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Endogenous glucagon-cells are necessary for the glucose-responsiveness of the HIT-T15 hamster β-cell.. Väänänen, Jeffrey Eric 1993-08-26

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ENDOGENOUS GLUCAGON-CELLS ARE NECESSARY FOR THE GLUCOSE-RESPONSIVENESS OF THE HIT-T15 HAMSTER B-CELL LINEbyJeffrey Eric VaananenB.Sc., Simon Fraser University, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Physiology)We accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1993© Jeffrey Eric Vaananen, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of ^P I.-4—(S Cj(_66.,The University of British ColumbiaVancouver, CanadaDate ^\_,IC)^Qt 93 .DE-6 (2/88)ABSTRACTThe role of glucagon in glucose-stimulated insulin release was investigated using thehamster insulinoma cell line, HIT-T15, as a model of the pancreatic I3-cell.Glucose-stimulated insulin release from HIT-T15 cells was concentration-dependent overa range of glucose concentrations between 0 and 15 mM, with immunoreactive-insulin release(IRI) rising significantly above the basal level (zero glucose) when cells were exposed to glucoseconcentrations of 5, 10 and 15 mM (p < 0.02). Minimum (zero glucose) and maximum (15 mMglucose) insulin release was 1.6 ± 0.5 % and 12.0 ± 2.9 % of total cell content (TCC),respectively. In HIT-T15 cells, glucagon secretion was 5-9 % of total cell content during a 1 hrelease experiment. There was no significant difference between glucagon released in thepresence of zero- or high (15 mM)-glucose.The addition of a glucagon antibody completely abolished glucose-stimulated insulinrelease, while antibodies raised against somatostatin and glucose-dependent insulinotropicpolypeptide (GIP) had no effect. When HIT-T15 cells were incubated in conditions of zero-glucose and glucose plus glucagon-Ab there was no significant difference in insulin release (p >0.05). Furthermore, glucagon-Ab inhibited glucose-stimulated insulin secretion over the fullrange of glucose responsiveness (5 to 15 mM; p < 0.03). The abolition of glucose-stimulatedinsulin secretion by an antibody to glucagon indicates that glucose-stimulated insulin secretion isdependent on glucagon, probably acting via a receptor-dependent pathway. HIT-T15 cells thatwere co-cultured with glucagon producing InR1-G9 cells, displayed basal insulin release thatwas elevated, with an absence of glucose potentiation.HIT-T15 cells were immunoperoxidase stained for the following peptide hormones:insulin, somatostatin, glucagon, glucagon-like polypeptide-I, GIP, secretin, pancreaticpolypeptide (PP) and peptide-tyrosine-tyrosine (PYY). Staining was positive for all peptidehormones tested except somatostatin, secretin and GIP. Insulin, glucagon and PYY were theiidominant peptides. Double staining of HIT-T15 cells for insulin and glucagon demonstratedcolocalization of both peptides within single cells. Additionally, HIT-T15 cells wereimmunoreactive for the cell surface adhesion molecule uvomorulin.Immunocytochemical studies of InR1-G9 cells demonstrated the presence of glucagon,GLP-I and PP, with PP immunoreactivity present in every cell. However, no immunoreactivityfor insulin, somatostatin, GIP , secretin, PYY or uvomorulin was seen. Equal amounts of HIT-T15 and InR1-G9 cells were co-cultured, and stained with fluorescent and immunoperoxidasetechniques. HIT-T15 cell clusters appeared to be surrounded by InR1-G9 cells. These cell linesappeared healthy when cultured together, in spite of the fact that these two cell lines are derivedfrom different strains of hamsters, by different vectors.Electron microscopic examination revealed differences in HIT-T15 cell insulin secretorygranule number with different culture conditions, including: low-glucose media (0.8 mM), high-glucose media (11.1 mM) and high-glucose media (9.1 mM) plus InR1-G9 cells. Mostimportantly, HIT-T15 cells cultured in high-glucose (11.1 mM) were extensively granulated,while those cultured in high-glucose (9.1 mM) in the presence of InR1-G9 cells were agranular.In conclusion, glucose-stimulated insulin secretion from the HIT-T15 cell line isglucagon-dependent, and endogenous glucagon containing cells (HIT-T15-G) can provide theglucagon. The addition of glucagon secreting cells (in large numbers) led to chronic stimulationof HIT-T15 cell secretion, and secretion could not be potentiated by glucose. Furthermore, co-culturing HIT-T15 cells with equal numbers of InR1-G9 cells led to a complete degranulation ofHIT-T15 cells, most likely by continuous stimulation. The HIT-T15 cells are a heterogeneouscell population, expressing multiple peptides, some of which are colocalized, as in the case ofinsulin and glucagon. InR1-G9 cells appeared to be relatively homologous with respect topeptide production. These cells extended neuron-like processes, which contained the majority ofthe secretory granules.TABLE OF CONTENTSPageABSTRACT^ iiTABLE OF CONTENTS^ ivLIST OF TABLES viiiLIST OF FIGURES^ ixACKNOWLEDGMENTS xiHYPOTHESIS AND SPECIFIC OBJECTIVES^ 1RATIONALE^ 2CHAPTER ONE - BACKGROUND^ 3I.^Islet of Langerhans Background 3A. Islet Structure and Cell Distribution^ 3B. Islet-Cell Ultrastructure^ 51.Beta Cells 52. Alpha Cells^ 6C. Insulin, Glucagon and Somatostatin Receptors in the Islet. ^6D.Paracrine Interactions Between a-, 13- and 8-cells^7II.^The Structure and Function of Insulin and Glucagon 7A. Insulin^ 8B. Glucagon 8Ill.^Insulin and Glucagon Secretion^ 10A. Insulin Secretion from the B-Cell^ 101.Secretory Stimuli 102. Inhibitors of Secretion^ 123. Modes of Secretion 134. Heterogeneities of Secretion^ 14B. Glucagon Secretion from the a-Cell 15C. Insulin/Glucagon Interactions^ 16ivIV.^Islet Tumor Cell Background^ 17A. HIT-T15 Cells 17B. InR1-G9 Cells^ 20V.^The Immunoneutralization Technique^ 20CHAPTER TWO - MATERIALS AND METHODS 23I.^Cell Culture^ 23A. HIT-T15 Cells^ 23B. InR1-G9 Cells 23C. HIT-T15 and InR1-G9 Cell Co-cultures^24II.^Static Incubation Studies^ 24III.^Microscopy^ 25A. Light Microscopy^ 251.Fixation 252. Staining Procedure^ 253. Fluorescence Staining 254. Immunoperoxidase Staining^ 265. Photography^ 26B. Electron Microscopy 281.Tissue Preparation^ 282. Staining of EM Sections 293. Equipment and Photography^ 29IV.^Radioimmunoassay (RIA)^ 30A. Insulin Antibody 30B. Insulin RIA^ 30C. Insulin Solid Phase RIA^ 30D. Glucagon RIA^ 31E. Acid Extraction Protocol^ 31F. Analysis of RIA Results 32viCHAPTER THREE - STATIC INCUBATION STUDIES ^ 33I. HIT-T15 Glucose Stimulated Insulin Release 33II. Glucose-Stimulated Insulin Release in the Presence of Antibodies^33III. Glucose-Stimulated Insulin Release Abolished by Glucagon-Antibody ^37IV. Glucose-Stimulated Insulin Release from HIT-T15/InR1-G9 Co-cultures 37V. Glucagon Released from HIT-T15 cells in the Presence of Glucose ^37VI. Cell Viability Following Static Release^ 38CHAPTER FOUR - LIGHT MICROSCOPY RESULTS 45I.^HIT-T15 Cells^ 45A. Single Staining^ 45B. Double Staining 45C. Morphological Observations^ 45II.^InR1-G 9 Cells^ 45A. Staining 45B. Morphological Observations^ 46III.^HIT-T15 and InR1-G9 Cell Co-cultures 46CHAPTER FIVE - ELECTRON MICROSCOPY RESULTS^ 66I. Immunogold Staining^ 66II. HIT-T15 Cell Morphology 66III.^InR1-G9 Cell Morphology^ 67CHAPTER SIX - DISCUSSION 75I.^Morphology^ 75A. Immunocytochemistry^ 75B. HIT-T15 Cell Ultrastructure 76II.^Cell Culture^ 76A. Growth Pattern of HIT-T15 Cells in Culture^76B. Effect of Low-Glucose Culture Conditions on Cell Viability 77C. Insulin Secretion from HIT-T15 Cells ^ 77D. Glucagon Secretion^ 79viiE. Co-Culture HIT-T15 and InR1-G9 Cells^801. Insulin Secretion from Co-cultures 802. Morphology of Co-cultures^ 80III.^Future Directions^ 82CONCLUSIONS 83REFERENCES^ 84LIST OF TABLESNumber^Title^ Page1^Details of Primary Antibodies^ 272^Secondary Antibodies used in Fluorescent Immunostaining^273^Biotinylated Secondary Antibodies used in Peroxidase Immunostaining 274^Summary of HIT-T15 and InR1-G9 Cell Staining^ 485^HIT-T15 Cell Secretory-Granulation Under Varied Culture Conditions^68viiiLIST OF FIGURESNumber Title Page1 IR-Insulin Released from HIT-T15 Cells in Response to Glucose 342 HIT-T15 Cell Extract Values 353 HIT-T15 Cell Glucose-Response in the Presence of Antibodies 364 HIT-T15 Cell Insulin-Response to GlucoseImmunoneutralized by Glucagon-Antibody 395 Comparison of HIT-T15 Cell Extract Valueswith/without Glucagon-Antibody 406 RIA Non-Specific Binding 417 Glucose-Response of HIT-T15/InR1-G9 Cell Co-cultures 428 HIT-T15 and InR1-G9 Co-culture Extract Values 439 Glucagon Release from HIT-T15 Cells in the Presence of Glucose 4410 HIT-T15 Cells Stained for Insulin 4911 HIT-T15^,, 11^Somatostatin 5012 HIT-T15^„ ,, Glucagon 5113 HIT-T15^II^II^II^Glucagon-Like Polypeptide-I 5214 HIT-T15^,, ,, Peptide YY 5315 HIT-T15^II^II^Pancreatic Polypeptide 5416 HIT-T15^,, " Uvomorulin 5517 HIT-T15 Immunoperoxidase Control 5618 HIT-T15 Cell Colocalization of Insulin and Glucagon 5719 InR1-G9 Cells Stained for Glucagon 5820 InR1-G9^"^„^"^Glucagon-Like Polypeptide-I 5921 InR1-G9^" "^Pancreatic Polypeptide 6022 InR1-G9^" "^Insulin 61ix23 InR1-G9 Immunoperoxidase Control 6224 Co-cultured HIT-T15 and InR1-G9 Cells Stained for Insulin 6325 Co-cultured HIT-T15 and InR1-G9 Cells Stained for Glucagon 6426 Co-cultured HIT-T15 and InR1-G9 Cells Stained for Insulin/Glucagon 6527 HIT-Cells Cultured in 11.1 mM-Glucose (2 days) 6928 HIT-Cells Cultured in 11.1 mM-Glucose (10 days) 7029 HIT-Cells Cultured in 0.8 mM-Glucose (2 days) 7130 HIT-Cells Cultured in 0.8 mM-Glucose (10 days) 7231 HIT-T15/InR1-G9 Cells Co-cultured in 9.1 mM-Glucose (2 days) 7332 InR1-G9 Cell Projections and Hormone Granule Localization 74ACKNOWLEDGMENTSHad it not been for Professor Margaret Savage at Simon Fraser University, I would neverhave been kindly introduced to my Masters Supervisor, Dr. Alison M.J. Buchan. Thank you,Professor Savage. I would like to thank Dr. Buchan for taking me on as a Masters student, herenthusiasm and drive served as excellent examples of how to succeed in science. In writingpapers Dr. Buchan has assisted me in presenting my arguments clearly, without changing mywords to fit her writing style. Furthermore, I would like to thank Dr. Buchan for providing mewith the freedom to create and solve problems in an independent fashion.All of the graduate students are experts in one area or another, and because of that theywere an excellent source of information. Although all of the graduate students deserve somecredit for my success, four individuals stand out in my mind, they are Dr. Bruce Verchere, Dr.Eric Accili and Doctors to be: Tim Kieffer and Xiaoyan Jia. Bruce's brilliant success gave me agoal to strive towards. Eric was a constant source of advice on an interpersonal and a scientificlevel. Tim was a supportive and steadfast friend, and my friend Xiaoyan Jia kindly providedassistance with assays.The technical staff in the Department have been an irreplaceable source of expertise.Narinder Dhatt's assistance and instruction on ICC and EM; Leslie Cheqnita's assistance withassays; Joe Tay's expert photo developing; and, Jeanie Kehl's assistance taking blood samplesfor a glucose tolerance test, all deserve special recognition.I wish to express thanks to is Dr. Stephen Kehl, who I found an excellent source ofinformation when I was doing my course work. Dr. Kehl clearly and patiently explained theinner workings of calcium channels to me. Furthermore, Dr. Kehl provided me with muchneeded constructive criticism regarding my original project.Lisa Oliver has supported, encouraged, 'prodded' and entertained me, at the right times.Finally, my parents deserve the final words of appreciation. First, I would like to thank mymother for always being there when I need her. My mother gave me the determination andpersistence to continue with my studies no matter how trying they were. My father deservescredit for being a teacher. I believe that the many hours he spent teaching me about black holes,and why the grass is green have sparked my interest in science, and culminated into this thesis.xiHYPOTHESISGlucose-stimulated insulin secretion, from the HIT-T15 hamster pancreatic B-cell clone,is influenced by endogenously secreted glucagon.SPECIFIC OBJECTIVES1. To demonstrate that insulin secretion from HIT-T15 cells can be regulated by glucagon. Toenhance this insulin release by introducing glucagon secreting cells. Also, to provide evidencefor paracrine or autocrine modulation of glucose-stimulated insulin release by glucagon in HIT-T15 cells, using the immunoneutralization technique.2. To characterize the HIT-T15 and InR1-G9 cell lines with respect to peptide production.3. To examine the ultrastructural appearance of HIT-T15 cells cultured in the presence orabsence of the glucagon secreting cell line InR1-G9.1RATIONALEHIT-T15 cells have been used extensively as a pancreatic 13-cell model since theircreation in 1981, however, they have not been thoroughly characterized during this time. Morerecently there has been the discovery of a 'contaminant' cell in the HIT-T15 cell line. This'contaminant' consists of glucagon producing cells which have been designated as the 'HIT-T15-G' cell (85). The purpose of these studies was three fold. Firstly, glucagon immuno-neutralization was used to determine the effect of HIT-T15-G cells on insulin secretion in theHIT-T15 cell line. Secondly, characterization of HIT-T15 cell line peptide production wasperformed in order to determine whether or not HIT-T15 cells express regulatory peptides, otherthan insulin and glucagon, which may also affect insulin secretion from this cell line. Thirdly,this peptide characterization in conjunction with studies of HIT-T15 cell ultrastructure were usedto determine the validity of the HIT-T15 cell line as a 13-cell model.2CHAPTER ONE - BACKGROUNDI. Islet of Langerhans Background.A. Islet Structure and Cell Distribution.Nutrient homeostasis is one of the most important of all physiological functions. Thetask of maintaining an adequate fuel supply for the body's cells falls mainly on the islets ofLangerhans. The islets of Langerhans (islets) are cell clusters of insulin (13), glucagon (a),somatostatin (8) and pancreatic polypeptide (PP) containing cells. These four islet cell types areprobably differentiated from a peptide-tyrosine-tyrosine (PYY) progenitor cell, as the four majorislet peptides are often co-localized with PYY during development (22, 73, 151). Islets are foundin the pancreas of mammals (40, 46, 64, 69) as well as the duodenal wall of rats (12). Theaverage adult human has approximately 500,000 islets, accounting for 1 to 2% of total pancreaticvolume (39). Islets range in size from 40 to 700 pm, with most being 100 to 200 ym in diameter(158). Within the islet, 13-, a-, 8- and PP-cells account for about 70%, 10-15%, 5-10% and 5% ofcells, respectively (39). In adult rats the core of the pancreatic islet contains mainly 13-cells,surrounded by a peripheral mantle of a-, 8- and PP-cells (108). However, in humans, a- and $5-cells penetrate the 13-cell core as intermixed cords (24); while, in the common tree shrew (Tupaiaglis), the a- and 6-cells compose the islet core, and are surrounded by 13-cells (7). Theproportions of specific islet cells varies between islets from different regions of the pancreas, asdemonstrated in the rat (147). Islets isolated from the rat dorsal pancreas are rich in a- and &-cells when compared to islets from the ventral pancreas which contain a greater proportion ofPP-cells.The complex arrangement of a-,13- and 8-cells is thought to be important in the normalfunctioning of the islet, possibly by facilitating gap-junction and/or paracrine communicationbetween cells (108). The highly ordered structure of the islet is partly due to differential cell3adhesion molecules among the islet cells. Different levels of Ca2+-dependent (uvomorulin) andCa2+-independent cell adhesion molecules are found on heterogeneous islet cell types (10, 51,125). These cell adhesion molecules allow dispersed islet cells to re-aggregate into organizedislet-like structures similar to non-disrupted islets (51, 125). Both glucose and cyclic-adenosine-monophosphate (cAMP) promote the adhesion of B-cells (87, 103). As suggested above, it isbelieved that islet adhesion and/or junctional communication play a role in the regulation ofhormone secretion from individual B-cells (133). Cell adhesion molecules are involved in theregulation of gap-junctional communication in other systems, such as mouse epidermal cellswhere communication is directly regulated by a Ca2+-dependent cell adhesion molecule (E-cadherin). E-cadherin is involved in post-translational assembly and/or function of the gap-junction protein connexin-43 (65).In addition to the complex arrangement of islet cells, there exists a complex isletmicrovasculature. Studies by Stagner and associates suggest that this microcirculation flowsfrom 13- to a- to 6-cells in the rat (19, 137) and dog islet (136). Additionally, human islet B-cellsmay also be perfused before a- and 6-cells (138). On the contrary, a study of the rat islet by Luiand associates describes blood flow from mantle to core to mantle, as follows (84):"The arterioles first reach the surrounding mantle of the islet where theydivided into capillaries that go to other portions of the mantle or the core of the islet,the flow then traverses the core and returns to different portions of the mantle. Theflow is nonhomogeneous in that flow in one portion of the islet can stop, then moveon, while other portions flow freely."Another study in rats suggested that different modes of microcirculation exist in differentsized islets, for example (158): (a) small islets (40-150 pm in diameter) had serial vasculatureonly, (b) midsize islets (160-250 pm) were found to have serial vessels, in addition topostcapillary collecting venules that were directly continuous with larger interlobular veins,4indicating parallel microcirculation, and (c) all large islets (260-700 pm) were also found to haveboth serial as well as parallel microcirculatory patterns within the same islet. In the tree shrew,the insular arteriole, a branch of the interlobular artery, penetrates deeply into the core of the isletbefore branching off into the glomerular capillary network supplying the islet (7). Thesecapillaries reunited at the periphery of the islets to become vasa efferentia and then gave offcapillaries to anastomose with those in the exocrine part of the pancreas.This ordered perfusion of cells within the islet suggests that islet hormones may actwithin the islet in an endocrine manner. This view is further supported by an electronmicroscopic study of rat islet 6-cells, where 75 % of granules were polarized to the capillary endof the cell, suggesting that pancreatic somatostatin is released into islet capillaries (5). Theprecise nature of these endocrine effects appears to differ with species and islet size.Glucagon, insulin, somatostatin and pancreatic polypeptide are also secreted from a-, B-,6- and PP-cells found within the pancreatic duct epithelial lining of the normal adult rat, andhuman (109, 162). Alpha- and 6-cells found in the mammalian gastrointestinal tract are anothersource of glucagon and somatostatin, respectively (8, 60, 74). It is possible that hormonessecreted from these duct and gastrointestinal endocrine cells play a role in the regulation ofpancreatic-islet secretion and/or nutrient metabolism throughout the body.B. Islet-Cell Ultrastructure.1. Beta-Cells.Beta-cells have numerous electron dense secretory vesicles that are about 300 nm indiameter (80, 98). The electron dense core is composed of rhomboidal or polygonal crystalloids,believed to be insoluble insulin (80, 98). A second insulin secretory vesicle, that is spherical andshows no subunit structure, exists (80, 98). These two secretory granule types probablyrepresent different stages of granule maturity (80, 98).5Beta-cells have nearly spherical mitochondria that are more numerous than those of thea-cell (80, 98, 126). The Golgi apparatus is also more extensive in the B- than the a-cell (80,98). Additionally, the rough endoplasmic reticulum (RER) is sparse in 8-cells when comparedwith acinar cells (80, 98), but is greater than that of a-cells (126). Microtubules andmicrofilaments within the cytoplasm are concentrated near the capillary pole of the cell, withmicrotubules arrayed in association with insulin secretory vesicles probably playing a role insecretory granule exocytosis (80, 98).2. Alpha Cells.Identifying features of the a-cell are filamentous mitochondria and small Golgi apparatus(80, 98). Alpha-cell nuclei are indented or lobulated (80, 98). Secretory vesicles are uniform indiameter, at approximately 250 nm, with a highly electron dense core embedded in a matrix ofless dense material. The secretory vesicles are closely surrounded by a limiting membrane (80).C. Insulin, Glucagon and Somatostatin Receptors in the Islet.Receptor binding and internalization of radiolabelled insulin, glucagon and somatostatinare time- and temperature dependent in islet cells (2). Endocytosis and lysosomal degradation ofradio-labeled peptides occurs freely in cells heterologous, but poorly in cells homologous for thepeptide. For example, receptor bound glucagon is rapidly endocytosed and degraded in 6- and 8-cells, but only slowly in a-cells (4, 155). There are, however, specific low-affinity receptors forinsulin, glucagon and somatostatin on cells homologous for these peptides (9). The presence ofinsulin, glucagon and somatostatin receptors on a-, 8- and 6-cells supports the possibility ofparacrine and/or autocrine regulation within the pancreatic-islet.6D. Paracrine Interactions Between a-, B- and b-cells.The existence of 'paralcrinen' (paracrine) hormones was suggested by Feyrter in 1953(42). Paracrine hormones are substances which act locally via the interstitial fluid rather than ina humoral (endocrine) manner. Somatostatin is thought to act, via cytoplasmic processes, as aparacrine inhibitor of gastrin and acid secretion within the mammalian stomach (60, 74, 76).Furthermore, somatostatin has also been suggested to be a paracrine inhibitor of a- and I3-cellswithin the islet (2). The cytoplasmic processes found on some 8-cells have been reported on thea-cells of amphibians (104, 166) and mammals (8, 109). Cells possessing these cytoplasmicprocesses are sometimes called pseudo-neurons, because of their neuron-like appearance.Paracrine interactions in the islet may be facilitated by tight-junctions, which mightfunction to prevent diffusion of released hormone away from its 'target' (106, 107). The numberof tight-junctions has been shown to increase with increasing glucose concentration and exposuretime, suggesting an involvement of tight-junctions in the regulatory process of glucose-inducedinsulin secretion (130). However, the existence of tight-junctions in normal islets has beenbrought into question, as they were not seen in one study, that used freeze-fracture replicas of insitu rat or human islets kept in long term culture (58). It was suggested that the disruption ofislets with collagenase and trypsin during isolation led to tight-junction formation, as a protectivemeasure, intended to seal and protect islet micro-domains against sudden perturbations in localinterstitial fluid (58).II. The Structure and Function of Insulin and Glucagon.The hormones insulin and glucagon appear to play the dominant role in nutrienthomeostasis, within the body. Insulin and glucagon oppose one another in almost all of theirrespective nutrient homeostatic actions.7A. Insulin.Insulin is a 51 amino acid polypeptide (m.w. 5800) which is secreted from the 13-cell(108). The insulin gene, which is located on human chromosome 11, is transcribed to yield aninsulin precursor (preproinsulin) with a molecular weight of 11,500 (16). Preproinsulin containsfour regions (signal, A, connecting and B). The signal region is cleaved, and the remainingregions fold upon one another, with the A and B regions aligning and forming disulfide bridges.The connecting region is then cleaved to yield insulin and C-peptide.Insulin release is stimulated under conditions of fuel excess and is inhibited underconditions of fuel deficiency. Fasting, basal and postprandial plasma insulin concentrations areapproximately 5, 10 and 30-100 pU/ml, respectively (16). Circulating insulin has a half-life of 6to 8 min (16). The main functions of insulin are to promote the uptake, utilization and storage ofnutrients, in liver, muscle and adipose tissues (150). Insulin acts through receptor-regulatedglucose transporters, to stimulate glucose uptake into muscle and adipose tissues (16). Inmuscle, fuel utilization is increased by an increase in muscle glycolysis and glucose oxidation(143). Increased fuel storage is accomplished by increasing glycogenesis and protein anabolism(29, 62, 143). Additionally, increased storage is promoted by a decrease in glycogenolysis andgluconeogenesis (37). Insulin further reduces circulating nutrients by acting as a CNS satietyfactor (16). Finally, hyperinsulinemia is often associated with obesity (63), and in the absence ofinsulin (diabetes) the body's cells literally starve as high levels of glucose in the blood stream cannot be utilized.B. Glucagon.Alpha-cells secrete glucagon (m.w. 3485), a 29 amino acid polypeptide, (107, 150), thatis a member of a highly conserved family of peptides. The glucagon family of peptides includesglucagon, glucagon-like peptide-I and II (GLP-I and GLP-II), glucose-dependent insulinotropicpolypeptide (GIP) and secretin (16, 35, 78, 153). These peptides are derived from the same8(glucagon, GLP-I and GLP-II), or closely related gene(s), and undergo post-translationalprocessing to their respective forms (35). The glucagon gene is expressed in a-cells of thepancreas, and in endocrine L-cells of the gastrointestinal tract (111), and in the brainstem andhypothalamus (33). The mRNA transcripts that arise from these genes are identical (33). Theapparent molecular weight of the preproglucagon molecules is approximately 16,000 Daltons(132). Preproglucagon is processed to proglucagon a polyprotein precursor containingglucagon, GLP-I and GLP-II, and an intervening peptide (111). The preproglucagon sequenceencodes a 20-amino acid signal sequence followed by a 69 amino-acid sequence for the peptideglicentin. Glicentin contains the amino-acid sequence for glucagon. The glicentin sequence isfollowed by two additional glucagon-like peptides (83).Basal plasma glucagon levels are approximately 100 pg/ml, with glucagon having a half-life of 6 min (16). Glucagon is important in normal promotion of fuel mobilization from liver,muscle and adipose tissues. It is also important in preventing hypoglycemia during seriousinjury, shock and starvation (150). In times of stress, it could be said that glucagon is theprotector of the central nervous system as neurons have an absolute need for glucose, or ketonesafter prolonged starvation, to survive (43). During starvation when glycogen stores areexhausted, glucagon-dependent mechanisms convert free fatty acids to ketones which can beused in place of glucose by the CNS (43). In the maintenance of normal nutrient homeostasisglucagon maintains cellular fuel levels via receptor mediated mechanisms that: 1) increaseglycogenolysis, gluconeogenesis and I3-oxidation of free fatty acids (FFA), and 2) reducetriacylglyceride synthesis and glycogenesis (16). Interestingly, glucagon, like insulin, has beenreported to regulate appetite by acting on the CNS (16). Antisera against glucagon lead to adecrease in food intake and body weights in rats (86), thus glucagon probably stimulatesappetite.9III. Insulin and Glucagon Secretion.A. Insulin Secretion from the 8-Cell.1. Secretory Stimuli.The regulation of islet B-cell secretion is multi-faceted, and involves numerous neural,hormonal and nutrient inputs.During the cephalic phase of digestion, the thought, sight, smell and taste of food cancause the autonomic nervous system (ANS), to stimulate insulin secretion (16). Thispredominantly vagal ANS stimulation involves sympathetic (B-adrenergic) and parasympathetic(acetylcholine, muscarinic) pathways (27, 44, 57, 77, 121). Adrenal medullary epinephrine alsostimulates B-adrenergic pathways (16).Following the cephalic phase, and during the gastric and intestinal phases of digestion,direct enteric innervation of the pancreas and/or islets may also play a role in the regulation ofinsulin secretion (70). The gastric and intestinal phases of digestion are partly regulated bypeptide hormones which are secreted from the gastrointestinal tract and the pancreas. Theseinsulinotropic enteric peptides, known as incretins, include the following: enteroglucagon (16),truncated forms of glucagon-like polypeptide-I (GLP-I; 7-36 amide and 7-37) (41, 121), GIP(25, 66, 105, 129), secretin (45) and cholecystokinin (121). Other hormones that stimulateinsulin secretion are pancreatic glucagon (127) and epinephrine secreted from the adrenalmedulla (16, 27, 44, 77).Circulating nutrients that stimulate insulin secretion include: glucose, amino acids, freefatty acids and keto acids. The amino acids that stimulate insulin secretion are leucine, lysine,alanine or arginine (114, 115, 127). Glucose is the primary nutrient stimulus for insulinsecretion, with half-maximal and maximal stimulation at glucose concentrations of 150 and 300-10500 mg/di, respectively. In order for glucose to act as a nutrient for the I3-cell it must first enterthe cell. Rat islets express the GLUT-2 glucose transporter isoform, which is a glucose carrierwith a low affinity for glucose but a high capacity for glucose transport (165). To a lesserdegree, a lower capacity glucose transporter isoform (GLUT-1) is expressed (165). In isolatedislets the levels of GLUT-1 and GLUT-2 mRNA are increased by high-glucose when comparedwith low-glucose culture conditions (165). Additionally, a decreased expression of glucosetransporters in diabetic rat B-cells is associated with impaired glucose sensing characteristics thatmay contribute to the diabetic state (144).Briefly, when glucose is metabolized, a rise in ATP leads to the closure of an ATP-sensitive potassium channel, which in-turn leads to depolarization of the 13-cell (18, 125). Thisglucose stimulated depolarization opens voltage-dependent Ca2+-channels, which in-turn leads toan increase in intracellular Ca2+ and subsequently insulin secretion (64).Receptor coupled 0-protein (Gs) pathways can lead to a rise in cAMP and protein kinase-A (PKA) activity, which potentiate glucose-stimulated insulin release in the B-cell (49, 122).Glucagon acts potently and rapidly through this cAMP pathway (127). However, glucose on itsown does not increase cAMP levels in purified B-cells, but can enhance glucagon-induced cAMPformation (127). Therefore, glucose-dependent rises in cAMP levels are not considered to be anutrient-induced mediator for hormone release but rather as a minor amplification of theglucagon-dependent signal. (127). Neither insulin nor PP affect cAMP formation in pancreatic Bcells (127).In addition to cAMP-PKA mechanisms, inositol triphosphate (IP3), diacylglycerol (DAG)and protein kinase-C (PKC) are also reported to be involved in the secretion of insulin from theB-cell (14, 120). Similar to PKA, PKC is also thought to act on voltage-gated Ca2+ channels toincrease intracellular Ca2+ levels, and activate calcium-dependent secretory mechanisms (163).Intracellular Ca2+ stores found primarily in the endoplasmic reticulum, are released via an 1P3-dependent pathway, to further increase free intracellular Ca2+ levels (15, 119, 124).112. Inhibitors of Secretion.The above second messenger systems act in concert to stimulate insulin secretion fromthe islet I3-cell, but are antagonized by the inhibitory systems described below. There arenumerous inhibitors of insulin secretion from the islet, including the following: somatostatin(27), PYY(17), insulin (16), norepinephrine- and epinephrine-activation of a-adrenergicreceptors (27, 67, 77), galanin (27, 77) and prostaglandin-E2 (77, 122).Somatostatin is an important intra-islet inhibitor of glucagon, insulin and somatostatinsecretion (2, 71, 139). Somatostatin inhibits insulin release at multiple sites of the I3-cellsecretory mechanisms, including: 1) reduced intracellular Ca2+ , 2) reduced cAMP, and 3) cellhyperpolarization. The reduction in intracellular Ca2+ is due to inhibition of dihydropyridine-sensitive (L-type)- and w-conotoxin-sensitive (N-type) voltage-dependent Ca2+-channels on theI3-cell. This inhibition is mediated via a pertussis toxin-sensitive inhibitory G-protein called Gi(1, 54, 97, 149). Pertussis toxin irreversibly ADP-ribosylates G1, rendering it nonfunctional (79,117, 122), for at least five days following toxin removal (28). Somatostatin counteractsglucagon-induced cAMP production in purified 13-cells (128), by the same pertussis toxin-sensitive Gi (27, 53, 54, 122, 149). Pertussis toxin acts at a common site to block somatostatin-induced Ca2+ inhibition, hyperpolarization and a reduction in cAMP levels (36, 72, 161).Hyperpolarization of I3-cells inhibits insulin release by closing voltage-sensitive Ca2+ channelsand possibly by closing gap junctions. In some systems somatostatin-dependent inhibition ofNa+-H+ exchange can cause an increase in extracellular and a decrease in intracellular pH (46).Interestingly, both of these pH alterations are known to reduce gap junction coupling in somesystems. Finally, Nilsson and associates report that there is a decreased sensitivity of thesecretory machinery of the 13-cell to Ca2÷, and a direct inhibition of the exocytotic process,mediated by a pertussis toxin-sensitive Gi (100).It is plausible that receptor mechanisms for prostaglandin-E2, a2-adrenoceptors, andgalanin involve a common G protein(s)/potassium channel complex which transduces the12inhibitory signals. A common property of receptors which activate potassium channels via Giand inhibit Ca2+ channels through Go and/or Gi is that they also inhibit adenylate cyclase (77).Gi mediates decreases in intracellular cAMP caused by inhibitors of insulin secretion, e.g.epinephrine, somatostatin, prostaglandin-E2, and galanin (36). G-proteins also regulate ionchannels, phospholipases and distal sites in exocytosis (122).3. Modes of Secretion.Insulin secretion occurs in two phases, as follows: phase-I is an initial peak which isfollowed by phase-II a sustained plateau (16). Possible explanations for this biphasic release areas follows: 1) two storage compartments that contain granules with different sensitivities toglucose, and 2) the second phase of insulin secretion is de novo synthesized insulin stimulatedby glucose. It is likely that both of these mechanisms are active.Additionally, protein secretion from endocrine cells can occur via several differentmeans. Constitutive release refers to the secretion of proteins as fast as they are synthesized,without intracellular storage in typical granules (68). Typically most post-translationalprocessing takes place prior to exit from the medial Golgi stack, however, hormones released viaconstitutive means do not undergo full post-translational processing, as occurs in the secretorygranule prior to secretion.Regulated secretion refers to secretion of previously produced proteins, that have beenstored at high concentrations in secretory vesicles (68). Hutton and associates state that theinsulin secretory granule of the pancreatic B-cell is a complex intracellular organelle comprisedof many proteins with different catalytic activities and messenger functions (55). Secretorygranules form at the trans-Golgi network (TGN) by envelopment of the dense-core aggregate ofregulated secretory proteins by a specific membrane. This dense-core may be created viaselective aggregation of the secretory protein, or sorting of the secretory protein by a sortase13(69). At this stage they are referred to as immature secretory granules (145). The immaturesecretory granule then undergoes a maturation process (post-translational modification) whichgives rise to the mature secretory granule (146). Proinsulin is cleaved to biologically activeinsulin by two distinct Ca2+-dependent endopeptidases that are found in the insulin secretorygranules (6, 30). Type I cleaves between the B-chain and the C-peptide junction; Type II on theC-peptide/A-chain junction. Davidson and associates, report that there are specific Ca2+ and pHrequirements for each of these proteases. These specific requirements indicate that Type I couldonly be active in the intragranular environment and type II in the Golgi apparatus or secretorygranule. In a cell where both constitutive and regulated protein secretion occur, mechanismsexist for sorting the correct secretory protein into the correct secretory vesicle (69).Following this, the secretory vesicle must then be delivered to the appropriate region ofplasma membrane (68). Targeting to the cell surface involves the actin-based cytoskeleton andsmall GTP-binding proteins. Calcium-dependent contractile events involving cytochalasin B-sensitive microfilamentous structures provide the motive force for both the intracellulartranslocation and exocytotic release of beta granules (154). Secretion occurs when maturesecretory granules fuse with the plasma membrane.4. Heterogeneities of Secretion.In addition to heterogeneities in cell types within the islet it has recently become apparentthat the population of I3-cells is not homogeneous. Beta-cell properties such as 1) hormoneresponsiveness, 2) insulin secretion, 3) insulin biosynthesis, 4) electrophysiology, and 5) ion(K+, Na-'- and Ca2+) current all show variability (20, 53, 99). Although insulin secretoryresponses may vary between I3-cells, they are usually homogeneous within a single I3-cell uponsuccessive stimulations (20, 47). Furthermore, 13-cells that are undergoing de novo proteinsynthesis release insulin preferentially during stimulation (20). Finally, differences inacetylcholine-stimulated rises in intracellular Ca2+ have been reported in normal rat pancreatic f3-14cells (156).The heterogeneity in biosynthesis and secretion of insulin is reduced with 13-cell contact(20). This reduction in heterogeneity could in part be due to a phenomenon termed 'recruitment',which refers to the coordination of 'units' of cells via the sharing of various intracellular signalsthrough gap-junctional communication. The less responsive cells are recruited into the secretingunit, where they act in a homologous manner, possibly by decreasing their average threshold forstimulation. Longo and associates report patterns of Ca2+ change within islets that are consistentwith the recruitment of cells (82). They state that there is a coexistence of oscillations withsimilar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+, withincommunicating 13-cell units. Additionally, stimulation of I3-cells appears to lead to an increase inthe size of coupled units. An example of altered coupling upon stimulation is seen withprolactin, a glucose-dependent insulin secretory hormone, which increases coupling (135).Prolactin appears to decrease the apparent glucose threshold for insulin release, while it increasesthe extent of dye coupling among 13-cells (96, 134).When compared with intact islets, single B-cells and re-aggregated I3-cells respondpromptly to glucose, but exhibit an elevated basal insulin secretion, and profoundly loweredpeak- and total-insulin secretion (81, 87). Although weaker than whole islets, re-aggregated B-cells have a slightly stronger glucose-stimulated insulin secretion than isolated B-cells. Animproved response is restored by simple cell-to-cell contact, but a lowered basal insulin secretionrequires gap-junctional contacts between re-aggregated cells (113). Conversely, heterologous B-cell to non-B-cell contact was not effective in enhancing the recruitment of B-cells or increasingtheir individual secretion (21).B. Glucagon Secretion from the a-Cell.Islet a-cells are electrically active, in the absence of glucose (159), and require Ca2+ forglucagon release (114, 115). Glucagon secretion is stimulated by amino acids, enteric peptidesand neural pathways. Stimulatory amino acids include arginine, alanine, and glutamine (114,15115, 128). Oral nutrients stimulate glucagon release via enteric hormones (GIP, gastrin andCCK). Additionally, cAMP analogues and epinephrine can further amplify nutrient-stimulatedglucagon release from a-cells (114, 115). Stressors, including infection, toxemia, burns, tissueinfarction and major surgery, all promptly increase glucagon secretion. This phenomenon isprobably mediated by the adrenergic nervous system via sympathetic outflow from theventromedial hypothalamus to a-adrenergic receptors in the I3-cells. Rat islets are innervated byenteric neurons, which probably play a role in normal islet regulation (70).Amino acid-stimulated glucagon release is inhibited by glucose through directmechanisms that potentiate insulin's inhibitory actions (16, 114, 115). Free fatty acids act tofurther inhibit glucagon release. Similarly, pancreatic somatostatin is an important mediator ofthe suppression of glucagon secretion (71). In addition to glucose, somatostatin and insulin,PYY also exerts inhibitory effects on a -cell glucagon secretion in mammals (22) .Most newly synthesized proglucagon is membrane-associated (86-88%), while glucagonexhibits much less membrane-association (24-31%). Suggesting that association of newlysynthesized proglucagon with intracellular membranes could be related to the facilitation ofproteolytic processing of proglucagon and/or transport from the site of synthesis to the secretorygranules (102).C. Insulin/Glucagon Interactions.Pipeleers and associates showed that isolated 13-cells were less glucose-responsive than 13-cells coupled with other 13- or a-cells, and that glucose-responsiveness was restored by addingglucagon or a-cells (113, 127). Alpha-cells may influence I3-cell secretion via endocrine (19) orparacrine (150) pathways. Glucagon cell cytoplasmic processes originally demonstrated in thegut, have more recently been demonstrated on islet a-cells (8, 104, 109, 166). Thesecytoplasmic processes may be reaching out to 'pass a message on', as is suspected for gut16somatostatin cells, which also extend cytoplasmic processes (76). Thus, it is possible thatglucagon stimulates insulin release via a paracrine mechanism. Furthermore, a-cells maymodulate B-cells via electrical- or second messenger-coupling through gap-junctions (89-93). Inthe rat islet cAMP has been shown to increase gap-junction surface expression (58), anotherstimulus of insulin secretion. Glucagon is known to increase cAMP levels in B-cells (127). Gap-junction coupling has also been shown between B- and a-cells within the islets of various species(96, 108). Thus it is probable that B- and a-cells regulate one another through electrical andsecond messenger coupling through gap junctions.Local intracellular rises in Ca2+ concentration have been observed with fluorescenceimaging. These local rises may induce a rise in Ca2+ from a second Ca2+ store, creating a waveof Ca2+. Calcium waves may traverse gap junctions to co-ordinate extended networks of cells, asis seen in pancreatic acinar units (94, 110). Gylfe and associates (50), reported the propagationof cytoplasmic Ca2+ waves in clusters of pancreatic B-cells in response to glucose. Similarly,cAMP waves are seen in the amoeba, Dictyostelium discoideum., which forms communicatingcolonies (110). There is the intriguing possibility that IP3, nucleotides and/or other intracellularmolecules may act in this manner, as well. Studies using the movement of fluorescent tracers(lucifer yellow) have estimated that small molecules with molecular weights up to 1000 daltons(approx. 1.5-2.0 nm) can pass through gap junctions (148). In the rat islet, the tracer dye, 6-carboxyfluorescein has been used to identify separate territories consisting of 2-8 coupled a—, B-and/or 6—cells (95). Beta-cells located in the periphery of the islet appear to have twice as manygap junctions per unit membrane area as the B-cells situated in the islet center (89). Thisapparently non-random clustering of gap junctions on the B-cell membrane may play asignificant role in regulation of insulin release.IV. Tumor Cell Background.17A. HIT-T15 Cells.The HIT-T15 cell line was established by Simian virus 40 (SV40) transformation ofSyrian hamster pancreatic islet cells (125). HIT-T15 cells secrete insulin in response to glucosewith reported half-maximal and maximal stimulation, at glucose concentrations of 1 mM (120)and 7.5 to 10 mM (125), respectively. Glucose-stimulated insulin secretion in the HIT-T15 cellsis passage dependent with a 30-fold reduction between passages 41 and 88 (125), thus it isimportant to either standardize experiments or perform experiments on a single passage. Theloss of HIT-T15 cell function is caused by a loss of insulin mRNA, insulin content, and insulinsecretion and is preventable by culturing HIT-T15 cells in low-glucose conditions (123). Insulinsecreted from HIT-T15 cells is reported to have a half-life of 36 h (32), significantly greater thanthe 6 to 8 min half-life of circulating insulin (16).In the HIT-T15 cell line there appears to be abnormal glucose handling, as there is anelevated level of GLUT-1 and GLUT-3, but a depressed level of the major glucose transporter(GLUT-2) found in islet 13-cells (23). Additionally, HIT-T15 cells chronically exposed to highglucose containing media, exhibit lowered glucose transporter mRNA levels, glucose transportand glucose-induced insulin secretion, when compared with cells cultured in low-glucose media(118). In HIT-T15 cells, GLUT-2 mRNA, glucose uptake activity, and the glucose-responsiveness of insulin secretion correlates with glucose-induced changes in glucose uptakeactivity (56). Although glucose handling does not appear to be normal in the HIT-T15 cell line,it remains a glucose-responsive cell line.Glucose stimulated depolarization of HIT-T15 cells increases intracellular Ca2+ (116,120), most likely by ATP-sensitive K+-channel associated depolarization, as described above inthe normal 13-cell (99). Similarly, glucagon stimulates the secretion of insulin from HIT-T15cells in the absence and presence of glucose (75), possibly via a rise in cAMP that leads to anincrease in protein kinase-A activity, and Ca2f. influx through voltage-dependent Ca2+ channels(116). GIP and GLP-I potentiate glucose-stimulated insulin secretion by increasing extracellularCa2+ influx through voltage-dependent Ca2+ channels (84). Increasing glucose leads to anincrease in preproinsulin mRNA (48). Similarly, glucagon, forskolin and dibutyryl cAMP also18increase content of preproinsulin mRNA approximately two fold, and stimulate insulin release inHIT-T15 cells in the presence and absence of glucose (48, 75, 125). Unlike normal B-cells, it isunlikely that DAG and PKC play an important role in insulin secretion from HIT-T15 cells(120).Glucose- and glucagon-stimulated insulin release from HIT-T15 cells is inhibited bysomatostatin and PP (75, 149). The somatostatin-dependent inhibition of insulin release occursvia G-protein mediated mechanisms (149). Furthermore, the predominant somatostatin receptorprefers somatostatin-28 (142).As mentioned above, cultured HIT-T15 cells have been shown to contain a subpopulationof glucagon expressing and secreting cells designated HIT-T15-G (32, 131). These HIT-T15-Gcells had a 2-fold increase in glucagon mRNA following forskolin or phorbol ester treatment(131). Glucagon levels in acid extracts were found to be 0.72 ± 0.15 pmol/mg protein, withsecreted glucagon having a half-life of 18 hr (32), significantly greater than the 6 min half-life ofcirculating glucagon (16). In addition to glucagon, HIT-T15-G cells also process proglucagoninto the peptides GLP-I and GLP-II, and the major proglucagon fragment (MPF) (131).Forskolin, adrenaline, arginine and KC1 stimulate glucagon release from HIT-T15-G cells (131),while arginine-stimulated glucagon secretion is inhibited by somatostatin in this subset of cells(32, 131). Shennan and associates, suggest that HIT-T15-G cells may represent a lessdifferentiated form of the parental HIT-T15 cell line in which the a-cell phenotype is dominantbut not complete (131). Finally, HIT-T15 cells are also known to transiently express and secretesecretin (160).It has been reported that HIT-T15 cells grow in dome shaped clusters, similar to hamsterislet monolayers (125). Unlike the normal B-cell, uvomorulin has not been reported on the HIT-T15 cell surface.19B. InR1-G9 Cell Background.The InR1-G9 cell line is a BK virus transformed hamster islet cell line, that undergoespost-translational processing of proglucagon to glucagon, GLP-I and 11 (35). Phorbol esters andsodium butyrate, agents that increase glucagon gene transcription in RIN1056A cells, have noeffect on glucagon mRNA levels in InR1-G9 cells, but secretion of glucagon and the glucagon-like peptides is stimulated by phorbol esters (35). Insulin negatively regulates glucagonsecretion as well as glucagon gene expression (112). The InR1-G9 cell line does not containsecretin, another member of the glucagon family of peptides (160), and they are not responsive toglucocorticoid stimulation (157). In the rat endocrine pancreas, the glucagon gene is regulatedby a PKA-dependent pathway, however, InR1-G9 cells lack PKA regulation of glucagonsecretion (34). In the InR1-G9 cells a 50 base-pair region in the 3'-flanking sequence of theglucagon gene is important for the accurate processing of proglucagon mRNA transcripts (78).Glucagon gene sequences are not amplified, but appear to be hypomethylated in the InR1-G9cells when compared to hamster liver or kidney gene sequences (35). The InR1-G9 glucagonmRNA species is 1300 base-pairs (35), similar to the 1200 base-pairs found in mammals (83).In the following studies, the InR1-G9 cell line provides a tool for studying HIT-T15 cellsecretion and ultrastructure in the presence of exogenous glucagon-secreting cells. The effects ofInR1-G9 cell secreted GLP-I and GLP-II were not studied.V. The Immunoneutralization Technique.Due to the non-availability of specific glucagon antagonists, immunoneutralizationbecomes the technique of choice when examining the role of glucagon in the 13-cell and the HIT-T15 cell line. The term immunoneutralization refers to the binding of an antibody (Ab) to the20active site of a molecule in order to abolish biological activity. The technique ofimmunoneutralization requires an Ab that will prevent antigen binding to the receptor. Whenusing an Ab to immunoneutralize a peptide such as glucagon it is important to ensure that cross-reactivity for glucagon-like peptides does not exist. Finally, an immunoglobulin control must beutilized to ensure that the effect is due to the specific Ab in use, and is not just a non-specificeffect of immunoglobulins.Studies using antisera to immunoneutralize glucagon and somatostatin have beenperformed on the islet (141) and perfused pancreas (126), of rats. In the isolated islet theaddition of anti-somatostatin serum to incubation media containing 5.5 mM or 20 mM glucose,significantly increased or had no effect on insulin secretion, respectively (141). In another study,anti-somatostatin gamma globulin augmented secretion of glucagon and insulin from pancreatica- and 13-cells, respectively (61).In the isolated islet the addition of glucagon antiserum to incubation media containing 5.5or 20 mM glucose, significantly increased or had no effect on insulin secretion, respectively(141). However, glucagon antiserum led to a marked increase in glucagon secretion from theislet (141). This marked increase in glucagon secretion may have been a compensatorymechanism, allowing the islet to overcome an artificially induced reduction in glucagon levels.In the perfused pancreas glucagon antiserum had no significant affect on either insulin orsomatostatin secretion, although it enhanced glucagon secretion. Another study in the arginineinfused rat, demonstrated a reduction in circulating C-peptide levels when glucagon antiseraprevented a rise in plasma immunoreactive glucagon levels (101). Anterograde infusion ofglucagon antibodies did not effect insulin release but did decrease somatostatin secretion byabout 50%, while retrograde infusion of glucagon-Ab decreased insulin secretion approximately30%, but had no effect on somatostatin secretion (137). Glucagon-Ab administration led to nochange in plasma glucose, immunoreactive insulin or immunoreactive somatostatin in rats (140).21The ability of glucagon antibodies to gain access to the inner core of the islet isquestionable. If paracrine or autocrine regulation is occurring in the islet, and tight junctionisolated inter-cellular spaces exist, it is unlikely that an Ab could easily enter these isolatedspaces. Thus, the immunoneutralizing ability of antibodies used in the vasculature or inperifusion experiments is questionable. Additionally, the above studies did not take nervous andhumoral factors into consideration. The finely balanced interactions between insulin, glucagon,and somatostatin within the islet may be able to compensate for disturbances created by theaddition of antisera to one of these three peptides. Therefore, studies using glucagon andsomatostatin antisera on isolated, dissociated islet cells are required to determine their effects inthe absence of nervous and humoral factors.22CHAPTER TWO - MATERIALS AND METHODSI. Cell Culture.A. HIT-T15 Cells. The insulin-secreting hamster B-cell line HIT-T15 (passage 60), waspurchased from American Tissue and Cell Culture (ATCC, Rockville, MD). Frozen HIT-T15cells were rapidly thawed in a 37°C water bath and seeded at 3 million cells / 50 ml media in a250 ml flask (Falcon, Becton Dickinson). Cells were cultured in Ham's F12K (Irvine Sci., SantaAna, Ca) containing glucose (7.0 mM), L-glutamine (Sigma, 2 mM), penicillin (Gibco; 100U/ml), streptomycin (Gibco; 100 pg/ml) and supplemented with 10% dialyzed horse serum(DHS; Gibco) and 2.5% heat inactivated fetal bovine serum (FBS; Gibco). Additionally, longterm cell culture experiments were performed using low- (0.8 mM) and high-glucose (11.1 mM)containing RPMI (Gibco) media that was supplemented with glutamine, penicillin, streptomycinand serum, as described above. HIT-T15 cell doubling time was one week, when cells were fedevery three days and grown in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Whencells reached greater than 90% confluency they were detached from culture flasks with a 10 minincubation in a trypsin/EDTA solution (Gibco), washed and re-seeded in new culture flasks at 5million cells per flask. Some trypsinized cells in approximately i00 pi of media were placed onmicroscope slides and observed at the light microscopic level, for the purposes of determining re-aggregation characteristics. In order to eliminate variation between passages, all experimentsused cells subcloned to a single passage (# 68) rather than using a series of passages.B. InR1-G9 Cells. The glucagon-secreting hamster a-cell line InR1-G9 (passage 19),was kindly provided by Dr. D. Drucker (University of Toronto). InR1-G9 cells were seeded at 3million cells/50 ml media in a 250 ml flask. Media contained high glucose (11.1 mM) RPMIsupplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 pg/ml) and5% heat inactivated fetal calf serum (FCS; Sigma). InR1-G9 cell doubling time was three days,when cells were fed every three days and grown in a humidified atmosphere of 95% air and 5%CO2 at 37°C. As above, all experiments used cells subcloned to a single passage (# 24).23C. HIT-T15 and InR1-G9 Co-cultures. Co-cultures of HIT-T15 (passage 68) andInR1-G9 (Passage 24) cells were grown in media composed of HIT-T15 and InR1-G9 culturemedia (1:1), under the conditions described above. Hence, glucose was 9.1 mM in the mixedmedia. Co-cultures were seeded at a density of one million cells per well (48 h prior to theexperiment), including the following ratios of HIT-T15:InR1-G9 cells: 100:0; 75:25; 50:50 and25:75.II. Static Incubation Studies.Static release experiments were performed on one million cells per well, cultured for 48 hon 12 well plates (Falcon, Becton Dickinson). After the culture period, medium was removedfrom HIT-T15 cells, and replaced with preincubation medium. Preincubation medium consistedof DMEM supplemented with glucose (3.0 mM). Preincubation was for 1 h at 37°C in anatmosphere of 95% air: 5% CO2. Preincubation medium was removed and DMEM, which wassupplemented with glucose (0, 3, 5, 10 or 15 mM) or glucose plus a purified Ab, was added andincubated for 1 hr under the same conditions as the preincubation. Antibodies that were specificto glucagon (26), somatostatin (164) or GIP (T.J. Kieffer and N. Dhatt, unpublishedobservations), were used to immunoneutralize their respective antigens. See Table 1 for furtherinformation regarding these antibodies. All test conditions were performed in duplicate.Following incubation, the medium was removed and insulin content was determined byradioimmunoassay (RIA) unless indicated as being determined by solid-phase RIA (SPRIA).Values for released insulin are expressed as a percentage of total cell insulin content, asdetermined by the extraction procedure described below.III. Microscopy.Tumor cells used for immunocytochemistry or electron microscopy were cultured for 48h on ethanol sterilized glass coverslips (25 mm x 1 mm; Fisher, Canada) in 6 well plates(Falcon, Becton Dickinson) at a density of one million cells per well.24A. Light Microscopy (LM)1. Fixation. Cells which adhered to coverslips were fixed in Bouin's solution for5 min and washed in phosphate buffered saline (PBS; pH 7.3) at room temperature beforeimmunostaining. The ability to stain cells fixed with the electron microscopic fixativesparaformaldehyde (2%) or glutaraldehyde (0.5%) with or without 1% osmium tetroxide wastested at the light microscopic level.2. Staining Procedure. Peptide antigens were localized with specific primaryantibodies (see Table 1). The bound primary antibodies were subsequently detected withsecondary antibodies to either rabbit, mouse or guinea pig immunoglobulins. Secondaryantibodies conjugated to a fluorescent label or peroxidase were visualized with fluorescence orlight microscopy, as described below. Prior to incubation with primary antibodies, endogenousperoxidase activity was blocked by incubating slides with 0.3% hydrogen peroxide in methanol,for thirty minutes. Fixed cells on coverslips were washed three times with phosphate bufferedsaline (PBS), then non-specific binding of Ab was blocked with 10% solution of normal swineserum (NSS), in PBS. The PBS solution was composed of NaC1 (137 mM), KH2PO4 (1.5 mM),Na2HPO4 (8.1 mM), KC1 (2.7 mM), NaN3 (3.0 mM), at pH 7.3. Excess blocking solution waswashed off with PBS. Cells were incubated with primary antibodies (for 48 hr, at 4°C), dilutedwith NSS:PBS (1:10). Unbound primary antibodies were removed by washing three times withPBS prior incubation with secondary antisera.3. Fluorescence Staining. Primary-Ab labeling for insulin and glucagon waslocalized using fluorescein isothyocyanate (FITC)-conjugated or rhodamine isothyocyanate(RITC)-conjugated secondary antibodies. Incubation with these secondary antibodies was for 1h at room temperature in a humid atmosphere. The stained cells were then examined under aZeiss Universal Microscope equipped for epifluorescence. The FITC and RITC conjugatedsecondary antibodies are listed in Table 2. Stained cells on coverslips were mounted on glassslides (VWR Sci. Inc., San Francisco, CA) with 10% glycerol in PBS, and photographed.254. Immunoperoxidase Staining. The diaminobenzidine (DAB)/glucose oxidaseimmunoperoxidase staining method, as described by Buchan et al. (26), was used to localizeprimary antibodies bound to desired antigens. Slides were washed three times with PBS,followed by a 30 min incubation with 1:10 NSS. Excess NSS was rinsed off with three washesof PBS and a biotinylated secondary-Ab (diluted in 1:10 NSS) was applied for 1 h. Biotinylatedsecondary antibodies are listed in Table 3. Following the secondary-Ab, the cells were washedthree times with PBS, and further incubated with complexed A and B (ABC Vectastain,Dimension Lab. Inc., Mississauga, Ont.) in 1:10 NSS, for 1 h. Three final washes with PBSwere performed prior to development of the staining with a DAB/glucose oxidase solution. TheDAB/glucose oxidase solution consists of dextrose (11.1 mM), ammonium chloride (0.4 mM),glucose oxidase (20 mg/1) and DAB (12.5 mg/ml) in a 0.1 M TRIS buffer. Staining developedwithin 10 to 60 minutes, and was then washed for 5 min in running tap water. Some coverslipswere counterstained (for 30 sec) with filtered hematoxylin. Following a wash in running tapwater the stain was differentiated in acid alcohol (30 sec), and again washed (5 min) in runningtap water. Coverslips were dehydrated through graded alcohols (70, 90, 100 and 100%),followed by 2 x 3 min in xylene. Finally, coverslips were permanently mounted on slides withEulcitt, and photographed.5. Photography. Stained cells were photographed with a Zeiss Axiophotmicroscope using a built-in 35 mm camera, or a Zeiss Axiovert 35M equipped with a 35 mmcamera (Contax 167 MT). The types of film used for black/white and colour photography were26Table 1 - Details of Primary AntibodiesAntigen Host Source Dilution Dilution-IN TypeGlucagon Mouse Gregor 10 pg/ml 10 pg/m1 AscitesGlucagon Rabbit Milab 1:2,500 Serum* GIP Mouse RPG 1:5,000 1:1000 AscitesGLP-I (C-terminal) Guinea pig RPG 1:1,000 SerumPP (C-terminal) Rabbit Buchanan 1:2,000 SerumPYY Rabbit McDonald 1:1,000 SerumSecretin 53 Rabbit Polak 1:500 SerumInsulin Guinea pig RPG 1:1,000 SerumSomatostatin Mouse CURE 1:10,000 1:1000 AscitesUvomorulin  Rat^ Sigma 1:500 MonoclonalDilution-IN - dilution/concentration of Ab used in immunoneutralization studies.Buchanan - Dr. K. Buchanan, Dept. Med., University Hospital, Belfast, Ireland.CURE - Dr. J. Walsh, Center for Ulcer Research and Education.Gregor - Dr. M. Gregor, Dept. Gastroenterology, University of Dublingen, Germany.McDonald - Dr. T. McDonald, University of Western Ontario, Ontario, Canada.Milab - Milab, Sweden.Polak - Dr. J. Polak, Royal Postgrad. Med. School, London, England.RPG - Regulatory Peptide Group, Dept. Physiology, University of British Columbia, Canada.Sigma - Sigma, St. Louis, MO, USA.* Antibodies used for immunoneutralizing target peptides.Table 2- Secondary Antibodies used in Fluorescence ImmunostainingAntigen Host Label Source DilutionGuinea pig IgG Goat FITC Jackson 1:500Mouse IgG Donkey FITC Jackson 1:1,000Guinea pig IgG Goat RITC Jackson 1:2,000Mouse IgG Rabbit^ RITC Vector^ 1:300Jackson - Jackson ImmunoResearch Laboratories Inc., Mississauga, Ontario, CanadaVector - Dimension Labs, Mississauga, Ontario, CanadaTable 3 - Biotinylated Secondary Antibodies used in Peroxidase ImmunostainingAntigen Host Source DilutionGuinea pig IgGMouse IgGRabbit IgGRat IgG GoatRabbitGoatRabbitVectorVectorVectorVector 1:3001:3001:3001:200Vector - Dimension Labs, Mississauga, Ontario, Canada27Ilford HP5 (ASA 400) and Fujichrome P1600 (ASA 800), respectively. Ilford HP5 wasdeveloped using Ilford developer (Ilford, Mobberly Cheshire, UK). The developer was appliedfor 10 min followed by a 5 min wash in running tap water. The film was fixed (Ilford fixer) for 5min, and again washed for 5 min in running tap water, then hung to dry. Fujichrome P1600 wasdeveloped using the Rapid E6 process (Photo Systems Inc., Dexter, Michigan), as described inthe supplied instructions.B. Electron Microscopy (EM).1. Tissue Preparation. HIT-T15 cell cultures (at 2, 4, 6, 8 and 10 days of culturein low- and high-glucose), and HIT-T15/InR1-G9 co-cultures were fixed for 15 min with 2.5 %glutaraldehyde in a 0.1 M phosphate buffer. Fixed tissue was washed three times with phosphatebuffer, removed from the bottom of the culture plate with a rubber policeman, and centrifuged ina microtube (Eppendorf, Sigma) for 5 minutes. Pelleted tissue was further fixed with 1%osmium tetroxide, and washed three times (10 min each wash) with phosphate buffer. At thisstage, tissue was stored at 4°C until embedding.Prior to embedding, tissue was dehydrated through graded alcohols (70, 80, 90, 100, 100and 100%; 15 min each). Dehydration was followed by a wash in propylene oxide (10 min),after which, the tissue was infiltrafed with propylene oxide:epon (described below) mixtures of1:1 and 1:3 (2 h each). After the 2 h periods the 1:3 propylene oxide:epon mixture was removedand replaced with pure epon overnight following which the epon was replaced. The eponinfiltrated tissue was then placed in a 70°C oven and allowed to harden overnight. Eponconsisted of Jembed-812-resin (6 ml) and dodecenyl-succinic-anhydride (12 ml) mixed withJembed 812 resin (7 ml) and nadic-methyl-anhydride (6 ml), and tri(dimethylaminomethyl)-phenol as a hardener. All epon reagents were purchased from JBS Chem, Dorval, Quebec.Tissue blocks were cut free from microtubes with a razor blade, and trimmed inpreparation for sectioning with an ultramicrotome. Semi-thin sections were cut to a thickness of285 p.m and stained with 1% toluene blue, for light microscopic examination. Following thisexamination tissue blocks that were determined to be embedded adequately were sectioned forEM. Sections cut for EM were 0.5 gm in thickness, as confirmed by the reflection of gold andsilver light when sections were floated on a water bath. Sections were picked up with EM grids(200 mesh Ni) and allowed to dry for two days before staining.2. Staining of EM Sections. All tissue sections were stained with a saturatedsolution of uranyl acetate (UA), by placing the EM grid on a drop UA solution for 5 minutes.The grid was rinsed thoroughly with distilled water, and further stained with a saturated solutionof lead citrate (LC) for 1 min (in the presence of a pellet of NaOH). Sections were again washedwith distilled water, and dried.Prior to UA/LC staining some sections were stained for insulin and glucagon using avariation of the immuno-gold staining technique (13). Briefly, EM grids were placed on drops of33% H202 (10 min) washed with distilled water, and left on drops of 1:10 NSS (30 min).Primary antibodies raised against insulin and glucagon (Table 1) were diluted in PBS containing0.1% bovine serum albumin (BSA, Sigma). Grids were incubated on drops of Ab solution for 48h, at 4°C. Following incubation with Ab, grids were washed in PBS, PBS-tween (PBScontaining 0.05% tween-20), and PBS-tween containing 0.5% BSA for 15 min each. A 30 minincubation on drops of 1:10 NSS followed. Gold particle-conjugated secondary antibodies raisedagainst guinea-pig (Sigma; 5 nm gold particle) and mouse (Sigma; 10 nm gold particle), werediluted 1:10 in 0.5% BSA/PBS-tween. Grids were incubated for 1.5 h on drops of Ab solution.Three 15 min washes with 0.5% BSA/PBS-tween, PBS-tween and distilled water, were followedby counterstaining with LC and UA, as described above.3. Equipment and Photography. Prepared tissue sections were examined andphotographed using a transmission electron microscope (Zeiss, High Resolution ElectronMicroscope, EM 10C/CR). Film negatives were developed for 3-4 min (Kodak, TMAX29developer), rinsed (1 min; running tap water), fixed (3 min; Kodak Fixer) and rinsed (30 min;running tap water), before being hung to dry. Negatives were printed in the PhysiologyDepartment with the assistance of Joseph Tay, and some kind suggestions from the staff at Lensand Shutter.IV. Radioimmunoassay.A. Insulin-Antibody. The insulin antibodies used for RIA and SPRIA were derived bysubcutaneous immunization of guinea pigs (Gp) with crystalline human insulin (UBC PhysiologyDept., Dr. R.A. Pederson, 1977). Blood was collected through cardiac puncture and plasma wasseparated by centrifugation. The antisera designated Gp-01 and Gp-07 were divided into 100 pialiquots, lyophilized and stored at -20°C until reconstituted in distilled H20 prior to use.B. Insulin-RIA. Insulin in the release medium or in acid extracts of HIT-T15 cells wasassayed using RIA, unless otherwise stated. The assay used guinea pig antiserum (Gp-01).Iodinated bovine insulin (2000 cpm/tube), and rat insulin standards were employed. The rangeof the assay standards was from 5 to 160 p.U/ml. A 0.01 M phosphate buffer (pH 7.5) containing5 % charcoal extracted human plasma was used for diluting samples and controls. Samples wereassayed in duplicate, and counted for 2 min on a Wallac 1277 Gammamaster. The RIA wassensitive to 10 i.tU/ml, when the sensitivity was determined by taking the insulin concentrationtwice the standard error of the zero-binding below the zero-binding value. Intra- and interassayvariations were less than 2.5 %.C. Insulin Solid Phase RIA. SPRIA for insulin was performed as outlined by Vaananenand co-workers (152). Prior to performing the insulin-SPRIA, Irnmulon-3 removawells in aremovawell holder (Canlab, Vancouver, BC) were coated with 100 41 of insulin-Ab (Gp-07) inCarbonate Coating Buffer (CCB), at least 18 h before use. The CCB (pH 9.6) is a solutioncomposed of Na2CO3 (1.5 mM), NaHCO3 (3.5 mM) and NaN3 (0.3 mM). After the 18 hincubation the plates were washed three times with PBS-tween, and dried by lightly tapping the30plate on a bed of paper towels. Nonspecific binding was blocked by incubation with 200 p.1 ofPBS-tween containing 5% fetal calf serum (Gibco, Burlington, Ont.) for 1 to 4 h at roomtemperature. Plates were again washed 3 times with PBS-tween then dried and either usedimmediately or stored at 4°C until use. Human insulin (Novo Research, Copenhagen) standards,from 0.5 to 256 pil/ml, were made up at pH 7.3 in DMEM containing 0.1 % BSA. Standardsand unknowns were then added to triplicate wells (100 Ill/well). One hundred p.1 of 125I-insulindiluted in PBS-tween to 2000 CPM was then added to the wells immediately following thestandards and samples. After a minimum 15 hr incubation at 4°C, plates were washed threetimes in PBS-tween and dried; the removawells were separated and placed in borosilicate culturetubes (Canlab, Vancouver, BC). Tubes were counted for 2 min on a Wallac 1277 Gammamaster.After counting, culture tubes were emptied and recounted; any contaminated tubes werediscarded, while non-contaminated tubes were stored and reused in subsequent assays.D. Glucagon RIA. Glucagon RIA was kindly performed by Dr. R.A. Pederson'stechnician Leslie Cheqnita. A 0.06 M phosphate buffering system (pH 7.4) was used. Highlypurified porcine glucagon (Novo) was used for standard controls which covered a range of 25 to6,400 pg/m1 in 1:2 serial dilutions. Iodinated glucagon was purchased from Amers ham, and usedin the assay at 2000 cpm/tube. Glucagon-Ab (Gregor MoAB 23-6-B4) was used at a finaldilution of 1:160,000. Samples were assayed in duplicate, and counted for 2 min on a Wallac1277 Gammamaster.E. Acid Extraction Protocol. Total cell hormone content was determined by assayingacid extracts of cultured cells. The extraction procedure consisted of removing cultured cells (in1 ml of 2N acetic acid) from the bottom of the culture plate with a rubber policeman, and placingthe suspension into a microtube. The suspended cells were then sonicated and boiled for 10minutes. Extracts were centrifuged (at 500 g) for 5 min, transferred to a fresh microtube andfrozen (-20°C) until assayed. Acid extracts of HIT-T15 cells were assayed using either RIA (20xdilution) or SPRIA (50x dilution). Cells extracted for glucagon were not boiled.31F. Analysis of RIA Results. Using RIA or SPRIA, samples were assayed in duplicateor triplicate, with values of duplicates or triplicates averaged. Experiments were each performedin duplicate and the duplicate measurements were averaged. The values of each separateexperiment were then averaged, and standard error of the mean (SEM) was calculated. Data setswith p <0.05 (student's t-test) were considered to be significantly different. Hormone releasedwas expressed as a percentage of total cell content (% TCC), as determined by the acid extractionprotocol above.32CHAPTER THREE - STATIC INCUBATION EXPERIMENT RESULTSI. HIT-T15 Glucose-Stimulated Insulin Release.Figure 1 depicts a concentration-dependent rise in IR-insulin released from HIT-T15 cells(passage 68), in response to glucose. Immunoreactive-insulin release (IRI) increasedsignificantly above the basal level (zero glucose) when cells were exposed to glucoseconcentrations of 5, 10 and 15 rnM (p <0.02). Release of IRI is expressed as a percentage oftotal cell content (% TCC), with TCC being 1300 ± 5111U/m1 (mean ± SEM; n=6). When theTCC from cells exposed to zero- and high-glucose (15 mM) was compared (Figure 2), however,there was slightly less intracellular-insulin in the high-glucose exposed cells. This reduction inintracellular-insulin was not significant (p > 0.05; n=6).H. HIT-T15 Insulin Release in the Presence of Antibodies.Antibodies raised against somatostatin and GIP had no effect on glucose-stimulatedinsulin release, while glucagon-Ab completely abolished insulin release (Figure 3).Somatostatin-Ab and GIP-Ab acted as immunoglobulin controls for the glucagon-Ab effect.Insulin released under conditions of zero-glucose and glucose with glucagon-Ab were notsignificantly different (p > 0.05). Insulin released in response to glucose was the same as that inresponse to glucose with somatostatin-Ab (p > 0.05) or glucose with GIP-Ab (p > 0.05).However, zero-glucose and glucose with glucagon-Ab conditions were both significantlydifferent from glucose, glucose with somatostatin-Ab, and glucose with OP-Ab (p <0.001).Values depicted in figure 3 are means ± SEM (n = 7).3320 -34*15 - illC.)C.)^10 -0 5 -0 0.10^3^5^10^15[Glucose] mMFigure 1. IR-Insulin Released from HIT-T15 Cells in Response to Glucose.IR-Insulin release is expressed as a percentage of total cell content (% TCC). Glucose-stimulated insulin secretion is significantly different from basal (0 mM glucose) insulin release(* p <0.02). Each value represents the mean ± SEM of six experiments.Zero^FifteenExtracted WellFigure 2. HIT-T15 Cell Extract Values.Both zero- and high (15 mM)-glucose exposed HIT-T15 cells showed similar total insulincontent. Each value represents the mean ± SEM (n = 6). No significant difference (p > 0.05)between zero- and high-glucose exposed cells was detected.35100 -,80 -60 -40 -20 -0CC1 115 mM GLUCOSE36None^None Glucagon SS^GIPAntibodyFigure 3. HIT-T15 Cell Glucose-Response in the Presence of Antibodies.Each value represents the mean ± SEM (n = 7). Insulin released from the zero glucosecondition was significantly different from that of the glucose, glucose/anti-somatostatin andglucose/anti-GIP conditions (p <0.001). However, no significant difference was detectedbetween zero-glucose and glucose/anti-glucagon conditions (p > 0.05); or, glucose, glucose/anti-somatostatin and glucose/anti-GIP conditions (p > 0.05). These data were obtained usingSPRIA.HI. Glucose-Stimulated Insulin Release Abolished by Glucagon-Antibodies.Glucagon-Ab inhibited glucose-stimulated insulin secretion over the full range of HIT-T15 cell glucose responsiveness (Figure 4), with significance in the 5, 10 and 15 mM glucoseconditions (p <0.03). The %TCC depicted in Figure 4 are means ± SEM of seven experiments.Figure 5 depicts insulin extract values from HIT-T15 cells (n = 6) and glucagon-Ab exposedHIT-T15 cells (n = 7), with no significant difference between the two groups (p > 0.05). Non-specific binding of 1251-insulin (and fragments) in the RIA, was unaffected by the addition ofsample, sample plus glucagon-Ab or 20 times diluted acid extracts (Figure 6).IV. Glucose-Stimulated Insulin Release from HIT-T15/InR1-G9 Co-cultures.The glucose responses of HIT-T15 and InR1-G9 co-cultures at HIT-T15:InR1-G9 ratiosof 75:25, 50:50 and 25:75 were similar, with all displaying basal insulin release that was highlyelevated over HIT-T15 cells alone (Figure 7). Additionally, a marked reduction in sensitivity toglucose is also observed. Acid extracts of co-cultured cells showed parallel changes in TCC of1R1 with changes in insulin cell (H1T-T15) percentage (Figure 8).V. Glucagon Release from HIT-T15 Cells in the Presence of Glucose.Glucagon released from HIT-T15 cells in the presence of glucose is depicted in Figure 9.The concentrations of glucagon released in response to zero-, 5- and 15 mM-glucose, were 264 ±72, 157 ± 66 and 165 ± 77 pg/ml, respectively. The mean glucagon concentration of wellsextracted into 1 ml of 2N acetic acid was 3062 ± 150 pg/tnl. No significant difference was seenbetween any of the glucose conditions studied (p > 0.05). Standard error for all conditions washigh at an average of 38.6% (n=4).37VI. Cell viability Following Static Release.Finally, prior to acid extraction, viability staining with trypan-blue confirmed that greaterthan 95% of HIT-T15 and InR1-G9 cells were viable following each of the static releaseexperiments. Visual inspection of viability stained cells, under the light microscope, with ahaemocytometer indicated that the viability of cells remained unchanged throughout theexperiments.380^3^5^10^15[Glucose] mMFigure 4. HIT-T15 Cell Insulin-Response to Glucose Immunoneutralizedby Glucagon-AntibodyInsulin release values in response to glucose (solid black) are the mean ± SEM of sixexperiments, while insulin release values in response to glucose/glucagon-Ab (hatched) are themean ± SEM of seven experiments. All values are expressed as a percentage of total cell content(% TCC). Glucagon-Ab significantly inhibited glucose-stimulated insulin release (* p <0.004;0 p < 0.03).391600 -1400 -_E 1200 ----M1000 -II^800 - C600 -Mu)C^400 -200II....1200 -0HIT^HIT + Glucagon Ab40Figure 5. Comparison of HIT-T15 Cell Extract Values with/withoutGlucagon-Antibody.This figure compares the extract values from HIT-T15 cells (n=6) and HIT-T15 cellsexposed to glucagon-Ab (n=7). Each value represents the mean ± SEM of experiments. Theseconditions are not significantly different (p > 0.05).41Figure 6. RIA Non-Specific Binding.The level of nonspecific binding (NSB) was unchanged by the presence of either sample,sample containing glucagon-Ab, or 20x diluted acid extracts when compared with the assay NSB(p > 0.05). Each value is the mean ± SEM of six experiments.250 100(n=6)Ea75/25 (n=2)50/50 (n=2)25/75 (n=5)1 00 -50 -0200 -,150 - 100(n=6)el 75/25(n=2)50/50(n=2)121 25/75(n=5)Zero^Three^Five^Ten^Fifteen42AGlucose (mM)20.0^0^17.5I-15.0 12.5O ^10.071;CC^7.5 5.0^Cl)^2.5C0.0Zero^Three^Five^Ten^FifteenGlucose (mM)Figure 7. Glucose-Response of HIT-T15/InR1-G9 Co-cultures.HIT-T15 and InR1-G9 cells were co-cultured at the HIT-T15:InR1-G9 ratios indicatedabove. Insulin release is displayed in: A) concentration (in pll/m1), or B) percentage of totalcell content (% TCC). Error bars represent SEM in the 100/0 and 25175 conditions, while theyrepresent the range in the 75/25 and 50/50 conditions.150012501000g—g^750cn^50025001 0 0/0^75/25^5 0/5 0^25/75HIT-Ti5/InR1-G943Figure 8. HIT-T15 and InR1-G9 Co-culture Extract ValuesTotal cell content of insulin was determined, in varying ratios of co-cultured HIT-T15and InR1-G9 cells. The HIT-T15/InR1-G9 ratios follow, with n values in brackets: 100/0 (6),75/25 (2), 50/50 (2) and 75/25 (5). Values represent means of experiments performed induplicate. Error bars represent SEM in the 100/0 and 25/75 conditions, while they representrange in the 75125 and 50/50 conditions.C.)C-)1744Zero^Five^Fifteen[Glucose] mMFigure 9. Glucagon Release from HIT-T15 Cells in the Presence of Glucose.Glucagon release from HIT-T15 cells in the presence of glucose. Release is expressed asa percentage of total cell content (% TCC). There was no significant difference between values(p > 0.05).CHAPTER FOUR - LIGHT MICROSCOPY RESULTSI. HIT-T15 Cells.A. Single Staining. Using the immunoperoxidase staining procedure described aboveHIT-T15 cells were stained for peptide hormones, including: insulin (Figure 10), somatostatin(Figure 11), glucagon (Figure 12), C-terminally amidated GLP-I (Figure 13), GIP (not shown),secretin (not shown), PYY (Figure 14) and PP (Figure 15). Additionally, HIT-T15 cells werestained for the cell surface adhesion molecule uvomorulin (Figure 16). Staining was positive forall peptides except somatostatin, secretin and GIP. It should be noted that cell surface staining ofuvomorulin allowed the irregular surface of HIT-T15 cells to be seen. As a control,immunoperoxidase staining was performed in the absence of a primary-Ab, using either an anti-mouse or an anti-guinea pig secondary-Ab (Figure 17). The immunoperoxidase controls wereboth negative. The results of the above HIT-T15 cell immunostaining are summarized (Table 4).B. Double Staining. Double staining of HIT-T15 cells for insulin and glucagondemonstrated co-localization of these peptides within single cells (Figure 18).C. Morphological Observations. While viewed under the light microscope (at 200-400x magnification) it was observed that trypsinized HIT-T15 cells re-aggregated into islet-likemasses, before attaching to the bottom of a culture plate. This re-aggregation occurred in as littleas 5 to 10 min, and it was difficult to physically separate these cells once they were aggregates.With time these domed HIT-T15 cell masses combined to cover the bottom of the culture plate.II. InR1-G9 Cells.A. Staining. InR1-G9 cells were stained in the same manner as the HIT-T15 cells.Positive staining for glucagon, GLP-I and PP is depicted in Figures 19, 20 and 21, respectively.Staining for insulin (Figure 22), somatostatin (not shown), GIP (not shown), secretin (not45shown), PYY (not shown) and uvomorulin (not shown) was negative. As a control,immunoperoxidase staining was performed in the absence of a primary-A b, with either an anti-mouse or an anti-guinea pig secondary-Ab (Figure 23). As with the HIT-T15 cells, theimmunoperoxidase controls were unstained. Table 4 provides a summary of InR1-G9 cellstaining.B. Morphological Observations. Unlike the HIT-T15 cells, trypsinized InR1-G9 cellsdid not re-aggregate with one another prior to attaching to the bottom of culture flasks.However, once attached to the plate, InR1-G9 cells grew toward neighboring cells by extendingneuron-like processes. In the InR1-G9 cells the intensity of fluorescent staining for glucagonwas greatest near the end of these processes. These neuron-like processes were not seen in theHIT-T15 cells and served as a good marker to distinguish between HIT-T15 and InR1-G9 cellsin co-culture.III. HIT-T15 and InR1-G9 Co-cultures.HIT-T15 and InR1-G9 cells were co-cultured at a HIT-T15:InR1-G9 ratio of 1:1 andstained with fluorescent and immunoperoxidase techniques. Figure 24 shows a co-culturestained for insulin while Figure 25 shows similar cells stained for glucagon. These two figuresclearly show (based on morphology), clustering of putative HIT-T15 cells, with putative InR1-G9 cells and their processes surrounding these clusters. Fluorescence staining for insulin andglucagon further revealed the spatial arrangement of co-cultured cells (Figure 26). Within oneweek, InR1-G9 cells filled in all empty spaces between HIT-T15 cell clusters.46Finally, these the two cell lines generated from hamster tissue were compatible in culture,as trypan blue viability staining indicated a greater than 95 % viability. Additionally, at theelectron microscopic level there was an absence of significant amounts of dead cells andphagocytic activity. These data are unlike co-cultures of BTC3 (murine derived) and InR1-G9(hamster derived) cells which were incompatible and destroyed one another (T. Kieffer,unpublished data).47Table 4 - Summary of HIT-T15 and InR1-G9 Cell StainingPeptide Relative AbundanceHIT-T15 InR1-G9Insulin +++ 0Somatostatin 0 0Glucagon -H- +++GLP-I ++ +++GIP 0 0Secretin 0 0Pancreatic Polypeptide + ++++Peptide-YY ++++ 0Uvomorulin +++ 0Control 0 0++++ - 100%^of cells were immunoreactive for peptide+++ -> 75% "^"^"++ -> 50% " If /I II II 11± - <50% "H H II H H0 - < 5%^" II 11 II II IIGLP-I - Glucagon-like Polypeptide-IGIP - Glucose-dependent Insulinotropic Polypeptide, orGastric Inhibitory Polypeptide48Figure 10. HIT-T15 Cells Stained for Insulin.Immunoperoxidase staining of insulin appears brown, and hematoxylin counterstaining ofnuclei is seen as blue. Magnification 400x49Figure 11. HIT-T15 Cells Stained for Somatostatin.Immunoperoxidase staining of somatostatin appears brown, and hematoxylincounterstaining of nuclei is seen as blue. Magnification 400x50Figure 12. HIT-T15 Cells Stained for Glucagon.Immunoperoxidase staining of glucagon appears brown, and hematoxylin counterstainingof nuclei is seen as blue. Magnification 400x51Figure 13 HIT-T15 Cells Stained for Glucagon-Like Polypeptide-I (GLP-I).Immunoperoxidase staining of GLP-I appears brown, and hematoxylin counterstaining ofnuclei is seen as blue. Magnification 200xFigure 14. HIT-T15 Cells Stained for Peptide-YY (PYY).I minunoperoxidase staining of PYY appears dark. Magnification 400x54Figure 15. HIT-T15 Cells Stained for Pancreatic Polypeptide (PP).Immunoperoxidase staining of PP appears dark (arrows). Magnification 200xFigure 16. HIT-T15 Cells Stained for Uvomorulin.Immunoperoxidase staining of uvomorulin (large arrow) appears dark.Blebs protruding from the HIT-T15 cell surface (small arrow). Magnification 400x.Figure 17. HIT-T15 Immunoperoxidase Control.HIT-T15 cells stained with the immunoperoxidase method in the absence of a primary-Ab, but using a secondary-Ab to mouse immunoglobulins. Haematoxylin staining of nucleiappears blue. Note that secondary-Ab to guinea-pig showed similar results. Magnification 400x56ABFigure 18. HIT-T15 Cell Colocalization of Insulin and Glucagon.This figure depicts fluorescein isothyocyanate (FITC) staining of insulin (A), andimmunoperoxidase staining of glucagon (B). Magnification 1000x.5740LA.58Figure 19. InR1-G9 Cells Stained for Glucagon.Immunoperoxidase staining of glucagon appears dark. Magnification 400xFigure 20. InR1-G9 Cells Stained for Glucagon-Like Polypeptide-I (GLP-I).Immunoperoxidase staining of GLP-I appears dark. Magnification 400x59Figure 21. InR1-G9 Cells Stained for Pancreatic Polypeptide (PP).Immunoperoxidase staining of PP appears dark. Magnification 200x60Figure 22. InR1-G9 Cells Stained for Insulin.Immunoperoxidase staining of insulin (brown) is exceptionally rare (arrow).Haematoxylin counterstaining stained InR1-G9 cell nuclei (blue). Magnification 200x61Figure 23. InR1-G9 Immunoperoxidase ControlInR1-G9 cells stained with the immunoperoxidase method in the absence of a primary-Ab, but using a secondary-Ab to guinea-pig immunoglobulins . Haematoxylin staining of nucleiappears blue. Phase contrast allows cells to be visualized. Note that anti-mouse secondary-Abshowed similar results. Magnification 400x62Figure 24. Co-cultured HIT-T15 and InR1-G9 Cells Stained for Insulin.Immunoperoxidase staining of co-cultured cells for insulin appears brown, andhaematoxylin counterstaining of nuclei appears blue. Magnification 400x63Figure 25. Co-cultured HIT-T15 and InR1-G9 Cells Stained for Glucagon.Immunoperoxidase staining of co-cultured cells for glucagon appears brown, andhaematoxylin counterstaining of nuclei appears blue. Magnification 400x64ABFigure 26. Co-cultured HIT-T15 and InR1-G9 Cells Stained for Insulin/Glucagon.Staining of co-cultured cells for glucagon appears red (Rhodamine), while staining ofinsulin appears green (FITC). Magnification 1000x.65CHAPTER FIVE - ELECTRON MICROSCOPY RESULTSI. Immunogold Staining.Immunogold staining of HIT-T15 and InR1-G9 co-cultures for insulin and glucagonyielded no convincing labeling when viewed with the electron microscope. Cell cytoplasm wasdotted with gold particles, however, nuclei and epon embedding plastic also showed somebackground. Staining of prepared HIT cell sections was complicated by fixative interferencewith Ab binding. Fixative interference was confirmed with imrnunoperoxidase staining ofBouin's, paraformaldehyde (2%), glutaraldehyde (0.5%) and glutaraldehyde (0.5%) plus 1%osmium tetroxide fixed tissue (data not shown). At the light microscopic level, Bouin's fixedtissue was stained positively using the immunoperoxidase method (Figures 10-16). On thecontrary, the preferred EM-fixatives paraformaldehyde, glutaraldehyde and osmium tetroxideprevented staining, and were therefore not suitable for electron microscopic tissue preparation inthese experiments (data not shown). Following this result, further attempts at immunogoldstaining of HIT-T15 cultures and HIT-T15/InR1-G9 co-cultures were not made. Althoughstaining was not performed HIT-T15 and InR1-G9 cells were easily distinguished by their grossmorphology, such that, HIT-T15 cells appear round while InR1-G9 cells extend neuron-likeprocesses.H. HIT-T15 Cell Morphology.Electron microscopic examination revealed changes in HIT-T15 cell insulin granulationunder varied culture conditions. Specifically, cells cultured in media supplemented with high-glucose (11.1 mM) had large numbers of secretory-granules (Figure 27). Furthermore, thenumber of granules appeared to increase with culture time (compare Figures 27 and 28).Similarly, cells cultured in low-glucose (0.8 mM) supplemented media were also extensivelygranulated (Figure 29). As in the high-glucose condition, culture time appeared to increase theoverall number of granules (compare Figures 29 and 30). No evidence was obtained for polarity66in distribution of secretory granules, in either the low (0.8 mM)- or high (11.1 mM)-glucoseculture conditions. Unlike the two previous culture conditions, HIT-T15 cells co-cultured withInR1-G9 cells (50:50) in high-glucose (9.2 mM) media were agranular. For a summary of theseresults see Table 5.In all of the described culture conditions HIT-T15 cells appeared to store large amountsof glycogen (see small arrows in Figures 27-31).III. InR1-G9 Cell Morphology.InR1-G9 cells appear to release secretory granule contents into the interstitial spacebetween adjacent cells (Figure 31). This directed release of granule content is further enhancedby the neuron-like projections that InR1-G9 cells extend toward other cells, as these projectionscontain the majority of secretory granules (Figure 32). This finding supports the lightmicroscopic observation that fluorescent staining for glucagon is most intense at the tip of InR1-09 projections.67Table 5 - HIT-T15 Cell Secretory-Granulation Under Varied Culture ConditionsCulture ConditionsGlucose (m1VI)^Duration (days)Secretory-granuleNumber/(1 section)^CharacteristicsPercentage oflive cells11.1 2 25-100 Clustered > 9511.1 10 25-100 Clustered > 950.8 2 25-100 Unclustered 75 - 900.8 10 25-75 Unclustered 50 - 75*9.2 2 0-5 >95* This condition consisted of HIT-T15 cells co-cultured with equal numbers of InR1-G9 cells.6869AFigure 27. HIT-Cells Cultured in 11.1 mM-Glucose (2 days).Secretory-granule (large arrow) and glycogen-granule (small arrow)A. Magnification 6,500 xB. Magnification 21,500 xAFigure 28. HIT-Cells Cultured in 11.1 mM-Glucose (10 days).Secretory-granule (large arrow) and glycogen-granule (small arrow)A. Magnification 10,500 xB. Magnification 21,500 x70;A71Figure 29. HIT-Cells Cultured in 0.8 mM-Glucose (2 days).Secretory-granule (large arrow) and glycogen-granule (small arrow)A. Magnification 5,200 xB. Magnification 21,500 x72AFigure 30. HIT-Cells Cultured in 0.8 mM-Glucose (10 days).Secretory-granule (large arrow) and glycogen-granule (small arrow)A. Magnification 10,500 xB. Magnification 21,500 xFigure 31. HIT-T15/InR1-G9 Cells Co-cultured in 9.1 mM-Glucose (2 days).InR1-G9 projection (middle cell) in close contact with an adjacent cell. The position ofsome glucagon-granules (large arrow) suggests that glucagon may be released into the spacebetween cells.Magnification 10,500 x74AFigure 32. InR1-G9 Cell Projections and Hormone Granule Localization.A. InR1-G9 projecting a process toward an adjacent cell. Magnification 7,000 xB. Hormone granule localization within the process above. Magnification 20,500 xCHAPTER SIX - DISCUSSIONI. MorphologyA. Immunocytochemistry.HIT-T15 cells (passage 68) stained with the immunoperoxidase method were positive 101-the peptide hormones insulin, glucagon, GLP-1, PYY and PP. Of these peptides glucagon andGLP-I are known to have a regulatory role in insulin secretion, raising the possibility that PYYand PP may also exert effects on the HIT-T15 cells. The presence of glucagon and GLP-Iimmunoreactivity in the HIT-T15 cell line suggested that other glucagon family members such asGIP and secretin may also be expressed. However, no immunoreactivity for the glucagon familymembers GIP and secretin was found in the HIT-T15 cell line, suggesting that expression waslimited to the preproglucagon gene. Another product of this gene is GLP-II, but lack of aspecific antibody precluded staining for the peptide. A small percentage (< 5%) of HIT-T15cells demonstrated immunoreactivity for somatostatin.Interestingly, insulin and glucagon were colocalized within single cells, as demonstratedby double staining with fluorescent and immunoperoxidase techniques. Some cells showed noco-localization. However, cells with co-localization varied, such that, some stained f or bothpeptides with one at a greater intensity, while others stained with equal intensity for bothpeptides.All HIT-T15 cells stained positively for PYY, however, as with insulin and glucagon,some cells stained more intensely than others. It has been reported that the midgestational fetalpancreas (human and porcine) contains cells that co-express insulin, glucagon, somatostatin andPP/PYY (31, 85). Additionally, these hormones were localized in separate secretory granules(31) or colocalized i n i ndividual  secretory granules (85), however, this co-expression was not75found in the newborn pancreas of either species (31, 85). As the HIT-T15 cells display multiplepeptide expression and colocalization, in a manner similar to the fetal pancreas (31, 85) they mayprovide a model for the study of fetal islets and/or islet development. Additionally, thesecharacteristics are also similar to those of human and rodent islet tumors (3, 11, 13, 38), thusHIT-T15 cells may also be a good model for studying these tumors.The InR1-G9 cell line was immunoreactive for glucagon, GLP-I and PP. The presence ofPP immunoreactivity in all cells suggests a potential regulatory role in this cell line.B. HIT-T15 Cell Ultrastructure.Ultrastructural studies demonstrated that HIT-T15 cell secretory-granule content variedwith condition and duration of culture. Glucose levels appeared to have only minor effects onsecretory-granule characteristics, however, in all culture conditions the HIT-T15 cells storedlarge amounts of glycogen. Hyperglycaemic mice and rat models exhibit glycogen stores thatare reduced by lowering circulating glucose levels (52). It was thought that HIT-T15 cellglycogen could be due to chronic exposure to high-glucose culture conditions shunting glucose'into the glycogenic pathway. However, after 10 days in low-glucose culture conditions nosignificant depletion of intracellular glycogen was observed.II. Cell CultureA. Growth Pattern of HIT-T15 Cells in Culture.Trypsinized HIT-T15 cells re-aggregate into islet like masses. This re-aggregation occursprior to cell attachment to the culture vessel, and can be observed under the light microscope.Within five to ten minutes clusters of three to ten cells have formed. Uvomorulin, a cellularadhesion molecule (cadherin), is probably responsible tbr this aggregation, as uvomorulinimmunoreactivity was clearly localized to the external surface of the HIT-T IS cells. As76uvomorulin is a Ca2+-dependent adhesion molecule, studies comparing the re-aggregation ofcells in Ca 2+ containing and Ca2+ free media, would be expected to further confirm the role ofCa2+-dependent uvomorulin in HIT-T15 cell clustering.B. Effect of Low-Glucose Culture Conditions on Cell Viability.As mentioned above, HIT-T15 cells are reported to regain some of their responsiveness toglucose when cultured in low-glucose (118). Thus, it was important to determine the ability ofthese cells to grow and survive in low-glucose media. Low-glucose (0.8 mM) media cultureadversely affected HIT-TI5 cell viability in a time-dependent fashion. Evidence for this was, asfollows: 1) following one week of culture, there were approximately four times more floatingcells in culture media containing low-glucose than in culture media containing high-glucose, 2)floating cells were dead, as indicated by an inability of these cells to exclude the viability staintrypan blue, 3) electron microscopic examination revealed a greater number of dead cells inlow-glucose compared to high-glucose cultures, and 4) cell doubling time was almost twice aslong in the low-glucose cultured cells. These results indicate that growing HIT-T15 cells in low-glucose containing media may not be practical. However, the reported (118) restoration ofglucose responsiveness may over-ride any extension in time required to perform experiments.C. Insulin Secretion from HIT-T15 Cells.The glucose responsiveness of HIT-T15 cells (passage 68), with maximal glucose-stimulated insulin secretion at around 10 mM glucose, corresponded well with the maximalglucose-stimulated insulin secretion for hamster 13-cells (120) and HIT-T15 cells in the hands ofother investigators (120, 125). However, as previously reported, when compared with hamster 13-cells, HIT-T15 cells show a blunted insulin secretory response, due primarily to a low total cellcontent (120, 125). The total cell content of HIT-T15 cells at passage 68 (3.2 mU/m1) was onefifth of the reported value for cells at passage 41 (15.0 mU/m1), but was six times greater thanthat reported at passage 88 (0.5 mU/m1) (125). Thus, in view of the decreasing total cell content77previously reported, the values appeared to be within the range expected for cells at passage 68.There is a reduction in HIT-T15 cell insulin secretion with increasing passage, which parallelsthe reduced content. It is possible that this reduction in secretion is due to alterations in HIT-T15cell metabolism, or changes in the ratio of insulin- to other peptide-containing cells withsuccessive passages.The morphological studies demonstrated co-production of glucagon within the HIT-T15cells. Therefore, because of its known stimulatory action on insulin release, the ability ofglucagon to regulate HIT-T15 cell secretion was examined.Addition of a monoclonal glucagon-antibody, to passage 68 cells, completely abolishedglucose-stimulated insulin secretion over all glucose concentrations tested. The glucagon-antibody did not affect total insulin content of HIT-T15 cells, as determined by extract values.Control experiments demonstrated that addition of either a somatostatin or GIP monoclonalantibody did not alter secretion indicating the specificity of the technique. No interferance of theglucagon antibody with the insulin RIA was observed either, with no change in non-specificbinding levels. Furthermore, samples from the two immunoneutralization studies were assayedusing different techniques, and their results corroborated one another. These studies could befurther supported by performing experiments using varying concentrations of the glucagon-Ab,and by using other glucagon-antibodies. Finally, in order to rule out this effect as a secondaryone, immunoneutralization of other peptides found in the HIT-T15 cell line (GLP-I, GLP-II,PYY and PP), should be performed.Of the remaining peptides localized to the HIT-T15 cells only the activity of somatostatinhas been investigated. An antibody to somatostatin, added in the same manner as the glucagonantibody, had no effect on glucose-stimulated insulin release. These data indicate that theamount of somatostatin released into the medium was insufficient to affect insulin release.78The abolition of glucose-stimulated insulin secretion by a glucagon-antibody indicatesthat glucose-stimulated insulin secretion in HIT-T15 cells may be dependent on glucagon co-secreted from the cells acting through a receptor-dependent pathway. Presumably, the antibodyinhibits-insulin secretion by binding the functional domain of the antigen, thus rendering itincapable of binding to its receptor. It is unlikely that gap-junctional pathways could be blockedby an antibody, as it would not traverse the cell membrane and interfere with intracellularmechanisms. No attempt was made in the present studies to define the nature of potentialreceptor-mediated mechanisms, however, previous studies of the effect of glucagon on HIT-T15cells indicates that they respond to glucagon via a G-protein mediated rise in cAMP whichcauses the release of insulin (75, 116). These data support a receptor-dependent mechanism forglucagon's effect because, it is unlikely that glucagon released from HIT-T15-G cells acts in amanner different from exogenously applied peptide. It is also unlikely that glucagon actsthrough cytosolic receptors, as once again, antibodies would not block this effect.Recent studies, of B-cell clones (B-TC3), have demonstrated the presence of receptors thatbind GLP-I more readily than glucagon (H. Kofod, NOVO, unpublished data). However, asmentioned in the background section, paracrine communication between cells, may not require ahigh-affinity receptor. The high concentrations of peptide that receptors are in contact with maybe sufficient, if the peptide release is directed toward 'target cells, rather than being released intothe general circulation, thus eliminating the need for a high-affinity receptor. The effects ofendogenous GLP-I on HIT-T15 cell insulin secretion were not examined in this investigation.D. Glucagon Secretion.Glucagon release from HIT-T15 cells was measured in response to glucose, and nosignificant difference was seen between zero-, low- and high-glucose conditions. This suggeststhat glucagon was differentially released from the HIT-T15 cells and was not regulated byglucose. It also demonstrates that glucagon and insulin are stored in separate secretory granules,because, if insulin and glucagon were stored in the same granule a parallel rise in glucagon79secretion would follow the glucose-stimulated insulin secretion. The most effectivesecretagogues for glucagon secretion are amino acids. However, their effect was not examinedin the present study. These data do, however, provide evidence for a basal level of glucagonrelease from HIT-T15 cells which provides a basis for the requirement of glucagon for glucose-stimulated insulin release.E. Co-Culture HIT-T15 and InR I-G9 Cells.I. Insulin Secretion from Co-cultures.Insulin release from HIT -T15 cells was stimulated by the addition of exogenousglucagon-secreting cells (InRI-09; passage 24). The InR1-G9 cell line stimulated insulinsecretion was not enhanced by glucose in any of the concentrations tested. The 25:75 conditionclearly demonstrates an absence of glucose-potentiation of glucagon-stimulated insulin release.It also demonstrates a glucose-independent, glucagon-stimulated insulin secretion, as shown bythe high level of insulin released in the zero-glucose condition. Glucagon concentrations werenot measured in these experiments. The usefulness of this data would, however, be limited as allconcentrations of InR1-G9 cells tested exhibited the same degree of insulin stimulation. Thisindicates that even at 25% of the cells, a maximal stimulation of insulin secretion was present.Co-cultures with fewer I nR I-G9 cells would be useful in determining if HIT -T15 cells alreadypossess the optimal glucagon conditions for maintaining glucose-stimulated insulin secretion.2. Morphology of Co-cultures.Fortunately HIT -T15 cells were easily distinguished from InR1-09 cells by their grossmorphology and growth patterns. For example, HIT-T15 cells were circular, while InR1-G9cells extended neuron-like processes (almost without exception). In addition, HIT-T15 cellsgrew in dome shaped clusters, probably mediated by uvomorulin (as suggested by the presenceof uvomorulin immunorcaetivitv). On the contrary, InR1-09 cells, which were not80immunoreactive for uvomorulin, grew evenly spaced apart until cell density caused them to growtogether after approximately one week. These growth patterns were maintained even when co-cultured. Interestingly, InR1-G9 cells appeared to direct hormone release toward adjacent cells,by concentrating secretory granules within projections that were reaching toward neighboringcells. Further evidence for this apparent polarization of secretory granules is provided by thehigh intensity of fluorescent staining found at the tips of InR1-G9 cell projections. These datasuggest that InR1-G9 cells, like somatostatin cells of the gut, may act in a directed paracrinefashion (74). This directed release may reduce the total amount of hormone required, for aspecific effect, by concentrating the peptide to sites of action.In co-culture, HIT-T15 cells appear to be essentially agranular, while, InR1-G9 cells hadgranules. The absence of secretory granules in co-cultured HIT-T15 cells is likely due todepletion caused by chronic stimulation, and an inability to replenish stores. The inability toreplenish these stores could be due to the length of glucagon exposure, or an absence of phasicglucagon release. It is unknown why high glucose concentrations do not cause a similardegranulation of the HIT-T15 cells, however, this difference indicates that glucagon is probablya more potent stimulus to insulin secretion than glucose alone in these experimental conditions.In a fashion similar to the normal B-cell, glucose may play a role as a potentiator of glucagon-stimulated insulin secretion (127). It would be interesting to see if granules were depleted inlow-glucose media co-cultures. Studies using high-performance liquid chromatography, couldbe used to determine whether or not the proinsulin/insulin ratio of degranulated co-cultures waselevated. If elevated this might provide evidence for constitutive release of insulin in thisexperimental condition.SiIII. Future Directions.Studies to quantify relative peptide abundance between early and late HIT-T15 cellpassages, would be useful. Especially if glucagon expression rose with increasing passages, as arise in glucagon expression could be the cause of the elevated basal and reduced insulin secretoryresponse.Subcloning of pure insulin and pure glucagon secreting cells from the HIT-T15 cell line,and examining their peptide secretion alone and when mixed at fixed ratios, could further assistin understanding HIT-T15 cell function. Performing these experiments would require HIT-T15cells of a very early passage, as growing subcloned cells would take them into later passageswhere their usefulness as a model is in question. This study would be complicated by the co-expression of peptides in the HIT-T15 cell line, however, some cells do not co-express insulinand glucagon, and these should be the cells targeted for further study.And finally, studies of insulin release in co-cultures could also be done in a time courseexperiment, with observations being taken at hour intervals. These studies would help todetermine how long these conditions had to exist for degranulation to occur.82CONCLUSIONSIn conclusion, glucose-stimulated insulin secretion from the HIT-T15 cell line isglucagon-dependent, and endogenous glucagon-cells (HIT-T15-G) provide a ready source ofglucagon. The addition of large numbers of exogenous glucagon secreting cells (InR1-G9) led toa high-level stimulation of HIT-T15 cell insulin secretion, and this secretion was not potentiatedby glucose. The absence of glucose-potentiation of glucagon-stimulated insulin secretion wasprobably due to degranulation of cells, due to chronic high-level stimulation by glucagon. TheHIT-T15 cells appear to be different from normal 13-cells in that B-cells are only partiallydependent on glucagon for full glucose-responsiveness, while HIT-T15 cells are completelydependent on glucagon's presence. Further, studies are required to determine whether or not thisapparent complete dependence on glucagon is a property of only the HIT-T15 cells, or a propertyof other cell types such as a fetal 13-cell precursor. These studies suggest that the HIT-T15 cellline is not a good adult B-cell model, but may be a good developmental or cancer model.Additionally, HIT-T15 cells are a heterogeneous cell population, expressing multiplepeptides. Some of these peptides were colocalized, as in the case of insulin and glucagon. 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