the balance in favor of dephosphorylation in part by inhibiting the enzyme, glycogen synthase kinase 3 (GSK-3), and in part by activating a phosphatase. Dephosphorylation of glycogen synthase not only increases its activity directly, but also increases its responsiveness to stimulation by its substrate, glucose-6-phosphate. Hence the powerful effects of insulin on muscle glycogen synthesis are achieved by the complementary effects of increased glucose transport, increased glucose phos- phorylation, and increased glycogen synthase activity. The alternative fate of glucose-6-phosphate, metabolism to pyruvate in the glycolytic pathway, is also increased by insulin. Access to the glycolytic pathway is guarded by phosphofructokinase, whose activity is precisely regulated by a combi- nation of allosteric effectors, including ATP, ADP, and fructose-2,6-bisphosphate. This complex enzyme behaves differently in intact cells and in the broken cell preparations typically used by biochemists to study enzyme regulation. Because conflicting findings have been obtained under a variety of experimental circum- stances, no general agreement has been reached on how insulin increases phos- phofructokinase activity. In contrast to the liver, the isoform of the enzyme that forms fructose-2,6-bisphosphate in muscle is not regulated by cyclic AMP. The effects of insulin are likely to be indirect. It should be noted that oxidation of fat profoundly affects the metabolism of glucose in muscle and that insulin also increases all aspects of glucose metabolism in muscle as an indirect consequence of its action on adipose tissue to decrease FFA production.When insulin concentrations are low, increased oxidation of fatty acids decreases oxidation of glucose by inhibiting the decarboxylation of pyruvate and the transport of glucose across the muscle cell membrane. In addition, products of fatty acid oxidation appear also to inhibit hexokinase, but recent studies have called into question the relevance of earlier findings that fatty acid oxidation may inhibit phosphofructokinase. Insulin not only limits the availability of fatty acids, but also inhibits their oxidation. Insulin increases the formation of malonyl CoA, which blocks entry of long-chain fatty acids into the mitochondria as described for liver (Figure 7).These effects are discussed in Chapter 9. Protein synthesis and degradation are ongoing processes in all tissues, and in the nongrowing individual are completely balanced so that on average there is no net increase or decrease in body protein (Figure 14). In the absence of insulin there is net degradation of muscle protein and muscle becomes an exporter of amino acids, which serve as substrate for gluconeogenesis and ureogenesis in the liver.As with its effects on carbohydrate and fat metabolism, insulin intercedes in protein synthesis at several levels, and has both rapidly apparent and delayed effects. Insulin increases uptake of amino acids from blood by stimulating their transport across the plasma membrane. Insulin increases protein synthesis by promoting phosphoryla- tion of the initiation factors (e.g., eIF-2, eukaryotic initiation factor-2) that govern translation of mRNA. Under the influence of insulin, attachment of mRNA to ribosomes is enhanced, as reflected by the higher content of polysomes compared Insulin 187 to monosomes.This effect of insulin appears to be selective for mRNAs for spe- cific proteins. On a longer time scale, insulin increases total RNA in muscle by increasing synthesis of ribosomal RNA and proteins. Understanding of how insulin decreases protein degradation is incomplete, but it appears that insulin decreases ATP-dependent protein degradation both by decreasing expression of various elements of the proteosomal protein degrading apparatus and by modulat- ing the protease activity of its components. Effects on Liver Insulin reduces outflow of glucose from the liver and promotes storage of glycogen. It inhibits glycogenolysis, gluconeogenesis, ureogenesis, and ketogenesis, and it stimulates the synthesis of fatty acids and proteins.These effects are accom- plished by a combination of actions that change the activity of some hepatic enzymes and rates of synthesis of other enzymes. Hence not all the effects of insulin occur on the same time scale. Although we use the terms “block” and “inhibit” to describe the actions of insulin, it is important to remember that these verbs are used in the relative and not the absolute sense. Rarely would inhibition 188 Chapter 5. The Pancreatic Islets sarcoplasm blood p lasma membrane amino acids Na + proteins amino acids ribosomes proteosome & lysosomes mRNA binding initiation & elongation factors synthesis & assembly Figure 14 Effects of insulin on protein turnover in muscle. Reactions stimulated by insulin are shown in blue. The dashed arrows indicate inhibition. of an enzymatic transformation be absolute. In addition, all of the hepatic effects of insulin are reinforced indirectly by actions of insulin on muscle and fat to reduce the influx of substrates for gluconeogenesis and ketogenesis.The actions of insulin on hepatic metabolism are always superimposed on a background of other regulatory influences exerted by metabolites,glucagon, and a variety of other regu- latory agents.The magnitude of any change produced by insulin is thus determined not only by the concentration of insulin, but also by the strength of the opposing or cooperative actions of these other influences. Rates of secretion of both insulin and glucagon are dictated by physiological demand. Because of their antagonistic influences on hepatic function, however, it is the ratio,rather than the absolute con- centrations, of these two hormones that determines the overall hepatic response. Glucose Production In general, liver takes up glucose when the circulating glucose level is high and releases it when the level is low. Glucose transport into or out of hepatocytes depends on a high-capacity insulin-insensitive isoform of the glucose transporter, GLUT 2. Because the movement of glucose is passive, net uptake or release depends on whether the concentration of free glucose is higher in extracellular or intracellular fluid.The intracellular concentration of free glucose depends on the balance between phosphorylation and dephosphorylation of glucose (Figure 2, cycle II). The two enzymes that catalyze phosphorylation are hexokinase, which has a high affinity for glucose and other six-carbon sugars, and glucokinase, which is specific for glucose. The kinetic properties of glucokinase are such that phos- phorylation increases proportionately with glucose concentration over the entire physiological range. In addition, glucokinase activity is regulated by glucose.When glucose concentrations are low, much of the glucokinase is bound to an inhibitory protein that sequesters it within the nucleus.An increase in glucose concentration releases glucokinase from its inhibitor and allows it to move into the cytosol, where glucose phosphorylation can take place. Phosphorylated glucose cannot pass across the hepatocyte membrane. Dephosphorylation of glucose requires the activity of glucose-6-phosphatase. Insulin suppresses synthesis of glucose-6-phosphatase and increases synthesis of glucokinase, thereby decreasing net output of glucose while promoting net uptake. This response to insulin is relatively sluggish and contributes to long-term adaptation rather than to minute-to-minute regulation.The rapid effects of insulin to suppress glucose release are exerted indirectly through decreasing the avail- ability of glucose-6-phosphate, hence starving the phosphatase of substrate. The process of uptake and phosphorylation by glucokinase is only one source of glucose-6-phosphate. Glucose-6-phosphate is also produced by gluconeogenesis and glycogenolysis. Insulin not only inhibits these processes, but it also drives them in the opposite direction. Insulin 189 Most of the hepatic actions of insulin are opposite to those of glucagon, as discussed earlier, and can be traced to inhibition of cyclic AMP accumulation. Rapid actions of insulin largely depend on changes in the phosphorylation state of enzymes already present in hepatocytes. Insulin decreases hepatic concentrations of cyclic AMP by accelerating its degradation by cyclic AMP phosphodiesterase, and may also interfere with cAMP formation and, perhaps, activation of protein kinase A.The immediate consequences can be seen in Figure 15 and are in sharp contrast to the changes in glucose metabolism produced by glucagon shown in Figure 2. Insulin promotes glycogen synthesis and inhibits glycogen breakdown. These effects are accomplished by the combination of interference with cyclic AMP-dependent processes that drive these reactions in the opposite direction (see Figure 3), inhibition of glycogen synthase kinase (which, like protein kinase A, inactivates glycogen synthase), and by activation of the phosphatase that dephosphorylates both glycogen synthase and phosphorylase.The net effect is that glucose-6-phosphate is incorporated into glycogen. By lowering cAMP concentrations, insulin decreases the breakdown and increases the formation of fructose-2,6-phosphate, which potently stimulates phosphofructokinase and promotes the conversion of glucose to pyruvate. Insulin affects several enzymes in the PEP substrate cycle (Figure 2, cycle IV) and in so doing directs substrate flow away from gluconeogenesis and toward lipogenesis (Figure 16).With relief of inhibition of pyruvate kinase, PEP can be converted to pyruvate, which then enters mitochondria. Insulin activates the mitochondrial enzyme that catalyzes decarboxylation of pyruvate to acetyl CoA and indirectly accelerates this reaction by decreasing the inhibition imposed by fatty acid oxida- tion. Decarboxylation of pyruvate to acetyl CoA irreversibly removes these carbons from the gluconeogenic pathway and makes them available for fatty acid synthesis. The roundabout process that transfers acetyl carbons across the mitochondrial membrane to the cytoplasm, where lipogenesis occurs, requires condensation with oxaloacetate to form citrate. Citrate is transported to the cytosol and cleaved to release acetyl CoA and oxaloacetate. It might be recalled from earlier discussion that oxaloacetate is a crucial intermediate in gluconeogenesis and is converted to PEP by PEP carboxykinase. Insulin bars the flow of this lipogenic substrate into the gluconeogenic pool by inhibiting synthesis of PEP carboxykinase. The only fate left to cytosolic oxaloacetate is decarboxylation to pyruvate. Finally, insulin increases the activity of acetyl CoA carboxylase, which cat- alyzes the rate-determining reaction in fatty acid synthesis. Activation is accom- plished in part by relieving cyclic AMP-dependent inhibition and in part by promoting the polymerization of inactive subunits of the enzyme into an active complex.The resulting malonyl CoA not only condenses to form long-chain fatty acids but also prevents oxidation of newly formed fatty acids by blocking their entry into mitochondria (Figure 7). On a longer time scale, insulin increases the synthesis of acetyl coA carboxylase. 190 Chapter 5. The Pancreatic Islets Insulin 191 glycogen glucose-1-P I glucose-6-Pglucose II fructose-6-P fructose-1,6-P III hexose monophosphate shunt PEP pyruvate IV acetyl CoA fatty acids ketone bodies V TCA Cycle CO 2 CO 2 Figure 15 Effects of insulin on glucose metabolism in hepatocytes. Blue arrows indicate reactions that are increased, and broken arrows indicate reactions that are decreased. It may be noted that hepatic oxidation of either glucose or fatty acids increases delivery of acetyl CoA to the cytosol, but ketogenesis results only from oxidation of fatty acids.The primary reason is that lipogenesis usually accompanies glucose utilization and provides an alternate pathway for disposal of acetyl CoA. There is also a quantitative difference in the rate of acetyl CoA production from the two substrates: 1 mole of glucose yields only 2 moles of acetyl CoA compared to 8 or 9 moles for each mole of fatty acids. MECHANISM OF INSULIN ACTION The many changes that insulin produces at the molecular level—membrane transport, enzyme activation, gene transcription, and protein synthesis—have been described. The molecular events that link these changes with the interaction of insulin and its receptor are still incompletely understood but are the subjects of intense investigation. Many of the intermediate steps in the action of insulin have been uncovered, but others remain to be identified. It is clear that transduction of 192 Chapter 5. The Pancreatic Islets oxaloacetate citrate TCA cycle acetyl CoA 2 pyruvate cytosol mitochondrial matrix malonyl CoA acetyl CoA citrate pyruvate PEP long chain fatty acids 1 3 4 malate oxaloacetate Figure 16 Effects of insulin on lipogenesis in hepatocytes. Blue arrows indicate reactions that are increased, and broken arrow indicates reaction that is decreased. (1) Pyruvate kinase; (2) pyruvate dehy- drogenase; (3) acetyl CoA carboxylase; (4) fatty acid synthase. the insulin signal is not accomplished by a linear series of biochemical changes, but rather that multiple intracellular signaling pathways are activated simultane- ously and may intersect at one or more points before the final result is expressed (Figure 17). The insulin receptor is a tetramer composed of two alpha and two beta glycoprotein subunits that are held together by disulfide bonds that link the alpha subunits to the beta subunits and the alpha subunits to each other (Figure 18). The alpha and beta subunits of insulin are encoded in a single gene that contains Insulin 193 glucose transport glycogen synthesis protein synthesis mitogenesis insulin receptor IRS proteins Shc GRB2 GRB2 p110 SHP-2 SOS SOS p85 Y Y Y Y Y Y Figure 17 Current model of the insulin receptor signaling. Phosphorylated tyrosine residues (Y) on the insulin receptor serve as anchoring sites for cytosolic proteins (IRS proteins and Shc),which in turn are phosphorylated on tyrosines (dark blue circles) and dock with other proteins. IRS, Insulin receptor substrate; Shc, Src homology-containing protein; SHP-2, protein tyrosine phosphatase-2; GRB2, growth factor receptor binding protein 2; SOS, son of sevenless (a GTPase-activating protein); p85 and p110 are subunits of phosphoinositol-3-kinase. (From Virkamäki,A., Ueki, K., and Kahn, C. R., J. Clin. Invest. 103, 931, 1999, with permission.) 22 exons.The alpha subunits are completely extracellular and contain the insulin- binding domain. The beta subunits span the plasma membrane and contain tyrosine kinase activity in the cytosolic domain. Binding to insulin is thought to produce a conformational change that relieves the beta subunit from the inhibitory effects of the alpha subunit, allowing it to phosphorylate itself and other proteins at tyrosine residues.Autophosphorylation of the kinase domain is required for full activation.Tyrosine phosphorylation of the receptor also provides docking sites for other proteins that participate in transducing the hormonal signal. Docking on the phosphorylated receptor may position proteins optimally for phosphorylation by the receptor kinase. Among the proteins that are phosphorylated on tyrosine residues by the insulin receptor kinase are four cytosolic proteins called insulin receptor substrates (IRS-1, IRS-2, IRS-3, and IRS-4).These relatively large proteins contain multiple tyrosine phosphorylation sites and act as scaffolds, on which other proteins are assembled to form large signaling complexes. IRS-1 and IRS-2 appear to be present in all insulin target cells, whereas IRS-3 and IRS-4 have more limited dis- tribution. Despite their names the IRS proteins are not functionally limited to transduction of the insulin signal, but are also important for expression of effects of 194 Chapter 5. The Pancreatic Islets -S-S- -S-S -S-S- COOH COOH insulin binding domain tyrosine kinase domain membane α-subunits β-subunits NH 2 NH 2 - Figure 18 Model of the insulin receptor. other hormones and growth-promoting factors. Moreover, they are not the only substrates for the insulin receptor kinase. A variety of other proteins that are tyrosine phosphorylated by the insulin receptor kinase have also been identified. Proteins recruited to the insulin receptor and IRS proteins may have enzymatic activity or they may in turn recruit other proteins by providing sites for protein:protein interactions.The assemblage of proteins initiates signaling cascades that ultimate express the various actions of insulin described above. One of the most important of the proteins that is activated is phosphatidylinositol-3 (PI-3) kinase. PI-3 kinase plays a critical role in activating many downstream effector molecules, including protein kinase B, which is thought to mediate the effects of insulin on glycogen synthesis and GLUT 4 translocation. PI-3 kinase, however, is also activated by a variety of other hormones, cytokines, and growth factors whose actions do not necessarily mimic those of insulin.The uniqueness of the response to insulin probably reflects the unique combination of biochemical consequences produced by the simultaneous activity of multiple signaling pathways and the particular set of effector molecules expressed in insulin target cells. Although insulin is known to regulate expression of more than 150 genes, few of the nuclear regulatory proteins that are activated by insulin are known, and precisely how the insulin receptor communicates with these regulatory proteins is unknown. A more detailed discussion of the complex molecular events that govern insulin action can be found in the suggested readings listed at the end of this chapter. REGULATION OF INSULIN SECRETION As might be expected of a hormone whose physiological role is promotion of fuel storage, insulin secretion is greatest immediately after eating and decreases during between-meal periods (Figure 19). Coordination of insulin secretion with nutritional state as well as with fluctuating demands for energy production is achieved through stimulation of beta cells by metabolites, hormones, and neural signals. Because insulin plays the primary role in regulating storage and mobiliza- tion of metabolic fuels, the beta cells must be constantly apprised of bodily needs, not only with regard to feeding and fasting, but also to the changing demands of the environment. Energy needs differ widely when an individual is at peace with the surroundings and when fighting for survival. Maintaining constancy of the internal environment is achieved through direct monitoring of circulating meta- bolites by beta cells.This input can be overridden or enhanced by hormonal or neural signals that prepare an individual for rapid storage of an influx of food or for massive mobilization of fuel reserves to permit a suitable response to envi- ronmental demands. Insulin 195 Metabolite Control Glucose Glucose is the most important regulator of insulin secretion. In the normal individual its concentration in blood is maintained within the narrow range of about 70 or 80 mg/dl after an overnight fast to about 150 mg/dl immediately after a glucose-rich meal. When blood glucose increases above a threshold value of about 100 mg/dl, insulin secretion increases proportionately. At lower concentra- tions adjustments in insulin secretion are largely governed by other stimuli (see below) that act as amplifiers or inhibitors of the effects of glucose.The effective- ness of these agents therefore decreases as glucose concentration decreases. Other Circulating Metabolites Amino acids are important stimuli for insulin secretion. The transient increase in plasma amino acids after a protein-rich meal is accompanied by increased secretion of insulin. Arginine, lysine, and leucine are the most potent 196 Chapter 5. The Pancreatic Islets 120 110 40 30 20 10 100 90 80 meal mealmeal 0.2 0.1 8 a.m. 10 12 2 p.m. 4 6 8 10 12 2 a.m. 4 6 8 time insulin (µ units/ml) glucagon (ng/ml) glucose (mg/DL) Figure 19 Changes in the concentrations of plasma glucose, immunoreactive glucagon and immunore- active insulin throughout the day.Values are the mean ± SEM (n = 4). (From Tasaka,Y., Sekine, M., Wakatsuki, M., Ohgawara, H., and Shizume, K., Horm. Metab. Res. 7, 205–206, 1975, with permission.) [...]... Integration fold change in hormone concentration required for 50 % maximum response 6.0 down-regulation up-regulation 5. 0 4.0 3.0 2.0 1.0 0 -5 0 -4 0 -3 0 -2 0 -1 0 0 +10 +20 +30 +40 +50 percent change in receptor number Figure 10 The effects of up- or down-regulation of receptor number on sensitivity to hormonal stimulation down-regulation of receptors Down-regulation may result from inactivation of the receptors... hormone from blood 218 Chapter 6 Principles of Hormonal Integration hormone binding 100 percent of maximum 75 spare receptors 50 biological response 25 100 50 25 0 1 5 10 35 100 1,000 hormone concentration (ng/ml) Figure 11 Spare receptors Note that the concentration of hormone needed to produce a half-maximal response is considerably lower than that needed to occupy half of the receptors Sensitivity to... stimulatory effect of glucose Some evidence suggests that the voltage-sensitive calcium channels may be substrates for 200 Chapter 5 The Pancreatic Islets A glucose glucose GK glucose-6-P ADP (+) K+ pyruvate K+ mitochondrion ASKC ATP Ca2+ Ca2+ CO2 + H2 0 VSCC K+ secretory granules CSKC B glucose glucose GK K+ glucose-6-P pyruvate ADP K+ mitochondrion (-) ATP Ca2+ Ca2+ CO2 + H2O K+ K+ Figure 21 Regulation of insulin... high capacity glucose amino acids fatty acids ketones acetylcholine VIP GLP-1 GIP glucagon somatostatin epinephrine norepinephrine - + beta cell + growth hormone cortisol insulin Figure 20 Metabolic, hormonal, and neural influences on insulin secretion.VIP, vasoactive intestinal peptide; GLP-1, glucagon-like peptide-1; GIP, glucose-dependent insulinotropic peptide Insulin 199 but relatively low affinity... needed to produce a half-maximal response by about a factor of 2, while decreasing the number of receptors by 40% increases the needed concentration of hormone by a factor of about 5 Many hormones decrease the number of their own receptors in target tissues This so-called down-regulation was originally recognized as a real phenomenon 2 15 Modulation of Responding Systems 100 A half-maximum biological response... so that ATP-sensitive potassium channels (ASKC) are open, and the membrane potential is about −70 mV.Voltage-sensitive calcium channels (VSCC) and calcium-sensitive potassium channels (CSKC) are closed (B) Beta cell response to increased blood glucose In response to increased glucose entry and metabolism, the ratio of ADP/ATP decreases, and ATP-sensitive potassium channels close.Voltage-sensitive calcium... by insulin Annu Rev Physiol 56 , 321–348 Miller, R E (1981) Pancreatic neuroendocrinology: Peripheral neural mechanisms in the regulation of the islets of Langerhans Endocr Rev 2, 471–494 Suggested Reading 203 Pilkis, S J., and Granner, D K (1992) Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis Annu Rev Physiol 54 , 8 85 909 Rajan,A S.,Aguilar-Bryan, L., Nelson, D.A.,Yaney,... Accili, D., Barbetti, F., Quon, M J., de la Luz Sierra, M., Suzuki,Y., Koller, E., Levy-Toledano, R., Wertheimer, E., Moncada, V Y., Kadowaki, H., and Kadowaki, T (1992) Mutations in the insulin receptor gene Endocr Rev 13, 56 6 59 5 Unger, R H., and Orci, L (1976) Physiology and pathophysiology of glucagon Physiol Rev 56 , 778–838 CHAPTER 6 Principles of Hormonal Integration Redundancy Reinforcement Push–Pull... degradation or synthesis Down-regulation is not limited to the effects of a hormone on its own receptor, or to the surface receptors for the water-soluble hormones One hormone can down-regulate receptors for another hormone This appears to be the mechanism by which T3 decreases the sensitivity of the thyrotropes of the pituitary to TRH (see Chapter 3) Similarly, progesterone may down-regulate both its own... glucagon-like peptide, and glucose-dependent insulinotropic peptide (GIP), can evoke insulin secretion when tested experimentally, but of these hormones, only GLP-1 and GIP appear to be physiologically important incretins Secretion of insulin in response to food intake is also mediated by a neural pathway The taste or smell of food or the expectation of eating may increase insulin secretion during this so-called . of effects of 194 Chapter 5. The Pancreatic Islets -S-S- -S-S -S-S- COOH COOH insulin binding domain tyrosine kinase domain membane α-subunits β-subunits NH 2 NH 2 - Figure 18 Model of the insulin. the synthesis of acetyl coA carboxylase. 190 Chapter 5. The Pancreatic Islets Insulin 191 glycogen glucose-1-P I glucose-6-Pglucose II fructose-6-P fructose-1,6-P III hexose monophosphate shunt PEP pyruvate IV acetyl. long-term adaptation rather than to minute-to-minute regulation.The rapid effects of insulin to suppress glucose release are exerted indirectly through decreasing the avail- ability of glucose-6-phosphate,