Insulin Action and Its Disturbances in Disease - part 3 pptx

108 THE EFFECT OF INSULIN ON PROTEIN METABOLISM mechanisms whereby insulin selectively affects mRNA stability have not been well defined. Initiation of mRNA translation into protein begins with formation of the 7- methyl guanine cap at the 5-prime end of the RNA. A number of cap-associated proteins including eukaryotic initiation factor 4E (eIF-4E), eIF-4G and phospho- rylated heat–acid-stable protein (PHAS-1) are influenced by insulin. PHAS-1 binds to the eIF-4E cap binding protein, insulin enhances phosphorylation of PHAS-1 and favours dissociation of eIF-4E and PHAS-1. 14 This allows for binding of eIF-4E to eIF-4G and hence favours association with the 40S ribo- somal subunit and translation initiation. 15, 16 Also important in the binding of the 40S ribosomal subunit is eIF-2, and the binding of this initiation factor is dependent on its association with GTP. Controlling the recycling of the GTP/GDP-bound state of eIF-2 is eIF-2B. Insulin increases the activity of eIF- 2B and favours the GTP-bound (active) state of eIF-2, which in turn enhances translation initiation. 16, 17 Protein elongation depends on the action of multiple elongation factors. Among these are elongation factor 2 (eEF-2). This factor is important for move- ment of the ribosomal complex along the mRNA and for the migration of the amino acyl-tRNA from the acceptor site to the peptidyl site of the ribosome. 18 Insulin enhances eEF-2 activity by reducing its phosphorylation via inhibition of its kinase. 19 A comprehensive description of the molecular mechanisms of insulin’s effect on translation is available in review form. 20 The abundance of ribosomes and RNA content in part determines the cellu- lar capacity to synthesize protein. 21 Ribosomes are made up of approximately 80 proteins and 4 ribosomal RNA (rRNA) species. Production and assembly of ribosomes takes place in the nuclei. In chick embryo fibroblasts insulin has been shown to induce a fourfold increase in the synthesis of ribosomal proteins. 22 Similar findings have been made in mouse myoblasts. 23 This appears to in part be due to post-transcriptional events. Messenger RNAs that encode ribosomal proteins appear to be preferentially associated with polysomes in mouse myoblasts treated with insulin. 23 The synthesis of rRNAs has been shown to increase after insulin treatment in a variety of cell types including fibroblasts, 22, 24 myoblasts 23 and hepatocytes. 25 Finally, insulin may also reduce the rate of ribosome degradation. 25–27 Effect of insulin on intracellular events controlling protein breakdown Cellular protein breakdown is a tightly controlled and highly specific process. In catabolic states such as starvation, sepsis or insulin deprivation, protein break- down can markedly increase. At the intracellular level, proteins can be degraded through several pathways including the lysosomal pathway, the calcium-depen- dent protease pathway or the ubiquitin–proteosome path. 28 The majority of proteins in mammalian cells are degraded through the ubiquitin–proteosome MOLECULAR MECHANISMS OF INSULIN’S EFFECT ON PROTEIN TURNOVER 109 pathway. Proteins are targeted for breakdown by covalent conjugation to ubiq- uitin. This is an ATP-dependent process, and multiple ubiquitin molecules are added such that a ubiquitin chain is formed. 29 Proteins with a ubiquitin chain attached are degraded by the ATP-dependent 26S proteosome complex. The rate-limiting step in this process is ubiquitin conjugation. Indirect evidence from animal studies suggests that ubiquitin-dependent protein degradation is important in states of insulin deprivation. Protein breakdown rates increase markedly in rats that are made insulinopenic by treatment with streptozotocin. Treatment with selective inhibitors of the lysosomal or calcium-dependent protease pathways did not affect protein breakdown. When ATP synthesis was blocked, how- ever, protein breakdown declined. 30 This suggests that ATP-dependent ubiqui- tin–proteosome-mediated protein breakdown is important in insulin deficiency. Others have shown that mRNAs for ubiquitin–proteosome proteins are increased in the insulin-deficient state. 31 If diabetic rats are treated with insulin, pro- tein breakdown is reduced, and ubiquitin–proteosome mRNAs are reduced to control levels. 32 Acidosis and increased cortisol levels, which occur following insulin deprivation, stimulate protein degradation in the ubiquitin–proteosome pathway. 32, 30 In summary, insulin deficiency in a diabetic animal model shows coordinate time-dependent changes in different proteolytic pathways in muscle, resulting in increased overall proteolysis. Only the capacity of non-lysosomal processes seems to be altered in muscle in response to insulin deficiency. The many intracellular mechanisms of insulin action to affect protein turnover are summarized in Figure 4.2. Insulin effect on protein synthesis ribosomes mRNA transcription -IRE promoter elements Insulin effect on protein breakdown Ubiquinone–proteosome path activity mRNA stability Translation initiation -PHAS-1 phosphorylation eIF-2B activity Elongation eEF-2 phosphorylation - - mRNA - - Figure 4.2 Effect of insulin on protein turnover 110 THE EFFECT OF INSULIN ON PROTEIN METABOLISM Insulin as a regulator of protein turnover in vitro and in situ Studying the effect of insulin on protein turnover in humans is complicated. The body is an intricate system with many hormonal mechanisms that interact with one another. Therefore, altering a single hormone such as insulin can lead to changes in other hormones, including growth hormone, glucagon, cortisol and epinephrine to name a few. These in turn can lead to changes in concentrations of substrates, such as amino acids, glucose and fatty acids, in heart rate and in blood flow or have more direct effects on protein synthesis and breakdown. Because of these complexities several in vitro and in situ systems have been used to study insulin’s effect on protein turnover. By using an in vitro or in situ model one can simplify the experiment by removing confounding factors like other hormones and alterations in other parameters such as blood flow. The simplest model is an in vitro cell culture system. In this system a homoge- neous population of cells can be studied under very controlled conditions. Using a specific cell line such as L6, a rat skeletal muscle myoblast line, allows one to determine insulin’s effect on protein turnover within a single cell type. The components of the cell medium and the insulin concentration can be well defined. Using this model, it has been shown that insulin stimulates protein synthesis in L6 myoblasts. 33, 34 This type of model is ideal for studies of signal transduction path- ways stimulated by insulin. 35, 36 The biggest disadvantage of a cell culture model is that it may not be representative of the whole body system. Cell lines are gener- ally transformed in some manner and even when differentiated the cells lack some characteristics of cells in vivo. For example, even when L6 myoblasts are differen- tiated into myotubes, they do not express the same myosin heavy chain isoforms as adult skeletal muscle. 37 In addition, within a tissue such as skeletal muscle, there are many different cell types such as fibroblasts, vascular muscle cells, vascular epithelium etc. These may be important modulators of skeletal muscle cells and the effect would be missed in a simple cell culture system. In order to account for these parameters but to still maintain a very con- trolled system, several investigators have used in situ methods to study the effect of insulin on protein turnover. These models have utilized perfused animal diaphragm, heart, skeletal muscle, or whole limbs. 38–40, 16 Consistently, insulin reduces tissue protein breakdown. Although in situ studies provide a simpli- fied system that may be optimal for understanding mechanisms behind insulin’s effect on protein metabolism, they too may not fully represent the in vivo situa- tion. Ultimately, to fully understand insulin’s regulation of protein metabolism in humans, one must study an in vivo system. A number of methods have been used to measure protein turnover in animal models and in human subjects. Animal studies Rodent models have been extensively used to study insulin effect on protein metabolism. In studies performed in growing rodents indicated that insulin MEASUREMENT OF PROTEIN METABOLISM 111 deficiency was associated with reduced synthesis rates of muscle proteins, where- as in fully grown rodents insulin failed to stimulate muscle protein synthesis. 41 Similarly, in piglets insulin stimulates muscle protein synthesis rates 42 and with increasing age the magnitude of synthesis rates decreases. 43, 44 These mea- surements were performed on mixed tissue proteins, representing the average fractional synthesis rates of many proteins. Recent studies in sexually matured miniature pigs demonstrated that when the insulin effect was determined on different subfractions of muscle proteins a specific stimulatory effect on mus- cle mitochondrial protein synthesis was observed, with no significant effect on synthetic rates of sarcoplasmic and myosin heavy chain proteins. 45 In contrast, the insulin effect on liver proteins in mini-pigs is variable, showing no effect on liver tissue protein synthesis whereas synthesis rate of fibrinogen was inhibited. 46 Since human adult life is much longer than that of rodents and pigs it is impor- tant to study the insulin effect on adult humans to understand the regulation of protein turnover in humans after the genetic potential for growth is passed. 4.3 Measurement of protein metabolism (synthesis and breakdown or turnover) in human subjects Measurement of protein turnover Net protein turnover, a result of both synthesis and breakdown, can be quanti- fied using a number of different methods. Some of the more global techniques include whole body nitrogen balance, 3-methylhistidine excretion (specifically for myofibrillar protein breakdown), regional amino-acid balance and systemic amino-acid tracer incorporation. By using biopsies or separation techniques, protein synthesis can also be measured within a specific tissue or for a specific protein. Ultimately, the regulation of protein concentrations may be a result of many factors including changes in gene expression, mRNA stability and trans- lation efficiency. Assessment of changes in protein turnover induced by insulin can take place at many levels: (1) the cellular level, where one may observe the mRNA changes and changes in translation efficiency; (2) the tissue level, where one can study the effect of insulin on a specific tissue or set of proteins (such as skeletal muscle on myofibrillar proteins); (3) the regional or whole body level, where one can more globally assess insulin’s effects (Figure 4.3). In this section, the various methods of studying protein turnover in human subjects will be discussed. Following the description of each method, we shall review the use of the method to assess the effect of insulin on protein turnover. Whole body nitrogen balance When protein is broken down, free amino acids and their metabolites are released into the circulation. All amino acids contain at least one nitrogen molecule. Transamination is a critical process necessary to transfer nitrogen for synthesis 112 THE EFFECT OF INSULIN ON PROTEIN METABOLISM Whole body Regional Tissue Amino-acid availability Specific proteins Transcriptional and translational regulation DNA and mRNA Figure 4.3 Sites to assess insulin effect on protein metabolism of non-essential amino acids. Amino acids that are oxidized or transaminated can give rise to ammonia. Most of this circulating ammonia is converted to urea in the liver via the ornithine cycle and can be excreted in the urine. Urinary nitrogen is composed of 80–85 per cent urea and ammonia. Another 5–10 per cent of urinary nitrogen is accounted for by creatine, creatinine, uric acid and free amino acids. 47 By collecting urine and stool for 24 hours, one can quantify total body nitrogen loss. To determine net nitrogen loss daily nitrogen intake also has to be measured. This reflects the summation of multiple processes including changes in protein breakdown, protein synthesis, dietary protein intake, and alterations in the recycling of amino acids. Although this method seems straightforward in concept, there are several problems with it. First, results can be affected by changes in renal function, hydration status, certain medications and the amount of protein that is ingested. Generally, subjects are asked to maintain a specific diet (normalized for protein intake) for several days before a study. This reduces the variability in nitro- gen generated by dietary protein intake. In diabetic patients with reduced renal function, proteinuria or renal tubular acidosis, the results of whole body nitrogen balance can be unreliable. Insulin effect as measured by nitrogen balance and free amino-acid concentrations Early studies on diabetic patients used whole body nitrogen balance to assess the effect of insulin on whole body protein metabolism. Withdrawal of insulin MEASUREMENT OF PROTEIN METABOLISM 113 treatment has been shown to increase urinary nitrogen losses and to increase the concentrations of several essential amino acids, especially branched chain amino acids. 48, 1, 49 Insulin treatment normalizes the increased urinary nitrogen loss and the increased circulating amino-acid concentrations. 5, 49, 50 In 1976, Walsh and colleagues 51 studied 18 uncontrolled diabetic patients before and after 6–8 weeks of treatment. This group was a mixture of type 1 and type 2 diabetic patients. In subjects who were given insulin to control blood sugars, there was an average weight gain of 8.7 per cent and average nitrogen balance of +13 per cent. In the diabetic patients treated with diet alone or with diet and an oral agent there was no change in weight, and only a +3.8 per cent nitrogen balance. 51 This increase in body mass and a positive nitrogen balance shows that in patients who are relatively insulin deficient (diabetic patients) treatment with insulin has an anabolic effect. 3-methylhistidine quantification Skeletal muscle actin and myosin contain 3-methylhistidine (3-MH). This mod- ified amino acid is not further metabolized or reutilized after release from actin or myosin. The only fate is urinary excretion. These properties make 3-MH a potential surrogate for muscle protein breakdown. 3-MH measurements com- paring arterial versus venous concentrations have been made across local tissue beds (the forearm or leg). In this type of study, the increase in venous concentra- tion of 3-MH can provide good estimates of muscle protein breakdown. Whole body studies quantifying urinary excretion of 3-MH are difficult to interpret and do not necessarily reflect only skeletal muscle protein breakdown because smooth muscle (particularly intestinal) can give rise to as much as 10 per cent of urinary 3-MH. 47 Moreover, myofibrillar proteins have slow turnover (approxi- mately 1–2 per cent/day), which makes it difficult to perform short term studies on the effect of insulin on myofibrillar protein breakdown. Insulin effect as measured by 3-MH In healthy volunteers, insulin infusion does not change the flux of 3-MH across the leg or forearm. 52, 53 In contrast, in a study of poorly controlled diabetic patients there was a substantially greater excretion of urinary 3-MH as com- pared with healthy volunteers. When the same diabetic patients were restudied after achieving satisfactory glycemic control, urinary 3-MH excretion was not different from that of healthy volunteers. 54 This suggests that insulin deficiency results in increased muscle (we cannot differentiate between skeletal and smooth) protein breakdown and that replacement of insulin inhibits this breakdown. The available techniques to measure 3-MH have widely varying coefficients of vari- ation, which makes these measurements insensitive to small differences. 114 THE EFFECT OF INSULIN ON PROTEIN METABOLISM 4.4 Whole body and regional protein turnover The effect of amino-acid availability Amino acids are the building blocks of proteins. The availability of these build- ing blocks can determine whether protein synthesis can take place. Based on the k m value of amino-acyl tRNA ligase it was argued that normal physiologi- cal changes in free amino acids have little effect on protein synthesis. However, recent studies have clearly demonstrated that amino acids by themselves enhance translational efficiency of gene transcripts. 55, 56 Amino acids can be provided by reuse of amino acids provided by protein breakdown or they can be provided in the form of a meal or infusion. Amino-acid availability is of great impor- tance when considering insulin’s effect on protein turnover, because amino-acid availability has been shown to be a major factor controlling muscle protein synthesis. 57–59 Systemic or regional infusion of insulin has been shown to reduce blood concentrations of amino acids (hypoaminoacidemia). 60, 48, 61, 62 A reduced rate of protein breakdown by insulin is the likely cause of this insulin-induced hypoamino acidaemia. Another potential site of the insulin effect is on transmembrane transport of amino acids. Transmembrane transport of neutral amino acids in skeletal muscle is mediated by at least four different sys- tems (A, ASC, L and N m ). Regional studies of forearm skeletal muscle using methylaminoisobutyric acid (MeAIB), a non-metabolizable amino-acid analogue specific for system A amino-acid transport, showed that physiologic hyperinsuli- naemia stimulates the activity of system A amino-acid transport. 63 This effect may play a role in determining the response of muscle amino-acid transport and protein metabolism in response to insulin. When trying to reconcile the results of whole body and regional studies in humans, it is important to note whether blood amino-acid concentrations were monitored and/or clamped during the study. When discussing results below, we shall note this. Amino-acid tracer techniques Use of a labelled amino-acid tracer allows simultaneous determination of pro- tein synthesis and breakdown rates at the whole body level and across tissue beds. Quantifying incorporation of the tracer into a specific protein or protein fraction or mixed proteins can yield the synthesis rate. Measuring the dilution of the tracer (provided it is a labelled essential amino acid) in the free tracee (amino-acid) pool in the steady state is extensively used for calculation of pro- tein breakdown rates. During a steady state condition the rate of appearance of an essential amino acid such as leucine is the same as its disappearance rate. Therefore, in a fasted state, rate of appearance is equivalent to protein breakdown because essential amino acids only appear from protein breakdown, and rate of disappearance (sum of catabolism and incorporation into protein) can be estimated. Once the catabolic rate (e.g. leucine oxidation, phenylalanine WHOLE BODY AND REGIONAL PROTEIN TURNOVER 115 hydroxylation to tyrosine etc.) and flux (appearance or disappearance rate) are measured, rate of incorporation of amino acid into protein (protein synthesis) can be calculated by subtracting the catabolic rate of the amino acid from its flux 7 (Figure 4.3). In addition, from tracer and tracee measurements in artery and vein (e.g. femoral vein for leg or hepatic vein for splanchnic bed) as well as blood flow measurements (usually based on indicator dye dilution) the kinetics of protein (breakdown and synthesis) and net balances can be estimated in the respective tissue beds. 49 In addition, serial needle biopsy of skeletal muscle and infusion of an isotopic tracer and measurements of isotopic abundance of the tracer in muscle protein or proteins will allow the estimation of fractional synthesis rates of mixed proteins or specific proteins. 64 Similar approaches can be applied to measure fractional synthesis rates of circulating plasma proteins. 65, 66 The tracer technique, therefore, can be used to determine whole body, regional and specific protein (such as myosin heavy chain) synthesis rates. In most cases, if the appropriate samples are taken (including blood, breath samples and tissue biopsies), a single experiment can determine all of these parameters. Two tracer methods are widely used for determination of tissue protein synthesis rates in humans – flooding dose and continuous infusion. The flooding dose technique With the flooding dose a large amount of unlabelled amino acid (tracee) is injected as a bolus along with the labelled amino acid (tracer). 67 The goal of infusing this large dose is to quickly achieve an equilibrium of tracer concentra- tion between the plasma and the intracellular ‘precursor pool’. The obligatory ‘precursor pool’ is the amino acid acylated to its transfer RNA (amino-acyl tRNA). This is the step just prior to incorporation of the amino acid into a protein. To accurately calculate synthesis rates based on extracellular tracer enrichment, the extracellular tracer enrichment and intracellular ‘precursor pool’ enrichment must be in equilibrium. The primary advantage of this technique is that protein synthesis rates can be determined in a short period of time (10–30 minutes). Since a large amount of tracer is infused, it will make up a greater percentage of the amino acids incor- porated into protein. This is particularly useful in studies of acute interventions such as short term infusion of a compound. The main disadvantage of this technique is that a number of assumptions need to be made. First, the large bolus of amino acid must be assumed to have no effect on protein dynamics. Second, in order for rates of synthesis and breakdown to be calculated, one must assume that enrichment is at steady state during the study period, which may not be the case during a declining phase of both tracer and tracee. These assumptions can be incorrect if certain requirements of the flooding dose condition are not met, particularly if the concentration of 116 THE EFFECT OF INSULIN ON PROTEIN METABOLISM the ‘flooding dose’ is too low or the study period is too long. Either of these can cause non-equilibrium conditions in tracer enrichment between extracellular and intracellular compartments. The tracer in this approach is not truly in the ‘tracer amount’ and the high concentration of ‘tracer’ may affect the protein synthesis measurements. 68 The advantages and disadvantages of this technique have been described in detail elsewhere. 69–74 The continuous infusion technique With the continuous infusion technique, a continuous lower level infusion of tracer is given. In order to reach a steady state more quickly, the continuous infusion is typically preceded by a priming bolus of tracer. 75 The continuous infusion technique allows study over a long period of time (several hours). Hence, this technique is better suited to the study of proteins that have a slow rate of turnover. Most skeletal muscle proteins fall into this category. On the other hand, this technique is not ideal for quick turn over proteins because of the amino-acid recycling that can occur over a prolonged time period. Another disadvantage is that in most cases a surrogate measure of the oblig- atory precursor (amino-acyl tRNA) has to be used for calculation of protein synthesis. This results in underestimation of protein synthesis calculation. 76 For whole body measurements surrogate measures of intracellular pool, such as ketoisocaprioate in the case of leucine tracer, have been used with some strong theoretical reasons. 77 However, this approach is not practical with every amino-acid tracer. Amino-acid tracers In the past, radiolabelled amino acids were used as tracers. More recently, sta- ble isotope amino-acid tracers have been more widely used, which has many theoretical advantages and is more acceptable for volunteers for studies and institutional ethical committees. The incorporation of the tracer into protein can then be quantified by mass spectrometry. The amount of incorporated tracer is a reflection of the amount of newly synthesized protein over the time of the infusion. 7 The amino acid chosen for the tracer varies from study to study, and it is not uncommon to use more than one tracer within a single study. 7 For whole body studies tracers such as L[1- 13 C] leucine and labelled pheny- lalanine (e.g. L[ 15 N] phenylalanine L[ 2 H 5 ] phenylalanine) are extensively used. For regional studies involving skeletal muscle bed phenylalanine has many advantages, which include its small intracellular pool and thus the shorter period needed to equilibrate with the free amino-acid pool. Within skeletal muscle and the other tissues of the forearm or leg, phenylalanine is not metabolized. Protein synthesis rates can be determined by measuring the rate of disappear- ance of phenylalanine. However, if one is studying the splanchnic bed (liver and intestine), it is important to account for the conversion of phenylalanine WHOLE BODY AND REGIONAL PROTEIN TURNOVER 117 to tyrosine within the liver using an independent tracer of tyrosine. 7 Recent studies have also demonstrated that phenylalanine is converted to tyrosine in the kidney 78 besides in the liver. Therefore, for regional studies involving kid- ney as well, phenylalanine and tyrosine tracers have to be used to measure protein turnover. Leucine is an essential amino acid that composes 6–8 per cent of protein. Because leucine concentrations are high within most proteins, it provides large enrichment when used as a tracer. This is particularly useful when studying synthesis rates of proteins that have slow rates of turnover. Use of leucine in regional studies, however, can make calculations more complicated because it can be either directly incorporated into protein (non-oxidative metabolism) or reversibly transaminated to form ketoisocaproic acid (KIC). KIC can then be further oxidized to carbon dioxide and isovaleryl CoA (Figure 4.3) or reami- nated back into leucine. If leucine is labelled at the carboxyl carbon (e.g. 13 C) and the amino group with 15 N, it is possible to quantify leucine transamination rates. Leucine tracers with both labels have been used to measure transami- nation rates at the whole body 79 and regional levels. 80, 49 In order to account for the metabolic products one must collect breath samples for measurement of label within expired carbon dioxide or 13 CO 2 production across tissue beds in regional studies. 80 Measurement of 13 C-KIC enrichment can be a useful sur- rogate of the precursor pool leucyl-tRNA enrichment. Measurement of this compound requires far less muscle tissue and labour than does direct mea- surement of leucyl tRNA. It has been demonstrated in human studies to be a good surrogate 81 although muscle tissue fluid is closer to tRNA enrichment. 81 For studies involving liver proteins (plasma proteins such as albumin, fibrino- gen, APOB 100 etc.) plasma [ 13 C] KIC is an excellent surrogate measure of liver leucyl-tRNA enrichment. For skeletal muscle, muscle tissue fluid leucine enrich- ment is a better indicator of leucyl tRNA. Amino-acid tracers can thus be used to study protein kinetics of the whole body, of a region, of a certain tissue or of specific proteins. Insulin and protein turnover in type 1 diabetic patients using whole body leucine flux Type 1 diabetic patients are deficient in insulin, so perhaps the most dramatic effects of insulin can be observed in these subjects. One can study these patients in the insulin deficient state and compare these results to the insulin replete state. Using the whole body leucine flux technique, several groups have con- firmed that insulin deprivation in type 1 diabetic patients results in increased protein breakdown as demonstrated by increased leucine flux, phenylalanine and tyrosine flux. 82–85, 62, 49, 86–89 In the majority of studies, insulin infusion nor- malized leucine flux, providing strong evidence that insulin suppresses protein breakdown. Somewhat surprisingly, whole body protein synthesis also increased [...]... Increased insulin sensitivity Increased insulin resistance Obesity/maintained insulin sensitivity Lethal Lean/increased insulin sensitivity Lethal 70% Transgenic lipodystrophic adipose tissue Transgenic muscle 65 66 38 FIRKO LIRKO BIRKO NIRKO 32 35 36 37 63 64 55 55 67 68 34 , 33 136 GENETICALLY MODIFIED MOUSE MODELS OF INSULIN RESISTANCE 5.4 Insulin signalling network The insulin signalling network... acids are provided Insulin- induced fall in circulating amino acids blunts the stimulatory effect of insulin on synthesis of muscle proteins Insulin s primary effect on muscle appears to be an inhibition of protein breakdown While amino acids have a key role in modulating insulin effect on muscle protein synthesis, amino acids are the main regulators of splanchnic protein synthesis Insulin, however, has... leptin action. 26 Ablation of another phosphatase, SH2 domain-containing inositol 5-phosphatase (SHIP2), also led to a dramatic increase in insulin sensitivity, resulting in severe neonatal hypoglycaemia, decreased gluconeogenesis and perinatal death.27 Other potential insulin signalling negative modulators include PKCθ, a serine kinase potentially involved in lipotoxicity-associated insulin resistance, and. .. results in insufficient insulin secretion to compensate for the insulin resistance The knockouts of IRS3 and IRS4 did not result in any phenotype that suggested that these substrates of the insulin receptor are involved in carbohydrate metabolism.14, 15 The third line in the insulin signalling cascade, regulating more especially carbohydrate metabolism, involves phosphoinositide 3 kinases (PI3kinase)... that plasma amino acids have a critical role in stimulating muscle protein synthesis and insulin alone reduces circulating amino acids in vivo, which may explain some of the discrepancies between in vivo and in vitro studies Insulin also has a specific effect on synthesis of certain muscle protein fractions such as mitochondrial proteins and plasma proteins such as albumin Effect of insulin in type 2 diabetics... determine whether and how insulin may affect energy homeostasis through the brain The first strategy was to target the insulin receptor in neurons, creating a neuronal-specific insulin receptor knockout mouse (NIRKO) This animal model has provided direct evidence that insulin controls energy homeostasis through its actions in the brain NIRKO mice have increased food intake and develop obesity and insulin. .. tracer L-[1– 13 C]leucine Diabetologia 33 , 43 51 83 Bennet, W M., Connacher, A A., Jung, R T., Stehle, P and Rennie, M J (1991) Effects of insulin and amino acids on leg protein turnover in IDDM patients Diabetes 40, 499–508  130 THE EFFECT OF INSULIN ON PROTEIN METABOLISM 84 Luzi, L., Castellino, P., Simonson, D C., Petrides, A S and DeFronzo, R A (1990) Leucine metabolism in IDDM Role of insulin and substrate... Bailey, J L and Goldberg, A L (1994) Metabolic acidosis stimulates muscle protein REFERENCES 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 127 degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes J Clin Invest 93, 2127–2 133 Kimball, S R., Horetsky, R L and Jefferson, L S (1998) Signal transduction pathways involved in the regulation of protein synthesis... from the insulin receptor: (a) the mitogenic Grb2/Sos and the Ras-MAP kinase pathway and (b) the PI3kinase pathway, which exerts most of the metabolic actions of insulin Figure 5.1 shows a representative scheme of insulin signalling pathways Upon insulin binding to the insulin receptor there is a divergent cascade of coupled phosphorylation/dephosphorylation processes modulated by kinase and phosphatase... MEK Glut-4 c Sh Raf-1 GLUCOSE TRANSPORT IRS1,2 ,3, 4 p85 MAPK JKN PKC Akt PKB PI3K p90 rsk p110 GskPTP-1B PP-1G PDK-1 p70S6 kinase Glycogen synthase Transcription factors (e.g FOXO1) MITOGENESIS GLYCOGENESIS Figure 5.1 Insulin signalling pathways PROTEIN SYNTHESIS INSULIN SIGNALLING AND GLOBAL KNOCKOUTS 5.5 137 Factors leading to insulin resistance Insulin resistance may arise from abnormalities in the . (PHAS-1) are in uenced by insulin. PHAS-1 binds to the eIF-4E cap binding protein, insulin enhances phosphorylation of PHAS-1 and favours dissociation of eIF-4E and PHAS-1. 14 This allows for binding. eEF-2 phosphorylation - - mRNA - - Figure 4.2 Effect of insulin on protein turnover 110 THE EFFECT OF INSULIN ON PROTEIN METABOLISM Insulin as a regulator of protein turnover in vitro and in situ Studying the effect of insulin. mixed muscle pro- tein, myosin heavy chain and mitochondrial protein were unchanged in diabetic patients infused with insulin. 120 It is interesting that intensive insulin treatment in type 2 diabetic