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Glutamine metabolism and function in skeletal muscle Zhou, Xiwu 1996

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G L U T A M I N E M E T A B O L I S M A N D FUNCTION IN S K E L E T A L M U S C L E by  XIWUZHOU B. Med., The First Medical College of PLA, Conton, 1986 M . S c , Institute of Space Medico-Engineering, Beijing, 1989  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF T H E REQUIREMENT FOR T H E D E G R E E OF D O C T O R OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Animal Science)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A June 1996 © Xiwu Zhou,  1996  In  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  the  requirements  British Columbia, I agree that the  for  an advanced  Library shall make it  freely available for reference and study. I further agree that permission for extensive copying  of  department  this thesis for scholarly purposes may be granted or  by  his  or  her  representatives.  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia i Vancouver, Canada I  Date  DE-6 (2/88)  11  ABSTRACT Glutamine is an amino acid possessing a number of physiological functions in addition to its contribution as a substrate for protein synthesis.  Skeletal muscle is a major  source of glutamine synthesized in vivo. The main objectives of this thesis were to study regulation of the glutamine synthesis and to characterize glutamine's effect on protein turnover in skeletal muscle. Regulation of glutamate dehydrogenase (GDH) by branched-chain amino acids (BCAA) was first investigated. Leucine and isoleucine were found to increase the activity of G D H in rat skeletal muscle in a dose-dependent manner [45% (P<0.01) and 27% (P<0.05) by 1 mM leucine and 1 mM isoleucine, respectively].  The effects of leucine  and isoleucine on G D H activity are additive, suggesting different regulatory sites for each amino acid. Leucine, but not isoleucine, was also found to increase G D H activity in chick skeletal muscle (36% at 1 mM leucine, P<0.05). Acidosis, a condition under which glutamine requirement by the kidney is substantially increased, upregulated the activities of both G D H and glutamine synthetase (GS) in rat skeletal muscle. 1.5%  NH C1, 4  Following 5-d feeding  the activity of G D H and GS increased 88% and 66%, respectively  (P<0.01). Acidosis also increased the sensitivity of rat skeletal muscle G D H to regulation by all three B C A A .  These data provide information to complete our understanding in  regulation of glutamine synthesis in skeletal muscle. In the second part of this thesis, the effect of glutamine on protein turnover in rat skeletal muscle cells was characterized using myotubes differentiated from L8 rat skeletal myoblasts. Glutamine (0.65 - 15 mM) was found to increase the rate of protein synthesis  Ill  in stressed myotubes ( 4 3 ° C for 45 min) (P<0.05) but not in normal-cultured myotubes. Glutamine (0.65 - 15 mM) decreased the rate of degradation of long-lived proteins in both heat-stressed and normal-cultured myotubes (P<0.05) but had no influence on the rate of degradation of short-lived proteins.  In an attempt to study the mechanism underlying the  effects of glutamine on protein turnover in myotubes, glutamine was found to increase the expression of heat-shock protein 70 (HSP70) at both the protein level (7.5-fold increase by 10 m M glutamine, P<0.001) and the mRNA level (7.8-fold  increase by 10 m M  glutamine, P < 0.001) in heat-stressed myotubes but not in normal-cultured myotubes. This effect of glutamine may be specific to HSP70 as the expression of HSP27 was not altered by glutamine. In spite of an increase in the abundance of hsplO mRNA, the D N A binding activity of heat shock transcription factor was not influenced by glutamine.  It is  concluded that the effect of glutamine on protein turnover in skeletal muscle is conditiondependent, and that HSP70 may play a role in the mechanism underlying the anabolic effect of glutamine in skeletal muscle.  iv TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Figures  ix  Abbreviations  xi  Acknowledgement  xii  CHAPTER ONE  G E N E R A L INTRODUCTION  I. Glutamine Metabolism 1.1. Glutamine metabolism in skeletal muscle  1 2. 2  1.1.1. Glutamine synthesis  3  1.1.2. Glutamine degradation  6  1.1.3. Glutamine transport  6  1.2. Glutamine metabolism in other tissues  7  1.2.1. Glutamine metabolism in the kidney  7  1.2.2. Glutamine metabolism in the gastrointestinal tract  8  1.2.3. Glutamine metabolism in the liver  8  1.2.4. Glutamine metabolism in immunocytes  9  II. Protein Turnover in Skeletal Muscle  9  II. 1. Protein synthesis  10  II.2.  12  Protein degradation II. 2.1. Lysosomal system  13  II.2.2. Nonlysosomal system  13  11.2.2.1. Ubiquitin-dependent pathway  13  11.2.2.2. Calcium-dependent pathway  13  II. 3. Regulation of protein turnover in skeletal muscle II. 3.1.  Effects of hormones  14 14  11.3.2. Effects of amino acids  15  11.3.3. Effects of stressors  17  III. Heat-Shock Proteins and Their Expression in Skeletal Muscle  18  III. 1. HSP classification  19  111.2. Role of HSP as molecular chaperones  19  111.3. The mechanism underlying HSP induction  20  111.4. HSP expression in skeletal muscle  22  111.4.1. Skeletal muscle stress models  23  111.4.2. HSP expression in skeletal muscle  24  IV. Objectives of the Thesis  25  V . References  26  CHAPTER TWO  R E G U L A T I O N OF G L U T A M A T E D E H Y D R O G E N A S E B Y B R A N C H E D - C H A I N A M I N O ACIDS IN R A T A N D CHICK SKELETAL MUSCLE 37  I. Introduction  37  II. Materials and Methods  38  II. 1. Chemicals  38  11.2. Experimental animals  38  11.3. Preparation of mitochondria from skeletal muscle  38  11.4.  Measurement of enzyme activity  39  11.5.  Statistical analyses  40  III. Results  40  III. 1. Effects of leucine and isoleucine on glutamate dehydrogenase activity in skeletal muscle from rats and chicks  40  III.2. Additive effects of leucine and isoleucine on glutamate dehydrogenase activity  41  IV. Discussion  42  V . References  50  CHAPTER THREE  R E G U L A T I O N O F E N Z Y M E S F O R G L U T A M I N E SYNTHESIS IN R A T S K E L E T A L M U S C L E DURING M E T A B O L I C ACIDOSIS 52  I. Introduction  52  II. Materials and Methods  53  II. 1. Chemicals  53  11.2.  Experimental animals  53  11.3.  Preparation of muscle samples for enzyme assay  53  11.4.  Measurement of G D H activity  54  11.5.  Measurement of GS activity  55  11.6.  Statistical analyses  55  III. Results  55  III. 1. Effect of acidosis on G D H and GS activity in rat skeletal muscle  56  III.2. Regulation of G D H activity by B C A A in skeletal muscle during acidosis IV. Discussion  56 57  V . References CHAPTER FOUR  62 R E G U L A T I O N O F PROTEIN T U R N O V E R B Y G L U T A M I N E IN STRESSED A N D N O R M A L - C U L T U R E D R A T S K E L E T A L MYOTUBES 63  I. Introduction  63  II. Materials and Methods  65  II. 1. Materials  65  11.2.  Cell culture  65  11.3.  Measurement of the rate of protein synthesis  66  11.4.  Measurement of the rate of protein degradation  67  11.5.  Heat-shock treatment and measurements of protein synthesis and degradation in heat-stressed myotubes  68  Statistical analyses  70  11.6. III. Results  70  III. 1. Effect of glutamine on protein turnover in non-stressed myotubes  70  III.2. Effect of glutamine on protein turnover in stressed myotubes  71  IV. Discussion  72  V . References  80  C H A P T E R FIVE  G L U T A M I N E INCREASES H E A T S H O C K PROTEIN EXPRESSION IN STRESSED R A T S K E L E T A L MYOTUBES  84  I. Introduction  84  II. Materials and Methods  85  ILL  Materials  85  II.2.  Cell culture and heat-shock treatment  86  Vlll  11.3. Western blot analysis  87  11.4. R N A preparation and Northern blot analysis  88  11.4.1. R N A preparation  88  11.4.2. Preparation of riboprobe  88  11.4.3. Northern blotting and hybridization  89  11.5. Nuclei extract preparation and gel mobility shift assay  90  11.5.1. Nuclei extract preparation from myotubes  90  11.5.2. Preparation of oligonucleotides  90  11.5.3. Gel mobility shift assay  91  11.6. Statistical analyses III. Results  91 92  III. 1. Regulation of HSP70 and HSP27 expression by glutamine 111.2. Effect of glutamine on the abundance of hsplO mRNA 111.3. The D N A binding activity of HSF is not altered by glutamine  92 92 93  IV. Discussion  93  V . References  102  C H A P T E R SIX  G E N E R A L DISCUSSION A N D SUGGESTIONS F O R F U T U R E STUDIES  104  I. General Discussion  104  II. Conclusions  110  III. Suggestions for Future Studies  111  IV. References  113  IX  LIST O F FIGURES Fig. 1-1.  Glutamine synthesis pathway in skeletal muscle and degradation pathway in the kidney.  4  Fig. 1-2.  Initiation pathway of protein synthesis in eukaryotic cells.  11  Fig. 1-3.  A model for induction of heat shock proteins by stressors.  22  Fig.2-1. Effect of leucine on glutamate dehydrogenase activity in rat and chick skeletal muscle. Fig.2-2. Effect of isoleucine on glutamate dehydrogenase activity in rat and chick skeletal muscle.  44 45  Fig.2-3. Effects of valine and 2-oxoisocaproate on glutamate dehydrogenase activity in rat and chick skeletal muscle.  46  Fig.2-4. Additive effects of leucine and isoleucine on glutamate dehydrogenase activity in rat skeletal muscle.  47  Fig.3-1. Effect of metabolic acidosis on glutamate dehydrogenase activity in rat skeletal muscle.  59  Fig.3-2. Effect of metabolic acidosis on glutamine synthetase activity in rat skeletal muscle.  60  Fig.3-3. Regulation of glutamate dehydrogenase activity by the branched-chain amino acids in rat skeletal muscle during acidosis. 61 Fig.4-1. The rate of protein synthesis in normal-cultured and heat-stressed myotubes in the presence of various concentrations of glutamine.  76  Fig.4-2. Degradation of short-lived proteins and long-lived proteins in normalcultured myotubes.  77  Fig.4-3. The half-life of short-lived proteins in normal-cultured and heat-stressed myotubes in the presence of various concentrations of glutamine.  78  Fig.4-4. Effect of glutamine on the half-life of long-lived proteins in normalcultured and heat-stressed myotubes.  79  Fig.5-1. Glutamine increases HSP72 expression in heat-stressed L8 skeletal myotubes. 98 Fig.5-2. Expression of HSP27 is not regulated by glutamine in L8 skeletal myotubes.  99  X  Fig.5-3. Glutamine increases hsplO mRNA expression in heat-stressed L 8 skeletal myotubes.  100  Fig.5-4. The D N A binding activity of heat shock transcription factor is not regulated by glutamine in myotubes.  101  ABBREVIATIONS BCAA:  branched-chain amino acids.  BSA:  bovine serum albumin.  CBP:  C C A A T binding protein.  DMEM:  Dubbeco's modified Eagle medium.  DTT:  dithiothreitol.  EDTA:  ethylenediaminetetraacetic acid.  eIF-2a:  eukaryotic initiation factor 2a.  FBS:  fetal bovine serum.  GDH:  glutamate dehydrogenase.  GS:  glutamine synthetase.  HEPES:  A/-2-hydroxyethylpiperaxine-iV-2-ethanesulfonic acid.  HSE:  heat shock element.  HSF:  heat shock transcription factor.  HSPs:  heat shock proteins.  hsp:  heat shock gene.  NP-40:  Nonidet P-40.  PAGE:  polyacrylamide gel electrophoresis.  PBS:  phosphate buffered saline.  PMSF:  phenylmethylsulfonyl fluoride.  SDS:  sodium dodecyl sulfate.  TCA:  trichloroacetic acid.  Xll  ACKNOWLEDGMENTS I would like to thank my supervisor Dr. James R. Thompson for his support, encouragement, and guidance throughout my Ph.D study at The University of Alberta (1991-1992) and The University of British Columbia (1992-1996). With Dr. Thompson and his family, Edmonton was no more chilly, and Vancouver is no more rainy. I would also like to thank Dr. E . Peter M . Candido and Dr. George K. Iwama for serving on my Ph.D committee and sharing with me their expertise in molecular biology and particularly the biology of stress proteins. Last but not least, I must express my deepest appreciation to my parents and my wife Jennifer for their love, support, and understanding. Jennifer always shares with me my enjoyment and frustration, and shoulders the heavy burden of the whole family while I have to stay in the laboratory for countless evenings and weekends. I promise, Jennifer, next life I stay at home and you go to do a Ph.D.  1 CHAPTER ONE  General Introduction  This thesis addresses issues concerning the metabolism and physiological function of glutamine in skeletal muscle.  Glutamine is the most abundant amino acid in the free  amino acid pool in both skeletal muscle and blood.  It has a number of unique  physiological functions in addition to its contribution as a precursor for protein synthesis (1). The importance of glutamine in humans and animals under various pathophysiological conditions has been well documented (1,2).  In humans and various animals, dietary  protein provides only a small portion of daily glutamine requirements.  The majority of  glutamine is synthesized in the skeletal musculature and released into the bloodstream to meet requirements of other tissues. Regulation of glutamine metabolism in skeletal muscle is crucial to ensure that appropriate amount of this amino acid is released to meet the requirements of other tissues in the body (1). One prominent physiological function of glutamine is its regulation of protein turnover in skeletal muscle.  Skeletal muscle comprises the largest protein pool (about  50%) in the mammalian body. Regulation of protein turnover in this tissue determines, to a large extent, the status of protein deposition in the whole body as well as in skeletal muscle per se (i.e. the growth or atrophy of skeletal muscle). A general anabolic effect of glutamine on skeletal muscle protein has been reported both in vitro and in vivo (3,4,5,6). However, the mechanism(s) underlying this effect of glutamine is unclear and requires further study. A thorough understanding of the effect of glutamine on protein turnover in skeletal muscle has the potential to benefit human health and animal production.  2 In this chapter, glutamine and protein metabolism in skeletal muscle will be outlined.  The biology of heat-shock proteins will be briefly reviewed, because in this  research the effect of glutamine on heat-shock protein expression in muscle cells was studied in an effort to explore the mechanism underlying the anabolic effect of glutamine on skeletal muscle protein.  Finally, the research projects presented in this thesis will be  introduced.  I. Glutamine Metabolism Glutamine is the most abundant amino acid in the blood plasma and intramuscular free amino acid pools in mammalian and avian species (7,8,32).  Glutamine can be  provided from digestion of dietary protein, however, studies show that most dietary glutamine is metabolized in the enterocytes with very little released into the bloodstream (2).  The glutamine requirements of various tissues are largely met by its synthesis in and  release from skeletal muscle.  1.1. Glutamine metabolism in skeletal muscle. Glutamine accounts for less than 5% of the amino acids that comprise skeletal muscle proteins (9), but accounts for more than 20% of the free amino acids released from skeletal muscle into the circulation (1).  This indicates that a large portion of the released  glutamine must be synthesized within skeletal muscle. The intramuscular concentration of glutamine is about 20 m M , approximately 30 times that of plasma (10).  Considering its  3  mass, skeletal muscle appears to be the major site of glutamine synthesis as well as the largest free glutamine reservoir in humans and animals.  1.1.1.  Glutamine synthesis. Glutamine synthesis from glutamate and ammonia is  catalyzed by the enzyme glutamine synthetase (GS, E . C . 6.3.1.2.) (see Fig.l). The synthetic reaction reaches equilibrium in vitro when about 90% of the glutamate is converted to glutamine in a reaction system including L-glutamate, ammonium ions, M g , and A T P , each at a concentration of 10 mM, at pH 7.0 and 3 7 ° C (11). 2 +  As a key precursor for synthesis of glutamine, glutamate is formed from the tricarboxylic acid cycle (TCA cycle) intermediate a-ketoglutarate via either reductive amination or transamination.  Reductive amination is catalyzed by the enzyme glutamate  dehydrogenase (GDH, E . C . 1.4.1.3.) (see Fig.l). Regulation of G D H can alter the rate of production of glutamate thus influencing the rate of glutamine synthesis. dehydrogenase is located within the mitochondria (12).  Glutamate  Although the G D H reaction is  reversible, the equilibrium of the reaction in skeletal muscle strongly favors glutamate synthesis (13). Glutamate dehydrogenase has been shown to be subject to regulation by a number of factors.  In mitochondria isolated from the rat liver, G D H activity is increased  by the branched-chain amino acids (BCAA) of which leucine has the greatest effect (14). Glutamate dehydrogenase in rat islets of Langerhans has been shown to be activated by leucine and A D P and to be inhibited by magnesium (15). Tumor necrosis factor has an inhibitory effect on the activity of this enzyme in the rat liver (16).  Glutamate  dehydrogenase activity has recently been reported to be 68 % lower in brown adipose tissue from obese rats compared to normal rats (17). Regulation of G D H activity has also been  4  Skeletal muscle  Fig. 1-1. Glutamine synthesis pathway in skeletal muscle and degradation pathway in the kidney. In skeletal muscle, G D H catalyzes the conversion of a-ketoglutarate from the T C A cycle to glutamate. Glutamate is then converted to glutamine which is catalyzed by GS. The kidney is the major consumer of glutamine, in which glutamine is converted to a-ketoglutarate with production of two moles of N H . N H then combines with H with formation of N H which will be excreted with urine. Note the production of N H during glutamine metabolism in the kidney results in the removal of two moles of H from the body. 3  4  3  +  +  4  +  +  5  studied in purified enzyme preparations from bovine liver and brain (18,19).  However,  there is a paucity of information available regarding the activity and regulation of G D H in skeletal muscle.  The first objective of this thesis is to study the regulation of G D H in  skeletal muscle from either normal or stressed animals. In the pathway of glutamine synthesis, another site of regulation is the reaction catalyzed by GS.  Regulation of GS has been extensively studied.  The activity of this  enzyme can be regulated by hormones and glutamine by different mechanisms. skeletal muscle (20)  and cultured muscle cells (21),  dexamethasone and hydrocortisone-21-acetate. on GS activity can be blocked by androgens (22).  GS activity  In rat  is increased by  The stimulatory effect of glucocorticoids Studies have revealed that an increasing  transcriptional rate of the GS gene and an increase in the abundance of its mRNA are responsible for the effect of glucocorticoids on GS in skeletal muscle (23).  In addition, GS  activity can be regulated by glutamine. Removal of glutamine from culture media results in an increase in GS activity without altering GS mRNA levels in skeletal muscle cells. This upregulation of GS activity can be blocked by cycloheximide but not by actinomycin D (24), suggesting that regulation of GS activity by glutamine is at the post-transcriptional level. Regulation of glutamine production in skeletal muscle is critical during some pathophysiological states.  During metabolic acidosis, the glutamine requirements by the  kidney substantially increase (25).  Accordingly, GS activity, glutamine production, and  glutamine release by skeletal muscle are elevated during acidosis (26).  During catabolic  states, such as trauma, post-surgery, or sepsis, skeletal muscle releases glutamine at a high  6 rate, resulting in a substantial reduction (about 50%) of intramuscular free glutamine concentration  (10,27).  Interestingly,  chronic  exercise  can reduce  the effect of  glucocorticoids by attenuating the increase of GS mRNA and enzyme activity in skeletal muscle (29). 1.1.2. Glutamine degradation  Glutamine degradation was thought not to be  important in skeletal muscle due to negligible or complete absence of glutaminase in this tissue (30,31). tissue.  Recently, Wu et al. (32) reported extensive glutamine degradation in this  Two enzymes are responsible for the degradation of glutamine. One is glutamine  aminotransferase  located in the cytosol and the other is glutaminase located in the  mitochondrion. Glutamine aminotransferase catalyses transamination of glutamine to form a-ketoglutaramate, which can undergo deamidation catalyzed by co-amidase to form ccketoglutarate.  This sequence of reactions is responsible for degradation of glutamine in rat  skeletal muscle.  Glutaminase catalyses oxidative deamidation of glutamine with the  formation of glutamate.  This enzyme is responsible for degradation of glutamine in chick  skeletal muscle. The enzyme activity of both glutamine aminotransferase and glutaminase can be increased by increasing intramuscular concentrations of glutamine (32). 1.1.3.  Glutamine transport  The contribution of skeletal muscle to whole body  glutamine production depends not only upon its rate of intramuscular synthesis but also upon the rate of efflux of this amino acid from the muscle to the bloodstream. In fact, it has been proposed that the efflux process is the flux-generating step in the pathway by which skeletal muscle supplies glutamine to other tissues (33). A specific transport system for glutamine in skeletal muscle termed system N has been found, which is different from m  7 its counterpart in the liver, system N (34).  Although the gene for system N  m  has not been  cloned yet, its physiological features have been demonstrated by in vivo and in vitro studies. System N has a high affinity and high capacity for glutamine, and it seems to be m  one of the most rapid amino acid transport systems in the body. Two other amino acids, asparagine and histidine, competitively inhibit glutamine transport in skeletal muscle thus appearing to be substrates for system N as well. m  Glutamine efflux from skeletal muscle is increased by the synthetic glucocorticoid dexamethasone (35).  This may contribute to the elevated rate of glutamine release from  skeletal muscle during catabolic states (36).  1.2. Glutamine metabolism in other tissues Glutamine is metabolized in almost all tissues, and its metabolism plays different physiological roles in different tissues. 1.2.1. Glutamine metabolism in the kidney glutamine metabolism in the body.  The kidneys are a major site of  Circulating glutamine is taken up by the distal renal  tubular cells and degraded by glutaminase to produce ammonia (NH ) and glutamate. 3  Glutamate is further degraded into N H and a-ketoglutarate due to the action of renal 3  GDH.  It is important to note that unlike muscle G D H , renal G D H catalyzes the  deamination of glutamate.  Two mols of N H produced from one mole of glutamine 3  diffuses into the tubular lumen via the brush border membrane, and combines with protons to form ammonium ions ( N H ) which are readily excreted in the urine. In addition, for 4  +  each mol of glutamine metabolized, one mol of H C 0 " is produced tying up one more mole 3  8 of H . Therefore, in the kidney, degradation of one mol of glutamine will contribute to +  removal of three mols of H  +  (Fig.l). Because of this feature, glutamine metabolism in the  kidney becomes very important during metabolic acidosis, during which large amounts of H  +  accumulate in the bloodstream. 1.2.2. Glutamine metabolism in the liver  The liver is an organ which not only  consumes glutamine quantitatively but can also synthesize this amino acid at a high rate. This feature  is due to the heterogeneity  of glutamine metabolism in hepatocytes.  Glutaminase is located in the periportal hepatocytes, whereas glutamine synthetase is located in the perivenous hepatocytes (about 7% of all hepatocytes in an acinus) (37). As a result of the specific distribution of these enzymes in the liver, glutamine synthesis occurs in the perivenous hepatocytes hepatocytes.  while glutamine degradation occurs  in the periportal  The liver is a net consumer of glutamine under normal conditions (38).  There is a switch in glutamine metabolism in the liver in response to whole body glutamine requirements during certain pathophysiological states.  During the normal  postabsorptive state, the liver consumes a small amount of glutamine from the plasma pool. Short-term starvation, however, induces a switch from uptake to net release of glutamine from the liver of animals (39). Similarly, metabolic acidosis induces the liver to become a net glutamine-producer rather than a net glutamine-consumer (40). In both cases, more glutamine is utilized by other organs, particularly the gut and the kidney. 1.2.3. Glutamine metabolism in the gastrointestinal tract found to be the major respiratory fuel for enterocytes.  Glutamine has been  Nearly 40% of the C 0 produced in 2  the isolated or perfused rat jejunum preparation comes from oxidation of glutamine  9 (41,42). During the absorptive state, glutamine is taken up by the enterocytes from dietary protein; while during the postabsorptive state, a substantial amount of circulating glutamine (about 25%) has been shown to be extracted and oxidized by the rat small intestine (43). Two features facilitate glutamine's role to serve as a major energy source for enterocytes.  One is that the glutamine transport system in these cells has a high capacity  and high affinity for this amino acid (2). The other is that glutaminase activity is very high in enterocytes (2).  In addition to serving as an energy source, glutamine is also a  precursor for synthesis of purines and pyrimidines which are required in particularly large amounts for nucleic acid synthesis in the rapidly proliferating enterocytes.  1.2.4. Glutamine metabolism in immunocytes respiratory  fuel of immunocytes  including  Glutamine is an obligatory  lymphocytes and macrophages  (44,45).  Glutamine provides about 40% of the energy requirements of rat immunocytes (2). It has been shown that like enterocytes,  immunocytes have both a high capacity glutamine  transport system and high glutaminase activity (45).  The consumption of glutamine in  proliferating lymphocytes has been reported to increase (46). Like in enterocytes, in immunocytes glutamine is also a precursor for synthesis of purines and pyrimidines which are required in particularly large amounts for nucleic acid synthesis in the rapidly proliferating immunocytes. Glutamine has been suggested to be critical for proper function of lymphocytes during immune challenges (47).  n. Protein Turnover In Skeletal Muscle  10 Protein turnover is a dynamic process involving protein synthesis and protein degradation.  The balance between the rates of protein synthesis and protein degradation  within a tissue determines its protein mass.  Skeletal muscle constitutes about half the  protein in the whole body. Alterations in protein turnover in this tissue have a significant effect on whole body protein turnover and on metabolism in other tissues such as the gut and immune system.  n.l. Protein synthesis The pathways of protein synthesis in eukaryotic cells have been well studied (see Fig.2).  In the nucleus of eukaryotic cells, mRNA transcription starts when transcriptional  activators act on specific sequences in the promoter region of genes and subsequently R N A polymerase II binds with the promoter.  Before moving to the cytosol to serve as a  template for protein translation, newly synthesized mRNA undergoes splicing to remove the non-coding sequences (introns).  In the cytosol, the aminoacyl-tRNA synthetase  catalyzes covalent binding of amino acids to its specific tRNA with the formation of aminoacyl-tRNA.  Methionyl-tRNA binds to the eukaryotic initiation factor 2 (eIF-2),  consisting of eIF-2ct and eIF-2|3, and GTP forming a ternary complex, which then binds to the 40S ribosomal subunit to form a 43S subunit.  The binding of the 43S subunit to an  mRNA molecule results in formation of a 48S complex.  A protein molecule, called cap  binding protein, is necessary for the formation of 48S complex. Addition of a 60S ribosomal subunit to the 48S complex results in the formation of the 80S initiation complex. Following formation of the 80S ribosome, aminoacyl-tRNAs  11  —, I aminoacyl-tRNA  Met-tRNA  \ribosomty  ^  43 S preinitiation| complex  I  ribosorne)  ^ <^CIF-2^S)P)  <^IF-2P>  80 S initiation complex  Fig. 1-2 Initiation pathway of protein synthesis in eukaryotic cells. The initiation pathway starts with methionyl-tRNA binding to the complex of eIF-2 and G T P forming a ternary complex. The complex of eIF-2 and GDP is released during the formation of 80 S initiation complex. eIF-2 is to be recycled after an exchange of G D P with G T P .  12 bring the aminoacyl group to the ribosome and transfer it to the last amino acid residual in the growing polypeptide chain. As the ribosomes shift along the mRNA from the 5' to the 3'end, the polypeptide undergoes elongation. A number of elongation factors are required for the elongation process. the sequence of mRNA.  The elongation process stops when a stop codon is present in  The nascent polypeptides are then released from the ribosomes.  To be functional, the nascent polypeptides must be modified and folded through which they obtain a stable functional conformation. reticulum.  Most modifications occur in the endoplasmic  The modifications usually include phosphorylation, methylation,  attachment of oligosaccharides.  and/or  The folding process may require help from other factors  such as heat shock proteins (HSP) (49). The subunits of eIF-2 have been shown to be major sites of regulation of protein translation . For example, eIF-2a can be phosphorylated by a number of agents (e.g. heat shock or depletion of serum in cell culture), resulting in inability of conversion of elF2-GDP to eIF-2-GTP thus blocking the consecutive translation process (48) (see Fig.2).  II.2. Protein degradation Skeletal muscle contains multiple proteolytic pathways playing distinct roles in degradation of its constituent proteins. Based on the location of the proteases, they can be classified into lysosomal and nonlysosomal systems.  However, the relative contribution  that each proteolytic system makes to skeletal muscle protein degradation remains largely unknown.  13 II.2.1. Lysosomal system  In muscle cells,  cathepsins B, D , H , and L (50,51).  the lysosomal proteases include  The lysosome is a site for degradation of both  myofibrillar and non-myofibrillar proteins (52,53).  Before entering the lysosome,  myofibrils are disassembled to individual myofilaments (52). Once the proteins enter the lysosome, they are denatured by low pH and cleaved into polypeptides by cathepsins. It is unlikely that the lysosomal system makes a significant contribution to protein degradation in normal skeletal muscle.  n.2.2. Nonlysosomal systems n.2.2.1.  Ubiquitin pathway  The ubiquitin-dependent protein degradation  pathway constitutes a major pathway for protein degradation in eukaryotes.  It has been  well reviewed by Jentsch (55) and Ciechanover (126). Ubiquitin is a highly conserved and abundant cytosolic protein. It becomes linked to specific internal lysyl residues of target proteins by an isopeptide bond with the help of ubiquitin-conjugating enzymes. Subsequently,  the ubiquitin-protein conjugates  are degraded  by the proteasome  (multicatalytic proteinases, MCP). This process is energy dependent, requiring hydrolysis of A T P . The ubiquitin-dependent pathway has been identified in skeletal muscle (56). It is implicated in degradation of myofibrillar proteins (57). It has also been suggested to be involved in programmed cell death of insect muscle (127). II.2.2.2. Calcium-dependent pathway The Ca  -dependent proteolytic pathway 2+  exists widely in mammalian cells. It usually includes two types of proteases, a high Ca requiring type (m calpain) and a low Ca -requiring type (p. calpain). 2+  Both types are  14 present in skeletal muscle (57).  A third Ca -dependent protease, p94, has been identified 2+  and is thought to be specifically expressed in skeletal muscle (58).  This patheay is  involved in degradation of proteins making up the Z-disk (52) in addition to some sarcoplasmic proteins, such as protein kinase C (59).  II.3. Regulation of protein turnover in skeletal muscle A number of factors have been identified that are capable of regulating protein turnover in skeletal muscle in vivo and in vitro. Because of the mass of skeletal muscle in the body, these regulators play an important role in regulating energy homeostasis and body mass, and in adapting the organism to various pathophysiological conditions.  n.3.1. Effects of hormones n.3.1.1. Insulin  The effect of insulin on protein turnover in skeletal muscle has  been extensively studied both in vitro and in vivo.  In skeletal muscle preparations  incubated in vitro, insulin at physiological concentrations has been shown to stimulate protein synthesis (up to 100%) and inhibit protein degradation (up to 50%) (61).  In vivo,  insulin deficiency results in an inhibition of protein synthesis in rat skeletal muscle (65). Kimball and Jefferson (66) and Russo and Morgan (67) have suggested that insulin acts by increasing the initiation rate of protein synthesis. On the other hand, insulin deficiency has been shown to lead to an inhibition of translation initiation in skeletal muscle (68). In addition to its effect on protein synthesis, insulin also influences protein degradation in skeletal muscle.  By using cultured skeletal muscle cells, insulin has been  shown to inhibit the rate of degradation of long-lived proteins without affecting the rate of  15 degradation of short-lived proteins (62).  In vivo,  it has been shown that insulin at  physiologically high concentrations inhibits proteolysis in skeletal muscle in man (64). The mechanism underlying the effect of insulin on protein degradation includes a reduction in the number and size of autophagic vacuoles and an increase in the proportion of the latent form cathepsin D (69). Besides insulin, other hormones such as insulin-like growth factors -I and -II (IGF-I and IGF-II) and growth hormone have similar effects on protein turnover (62,63). Recently, it has been shown that IGF-I inhibits protein degradation in muscle cells by inducing autocatalytic inactivation of lysosomal cathepsins B and L (51).  n.3.1.2. Glucocorticoids In contrast to insulin, glucocorticoids can increase the rate of protein degradation in skeletal muscle in vivo (70,71).  Glucocorticoids increase  protein degradation in skeletal muscle by increasing expression of mRNA for the lysosomal proteases as well as for ubiquitin and proteasome subunits (72,73).  Because the plasma  level of glucocorticoids increases during stress, the effects of glucocorticoids are important contributors to the general catabolic state of protein metabolism in skeletal muscle during stress. H.3.2. Effects of amino acids  In addition to their role as substrates for  protein synthesis, two amino acids, leucine and glutamine, have also been reported to possess regulatory effects on protein synthesis and degradation in skeletal muscle. n.3.2.1.  Leucine  Leucine has been reported to stimulate protein synthesis and  inhibit protein degradation in skeletal muscle in vitro (78) and to stimulate protein synthesis in skeletal muscle in vivo (77).  Leucine's transamination product, a-ketoisocaproate (a-  16 KIC), has a similar effect on muscle protein degradation both in vitro and in vivo (78,79). The anabolic effect of leucine has led to studies on its possible application in clinical efforts to maintain muscle mass thus improving conditions of catabolic patients(80). II.3.2.2. Glutamine Glutamine was first shown to inhibit protein degradation in cultured skeletal muscle cells by Smith in 1985 (74). He demonstrated that physiological concentrations of glutamine can reduce the rate of protein degradation in muscle cells incubated for 30 h but not in muscle cells incubated for 2.5 h. In both the rat hindlimb perfusion preparation (3,4) and in the isolated chick skeletal muscle preparation incubated in vitro (6), glutamine has been reported to stimulate the rate of protein synthesis and inhibit the rate of protein degradation.  It has been demonstrated that in vivo, under a  variety of catabolic (stress) conditions, glutamine exerts an anabolic effect on skeletal muscle protein turnover thus preserving protein in the skeletal musculature.  For example,  a positive relationship has been demonstrated between the rate of muscle protein synthesis and the intramuscular concentration of glutamine in rats during starvation, consuming protein-deficient diets, and during endotoxemia (5).  Intravenous administration of  glutamine to surgical patients and to septic animals helps retain skeletal muscle glutamine concentration and minimize muscle protein loss.  Thus, glutamine appears to have an  anabolic effect on protein turnover in skeletal muscle (75,76). Although the anabolic effects of glutamine on protein turnover in skeletal muscle have been identified both in vitro and in vivo, little is known about the mechanisms underlying these effects.  Two mechanisms have been suggested in other tissues.  In  Ehrlich tumour cells, starvation of glutamine results in phosphorylation of the initiation  17 factor eIF-2a, which leads to inhibition of protein synthesis (48).  In rat hepatocytes,  glutamine has been shown to inhibit protein degradation through increasing cell volume thus alkalizing lysosomes (28).  Research designed to explore the effect of glutamine on  phosphorylation of initiation factors in translation warrants immediate attention.  It is  unlikely that glutamine affects protein metabolism in skeletal muscle through its action on lysosomes as this tissue contains few of these organelles. It is interesting to note that all in vivo experiments showing an anabolic effect of glutamine on muscle protein have been performed on stressed animals (5,118); in normal healthy animals, glutamine has been reported not to influence protein synthesis in skeletal muscle (e.g. 77).  In contrast to in vivo experiments, in vitro experiments clearly  demonstrate an anabolic effect of glutamine on muscle protein metabolism from normal animals (3,4,6). However, it must be noted that tissues used in those in vitro experiments (3,4,6) were subjected to a variety of stressors (e.g. surgery and hypoxia) (120,121).  It  remains unclear whether the effect of glutamine on protein turnover in skeletal muscle is a general phenomenon or is restricted to muscle exposed to stressors.  Addressing this  question is the second objective of this thesis. n.3.3  Effect of stressors  When cells are exposed to stressors, the rate of protein  synthesis is commonly decreased while the rate of protein degradation is increased (81). In stressed cells, several protein kinases, including the heme-regulated protein kinase (HRI), are activated, leading to phosphorylation of the initiation factors eIF-2oc, eIF-4B, and elF4E (84,85,86), and to the inhibition of protein synthesis. skeletal muscle cells share a similar mechanism.  It is not known yet whether  18 Although general protein synthesis is inhibited during stress, a family of heat-shock proteins (HSP) are selectively synthesized (81). The production of HSP has been shown to be quantitatively correlated with the degree of stress (82,83). It has been well documented that HSP. protect proteins  from stress-induced  denaturation and degradation  (81).  Overexpression of HSP70 in rat fibroblasts increases the rate of dephosphorylation of elF2a following heat-shock treatment, thus facilitating recovery of protein synthesis in these cells (119,122).  These results suggest an explanation for the observation that HSP  accumulation in cells is required before they recover from stress (82,83).  HI. Heat-Shock Proteins and Their Expression in Skeletal Muscle When cultured cells or whole organisms are exposed to temperatures a few degrees above their normal growth temperature, they respond by switching off the synthesis of most proteins and by initiating or increasing the synthesis of a small number of specific proteins, the HSPs.  The heat shock response was first noted in 1962 by Ritossa (87) who observed the  appearance of a new puffing pattern in Drosophila cell chromosomes in response to temperature shock.  The synthesis of HSPs was subsequently noted to coincide with the appearance of  chromosomal puffing (88).  In addition to heat shock,  other types of physical and chemical  stressors, e.g. hypoxia, heavy metals, and amino acid analogues, have subsequently been found to induce HSP synthesis (81).  Heat shock proteins are therefore also called stress proteins.  Some HSPs are expressed at a low level in cells under normal conditions and are involved in a chaperone-like function. These proteins are called constitutive HSPs.  19 Heat shock proteins are expressed in both prokaryotes and eukaryotes. The amino acid sequences of HSPs are highly conserved among species (60). In addition, the mechanism by which HSPs are induced is identical in most, if not all, species.  m.l. HSP classification Heat shock proteins are usually classified according to their molecular weight (60). In eukaryotic cells, HSPs are classified into four families. The HSP90 family contains HSP90 and glucose-regulated protein 94 (GRP94), which are found in the cytosol. contains GRP78, HSP73, and HSP72.  The HSP70 family  GRP78 is a mitochondrial membrane protein, while  HSP73 and HSP72 reside in the cytosol.  The HSP60 family is a group of proteins residing  within mitochondria and chloroplasts. The small HSP family consists of HSP32, HSP27, and HSP16.  The amino acid sequences of members of the small HSP family are less conserved  than those of other HSP families.  m.2. Role of HSPs as molecular chaperones One of the most prominent functions of HSPs is their role in protecting proteins from denaturation.  This role is referred to as molecular chaperoning.  As molecular chaperones,  HSPs help other proteins achieve proper folding and/or translocation into the appropriate cellular organelle.  It was noted a decade ago that abnormal proteins produced due to  incorporation of amino acid analogues or normal proteins released prematurely from the translation machinery can induce HSP expression (98,99). Heat shock protein production can also be induced by injection of denatured proteins into living cells (100). These observations,  20 along with others, led to investigations of the role of HSPs in cells which possess abnormal proteins.  Recently, it has been found that HSP70 associates with nascent polypeptides to  prevent abnormal aggregation of partially folded nascent polypeptides and to ensure their proper folding (92).  HSP70 has been implicated in the process of posttranslational translocation of  proteins into mitochondria and their subsequent refolding in this organelle (101). The HSP60 family, the so called "chaperonins", is a group of mitochondrial proteins that promote protein folding and assembly within mitochondria and protect mitochondrial proteins from thermal inactivation (94,102).  HSP90 has been found to bind to steroid hormone receptors in the  cytosol (103). Binding of HSP90 to hormone receptors maintains their high hormone affinity while making them unable to bind to D N A (90).  When the ligand (hormone) is available,  HSP90 is released from the HSP90-receptor complex, and the conformation of the receptor is changed to a form that has a high affinity for D N A (90,104). HSP27, a member of the small HSP family, is thought to participate in actin polymerization leading to stabilization of the intracellular microfilament network (96,97).  III.3 The mechanism underlying HSP induction In recent years, extensive research has led to the development of a model explaining how HSP are induced in stressed cells (105) (Fig.3). A transcription factor, called heat-shock transcription factor (HSF), is a key molecule mediating the HSP induction. The HSF is preexisting in non-stressed cells as a monomer without D N A binding activity.  When cells are  exposed to stressors, HSF is activated to form trimers and subsequently bind to a specific motif within the hsp gene promoter region called the heat-shock element (HSE).  The HSE is  21  Stressors (elevated temparature etc.)  Fig. 1-3 A model for the induction of HSP by stressors. Stressor activates HSF through yet unidentified mechanism(s). Activated HSF forms trimers and binds to H S E in the promoter region of hsp genes. HSF is phosphorylated and this phosphorylated form of HSF activates transcription of hsp genes. With increasing synthesis and accumulation of HSP, hsp gene transcription slows down and eventually stops. HSP70 may play a role in this negative feedback.  22 composed of contiguous alternating repeats of the 5-basepair sequence N G A A N (105). The DNA-bound HSF is then phosphorylated, a step necessary for the activation of hsp gene transcription (54). As a result of the activation of hsp gene transcription, HSP synthesis will be increased (Fig.3). There are, however, still a few questions in the model described above. For example, it is unclear what mechanism(s) links extracellular stressors to the activation of HSF.  For a  decade, HSP70 have been suggested to play such a role (105). As summarized by Morimoto (105), under normal conditions the constitutive level of HSP70 binds to the latent HSF monomer. Upon stress, the decrease of free HSP70 results in release of HSP70 from HSF, and the free HSF goes to form timers.  When sufficient HSP70 has been synthesized and  accumulated, it binds to HSF again and diminishes the D N A binding activity of HSF (Fig.3). Recently, the concept of HSP autoregulatory loop has been challenged by the workfromWu and his colleagues (125). They found that upon stress, HSP70 is still bound to HSF and this HSP70-HSF complex does not influence the DNA-binding activity of HSF. On the other hand, they found HSP70 may play a role in disassembly of HSF trimers (125). The molecular events linking stressor to activation of HSF are still to be characterized. It has to be pointed out that the DNA-binding activity of HSF does not always correlate with transcriptional activity (123). Moreover, increasing D N A binding activity of HSF may not activate the transcription of all hsp genes (124). In fact, in additiontoHSF, there are other transcription factors which may also mediate the induction of HSPs (105).  III.4 HSP expression in skeletal muscle  23 Like all eukaryotic cells, skeletal muscle cells express a number of HSPs, including inducible and constitutive forms.  Skeletal muscle is often subjected to stressors, e.g. high  temperature or low pH due to elevated rates of metabolism during exercise.  To date, seven  HSPs have been identified and studied in the skeletal musculature or in cultured muscle cells (Tabel 1-1).  Table 1-1 Heat-Shock Proteins Identified in Skeletal Muscle HSP  Source  References  HSP27 HSP31 GP46 HSP65 HSP73 * HSP72 GRP78  rat muscle rat L6 cell culture rat L6 cell culture rat muscle rat muscle rat muscle rat muscle  106 107 108 109 109 110 111  * constitutively expressed protein.  m.4.1 Skeletal muscle stress models A number of stress models have been employed to induce HSP expression in skeletal muscle in vivo and in vitro. Exercise is a useful in vivo model since it can result in elevated muscle temperature (43-44°C) (112) and quantitative production of oxygen radicals and other free radicals in muscle (109,113).  Heat shock (42°C for 2 h) and oxidative stressors (0 \ 2  H 0 , and O H ' generated by xanthine oxidase plus xanthine) have been used in in vitro skeletal 2  2  muscle incubation systems (114).  To introduce stress in cultured muscle cells, a number of  24 treatments, such as heat shock (43°C) (107), glucose deprivation, and intracellular acidification (111,115) have been used.  III.4.2 HSP expression in skeletal muscle Among the HSP families, HSP70 (HSP72/73) are the most abundant HSPs in all eukaryotic cells. HSP72 is a stress-inducible protein while HSP73 is constitutively expressed but its synthesis also increases upon stress. HSP70 expression has been shown to be induced in skeletal muscle by stressors. Following intermediate amounts of exercise, synthesis of HSP70 in rat skeletal muscle in vivo is significantly enhanced, and the amounts of these proteins are proportional to the extent of exercise (109). A corresponding increase in hsp gene transcription is also apparent with increased exercise.  For example, Salo et al. (114) reported that hsplQ  mRNA reaches its maximal level in the soleus, E D L , and plantaris muscles 1 h after exercise, and remains at a high level for several hours before returning to pre-exercise level.  It is  interesting to note the features of HSP expression in different types of muscle fibres. In type I fibres, both HSP73 and HSP72 are constitutively expressed. HSP73 has been shown to be constitutively expressed (110).  In type II fibres, however, only The significance of the fibre-  specific pattern of expression of HSP70 in skeletal muscle remains unknown. The small HSP family has also been identified in skeletal muscle cells.  Unlike other  families of HSPs, some of the small HSPs are expressed in a development-dependent manner. In cultured rat L6 myoblasts, hsp3l gene transcription is maximally induced after 2 h of heat shock or sodium arsenite treatment.  Once myocytes differentiate to myotubes, no HSP31 is  inducible by stressors (107). GP46, another member of the small HSP family, is constitutively  25 expressed in myoblasts.  However, in myotubes, its expression is no longer constitutive but  becomes stress-inducible (108). Skeletal muscle HSPs have been implicated in the protection of muscle cells from stress damage (116). This protection is likely mediated through the chaperoning effect of HSPs on cellular proteins.  For example, Eichler et al. (117) showed that acetylcholinesterase (AchE)  activity in primary chick muscle cultures drops sharply during heat shock (45 °C).  With the  accumulation of HSPs, the cells regain AchE activity which is not due to de novo synthesis of AchE.  IV. Objectives of This Thesis The objectives of this thesis are to study the regulation of the pathway of glutamine synthesis in skeletal muscle and to further characterize glutamine's role in skeletal muscle protein turnover. 1. to study the regulation of G D H by B C A A in skeletal muscle from both rats and chicks. 2. to investigate the effect of acidosis on G D H and GS activity and on the regulatory effect of B C A A on G D H activity. 3. to characterise the effect of glutamine on protein turnover in skeletal muscle under normal and stress conditions. 4. to explore the mechanism underlying the effect of glutamine on protein turnover in skeletal muscle.  26 References 1. Lacey, J . M . and Willmore, D . W . (1990) Is glutamine a conditionally essential amino acid? Nutr. Rev. 48, 297-309. 2. Souba, W . W . (1991) Glutamine: a key substrate for the splanchnic bed. Ann. Rev. Nutr. 11, 285-308. 3. MacLennan, P . A . , Brown, R . A . , and Rennie, M . J . 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M . , Myer, A . , Kosz, L . , Englestein, M . , and Muier, C . (1990) Activation of polyubiquitin gene expression during development programmed cell death. Neuron 5, 411-419.  37  CHAPTER TWO  Regulation of Glutamate Dehydrogenase by Branched-Chain Amino Acids in Rat and Chick Skeletal Muscle  I. Introduction Glutamate is an amino acid extensively metabolised in a number of tissues including the skeletal musculature.  It is the immediate precursor for synthesis of glutamine, an  amino acid produced and released in large amounts by the skeletal musculature with a number of important physiological functions throughout the body (1,2). Glutamate can be synthesized from -ketoglutarate via reactions catalyzed by either glutamate dehydrogenase (GDH, E . C . 1.4.1.3.) or the -ketoglutarate transaminases.  G D H is located exclusively  within mitochondria (3). Although the G D H reaction is reversible, the equilibrium of the reaction strongly favours glutamate synthesis (4).  G D H has been shown to be subject to  regulation by a number of factors. In mitochondria isolated from rat liver, G D H activity is increased by the branched-chain amino acids (BCAA) of which leucine has the greatest effect (5).  G D H in rat islets of Langerhans has been shown to be activated by A D P and  leucine and to be inhibited by magnesium (6).  Tumor necrosis factor has an inhibitory  effect on the activity of this enzyme in the rat liver (7).  In obese rats, G D H activity has  recently been reported to be reduced by 68% in brown adipose tissue (8).  Regulation of  G D H activity has also been studied in purified preparations from bovine liver and brain (9,10).  However, there is a paucity of information available regarding the activity and  regulation of G D H in skeletal muscle.  38 Skeletal muscle is a major site for B C A A metabolism (11).  In addition to serving  as an energy substrate for the T C A cycle, the B C A A exert regulatory effects on protein turnover and glucose metabolism in skeletal muscle (12,13).  The work in this chapter  comprises the study of whether B C A A also have a regulatory effect on G D H activity in skeletal muscle.  Given that there is a species difference in glutamine-degrading enzyme  activity between rat and chick skeletal muscles (14), we also studied the regulation of G D H by B C A A in both rat and chick skeletal muscles.  n . Materials and Methods n . l . Chemicals. P-NADH, P-NAD, a-ketoglutarate,  a-ketoisocaproate,  L-amino acids and other  chemicals were purchased from Sigma (St. Louis, USA).  n.2. Experimental Animals The experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care.  Male Sprague-Dawley rats (270-290 g) were fed rat chow  containing 24% (w/w) crude protein. Male broiler chicks of the Hubbard strain (2 wk old) were fed a standard chick diet containing 23 % (w/w) crude protein. Water was provided ad libitum to all animals.  II.2. Preparation of Mitochondria from Skeletal Muscle  39 The soleus muscle from rats and the sartorius muscle from chicks were carefully dissected while the animals were under mild halothane induced anaesthesia.  Following  removal of visible fat and connective tissue, muscles were gently minced with scissors in ice-cold 0.15 M KC1. Mitochondrial preparations were obtained by the method of Miller et al. (15) with minor modifications.  Briefly,  the finely minced muscles were  homogenized using a motor driven Teflon pestle homogenizer in isolation buffer containing 100 mM KC1, 50 m M Tris-HCl (pH 7.4), 5 mM M g C l , and 2 m M E G T A . 2  The  homogenate was centrifuged at 1,000 x g for 10 min at 2 ° C , and the resultant supernatant was recovered and centrifuged at 10,000 x g for 10 min at 2 ° C .  The pellet was  resuspended in the isolation buffer and disrupted using a Sonic 300 dismembrator at the level of 5 % for a total of 90 sec (three 30 sec intervals separated by 15 sec) in an ice bath. Ninety sec of sonication was found to result in optimal G D H activity. solution was used as the mitochondrial preparation.  The resultant  The protein concentration of  mitochondrial preparations averaged approximately 150 tig/ml when measured by the method of Bradford (16).  n.3. Measurement of Enzyme Activity G D H activity was analyzed in a Gilford 2600 spectrophotometer at 25 °C by measuring the change of absorbency at 340 nm, corresponding to the change of N A D H concentration in the reaction mixture. The reaction mixture, made up to 500 /xl in a semimicro cuvette, contained 50 mM triethanolamine (pH 7.5), 2.5 m M E D T A , 200 /xM N A D H , 100 mM NH C1, 7 mM a-ketoglutarate, 200 /xl of sample, and 250 /xl of the test 4  40 compounds (leucine, etc.) at various concentrations. The enzyme activity was measured as the initial reaction rate which was calculated using the millimolar extinction coefficient for N A D H of 6.22 cm" at 340 nm. 1  II.4. Statistical analyses The experimental results are given as means ± S E M .  The results were analyzed for  statistical significance by using one-way A N O V A and Student's t tests.  m . Results DLL  Effects of Leucine and Isoleucine on GDH Activity in Skeletal Muscle from  Normal Rats and Chicks G D H activity in the oxidative direction, i.e. deamination of glutamate, was found to be negligible in mitochondria from both rat and chick skeletal muscles in the presence or absence of B C A A (1-2 nmol/mg protein/min). The activity of G D H is therefore reported only for the direction of reductive amination, i.e. glutamate formation. The basal activities of G D H in skeletal muscles from rats and chicks were 62.5 ± 4.4 and 40.7 ± 3.3 nmol/mg protein/min, respectively (Figure 2-1). maximum of 45%  Leucine increased the activity of G D H by a  (P< 0.001) and 36% (P<0.05) in rat and chick skeletal muscles,  respectively. The maximum effect of leucine was obtained at a concentration of 1 mM for both species. The apparent Km for half-stimulation was 0.33 mM (Table 1) for rat muscle and 0.37 mM for chick muscle. Isoleucine stimulated the activity of G D H in rat muscle by  41 27% (P<0.05) at 1 mM and the apparent Km for half-stimulation was 0.41 mM (Table 1). Both leucine and isoleucine increased G D H activity in a dose-dependent manner. However, isoleucine did not significantly increase G D H activity in chick muscle (Figure 2-2). Valine did not have a significant effect (P>0.05) on G D H activity in skeletal muscle from either rats or chicks (Figure 2-3). To further investigate the characteristics of regulation of G D H by B C A A in skeletal muscle, the effect of a-ketoisocaproate,  the product of leucine transamination, was  examined. In contrast to leucine, increasing the concentration of a-ketoisocaproate from 0 to 5 mM did not affect rat muscle G D H activity (Figure 2-3). Animal age did not appear to affect G D H activity and its regulation by B C A A in skeletal muscle from rats. G D H activity in skeletal muscles from young rats (60 g) in the presence or absence of leucine or isoleucine was similar to that measured in muscles from adult rats (Table 2).  in.2.  Additive Effects of Leucine and Isoleucine on GDH Activity in Rat Skeletal  Muscle To determine if the effects of leucine and isoleucine on G D H activity in rat skeletal muscle are additive, leucine and isoleucine were added together in equal molar amounts to the reaction mixture and G D H activity was determined. Figure 2-4 shows that G D H activity was increased by a maximum of 81% in the presence of leucine plus isoleucine (P<0.001). The apparent Km for half-stimulation was 0.50 mM for the combination of equal molar amounts of leucine plus isoleucine (Table 1). The maximum additive effect  42 was achieved when both amino acids were present at 1 m M , the same concentration at which the amino acids individually had their maximum effect. The maximum increase in G D H activity measured in the presence of both amino acids (48.1 nmol/mg protein/min) was approximately the sum of the maximum increases in activity when each amino acid was added alone (28.0  and 18.9  nmol/mg protein/min by leucine and isoleucine,  respectively). These data suggest an additive effect of leucine and isoleucine on G D H activity in rat skeletal muscle.  IV. Discussion The present study provids the first evidence that B C A A can regulate G D H activity in skeletal musculature.  In skeletal muscle from rats, leucine and isoleucine both  significantly increased G D H activity. The maximum effect of leucine was greater than that of isoleucine (45% versus 27%).  The relative effects of these two amino acids on G D H  activity in rat skeletal muscle are similar to their relative effects on the enzyme in rat liver (5).  In the present study, we have also shown that the stimulatory effects of leucine and  isoleucine on G D H are additive, suggesting that the regulatory binding sites for these two amino acids differ, despite their structural similarity. Unlike leucine and isoleucine, valine did not increase G D H activity in rat skeletal muscle. Similarly, Aftring et al. (17) showed that leucine and isoleucine, but not valine, increased the activity of branched-chain a-keto acid dehydrogenase in vivo in rat skeletal muscle. The product of leucine transamination, a-ketoisocaproate, did not affect G D H activity (Figure 2-3), suggesting that the a-amino group is required for leucine's effect on this enzyme.  43 In the present work, it was found that the activity of skeletal muscle G D H and its regulation by B C A A were different between rats and chicks. The basal activity of G D H in rat soleus muscles was similar to that reported by Yoshino et al. (18). It was significantly higher (P<0.01) than the basal activity of G D H in chick sartorius muscles.  Leucine  stimulated G D H activity in skeletal muscles from both rats and chicks, whereas isoleucine increased the activity of this enzyme in rat muscles, but not in chick muscles (Figure 2-2). The  reasons for these differences are unknown.  Species differences in glutamine  metabolism between rat and chick skeletal muscle have been previously demonstrated by our laboratory (14), which showed that in rat skeletal muscle, glutamine degradation is catalyzed mainly by glutamine aminotransferase while in chick skeletal muscle it is catalyzed mainly by glutaminase. The activity of G D H in chick skeletal muscle during development and under different feeding conditions has been studied.  Its activity in chick leg muscle increases  during development in ovo, but does not significantly change after one day posthatch (19). Similarly, in the present work, we found that the activity of this enzyme in rat soleus muscle was not different in rats weighing 60 g or 280 g. Although G D H activity in chick skeletal muscle is not altered by a, number of factors such as starvation, high-protein feeding, or postnatal development (19,20), it is regulated by leucine.  This suggests that  leucine plays an important role in glutamate synthesis in skeletal muscle. In summary, this study demonstrated the regulation of G D H by B C A A in skeletal muscles from rats and chicks. This study provides new information on the regulation of the pathway of glutamine synthesis in skeletal muscles.  44  100 •En  90  • rH  >  • r-H -4-»  3 <D  6 e ~c  80  on  CD  '& rH  mat dehy  (nmol/mg  o o rH  CD  $  O  OH  70 60 50 40 30 20 0  5  6  Leucine (mM)  Figure 2-1 Effect of leucine on G D H activity in rat and chick skeletal muscle. Muscle mitochondria were prepared as described in the Materials and Methods. The reaction mixture contained: 50 mM triethanolamine (pH 7.5), 2.5 mM E D T A , 200 ttM N A D H , 100 mM ammonium acetate, 200 /xl sample, 250 /xl leucine at various concentrations as indicated, and 7 mM a-ketoglutarate which was added to start the reaction. The reaction was performed at 2 5 ° C . The initial velocity was recorded after 2 min. Data are means ± S E M with n=5 or 6 muscles. • : rat skeletal muscle; •: chick skeletal muscle, a: P<0.001 in comparison with 0 mM leucine; b: P<0.05 in comparison with 0.25 mM leucine; c: P<0.05 in comparison with 0 mM leucine.  45  0  2  3  4  5  Isoleucine (mM)  Figure 2-2  Effect of isoleucine on G D H activity in rat and chick skeletal muscle.  The reaction conditions were the same as those in Figure 1 with isoleucine replacing leucine.  Data are means ± S E M with n=5 or 6 muscles. The legends are the  same as in Figure 1. a: P<0.05 in comparison with 0 mM leucine.  46  CD CO  § .s cu  53  o 2 CD  E  o  6  8  6 C3  =J  O  0  4  5  Amino acids (mM)  Figure 2-3 Effects of valine and a-ketoisocaproate on G D H activity in rat and chick skeletal muscle. The reaction conditions were the same as those in Figure 1 with valine or a-ketoisocaproate replacing leucine. Data are means ± S E M with n=5 or 6 muscles; • : valine on rat muscle; •:2-oxoisocaproate on rat muscle; o: valine on chick muscle.  47  0  1 Leucine and isoleucine (mM)  Figure 2-4 Additive effects of leucine and isoleucine on G D H activity in rat skeletal muscle. The reaction conditions were the same as those in Figure 1 except that equal molar amounts of leucine plus isoleucine were present in the reaction mixture. The values on the x-axis represent the concentration of each individual amino acid present in the reaction mixture. Data are means ± S E M with n=5 or 6 muscles, a and b: P<0.05 and P < 0.001, respectively, in comparison with 0 mM amino acids; c: P<0.01 in comparison with 0.125 mM amino acids.  48  Apparent kinetic parameters of activation of glutamate dehydrogenase in rat skeletal muscle by leucine and isoleucine  Table 1  Activator  Apparent Km (mM)  leucine  0.33 ± 0.03  90.45 ±  4.31  isoleucine  0.41 ± 0.16  79.16 ±  3.49  leucine-l- isoleucine  0.50 ± 0.13  106.71 ± 3.53  Apparent Vmax (nmol/mg protein/min)  1. The enzyme assay is described in the Materials and Methods. The initial velocity was recorded 2 min after addition of a-ketoglutarate and rapidly mixing the reaction mixture. Km was calculated according to the concentration of leucine or isoleucine or both of them at which the one-half maximum activation was achieved. Vmax was calculated according to the maximum activation achieved by leucine or isoleucine or both of them. 2. Each value is the mean ± S E M , n=5 or 6 muscles.  Glutamate dehydrogenase activity in skeletal muscle from young and old rats.  Table 2.  Basal  1 mM leucine  1 mM isoleucine  young rats (60 g)  68.3 + 3.9  89.2 ± 3.6  80.8 ± 4 . 7  old rats (270-290 g)  62.5 + 4.4  87.7 ± 2 . 2  79.1 ± 3 . 5  1. The enzyme assay is described in the Materials and Methods. The initial velocity was recorded 2 min after addition of a-ketoglutarate and rapidly mixing the reaction mixture. The enzyme activity is expressed as nmol/mg protein/min. 2. Each value is the mean ± S E M , n=5 or 6 muscles.  50 V.  References  1. Windmueller, H . G . , and Spaeth, A . E . (1975) Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from blood. Arch. Biochem. Biophys. 171, 662-672. 2. Krebs, H . (1980) Glutamine metabolism in the animal body. In Glutamine: Metabolism, Enzymology and Regulation (Edited by Mora J. and Palacios R.), pp.319-329. Academic Press, New York. 3. Smith, E . L . , Austen, B . M . , Blumenthal, K . M . , and Nyc, J.F. (1975) Glutamate dehydrogenase. In The Enzymes (Edited by Boyer, P.D.), V o l . 11, pp293-267. Academic Press, New York. 4. Hudson, R . C . and Daniel, R . M . (1993) L-GDHs: mechanism. Comp. Biochem. Physiol. 1 0 6 B , 767-792.  distribution, properties and  5. McGiven, J . D . , Bradford, N . M . , Crompton, M . , and Chappell J.B. (1973) Effect of L-leucine on the nitrogen metabolism of isolated rat liver mitochondria. Biochem. J. 134, 209-215. 6. Bryla, J., Michalik, M . , Nelson, J., and Erecinska, M . (1994) Regulation of the G D H activity in rat islets of Langerhans and its consequence on insulin release. Metabolism 43,1187-1195. 7. Yasmineh, W . G . , Tsai, M . Y . , and Theologides, A . (1995) Hepatic mitochondrial enzyme activity and serum amino acid composition in rats treated with tumor necrosis factor. Life Sci. 5 6 , 621-627. 8. Serra, F . , Gianotti, M . , Pons, A . , and Palou A . (1994) Brown and white adipose tissue adaptive enzymatic changes on amino acid metabolism in persistent dietary-obese rats. Biochem. M o l . Biol. Intl. 3 2 , 1173-1178. 9.  Bailey, J., Bell,  E . T . , and Bell, J.E. (1982) Regulation of bovine glutamate  dehydrogenase. J. Biol. Chem. 2 5 7 , 5579-6683. 10. Wrzeszczynski, K . O . and Colman, R . F . (1994) Activation of bovine liver glutamate dehydrogenase by covalent reaction of adenosine 5-0-[S-(4-bromo-2,3-dioxobytyl) thiophosphate] with arginine-459 at an A D P regulatory site. Biochemistry 3 3 , 1154411553. 11.  Goldberg, A . L . and Odessey, R. (1972) Oxidation of amino acids by diaphragms  from fed and fasted rats. A m . J. Physiol. 2 2 3 , 1384-1391.  51 12. Chang, T . C . and Goldberg, A . L . (1978) Leucine inhibits oxidation of glucose and pyruvate in skeletal muscles during fasting. J. Biol. Chem. 2 5 3 , 3696-3701. 13. Goldberg, A . L . and Chang, T . W . (1978) Regulation and significance of amino acid metabolism in skeletal muscle. Fed. Proc. 3 7 , 2301-2307. 14. Wu, G . , Thompson, J.R., and Baracos, V . E . (1991) Glutamine metabolism in skeletal muscles from the broiler chick (Gallus domesticus) and the laboratory rat (Rattus norvegicus). Biochem. J. 274, 769-774. 15. Miller, R . H . , Eisenstein, R.S., and Harper, A . E . (1988) Effect of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. J. Biol. Chem. 2 6 3 , 3454-3461. 16. Bradford, M . (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protective binding. Anal. Biochem. 7 2 , 248254. 17.  Aftring, P.R., Block, K . P . , and Buse, M . G . (1986) Leucine and isoleucine activate  skeletal muscle a-keto acid dehydrogenase in vivo. A m . J. Physiol. 2 5 0 , E599-E604. 18. Yoshino, M . , Kato, K . , Murakami, K . , Katsumata, Y . , Tanaka, M . , and Mori, S. (1990) Shift of anaerobic to aerobic metabolism in rats acclimatized to hypoxia. Comp. Biochem. Physiol. 9 7 A , 341-344. 19. Garcia-Palmer, F.J., Pons, A . , and Palou, A . (1985) Patterns of amino acid enzyme in domestic fowl and leg muscle during development. Comp. Biochem. Physiol. 8 2 B , 143146. 20. Pons, A . , Garcia, F.J., Palou, A . , and Alemany, M . (1986) Effect of starvation and a protein diet on the amino acid metabolism enzyme activities of the organs of domestic fowl hatchlings. Comp. Biochem. Physiol. 8 5 B , 275-278.  52  CHAPTER THREE  Regulation of Enzymes for Glutamine Synthesis in Rat Skeletal Muscle During Metabolic Acidosis  I. Introduction In humans and animals, the kidney is a major organ that consumes glutamine. The pathway for metabolism of glutamine in the kidney includes deamination of glutamine to glutamate followed by conversion to a-ketoglutarate.  These two reactions generate  ammonia which binds H , a process facilitating the removal of H +  +  from the body. During  acidosis, there is a substantial increase in the flux through the glutamine degradation pathway in the kidney, which is critical for the clearance of H equilibrium (1).  +  so as to restore acid-base  As a result, the renal extraction of glutamine from the blood increases.  The increase in renal extraction has been shown to be correlated with an increase in the plasma concentration of glutamine (2). Skeletal muscle is a major source of circulating glutamine. It has been shown that during acidosis skeletal muscle releases elevated amounts of glutamine to the bloodstream (3).  This is accomplished by an increase in the activity of glutamine synthetase (GS) in  skeletal muscle which leads to a decrease in intramuscular concentrations of both glutamate and a-ketoglutarate (3,4). skeletal muscle.  Glutamate is an essential substrate for glutamine synthesis in  The synthesis of glutamate in this tissue via glutamate dehydrogenase  (GDH) was shown in the preceding chapter to be regulated by the branched-chain amino acids (BCAA).  However, it remains unknown how glutamate dehydrogenase responds to  the challenge during acidosis which requires more glutamine to be produced by skeletal muscle, and whether the regulation of G D H is correlated with the up-regulation of  53 glutamine synthesis in skeletal muscle during acidosis. It was hypothesized that similar to the activity of GS, the activity of G D H in skeletal muscle will increase in responding to acidosis. The specific aim of this study was to characterize the regulation of G D H activity by B C A A and to determine if the regulation of G D H is correlated with the regulation of glutamine synthetase in skeletal muscle during metabolic acidosis.  11. Materials and Methods n . l . Chemicals p - N A D H , P-NAD, a-ketoglutarate, a-ketoisocaproate,  L-amino acids and other  chemicals were purchased from Sigma (St. Louis, USA). AG1-X8 and BioRex 70 ( H ) +  were purchased from Bio-Rad Laboratories (Richmond, C A , USA).  n.2. Experimental Animals The experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care.  Male Sprague-Dawley rats (270-290 g) were fed rat chow  containing 24% (w/w) crude protein. A standard procedure was used to induce chronic metabolic acidosis by providing 1.5% NH C1 (w/v) as the sole source of drinking water for 4  2 or 5 d (5).  During recovery from metabolic acidosis, rats were provided access to water  ad libitum.  II.3. Preparation of Muscle Samples for Enzyme Assay  54 The soleus muscles from rats were carefully dissected while the animals were under mild halothane induced anaesthesia.  Following removal of visible fat and connective  tissue, muscles were gently minced with scissors in ice-cold 0.15 M KC1.  The finely  minced muscles were homogenized using a motor driven Teflon pestle homogenizer in isolation buffer containing 100 mM KC1, 50 mM Tris-HCl (pH 7.4), 5 mM M g C l , and 2 2  mM E G T A .  The homogenate was centrifuged at 1,000 x g for 10 min at 2 ° C . The  resultant supernatant was recovered.  One fourth of the supernatant was used for  measurement of GS activity. The remaining supernatant was centrifuged at 10,000 x g for 10 min at 2 ° C . The pellet was resuspended in the isolation buffer and disrupted using a Sonic 300 dismembrator at a level of 5% for a total of 90 sec (three 30 sec intervals separated by 15 sec) in an ice bath. optimal G D H activity.  Ninety seconds of sonication was found to result in  The resultant solution was used as the mitochondrial preparation  for measurement of G D H activity.  The protein concentration in each preparation was  determined by the method of Bradford (7).  n.4. Measurement of G D H Activity G D H activity was analyzed in a Gilford 2600 spectrophotometer at 25 °C by measuring the change of absorbance at 340 nm, corresponding to the change of N A D H concentration in the reaction mixture. The reaction mixture, made up to 500 fil in a semimicro cuvette, contained 50 mM triethanolamine (pH 7.5), 2.5 m M E D T A , 200 /xM N A D H , 100 mM NH C1, 7 mM a-ketoglutarate, 200 /xl of sample (30 fig protein), and 4  250 /xl of the test compounds (leucine, etc.) at various concentrations. The enzyme activity  55 was measured as the initial reaction rate which was calculated using the millimolar extinction coefficient for N A D H of 6.22 cm" at 340 nm. The enzyme activity is expressed 1  as nanomoles of N A D H converted per mg protein per min.  n.5. Measurement of GS activity Glutamine synthetase activity was determined by following the conversion of [ C]14  glutamate to [ C]-glutamine as described by Falduto et al. (8).  The reaction mixture,  14  containing 50 /xl of sample (approximately 50 /xg protein), 10 mM glutamate, 0.25 /xCi L [U- C]-glutamate, 50 mM imidazole-HCl (pH 7.2), 15 mM M g C l , 10 mM A T P , 4 mM 14  2  NH C1, and 1 mM 2-mercaptoethanol in a final volume of 100 /xl, was incubated at 3 7 ° C 4  for 30 min. The reaction was stopped by adding 1 ml of ice-cold water and placed on ice. The [ C]-glutamine formed was separated from [ C]-glutamate by passing the reaction 14  14  mixture through AG1-X8 (acetate form) column (6 ml) fitted on the top of a BioRex 70 ( H ) column (6 ml). The columns were eluted with 6 ml water. +  eluates associated with glutamine was measured.  Radioactivity of the  The enzyme activity is expressed as  nanomoles of glutamine formed per mg protein per min.  II.6. Statistical analysis The experimental results are given as mean ± S E M .  The results were analyzed for  statistical significance by using one-way A N O V A and Student's t test.  m . Results  56  m . l . Effect of acidosis on GDH and GS activity in rat skeletal muscle Acidosis increased the activity of G D H in skeletal muscle from rats.  In skeletal  muscle from 2-d and 5-d acidotic rats, the activity of G D H increased 68% (P<0.05) and 88% (P<0.01), respectively. levels (Figure 3-1). rats.  After 2 d of recovery, G D H activity returned to control  The activity of GS in skeletal muscle was also increased in acidotic  In skeletal muscle from 2-d acidotic rats, GS activity increased 23%.  muscle from 5-d acidotic rats, the activity of GS increased 66% (P<0.01).  In skeletal  Unlike G D H ,  GS activity remained significantly higher in muscle after 2 d of recovery than in the control muscle (P<0.05), though it was significantly lower than that in muscles from 5-d acidotic rats (P < 0.05) (Figure 3-2).  rn.2. Regulation of GDH activity by BCAA in skeletal muscle during acidosis Of the three B C A A , leucine and isoleucine have been shown in the preceding chapter to regulate the activity of G D H in skeletal muscle from normal rats.  In acidotic  rats, the regulatory effects of leucine and isoleucine on G D H were retained in skeletal muscle from 5-d acidotic rats (Figure 3-3).  Leucine increased G D H activity by 86%  (P<0.001), while isoleucine increased its activity by 55% (P<0.01).  The concentrations  of leucine and isoleucine at which maximum stimulatory effect on G D H activity was achieved shifted from 1 mM in the normal rats to 2 mM in the acidotic rats. Interestingly, unlike the situation in normal rats, valine significantly increased G D H activity (maximally 33% at 1 m M , P<0.05) in skeletal muscle from acidotic rats (Figure 3-3).  57  rv. DISCUSSION Chronic metabolic acidosis is well known to increase glutaminase (9) and G D H oxidative deamination activities (10) leading to increased production of N H in the kidney, which facilitates a reduction in whole body H  +  4  +  and H C 0  concentrations.  3  During  chronic acidosis, the activity of GS'has been shown to increase in the skeletal musculature resulting in the release of large amounts of glutamine to the circulation (11).  One would  anticipate that reactions catalysed by enzymes such as G D H would also increase during acidosis to provide substrate for glutamine synthesis. In the present work, we demonstrate for the first time that chronic acidosis results in an increase in G D H activity in skeletal muscle.  The increase in G D H activity is coordinated with an increase in GS activity in  skeletal muscle.  This coordination is of physiological importance, because it has been  shown that the intramuscular concentration of glutamate decreases during acidosis which could result in a decrease in glutamine synthesis.  (3),  However, with the increase in  G D H activity, more glutamate can be produced to meet the requirement of an increasing rate of glutamine synthesis resulting from the increase of GS activity. In the preceding chapter, we found that the activity of G D H in skeletal muscle increases in response to increasing concentrations of leucine and isoleucine but not valine. In this chapter, we show that during metabolic acidosis the increase in skeletal muscle G D H activity in response to elevated concentrations of leucine or isoleucine is greater than in normal rats. In addition, it is interesting to find that G D H activity increased in response to increased concentrations of valine in skeletal muscles from acidotic rats.  While the  mechanism underlying this phenomenon is unknown, it may involve the change in the  58 conformation of G D H during acidosis which might then increase the binding of valine to this enzyme and subsequently activation of the activity of this enzyme.  These data  demonstrate that metabolic acidosis increases skeletal muscle G D H activity as well as the sensitivity of this enzyme to all three B C A A . In summary, the present study demonstrates a coordinated response involving G D H and GS in rat skeletal muscle during metabolic acidosis to provide additional circulating glutamine.  It also shows that the regulation and sensitivity of G D H to B C A A increases  during acidosis.  These changes  are of physiological importance to the animal's  coordinated interorgan response to decrease elevated concentrations metabolic acidosis.  of H  +  during  59  Figure 3-1. Effect of metabolic acidosis on glutamate dehydrogenase activity in rat skeletal muscle. Rats were fed 1.5 % NH C1 to induce metabolic acidosis for 2 or 5 d as described in the Materials and Methods. One group of rats was allowed to recover from metabolic acidosis for 2 d. Data are means ± S E M with n=5 or 6 rats, a and b: P<0.05 and P<0.01, respectively, in comparison with 0-d (control) or 2-d recovery. 4  60  water  NH4 Cl  I  0 Days  Figure 3-2. Effect of metabolic acidosis on glutamine synthetase activity in rat skeletal muscle. Rats were treated the same as in the Figure 3-3-1. Data are means ± S E M with n=5 or 6 rats, a and b: P<0.05 and P<0.01, respectively, in comparison with 0-d (control).  61  270  0 Amino acids (mM)  Figure 3-3. Regulation of glutamate dehydrogenase activity by branched-chain amino acids in rat skeletal muscle during metabolic acidosis. The reaction mixture was the same as in Figure 3-3-1 containing leucine (•), isoleucine (•), or valine (o). Data are means ± S E M with n=5 or 6 muscles, a, b, and c: P<0.05, P<0.01, and P<0.001, respectively, in comparison with 0 mM branched-chain amino acids.  62 V.  References  1. Tannen, R . L . and Sastrasinh, S. (1984) Response of ammonia metabolism to acute acidosis. Kidney Int. 25, 1-10. 2. Hughey, R.P., Rankin, B.B. and Curthoys, N.P. (1980) Acute acidosis and renal arteriovenous differences of glutamine in normal and adrenalectomized rats. A m . J. Physiol. 238, F199-F204. 3. Goldstein, L . , Perlman, D . F . , McLaughlin, P . M . , King, P . A . and Cha, C.J. (1983) Muscle glutamine production in diabetic ketoacidotic rats. Biochem. J. 214, 757-767. 4. King, P . A . , Goldstein, L . and Newsholme, E . A . (1983) Glutamine synthetase activity of muscle in acidosis. Biochem. J. 216, 523-525. 5. Hwang, J.J. and Curthoys, N.P. (1991) Effect of acute alterations in acid-base balance on rat renal glutaminase and phosphoenolpyruvate carboxykinase gene expression. J. Biol. Chem. 266, 9392-9396. 6. Miller, R . H . , Eisenstein, R.S. and Harper, A . E . (1988) Effect of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. J. Biol. Chem. 263, 34543461. 7. Bradford, M . (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protective binding. Anal. Biochem. 72, 248254. 8. Falduto, M . T . , Young, A . P . and Hichson, R . C . (1992) Exercise inhibits glucocorticoid-induced glutamine synthetase expression in red skeletal muscles. A m . J. Physiol. 262, C214-C220. 9. Welbourne T . C , Phromphetcharat V . , Givens G . and Joshi S. (1986) Regulation of interorganal glutamine flow in metabolic acidosis. A m . J.Physiol. 250,F457-F463. 10. Wright P. A . and Knepper M . A . (1990) Glutamate dehydrogenase activities in microdissected rat nephron segments: effects of acid-base loading. A m . J. Physiol. 259, F53-F59. 11. Welbourne T . C . (1986) Effect of metabolic acidosis on hindquarter glutamine and alanine release. Metabolism 35, 614-618.  CHAPTER FOUR  Regulation Of Protein Turnover In Stressed And Normal-  63  Cultured Rat Skeletal Myotubes  I. Introduction Protein in skeletal muscle undergoes continuous synthesis and degradation, or protein turnover, which is precisely regulated. This regulation not only determines protein homeostasis in skeletal muscle per se, but also influences energy metabolism and the physiological status of the whole body. A number of factors, including amino acids (1), hormones (2,3), and physical stretch (4), have been identified to have regulatory effects on protein turnover in skeletal muscle. The amino acid glutamine has been reported to regulate protein turnover in skeletal muscle.  In rats under stressful conditions (e.g. endotoxemia or starvation), a positive  correlation has been observed between the concentration of glutamine and the rate of protein synthesis in skeletal muscle (5). Similar results have been demonstrated in humans during sepsis or following surgery or trauma, conditions during which the intramuscular free glutamine pool is depleted (6,7,8).  Parenteral administration of glutamine to these  patients can increase the rate of protein synthesis, thus improving the nitrogen balance in skeletal muscle (7).  However, experiments with normal subjects have failed to show a  stimulatory effect of glutamine on the rate of protein synthesis in skeletal muscle (10,11,12). The latter observations are in contrast to results obtained with in vitro skeletal muscle preparations from normal animals.  For example, results obtained from rat  hindlimb perfusion preparations or isolated chick skeletal muscle preparations have  demonstrated that glutamine stimulates the rate of protein synthesis in these tissues from normal animals in a concentration-dependent manner (13,14).  64  Therefore, it is unclear  whether the effect of glutamine on protein synthesis in skeletal muscle is a general phenomenon or a stress-dependent phenomenon. In addition to its effect on protein synthesis, glutamine has also been demonstrated to regulate the rate of protein degradation in skeletal muscle.  In humans subjected to  stressors, supplementation of glutamine decreases urinary excretion of 3-methylhistidine, an index of myofibrillar protein degradation (15). In rat hindlimb perfusion (1) and chick skeletal muscle incubation preparations (14), glutamine has also been found to decrease the rate of protein degradation. However, again, it is unclear whether the effect of glutamine on protein degradation in skeletal muscle is a general or stress-associated phenomenon. Furthermore, different protein fractions, i.e. the short-lived proteins and the long-lived proteins, in skeletal muscle have different responses to regulators of protein turnover such as insulin and insulin-like growth factor (IGF-1) (16,17). It remains unknown if glutamine decreases the rate of degradation of short-lived and/or long-lived skeletal muscle protein fraction. To address these questions, we set out to characterize the effect of glutamine on protein turnover in skeletal muscle under normal and stress conditions using L8 skeletal myotubes differentiated from the L8 myoblast cell line derived from rat skeletal muscle. This type of myotube expresses most of the muscle specific proteins (18) and has been previously used as a model for studies of protein metabolism in skeletal muscle (2,17). A n advantage of using the myotube culture system is that the experimental conditions can be  clearly controlled, and the cultures can be used for prolonged experimental periods  65  without many of the problems encountered with hindlimb perfusion and whole muscle incubation preparations (e.g. surgical stress and hypoxia (19)).  n . Materials and Methods n . l . Materials The L8 skeletal muscle cell line was purchased from America Type Culture Collection (Rockville, U . S . A . ) .  Dulbecco's modified Eagle's medium (DMEM), fetal  bovine serum (FBS), dialyzed horse serum (HS),  L-tyrosine, and antibiotics were  purchased from Life Technologies (Burlington, Canada). L-[rcng-3,5- H]-L-tyrosine 3  Ci/mmol) was purchased  from DuPont N E N (Markham, Canada).  (46  L-glutamine,  bicinchoninic acid (BCA) protein assay kit, and all other chemicals were purchased from Sigma (St. Louis, USA).  H.2. Cell culture L8 cells were grown in 6-well tissue culture plates in D M E M supplemented with 3 mM  glutamine,  10% FBS and antibiotics  (100  U/ml penicillin  and 100 mg/ml  streptomycin) (growth medium) at 3 7 ° C in a humidified atmosphere of air/C0 v/v).  2  (19:1,  When cells reached about 70% confluence, they were subcultured onto six-well  tissue culture plates (35 mm /well). When cells reached 80% confluence, the medium was 2  replaced with differential medium ( D M E M containing 4% HS and the same concentrations of glutamine and antibiotics as the growth medium).  The cells were grown for an  additional 3 to 4 d to obtain maximum fusion (about 90%).  The media for all cultures  66  were replaced with glutamine-free growth media for 2 h in an effort to bring the intracellular glutamine concentrations for all cultures to a similar level before onset of treatments. Glutamine solutions were freshly prepared and added to culture media immediately before use in each experiment.  II.3. Measurement of the rate of protein synthesis The rate of protein synthesis was measured by determining the rate of incorporation of  [ H]tyrosine into the trichloroacetic acid (TCA)-insoluble materials as described by  Gulve and Dice (2) with minor modifications to the procedures used for harvesting the samples. The cultures were incubated with experimental media (growth media containing 0, 0.65, 5, 10, or 15 mM glutamine) for various periods of time. One hour before the end of each incubation period, the media were replaced with fresh experimental media containing 1 mM L-[ H]tyrosine (2 itCi/ml). 3  After 1-h incubation with the radioactive  experimental media, the cultures were washed four times with ice-cold phosphate buffered saline containing 2 mM L-tyrosine (PBS-tyr). The cultures were then fixed with 1 ml 15% T C A for 20 min on ice and washed twice for 5 min each with 15% T C A on ice. Following washing with T C A , the cultures were washed with ice-cold 100% ethanol twice for 3 min and once for 5 min. The cultures were briefly air dried and then lysed with 300 ml of lysis buffer (2% SDS, 40 mM Tris (pH 7.4), 1 mM E D T A , 10 mg/ml leupeptin, and 100 mM phenylmethylsulfonyl fluoride (PMSF)). The lysates were incubated at 7 0 ° C for  5 min and stored at - 7 0 ° C . This procedure reduced the amount of radioactivity from free [ H]-tyrosine to less than 0.5% of total radioactivity in the acid-insoluble material. 3  this procedure was found to minimize protein loss from the preparation.  67  Also, Protein  concentrations in the lysate were measured using the B C A method (15) with bovine serum albumin (BSA) as the standard. Radioactivity in the lysate was measured using a Beckman LS6500 liquid scintillation counter. The rate of protein synthesis is expressed as the amount of tyrosine incorporated per mg of protein per h.  n.4. Measurement of the rate of protein degradation The rate of protein degradation was measured by determining the rate of release of TCA-soluble radioactivity into the culture media during various periods of time by myotubes prelabelled with [ H]tyrosine. 3  For measurement of the rate of degradation of  short-lived proteins, the cultures were labelled with [ H]tyrosine (3 iiCi/ml D M E M ) for 3  1.5 h.  The cultures were then washed with PBS-tyr and incubated in the growth media  containing 2 mM L-tyrosine for 10 min to remove the effect of degradation of very shortlived proteins (2).  The cultures were washed twice with PBS-tyr and then incubated in the  experimental media containing 0, 0.65, 5, 10, or 15 mM glutamine plus 2 mM L-tyrosine for 2 h. At 30 min intervals, a 300 ml aliquot of medium was removed from each culture. To each aliquot, 50 mg of BSA was added followed by addition of T C A to a final concentration of 15%.  After standing on ice for 1 h, samples were centrifuged at 14,000 x  g for 5 min. A sample of the supernatant was taken to measure the radioactivity associated with free [ H]tyrosine. 3  Negligible radioactivity was found in the TCA-insoluble material  (the pellet).  At the end of the incubation period, the cultures were washed four times  68  with ice-cold PBS-tyr and the cell lysate was prepared for measurement of the remaining radioactivity in the myotubes using procedures similar to those used for measurement of protein synthesis. In experiments designed to measure the rate of degradation of long-lived proteins, the cultures were labelled with [ H]tyrosine (1.5 itCi/ml D M E M ) for 48 h in growth 3  media.  The cultures were then washed twice with PBS-tyr and placed in growth media  containing 2 mM L-tyrosine for 6 h to ensure degradation of short-lived proteins. The cultures were washed twice with PBS-tyr and incubated in the experimental media containing 0, 0.65, 5, 10, or 15 mM glutamine and 2 mM L-tyrosine for 24 h. A 300 ml aliquot of the culture media was removed every 8 h from each culture. At the end of the incubation, the cultures were washed four times with ice-cold PBS-tyr.  Radioactivity  associated with [ H]tyrosine in the incubation medium aliquots and in the cultures was measured by the same procedure used for measurement of the degradation of the shortlived proteins. The half-lives of short-lived proteins and long-lived proteins were calculated based on regression analysis described previously (2,20).  II.5. Heat shock treatment and measurement of protein synthesis and degradation in heat-stressed myotubes Heat shock treatment in all experiments consisted of incubating the myotube cultures at 4 3 ° C for 45 min in a humidified atmosphere of a i r / C 0  2  (19:1, v/v). In  experiments designed to measure the rate of protein synthesis, myotubes were heat shocked in experimental media (i.e. growth media containing 0, 0.65, glutamine).  69  5, 10 or 15 mM  Following heat shock, myotubes were immediately returned to 3 7 ° C and  incubated for either 1 or 2 h.  One hour before the end of each incubation period, the  media were replaced with experimental media containing 1 m M L-[ H]tyrosine (2 ttCi/ml). The procedure for measurement of the rate of incorporation of [ H]tyrosine was similar to those described above. In experiments designed to measure the rate of degradation of short-lived proteins in heat-stressed myotubes, the cultures were labelled with L-[ H]tyrosine (3 itCi/ml D M E M ) in growth medium for 1.5 h at 3 7 ° C before they were subjected to heat shock. The cultures were then washed with PBS-tyr and incubated in the growth media containing 2 mM L-tyrosine for 10 min to allow for degradation of very short-lived proteins (2). They were then placed in the experimental media and subjected to heat shock treatment. Immediately following heat-shock, the cultures were returned to 37 °C and incubated for 2 h.  A 300 til aliquot of media was removed immediately following heat-shock to correct  for initial background radioactivity associated with free [ H]tyrosine released during heat3  shock.  Aliquots were removed every 30 min during the 2-h incubation period.  Radioactivity in the aliquots and the cell lysates was measured as described above.  In  experiments designed to measure the rate of degradation of long-lived proteins, the cultures were labelled with L-[ H]tyrosine (1.5 itCi/ml D M E M ) in growth medium for 48 h at 3  37°C.  The cultures were washed twice with PBS-tyr and incubated in growth media  containing 2 mM L-tyrosine for 6 h to allow for degradation of short-lived proteins. The  cultures were then washed twice with PBS-tyr and placed in experimental media before heat shock treatment.  70  Immediately following heat shock, the cultures were returned to  3 7 ° C and incubated for 3 h. A 300 id aliquot of medium was sampled immediately after heat shock and then every 1 h during the 3-h incubation period.  Radioactivity in the  aliquots and the cell lysates was measured as described above.  11.6. Statistical analysis All values are expressed as means ± S E M .  Differences between means were  assessed using Student's t tests.  m . Results m . l . Effect of glutamine on protein turnover in normal-cultured myotubes We examined the effect of glutamine on the rate of protein synthesis in normalcultured (non-stressed) myotubes during 1 and 2 h of incubation.  The rate of protein  synthesis was not significantly affected by glutamine concentration in the culture media during 1 or 2 h of incubation (Figure 4-1A).  To examine if glutamine would have an  effect after a longer incubation period, we extended the incubation time to 4 or 6 h, and we still did not find a significant effect of glutamine on the rate of protein synthesis (data not shown).  However, the rate of protein synthesis was higher (P<0.05) in the myotubes  incubated for the longer time periods (4 h or 6 h, average 2.94  nmol tyrosine  incorporated/mg protein/h) than in myotubes incubated for the shorter time periods (1 h or 2 h, average 2.16 nmol tyrosine incorporated/mg protein/h).  71 In the present work, the rate of protein degradation is expressed as the half-life of the proteins which was calculated using regression analysis.  Representative regression  curves used to calculate the half-life of short-lived proteins and long-lived proteins in nonstressed myotubes are shown in Figure 4-2A and Figure 4-2B, respectively.  The  regression coefficients ranged from 0.991 to 0.997. Figures 4-3A and 4-4A show that in non-stressed myotubes, glutamine did not alter the half-life of short-lived proteins but increased the half-life of long-lived proteins in a concentration-dependent manner. The half-life measured in the absence of glutamine was 7.8 ± 0.2 h for the short-lived proteins and 52.8 ± 1.6 h for the long-lived proteins. These values are similar to those reported by other laboratories [2,17].  When the glutamine  concentration was increased from 0 to 0.65 m M , the physiological concentration of glutamine in blood, the half-life of long-lived proteins increased 16% (P<0.05). the glutamine concentration  When  was increased from 0 to 15 m M , the physiological  concentration of intramuscular glutamine, the half-life increased 35% (P< 0.001).  ni.2. Effect of glutamine on protein turnover in stressed myotubes Since glutamine did not have a stimulatory effect on the rate of protein synthesis in normal cultured non-stressed myotubes, we determined if glutamine influenced the rate of protein synthesis in stressed myotubes.  Myotubes were heat shocked at 4 3 ° C for 45 min  and then incubated at 3 7 ° C for recovery periods of 1 or 2 h. Heat stress significantly decreased the rate of protein synthesis. During the first hour following heat stress, the rate of protein synthesis was 68-75% lower than the rate measured in normal-cultured  myotubes, while during the second hour it was 31-46% lower (P<0.001) (Figure 4-1B).  72  In contrast to the response to glutamine observed in normal-cultured myotubes, there was a significant increase in the rate of protein synthesis with increasing concentration of glutamine in the stressed myotubes (Figure 4-IB).  During the first hour after heat shock  treatment, the rate of protein synthesis was increased 21% (P<0.05) by increasing the glutamine concentration from 0 to 15 m M .  During the second hour after heat shock  treatment, the rate of protein synthesis was increased 10% (P<0.01) by increasing the glutamine concentration by the same amount. In the stressed myotubes, the rate of degradation of short-lived proteins was not influenced by glutamine concentration (Figure 4-3B).  The basal half-life of short-lived  proteins in the stressed myotubes was 5.9 ± 0.2 h, 76% of that in the non-stressed myotubes.  In contrast to the short-lived proteins, the degradation of long-lived proteins  was significantly inhibited by glutamine in a concentration-dependent manner (P<0.05) (Figure 4-4B). The basal half-life of long-lived proteins in the stressed myotubes was 43.3 + 0.9 h, 82% of that in the normal myotubes.  When glutamine concentration was  increased from 0 to 15 m M , the half-life of long-lived proteins increased 27% (P< 0.001).  IV. Discussion The present study clearly demonstrated that glutamine has a stimulatory effect on the rate of protein synthesis in skeletal myotubes subjected to stress, while it has no effect on the rate of protein synthesis in normal-cultured skeletal myotubes.  To the author's  knowledge, this is the first report to definitively show that the effect of glutamine on protein synthesis in skeletal muscle is condition-dependent.  73  The data obtained in the  present study are consistent with data obtained from a number of studies performed with either individuals subjected to various stressors or with non-stressed normal individuals. Administration of glutamine or glutamine-containing dipeptides to individuals subjected to stressors, such as sepsis or surgery, has been consistently reported to increase the depressed rate of skeletal muscle protein synthesis (7).  On the other hand, glutamine administration  to relatively non-stressed normal individuals has been reported not to affect the rate of skeletal muscle protein synthesis in these individuals (6,10).  However, researchers using  rat hindlimb perfusion or in vitro chick skeletal muscle incubation preparations have suggested that glutamine has a stimulatory effect on protein synthesis in these preparations obtained from normal animals.  In fact, skeletal muscles studied under these conditions  have been and continue to be subjected to a range of stresses, including surgical manipulation (20) and hypoxia (19).  Therefore, the effect of glutamine demonstrated in  these in vitro experiments may actually mimic the effect of glutamine on skeletal muscle protein synthesis in stressed individuals in vivo. The myotube culture system used in the present work provides an ideal system to differentiate between the effects of glutamine on protein metabolism in skeletal muscles under non-stressed and stressed states because optimal conditions for normal cell growth and differentiation (e.g. temperature, nutrient concentration, and growth state etc.) can be precisely controlled and maintained and the cells are not subjected to stressors such as hypoxia.  The mechanism underlying the effect of glutamine on protein synthesis in stressed skeletal muscle remains unknown.  Murtha-Riel et al. (21)  74  have suggested that the  inhibition of protein synthesis due to heat stress is largely due to phosphorylation of the eukaryotic initiation factor 2a (eIF-2a).  Phosphorylation of eIF-2a leads to inhibition of  the initiation machinery of mRNA translation.  Heat shock protein 70 (HSP70) induced  during heat stress facilitates dephosphorylation of eIF-2a thus removing the inhibition on protein synthesis (22).  Recently, we found that glutamine increases HSP70 expression in  heat-stressed myotubes (see Chapter 5).  Therefore, HSP70 may play a role in the  mechanism by which glutamine enhances the rate of protein synthesis in stressed myotubes, though a direct involvement of HSP70 remains to be demonstrated. In the present work, glutamine was found to inhibit the rate of degradation of longlived proteins but not short-lived proteins in skeletal myotubes.  Previously, a general  inhibitory effect of glutamine on the rate of protein degradation had been demonstrated in perfused rat hindlimb (1) and in chick skeletal muscle incubation preparations (14).  Smith  (23) measured the rate of protein degradation in L6 myotubes cultured in amino acid-free media for either 2.5 h or 30 h. Inclusion of 2 mM glutamine in the incubation media was found to decrease the rate of protein degradation in myotubes cultured for 30 h but not for 2.5 h. Unfortunately, the experimental approach used in this study does not allow one to clearly differentiate between the degradation of long-lived proteins and short-lived proteins.  Moreover, the amino acid-free media likely put the myotubes in a non-  physiological environment. In spite of these shortcomings, the data reported by Smith (23)  appear to be consistent with the results obtained in the present study because the  75  degradation of proteins during 2.5-h incubation period likely reflects the degradation of short-lived proteins, while the degradation of proteins during 30-h incubation period likely reflects the degradation of long-lived proteins.  Other anabolic agents such as insulin and  IGF-1 have also been reported to inhibit the rate of protein degradation of the long-lived fraction but not of the short-lived fraction (2,16). Because the long-lived proteins account for the majority of myotube proteins (2), these results suggest that glutamine can confer a physiologically meaningful anabolic effect on protein metabolism in skeletal muscle by inhibiting its rate of degradation. The mechanism underlying the effect of glutamine on protein degradation in skeletal muscle remains unknown.  In rat hepatocytes, glutamine has been reported to  inhibit protein degradation by inducing cell swelling leading to lysosomal alkalization (24). However, in skeletal muscle cells it has been suggested that the lysosomal activity is very low and likely does not play a major role in the anabolic effect of glutamine on protein degradation in this tissue. HSP70 can bind to proteins which undergo conformation change and protect them from denaturation and degradation (9).  Since glutamine increases HSP70  expression in stressed myotubes (see chapter 5), it is reasonable to assume that HSP70 may be involved in the inhibitory effect of glutamine on muscle protein degradation. In summary, the present work demonstrated that increasing the concentration of glutamine increases the rate of protein synthesis in heat-stressed myotubes but not in normal-cultured myotubes; in both normal and stressed myotubes, the rate of degradation of short-lived proteins is not altered but the rate of degradation of long-lived proteins was  76  A: Normal-cultured myotubes  • 0 mM • 0.65 m M • 5 mM • 10 m M • 15mM  Time (h)  B: Stressed myotubes  • OmM • 0.65 mM • 5 mM EH 10 mM  • 15mM  1  Time (h)  2  Figure 4-1 The rate of protein synthesis in normal myotubes and stressed myotubes in the presence of various concentrations of glutamine Myotubes were grown on 6-well  tissue culture plates. They were incubated in the experimental media containing different concentrations of glutamine for 1 or 2 h without (A) or with (B) 45-min of pretreatment with heat-shock (43°C for 45 min). Myotubes were labelled with [H]-tyrosine (2 /xCi/ml) during the last hour of incubation. The rate of protein synthesis is expressed as nmol tyrosine incorporated/mg of protein/h. Values are means ± SEM (n=4). a and b, different from 0 mM glutamine at P<0.05 and P<0.01, respectively; c, different from 0.65 mM glutamine at P<0.05. 3  77  Time (h)  Time (h)  Figure 4-2 Degradation of short-lived proteins and long-lived proteins in normal myotubes Myotubes grown under normal conditions were prelabelled with [ H]tyrosine (A) 3 iiCi/ml for 1.5 h for measuring degradation of short-lived proteins or (B) 1.5 /xCi/ml for 48 h for measuring degradation of long-lived proteins as described in the Materials and Methods section. Curves best fitting the datum points (mean ± S E M , n=4) are shown. 3  78  A : Normal-cultured myotubes 10  T  Glutamine (mM)  B: Stressed myotubes  10 j  0  0.635  5  10  15  Glutamine (mM)  Figure 4-3 The half-life of short-lived proteins in normal and stressed myotubes in the presence of various concentrations of glutamine Myotubes grown on 6-well tissue culture plates were prelabelled with [ H]-tyrosine (3 itCi/ml) for 1.5 h. Cells were washed and chased for 10 min. (A) Myotubes were incubated in the experimental media containing various concentrations of glutamine as indicated at 37 °C for 2 h. (B) Myotubes were incubated in the experimental media containing various concentrations of glutamine as indicated at 4 3 ° C for 45 min following incubation at 3 7 ° C for 2 h. The half-life is calculated by using regression analysis. Values are means ± S E M (n=4). No differences were observed among the groups.  A : Normal-cultured myotubes  79  Figure 4-4 Effect of glutamine on the half-life of long-lived proteins in normalcultured and stressed myotubes Myotubes grown on 6-well tissue culture plates were prelabelled with [ H]-tyrosine (1.5 /xCi/ml) for 48 h. Myotubes were washed and incubated for 6 h to ensure degradation of short-lived proteins. Measurement of the halflive of long-lived proteins in normal-cultured (A) and stressed (B) myotubes was described in the Materials and Methods section. Values are means ± S E M (n=4). a,b, and c, different from the 0 mM glutamine group at P<0.05, P<0.01, and P<0.001, respectively; d , different from the 0.65 and 5 mM glutamine groups at P<0.05. 3  V . References  80  1 MacLennan, P . A . , Brown, R . A . , and Rennie, M . J . (1987) A positive relationship between protein synthesis rate and intracellular glutamine concentration in perfused rat skeletal muscle. FEBS Letter 215, 187-191. 2 Gulve, E . A . and Dice, J.F. (1989) Regulation of protein synthesis and degradation in L8 myotubes. Biochem. J. 260, 377-387. 3 Price, S.R., England, B . K . , Bailey, J.L., Vreede, K . V . , and Mitch, W . E . (1994) Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle. A m . J. Physiol. 267, C955-C960. 4 Howard, G . , Steffen, J . M . , and Geoghegan, T . E . (1989) Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading. J. Appl. Physiol. 66, 10931098. 5 Jepson, M . M . , Bates, P . C . , Broadbent, P., Pell, J . M . , and Millward, D . J . (1988) Relationship between glutamine concentration and protein synthesis in rat skeletal muscle. Am. J. Physiol. 255, E166-E172. 6 Hammarqvist, F . , Wernerman, J., von der Decken, A . , and Vinnars, E . (1991) Alphaketoglutarate preserves protein synthesis and free glutamine in skeletal muscle after surgery. Surgery 109, 28-36. 7 Blomqvist, B.I., Hammarqvist, F . , von der Decken, A . , and Wernerman, J. (1995) Glutamine and a-ketoglutarate preserve the decrease in muscle free glutamine concentration and influence protein synthesis after total hip replacement. Metabolism: Clin. Exp. 44, 1215-1222. 8 Rennie, M . J . , Hundal, H . S . , Babij, P., Maclennan, P., Taylor, P . M . , Watt, P.W., Jepson, M . M . , and Millward, D.J. (1986) Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection, and clinical disease. Lancet 2, 1008-1012. 9 Georgopoulos, C . and Welch, W.J. (1993) Role of the major heat shock proteins as molecular chaperones. Ann. Rev. Cell Biol. 9, 601-634. 10 Hickson, R . C . , Czerwinski, S . M . , and Wegrzyn, L . E . (1995) Glutamine prevents downregulation of myosin heavy chain synthesis and muscle atrophy from glucocorticoids. Am. J. Physiol. 268, E730-E734.  81 11 Wusteman, ML, Tate, H . , and Elia M . (1995) The use of a constant infusion of [ H]phenylalanine to measure the effects of glutamine infusion on muscle protein synthesis in rats given trupentine. Nutrition 11, 27-31. 3  12 Garlick, P.J. and Grant, I. (1988) Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Biochem. J. 254, 579-584. 13 MacLennan, P . A . , Smith, K . , Weryk, B . , Watt, P.W., and Rennie, M . J . (1988) Inhibition of protein breakdown by glutamine in perfused rat skeletal muscle. FEBS Lett. 237, 133-136. 14 Wu, G . and Thompson, J.R. (1990) The effect of glutamine on protein turnover in chick skeletal muscle in vitro. Biochem. J. 265, 593-598. 15 Hasselgren, P . O . , Zamir, O . , James, J . H . , and Fischer, J.E. (1990) Prostaglandin E does not regulate total or myofrillar protein breakdown in incubated skeletal muscle from normal or septic rats. Biochem. J. 270, 45-50.  2  16 Gulve, E . A . , Mabuchi, K . , and Dice, J.F. (1991) Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes. J. Cell. Physiol. 147, 37-45. 17 Hong, D . - H . and Forsberg, N . E . (1994) Effects of serum and insulin-like growth factor I on protein degradation and protease gene expression in rat L8 myotubes. J. Anim. Sci. 72, 2279-2288. 18 Toyota, N . and Shimada,Y. (1983) Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves. Cell 33, 297-304. 19 Martin, C A . and Harris, C I . (1985) Morphological observation and rates of protein synthesis in rat muscle incubated in vitro. Biochem. J. 232, 927-930. 20 Udelsman, R. and Holbrook, N.J. (1994) Endocrine and molecular responses to surgical stress. Curr. Problem Surg. 31, 653-720. 21 Murtha-Riel, P., Davis, M . V . , Scherer, B.J., Choi, S.-Y., Hershey, J.W.B., and Kaufman, R.J. (1993) Expression of a phosphorylation-resistant eukaryotic initiation factor 2a subunit mitigates heat shock inhibition of protein synthesis. J. Biol. Chem. 268, 1294612951. 22 Chang, G . C , Liu, R., Panniers, R., and L i , G . C (1994) Rat fibroblasts transfected with the human 70 kDa heat shock gene exhibit altered translation and eukaryotic initiation factor 2a phosphorylation following heat shock. Int. J. Hypertherm. 10, 325-337.  82 23 Rennie, M . J . , Hundal, H . S . , Babij, P., Maclennan, P., Taylor, P . M . , Watt, P.W., Jepson, M . M . , and Millward, D.J. (1986) Characteristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen loss in injury, infection, and clinical disease. Lancet 2, 1008-1012. 24 Georgopoulos, C . and Welch, W.J. (1993) Role of the major heat shock proteins as molecular chaperones. Ann. Rev. Cell Biol. 9, 601-634. 25 Hickson, R . C . , Czerwinski, S . M . , and Wegrzyn, L . E . (1995) Glutamine prevents downregulation of myosin heavy chain synthesis and muscle atrophy from glucocorticoids. Am. J. Physiol. 268, E730-E734. 26 Wusteman, M . , Tate, H . , and Elia M . (1995) The use of a constant infusion of [ H]phenylalanine to measure the effects of glutamine infusion on muscle protein synthesis in rats given trupentine. Nutrition 11, 27-31. 3  27 Garlick, P.J. and Grant, I. (1988) Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Biochem. J. 254, 579-584. 28 MacLennan, P . A . , Smith, K . , Weryk, B . , Watt, P.W., and Rennie, M . J . (1988) Inhibition of protein breakdown by glutamine in perfused rat skeletal muscle. FEBS Lett. 237, 133-136. 29 Wu, G . and Thompson, J.R. (1990) The effect of glutamine on protein turnover in chick skeletal muscle in vitro. Biochem. J. 265, 593-598. 30 Hasselgren, P . O . , Zamir, O . , James, J . H . , and Fischer, J.E. (1990) Prostaglandin E does not regulate total or myofrillar protein breakdown in incubated skeletal muscle from normal or septic rats. Biochem. J. 270, 45-50.  2  31 Gulve, E . A . , Mabuchi, K . , and Dice, J.F. (1991) Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes. J. Cell. Physiol. 147, 37-45. 32 Hong, D . - H . and Forsberg, N . E . (1994) Effects of serum and insulin-like growth factor I on protein degradation and protease gene expression in rat L8 myotubes. J. Anim. Sci. 72, 2279-2288. 33 Toyota, N . and Shimada,Y. (1983) Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves. Cell 33, 297-304. 34 Martin, C A . and Harris, C I . (1985) Morphological observation and rates of protein synthesis in rat muscle incubated in vitro. Biochem. J. 232, 927-930.  35 Udelsman, R. and Holbrook, N.J. (1994) Endocrine and molecular responses to surgical stress. Curr. Problem Surg. 31, 653-720.  83  36 Smith, R.J. (1985) Regulation of protein degradation in differentiated skeletal muscle cells in monolayer culture. In "Intracellular Protein Catabolism", pp 633-635. Alan R. Liss, Inc., New York. 37 Volkl, H . , Busch, G . L . , Haussinger, D . , and Lang, F. (1994) Alkalization of acidic cellular compartment following cell swelling. FEBS Lett. 338, 27-30.  CHAPTER FIVE  Glutamine Increases Heat Shock Protein Expression in Stressed Myotubes  84  I. Introduction In the preceding chapter, it was shown that elevated concentrations of glutamine stimulate the rate of protein synthesis in heat-stressed myotubes but not in normal-cultured myotubes, and that glutamine inhibits the rate of degradation of long-lived proteins in both stressed and normal-cultured myotubes.  A number of in vivo studies have also demonstrated that  intravenous administration of glutamine in humans and animals subjected to various stressors, such as surgery and sepsis, also increases the rate of protein synthesis (1,2).  Despite the  potential clinical importance of these observations, the mechanism(s) underlying the effect of glutamine on protein turnover in skeletal muscle remains unknown. A prominent feature of stressed cells is the synthesis of a specific group of proteins called HSPs.  Heat shock proteins have been suggested to confer a variety of protection to  stressed cells. It has been observed that the levels of HSPs, particularly their major subfamily, HSP70, are positively correlated with the rate of protein synthesis in rabbit reticulocyte lysates (3).  By overexpressing HSP70 in rat fibroblasts, Liu et al. (4) have shown that HSP70 can  relieve the inhibition on protein synthesis by heat stress.  This effect of HSP70 has been  attributed to its ability to increase the rate of dephosphorylation of the initiation factor eIF-2a thus removing the inhibition on the initiation machinery of translation due to stress (5).  HSP70  can also bind to proteins and protect them from stress-induced denaturation and degradation, and for this reason HSP70 has been referred to as a molecular chaperone (6).  These  85 observations, along with the report that glutamine increases HSP70 expression in opossum kidney cells (20), lead us to examine whether glutamine regulates the expression of HSP70 in skeletal muscle cells, which then might contribute to our understanding of the mechanism underlying glutamine's beneficial effects on protein turnover in muscle cells. Regulation of HSP induction has been suggested to be at the transcriptional level (21). Upon stress, heat shock transcription factors (HSF) are activated to form trimers in the cytoplasm. The HSF trimers are translocated into nuclei and bind to a specific D N A sequence, the heat shock element (HSE), in the promoter regions of hsp genes. The DNA-bound HSF is phosphorylated followed by activation of the transcription of hsp genes (7).  In this chapter, the  effect of glutamine on HSP70 protein levels and on hsplO gene transcription in myotubes was investigated.  n . Materials and Methods II.1. Materials The L8 skeletal muscle cell line was purchased from American Tissue Culture Collection (Rockville, USA).  Fetal bovine serum (FBS), TriZol reagent, and Dulbecco's  modified Eagle medium (DMEM) without glutamine were purchased from Gibco B R L (New York, USA). SPA-801 and SPA-810, antibodies raised against rodent HSP27 (polyclonal, from rabbit) and human HSP72 (the inducible form of HSP70) (monoclonal, from mouse), respectively, and P17, a 4.6 kb hspll cDNA probe, were purchased from StressGen (Victoria, Canada).  The subclone system containing a transcriptional vector pGEM-3Z was purchased  from Promega (Madison, USA).  86 A Dig R N A labelling and detection system and nylon  membranes were purchased from Boehringer Mannheim (Laval, Canada). A gel mobility shift assay kit was purchased from Pharmarcia (Missauga, Canada). Oligonucleotides corresponding to the sequences of human HSE [the sequence between -107 to -84 of human hsplO gene, 5' T A T G C G A A A C C C C T G G A A T A T T C C 3' (the underlined nucleotide sequnces denote the consensus nGAAn sequence of HSE) and its complementary strand] and a mutant form of the HSE (HSE-M) [5'  TATGCrAAACCGCTGCAATACTCC  3' (the italic bold letters denote the  mutant positions) and its complementary strand] were synthesized in the Department of Biochemistry at The University of British Columbia.  Polyclonal antibodies raised in rabbits  against rat actin (Ab-actin), glutamine, and all other chemicals were purchased from Sigma (St. Louis, USA).  Sep-Pak C18 columns were purchased from Pharmacia (Montreal, Canada).  II.2. Cell culture and heat shock treatment L8 skeletal myoblasts were cultured as described in Chapter 3. All experiments in this study were done after approximately 90% of the myoblasts were fused into myotubes. The culture media were replaced with D M E M plus 10% FBS containing 0, 0.65, 5, or 10 mM glutamine before heat-shock treatment. Heat-shock was performed by incubating the myotubes at 43°C for 45 min in an atmosphere of air/C0 (95%/5%). For studying the binding activity 2  of HSF, cells were harvested immediately after heat shock.  For studying the expression of  hsplO mRNA and HSP proteins, cell cultures were returned to a 3 7 ° C incubator after heat shock and harvested 2 h later for RNA preparation or 4 h later for protein preparation.  87  n.3.  Western blot analysis Myotube cultures were washed twice with ice-cold phosphate-buffered saline (PBS) and  scraped off the culture dishes 4 h after heat-shock. Tris-HCl (pH 7.5),  Cells were lysed in a lysis buffer [20 mM  150 mM NaCl, 2 mM E D T A , 1% NP-40, 1 mM phenylmethylsulfonyl  fluoride (PMSF), and 2 mM DTT] on ice for 30 min, followed by centrifugation at 14,000 x g for 15 min at 4 ° C . Protein concentrations in the supernatant were measured by the method of Bradford (8).  Equal amounts of protein (40 fig) from each sample were fractionated using  SDS-polyacrylamide gel electrophoresis  (SDS-PAGE) (10%  separating gel).  Following  electrophoresis, proteins were transferred onto nitrocellulose membranes in transfer buffer containing 192 mM glycine, 25 mM Tris, and 20% methanol using a Bio-Rad Transblot apparatus (9).  The membrane was blocked with a blocking solution (3% non-fat dry milk in  PBS) for 1 h at room temperature and incubated with the primary antibody (SPA-801, SPA810, or Ab-actin ) for 2-3 h.  The membrane was then washed three times with a washing  buffer (0.1 % Tween-20 in PBS), blocked with the blocking solution for 1 h, and incubated with horseradish peroxidase (HRP) conjugated anti-rabbit IgG secondary antibody (for SPA-801 and Ab-actin) or alkaline phosphatase (AP) conjugated anti-mouse IgG secondary antibody (for SPA-810) for 2 h. The membrane was washed three times with the washing buffer followed by detection using an enhanced chemiluminesence (for HRP-conjugated secondary antibody) or a colourmetric method (for AP-conjugated secondary antibody). Protein bands on the membrane were quantitated using a Molecular-Dynamics densitometer.  88  II.4. RNA preparation and Northern blot analysis  n.4.1. RNA preparation: PBS 2 h after heat-shock.  Myotube cultures were washed three times with ice-cold  Myotubes were then scraped off the dishes and collected in  Eppendorf tubes. One millilitre of TriZol reagent was added for every 5 x 10 cells. Total 6  RNA was isolated according to the manufacturer's manual (Gibco-BRL). Briefly, cells were disrupted by repeated pipetting, followed by addition of 0.5 ml chloroform and centrifugation for 15 min at 14,000 x g at 4 ° C .  The top layer was collected and mixed with 1 ml of  isopropanol. The mixture was again centrifuged at 14,000 x g for 15 min at 4 ° C . The pellet containing total R N A was collected and dissolved in formaldehyde. R N A was quantitated by measuring its absorbency at 260 nm. To confirm the concentration of each RNA sample, RNA samples were run on a minigel and the levels of 18S rRNA and 28S rRNA from each sample were assessed. n.4.2. Preparation of riboprobe: To synthesize a RNA probe with a high specificity for hsp72, a 760 bp Hind Ill/Hind III fragment was excised from the P17 D N A probe (4.6 kb) and subcloned into the transcriptional vector pGEM-3Z in such an orientation that the antisense strand was downstream of the T7 promoter. In vitro transcription of the engineered pGEM-3Z was performed using the T7 RNA polymerase and the mixture of ribonucleotides (10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM UTP, and 3.5 mM digoxigenin-ll-UTP) in a buffer containing 40 mM Tris-HCl (pH 8.0), 6 mM MgCl , 10 mM D T T , 10 mM NaCl, 20 mM 2  89 spermidine, and 1 U//xl of RNase inhibitor. The resulting digoxigenin labelled RNA transcripts were used for probing the hsplO mRNA. n . 4 . 3 . Northern blotting and hybridization: Twenty micrograms of total R N A from each sample was denatured at 65°C for 15 min and fractionized using a 1.2% formaldehyde gel.  agarose-  RNA on the gel was transferred onto a nylon membrane by the alkaline  downward transfer method described by Chomczynski (10).  Briefly, a stack of paper towels (3  cm) was covered by five sheets of 3 M M filter paper with the top one soaked with the transfer solution (3 M NaCl and 8 mM NaOH).  A nylon membrane which had been soaked in  demineralized water for 20 min was placed on top of the filter papers and then was covered with the agarose gel. The gel was covered with three sheets of filter paper with the size same with the gel and with two sheets of long filter paper (all soaked with the transfer solution) forming a bridge. The distal parts of the bridge were placed in the transfer solution. Transfer was performed for 2 h.  Following transferring, the membrane was stained with  0.02%  methylene blue for 3 min to visualize 18S rRNA and 28S rRNA, which were photographed and used as an internal control for each sample, and as evidence of the integrity of R N A and its successful transfer. membrane.  The membrane was baked at 80°C for 30 min to fix R N A to the  The membrane was incubated in a prehybridization solution [750 mM NaCl, 15  mM sodium citrate (pH 7.0), 50% formamide, 0.02% SDS, 0.1% N-lauroylsarcosine, and 2% Boehringer-Mannheim blocking reagent] for 2 h at 65°C.  Subsequently, the membrane was  incubated in a hybridization solution (prehybridization plus the riboprobe at a concentration of 100 ng/ml) for 14 h at 65°C in a shaking water bath.  RNA signals were detected by a  chemiluminescent method using Lumigen-PPD as a substrate.  90 The hsplO mRNA levels  recorded by autoradiography were quantitated using a Molecular-Dynamics densitometer and normalized with the 28S rRNA level of each sample.  n.5. Nuclear extract preparation and gel mobility shift assay n.5.1. Nuclear extract preparation from myotubes: Myotube cultures were washed three times with ice-cold PBS and scraped off from the culture dishes immediately after heatshock.  Nuclear extracts were prepared according to the method of L i et al. (11). Briefly,  approximately 5 x 10 cells were washed with buffer A [10 mM HEPES (pH 7.9), 1.5 mM 6  MgCl , 10 mM KC1, 0.5 mM DTT, and 1 mM PMSF] and lysed in 200 td of buffer A by 2  gently passing the cell suspension through a 30-gauge needle 15 times.  The lysate was  centrifuged at 14,000 x g for 8 sec, and the pellet was collected. The pellet was resuspended in 30 pi of buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM KC1, 1.5 mM MgCl , 0.2 2  mM E D T A , 0.5 mM DTT, 1 mM PMSF] and placed on ice for 30 min. A n equal volume of buffer D (buffer C minus KC1) was added, and the mixture was centrifuged at 14,000 x g for 15 min at 4 ° C .  The supernatant collected represented the nuclear extract.  Protein  concentrations in the nuclear extract were determined by the method of Bradford (8). H.5.2.  Preparation  of the oligonucleotide probe:  The H S E and HSE-M  oligonucleotides were purified by dissolving in acetonitrile and passing through Sep-Pak C18 columns (9). Complementary oligonucleotides were annealed in a buffer containing 150 mM KC1, 5 mM E D T A , and 50 mM Tris-HCl (pH 7.5) at 100°C for 2 min, followed by 15 min of  incubation at each of the following temperatures:  65°C, 37°C, 2 5 ° C , and 4 ° C .  91 Fifty  nanograms of annealed oligonucleotides was labelled with P by the 5' filling-in method using 32  the Klenow enzyme. Following the labelling reaction, the oligonucleotides were washed once with 75 % ice-cold ethanol to remove unincorporated nucleotides. The labelled oligonucleotides were eventually dissolved in 50 itl of water, and their radioactivity was measured (approximately 120,000 cpm/til) using a Beckman LS6500 liquid scintillation counter. n.5.3.  Gel mobility shift assay:  mobility shift assay was performed.  To assess the D N A binding activity of HSF, a gel  Ten micrograms of nuclear extract protein from each  32  '  sample was incubated with 1 id of P labelled HSE oligonucleotides in a buffer containing 10 mM HEPES (pH 7.9), 2 mM E D T A , 10% glycerol, 1% NP-40, 3 /xg of poly (dI-dC)(dI-dC), and 1 /xg of calf thymus D N A for 25 min at room temperature. To determine the specificity of HSF binding to HSE, a competition experiment was performed by including 25-mol or 100mol excess of unlabelled HSE oligonucleotides or 100-mol excess of unlabelled HSE-M oligonucleotides  in the reaction  mixture  before  addition of the  32  P labelled H S E  oligonucleotides. The reaction mixtures were loaded on a 4.5 % native polyacrylamide gel (16 cm long and 1 mm thick) and electrophoresed in 0.5 x T B E buffer (1 x T B E buffer: 90 mM Tris-borate, 2 mM EDTA) at 150 V for 2.5 h. The gel was dried and exposed to X-ray film. The bands representing the specific HSF-HSE binding were quantitated using a MolecularDynamics densitometer.  n.6. Statistical analysis  92 One-way A N O V A was used to test the statistical significance of the results,  m . Results rfLl. Regulation of HSP70 and HSP27 expression by glutamine Figure 1 shows that the expression of HSP72, the stress-inducible form of HSP70, was induced by heat shock treatment in L8 myotubes. Increasing the concentration of glutamine significantly increased the amount of HSP72 in stressed myotubes in a concentration-dependent manner.  When glutamine increased from 0 to 10 mM in the culture media, the amount of  HSP72 increased 7.5-fold (P< 0.001). The equality of the amount of protein loaded among the samples was verified using the anti-actin antibody (Fig. IB). To determine if glutamine has a universal effect on HSP expression, we determined the amount of HSP27, a member of the small HSP family, in myotubes treated the same way as above. HSP27 was constitutively expressed in control (non-stressed) cells. Its expression was increased by heat-shock treatment.  However, unlike HSP72, the amount of HSP27 in the  myotubes did not change in response to the concentration of glutamine in the culture media (Fig.2).  m.2. Effect of glutamine on the abundance of hsplO mRNA Northern blot analysis showed that the expression of hsp72 mRNA (2.8 kb) was induced in stressed myotubes compared to normal-cultured myotubes.  Glutamine had a  stimulatory effect on hsp!2 mRNA abundance in a concentration-dependent manner. When glutamine concentration was increased from 0 to 10 mM, hspll mRNA abundance increased  93 7.8-fold (P<0.001) (Fig.3). The pattern of change of hsp!2 mRNA was similar to that of HSP72 protein. Hspl2> mRNA (a 2.3 kb band) was also detected by the probe (Fig.3).  Its  expression was greater in stressed myotubes compared to normal-cultured myotubes. However, unlike hspll mRNA, hsp!3 mRNA abundance was only increased by 10 mM glutamine. The magnitude of increase was also much smaller (1.2-fold) than that of hspll mRNA (7.8-fold), though it was also statistically significant (P<0.01).  H1.3. The DNA binding activity of HSF is not altered by glutamine The binding activity of HSF to HSE in stressed myotubes is shown in Fig. 4. There was no D N A binding activity of HSF in the control (non-stressed) myotubes regardless of the concentration of glutamine in the culture media. In stressed myotubes, there was a specific binding of HSF to HSE oligonucleotides as shown by the competition experiment (Fig. 4A). The HSF-HSE binding was completely inhibited by addition of 100-fold more of unlabelled HSE oligonucleotides, but was not affected by addition of 100-fold more of unlabelled HSE-M oligonucleotides (Fig. 4A). There was no significant change in the HSF-HSE binding activity regardless of the concentration of glutamine in the culture media (Fig. 4B).  TV. Discussion As shown in other types of cells (14), in the present work skeletal myotubes synthesized HSP72 in response to heat stress. This induction of HSP72 synthesis by heat stress was well correlated with the induction of its mRNA synthesis. These suggest that the myotube culture  94 system and heat-shock treatment used in the present work are suitable for studying HSP expression. In this chapter, glutamine was shown to regulate HSP expression in heat-stressed skeletal myotubes at both the protein and mRNA levels. This finding provides an avenue of research for exploring the mechanism underlying the effect of glutamine on protein turnover in skeletal muscle. It was shown in the preceding chapter that heat stress results in an inhibition of protein synthesis, and that glutamine can partially overcome this inhibition in myotubes. L i et al. (12) have demonstrated that the inhibition of protein synthesis by heat stress is largely due to increased phosphorylation of eIF-2a which results in inhibition of the initiation of protein synthesis.  Recently,  Chang et al. (5)  reported that HSP70  increases  the rate of  dephosphorylation of eIF-2a in heat-stressed rat fibroblasts, which may therefore be a mechanism by which HSP70 protects the rate of protein synthesis from inhibition by heat stress in these cells (4). It is reasonable to assume that HSP70 has a similar effect on heat-stressed rat skeletal myotubes.  Therefore, since glutamine increases the expression of HSP70, it may  relieve the inhibition of general protein synthesis in stressed myotubes by increasing dephosphorylation  of  eIF-2oc.  The  direct  effect  of  glutamine  on  phosphorylation/dephosphorylation of eIF-2ot remains to be determined in skeletal muscle. In addition to its role in protecting protein synthesis, HSP70 has also been suggested to protect proteins from degradation in stressed cells (6).  Since glutamine increases the expression  of HSP70 in stressed muscle cells, it is possible that HSP70 may play a role in accounting for  the inhibitory effect of glutamine on protein degradation.  95 However, in the preceding chapter,  glutamine was shown to inhibit the rate of degradation of long-lived proteins in both normalcultured and heat-stressed myotubes, but glutamine was shown to increase HSP70 expression only in stressed muscle cells.  These observations suggest that there must be additional  mechanisms underlying the inhibitory effect of glutamine on protein degradation. It is of interest to note that unlike HSP70, the amount of HSP27 is not regulated by glutamine in stressed muscle cells, though HSP27 also confers protection to cells from stress damage (13). by others.  The discordance in the regulation of HSP70 and HSP27 has also been reported For example, in certain rat tissues (e.g. brain and lung) HSP27 is regulated  differently from HSP70 by heat stress (14).  Physical restraint of rats induces the expression of  HSP70 but not HSP27 in the adrenal cortex (15).  The hspll gene, like the hsplO gene,  contains the consensus sequences for the HSE. Taken together, these observations suggest a distinct pathway for HSP27 induction, and the transcriptional regulation at the HSF-HSE binding activity seems not to be the sole mechanism for HSP induction. These findings also suggest that the stimulatory effect of glutamine on HSP70 expression may be specific. In the present work, HSP27 is found to be constitutively expressed in skeletal myotubes, and its expression is increased by heat stress. This is in agreement with results obtained from other type of mammalian cells (18). In a preliminary experiment with L6 myotubes, we observed that the rate of incorporation of S-methionine into HSP70 is increased by increasing the concentration of 35  glutamine.  This preliminary observation suggests that the increase in the amount of HSP70  96 shown in the present work is, in part, due to an increase in the rate of synthesis of this protein. This concept is also supported by the present study which shows that the abundance of hsplO mRNA is also increased by glutamine. It is interesting to note that, unlike HSP70 protein and mRNA, the D N A binding activity of HSF is not altered by glutamine in stressed muscle cells. Similarly, Engleberg et al. (19) have reported that ras, a regulatory protein in the Ras signalling pathway, downregulates the transcription of hsplO gene without affecting the D N A binding activity of HSF.  These data  suggest that certain pathway(s) other than activation of HSF may be responsible for the effect of glutamine on HSP70 expression. Besides the HSE, there is at least another regulatory element, the C C A A T box, in the hsp gene promoter region. Binding of the C C A A T binding protein (CBP), a transcription factor, to the C C A A T box can activate hsplO gene transcription (16).  It  is not known whether glutamine regulates other transcription factors such as CBP which in turn would regulate hsp gene transcription. In addition to providing a possible explanation for the mechanism of the regulation of protein turnover, the stimulatory effect of glutamine on HSP70 expression suggests a potential beneficial effect of glutamine to humans and animals. Though a number of biological functions of HSP have been elucidated, little effort has been made to study how cells' ability to express HSP can be enhanced to help mem cope with stress more successfully. In the present study, it has been clearly demonstrated that glutamine has a stimulatory effect on HSP70 expression at both protein and mRNA levels in stressed skeletal myotubes. These data are of physiological relevance, because skeletal muscle is a tissue often subjected to stressors. For example, during  exercise the temperature of skeletal muscle can reach as high as 4 2 ° C (17).  97 In addition, HSP70  expression in various tissues from rats has been reported to decrease with increasing animal's age (15); therefore, provision of glutamine may increase HSP expression in muscle as well as other tissues thereby protecting them from stress damage. In summary, the present work demonstrates that glutamine specifically increases HSP72 expression at both protein and mRNA levels in heat-stressed skeletal myotubes. This provides an avenue for exploring the mechanism by which glutamine regulates protein turnover in skeletal muscle cells. This work also suggests that HSP expression in mammalian cells can be modulated via a nutritional approach, which would be of clinical interest.  98  - HSP72  1  Control  Heat-stressed  B actm  C  0  0.65  5  10  Glutamine (mM) Figure 1. Glutarnine increases HSP72 expression in heat-stressed L8 skeletal myotubes. Western blotting analysis of HSP72 using a monoclonal antibody against rat HSP72 was performed as described in "Materials and Methods" section. In panel A , l,2,3,and 4 represent 0, 0.65, 5, and 10 m M glutamine, respectively. Panel B shows the levels of actin of the same samples in panel A . In panel C , the relative abundance of HSP72 in heat-stressed groups was calculated based on the densitometric results. Data are means ± S E M (n=3). a and b: significantly higher at P<0.01 and P < 0.001, respectively, than the 0 m M glutamine group.  99 -HSP27  1  2  3  Control  4  4 Heat-stressed 1  2  3  B -actin  C  2.5  T  B  T  fl  •9  i_T_ 9 0 mM • 0.65 mM • 5 mM ELMOmM  1.5  > i3  1  (0  o  CN  OH  -  0.5  Control myotubes  Stressed myotubes  Figure 2. Expression of HSP27 is not regulated by glutamine in L8 skeletal myotubes.  Western blotting analysis of HSP27 using a polyclonal antibody against rat HSP27 was performed as described in "Materials and Methods" section. In panel A , 1, 2, 3, and 4 represent 0, 0.65, 5, and 10 m M glutamine, respectively. Panel B shows the levels of actin from the same samples as in panel A . Panel C shows the relative levels of HSP27 in samples from control and heat-stressed myotubes. Data are mean ± S E M (n=3).  100  A - hspll - hsp!3  - 18S r R N A  1  2  3  Control  4  1  2 3 Heat-stressed  4  B  0  0.65  5  10  Glutamine (mM)  Figure 3.  Glutamine  increases hsplO m R N A expression in heat-stressed L8 skeletal  myotubes. Northern blotting analysis of hsplO mRNA was performed as described in "Materials and Methods" section. In panel A , 1,2,3, and 4 represent 0, 0.65, 5, and 10 m M glutamine, respectively. Positions of hspll and hspll mRNA as well as 18S r R N A are denoted. In panel B , the relative abundance of hspll mRNA in heat-stressed groups was calculated based on the densitometric results. Data are means ± S E M (n=3). a and b: significantly higher than the group of 0 m M glutamine at P<0.05 and P<0.001, respectively.  101  - HSF-HSE  - non-specific binding  - free probes 1  Figure 4.  2  3  4  1  2  3  4  The D N A binding activity of heat shock transcription factor is not regulated by  glutamine in L8 skeletal myotubes. Gel mobility shift assay was performed as described in "Materials and Methods" section. In panel A , all lanes contained the same amount of nuclei extract protein from the same heat-stressed sample. Lane 2 contained 25-mol excess of unlabelled H S E . Lane 3 contained 100-mol excess of unlabelled H S E . Lane 4 contained 100mol excess of unlabelled H S E - M . In panel B, lanes 1, 2, 3, and 4 represent 0, 0.65, 5, and 10 m M glutamine, respectively. The specific binding of H S F - H S E as well as non-specific binding and free probes are denoted.  V. References  102  1. Hammarqvist, F . , Wernerman, J., and A l i , R. (1989) Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis, and improves nitrogen balance. Ann. Surg. 209, 455-461. 2. Ardawi, M . S . M . (1992) Effects of xylitol- and/or glutamine-supplementation parenteral nutrition in septic rats. Clin. Sci. 82, 419-427. 3. Marts, R . L . and Hurst, R. (1992) The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates. J. Biol. Chem. 267, 18168-18174. 4. Liu, R A Y . , L i , X . , L i , L . and L i , G . C . (1992) Expression of human HSP70 in rat fibroblasts enhances cell survival and facilitates recovery from translational and transcriptional inhibition following heat shock. Cancer. Res. 52, 3667-3673. 5.  Chang, G . C , Liu, R., Panniers, R., and L i , G . C . (1994) Rat fibroblasts transfected with  the human 70-kDa heat shock gene exhibit altered translation and eukaryotic initiation factor 2a phosphorylation following heat shock. Intl. J. Hyperthermia 10, 325-337. 6. Georgopoulos, C . and Welch, W.J. (1993) Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, 601-634. 7. Cotto, J.J., Kline, M . , and Morimoto, R.I. (1996) Activation of heat shock factor 1 D N A binding precedes stress-induced serine phosphorylation. J. Biol. Chem. 271, 3355-3358. 8. Bradford, M . M . (1976) A simple method for protein concentration measurements. Anal. Biochem. 72, 248-254. 9.  Sambrook, J.E.F., Fritsch, F., Maniatis, T . (1989) Molecular Cloning: A Laborotary  Manual. Cold Spring Harbor Laboratory Press, N.Y. 10. Chomczynski, P. (1992) One-hour alkaline downward D N A and R N A transfer. Anal. Biochem. 201, 134-139. 11. L i , Y . , Ross, J., Scheppler, J.A., and Franza, B.R. (1991) A n in vitro transcription analysis of early response of the human immunodeficiency virus type 1 long terminal repeat to different transcription activators. Mol. Cell. Biol. 11, 1883-1893. 12. Murtha-Riel, P., Davies, M . V . , Scherer, B.J., Choi, S.-Y., Hershey, J.W.B., and Kaufman, R.J. (1993) Expression of a phosphorylation-resistant eukaryotic initiation factor 2asubunit mitigates heat shock inhibition of protein synthesis. J. Biol. Chem. 268, 12946-12951.  103 13. Lavoie, J . N . , Gingras-Breton, G . , Tanguay, R . M . , and Landry, J. (1993) Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization. J. Biol. Chem. 268, 3420-3429. 14. Blake, M . J . , Gerbson, D . , Fargnoli, J., and Holbrook, N.J. (1990) Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats. J. Biol. Chem. 265, 15275-15279. 15. Blake, M . J . , Udelsman, R., Feulner, G.J., Norton, D . D . , and Holbrook, N.J. (1991) Stress-induced heat shock protein 70 expression in adrenal cortex: A n adrenocorticotropic hormone-sensitive, age-dependent response. Proc. Natl. Acad. Sci. USA 88, 9873-9877. 16. Agoff, S . N . , Hou, J., Linzer, D.I.H., and Wu, B. (1993) Regulation of the human hsp70 promoter by p53. Science 259, 84-87. 17. Locke, M . , Noble, E . G . , and Atkinson, B . G . (1990) Exercising mammals synthesize stress proteins. Am. J. Physiol. 258, C723-C729. 18. Satoh, J. and Kim, S.U. (1995) Cytokines and growth factors induce HSP27 function in human astrocytes. J. Neuropathol. Exp. Neurol. 54, 504-512. 19. Engelberg, D . , Zandi, e., Parker, C.S., and Karin, M . (1994) The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor. Mol. Cell. Biol. 14, 4929-4937. 20. Nissim, I., States, B., Hardy, M . , Pleasure, J., and Nissim, I. (1993) Effect of glutamine on heat-shock-induced mRNA and stress proteins. J. Cell. Physiol. 157, 313-318. 21. Morimoto, R.I. (1993) Cells in stress: transcriptional activation of heat shock genes. Science 259, 1409-1410.  104  CHAPTER  I.  SIX  General Discussion A n d Suggestion F o r Future Studies  General Discussion  After decades of research work, it is now well recognized that certain amino acids such as glutamine and arginine have well-defined biochemical functions that are distinct from their participation in protein synthesis. The work reported in this thesis has focused on the metabolic features of glutamine in skeletal muscle.  The study was focused on  skeletal muscle because, (1) in humans and animals, skeletal muscle is the major site of glutamine synthesis in the body; and (2) skeletal muscle protein metabolism has been shown to be regulated by glutamine. A review of the literature dealing with glutamine metabolism in skeletal muscle revealed that all enzymes responsible for glutamine synthesis in this tissue have been well characterized with the exception of the enzyme G D H . The first part of this thesis was therefore directed towards determining whether this enzyme in skeletal muscle is subjected to metabolic regulation as shown in other tissues (16) and whether its activity changes during increased demands for skeletal muscle glutamine production during exposure to stress. It was demonstrated in this thesis that the G D H activity is increased by increasing the concentration of BCAA in skeletal muscle mitochondrial preparations from both rats and chicks. This result is of physiological significance because BCAA in the circulation are largely taken up by skeletal muscle (3). regulate the activity of G D H in the liver (16).  Similarly, BCAA have also been shown to  105 Despite the similarity in structure of the B C A A , their ability to increase G D H activity varied considerably.  Leucine was shown to increase G D H activity in muscle in  both rats and chicks, while isoleucine increased G D H activity in rat muscle but not in chick muscle, and valine was shown not have a significant effect on G D H activity in either rat or chick skeletal muscle. Although the mechanism accounting for these differential effects of B C A A remains to be studied, it was shown that the oc-amino group is required for leucine to have an effect.  Similarly, B C A A have been reported to have differential stimulatory  effects on other enzymes  in skeletal muscle such as branched-chain cc-keto acid  dehydrogenase (4). To biochemically convert one substrate to its end product, different species may possess different metabolic pathways. For example, it has been reported that the pathway for glutamine degradation in skeletal muscle is different in rats than in chicks (2).  The  present study showed another species difference between rats and chicks in terms of the effect of B C A A on G D H activity in skeletal muscle. difference remains to be determined.  The mechanism underlying this  To date, studies on glutamine metabolism and its  functions have been conducted largely in mammals.  There is an obvious need of further  work to characterize glutamine metabolism and function in skeletal muscles from other types of animals. During metabolic acidosis, increased rates of renal glutamine metabolism play an important role in the kidney's ability to clear increased amounts of H  +  from the body. The  rate of glutamine synthesis is considerably increased due to the increased activity of GS during metabolic acidosis (17).  In this thesis, another regulatory site at which acidosis  106 upregulates glutamine synthesis in muscle has been identified. increase skeletal muscle G D H activity.  Acidosis was found to  In addition, it was found to increase G D H  responsiveness to BCAA-induced upregulation. While the molecular basis for these effects remains unknown, a possible change in the protein conformation of G D H owing to the low pH during acidosis may constitute one of the mechanisms. It is important to note the coordination in glutamine metabolism between different organs in the body during metabolic acidosis.  In skeletal muscle, increased glutamine  synthesis is facilitated by increases in both G D H and GS activity. The liver switches from consuming glutamine under normal conditions to releasing glutamine during acidosis. The production and release of glutamine by these organs is designed to meet the increased glutamine requirements of the kidney in which the activities of glutaminase and G D H (in the direction of oxidative deamination) are increased, leading to the removal of 3 moles of H  +  from the body by degradation of every mole of glutamine.  Although both renal and  muscle G D H activity increase, these two enzymes catalyze the reaction in opposite directions. This coordination is critical for the body to combat elevated H  +  concentrations.  While the mechanism(s) for this inter-organ coordination remains unclear, a couple of possibilities can be raised: (1) humoral factors induced by acidosis may act on skeletal muscle cells and modulate GS and G D H activity. For example, glucocorticoids may be an external signal which will upregulate glutamine production in skeletal muscle; (2) low pH in skeletal muscle cells activates a certain signal transduction pathway which results in activation of transcription of GS and G D H genes; (3) increasing concentration of H  +  in the  liver decreases glutamine degradation by inhibiting hepatic glutaminase activity, which  107 results in a decrease in urea production and saves H C 0 ~ from urea biosynthesis. 3  saved H C 0 " then combines with H , with production of H 0 and C 0 , and C 0 +  3  2  2  2  The  will be  excreted via respiration. In addition to metabolic acidosis, a number of other stressors also cause a substantially increased demand for glutamine production in skeletal muscle (18).  This  increased demand for glutamine is partially met by an increase in the rate of glutamine synthesis (Chapter 2) and partially met by a drain of the relatively large intramuscular glutamine pool (e.g. ref. 5).  Intramuscular concentrations of glutamine have been shown  to be positively correlated with the rate of protein synthesis and negatively correlated with the rate of protein degradation in skeletal muscle (5,6).  However, to date, a very  fundamental but important issue, the nature of these effects of glutamine on protein turnover, remains unclear.  The second part of this thesis was therefore directed towards  further characterizing glutamine's regulatory effects on skeletal muscle protein turnover. This thesis presents the first evidence that glutamine stimulates the rate of protein synthesis in stressed muscle cells but not in normal-cultured cells. This result indicates that previous work which showed that glutamine stimulates the rate of muscle protein synthesis in rat hindlimb perfusion and in in vitro skeletal muscle incubation preparations (7,8) may mimic the effect of glutamine in stressed skeletal muscle, because the experimental techniques used in these models may have introduced stressors into the experimental conditions as reported by Udelsman and Halbrook et al. (9).  In the present work, we  employed a skeletal muscle cell culture system in which experimental conditions can be precisely controlled.  The results presented are consistent with clinical observations that  108 during stress (trauma, operation, and sepsis etc.), a loss of skeletal muscle mass is accompanied by a decrease in intramuscular concentration  of glutamine, and that  administration of glutamine can inhibit the wasting of skeletal muscle. In this thesis it was shown, for the first time, that the inhibitory effect of glutamine on muscle protein degradation exists only on long-lived proteins but not short-lived proteins.  Most studies on skeletal muscle protein degradation have involved examination  of changes in total protein.  The total muscle protein pool is comprised of short-lived  proteins and long-lived proteins, which consist of the non-myofibrillar proteins and the myofibrillar proteins, respectively.  In recent years, it has been found that degradation of  these two fractions of protein may be differently regulated by endocrine factors (10).  In  the present study, it was found that glutamine inhibits the degradation of long-lived proteins but not short-lived proteins in muscle cells. The long-lived proteins comprise the vast majority of muscle proteins.  Therefore, the data suggest that glutamine confers a  protection on the major fraction of total muscle proteins.  Similarly, it has been reported  that insulin and IGF-I also inhibit the degradation of long-lived proteins (e.g. myosin) but not short-lived proteins in skeletal muscle cells (10). Based on glutamine's effect on muscle protein turnover and its other functions such as increasing the function of immunocytes, and the fact that glutamine concentrations in the plasma pool and intramuscular pool decrease substantially during disease, there is a considerable clinical interest in supplementing this amino acid to help retain muscle protein mass thereby improving nitrogen balance of the whole body as well as to reduce clinical infection and increase lymphocyte recovery following clinical treatments such as bone-  109 marrow transplantation (11).  Because free glutamine in aqueous solution is unstable, in  recent years, a dipeptide L-analinyl-L-glutamine has been proven to have the same physiological effects as glutamine but is more stable and more soluble than free glutamine (12).  Therefore, this dipeptide holds a very promising potential for future clinical  application. Following the finding that glutamine's anabolic effect on protein metabolism may be more important in stressed muscle cells than in normal muscle cells, it was decided to study possible mechanisms underlying it. It was decided to explore the effect of glutamine on HSP expression in skeletal muscle, because (1) these proteins are well known to be selectively expressed during stress and play important roles in protecting proteins from stress-induced damage, and (2) glutamine has been shown to regulate HSP70 expression in Drosophila Kc cells and opposum kidney cells (13,14).  We found that in heat-shocked  skeletal muscle cells but not in normal-cultured cells, the amount of HSP70 increases as the concentration of glutamine increases in the culture media. The increase in the amount of HSP70 by glutamine means that glutamine may play a role in regulating the HSP70associated processes in stressed muscle cells.  This may constitute one mechanism by  which glutamine regulates protein turnover in stressed muscle cells.  Nevertheless, the  precise role of HSP70 in the mechanism of action of glutamine requires more direct evidence, which could be provided by experiments such as blocking hsplO gene transcripts by either the antisense R N A technique or the gene knockout technique and then examining the effect of glutamine on muscle protein metabolism.  110 In an attempt to examine the mechanism by which glutamine increases HSP70 expression, it was found that glutamine increases hsplO mRNA expression but does not influence the DNA-binding activity of HSF in stressed muscle cells. Similarly, Engleberg et al. (15) have reported that Ras, a regulatory protein in the ras signalling pathway, downregulates the transcription of the hsplO gene without affecting the D N A binding activity of HSF.  It appears that glutamine increases hsplO gene expression by other means.  For  example, glutamine may have an effect on the C C A A T binding proteins which bind to the C C A A T box in the hsplO promoter and activate gene transcription.  II. Conclusions In summary, the present work demonstrated that skeletal muscle G D H activity is differentially upregulated by B C A A in rats and chicks. among the B C A A s .  Leucine has the greatest effect  The effect of leucine and isoleucine on rat muscle G D H is additive.  During metabolic acidosis, both G D H and GS activity in skeletal muscle are increased. Furthermore, during acidosis muscle G D H is more responsive to regulation by B C A A . This work also demonstrated that glutamine stimulates the rate of skeletal muscle protein synthesis in a condition-dependent manner. Glutamine exhibits an inhibitory effect on the rate of degradation of long-lived proteins but not short-lived proteins in muscle cells. HSP70 may be a mediator of the effect of glutamine on muscle protein metabolism, as both protein and mRNA levels of HSP70 are increased by increasing the concentration of glutamine in stressed muscle cells.  Ill  HI. Suggestions for Future Studies Although the objectives of this thesis have been achieved, many additional interesting issues arise and need to be studied.  A number of suggestions are therefore  made for future studies. (1) There is a need to study signal transduction pathways which increase the activities of G D H and GS in skeletal muscle during metabolic acidosis.  The downstream  events of low cytosolic pH and the changes of G D H and GS gene transcription in response to acidosis in muscle cells also need to be investigated. (2) The mechanism by which skeletal muscle G D H becomes more sensitive to regulation by B C A A in skeletal muscle during acidosis needs to be investigated. A change in conformation of G D H may be involved in such a mechanism. (3) A study is needed to determine whether glutamine influences cell volume and lysosomal activity of muscle cells. (4) The effect of glutamine on the translational machinery in muscle cells needs to be  examined.  For  example,  one  can  determine  how  glutamine  regulates  the  phosphorylation/dephosphorylation status of eIF-2a, and which protein kinase pathway or phosphorylase system is activated by glutamine in skeletal muscle. (5) The role of HSP70 in the effect of glutamine on muscle protein turnover needs to be studied. This study could be performed by blocking the hsplO gene transcripts and then examining the effect of glutamine on protein turnover in skeletal muscle cells. (6) Mechanism(s) underlying the effect of glutamine on hsplQ gene transcription in stressed muscle cells needs to be explored.  To carry out this study, one can make an  112 expression vector containing a hsplO gene promoter ligated with a reporter gene, transfer the engineered plasmid into muscle cells, and then examine the effect of glutamine on the expression activity of the transferred plasmid. It could also be of value to determine the effect of glutamine on the function of transcription factors other than HSF which are involved in activation of hsp gene transcription (e.g. the C C A A T binding proteins) in skeletal muscle cells. In conclusion, glutamine has a very promising role in improving the health of humans and animals. There is a need to further investigate the mechanism underlying the diverse biochemical effects of this amino acid.  113  IV. References 1. Krebs, H . (1980) Glutamine metabolism in the animal body. In: Mora, J., Palacios, R. eds. Glutamine: metabolism, enzymology, and regulation, pp. 319-329, Academic Press, New York. 2. Wu, G . , Thompson, J.R., and Baracos, V . E . (1991) Glutamine metabolism in skeletal muscles from the broiler chick (Gallus domesticus) and the laboratory rat (Rattus norvegicus). Biochem. J. 274, 769-774. 3. Goldberg, A . F . and Chang, T . W . (1978) Regulation and significance of amino acid metabolism in skeletal muscle. Fed. Proc. 37, 2301-2307. 4. Aftring, R.P., Block, K . P . , and Buse, M . G . (1986) Leucine and isoleucine activate skeletal muscle branched-chain a-keto acid dehydrogenase in vivo. A m . J. Physiol. 250, E599-E604. 5. Jepson, M . M . , Bates, P . C . , Broadbent, P., Pell, J . M . , and Millward, D.J. (1988) Relationship between glutamine concentration and protein synthesis in rat skeletal muscle. Am. J. Physiol. 255, E166-E172. 6. Ardawi, M . S . M . (1992) Effects of xylitol- and/or glutamine- supplementation parenteral nutrition in septic rats. Clin. Sci. 82, 419-427. 7. Maclennen, P . A . , Brown, R . A . , and Rennie, M . J . (1987) A positive relationship between protein synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle. FEBS Lett. 215, 187-191. 8. Wu, G . and Thompson, J.R. (1990) The effect of glutamine on protein turnover in chick skeletal muscle in vitro. Biochem. J. 265, 593-598. 9. Udelsman, R. and Holbrook, N.J. (1994) Endocrine and molecular responses to surgical stress. Curr. Problem Surg. 31, 653-720. 10. Gulve, E . A . , Mabuchi, K . , and Dice, J.F. (1991) Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes. J. Cell. Physiol. 147, 37-45. 11. Ziegler, T . R . , Young, L . S . , and Benfell, K.(1992) Clinical and metabolic efficacy of glutamine-supplemented parenteral nutrition after bone marrow transplantation. Ann. Intern. Med. 116, 821-828. 12. Stehle, P., Zander, J., and Mertes, N . (1989) Effect of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance after major surgery. Lancet I, 231-233.  114  13. Sanders, M . M . and Kon, C . (1991) Glutamine is a powerful effector of heat shock protein expression mDrosophila Kc cells. J. Cell. Physiol. 146, 180-190. 14. Nissim, I., States, B . , Hardy, M . , Pleasure, J., and Nissim, I. (1993) Effect of glutamine on heat-shock-induced mRNA and stress proteins. J. Cell. Physiol. 157, 313318. 15. Engelberg, D . , Zandi, E . , Parker, C.S., and Karin, M . (1994) The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor. Mol. Cell. Biol. 14, 4929-4937. 16. McGiven, J . D . , Bradford, N . M . , Crompton, M . , and Chappell, J.B. (1973) Effect of L'-leucine on the nitrogen metabolism of isolated rat liver mitochondria. Biochem. J. 134, 209-215. 17. Souba, W . W . and Wilmore, D . W . (1991) Interorgan glutamine metabolism during acidosis. JPEN 14, 77S-85S. 18. Max, S.R., Mill, J., Mearow, K . , Konagaya, M . , Konagaya, Y . , Thomas, J.W., Banner, C , and Vitkovic, L . (1988) Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. A m . J. Physiol. 255, E397-E403.  

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