained using lead citrate and uranyl acetate. Grids were then washed in PBS, containing 0.1 % Tween-20 and 0.5 % BSA (15 min), in PBS (15 min), water (15 min) then air dried and observed with an electron microscope (Zeiss E . M . 10). Double staining of IGF-I and insulin was attempted following the above steps with different sized colloidal gold particles linked to the second antibodies for detection of immunoreactive IGF-I (5 nM) and insulin (10 nM).  71  e. Specificity testing of the IGF-I antibody The IGF-I monoclonal antibody, at the dilution used for immunostaining was incubated (12 hr, 4 ° C ) with glucagon, pancreatic polypeptide, somatostatin, insulin, proinsulin, IGF-II and C-peptide at concentrations ranging from 10 p M to 1 tiM, and with IGF-I (recombinant human) at dilutions of 0.01, 0.1, 1.0 and 10 nM. IGF-I immunostaining of rat and human pancreas sections was then performed using the peroxidase anti-peroxidase method described in section 2.3.2.C. Displacement studies were also performed on the ultra-thin electron microscope sections. The IGF-I antibody was incubated as above with insulin (100 nM) or with IGF-I (100 pM). Immunodetection of IGF-I was performed as in section 2.2.3.B.d). The specificity of the IGF-I antibody was also determined in an immunoblotting study. IGF-I, insulin, proinsulin and C-peptide (200 til, 100 nM each in PBS) were immobilized on a nylon membrane using a blotting apparatus (Bio-dot SF, Bio-Rad Laboratories, Richmond, C A ) . Membranes containing each hormone were washed in Tris buffered saline (TBS, 0.05 % Tween-20) then incubated in BSA (5 % in TBS, 12 h, 4 ° C ) to prevent non specific adhesion of the primary antibodies. Membranes were incubated with either guinea pig anti-insulin, or monoclonal IGF-I antibody in TBS (24 h, 4 ° C ) then with alkaline phosphatase conjugated goat anti-guinea pig and rabbit anti-mouse second layers in TBS (1 h, RT). Immunoreactive insulin and IGF-I were detected with an alkaline phosphatase substrate package containing 5bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) and nitroblue tetrazolium chloride (NBT).  72  2.4. E F F E C T O F IGF-I O N T H E R A T E N D O C R I N E P A N C R E A S  2.4.1. IGF-I R E C E P T O R BINDING a. Autoradiographic detection of IGF-I receptors (i) Preparation of tissues Freshly excised pancreatic tissue from male Wistar rats (250 - 300 g) was covered in embedding medium (OCT) on a cork block and put in 2-methyl butane (- 90 ° C , 5 min) in liquid nitrogen as quickly as possible. Sections (8 - 10 itm) were cut on a cryostat (Microm, Heidelberg, Germany, -20 ° C ) , heat (RT) mounted on glycine coated glass slides and kept in a desiccator (-70°C) overnight to improve adhesion to the slide.  (ii) Autoradiography Pancreatic sections were warmed in PBS (2 x 10 min, ice cold) to remove embedding medium, then incubated in PBS (0.1 % BSA, 1 mg/ml bacitracin, pH 7.4, 15 min) to reduce non specific binding of  125  I-IGF-I. Sections were then incubated with  125  I-IGF-I (130 pg/100  til, 6 h, 4 ° C ) , in the presence or absence of unlabelled IGF-I (300 pg/100 /xl) washed in PBS ( 3 x 5 min), fixed in methanol (ice cold) and dried (RT). In a dark room, slides were dipped in photographic emulsion (1:1 distilled water, 0.01 % Tween-20, 37 ° C ) and allowed to dry (30 min, RT) in a vertical position allowing a uniform distribution of emulsion. Slides were sealed in the dark with desiccant and film was exposed (4 days, 4 ° C ) then developed (Kodak D-19, 1:4 with water, 5 min), washed in water ( 2 x 3 min) and fixed (Nitrophen, 1:3 with water, 3 min). After washing under running water (10 min) sections were dehydrated in ethanol, counterstained with hematoxylin, mounted in xylene and observed under a microscope.  b. Receptor binding with isolated rat islets Rat islets were prepared and cultured as in section 2.3.l.b Following culture, islets were centrifuged (100 x g, 10 min) and the culture medium was removed. Islets (100/vial) were placed in a polypropylene vial and were incubated (18 h, 4 ° C ) with ^ I - I G F - I (total 68 x 10^ cpm) in PBS (3 % BSA, 5 mM glucose, 1 mg/ml bacitracin) in a total volume of 500  73 ul. Specificity of binding was determined by including unlabelled IGF-I (88 nM) in the buffer. Following incubation, the vials were centrifuged, the supernatant removed by pipette, and the radioactivity in the pellet determined. The percentage binding was determined by dividing the radioactivity in the pellet by the total radioactivity in the incubation.  2.4.2. CONDITIONS F O R T H E S T U D Y O F INSULIN S E C R E T I O N F R O M I S O L A T E D R A T ISLETS a. Islet insulin secretion studies Following the washes in culture medium, indicated in section 2.3. l.b, islets were picked in a laminar flow hood with a sterile pipette and plated (12 islets/well) in a 48 well plate in D M E M culture medium (500 ul). Wells were coated with rat tail collagen which was prepared by dissolving rat tail collagen fibers in acetic acid (0.02 M , approximately 3.5 g/1) over a 48 h period under centrifugation (4 ° C ) . Collagen was centrifuged (20 min, 1000 x g) and the supernatant stored (4 ° C ) until use. Wells were coated by covering the surface with collagen (45 min under U . V . light), aspirating excess collagen, drying (1 h, RT) then storing the plates (4 ° C ) until use. Islets were cultured in an incubator (95 % air, 5 % C O 2 , 48 h) in order to allow them to recover from the isolation procedure and to adhere to the wells. Each well was observed immediately prior to secretion studies under a microscope and those in which islets had not adhered or where contamination was evident were not used. Media removal and replacement involved careful pipetting so as not to disturb the islets. Incubation under study conditions was preceded by a preincubation (95 % air, 5 % C O 2 , 37 ° C ) with D M E M (4.4 mM glucose, 1 h, 500 id). This medium was then replaced with D M E M (600 ul) containing the substances to be tested for the appropriate time period. Following the study period, medium was removed under a dissecting microscope in order to avoid taking up islets which had become dislodged. Media were put in polypropylene vials and centrifuged (500 x g, 4 ° C , 10 min) in order to remove any cell debris which might interfere with the RIA, and the supernatant stored (-20 ° C ) until RIA. Acetic acid (2 M , 400 ill) was added to each well and islets were transferred to another vial, boiled (10 min), centrifuged (500 x g, 10 min) and the supernatant stored (-20 ° C ) until RIA.  74  b. Measurement of insulin in medium and cell extracts Insulin was measured using a disequilibrium radioimmunassay.  (i) Buffer The assay buffer consisted of 0.04 M phosphate (pH 7.5) stored as stock (0.4 M , 4 ° C ) to which charcoal extracted human plasma (5 %) was added on the day of assay. Charcoal extracted plasma was made from outdated human plasma which was centrifuged (30 min, 1000 x g) and passed through a sharkskin filter (15 cm) and stirred with charcoal (1 %, 1 h, 4 ° C ) . Charcoal was removed by centrifugation (30 min, 1000 x g) and the supernatant divided into aliquots (10 ml) and stored (4 ° C ) .  (ii) Antiserum Guinea pig anti-rat insulin serum was used at a final dilution of 1/1 x 10^. The antiserum was diluted (1/10) and stored (-20° C) in a lyophilized form. When required, an aliquot (100 /xl) was reconstituted (500 ml) in insulin buffer, divided into aliquots (2 ml) and stored (4 ° C ) until use in the RIA.  (iii) Iodinated insulin Porcine insulin (5 or 10 /tg) was reconstituted in H C l (0.01 M , 10 /xl) and phosphate buffer (0.2 M , pH 7.4) to a final concentration of 5 /ig/10 /xl. N a  125  I (1 mCi, 10 /xl) was  mixed with the insulin (5 /xg) in a glass test tube and the oxidation reaction started with the addition of chloramine-T (25 /xl, 4 mg/ml in 0.2 M phosphate buffer), and allowed to continue with light vortexing (10 s). The reaction was terminated with the addition of sodium metabisulphite (100 /xl, 2.4 mg/ml in 0.2 M phosphate buffer), the mixture incubated (45 s), and excess iodine added in the form of potassium iodide (50 /xl, 10 mg/ml 0.2 M in phosphate buffer). The reaction mixture was diluted in phosphate buffer (1.8 ml, 0.04 M ) and insulin purified from unbound N a  125  1 2 5  I-  I by transferring the mixture to a glass test tube containing  microfine silica (10 mg), vortexing and centrifuging (30 s, 100 x g). The supernatant was  discarded and the pellet containing the I-insulin washed and centrifuged twice with distilled 125  water to remove residual N a  125  I . Acid ethanol (3 ml) was added to the pellet to elute the  125  I-  insulin from the silica and, following centrifugation, the supernatant was diluted with acid ethanol (2 ml) and distilled water (1.5 ml). This solution was stored (-20 ° C , 4 weeks) and diluted to the appropriate concentration (2000 cpm/100 id) in assay buffer. The percent incorporation (40 - 70 %) of N a  125  I was determined according to the following equation:  % I n c o r p o r a t i o n = a c i d ethanol (cpm) + s i l i c a p e l l e t (cpm) t o t a l (cpm)  (iv) Standards Lyophilized rat insulin (100 ug, approximately 25 U/mg) was reconstituted (4.26 U/ml, approximately 200 ng/ml) in distilled water and phosphate buffer (0.04 M , 6 % BSA, 0.24 g/1 sodium merthiolate, 6 g/1 NaCl), divided into aliquots (1 ml) and stored (-20 ° C ) . When required these aliquots were further diluted (160 iiU/ml) in assay buffer, divided into aliquots (2 ml) and stored (-20 ° C ) until the day of assay. Serial dilutions were made in assay buffer to give concentrations in the standard curve of 160, 80, 40, 20, 10, and 5 /iTJ/ml.  (v) Controls The inter- and intra-assay variations were determined by including a control sample in each assay. The control consisted of samples from a glucose (17.8 mM) and arginine (10 mM) stimulated rat pancreas perfusion (30 min) as in section 2.2.3.A.a). The immunoreactive insulin level of the pooled samples was determined and the preparation was diluted in assay buffer (60 iiU/ml), divided into aliquots (1 ml) and stored (-20 ° C ) for use in the assay.  (vi) Separation Dextran coated charcoal was used to separate the free and bound I-insulin. Charcoal 125  was prepared by dissolving dextran (T-70) (5.0 g/1) in phosphate buffer (0.04 M , pH 7.4) and  76  adding charcoal (50.0 g/1). This slurry was stored (4 ° C ) and was stirred for a minimum of 1 h before use.  (vii) Procedure Assays were performed in glass tubes (12 x 75 mm) under refrigeration. Buffer, sample, and antisera (700 til, 100 til and til respectively) were added to the tubes, vortexed and incubated (4 ° C , 24 h).  125  I-insulin (100 til) was added to each tube vortexed and  incubated (4 ° C , 24 h). Dextran coated charcoal (200 til) was then added, vortexed, centrifuged (1000 x g, 4 ° C ) , decanted and the pellet was allowed to air dry (minimum 4 h) before counting on a gamma spectrometer. The assay protocol was identical to that for the IGF-I assay as was the calculation of the immunoreactive insulin content in each sample.  c. Statistical Analysis Statistical analysis was performed on the results of the islet secretion studies. In all studies, results were expressed as the percent of total cell content (TCC) and were given as the mean T C C 4 7 - standard error of the mean (SEM). The minimum number of replications in all experiments was three, and each experiment was done in triplicate. Significance was first determined, using either one or two way A N O V A . Dunnett's test was used to make multiple comparisons to the same control. Student's t-test was used to determine comparisons between two treatments.  77  3. R E S U L T S  3.1. M E A S U R E M E N T O F IGF-I IN R A T TISSUES A N D FLUIDS Since the IGF-I RIA was newly developed in this laboratory, it was necessary to optimize each step in the assay. Similarily, for reasons indicated in the Introduction, a method of IGF-I binding protein separation had to be verified in the laboratory in order to measure IGF-I levels in physiological samples.  3.1.1 T H E R A T IGF-I RIA The iodination of IGF-I yielded  125  I-IGF-I with a specific activity of 290 uCi/ug as  determined by a self displacement assay (Figure 2, p 57). The specific activity between iodinations varied by +/- 5.5 % (n=7). Elution profiles following column chromatography (PD-10) of the iodination reaction mixture were consistent between iodinations, with iodinated IGF-I eluting in fractions 2 and/or 3 and free N a  125  I eluting in fractions 6-8 (Figure 4).  Further purification of the iodinated hormone by column chromatography (G-50 Sephadex) also yielded consistent profiles over consecutive purifications, a representative example of which is shown in Figure 5. The proportion of peak #2 decreased as expected over a 5-6 week period. A rabbit anti-human IGF-I antiserum dilution of 1/18000 was used in the RIA since this dilution bound at least 50 % of  125  I-IGF-I with all levels of  125  I-IGF-I and normal rabbit  serum (NRS) included in titre assays (Figure 6). Precipitation of antibody bound I-IGF-I varied widely with combinations of 125  increasing concentrations of goat anti-rabbit antibody (GARG) and NRS (Figure 7). Based on the level of binding achieved with the above dilutions, inclusion of G A R G at a 1/50 dilution (final concentration 1/250 in assay) with a NRS concentration of 0.5 % was tested, and gave optimal precipitation of bound I-IGF-I (30 %), with low non-specific binding (2-3 %). 125  These concentrations of antisera and serum carrier were used for the IGF-I assay and  78  1 ml fractions  Figure 4 : Purification of I - I G F - I by column chromatography (G-25 Sephadex). Elution profile of I-IGF-I applied to a PD-10 prepacked column immediately following iodination. Iodinated IGF-I eluted in fractions 2 and or 3 (#1), and free N a I eluted in fractions 6 through 8 (#2). 125  125  125  79  40 -t  o o o E  a.  Figure 5: Purification of I - I G F - I by column chromatography (G-50 Sephadex). Elution profile of I-IGF-I purified within 12 hr prior to use in RIA or binding assay. I-IGF-I (peak #2) eluted within fractions 17-25. A minor peak eluting at the void volume (Vo, #1) may represent BSA associated, or polymerized I-IGF-I. Free N a I eluted after fraction 30 (peak #3). 125  125  125  125  125  80  Figure 6: IGF-I antibody binding of I-IGF-I with increasing primary antibody dilutions. The antibody dilution to be used in the IGF-I RIA was determined by comparing the binding ( B = I-IGF-I bound/total I-IGF-I x 100) of I-IGF-I (10000 or 7000 cpm) with increasing dilutions of antisera (1/500, 1/5000, 1/10000, 1/18000). The hormone/antibody complex was precipitated by centrifugation after incubation with G A R G (1/50) and NRS (0.4 or 0.7 %). Non specific binding was similar (2.0 - 3.0%) for all conditions and was subtracted in the calculation of B/Bo. 125  125  0  125  125  81  50  -i  GARG(1/10) GARG (1/25) 40 GARG (1/50) GARG (1/100) 30 -  0.8  1.0  Normal rabbit serum (%) Figure 7: Effect of second antibody dilution on precipitation of antibody bound I G F - I . Precipitation of antisera (1/18000) bound I-IGF-I in the presence of increasing concentrations of second antibody (GARG) and carrier (NRS). B = I-IGF-I bound/total I-IGF-I x 100. 125  125  125  0  125  82  yielded a standard curve with a sensitivity of 10 pg/ml and a maximum displacement of 250 pg/ml IGF-I (Figure 8), based on the E D  8 0  and E D  2 0  respectively, calculated using the RIAPC  data analysis programme (Rieger et al, 1988). The intra- and inter-assay variations were 5.5 % and 10.9 % respectively (n=12), based on the measurement of formic acid/acetone extracted rat serum control samples included in each assay.  3.1.2. S E P A R A T I O N O F IGF-I BINDING PROTEINS A . Reverse phase C  1 8  Sep-Pak extraction  Following incubation of  125  I-IGF-I in perfusate and phosphate buffer, the binding  protein extraction method of Davenport et al, (1988), yielded inconsistant and low recoveries (0.1 - 5.6 %, n=5) and was therefore not pursued further. Following incubation in phosphate buffer, elution of  125  I-IGF-I with acetonitrile (60  %), resulted in an inconsistantly high recovery (77 and 98 %). Identical samples extracted sequentially (the cartridge was primed between samples) over the same SepPak (4 x) varied by 30 %. Recovery of IGF-I in the IGF-I RIA was dependent on the sample media. Recovery of IGF-I (500 pg/ml) was low (0 - 10 %) in assay buffer and was inconsistently high (100 - 250 %) in perfusate (Krebs, section 2.3. La). The level of IGF-I detected in rat serum following the extraction procedure was consistently less (< 130 ng/ml) than that reported in the literature (> 300 ng/ml). This method was therefore not pursued further.  8 3  100  -i  80 -  o  60 -  CD  m  40 -  20 -  •  10  ' ' ' ' I—  I  I  100  I I I I  1000  IGF-I (pg/ml, log)  Figure 8: The IGF-I RIA standard curve. Increasing dilutions of human IGF-I in the RIA (2.5, 5.0, 10.0, 25.0, 50.0, 100.0, 250.0, 500.0 pg/ml IGF-I) demonstrated by plotting B / B against the standard concentration (log scale). The RIA yielded approximately 50 % displacement of I-IGF-I (7000 cpm) with 50.0 pg/ml unlabelled IGF-I, had a sensitivity of 10.0 pg/ml and a maximal displacement of 250 pg/ml, based on the E D and E D of the assay, respectively (see text). The intra- and inter-assay variation, determined using a rat serum sample included in all assays were 5.5 % and 10.9 %, respectively (n=18 assays). 125  8 0  2 0  0  84 B. Formic acid/acetone extraction a. Recovery and measurement of rat IGF-I Incubation and extraction of  125  I-IGF-I from perfusate (see section 2.2.2.b) and rat  serum resulted in consistently high recoveries (Table 4.).  Replicate  T o t a l (cpm)  Krebs p e r f u s a t e 1. 2. 3.  Supernatant  % Recovery  7129 8225 7055  6749 7778 6594  95 95 93  Krebs p e r f u s e d through i s o l a t e d r a t pancreas 5582 1. 2. 6623 5713 3.  5335 6342 5492  96 96 96  Rat serum 1. 2. 3.  6101 8310 5968  81 79 81  7560 10552 7331  Table 4. Recovery of I-IGF-I from formic acid/acetone extracted perfusate and rat serum 125  Similar recoveries of  125  I-IGF-I were determined in islet culture medium.  The formic acid/acetone extraction yielded consistently high recovery of unlabelled IGF-I in the RIA as follows:  Medium  IGF-I added  % Recovery (n=3)  Rat serum ( d i l u t e d i n RIA) 500 pg/ml 250 pg/ml  82 +/- 7% 66 +/- 5%  Krebs p e r f u s e d through i s o l a t e d r a t pancreas 500 pg/ml 250 pg/ml  91 +/- 10% 66 +/- 6%  Table 5 . Recovery of IGF-I from formic acid/acetone extracted serum and perfused pancreas  85  Serially diluted rat serum, with binding proteins extracted by the formic acid/acetone method, yielded a curve almost parallel with that of IGF-I standards in the IGF-I RIA (Figure 9). Levels of IGF-I in rat serum following extraction of the binding protein by this method were consistent with those reported by other investigators (> 300 ng/ml) and were consistent between extractions (7.1 % variance, n=7, rat sera). In the process of evaluating this extraction procedure it was determined that it was essential that the extraction be done over ice, or at 4 ° C with ice cold formic acid and acetone. At warmer temperatures there was a smaller pellet following centrifugation of the sample, and recovery of IGF-I was low (< 200 ng/ml). It was also determined that formic acid/acetone in the concentrations used in the extraction procedure, in the absence of sample, gave false positive IGF-I values (> 500 ng/ml in serum). Samples were therefore diluted (at least 1/20) prior to the RIA as suggested previously by Bowsher and coworkers (1991). If IGF-I was not detectable in samples at this dilution, samples were lyophilized and concentrated for RIA. Extraction of IGF-I from rat liver and pancreatic tissue, following the method of Lee and coworkers (1991) yielded mean levels of 600 +/- 156 and 257 +/- 63 ng/g IGF-I in these tissues respectively (n=4).  b. Gel filtration chromatography of tissues and serum The Sephacryl-200 (1 X 30 cm) column was selected to distingish between IGF-I (7649 D) and IGF-I bound to binding proteins (20 X 10 - 150 X 10 D) in physiological fluids and 3  3  tissue extracts, due to its fractionation range (5 X 10 - 2.5 X 10^ D) and high resolution (see 3  section 2.2.2.c). Molecular weights of radioactive and absorbance peaks resulting from the elution of samples were determined using the best fit profile resulting from chromatography of the molecular weight standards (see section 2.2.2.C, Figure 3, p 63). A l l experiments were repeated at least once and one representative profile is presented.  86  ;  IGF-I (pg/ml, log) serum dilutions (X100)  1  1  2.5  5  0  10  1  20  40  80  0  0  1  160  0  0  °  320  Figure 9: IGF-I RIA of serial dilutions of formic acid/acetone extracted rat serum. IGF-I levels were determined in serially diluted rat serum (1/250 - 1/32000) treated with the formic acid/acetone binding protein extraction method (see text). The displacement of I-IGF-I (B/Bo) with serum IGF-I paralleled that of displacement by the IGF-I standards (2.5 - 500 pg/ml) in the RIA as plotted above. 125  87  (i) Pancreatic extract Column chromatography of rat pancreatic extract, preincubated with  125  I-IGF-I,  yielded a radioactive elution profile with peaks in the high (> 12 kD, fractions 15 - 23) molecular weight range, as well as in the range of free  125  I-IGF-I (8 kD, fraction 29) (Figure  10). Radioactive peaks 1 and 2 were present in the molecular weight region predicted for IGFI associated binding proteins (30 - 150 kD). The molecular weights of these peaks were approximately 200 kD and 60 kD respectively, probably representing the association of  1 2 5  I-  IGF-I with IGF-IBP-3 (150 Kd) and with its acid stable subunit (50 kD). A higher proportion of  125  I G F - I was associated with the acid stable subunit (peak 2). A third radioactive peak (#3)  eluting in the same fraction as free  125  I-IGF-I was indicative of incomplete binding to  pancreatic binding proteins. A relatively high level of radioactivity existed in the fractions between peak 2 and peak 3, perhaps representing lower molecular weight binding proteins. Pancreatic extracts incubated with  125  I G F - I followed by treatment with the formic  acid/acetone IGF-I binding protein extraction method, and eluted over the column (Figure 10), exhibited little radioactivity in the high molecular weight range (fractions 15 -23) in comparison to unextracted samples, with the majority eluting as free  125  I-IGF-I (fraction 29).  Two radioactive peaks were once again discernable in the high molecular weight range (> 20 kD) but they occurred in differing proportions to those in the unextracted samples, with a larger proportion of  125  I-IGF-I eluting in the size range of IGF-IBP-3 (200 kD, peak 1). A  lower level of radioactivity was present in the fractions between peaks 2 and 3 than was evident in untreated pancreatic extracts suggesting a reduction in lower molecular weight binding proteins following formic acid/acetone treatment.  (ii) Serum Rat serum incubated with  125  I-IGF-I exhibited a similar elution profile to that of  pancreatic extract when applied to the same G-200 column (Figure 11). As was the case with the pancreatic extracts, untreated samples exhibited a greater proportion of radioactivity in the high molecular weight (30 - 150 kD) range of IGF-I associated binding proteins than in formic  88  6000 -i Pancreas extract (iodinated IGF-I)  5000 -  Pancreas extract (iodinated IGF-I) (following formic acid acetone extraction)  4000 E  Q. O  3000 -  2000 -  1000 -  Fractions (0.6 mL)  Figure 1 0 : Elution profile of rat pancreatic extracts incubated with I-IGF-I separated by column chromatography (Sephacryl S-200 HR). Radioactive elution profile of pancreatic extracts preincubated with I-IGF-I (approximately 15,000 cpm in 200 ttl), either untreated or extracted with the formic acid/acetone binding protein method. Arrows indicate radioactive peaks in the high (#1, #2, > 20 kD) and low (#3, 10 kD) molecular weight ranges as described in the text. Arrow #3 indicates the elution peak of free I-IGF-I. 12S  125  125  89  Figure 11: Elution profiles of rat serum incubated with I-IGF-I separated by column chromatography (Sephacryl S-200 HR). Radioactive elution profile of serum preincubated with I-IGF-I (approximately 10,000 cpm in 200 id), either untreated or extracted with the formic acid/acetone binding protein method. Arrows indicate radioactive peaks in the high (#1, #2, > 20 kD) and low (#3, 10 kD) molecular weight ranges as described in the text. Arrow #3 indicates the elution peak of free I-IGF-I. 125  125  125  90  acid/acetone treated samples. As for pancreatic extract, two peaks were apparent in the high molecular weight region (1, 2) in both untreated and in formic acid/acetone treated serum samples. In contrast to the pancreatic profiles, however, a higher proportion of  125  I-IGF-I  activity was associated with peak 1 (probably primarily IGFBP-3) in the unextracted serum, and with the acid stable subunit (peak 2), in the formic acid/acetone treated serum. Formic acid/acetone treated serum also decreased the amount of radioactivity eluting between peaks 2 and 3 which, as with pancreatic extract, may indicate a removal of low molecular weight IGFI binding proteins.  (iii) Liver Formic acid/acetone treated liver extracts also exhibited little radioactivity in the high molecular weight range with the majority eluting in the region of free  125  I-IGF-I (Figure 12).  As with pancreatic extract and serum, the elution profile of liver extract exhibited two peaks (1 and 2) in the high molecular weight range. A slightly higher proportion of  125  I-IGF-I activity  was associated with the acid stable subunit (peak 2) as seen in the formic acid/acetone treated serum samples.  (iv) Serum IGF-I immunoreactivity The effectiveness of the removal of IGF-I binding proteins from samples was further indicated in the determination of IGF-I immunoreactivity (by RIA) following column chromatography of formic acid/acetone treated serum (Figure 13). Peak immunoreactivity (above assay maximum) eluted in the molecular weight region associated with free  125  I-IGF-I  (8000 D , fraction 29). IGF-I immunoreactivity was also detectable in the high molecular weight region, perhaps indicating an incomplete removal of binding proteins. Decreasing IGFI concentrations evident in the lower molecular weight regions (fractions 32 - 55, < 7000 D) may be due to adsorption of some IGF-I to the column.  91  Fraction size (0.6 ml)  Figure 12: Elution profile of rat hepatic extract incubated with I-IGF-I separated by column chromatography (Sephacryl S-200 HR). Rat hepatic extract was incubated with I IGF-I, treated with the formic acid/acetone binding protein extraction method, and eluted over a Sephacryl S-200 HR column (1 X 30 cm). Arrows indicate radioactive peaks in the high (#1, #2, > 20 kD) and low (#3, 10 kD) molecular weight ranges as described in the text. Arrow # 3 indicates the elution peak of free I-IGF-I. 125  125  125  92  450 -  400 -  350 -  300 -  Fraction size (0.6 ml)  Figure 13: Immunoreactive IGF-I in rat serum following column chromatography (Sephacryl S-200 H R ) . Rat serum (200 ul) was treated with the formic acid/acetone binding protein extraction method, then subjected to chromatography over a Sephacryl S-200 HR column (1 X 30 cm). IGF-I immunoreactivity was measured in aliquots from each fraction (100 ul) using the IGF-I RIA. The arrow indicates peak immunoreactivity of IGF-I as well as peak elution of free I-IGF-I. 125  93 3.1.3. R E L E A S E OF IGF-I FROM RAT ENDOCRINE PANCREAS A. In situ perfusion of rat pancreas Samples resulting from the in situ perfusions of glucose (300 mg %) alone, with G H (0-5 i t g / ) or with GIP (0-1 nM) gradient) were treated with the formic acid/acetone ml  extraction method prior to inclusion in the IGF-I RIA. IGF-I was not detectable in the RIA, under any perfusion conditions, even when concentrated (2 x). Perfusate alone did not exhibit IGF-I immunoreactivity, either with or without the formic acid/acetone extraction. In order to verify that the pancreas was responsive to the various stimuli, insulin levels in the samples from glucose and the G H and GIP gradients were determined. These were consistent with levels following stimulation with a high glucose concentration (50-150 uU/min) and a high glucose concentration with GIP (300 - 700 iiU/min), as indicated previously (Pederson et al, 1990). Further attempts at detecting IGF-I in perfusate samples were made following acidification and elution (with 60 % acetonitrile) through C  l g  cartridges. Although IGF-I  immunoreactivity was detectable (0 - 15 pg/ml), levels did not vary throughout the perfusion period, and were likely a result of falsely elevated levels detected in Krebs perfusion buffer alone (see section 3.1.2.A.).  B. Isolated rat islet secretion studies IGF-I immunoreactivity was not detectable in the incubation medium following formic acid/acetone treatment (concentrated 1.5 x), under any of the following conditions:  Conditions: Peptide  Glucose  Time  I s l e t s per well  GH ( r a t ) (0.1, 1.0 uK)  17.8 mM  3, 24 hr  20, 50, 80  GIP (5.0 nM)  17.8 mM  2, 4, 24 h r  10, 50, 80  Table 6. Islet incubation conditions for the detection of secreted IGF-I  Inconsistent, low levels of IGF-I (0 - 50 pg/ml) were detected in assays of neat, unextracted release media from secretion studies. These results were considered unreliable due to the potential influence of binding proteins. Insulin release was determined in order to verify that the islets were viable and responding to stimuli. Insulin was determined following glucose (17.8 mM) and glucose and GIP (5 nM) stimulated secretion studies (2 hr, 10 islets per well):  release  2 hour  (n=6)  Treatment  Insulin  g l u c o s e (17.8 mM)  per w e l l (mean) % TCC (mean)  2523 /iU/ml +/5.4 +/- 0.5  g l u c o s e (17.8 mM) GIP (5 nM)  per w e l l (mean) % TCC (mean)  3750 jUU/ml +/8.7 +/- 2.3  Table 7. Islet incubation conditions for the detection of IGF-I and insulin  C. IGF-I in islet and B-cell extracts Given the absence of detectable immunoreactive IGF-I in the previous two studies, further attempts to measure IGF-I in rat islets were performed on islet and B-cell extracts. Wistar rat islets (approximately 500) and 6-cells (approximately 20,000) were extracted according to the method of Lee and coworkers (1991). Half of each sample was run in the IGF-I RIA, and the other half blotted onto a nylon membrane. Purified B-cells were kindly provided by B. Verchere and were obtained by cell sorting of dissociated islets by light scatter activity and fluorescence using a FACS-IV (Becton-Dickinson, Sunny Vale, C A ) .  a. Immunoreactive IGF-I determined by RIA Radioimmunoassay of an extract of 250 islets yielded a total of 79 pg of IGF-I. Immunoreactive IGF-I in an extract of 10,000 B-cells totalled 30 pg.  b. Immunoreactive IGF-I identified by slot blot B-cell extracts were blotted onto a nylon membrane and immunoreactive IGF-I was identified (Figure 14) using the immunocytochemical technique described in section 2.3.2.e  95 This procedure revealed the presence of immunoreactive IGF-I in the B-cell extract, as well as in the control well containing IGF-I (13 nM).  Figure 14: Detection of IGF-I in an immunoblot of B-cell extracts. The IGF-I monoclonal antibody (8 uM) indicated the presence of IGF-I in 6-cell (10,000) extract adsorbed to a nylon membrane (b). The positive control (IGF-I, 100 nM) was also detected by the monoclonal antibody (a), whereas the negative control (acetic acid extract of culture medium) indicated a lack of IGF-I immunoreactivity (c). All other wells were blocked with gelatin, and were immuno-negative.  96  3.1.4. HPLC CHARACTERIZATION OF RAT ISLET IGF-I A. Peptide standard elution Human IGF-I (2.5 tig), insulin (4.0 tig) and proinsulin (3.5 tig) standards were separated on a reverse phase C  1 8  column. A 33 - 45 % acetonitrile gradient yielded elution  times, based on absorbance (225 nm), as follows: Void volume IGF-I (rh) insulin (h) proinsulin (h)  : : : :  1.7 5.0 6.6 7.7  min (fraction min (fraction min (fraction min (fraction  4) 10) 14) 16)  Radioimmunoassay of the standard samples indicated that peak IGF-I immunoreactivity was above assay maximum and appeared in fractions 9-11. Peak insulin immunoreactivity, also above assay maximum, appeared in fractions 13-15, as well as in fraction 18, which perhaps indicated the detection of proinsulin in the insulin RIA. These peaks of immunoreactivity coincided with the peaks determined by U V absorbance, and yielded a separation of approximately 2 minutes (4 samples) between the elution of IGF-I and insulin, and 1 minute (2 samples) between the elution of insulin and proinsulin.  B. HPLC of rat islets High performance liquid chromatography of formic acid/acetone treated islet extracts, (100 til approximately 75 islets in duplicate RIA tubes) demonstrated the presence of a distinct IGF-I immunoreactive peak (fractions 9-11) eluting in the same position as the IGF-I standard (Figure 15). An approximation of the concentration of IGF-I was a minimum of 750 pg/ml, i.e. a minimum concentration of IGF-I of 1 pg/islet (based on 100 til volume/RIA tube). Some immunoreactivity was also detected in the void volume, perhaps associated with binding proteins not completely removed by the formic acid/acetone treatment. Lower levels of IGF-I immunoreactivity were also evident in the elution position of insulin and perhaps proinsulin. Insulin has been determined to cross react in the IGF-I RIA at a level of approximately 1 X 1 0 M , a level which was calculated to be present (3.8 tiM/75 islets) based 6  97  on the insulin content of an islet (40 ng or 6.3 nmoles per islet determined following islet secretion studies). Insulin immunoreactivity following H P L C of formic acid/acetone treated islet extract eluted in a broad peak in the position of insulin and proinsulin standards. No insulin immunoreactivity was evident in the region of the IGF-I standard.  C . H P L C of rat serum IGF-I immunoreactivity was also detected in H P L C fractions of formic acid/acetone treated serum (400 til) eluted under the same conditions as above (Figure 15). The IGF-I immunoreactive peak in rat serum, determined by RIA following H P L C , corresponded to the immunoreactive peaks found in the control and pancreatic H P L C profiles.  98  450  1  2  3  V  V  V  1  400 -  Time (0.5 min)  Figure 15: H P L C elution profile (33 - 45 % acetonitrile gradient) of IGF-I immunoreactivity detected by RIA in rat islet extracts and rat serum. Islet extracts and serum were treated with formic acid/acetone to remove binding proteins, then eluted over a reverse phase C column (3.9 X 30 mm). Peak IGF-I immunoreactivity eluted in fraction 10 in both islet extracts and serum. Peak immunoreactive IGF-I was above assay maximum in islet extracts. Arrows indicate the elution positions of IGF-I (arrow #1), insulin (arrow #2) and proinsulin (arrow #3) standards eluted under identical conditions and detected at 225 nm. 1 8  99  3.2. L O C A L I Z A T I O N O F IGF-I IN T H E P A N C R E A S  3.2.1. S P E C I F I C I T Y O F T H E IGF-I A N T I B O D Y A . Displacement of tissue IGF-I immunoreactivity Preliminary immunocytochemical studies using the IGF-I monoclonal antibody indicated widespread immunoreactivity within the rat endocrine islet tissue (Figure 16). The IGF-I antibody exhibited immunoreactivity in both rat (Figure 16), and human (Figure 19a) pancreatic sections at antibody concentrations ranging from 0.5 - 100 ug/ml, using the peroxidase/DAB method of demonstration. A concentration of 8 iig/ml or less was used throughout the study. At this dilution, preincubation of the antibody with hIGF-I (100 p M and greater) completely abolished immunodetection in rat pancreatic sections (Figures 17a,b). Incubation of the antibody with insulin (up to 10 ug/ml) slightly lessened the intensity of, but did not abolish, staining (Figures 18a,b,c). Identical results were obtained with human tissue sections (Figures 19a,b). Preincubation of the antibody with glucagon, pancreatic polypeptide, somatostatin, proinsulin, IGF-II and C-peptide did not affect immunoreactivity. Further control sections included the absence of first or second antibodies, which indicated a lack of non-specific binding. Similarly, at the electron microscope level, preincubation of the IGF-I antibody with IGF-I (10 nM) abolished immunostaining (Figure 26) in rat pancreatic sections, whereas preincubation with insulin (100 nM) did not (Figure 27), suggesting that at both the light and electron microscope levels, the IGF-I monoclonal antibody was specific for IGF-I detection using this technique.  100  Figure 16: Immunoreactive IGF-I in a section of Wistar rat pancreas. Immunodetection of IGF-I with an IGF-I monoclonal antibody (4 /xM), demonstrated with D A B , reveals the presence of immunoreactivity in the rat pancreas. Large arrows indicate immunoreactivity with a wide distribution, associated with the endocrine islet tissue, and individual cells. Small arrows indicate the absence of immunoreactivity in the exocrine tissue, as well as in ductular tissue (D).  101  mm  F I G U R E 17a, 17b: Absence of IGF-I immunoreactivity in rat pancreas sections following the incubation of the IGF-I antibody with IGF-I. The specificity of the antibody was tested by preincubating the IGF-I monoclonal antibody (4 uM) with MGF-I (a. 100 p M ; b. 1 nM), prior to incubation with rat pancreas sections. Arrows indicate islet tissue, indicating the absence of immunoreactivity following demonstration with D A B .  102  Figures 18a, 18b, 18c: Presence of IGF-I immunoreactivity in rat pancreas sections following the incubation of the IGF-I antibody with insulin. The specificity of the antibody was tested by preincubating the IGF-I monoclonal antibody (4 uM) with human insulin (18b 10 nM; 18c 1 uM), prior to incubation with rat pancreas sections. Arrows indicate the presence of IGF-I immunoreactivity following demonstration with D A B in both the control (no insulin preincubation; 18a) and test sections.  103  Figures 19a, 19b: Presence of IGF-I immunoreactivity in human pancreas sections, with and without incubation of the IGF-I antibody with insulin. The specificity of the IGF-I antibody was tested by preincubating the IGF-I monoclonal antibody (4 tiM) with insulin (100 nM; 19b) prior to incubation with human pancreas sections. Arrows indicate that insulin appears to decrease the intensity of IGF-I immunoreactive detection, demonstrated with D A B , particularly in certain areas of the islet, in comparison to the control (no insulin preincubation; 19a).  104 B. Immunoblotting studies The IGF-I monoclonal antibody recognised only IGF-I when incubated with membrane bound IGF-I, insulin, proinsulin and C-peptide using the identical detection techniques used for immunocytochemical studies (Figure 20). This further indicates a lack of recognition of closely related islet hormones by the IGF-I monoclonal antibody. Using the same technique, the insulin polyclonal antibody detected both insulin and proinsulin, but not C-peptide or IGFI.  105  Figure 2 0 : Specific detection of I G F - I by the I G F - I monoclonal antibody indicated b y a n immunoblotting study with homologous peptides. The IGF-I monoclonal antibody (8 uM)  detected only IGF-I as indicated by immunoblotting studies of proinsulin, C-peptide, insulin and IGF-I (200 ul, 100 nM) adsorbed to a nylon membrane and demonstrated with alkaline phophatase (panel b). The polyclonal insulin antibody, used for colocalization studies, recognized both insulin and proinsulin, but not C-peptide, or IGF-I (panel a).  106  3.2.2. LOCALIZATION OF IGF-I IN THE RAT PANCREAS  A. Immunolocalization of IGF-I at the light microscope level IGF-I immunoreactivity was evident in the endocrine pancreas as indicated by the peroxidase/DAB method of demonstration. (Figure 16, 18a). There was an absence of IGF-I immunoreactivity in the exocrine pancreas as well as in the vasculature and ducts (Figure 16). Double staining studies made use of fluorescence-linked second antibodies to indicate hormone immunoreactivity. The colour green indicates IGF-I (FITC) immunoreactivity and red indicates insulin, glucagon, somatostatin and pancreatic polypeptide (RITC) in sequential sections. These studies revealed that IGF-I immunoreactivity was widespread throughout the endocrine tissue (Figure 21a) as shown previously using peroxidase/DAB demonstration. Double antibody studies indicated colocalization with insulin, resulting in a yellow colour (Figure 21b). IGF-I was not localized with any other islet hormones as indicated by distinct red and green colours (Figures 21c, 21d, 21e), indicating that IGF-I was present only in the 8cells of the endocrine pancreas. Polyclonal antibodies (Dr. P.K.Lund #1, #2) also indicated IGF-I immunoreactivity in the rat islet B-cells (Figures 22a,b). Staining with both antibodies was displaced following preincubation with IGF-I (100 pM) and almost completely displaced with high concentrations (100 nM) of insulin. IGF-I immunoreactivity was also widespread in sections made from 1 and 7 day old rat pancreas (Figures 23a,b). Although double staining studies were not performed in order to localize the cell type(s), immunoreacivity was present in the core of the islets indicating the presence of IGF-I in the B-cell if not in other islet cells. This would indicate the absence of an age-dependent expression of IGF-I in the islet.  a  b  %  #  :%  ».••  r • -  *  ijj  •  #  •  Figures 21a, 21b, 21c, 21 d, 21e: Pancreatic localization of IGF-I and colocalization with other hormones determined using double staining techniques with fluorescent detection in serial sections of rat pancreas. Immunoreactive IGF-I (a) in a rat pancreatic islet demonstrated with FITC (green). Double staining using the IGF-I and insulin antibodies with IGF-I demonstrated with FITC, and insulin with RITC (red) indicating colocalization of the two peptides revealed by the yellow colour (b). Double staining using the IGF-I (FITC) antibody and the glucagon (RITC) (c), somatostatin (RITC) (d) and pancreatic polypeptide (RITC) (e) antibodies, indicated a lack of colocalization revealed by the distinct red and green colours.  108  Figure 22a, 22b: Immunoreactive IGF-I in the rat pancreas detected using two different polyclonal IGF-I antibodies. IGF-I immunoreactivity is indicated by D A B demonstration (arrows) using two different (a, #1; and b, #2) polyclonal rabbit anti-IGF-I antibodies. These antibodies revealed a similar localization of immunoreactivity to that demonstrated by the IGFI monoclonal antibody.  Figure 23a, 23b: Immunoreactive IGF-I in pancreatic sections from neonatal (day 1), and 7 day old rats. IGF-I immunoreactivity is indicated (arrows), using the IGF-I monoclonal antibody demonstrated with D A B , in pancreatic sections from neonatal (a) and 7 day old (b) Wistar rats.  110 B. Immunolocalization of IGF-I at the electron microscope level Immunodetection studies at the electron microscope level also revealed that IGF-I was concentrated in the B-cells (Figure 24a,b) and not in other islet cells (Figure 25a,b), nor in the exocrine pancreas (Figure 25b). There was little evidence of specific IGF-I immunoreactivity in any other cellular component but B-cell granules. It was apparent that IGF-I immunoreactivity was localized to some, but not all B-cell granules (Figure 24a,b). This IGF-I immunoreactivity was displaced by preincubation of the IGF-I antibody with IGF-I (1 nM) (Figure 26a), but unaffected by preincubation with insulin (100 nM) (Figure 26b). In particular, the mature B-cell granules, exhibiting a dark core and a light surrounding halo, demonstrated a concentration of IGF-I immunoreactivity. The immature B-cell granules, having a uniform appearance with the absence of a light halo, did not. All B-cell granules exhibited insulin immunoreactivity (Figure 27). The finding that both immmature as well as mature B-cell granules exhibited insulin immunoreactivity could be due to the detection of both insulin and proinsulin by the insulin antibody which was previously indicated in the immunoblotting study (Figure 20). Attempts at colocalization using the IGF-I and insulin antibodies, demonstrating each with different sizes of colloidal gold, were not successful as the gold particles appeared to attract one another and 'clumping' occurred (Figure 28). This was likely a result of the use of Protein A which recognizes IgG from many species including guinea pigs with a high affinity, however, a low affinity for mouse IgG resulted in the detection of both the insulin as well as the IGF-I antibodies.  I l l  Figure 24a, 24b: Thin sections of pancreas indicating IGF-I immunoreactivity in rat betacells. I m m u n o r e a c t i v e I G F - I i s indicated (arrows), u s i n g the I G F - I m o n o c l o n a l a n t i b o d y demonstrated w i t h c o l l o i d a l g o l d particles (5 n m ) , i n m i c r o t h i n sections o f rat pancreas (a a n d b ) . I m m u n o r e a c t i v i t y is evident o n l y i n mature granules (large a r r o w s ) e x h i b i t i n g a dense c o r e w i t h a s u r r o u n d i n g l i g h t h a l o , a n d i s absent i n the i m m a t u r e granules ( s m a l l a r r o w s ) a n d i n the nucleus ( N ) .  112  Figure 25a, 25b: Absence of IGF-I irnmunoreactivity in thin sections of non-fi-cells and  exocrine rat pancreas. IGF-I immunoreactivity is absent, as indicated by a lack of colloidal gold particles, in all areas of endocrine non-J3-cells including secretory granules (arrows) and nuclei (N) (a and b). IGF-I immunoreactivity is also absent in the exocrine pancreas (E) (b).  Figure 26a, 26b: Specificity of the IGF-I monoclonal antibody indicated by preincubation with IGF-I and insulin prior to application to thin rat pancreatic sections. IGF-I  immunoreactivity is almost absent in both mature and immature B-cell granules (arrows) following preincubation of the IGF-I antibody with IGF-I (10 nM), as indicated by an absence of colloidal gold particles (5 nm) (a). IGF-I immunoreactivity is evident in the mature B-cell granules (arrows) following preincubation of the IGF-I antibody with insulin (100 nm), (b).  114  Figure 27: Thin section of a rat B-cell exhibiting insulin immunoreactivity. Insulin immunoreactivity in a rat B-cell is demonstrated using colloidal gold particles (10 nm). Immunoreactivity is concentrated in both immature (large arrows) and mature (small arrow) 6cell granules. N = cell nucleus  115  Figure 28: Attempt to colocalize IGF-I and insulin immunoreactivity in the rat B-cell. A t t e m p t s to c o l o c a l i z e IGF-I a n d i n s u l i n i m m u n o r e a c t i v i t y i n the rat B - c e l l u s i n g t w o sizes o f c o l l o i d a l g o l d ( 1 0 n m a n d 5 n m respectively) resulted i n apparent ' c l u m p i n g ' o f the t w o sizes o f g o l d p a r t i c l e s i n both mature (large a r r o w ) a n d i m m a t u r e ( s m a l l a r r o w ) B - c e l l granules.  116 3.2.3. L O C A L I Z A T I O N O F IGF-I IN T H E P A N C R E A S O F O T H E R SPECIES IGF-I immunoreactivity was present in the beta-cells of swine, but not ovine pancreatic sections, using the peroxidase/DAB demonstration technique, with the same IGF-I antibody dilution as was used in the rat (Figure 29a,b). This may indicate an absence of the hormone in ovine endocrine pancreas, or may be due to a lack of recognition of ovine IGF-I by the IGF-I antibody. The porcine IGF-I amino acid sequence is identical to that of the human, whereas the ovine sequence exhibits one amino acid substitution.  117  Figure 29a, 29b: Presence of IGF-I immunoreactivity in sections of porcine, but not ovine pancreas. IGF-I immunoreactivity is indicated, using the IGF-I monoclonal antibody demonstrated with D A B , in a widespread area of the porcine (a) endocrine pancreas (arrows). There is an absence of IGF-I immunoreactivity in the ovine pancreatic section (b).  118 3.3. IGF-I RECEPTORS IN RAT PANCREAS  3.3.1. PRESENCE OF IGF-I RECEPTORS The presence of IGF-I receptors in whole rat islets was investigated by measuring IGF-I binding. Total binding was 0.58 % (n=2) when 68000 cpm  125  1 2 5  I-  I-IGF-I (approximately  130 pg IGF-I) were incubated with 100 islets. Displacement with unlabelled IGF-I (132 pg), indicating specific binding, was 58.5 % (n=2). Further binding studies were not pursued since extensive binding studies were published by Van Schravendijk and coworkers (1987), indicating IGF-I binding to high affinity receptors in both beta and alpha cells isolated from rats. The binding obtained in the current assay agreed with results published in whole rat islet preparations indicating that the iodinated  125  I-IGF-I prepared was able to recognize receptors  in Wistar rat islets.  3.3.2. LOCALIZATION OF IGF-I RECEPTORS Autoradiographic techniques using  125  I-IGF-I on rat pancreatic sections indicated that  IGF-I bound predominantly at the periphery of the islets, as well as within the islet (Figure 30). The density on the periphery of the islet suggests a predominance of receptors in this region, however, this may be due to the maintenance of better tissue integrity throughout the procedure in comparison with that within the islet. Indeed, at high magnification, it appeared that the cellular integrity at the core of the islet was adversely affected by the multiple incubations and washes prior to tissue fixation. Binding at the periphery as well as within the islet would be consistent with results of radioreceptor assays indicating IGF-I specific binding on alpha and beta cells (Van Schravendijk et al, 1987). Extensive, yet unsuccessful, attempts were made to perform immunocytochemical techniques on these tissue sections in order to localize binding to (a) particular cell type(s). A low level of non-specific binding of IGF-I was demonstrated by displacement studies which included an excess of unlabelled IGF-I in the incubation of  125  I-IGF-I with the tissue  section. Specific IGF-I binding in the pancreatic sections was confirmed by parallel studies in  119 rat ovarian tissue sections (Figure 31) which demonstrated a similar distribution of IGF-I binding to that previously shown in sheep ovaries (Monget et al, 1989) where binding was evident in both granulosa and thecal cells. The binding evident on both the granulosa lutein cells and the theca lutein cells of the section of corpus luteum in this study is also consistent with the presence of Type 1 receptor mRNA found in human granulosa lutein cells (Hernandez et al, 1992).  M3SOX Figure 30: Localization of IGF-I receptors in rat pancreas demonstrated using autoradiographic techniques. The binding of I-IGF-I (black dots) was localized to discrete areas in pancreatic sections. Binding is located primarily at the periphery, as well as within endocrine tissue (arrow). 125  Figure 31: Localization of IGF-I receptors in sections of rat ovary demonstrated using autoradiographic techniques. IGF-I receptors binding is identified in the corpus luteum (large arrow) in a section of rat ovarian tissue. Receptor binding (black dots) is evident in both peripheral thecal (T) and core granulosa (G) cells.  121 3.4. EFFECT O F IGF-I O N INSULIN SECRETION F R O M ISOLATED R A T ISLETS Insulin secretion is reported as a percentage of insulin secretion of total islet cell content due to the variability encountered during the course of the islet secretion studies. There existed variability, as expected, in the sizes of the islets and, therefore, in the amount of total insulin extracted from well to well and therefore in the basal release from each well of islets. Although it is also possible to express release based on D N A content, this would not neccessarily indicate B-cell D N A due to the presence of inconsistent percentages of the different cell types found in islets, given that islets were extracted from the whole pancreas.  3.4.1. E F F E C T OF IGF-I O N INSULIN SECRETION WITH INCREASING GLUCOSE CONCENTRATIONS In order to determine whether IGF-I exerted an effect on insulin secretion in a glucose dependent manner, IGF-I (170 nM), was incubated with isolated rat islets for a two hour period. This concentration of IGF-I was determined to increase insulin secretion significantly in the presence of high glucose in preliminary studies. The effect of IGF-I was found to be glucose dependent, as IGF-I had little effect on glucose stimulated insulin secretion up to a concentration of 8.9 mM, and only significantly elevated secretion (2.2 X) at the highest level of glucose used (17.8 mM) (Figure 32). IGF-I significantly increased insulin secretion in the presence of the highest levels of glucose (8.9 and 17.8 mM ) over IGF-I stimulated insulin secretion in the absence of glucose (4.2 X and 19.0 X respectively), further indicating the glucose dependence of IGF-I action. As expected, elevated glucose concentrations (8.9 and 17.8 mM) significantly stimulated insulin secretion (3 X and 7.4 X respectively) above that in the absence of glucose.  3.4.2. EFFECT OF INCREASING CONCENTRATIONS OF IGF-I O N INSULIN SECRETION IN T H E PRESENCE OF GLUCOSE AND GD? STIMULATION GIP has previously been demonstrated to be a strong potentiator of insulin secretion during hyperglycemia. The effect of IGF-I on glucose stimulated (17.8 mM) insulin secretion, was studied in the presence of a maximal stimulatory level of GIP (10 nM) to determine whether these two secretogogues were capable of potentiating each others activity.  122  10 n  8 -  * * I  glucose alone  0  IGF4(170.0nM)  o o  I—  ^  6H  c o CD  o  CD  =3 (A C  4 -  2 -  glucose (mM)  4.4  8.9  17.8  Figure 32: Effect of IGF-I (170 nM) on insulin secretion from isolated rat islets in the presence of increasing glucose stimulation. Ten islets from Wistar rats were incubated (2 h) with one of four glucose concentrations (0.0, 4.4, 8.9, 17.8) in the presence, or absence of IGF-I (170.0 nM). Insulin secretion is expressed as a percent of total cell content (%TCC +/SEM). * p<0.005 Dunnett's (comparison to 0.0 mM glucose within each treatment group) ** p<0.005 Student's t-test  123 GIP (10 nM) significantly elevated glucose stimulated insulin secretion (1.7 X) as expected (Figure 33). IGF-I, at a concentration of 1.7 nM had no significant effect on insulin secretion. The combination of IGF-I (1.7 nM) and GIP, however, resulted in insulin secretion levels much higher than those achieved when each hormone was administered separately, (2 X that of GIP alone, 3.1 X of IGF-I alone), or than the sum of these levels (1.3 X). Similarly, the higher level of IGF-I (17.0) also potentiated the influence of GIP [1.3 X the sum of insulin secretion stimulated by GIP (10 nM) and IGF-I (17.0 nM)]. The highest level of IGF-I (170 nM) increased GIP stimulated insulin secretion in an additive manner [(0.9 X the sum of insulin secretion stimulated by GIP (10 nM) and IGF-I (170.0 nM)], indicating a close to maximal stimulation of insulin secretion.  3.4.3. EFFECT O F AN IGF-I MONOCLONAL ANTIBODY ON INSULIN SECRETION IN T H E PRESENCE OF GLUCOSE AND IGF-I STIMULATION A. Effect of an IGF-I monoclonal antibody on glucose stimulated insulin secretion To determine the influence of a possible paracrine or autocrine effect of IGF-I, increasing concentrations of the IGF-I monoclonal antibody were incubated with isolated islets in the presence of a stimulatory level of glucose (17.8 mM). The monoclonal IGF-I antibody was found to have no effect on glucose stimulated insulin secretion, at any concentration of antibody included (Figure 34). This suggested that there was no influence of IGF-I of islet origin, released into the medium, on glucose stimulated insulin secretion.  B. Effect of an IGF-I monoclonal antibody on IGF-I stimulated insulin secretion In order to determine whether the monoclonal antibody was effective in neutralizing the effect of exogenous IGF-I on insulin secretion from isolated islets, the antibody was incubated in the presence of a stimulatory level of IGF-I (85 nM). The IGF-I monoclonal antibody significantly reduced (-2.7 X) IGF-I (85 nM) stimulated insulin secretion (-1.8 X), but as seen previously, had no effect on glucose stimulated (17.8 mM) insulin secretion in the absence of IGF-I (Figure 35).  124  * *  IGF-I (nM) glucose (17.8 mM)  0.0 +  1.7 +  17.0 +  170.0 +  FIGURE 33: Effect of increasing concentrations of IGF-I on GIP stimulated insulin secretion from isolated rat islets. Ten islets from Wistar rats were incubated (2 h) in media containing glucose (17.8 mM) and increasing concentrations of IGF-I (0.0, 1.7, 17.0, 170.0 nM) in the presence, or absence of GIP (10 nM). Insulin secretion is expressed as a percent of total cell content (%TCC +/- SEM). * p < .005 Dunnett's (comparison to 0.0 n M IGF-I within each treatment group) ** p<0.1 *** p < .01 **** p < .0005 Student's t-test. n=4 in the absence of GIP. n=3 in the presence of GIP.  12  O O  o t  o  <D CO  Glucose (17.8 mM) IGF-I Ab  + 1/750  + 1/75  F I G U R E 34: Effect of increasing concentrations of a n I G F - I monoclonal antibody o n glucose stimulated insulin secretion f r o m isolated r a t islets. Ten islets from Wistar rats were incubated (2 h) in media containing glucose (17.8 mM) and decreasing dilutions of an IGF-I monoclonal antibody (1/750, 1/75). Insulin secretion is expressed as a percent of total cell content (%TCC +/- SEM). n=3 for all conditions.  126  O O cr-  g CD i_ O CD CO  Z3 CO  c  glucose (17.8 mM) IGF-I (85 nM) IGF-I Ab (1/75)  FIGURE 35: Effect of an IGF-I monoclonal antibody on IGF-I stimulated insulin secretion from isolated rat islets. Ten islets from Wistar rats were incubated (2 hr) in media containing glucose (17.8 mM) in the presence or absence of an IGF-I monoclonal antibody (1/75) and IGF-I (85.0 nM). Insulin secretion is expressed as a percent of total cell content (%TCC +/- SEM). * p<0.05 Dunnett's (in comparison to all other conditions) n=3 for all conditions.  127 3.4.4. E F F E C T O F IGF-I ON INSULIN SECRETION F R O M ISOLATED ISLETS OVER A 24 HOUR INCUBATION PERIOD In order to assess the effect of IGF-I on insulin secretion over a longer time period, increasing concentrations of IGF-I were incubated with isolated rat islets for 24 hours. These experiments were performed in order to determine the possible influence of locally produced IGF-I and binding proteins. In contrast to the stimulatory effect of IGF-I (170.0 nM) seen following a two hour incubation, IGF-I at this concentration had no effect on glucose stimulated insulin secretion from isolated islets in the 24 hour period (Figure 36). Insulin secretion was, on average, 8.5 X greater than that following a two hour incubation in the presence of a stimulatory level of glucose (17.8 mM). Although not significant, IGF-I decreased mean insulin secretion, particularly at the lowest concentrations used (1.7 nM, 1.2 X and 17.0 n M , 1.4 X). The islets were determined to be viable following the 24 hour incubation period following assessment both visually under the microscope, and quantitatively based on the consistent level of insulin secretion in the glucose stimulated wells in the absence of IGF-I.  3.4.5. E F F E C T O F AN IGF-I VARIANT (DES-AMIDO IGF-I) ON INSULIN SECRETION F R O M ISOLATED ISLETS IN T H E PRESENCE O F GLUCOSE STIMULATION Since it has been proposed that autocrine or paracrine influences of IGF-I may be mediated primarily by des-amido IGF-I, a variant exhibiting reduced binding protein association, this variant was included in glucose stimulated (17.8 mM) islet incubations at increasing concentrations. It was hoped that the results would clarify the potential influence of binding proteins on IGF-I stimulated insulin secretion in isolated islet preparations. Only the highest level of des-amido IGF-I (170.0 nM), increased (1.5 X) insulin secretion above that induced by glucose alone (17.8 mM) (Figure 37). This level of stimulation was not as high as that achieved with IGF-I at the same concentration (2 - 4 X that of glucose at 17.8 mM). These results suggest that the observed stimulatory effect of IGF-I on insulin secretion in isolated islets is not influenced by binding proteins.  128  FIGURE 36: Effect of long term IGF-I incubation (24 h) on insulin secretion from isolated rat islets. Ten islets from Wistar rats were incubated (24 h) in media containing glucose (17.8 mM) with increasing concentrations of IGF-I (0.0, 1.7, 17.0, 170.0 nM). Insulin secretion is expressed as a percent of total cell content (%TCC +/- SEM). n=6 for all conditions.  c  129  FIGURE 37: Effect of an IGF-I variant (des-amido IGF-I) on glucose stimulated insulin secretion from isolated rat islets. Ten islets from Wistar rats were incubated (2 h) in media containing glucose (17.8 mM) with increasing concentrations of des-amido IGF-I (0.0, 1.7, 17.0, 170.0). Insulin secretion is expressed as a percent of total cell content (%TCC +/SEM). * p<0.005 Dunnett's (in comparison to 0.0 nM des-amido IGF-I) n=4 for all conditions.  130 3.4.6.  E F F E C T O F A POLYCLONAL GLUCAGON ANTIBODY O N INSULIN SECRETION F R O M ISOLATED ISLETS IN T H E PRESENCE O F IGF-I AND G L U C O S E STIMULATION In order to clarify whether A-cell secretion might be involved in the pathway by which  IGF-I stimulates insulin secretion from isolated islets over a two hour period, a glucagon antibody was included in the incubation. The monoclonal glucagon antibody (10 ug/m\) significantly decreased glucose stimulated (17.8 mM) insulin secretion both in the presence (- 2.2 X) and absence of IGF-I (170.0 nM) (-1.4 X) (Figure 38). The effect of the antibody in the presence of glucose stimulation alone suggests that glucagon plays a role in the glucose stimulated secretion, but does not elucidate the potential role of glucagon in IGF-I stimulated insulin secretion.  131  FIGURE 38: Effect of a polyclonal glucagon antibody on IGF-I stimulated insulin secretion from isolated rat islets. Ten islets from Wistar rats were incubated (2 h) in media containing glucose (17.8 mM) in the presence or absence of IGF-I (170.0 nM). Insulin secretion is expressed as a percent of total cell content (%TCC +/- SEM). * p < 0.025 Student's t-test n=4 for all conditions.  132 4. DISCUSSION  The objectives of the studies presented in this thesis were to determine if IGF-I is present in the adult rat endocrine pancreas and, if so, to characterize its cellular distribution and establish its potential role in the regulation of insulin secretion. IGF-I has been shown to have a wide distribution in many tissue types (Rechler and Nissley 1992), and to exert its effects by endocrine, autocrine and paracrine routes of action (Phillips and VassilopoulousSellin, 1986; Underwood et al, 1986). Although IGF-I had been found to be an important local regulator of the B-cell population and of insulin synthesis in fetal islets (Swenne et al, 1988; Romanus et al, 1985; Rabinovitch et al, 1982), and previous evidence has suggested that IGF-I might be present in the endocrine cells of the adult pancreas (Maake and Reinecke 1993: Hansson et al, 1988a; 1988b), its cellular localization and potential role in the regulation of insulin secretion in adult pancreas remained unclear. Immunocytochemical techniques using a monoclonal IGF-I xantibody were used to study the localization of IGF-I in adult rat pancreas. The finding, in this study, that IGF-I is localized to B-cells of adult rat pancreas agrees with findings in fetal human islets (Hill et al, 1987), but contrasts with studies completed in adult mouse (Hansson et al, 1989) and rat (Hansson et al, 1988) using polyclonal antibodies. The results of the present study also conflict with those of more recent immunocytochemical studies, in which the localization of IGF-I and IGF-II were compared in the endocrine pancreas of rat, dog and man (Maake and Reinecke 1993). These investigators reported that immunoreactive IGF-I was absent in the 6-cells, but present in the A-cells of all species, and in some D-cells in the human, whereas IGF-II was present in B-cells in all species. It had been suggested previously that there existed an agedependent expression of IGF-I in the B-cell, the significance of which would require further investigation (Hansson et al, 1988). The present study suggests that IGF-I is present in rat islets at all ages. As in the adult, immunoreactive IGF-I was detected in the B-cells of rats at one and seven days of age, although the precise cellular localization of IGF-I in the fetal rat was not studied.  133 The present study also revealed a very specific colocalization of IGF-I with insulin in the fi-cells at the light microscope level, with a complete lack of colocalization with either glucagon or somatostatin in either rat or human pancreatic sections. This localization was further verified at the electron microscope level, and indicates the presence of IGF-I specifically in the mature fi-cell granules. The opposite results compared to those noted above, are likely a result of the specificity of the polyclonal antibodies used by Maake and Reinecke (1993), which demonstrated IGF-I immunoreactivity in the A-cells and some D-cells in the human as compared to the monoclonal antibody used in the present study. However, it was noted, by Maake and Reinecke, that one of the antibodies (K37) had previously been found to demonstrate the presence of immunoreactive IGF-I in the fi-cell, although this was said to be due to a lack of antibody specificity when used at high concentrations (Maake and Reinecke 1993). The monoclonal IGF-I antibody used in the present study was raised for use in RIA, and the sera from several species including human, sheep, and pig serum were found to displace  125  I-IGF-I from the antibody (Kerr et al, 1989). The antibody recognizes the amino  terminus of IGF-I, exhibiting 40 % cross reactivity with des-amido IGF-I. The specificity of an antibody does, however, vary based on the binding conditions used, both in solution and in situ (Cuello et al, 1983). Preliminary studies indicated that the antibody appeared to bind specifically within rat islets using classical immunocytochemical techniques on sections of rat pancreas. The specificity of this monoclonal IGF-I antibody for immunocytochemical purposes was therefore investigated, as there exists a strong potential for cross reactivity due to the sequence homology of IGF-I and insulin. The specificity of immunoreactive IGF-I detection was demonstrated by displacement studies using IGF-I, IGF-II and established islet peptides (insulin, proinsulin, somatostatin, pancreatic polypeptide, glucagon). Both rat and human pancreatic sections were tested as the antibody was raised to human IGF-I. Although insulin at a high concentration (10 ug/ml) reduced the intensity of staining on both human and rat tissues, this is likely due to the high concentration of total protein in the incubation. Similar antibody displacement studies using  IGF-I and insulin on microthin (EM) rat pancreas sections further indicated the specificity of the antibody for IGF-I. Preincubation of the antibody with IGF-I completely abolished staining on microthin rat sections while preincubation with insulin had no effect. The specificity of the IGF-I monoclonal antibody was tested further using an immunoblot of hormones showing structural homology with IGF-I (insulin, proinsulin, C peptide and IGF-I). This indicated that the monoclonal antibody recognized only IGF-I, further leading to the conclusion that the antibody was specific and suitable for the detection of IGF-I in rat tissues. These findings were also supported by the detection of immunoreactive IGF-I in B-cells with two polyclonal antibodies (kindly provided by Dr. P . K . Lund), although the characteristics of these antibodies for immunocytochemical purposes were not studied. It remains unclear as to whether the immunoreactive IGF-I found to be present in the 6cells is the result of B-cell synthesis, or to uptake by these cells. The presence of IGF-I in the mature B-cell granules is the first indication, in any tissue, that IGF-I might be secreted via a regulated pathway. The only reported finding of IGF-I synthesis and secretion occurring in this manner is in a mouse pituitary cell line (AtT-20) (Schmidt and Moore 1994). This A C T H secreting cell line has dense core secretory granules which contain A C T H as well as IGF-I, secreted at a 1500 fold lower concentration. Previous studies investigating IGF-I secretion pathways have been performed primarily in the liver, where there has been no report of any regulated secretion (Salamero et al, 1990; Saucan and Palade, 1992). Indeed, in all tissues studied to date, the secretion of IGF-I has been thought to be constitutive. As discussed by Schmidt and Moore (1994), it is therefore unclear as to whether in the case of IGF-I this is due to a lack of sorting pathways in particular cell types, or to the absence of a sorting domain in IGF-I. Findings in the AtT-20 cell line indicate that granular sorting of IGF-I is unlikely to result from the concentration and ? •+pH/Ca  dependent pathway proposed for secretogranins (Schmidt and Moore 1994). cDNA  transfection studies in this cell line, however, indicated that many prohormones (including proinsulin) can be targetted to secretory granules and all peptides studied contained similar targetting motifs (Kizer and Tropsha 1991). These characteristics include two or more leucines  135 occupying one side of an amphipathic alpha-helix, with a serine or threonine positioned N terminal to the leucines. It has been suggested that IGF-I could contain a sorting region given its sequence homology with insulin, which exhibits granular targetting and regulated secretion. This region would likely be contained within the A - and B-domains based on the similarity in the sequence of IGF-I and insulin and findings that the C-peptide region is not necessary for the granular targetting of insulin (Schimdt and Moore 1994). Since little is known regarding the intracellular transport of IGF-I, particularly in a regulated pathway, the means by which the hormone could be targetted to the B-cell secretory granule following synthesis are only speculative. It could be assumed that if the signalling sequence was similar to that of insulin, that upon entering the ER, IGF-I could be directed to the Golgi apparatus and on to immature and mature granules. This would likely occur in parallel with insulin itself, as all mature B-cell granules viewed in this study exhibited IGF-I immunoreactivity. Another possible explanation for the presence of IGF-I in the B-cell granules would be uptake of the hormone following IGF-I receptor binding at the plasma membrane. It has been suggested that the immunocytochemical detection of IGF-I in other tissues may represent sequestered hormone following receptor or binding protein mediated uptake, particularly if the mRNA is not detectable (Swenne 1992; Han et al, 1987). Indeed, IGF-I has been shown to be internalized following receptor binding and to be present in endosomes, golgi apparatus, lysosomes as well as in the nucleus of lens epithelial cells (Soler et al, 1990), and in endosomes and lysosomes of a kidney epithelial cell line (Fawcett and Rabkin 1995). The IGFI receptor is internalized to an intracellular pool and recycled in the presence of elevated IGF-I (Yamamoto et al, 1993), and it is therefore possible that receptor bound IGF-I could be seen in the endosomes, lysosomes and nuclei. Furthermore, it has been shown that some intermixing of endocytosed products with secretory products occurs in the trans-golgi network (Schmid et al, 1988; Stoorvogel et al, 1988). Although such a pathway for IGF-I uptake would go against current dogma, entry into insulin secretory granules via such a pathway remains a possibility. Indeed, the finding that insulin and C-peptide are not released in equimolar amounts has led to a theory that maturing fi-cells undergo fusion with other cellular granules through a clathrin-  136 mediated process (Hutton 1994). An extension of such a theory is that endocytosed IGF-I could enter the maturing B-cell granules even later than the trans-golgi processing stage. Although immunocytochemical results in this study indicated no specific detection of immunoreactive IGF-I in any area of the B-cell other than the dense granules, this may be a result of receptor bound IGF-I being undetected by the antibody, or to differential tissue fixation of the cellular apparatus. There remains one further possible source of intracellular IGF-I. Uptake to the nucleus is highly selective and is thought to require a specific signal sequence (Radulescu 1994). Although IGF-I does not contain sequences associated with nuclear uptake, IGFBP-3 has been shown to exibit a 'nuclear localization signal' (NLS), and may promote nuclear uptake of IGFI, thereby influencing transcription directly. The potential for nuclear uptake of IGF-I in an endocrine cell is as yet unclear, and IGF-I was not observed in the nucleus of the B-cell in this study. The significance of IGF-I localization to the B-cell granules suggests that in addition to possible endocrine influences, IGF-I might influence B-cell function via an autocrine or paracrine route. It is also possible that the release of IGF-I from the B-cell could parallel that of insulin, prompting the following studies on IGF-I secretion from the islets and the effects of IGF-I on insulin secretion.  An IGF-I RIA was established in order to attempt the identification and quantification of IGF-I in the endocrine pancreas. The method used was based on that suggested by NIH for the antibody # UBK487. The resulting sensitivity and range (10 - 250 pg/ml), accuracy ( 5.5 % intra-assay variance), and variability (10.9 % inter-assay variance) of the assay were determined to be suitable to attempt quantification of IGF-I. Since IGF-I binding proteins can interfere with the determination of IGF-I levels, a method of binding protein removal had to be verified prior to attempting the determination of total IGF-I levels (Daughaday et al, 1987). The acidification of samples, to dissociate IGF-I from its binding proteins, followed by separation of the two components, is the generally  137 accepted technique. However, no single method has been found to be suitable for all species and for all physiological states (Breier et al, 1991). Furthermore, for the purposes of this study, the intention was to use the method for a large number and a wide variety of samples including serum, tissue extracts, as well as defined medium. Based on previous studies, several IGF-I binding protein extraction procedures were investigated. The separation of acidified samples by conventional column chromatography (Daughaday and Rotwein 1989) is labour intensive and time consuming for a large number of samples and more rapid techniques were therefore investigated. Acidified samples (HC1, 0.5 M) were applied to C  1 8  Sep-Pak cartridges (Daughday et al, 1987), which are faster and now  widely used for separation. Since low recoveries of  125  I-IGF-I were obtained by the method of  Davenport (1988) involving elution of samples with acetic acid followed by methanol, this method was not pursued further. Modification of the procedure by elution of samples with water then acetonitrile yielded a higher recovery of  125  I-IGF-I but inconsistent recovery of  unlabelled IGF-I from various sample types including serum, perfusate and RIA buffer. It appeared that either residual binding proteins were at a level which caused erroneous results in the RIA or that IGF-I was retained, either bound or unbound, on the C  1 8  Sep-Pak column. It is  also possible that IGF-I reassociated with residual binding proteins following neutralization of pH prior to the RIA. High variability in the recovery of  125  I-IGF-I using Sep-Pak columns has  since also been reported by others (Breier et al, 1990). These methods were therefore not pursued further. Several studies reporting success with acid ethanol cryoprecipitation led to attempts at binding protein removal using a formic acid and acetone extaction. This technique has been validated for the separation of IGF-II from its binding proteins in both tissue extracts (Lee et al, 1991) and fluids (Bowsher et al, 1991). High and consistent recoveries of both  125  I-IGF-I  (80 - 95 %) and IGF-I (80 - 90 %) from several sample types (Krebs perfusate buffer, serum, pancreatic perfusate) suggested that this method might be appropriate for the removal of IGF-I binding proteins in this study. Verification of this technique was pursued using high resolution column chromatography (Sephacryl G-200) to assess binding protein separation.  138 Serum and pancreatic and liver extracts, preincubated with  125  I-IGF-I showed a  decrease in radioactivity in the higher molecular weight range (60 - 200 kD), with a defined peak in the molecular weight range of free IGF-I (7 kD), when treated with the formic acid/acetone extraction procedure prior to application to the column. This is consistent with IGF-I being dissociated from binding proteins in the molecular weight range of 30 - 150 kD, as are present in tissues and serum. It was apparent that some immunoreactivity was still associated with the high molecular weight region (no quantitative measurements were made), and this remains a problem with most methods used to date. It is likely that the low pH (approximately p H 2) of the acidification procedure did in fact dissociate IGF-I from the binding proteins, but it is possible that there were some proteins remaining in the sample following centrifugation and, as discussed earlier, these reassociated with IGF-I following neutralization of pH prior to assay. The extent of residual binding protein interference in the RIA was first assessed by measuring immunoreactive IGF-I in fractions collected following the elution of formic acid/acetone extracted rat serum. This indicated only one peak of activity in the molecular weight region of free IGF-I. The absence of immunoreactivity in the region where binding proteins would be expected to elute suggested the absence of their interference in the assay. Furthermore, it was found that formic acid/acetone extraction of serially diluted rat serum resulted in near parallel displacement with the IGF-I standards in the IGF-I RIA. The RIA and binding protein extraction method were therefore considered to be satisfactory for studies involving the quantification of IGF-I in the endocrine pancreas. A similar formic acid/acetone cyroprecipitation method has since been used by others in the separation of binding proteins for the detection of IGF-I in defined media following cell culture (Schmidt and Moore 1994). Despite immunocytochemical evidence for the existence of IGF-I within the islets, attempts at stimulating secretion of IGF-I from isolated pancreatic and isolated islet preparations were unsuccessful. G H is the primary regulator of IGF-I synthesis and secretion in the liver (Hynes et al, 1987; Murphy et al, 1987; Mathews et al, 1986) and other tissues (see section 1.1.1., p8) as well as being the primary regulator of IGF-I secretion in fetal rat  139 (Swenne et al, 1987b) and human (Hill et al, 1987; Swenne et al, 1987b) pancreases. G H was therefore investigated first as the most likely regulator of IGF-I secretion in adult rat islets. Incubations contained a high glucose concentration (16.8 mM) since findings in fetal pancreas indicated that glucose elevated islet IGF-I secretion (Hill et al, 1987; Swenne et al, 1985), particularly in the presence of G H (Swenne et al, 1985). As expected, glucose and GIP stimulated insulin secretion, which suggested that both the pancreatic perfusions and the isolated islet preparations were viable. G H did not, however, stimulate IGF-I secretion to detectable levels in either perfused isolated rat pancreas preparations or isolated islets under any conditions applied. This suggests that either a similar stimulatory relationship does not exist in the adult pancreas, or that stimulated levels were below the detection limit of the RIA. Direct attempts at measuring IGF-I in the islets included RIA of islet and B-cell extracts, which revealed the presence of low but detectable levels of IGF-I: approximately 80 pg IGF-I per 250 islets; approximately 30 pg IGF-I per 10,000 B-cells. IGF-I in B-cell extracts was also detectable following immunoblotting. In order to ensure that these low levels did indeed represent IGF-I, islet extracts were separated on H P L C and assayed in the IGF-I RIA. It was found that immunodetectable IGF-I was present in the islet extract which eluted in the same position as the IGF-I standard. The minimum IGF-I concentration per islet was determined to be approximately 1 pg which is in the same order as the levels found in extracts of formic acid/acetone treated islets noted above. These levels are much lower than those of islet insulin content. In the present study, the average islet, based on islet extracts following culture, contained approximately 40 ng (6.3 pmol) insulin/islet. On a molar basis this suggests approximately 50,000 fold more insulin than IGF-I per islet (6.3 pmol insulin, 0.13 fmol IGFI per islet). It is unlikely that the immunoreactive IGF-I eluting following H P L C came from nonislet contamination as islets were cultured prior to extraction, and non-endocrine (exocrine, ductal and epithelial) cells have been shown not to survive well under these culture conditions.  140 These results indicate that immunoreactive IGF-I is present in the endocrine pancreas, but that the level of hormone present is very low. The localization of IGF-I to B-cell granules would suggest a high level of secretion when insulin secretion was stimulated. This was not the case, however, in the present studies. The difficulty in detecting secreted peptide from the perfused pancreas and from static islet preparations is likely attributable to this low level, and perhaps also to inappropriate stimulatory conditions. The absence of immunodetectable IGF-I in secretion studies may also reflect pancreatic, islet and B-cell heterogeneity. It has indeed been shown that islets in different parts of the pancreas (from areas of differing embryonic origin) have differing islet cell populations (Baetens et al, 1979; Orci and Perrelet 1979), and variable insulin responses to stimuli such as glucose and amino acids (Samols 1983; Trimble et al, 1982). Cultures of individual B-cells also exhibit variable insulin responses to glucose (Hiriart and Ramirez-Medeles 1991). Different 6-cell populations have been found to exhibit differing concentrations of dense B-cell granules, with those exhibiting a greater number of light (proinsulin) granules exhibiting a greater sensitivity to glucose (Pipeleers 1994). It has been noted that in perfused B-cells, newly synthesized insulin is preferentially secreted (2-3 times greater) relative to stored insulin, when exposed to glucose and glucagon (Van Schravendijk et al, 1990). It is possible that IGF-I is stored to a greater extent in some B-cells than in others, as has been noted in the case of insulin (Hiriart and Ramirez-Medeles 1991), or that the IGF-I observed in storage granules responds to secretion stimuli other than the classical insulin secretogogues. Finally, losses of IGF-I during the binding protein extraction procedure may have eliminated chances of detection in secretion studies if levels of IGF-I were low to begin with. Although the absence of detectable secretion does suggest, not surprisingly, that the endocrine pancreas is not an important endocrine source of IGF-I, it does not preclude the possibility for autocrine or paracrine influences of IGF-I within the islet, particularly since, as discussed below, relatively low concentrations can potentiate responses to other stimuli.  141 Since IGF-I appeared to be present within islet B-cells, the possible site of action and the potential autocrine effects of IGF-I on insulin secretion were studied. It was demonstrated, by binding studies, that  125  I-IGF-I binding sites were present in islets, and further attempts to  localize the receptors using autoradiographic techniques revealed a high density of  125  I-IGF-I  over the islet region. There existed a higher density of grains over the periphery of the islet and a lower density within the B-cell core region. These results agree with those of Van Schravendijk and coworkers (1987) who detected high affinity IGF-I receptors on A and ficells in rat islets. It was noted, in the present studies, that there may have been a problem with tissue preservation in the islet core, as a loss of cellular integrity was observed under magnification, presumably a result of using fresh frozen tissues. This was evident following numerous unsuccessful attempts at localizing receptor binding to particular cell types using immunocytochemical techniques. The receptor binding studies of Van Schravendijk and coworkers (1987) indicated that a higher density of receptors existed on the fi-cell than on the A-cell, although their findings did not take into account possible heterogeneity of A - and ficells. The exposure of cells to different isolation procedures may result in differing recovery of membrane receptors. IGF-I receptors could also be present on other islet cell types including the somatostatin secreting D-cell, and this possibility has not been ruled out in this or other studies.  As a result of the demonstration that IGF-I was present in fi-cells, of IGF-I receptors in the islet and to previous observations, in other tissues, of locally acting IGF-I, local intra-islet effects of IGF-I were therefore thought to be possible. There potential exists for IGF-I to influence not only the 6-cell directly but also indirectly by affecting A - , D- and PP-cell secretions. Isolated islet preparations were used to assess the influence of IGF-I on insulin secretion, as such preparations have previously been used successfully to study the effects of factors, including hormones and nutrients, on islet hormone secretion. These preparations are  142 perhaps more likely to reflect the effects of locally synthesized hormones due to the static nature of islets incubated in media, than would exposure in a perfused or perifused system. The maximum level of IGF-I (170 nM) used throughout the experiments was based on previous observations in isolated adult rat islets (Sieradzki et al, 1987), and on preliminary studies which indicated that this level consistently achieved significant stimulation of insulin secretion. This level is approximately four times the physiological circulating level in the rat. The present study, however, was performed in order to investigate the potential role of locally synthesized IGF-I, and autocrine and paracrine release in combination with circulating IGF-I would presumably expose islet cells to a higher concentration of the hormone. Furthermore, a higher local concentration of intra-islet IGF-I would be likely under appropriate stimulatory conditions and with potential binding to locally derived binding proteins. In addition, higher levels of IGF-I might be necessary in the in vitro conditions used in order that access of IGF-I to all islet cells could be achieved. IGF-I was found to stimulate insulin secretion from isolated islets in a concentration dependent manner. As with other insulin secretagogues, it was found that the effect of IGF-I (170 nM) was glucose dependent. Basal and glucose-induced insulin secretion were similar to those reported in earlier studies (Verchere 1991). Based on the above observation, that the potentially high levels of locally available IGF-I could be involved in glucose stimulated insulin secretion, the effect of the IGF-I monoclonal antibody on glucose (17.8 mM) induced insulin secretion was determined. The absence of an influence of the antibody on glucose stimulated insulin secretion may indicate that locally produced IGF-I has no role in glucose mediated secretion. It is possible, however, that the IGF-I antibody was not able to penetrate the islet to influence intra-islet IGF-I activity, a problem which has been found in other studies using islet hormone antibodies (Mcintosh, 1995 - personal communication). The glucagon antibody did, however, influence islet insulin secretory activity (discussed below) suggesting that antibodies are capable of influencing islet secretory activity in isolated islet cultures. It is possible, however that glucagon from the A cells at the periphery of the islet, was more readily bound by the glucagon antibody than was  143 IGF-I, secreted from the 8-cells at the core of the islet. Since the incubation of an IGF-I monoclonal antibody in the release media completely inhibited the stimulatory effect of IGF-I on insulin secretion (and did not influence insulin levels in the insulin RIA), the influence is likely specific for IGF-I and not to other factors in the media. The results of the present study agree with those of an earlier report on isolated rat islets (Sieradzki et al, 1987). Their study was designed to investigate potential effects of IGF-I on the growth and function of islet cells, and IGF-I (50 nM, 230 nM) was incubated with islets for a relatively long time period (3 days). It was found that IGF-I stimulated cell replication, as well as insulin synthesis and secretion, particularly in the presence of high concentrations of glucose (16.7 mM) and of IGF-I (230 nM). The results of the present study, however, contrast with findings of subsequent studies performed on perifused B-cells (Van Schravendijk et al, 1990) and perfused pancreas (Leahy and Vandekerkhove 1990) in which IGF-I was found to inhibit glucose-stimulated insulin secretion. These studies investigated a range of IGF-I concentrations from supra-physiological to lower than physiological levels (2 ng/ml - 200 ng/ml) with the intention of identifying the effects of circulating IGF-I. Given that IGF-I inhibits insulin secretion from perifused B-cells, in the absence of other islet cell types (Leahy and Vandekerkhove 1990; Van Schravendijk et al, 1990) and the presence of high affinity IGF-I receptors on the B-cell (Van Schravendijk et al, 1987), it is likely that the direct action of IGF-I on the 6-cell, at least in the short term, is to inhibit insulin secretion. It is possible, however, that in the longer term, the multifactorial intracellular signalling system of IGF-I might result in the direct stimulation of the B-cell. The stimulation of insulin secretion by IGF-I in this study and in that by Sieradzki and coworkers (1987), in comparison to the above-mentioned inhibitory effects, are therefore likely to be a result of differences in the experimental conditions. The presence of IGF-I receptors on both A - and B-cells (and perhaps as well on the D-cells) could result in the accumulation of several secretory products in the event of stimulation by IGF-I. Insulin, glucagon, somatostatin as well as IGF-I binding proteins, among other factors could interact to  144 result in either the stimulation or inhibition of insulin secretion. Static incubations are also influenced to a lesser degree by flow of the islet microcirculation which in the rat is thought to perfuse fl-A-D cells, in that order. In addition, the high levels of IGF-I incubated with the islets, and the accumulation of any secretory products could result in down-regulation of receptors over the two hour incubation period, further influencing overall insulin secretion. Like other in vitro preparations, isolated islets lack an extrinsic neural supply and therefore the effects of IGF-I are considered independently of sympathetic and parasympathetic innervation. In an attempt to elucidate possible interactions of IGF-I with binding proteins and with other secretagogues, a number of islet secretion studies were performed. In order to assess the possible involvement of IGF-I binding proteins, the effect of desamido IGF-I on insulin secretion from isolated islets was determined. Studies in other tissues have shown that this variant of IGF-I does not bind as readily to IGF-I binding proteins, due to the missing 3 amino terminal amino acids. The effects of des-amido IGF-I on the stimulation of protein and D N A synthesis have been found to be more potent both in vitro (Francis et al, 1988) and in vivo ( Tomas et al, 1992; 1991; Ballard et al, 1987). IGF-I binding proteins have also been shown to inhibit IGF-I activity depending upon tissue and experimental conditions. In the present study, however, des-amido IGF-I significantly increased insulin secretion above that of glucose stimulation (17.8 mM), only at the highest concentration used (170 nM). In fact the stimulation of insulin secretion by this variant was somewhat lower at all concentrations than that of IGF-I at equimolar levels. The fact that the effect of des-amido IGF-I on insulin secretion was also stimulatory and did not differ greatly from that of IGF-I suggests that binding proteins did not play a major role in modulating the activity of IGF-I in this study. In order to assess whether the stimulatory effect of IGF-I on insulin secretion might be mediated by glucagon, a glucagon antibody was included in the incubation medium. Glucagon did indeed appear to be involved in the stimulation of insulin secretion. However, since both glucose as well as glucose in combination with IGF-I, stimulated insulin secretion were inhibited by the glucagon antibody, the significance is unclear. Leahy and coworkers (1990)  145 found that IGF-I had no effect on glucagon secretion in the perfused rat pancreas over a 20 min period, wheras i.v. infusion of IGF-I in humans resulted in decreased glucagon levels following three days of administration (Mauras et al, 1992). Unfortunately glucagon was not detectable in samples following storage. Therefore, although the potential for glucagon to act as the mediator of IGF-I stimulated insulin secretion remains a possibility, its influence is as yet unclear. In order to study the possible interactions between IGF-I and other insulin secretagogues, isolated islets were incubated with increasing concentrations of IGF-I with GIP at a concentration (10 nM) which has been shown to maximally stimulate insulin secretion in isolated islets (Verchere 1991). IGF-I potentiated GIP stimulated insulin secretion, as the level of insulin secreted was higher than expected based on the addition of 10 n M GIP and levels obtained with IGF-I alone, particularly at the two lower levels of IGF-I used (1.7 and 17.0 nM). It is therefore possible, that even very low levels of IGF-I secreted from the B-cell as predicted in this study, could potentiate GIP induced insulin secretion. The relative impact of endocrine and autocrine IGF-I on GIP (and perhaps tGLP-1) stimulated insulin secretion is unclear. In the presence of the relatively constant levels of circulating IGF-I, it is possible that even small increases in local IGF-I levels could potentiate GIP induced B-cell secretion. In view of this potentiation, it is likely that IGF-I is acting via a separate signalling system than GIP. This may be mediated via receptor IRS-1 activation of the IP pathway 3  (Chakravorty et al, 1993). As suggested previously, IGF-I could also be acting via an intermediate, possibly glucagon. Although glucagon also stimulates insulin secretion by increasing c A M P and activating PK-A thereby increasing intracellular C a  2 +  , it is possible that  GIP and glucagon act via separate pools of intracellular cAMP. The synthesis of insulin might also have been increased by IGF-I during the two hour islet incubation. In this case, IGF-I could act directly on insulin gene transcription via Ras and M A P kinases, either directly via receptor phosphorylation, or via IRS-1. IRS-1 has also been found to associate with proteins containing SH2 domains including Grb-2, Nek and Syp, which are involved in phosphotyrosine dephosphorylation and may mediate the mitogenic and  146 metabolic responses of IGF-I. In addition the activation of P K A by GIP may be influencing IGF-I in this pathway, as in certain cell types P K A has been found to stimulate growth factor stimulated M A P kinase pathways (Faure et al, 1994). This could result in a potentiating effect of IGF-I and GIP on insulin secretion. As suggested previously, the exposure time to elevated IGF-I might influence the overall insulin response, and the incubation period was therefore increased to 24 h. The lack of stimulated insulin secretion does suggest a time dependent effect. It is possible that over this period of time, in the presence of high levels of IGF-I, there was a down-regulation of the IGF-I receptor on the B- and/or A-cells, or perhaps that insulin synthesis and secretion, over this period of time, required further glucose or amino acid supplementation. These results contrast with those of Sieradzki and coworkers (1987) who found that IGF-I stimulated insulin secretion, even after a period of three days with no reported medium changes or supplementation. Their study, however, did not investigate insulin secretion levels within the three day period. It is possible that a longer incubation period (> 24 h) in the present study could have resulted in IGF-I stimulating insulin secretion above that induced by glucose alone.  From the present islet secretion studies, it can therefore be concluded that IGF-I can stimulate insulin secretion. The results of this study are attributable to pre-culture and culture conditions, and the static system used over a long time period in comparison to other in vitro studies showing IGF-I inhibition of insulin secretion. In a static incubation of islets, the potential exists for islet hormones and other secretory products to interact, whereas in a perfused system, intermediates may not play a major role. Many differences also exist in the culture conditions prior to and during experiments examining the presence of, and the influence of IGF-I on both adult and fetal islet cultures. Differences include the type of pre-culture and culture media and the glucose concentration included in each. Perhaps the most influential factor is the concentration and type of serum used. In all fetal studies demonstrating the presence of islet IGF-I, or a stimulatory effect of IGF-I on islet cell function, a relatively high level (10 - 20 %) of fetal calf serum (FCS) was  147 used in the preculture period (Hogg et al, 1993; Scharfmann et al, 1989; Hill et al, 1987; Swenne et al, 1986). Similarly, the two studies involving adult rat islets which indicated the presence of (Swenne and Hill 1989) and a stimulatory effect of IGF-I on islet cell function (Swenne and Hill 1989; Sieradzki et al, 1987), contained either a low concentration of FCS throughout the pre-culture and culture periods (1%), or a high concentration during pre-culture (20 %). In contrast, two studies, one on fetal human islets (Otonkoski et al, 1988) and the other on young rats (3-5 d) (Billestrup and Nielsen 1991) included adult human serum in culture medium (10 % and 2 % respectively) during both pre-culture and culture. The studies revealed neither the presence of IGF-I mRNA, nor a stimulatory effect of IGF-I on B-cell proliferation (Billestrup and Nielsen 1991), or on insulin biosynthesis or release (Otonkoski et al, 1988). In addition, although the inhibitory effect of IGF-I seen in perifused adult rat B-cells involved pre-culture in FCS (Van Schravendijk et al, 1990), the concentration was relatively low (5 %), and incubation time short (20 h) in comparison to the other studies mentioned (3-5 d). It is therefore possible that the factors in fetal serum influence, and result in conditions, even in cultures of adult tissue, similar to those of fetal pancreas in which IGF-I is thought to stimulate B-cells. Fetal serum contains high levels of many growth factors including IGF-I and IGF-II, in comparison to adult serum. Culture under these conditions, for example, might result in either the stimulation of B-cell IGF-I synthesis, the cellular uptake of IGF-I, and/or influence IGF-I, IGF-IBPs or IGF-I receptors, resulting in a stimulatory effect on insulin biosynthesis and secretion. The physiological significance of an IGF-I stimulatory effect on insulin secretion may therefore be dependent on the prevailing physiological status and the resulting islet environment, as indicated in the above in vitro studies. Several in vivo studies have investigated the influence of IGF-I on insulin secretion, in normal humans and rats, and these also indicate that the administration of IGF-I is associated with decreased insulin secretion. IGF-I was found to inhibit insulin as well as amylin secretion in a dose dependent manner in free moving rats under a hyperglycemic clamp (13.9 mM)  148 (Furnsinn et al, 1994). A similar study in normal humans indicated that IGF-I infusion inhibited insulin secretion, but that the extent of inhibition was reduced at higher glucose concentrations, and it was suggested that the suppression of insulin secretion was overcome at higher glucose levels (Rennert et al, 1993). In contrast to findings in rats, this study revealed that increasing concentrations of IGF-I stimulated glucose disposal over the two hour period although to a lesser extent at higher glucose concentrations. Further study in normal humans also indicated that after 28 hours of IGF-I infusion glucose levels remained stable, while plasma insulin was decreased, as were C-peptide and glucagon levels (Mauras et al, 1992). In addition it was found that plasma IGFBP-3 levels remained unaffected over the IGF-I infusion period. Several studies have also been performed in order to assess the potential for IGF-I therapy in diabetics. A five day infusion of IGF-I in normal humans showed a reduction in insulin and C-peptide, but normal glucose and meal tolerance tests and normal glucose curves (Zenobi et al, 1992a). It was found that, despite the reduced plasma insulin levels with a high level of IGF-I infusion (10 itg/kg/h), the insulin response to a glucose tolerance test was prompt. IGF-I bolus doses (120 itg/kg/d) in Type 2 diabetics revealed normalized fasting glucose levels after 3 days of treatment in the face of decreased plasma insulin (Zenobi et al, 1992b). These studies suggest that in normal and in Type 2 diabetic subjects, IGF-I inhibits insulin secretion, but, at least in normal subjects, there is a lesser degree of inhibition at higher glucose levels. This is reflected in the finding that a glucose tolerance test is not impaired in the presence of chronic IGF-I infusion. In contrast to the above findings, a study of patients with acromegaly found that increased fasting plasma insulin levels were correlated with elevated circulating IGF-I levels (Hopkins and Holdaway 1992). Patients with active acromegaly exhibit decreased insulin sensitivity and increased B-cell function, both of which were found to be significantly related to serum IGF-I concentrations. The authors suggest that this reflects either enhanced IGF-I production, IGF-I stimulation of insulin synthesis and release of insulin, or increased islet cell  149 mass. Similarly, infusion of G H resulting in similar plasma elevations in IGF-I have been found to result in hyperinsulinemia, insulin resistance and carbohydrate intolerance (Horber et al, 1991). It is possible that prolonged G H exposure is required to elevate islet IGF-I levels and produce an environment conducive to IGF-I 6-cell stimulation, as seen in islets precultured in F C S . The physiological significance of the presence of islet derived IGF-I and its stimulatory influence on the 6-cell, either alone, or in the potentiation of GIP stimulated secretion, is therefore uncertain. As indicated in the studies involving GIP, low concentrations of IGF-I (1.7, 17 nM) strongly potentiated GIP stimulated insulin secretion. It is possible, therefore, that 6-cell derived IGF-I plays a role in the potentiation of the insulin stimulatory effects of other secretagogues and is involved in the daily physiological regulation of insulin secretion. In particular, relatively small increases in IGF-I concentrations could increase insulin secretion in the presence of GIP and perhaps other insulin secretagogues. As discussed, the presence of fetal serum, as opposed to adult serum, may result in conditions appropriate for the stimulation of insulin secretion by IGF-I in vitro, perhaps due to high levels of growth factors including IGF-I and IGF-II. The significance of this effect, in light of the inhibitory influence of chronic IGF-I administration in vivo is uncertain. This potential means of regulating the influence of IGF-I could be of interest in pathological situations involving the disruption of islet cell activity. It is, perhaps, of particular interest that there exists the possibility for the stimulation of insulin synthesis, secretion, and perhaps 8-cell proliferation in the adult endocrine pancreas. This has already been indicated following pancreatectomy (90%), where high levels of IGF-I mRNA have been found and are associated with islet cell proliferation (Brockenbrough et al, 1988).  150 Conclusions: 1. Immunoreactive IGF-I is present in adult pancreatic islet tissue. This immunoreactivity is localized to the mature B-cell granules. It is possible that this indicates the presence of local IGF-I synthesis, but a pathway for IGF-I endocytosis into the B-cell is also proposed. 2. The level of IGF-I present in the endocrine pancreas is low. G H , which regulates IGF-I synthesis and secretion in many tissue types, does not appear to increase IGF-I secretion from the B-cell. Factors regulating IGF-I in this cell type remain unclear, but neither glucose nor GIP, which stimulate insulin secretion, have stimulatory effects on islet IGF-I secretion. 3. IGF-I receptors are present on adult islet endocrine cells. Receptor binding is displaced by IGF-I, and appears to be localized both within the islet core, as well as at the periphery.  4. IGF-I, under the culture conditions of the present study, has a glucose dependent stimulatory effect on insulin secretion. A significant stimulatory effect is present at an IGF-I concentration higher than circulating physiological levels, but at levels potentially present within the islet microcirculation, supporting a role as an autocrine regulator. It is possible that this stimulatory effect is mediated directly via the IGF-I receptor on the B-cell, or via an increase in glucagon secretion. It is unlikely that IGF-I binding proteins are important in the stimulatory effect of IGF-I on insulin secretion. 5. IGF-I can potentiate the stimulatory effect of GIP on insulin secretion. This potentiation is present within the physiological range of IGF-I concentrations.  Future directions: In view of the findings in this study, future studies would be performed in order to elucidate the origin of endocrine pancreatic IGF-I. Preliminary studies attempting to detect IGF-I mRNA in total islet RNA using Northern blot and in situ hybridization techniques yielded negative results. A definitive result regarding B-cell IGF-I synthesis would likely be obtained using PCR (polymerase chain reaction) techniques to amplify IGF-I mRNA in poly-A R N A extracts from isolated B-cells. An absence of detectable IGF-I mRNA would suggest that the IGF-I immunoreactivity seen in the present studies was a result of hormone uptake, perhaps via an endosomal route. The most likely route of uptake into the E R and Golgi sorting pathways might be via the association of IGF-I with the mannose-6 phosphate receptor, given its homology with the IGF-II receptor, and its location on cytoplasmic membrane vesicles.  151 Further studies would be done to elucidate the factors involved in determining whether IGF-I exerts an inhibitory or a stimulatory influence on the fi-cell. The potential for IGF-I stimulation of fi-cell function in the adult would be of interest in cases where insulin synthesis and secretion is compromised, such as diabetes. The present studies suggest that IGF-I only stimulates insulin secretion in vitro under certain conditions, such as, following preincubation with, or in the presence of fetal serum. 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Chem. 9:389-395.  APPENDIX I CHEMICAL SOURCES CHEMICAL  SOURCE  Acetic acid Acetonitrile Acetone Alkaline phosphatase substrate package Aprotinin (Trasylol) L-arginine Bacitracin Bovine serum albumin (iodination grade, RIA grade, fraction V) Calcium chloride Carbon decolourizing neutral (activated charcoal) Chloramine-T Collagenase (Type XI) D-19 fixative Dextran (clinical, industrial grade) Dextran (T-70) Diaminobenzidine Disodium ethydiaminetetraacetate (EDTA) Dulbecco's Modified Eagle's Medium (Powder) Dulbecco's Modified Eagle's Medium (Sterile solution) Ethanol Epon embedding medium Fetal calf serum Formic acid Formaldehyde (40 %) Gluteraldehyde Glucose (50 % solution) D-glucose Glutamine Hematoxylin Heparin Hydrochloric acid Hydrogen peroxide (30 %) Lead citrate Lithium carbonate Microphen fixative Magnesium sulphate 2-methyl butane Nitrogen (liquid) O . C . T . embedding compound Paraffin wax Penicillin/Streptomycin Petroleum ether Picric acid Potassium chloride Potassium phosphate  BDH BDH BDH Gibco Miles Sigma Sigma Sigma Fisher Fisher BDH Sigma Eastman Kodak Sigma Pharmacia BDH BDH Gibco Terry Fox Labs BDH JBS Chemicals Gibco Fisher BDH Fisher Abbot BDH Sigma Fisher Fisher Fisher BDH BDH Baker Ilford Fisher BDH U B C chem. Miles Inc. Oxford Labware Gibco BDH BDH Fisher BDH  189 QUSO (microfine silica, G-32) Sephacryl (S-200 HR) Sephadex (G-50 fine) Sodium azide Sodium bicarbonate Sodium merthiolate Sodium metabisulphite Sodium pentobarbital Pharmaceuticals Sodium phosphate (mono- and di-basic) Tris-HCl Tween-20 Xylene (histological grade)  PhiladelphiaQuartz Pharmacia Pharmacia Baker BDH Eastman Kodak Fisher MTCBDH Sigma BDH Fisher  190  APPENDIX n LABWARE SOURCES LABWARE  SOURCE  Cannulation tubing  BectonDickinson SimportPlastics BioRad BectonDickinson JBS Chemicals Millipore Corp. BectonDickinson Fisher Millipore Baxter Canlab BectonDickinson BectonDickinson MacMillanBathurst BiopacificDiagnostics Brinkmann  Cassettes (tissue embedding) Column (chromatographic, glass 1X30 cm) Culture plates (Costar 48 well) Grids (EM tissue mounting) Immobilon (nylon blotting paper) Petri dishes Pipettes (10 ml, polystyrene) Sep-Pak C\$ cartridges Slides (glass) Tubes (50 ml, conical polypropylene) Tubes (15 ml, conical polystyrene) Tubes (12X75 mm, glass) Tubes (12X75 mm, polystyrene) Vials (0.5 ml, 1.5 ml, polypropylene)  191 APPENDIX m IMMUNOREAGENTS IMMUNOREAGENT  SOURCE  Avidin fluorescein isothiocyanate C-peptide (human) Des(l-3)IGF-I GIP (purified porcine)  Vector laboratories Eli-Lilly (gift) GroPep Regulatory Peptide Group, U B C  Glucagon antibody (mouse anti-human monoclonal) Glucagon antiserum (rabbit anti-human) Goat anti-rabbit gamma globulin Growth hormone (purified human) IGF-I antibody (mouse anti-human) IGF-I antiserum (rabbit anti-human, lot UBK487; for RIA) IGF-I (recombinant human, iodination) IGF-I (recombinant human) IGF-I (recombinant human) IGF-II (recombinant human) IgG (alkaline phosphatase conjugated: rabbit anti-mouse, goat anti-rabbit) IgG (biotinylated horse anti-mouse) IgG (colloidal gold conjugated: goat anti-mouse, Protein-A) IgG (tetramethylrhodamine isothiocyanate conjugated: donkey anti-rabbit, goat anti-guinea pig) Insulin (human) Insulin antiserum (guinea pig anti-human) Normal rabbit serum Pancreatic polypeptide antiserum (rabbit anti-mouse) Pro-insulin (human) Somatostatin-14 Somatostatin antibody (rabbit anti-human) Vectastain (ABC kit)  Dr. M . Gregor Berlin Milab Calbiochem NIH (gift) Dr. D . Kerr (U. of S.) NIH (gift) Amersham GroPep Eli-Lilly (gift) Eli-Lilly (gift) Vector Laboratories Vector Laboratories Peninsula  JacksonImmunoresearch Eli-Lilly (gift) Regulatory Peptide Group,UBC Calbiochem K . G . Buchanan U . of Belfast Eli-Lilly (gift) Peninsula Peninsula Vector Laboratories  

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