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Insulin Action, Post-Receptor Mechanisms
Insulin Action, Post-Receptor Mechanisms Shuichi Okada Gunma University Graduate School of Medicine, Gunma, Japan
Sean Crosson University of Michigan Medical Center, Ann Arbor, Michigan, United States
Masatomo Mori Gunma University Graduate School of Medicine, Gunma, Japan
Alan R. Saltiel University of Michigan Medical Center, Ann Arbor, Michigan, United States
Jeffrey E. Pessin State University of New York, Stony Brook, New York, United States
Glossary hepatic Relating to or affecting the liver. hyperglycemia An abnormally high concentration of glucose (sugar) in the blood. insulin A polypeptide hormone consisting of two peptide chains linked by a disulfide bridge, produced in the beta cells of the endocrine pancreas and released in response to elevated levels of blood glucose. phosphorylation A process in which a phosphate group is combined with an organic molecule; occurs naturally in cellular metabolism.
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nsulin is the most potent hormonal regulator of glucose, lipid, and protein metabolism. In states of glucose excess, insulin prevents hyperglycemia through the suppression of liver glucose production and the stimulation of adipose and skeletal muscle glucose uptake. In states of nutrient deprivation, the decrease in insulin levels results in enhanced liver glucose output (glycogenolysis and gluconeogenesis) and reduced adipose and skeletal muscle glucose uptake. The coordination of these cell type-specific responses maintains glucose levels within a narrow range. However, disruption of these homeostatic mechanisms leads to peripheral tissue insulin resistance, a hallmark for the development of type 2 diabetes.
THE INSULIN RECEPTOR The insulin receptor is a transmembrane protein composed of two a-subunits and two b-subunits that are disulfide-linked in an a2b2 heterotetrameric structure. The a-subunits are extracellular and contain the
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insulin-binding domains, whereas the b-subunits contain a single-transmembrane region with an intracellular tyrosine kinase domain. Insulin binding triggers activation of the b-subunit protein kinase activity via transphosphorylation, resulting in increased phosphorylation of several intracellular substrates. Although the insulin receptor is expressed in virtually all tissues, three have classically been considered as targets for the metabolic actions of the hormone: liver, muscle, and fat. Recent studies using genetically altered mice have challenged these assumptions and, in the process, have provided important clues to understanding the complexity of specific signaling molecules that control biological responsiveness to insulin.
Liver The fasting hyperglycemia that occurs in type 2 diabetes results from increased hepatic glucose production, through increased gluconeogenesis and glycogenolysis, as well as reduced glycogen synthesis. Disruption of insulin action in the liver by tissuespecific knockout of the insulin receptor in mice (LIRKO mice) produces severe glucose intolerance with insulin resistance. These data demonstrate that the liver plays a direct role in the regulation of postprandial glucose homeostasis by insulin and occurs due to a suppression of hepatic glucose production rather than an increase in muscle glucose uptake in mice. LIRKO mice also exhibit compensatory hyperinsulinemia, similar to transgenic mice overexpressing the insulin receptor. In addition, lipotropic mice display chronic hyperinsulinemia, leading to decreased IRS2 expression in liver concomitant with insulin
Encyclopedia of Endocrine Diseases, Volume 3. ß 2004 Elsevier Inc. All rights reserved.
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resistance of gluconeogenesis. Thus, hepatic insulin resistance in type 2 diabetes occurs secondary to chronic hyperinsulinemia.
Adipose Tissue White adipose tissue accounts for less than 10% of glucose uptake but plays a major role in whole body insulin sensitivity by releasing into the circulation various modulators of insulin action, including lipid products and hormones. Free fatty acids (FFAs) derived from adipocytes are elevated in obesity and type 2 diabetes and accumulate in muscle and liver, contributing to insulin resistance in these tissues. Mice with a selective deletion of the insulin receptor in brown adipocytes experience an age-dependent loss of intrascapular brown fat but increased expression of uncoupling proteins-1 and -2. In parallel, these mice develop a defect in insulin secretion, resulting in progressive glucose intolerance, without insulin resistance. These data demonstrate that the insulin receptor is required for brown fat formation throughout development and that brown fat regulates insulin secretion and glucose homeostasis. Histological, immunohistochemical, and ultrastructural analyses of white adipose tissue in newborn fat insulin receptor knockout mice (FIRKO mice) reveal a marked decrease in adipose tissue area. This fat cell depletion resulted from a reduction of adipocyte volume ( 90%), with a small decrease in the number of adipocytes. Electron microscopy analysis displays a normal pattern of adipogenesis in FIRKO mice, demonstrating that lack of insulin receptors is not associated with a selective impairment of the adipocyte differentiation process. Thus, the insulin receptor is dispensable for adipocyte differentiation but is necessary for the large extent of lipid storage in these cells. In addition, FIRKO mice display increased longevity, demonstrating an inverse relationship between high rates of adipose metabolism and life span. Adipocytes also secrete cytokines (adipokines) that modulate peripheral insulin sensitivity. Tumor necrosis factor-a (TNF-a) is elevated in obese humans and rodents and has been shown to reduce insulin signaling by decreasing insulin receptor and IRS1 tyrosine phosphorylation. Mice harboring deletion of the genes encoding TNF-a or the TNF-a receptor are refractory to insulin resistance associated with diet-induced obesity or when crossed with genetically obese ob/ob mice. Resistin is another adipokine secreted from adipocytes, and the levels of this hormone are increased in obese mice and reduced after administration of the insulin-sensitizing perioxisome proliferator-activated
receptor-g (PPAR-g)-activating thiazolidinediones. Resistin also inhibits adipogenesis and, thus, plays a negative feedback role in fat cell formation. Adipose tissue also secretes leptin, another hormone that positively influences metabolism and energy expenditure, and has profound effects on appetite. Inactivation of leptin production (ob gene in mice) or defects in the leptin receptor (db gene) results in autophagy, obesity, and severe insulin resistance. Similarly, transgenic ablation of white adipose tissue in mice leads to severe insulin resistance, elevated lipid levels, undetectable leptin concentrations, and diabetes, a phenotype reminiscent of generalized lipoatropic diabetes in humans. In these models, restoration of physiological serum leptin levels, either by leptin infusion, by transgenic overexpression, or by surgical implantation of white adipose tissue, reverses the diabetic and insulin-resistant phenotype. These results demonstrate that insulin resistance and diabetes in lipodystrophic mice can be caused by a deficiency of leptin secondary to a failure of adipocyte differentiation. Adiponectin is also secreted from adipocytes in a regulated fashion. The expression of this adipokine is decreased in obese humans and rodents, and increased expression positively correlates with insulin sensitivity. Acute treatment of obese mice with adiponectin decreases plasma FFA, muscle, and liver triglyceride content and improves insulin resistance. In a knockout model of lipoatropic diabetes, insulin resistance was completely reversed by a combination of leptin and adiponectin infusion but was only partially reversed by either leptin or adiponectin alone.
Skeletal Muscle Surprisingly, specific deletion of the skeletal muscle insulin receptor (MIRKO) yields mice with normal glucose and insulin levels but with dyslipidemia and increased adiposity. Although MIRKO mice display reduced insulin-stimulated muscle glucose uptake in vitro and during a euglycemic hyperinsulinemic clamp, glucose tolerance tests show near-normal glucose uptake. This discrepancy is likely due to increased insulin-independent glucose uptake. In this regard, muscle-specific ablation of the insulin receptor, coupled with a loss of insulin-like growth factor-1 (IGF-1) expression, results in severe insulin resistance in muscle. Interestingly, skeletal muscle insulinresponsive glucose transporter (GLUT4) null mice are also insulin resistant, demonstrating that downstream insulin action and glucose uptake, rather than
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Pancreatic Beta Cells Insulin receptor knockouts in pancreatic beta cells (b-IRKO) display a phenotype similar to that of type 2 diabetes, having a selective loss of glucose-stimulated first-phase insulin secretion and progressive impairment of glucose tolerance. The loss of normal secretory function results from decreased expression of the critical glucose-sensing enzyme glucose kinase. These data directly demonstrate the insulin receptor functions in an autocrine feedback loop necessary for maintaining appropriate beta cell insulin secretion.
Brain The insulin receptor is widely distributed throughout the central nervous system (CNS), especially in the hypothalamus and pituitary. Mice with CNS-specific disruption of the insulin receptor gene (NIRKO mice) show increased food intake, resulting in diet-induced obesity and insulin resistance. Thus, insulin action in the CNS appears to provide a negative feedback loop for postprandial inhibition of food intake and plays a central role in the regulation of body weight.
SIGNALING DOWNSTREAM OF THE INSULIN RECEPTOR On activation, the insulin receptor can catalyze the tyrosine phosphorylation of numerous cytoplasmic proteins. The best characterized of these is the insulin receptor substrate family, consisting of four closely related members (IRS1 to -4) and three members of the Grb2-associated-binding proteins (Gab1 to -3). The IRS and Gab proteins appear to provide overlapping functions given that each can participate in the actions of various growth factors, cytokines, and developmental signals. The insulin receptor phosphorylates all four IRS family members on numerous tyrosine residues, in the process generating recognition sites for the interaction of effector proteins containing Src homology (SH2) domains. For example, IRS1 contains 21 putative tyrosine phosphorylation sites, several of which are located in amino acid sequence motifs that bind to SH2 domain proteins, including the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, Grb2, Nck, Crk, Fyn, Csk, phospholipase Cg, and SHP2. IRS1 also contains more than 30 potential serine/threonine phosphorylation sites in motif
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recognized by various kinases such as casein kinase II, protein kinase C (PKC), protein kinase B/Akt, Jun amino-terminal kinase ( JNK), and mitogen-activated protein (MAP) kinase. The crucial role for these substrate proteins has been documented both in tissue culture model systems and by the use of homologous recombination in mice. In particular, disruption of the IRS1 gene results in growth retardation and mild insulin resistance, whereas IRS2 null mice display peripheral insulin resistance, impaired insulin secretion, and diabetes. Interestingly, the IRS1 null mice do not become diabetic due to pancreatic beta cell compensation. However, the IRS2 mice have a marked reduction in beta cell mass due to a marked inhibition of beta cell development. Although mice carrying an ablated IRS3 gene did not display any detectable phenotype, disruption of IRS4 resulted in modest growth retardation and mild insulin resistance. Both physiological and pathophysiological regulation of IRS protein function occur at multiple levels. Several studies have shown that serine/threonine phosphorylation of IRS proteins can reduce insulinstimulated tyrosine phosphorylation, demonstrating that serine/threonine phosphorylation functions in a feedback-inhibitory mechanism. In this regard, members of the MAP kinase superfamily are capable of catalyzing serine phosphorylations of the IRS proteins. Studies indicate that the stress-induced serine kinase JNK is one such kinase. JNK is stimulated during acute or chronic inflammation and in response to inflammatory cytokines such as TNF-a. JNK phosphorylates IRS1 and IRS2, Shc, and Gab1, and both IRS1 and IRS2 contain JNK-binding motifs. This motif mediates the specific association of JNK with IRS1, catalyzing the phosphorylation of a serine residue on the COOH-terminal side of the phosphotyrosine-binding (PTB) domain (Ser307 in [murine] IRS1, Ser312 in [human] IRS1). Phosphorylation of this residue inhibits the function of the PTB domain, disrupting the association between the insulin receptor and IRS1 and inhibiting tyrosine phosphorylation. This mechanism might explain, at least in part, the insulin resistance that occurs during trauma and obesity. IRS serine/threonine phosphorylation may occur downstream of other kinases activated by inflammatory signals. High doses of salicylates are shown to reverse hyperglycemia, hyperinsulinemia, and dislipidemia in obese rodents by sensitizing the insulinsignaling pathway, including IRS protein tyrosine phosphorylation. The effect of salicylates is attributed to inhibition of IkB kinase-b (IKK-b), resulting in decreased IRS serine/threonine phosphorylation. Importantly, heterozygous disruption of IKK-b protects
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against the development of insulin resistance during high-fat feeding and in obese, leptin-deficient (ob/ob) mice. Although there is no physical interaction between IRS proteins and IKK-b, salicylates increase insulin-stimulated phosphorylation of IRS proteins in the liver, demonstrating that IKK-b inhibits insulin receptor function through the activation of an intermediate kinase(s) or through activation of NFkB. Whereas this type of feedback via serine/threonine phosphorylation accounts for relatively short-term desensitization, the IRS proteins also undergo long-term down-regulation. Prolonged insulin stimulation substantially reduces IRS1 and IRS2 protein levels through a 26S proteosome degradative pathway. Furthermore, insulin stimulates the ubiquitination of IRS2, and reduction of IRS2 by ubiquitin/proteosome-mediated proteolysis in mouse embryo fibroblasts lacking IRS1 dramatically inhibits the activation of Akt and ERK1/2 in response to insulin or IGF-1. The activity of the ubiquitin/proteosome system is also elevated in diabetes, promoting the degradation of the IRS proteins and exacerbating insulin resistance.
PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY Tyrosine phosphorylated IRS proteins engage several effectors that contain SH2 domains. In particular, the SH2 domains of type 1A PI 3-kinase regulatory subunits directly interact with tyrosine phosphorylated IRS proteins, resulting in the targeting and activation of the PI 3-kinase catalytic subunit. Once activated and/or appropriately targeted, this kinase generates PI 3,4,5P3 in the plasma membrane and perhaps a small amount of PI 3,4,5P3 in other intracellular membrane compartments. Generation of PI 3,4,5P3 is essential for insulin-stimulated glucose uptake; glycogen, lipid, and protein synthesis; and changes in gene expression. There are several regulatory (p85a, p85b p55/AS53, p55PIK, and p50) and catalytic (110a and 110b) subunits of this lipid kinase. Surprisingly, genetic ablation of the p85a regulatory subunit results in enhanced insulin sensitivity, probably due to excess levels of the other regulatory subunits compared with the catalytic subunit. PI 3,4,5P3 functions as a signaling intermediate that can target and activate the serine/threonine kinase PIdependent kinase (PDK1). PDK1 contains a pleckstrin homology (PH) domain that has a high affinity and selectivity for PI 3,4,5P3. PI 3,4,5P3 also interacts with the PH domain of Akt. Akt is a substrate for PDK1, and phosphorylation on two carboxyl-terminal serine/threonine residues results in the activation of
the Akt kinase. Similarly, PDK1 can also phosphorylate an activating threonine residue in the atypical PKC isoforms, PKCl and PKCz. The Akt protein kinases exist as three isoforms, all of which are activated by phosphorylation on T308 and S473. On growth factor stimulation, Akt localizes near the plasma membrane, where it becomes phosphorylated on T308 by PDK1. Following phosphorylation and activation, Akt can remain associated with the plasma membrane or intracellular compartments, or it can translocate to the nucleus. Expression of constitutively active Akt stimulates glucose uptake in 3T3L1 adipocytes, whereas Akt inhibition through the use of dominant negative mutants partially inhibits the insulin-stimulated glucose transport. Although Akt1 knockout mice have no significant alteration in whole body glucose homeostasis, Akt2 gene ablation results in moderate insulin resistance and impaired glucose tolerance. These data demonstrate that Akt2 plays an important role in insulin-regulated glucose metabolism but that Akt1 can partially compensate for the loss of Akt2 function. PKCs are a large family of serine/threonine kinases that have also been implicated in several actions of insulin. There are three subgroups of PKCs: the classical cPKCs, the novel nPKCs, and the atypical aPKCs. Although these different family members appear to play overlapping but distinct roles, the aPKCs have been implicated in the stimulation of glucose uptake and protein synthesis by insulin in tissue culture model systems. Although the classical PKCa isoform has been ablated in mice and displays enhanced insulin sensitivity, atypical PKC knockout mice are not yet available.
PI 3-KINASE-INDEPENDENT PATHWAY Although numerous studies have demonstrated that PI 3-kinase activation is necessary, several lines of evidence have suggested that additional signals are required to fully stimulate GLUT4 translocation and glucose uptake. For example, activation of PI 3-kinase by platelet-derived growth factor (PDGF), interleukin-4 (IL-4), or engagement of integrin receptors does not induce GLUT4 translocation in adipocytes despite activation of PI 3-kinase. In addition, two insulin receptor mutants have been identified that are capable of inducing PI 3-kinase activation but not GLUT4 translocation. These data suggest that although the PI 3-kinase pathway is necessary for insulin-stimulated GLUT4 translocation, this pathway is not sufficient and at least one additional signaling pathway is required.
18 A potential clue toward identifying the PI 3-kinaseindependent arm of insulin action emerged from the idea that signal initiation might be segregated into discrete compartments in the plasma membrane. One candidate for such compartments is caveolae, that is, small invaginations of the plasma membrane containing caveolin that are a subset of lipid raft domains. These localized regions are enriched in lipid-modified signaling proteins, glycosylphosphatidylinositol (GPI)anchored proteins, glycolipids, sphingolipids, and cholesterol. Insulin stimulates the tyrosine phosphorylation of caveolin, the major structural protein in caveolae. Investigation of this pathway reveals the phosphorylation of another insulin receptor substrate, the proto-oncogene c-Cbl. The insulin-stimulated phosphorylation of Cbl occurs only in cell lines that respond to insulin by changes in glucose and lipid metabolism and not in other fibroblast lines, despite the presence of Cbl and an active insulin receptor. The phosphorylation of Cbl requires the presence of the adapter protein APS that recruits Cbl to the insulin receptor. APS is a member of the lnk family of adapter proteins, containing both a PH domain and an SH2 domain. Once phosphorylated, the insulin receptor recruits APS via an interaction between the activation loop of the receptor and the SH2 domain of APS. On binding to the receptor, APS undergoes phosphorylation on a single tyrosine residue. This, in turn, recruits Cbl to the receptor–APS complex via an interaction with the SH2 domain of Cbl and leads to the tyrosine phosphorylation of Cbl on three residues. The Cbl-associated protein (CAP) is identified in a two-hybrid screen using Cbl as bait and contains three carboxyl-terminal SH3 domains. CAP is expressed predominantly in insulin-sensitive tissues and in differentiated 3T3-L1 adipocytes but not in preadipocytes. Expression of the CAP gene is increased by the insulin-sensitizing thiazolidinedione (TZD) drugs. TZD activation of PPAR-g directly activates the transcription of CAP through a PPAR-g response element in the CAP promoter. Moreover, TZD-stimulated increases in CAP expression lead to a more robust phosphorylation of Cbl in response to insulin, establishing a potential primary link between TZD-induced insulin sensitization and insulin signal transduction. The CAP protein is recruited with Cbl to the insulin receptor due to an interaction of its third carboxylterminal SH3 domain with Cbl and another direct interaction with APS through its amino- and carboxyl-terminal SH3 domains. The localization and stabilization of this signaling complex in lipid rafts appear to result from its association with the hydrophobic protein flotillin. This interaction is localized to
Insulin Action, Post-Receptor Mechanisms
amino-terminal sequences of CAP that contain homology to the gut peptide sorbin, that is, the sorbin homology (SoHo) domain. Dominant interfering SH3 deletion mutants of CAP that bind to flotillin but not Cbl, or SoHo deletion mutants that bind to Cbl but not flotillin, interfere with the localization of Cbl to lipid rafts. Moreover, these mutants specifically block insulin-stimulated GLUT4 translocation and glucose uptake. The SH2 domain of the small adapter protein CrkII also associates with tyrosine-phosphorylated Cbl and thereby becomes recruited to plasma membrane lipid raft subdomains. In addition to its aminoterminal SH2 domain, CrkII contains two tandem SH3 domains, the first of which constitutively interacts with the proline-rich domain of the guanyl nucleotide exchange factor, C3G. Consistent with this mechanism, insulin also recruits C3G to lipid rafts, a process that is blocked by expression of the dominantinterfering CAP mutant lacking the SH3 domains. C3G can function as a guanine exchange factor (GEF) for several small guanosine triphosphate (GTP)-binding proteins, including Rap1 and R-Ras/ TC21. In screens for small GTP-binding proteins that could potentially regulate insulin-stimulated GLUT4 translocation, the Rho family member TC10 has been identified. Overexpression of this protein has profound effects on insulin-stimulated GLUT4 translocation and glucose uptake. Moreover, C3G activates the exchange of GTP for guanosine diphosphate (GDP) on TC10 both in vitro and in vivo. Unlike other members of the Rho family that undergo geranylgeranylation and interact with guanyl nucleotide dissociation inhibitors, TC10 is subjected to both farnesylation and palmitoylation in a manner analogous to that of H-Ras. These posttranslational modifications are responsible for the targeting of TC10 to lipid raft domains. Moreover, mistargeting of TC10 into non-lipid raft regions of the plasma membrane prevents its activation by insulin and alters the ability of TC10 to modulate insulin-stimulated GLUT4 translocation. In addition, disruption of the lipid raft with cholesterol-extracting drugs or by overexpression of inhibitory forms of caveolin completely blocks TC10 activation as well as the stimulation of glucose transport by insulin. Moreover, treatment of cells with the cholesterol-extracting drug b-cyclodextrin also blocks the stimulation of glucose transport. TC10 also interacts with one of the components of the exocyst complex, Exo70, in a GTP-dependent fashion. Exo70 translocates to the plasma membrane in response to insulin via the activation of TC10,
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where it assembles a multiprotein complex that includes Sec6 and Sec8 as well as other proteins that have not yet been identified. Overexpression of an Exo70 mutant blocks insulin-stimulated glucose uptake but not the trafficking of GLUT4 to the plasma membrane. However, this mutant does block the extracellular exposure of the GLUT4 protein. These data demonstrate that the exocyst may play a critical role in the targeting of the GLUT4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.
CYTOSKELETON AND GLUCOSE TRANSPORT It is well established that the cytoskeleton plays an important role in the stimulation of glucose uptake by insulin. Although the PI 3-kinase pathway can influence actin polymerization, changes occur in actin function that are PI 3-kinase independent. For example, insulin was reported to enhance the localization of the unconventional myosin (Myo1c) to GLUT4 compartments that are associated with actin filaments independent of PI 3-kinase activity. Overexpression of wild-type Myo1c enhances insulin-stimulated GLUT4 translocation, whereas expression of a dominant negative Myo1c mutant inhibits this action. Thus, Myo1c may function in a PI 3-kinase-independent insulinsignaling pathway controlling GLUT4 trafficking. Microtubules have also been implicated in the regulation of GLUT4 translocation. Kinesin KIF5B is partially colocalized with perinuclear GLUT4, and expression of dominant negative kinesin mutants blocks outward GLUT4 vesicle movements. These events are not affected by specific inhibition of PI 3-kinase activity. Thus, insulin signaling appears to target and/or initiate the movement of GLUT4containing membranes on microtubules via conventional kinesin (KIF5B) and subsequently actin through PI 3-kinase-independent mechanisms.
SNARE-MEDIATED GLUT4 VESICLE DOCKING/FUSION AND TRANSPORT ACTIVATION It is well established that vesicle-mediated fusion with target membranes requires a high-affinity interaction between specific pairs of target membrane SNAP receptors (t-SNAREs) and those of vesicle membrane SNAP receptors (v-SNAREs). Considerable progress has been made in identifying the v-SNAREs and
t-SNAREs that facilitate GLUT4 vesicle translocation. VAMP2 or syntaptobrevin 2 is the predominant v-SNARE found in GLUT4 vesicles, whereas syntaxin 4 and SNAP23 function as the major t-SNAREs. Many of the accessory components of GLUT4 vesicles have been identified, and intensive efforts are under way to isolate the component of the various subcellular GLUT4 compartments. Adapter molecules that regulate the interaction between VAMP2 and syntaxin 4 have been identified and include Munc18c, Synip, and tomosyn. As described previously, it is well established that insulin induces a redistribution of the GLUT4 protein from intracellular storage compartments to the plasma membrane. Although it was generally accepted that this was sufficient to account for the increase in glucose uptake, more recent studies have suggested that a second event is required for the acquisition of glucose transport activity, perhaps one dependent on the activation of the p38 MAP kinase.
INSULIN REGULATES GLUCOSE USE AND STORAGE In addition to stimulating uptake, insulin directly regulates the use and storage of glucose. Following its transport into the cell, insulin can activate enzymes responsible for the oxidation of glucose and its storage as glycogen or lipid. The hormone also can inhibit enzymes that promote the breakdown of lipid or glycogen. Intracellular glucose undergoes a regulated phosphorylation step and is directed to a storage pathway involving glycogen synthesis or an oxidative pathway culminating in the rate-limiting enzyme pyruvate dehydrogenase in the mitochondrion. The resulting two carbon intermediates serve as substrates for de novo FFA synthesis and subsequent esterification with glycerol to form triglyceride. Numerous enzymes that control these pathways are regulated by insulin through processes that involve covalent modifications such as phosphorylation, allosteric regulation, or changes in gene expression. The storage of glucose as glycogen allows for its rapid mobilization as a metabolic fuel. Glycogen can be metabolized to produce adenosine triphosphate (ATP) in the absence of oxygen and can produce more ATP per oxygen molecule used than does lipid when used under aerobic conditions. Thus, most tissues rely on glucose as their primary energy source under postprandial conditions and during times of stress or the initial stages of starvation. Insulin regulates glycogen metabolism primarily via the phosphorylation states
20 and activities of the enzymes that catalyze glycogen synthesis or breakdown. Glycogen synthase is inactivated by its phosphorylation at multiple residues. Phosphorylation of glycogen synthase occurs at nine different sites in a hierarchical fashion. These phosphorylations are catalyzed by a number of different kinases, including calmodulin-dependent kinases, casein kinase, cyclic adenosine monophosphate (cAMP)dependent protein kinase (PKA), AMP-activated kinase, and glycogen synthase kinase. Glycogen synthase kinase-3 (GSK3) plays a major role in the phosphorylation and inactivation of glycogen synthase. GSK3 is phosphorylated on Ser9 by the Akt protein kinase, which is activated by insulin via PDK1 as described previously. Phosphorylation at this site reduces the catalytic activity of the kinase. Thus, one mechanism by which insulin can increase the overall activity of glycogen synthase is through the inactivation of GSK3. Phosphorylation of glycogen synthase by GSK3 occurs in a sequential manner, beginning at Ser652 (site 4), followed by Ser648 (site 3c), Ser644 (site 3b), and Ser640 (site 3a). The phosphorylation of glycogen synthase by GSK3 can occur only after the initial phosphorylation of site 5 (Ser656) that is catalyzed by casein kinase II. Glycogen synthase can also be activated via direct dephosphorylation mediated by protein phosphatase 1 (PP1), the major phosphatase thought to stimulate glycogen synthase activation by insulin. PP1 can also dephosphorylate and inactivate glycogen phosphorylase and phosphorylase kinase. Because PP1 has relatively broad substrate specificity in vitro, a number of targeting subunits have been identified that facilitate the recruitment of the phosphatase to specific subcellular locales, including myofibrils, neuronal dendrites, nucleus, and glycogen particles. Four glycogentargeting subunits of PP1 have been described. Although these share certain conserved structural domains for the binding of PP1, glycogen, and PP1 substrates, they vary in their interactions with the enzymatic machinery of glycogen metabolism, tissue distribution, and roles in glycogen metabolism.
GM–Muscle Glycogen Targeting Subunit The expression of PPP1R3a (GM, RGL) is restricted to striated muscle fibers and cardiac muscle. GM is unique in that it possesses phosphorylation sites that have been implicated in the regulation of muscle glycogen synthesis by b-adrenergic stimuli and a large C-terminal region that is required for association with the sarcoplasmic reticulum. Phosphorylation of Ser67, which lies within the PP1-binding
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region, results in the dissociation of GM from PP1, and this may contribute to decreases in glycogen synthesis due to increased intramyocellular cAMP. Two mouse models of targeted disruption of the PPP1R3a gene have been reported, and both animal models exhibit greater than 80% decreases in the levels of steady-state muscle glycogen, paralleled by decreased activity of glycogen synthase and increased glycogen phosphorylase. These data demonstrate that GM plays a vital role in the regulation of glycogen metabolism in muscle. Paradoxically, one of the reported GM knockout models develops severe glucose intolerance and insulin resistance and becomes obese with aging, whereas the other does not. This discrepancy may be due to differences in genetic background, suggesting that dramatic decreases in muscle glycogen alone are not sufficient to produce insulin resistance without the contribution of other genetic or environmental factors.
GL PPP1R4 (GL) is a liver-specific glycogen-targeting subunit. This subunit is unique in that it does not bind glycogen synthase yet still appears to play a role in the regulation of hepatic glycogen synthase phosphatase activity. However, GL possesses a unique high-affinity glycogen phosphorylase a binding region at its C terminus. The binding of active phosphorylase by GL is thought to permit the allosteric inhibition of liver glycogen synthase phosphatase by phosphorylase a during periods of net glycogenolysis. Overexpression of GL in primary hepatocytes leads to significant increases in steady-state glycogen. The expression of GL is down-regulated during prolonged fasting and in streptozotocin diabetes and can be restored by refeeding or insulin treatment.
PTG Protein targeting to glycogen (PTG, PPP1R5) is expressed in a variety of tissues that store glycogen. PTG is expressed most highly in adipose, liver, heart, striated muscle, brain, and kidney. PTG is known to bind glycogen synthase, glycogen phosphorylase, and phosphorylase kinase in addition to PP1 and glycogen. The binding of enzymatic substrates to PTG can occur in the absence of glycogen in vitro and is mediated by a single binding region in the molecule. Overexpression of PTG in Chinese hamster ovary cells stably expressing the insulin receptor, primary hepatocytes, 3T3-L1 adipocytes, cultured human muscle cells, or intact rat liver results in dramatic
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increases in steady-state glycogen levels. However, unlike the other targeting subunits, overexpression of PTG seems to lock cells in a glycogenic mode, where they become resistant to the effects of glycogenolytic stimuli and glycogen feedback inhibition. Appropriate regulation of PTG expression is required to maintain the appropriate balance between glycogen synthesis and glycogenolysis. Targeted disruption of one allele of the gene for PTG results in moderate decreases in steady-state glycogen in adipose, heart, and liver, with some minor effects in white fiber striated muscle. Homozygous deletion of PTG results in embryonic lethality. PTG heterozygous mice develop glucose intolerance and mild insulin resistance, hyperinsulinemia, and hyperleptinemia with aging. This phenotype appears to arise from a repartitioning of fuel stores into lipid due to the decreases in whole body glycogen stores, thereby resulting in the accumulation of triglyceride in muscle and an attenuation of insulin signaling. The expression of PTG is downregulated in rat liver due to fasting or streptozotocin diabetes and can recover on refeeding or insulin treatment, much like GL. However, the changes in PTG expression occur much sooner on the commencement of fasting than do the changes in GL expression.
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The biosynthesis and breakdown of lipids are tightly regulated by insulin by modulating the activity of enzymes that catalyze the rate-limiting reactions of these pathways and by controlling expression of the genes encoding these enzymes. The hormone also stimulates the recruitment of fatty acids into tissues by hydrolysis of triacylglycerol derived from circulating very low-density lipoproteins and intestinal chylomicrons, which are by-products of hepatic lipid synthesis or dietary influx. Under normal conditions, lipogenesis occurs predominantly in adipose depots and in the liver. Two coupled processes are involved: de novo fatty acid synthesis from two carbon intermediates and the subsequent esterification of activated fatty acids to glycerol to form triglyceride (the final lipid storage form). The rate-limiting step of lipogenesis is the carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by the enzyme acetyl-CoA carboxylase (ACC). The activity of this enzyme depends on its state of polymerization, which is favored by binding of the allosteric activator citrate. Aggregation of the monomer into the active polymeric form is also stimulated by the phosphorylation at a specific site in response to
21 insulin. Conversely, phosphorylation at alternate sites by cAMP-dependent PKA or by the AMP-activated protein kinase results in the depolymerization and deactivation of ACC. Insulin also promotes the dephosphorylation of these sites. Oxidation of fatty acids occurs primarily in liver, as well as in cardiac and striated muscle, which uses fatty acids as a main fuel in the resting state. Insulin indirectly inhibits fatty acid b-oxidation by increasing malonyl-CoA production (via ACC activation). Malonyl-CoA allosterically inhibits the activity of carnitine palmitoyl transferase I, and this catalyzes the rate-limiting transport of activated fatty acids into the mitochondrion where oxidation takes place. In a similar fashion, feedback inhibition by malonyl-CoA and long-chain fatty acyl CoA esters also affects ACC activity by triggering depolymerization of the active polymer. The integrated coordination of lipogenesis and b-oxidation by insulin also occurs at the level of gene expression, mediated by two main transcription factors. These trans-acting factors include isoforms of the sterol receptor enhancer-binding proteins (SREBPs) and PPAR-g. SREBPs participate with other trans-acting factors, such as the upstream stimulatory factors, to mediate the effects of insulin on the expression of various genes involved in hepatic lipid metabolism, such as that encoding fatty acid synthetase. SREBP2 appears to be involved in hepatic cholesterol metabolism because its overexpression results in increased synthesis of hydroxymethylglutaryl (HMG)-CoA reductase and farnesyl pyrophosphate synthase. The role of SREBP1 in fatty acid/triglyceride metabolism seems to be restricted mainly to the liver, although it likely plays an important role in adipocyte cholesterol metabolism. SREBP null mice show no changes in adipose mass, or in the expression of acetyl-CoA carboxylase or fatty acid synthetase, although the up-regulation of hepatic lipogenic genes during fasting/refeeding is prevented. PPAR-g is a nuclear hormone receptor present mainly in adipose tissue, but it is also expressed at low levels in liver. PPAR ligands include prostaglandins, fatty acids, and the synthetically derived thiazolidinediones. PPAR-g expression is induced by insulin, and its promoter is itself trans-activated by SREBP1. PPAR-g expression is critical for adipocyte differentiation and is required for the trans-activation of adipocyte gene products involved in lipid metabolism, including acetyl-CoA synthetase, adipocyte fatty acid-binding protein, fatty acid transport protein, the glycerolneogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and lipoprotein lipase (LPL).
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This nuclear receptor also increases insulin sensitivity in cells and in vivo, explaining the antidiabetic effects of the PPAR-g-activating drugs. The circulating levels of insulin required to inhibit lipolysis are far below those required to stimulate glucose uptake into muscle. This physiological property results in an initial inhibition of fatty acid release into the circulation from adipose depots, and increased uptake of dietary-derived lipids by the peripheral tissues occurs following ingestion of a meal. Lipolysis in the fat cells is catalyzed by the enzyme hormone-sensitive lipase (HSL) in a mechanism that requires the translocation of the enzyme from the cytoplasm to the periphery of the lipid droplet. Upon phosphorylation by PKA, the enzyme undergoes catalytic activation and is translocated to the lipid droplet by a mechanism involving the PKA substrate perilipin. The phosphorylation of perilipin results in a dispersion of the normal perilipin ‘‘coat’’ around the lipid droplet, thereby potentially increasing the accessible surface area of the droplet to HSL activity. Insulin prevents the phosphorylation of HSL primarily by attenuating cAMP signaling via activation of cyclic nucleotide phosphodiesterase 3B, which catalyzes the conversion of cAMP to 50 -AMP. Insulin also increases the dephosphorylation of HSL and inhibits the phosphorylation of perilipin. Regulation of HSL localization also involves the docking protein lipotransin, and HSL is capable of interacting with lipotransin after phosphorylation by PKA, thereby allowing for its docking at the outer surface of the lipid droplet. Lipotransin possesses an intrinsic ATPase activity that allows for a dissociation of the complex after docking, permitting HSL to proceed with the catalysis. Insulin may freeze the HSL–lipotransin complex at the docking site, thereby preventing the dissociation step that is required for HSL to catalyze triglyceride hydrolysis. The release of dietary- or hepatic-derived fatty acids from circulating lipoprotein complexes is catalyzed
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by LPL, which catalyzes the rate-limiting step in the hydrolysis of lipoprotein-associated triglycerides to yield FFAs and 2-monoacylglycerol. Insulin appears to increase the expression and release of LPL from fat cells. Insulin stimulation of LPL activity occurs by both wortmannin-sensitive PI 3-kinase and rapamycin-sensitive p70 S6-kinase-signaling pathways.
See Also the Following Articles Insulin and Insulin-like Growth Factors, Evolution of Insulin Secretion: Functional and Biochemical Aspects Insulin Secretion, Physiology
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Further Reading Boss, A., Guilherme, A., Robida, S. I., Nicoloro, S. M., Zhou, Q. L., Jiang, Z. Y., Pomerleau, D. P., and Czech, M. P. (2002). Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature 420, 821–824. Brady, M. J., and Saltiel, A. R. (2001). The role of protein phosphatase-1 in insulin action. Recent Prog. Horm. Res. 56, 157–173. Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. Science 296, 1655–1657. Inoue, M., Chang, L., Hwang, J., Chiang, S. H., and Saltiel, A. R. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422, 629–633. Mauvais-Jarvis, F., Kulkarni, R. N., and Kahn, C. R. (2002). Knockout models are useful tools to dissect the pathophysiology and genetics of insulin resistance. Clin. Endocrinol. 57, 1–9. Mora, S., and Pessin, J. E. (2002). An adipocentric view of signaling and intracellular trafficking. Diab. Metab. Res. Rev. 18, 345–356. Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M. (2002). Transcriptional regulation of adipogenesis. Genes Dev. 14, 1293–1307. Saltiel, A. R., and Pessin, J. E. (2002). Insulin signaling pathways in time and space. Trends Cell. Biol. 12, 65–71. Unger, R. H., and Orci, L. (2001). Diseases of liporegulation: New perspective on obesity and related disorders. FASEB J. 15, 312–321. White, M. F. (2002). IRS proteins and the common path to diabetes. Am. J. Physiol. Endocrinol. Metab. 283, E413–E422.
Sean Crosson University of Michigan Medical Center, Ann Arbor, Michigan, United States
Masatomo Mori Gunma University Graduate School of Medicine, Gunma, Japan
Alan R. Saltiel University of Michigan Medical Center, Ann Arbor, Michigan, United States
Jeffrey E. Pessin State University of New York, Stony Brook, New York, United States
Glossary hepatic Relating to or affecting the liver. hyperglycemia An abnormally high concentration of glucose (sugar) in the blood. insulin A polypeptide hormone consisting of two peptide chains linked by a disulfide bridge, produced in the beta cells of the endocrine pancreas and released in response to elevated levels of blood glucose. phosphorylation A process in which a phosphate group is combined with an organic molecule; occurs naturally in cellular metabolism.
I
nsulin is the most potent hormonal regulator of glucose, lipid, and protein metabolism. In states of glucose excess, insulin prevents hyperglycemia through the suppression of liver glucose production and the stimulation of adipose and skeletal muscle glucose uptake. In states of nutrient deprivation, the decrease in insulin levels results in enhanced liver glucose output (glycogenolysis and gluconeogenesis) and reduced adipose and skeletal muscle glucose uptake. The coordination of these cell type-specific responses maintains glucose levels within a narrow range. However, disruption of these homeostatic mechanisms leads to peripheral tissue insulin resistance, a hallmark for the development of type 2 diabetes.
THE INSULIN RECEPTOR The insulin receptor is a transmembrane protein composed of two a-subunits and two b-subunits that are disulfide-linked in an a2b2 heterotetrameric structure. The a-subunits are extracellular and contain the
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insulin-binding domains, whereas the b-subunits contain a single-transmembrane region with an intracellular tyrosine kinase domain. Insulin binding triggers activation of the b-subunit protein kinase activity via transphosphorylation, resulting in increased phosphorylation of several intracellular substrates. Although the insulin receptor is expressed in virtually all tissues, three have classically been considered as targets for the metabolic actions of the hormone: liver, muscle, and fat. Recent studies using genetically altered mice have challenged these assumptions and, in the process, have provided important clues to understanding the complexity of specific signaling molecules that control biological responsiveness to insulin.
Liver The fasting hyperglycemia that occurs in type 2 diabetes results from increased hepatic glucose production, through increased gluconeogenesis and glycogenolysis, as well as reduced glycogen synthesis. Disruption of insulin action in the liver by tissuespecific knockout of the insulin receptor in mice (LIRKO mice) produces severe glucose intolerance with insulin resistance. These data demonstrate that the liver plays a direct role in the regulation of postprandial glucose homeostasis by insulin and occurs due to a suppression of hepatic glucose production rather than an increase in muscle glucose uptake in mice. LIRKO mice also exhibit compensatory hyperinsulinemia, similar to transgenic mice overexpressing the insulin receptor. In addition, lipotropic mice display chronic hyperinsulinemia, leading to decreased IRS2 expression in liver concomitant with insulin
Encyclopedia of Endocrine Diseases, Volume 3. ß 2004 Elsevier Inc. All rights reserved.
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resistance of gluconeogenesis. Thus, hepatic insulin resistance in type 2 diabetes occurs secondary to chronic hyperinsulinemia.
Adipose Tissue White adipose tissue accounts for less than 10% of glucose uptake but plays a major role in whole body insulin sensitivity by releasing into the circulation various modulators of insulin action, including lipid products and hormones. Free fatty acids (FFAs) derived from adipocytes are elevated in obesity and type 2 diabetes and accumulate in muscle and liver, contributing to insulin resistance in these tissues. Mice with a selective deletion of the insulin receptor in brown adipocytes experience an age-dependent loss of intrascapular brown fat but increased expression of uncoupling proteins-1 and -2. In parallel, these mice develop a defect in insulin secretion, resulting in progressive glucose intolerance, without insulin resistance. These data demonstrate that the insulin receptor is required for brown fat formation throughout development and that brown fat regulates insulin secretion and glucose homeostasis. Histological, immunohistochemical, and ultrastructural analyses of white adipose tissue in newborn fat insulin receptor knockout mice (FIRKO mice) reveal a marked decrease in adipose tissue area. This fat cell depletion resulted from a reduction of adipocyte volume ( 90%), with a small decrease in the number of adipocytes. Electron microscopy analysis displays a normal pattern of adipogenesis in FIRKO mice, demonstrating that lack of insulin receptors is not associated with a selective impairment of the adipocyte differentiation process. Thus, the insulin receptor is dispensable for adipocyte differentiation but is necessary for the large extent of lipid storage in these cells. In addition, FIRKO mice display increased longevity, demonstrating an inverse relationship between high rates of adipose metabolism and life span. Adipocytes also secrete cytokines (adipokines) that modulate peripheral insulin sensitivity. Tumor necrosis factor-a (TNF-a) is elevated in obese humans and rodents and has been shown to reduce insulin signaling by decreasing insulin receptor and IRS1 tyrosine phosphorylation. Mice harboring deletion of the genes encoding TNF-a or the TNF-a receptor are refractory to insulin resistance associated with diet-induced obesity or when crossed with genetically obese ob/ob mice. Resistin is another adipokine secreted from adipocytes, and the levels of this hormone are increased in obese mice and reduced after administration of the insulin-sensitizing perioxisome proliferator-activated
receptor-g (PPAR-g)-activating thiazolidinediones. Resistin also inhibits adipogenesis and, thus, plays a negative feedback role in fat cell formation. Adipose tissue also secretes leptin, another hormone that positively influences metabolism and energy expenditure, and has profound effects on appetite. Inactivation of leptin production (ob gene in mice) or defects in the leptin receptor (db gene) results in autophagy, obesity, and severe insulin resistance. Similarly, transgenic ablation of white adipose tissue in mice leads to severe insulin resistance, elevated lipid levels, undetectable leptin concentrations, and diabetes, a phenotype reminiscent of generalized lipoatropic diabetes in humans. In these models, restoration of physiological serum leptin levels, either by leptin infusion, by transgenic overexpression, or by surgical implantation of white adipose tissue, reverses the diabetic and insulin-resistant phenotype. These results demonstrate that insulin resistance and diabetes in lipodystrophic mice can be caused by a deficiency of leptin secondary to a failure of adipocyte differentiation. Adiponectin is also secreted from adipocytes in a regulated fashion. The expression of this adipokine is decreased in obese humans and rodents, and increased expression positively correlates with insulin sensitivity. Acute treatment of obese mice with adiponectin decreases plasma FFA, muscle, and liver triglyceride content and improves insulin resistance. In a knockout model of lipoatropic diabetes, insulin resistance was completely reversed by a combination of leptin and adiponectin infusion but was only partially reversed by either leptin or adiponectin alone.
Skeletal Muscle Surprisingly, specific deletion of the skeletal muscle insulin receptor (MIRKO) yields mice with normal glucose and insulin levels but with dyslipidemia and increased adiposity. Although MIRKO mice display reduced insulin-stimulated muscle glucose uptake in vitro and during a euglycemic hyperinsulinemic clamp, glucose tolerance tests show near-normal glucose uptake. This discrepancy is likely due to increased insulin-independent glucose uptake. In this regard, muscle-specific ablation of the insulin receptor, coupled with a loss of insulin-like growth factor-1 (IGF-1) expression, results in severe insulin resistance in muscle. Interestingly, skeletal muscle insulinresponsive glucose transporter (GLUT4) null mice are also insulin resistant, demonstrating that downstream insulin action and glucose uptake, rather than
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Pancreatic Beta Cells Insulin receptor knockouts in pancreatic beta cells (b-IRKO) display a phenotype similar to that of type 2 diabetes, having a selective loss of glucose-stimulated first-phase insulin secretion and progressive impairment of glucose tolerance. The loss of normal secretory function results from decreased expression of the critical glucose-sensing enzyme glucose kinase. These data directly demonstrate the insulin receptor functions in an autocrine feedback loop necessary for maintaining appropriate beta cell insulin secretion.
Brain The insulin receptor is widely distributed throughout the central nervous system (CNS), especially in the hypothalamus and pituitary. Mice with CNS-specific disruption of the insulin receptor gene (NIRKO mice) show increased food intake, resulting in diet-induced obesity and insulin resistance. Thus, insulin action in the CNS appears to provide a negative feedback loop for postprandial inhibition of food intake and plays a central role in the regulation of body weight.
SIGNALING DOWNSTREAM OF THE INSULIN RECEPTOR On activation, the insulin receptor can catalyze the tyrosine phosphorylation of numerous cytoplasmic proteins. The best characterized of these is the insulin receptor substrate family, consisting of four closely related members (IRS1 to -4) and three members of the Grb2-associated-binding proteins (Gab1 to -3). The IRS and Gab proteins appear to provide overlapping functions given that each can participate in the actions of various growth factors, cytokines, and developmental signals. The insulin receptor phosphorylates all four IRS family members on numerous tyrosine residues, in the process generating recognition sites for the interaction of effector proteins containing Src homology (SH2) domains. For example, IRS1 contains 21 putative tyrosine phosphorylation sites, several of which are located in amino acid sequence motifs that bind to SH2 domain proteins, including the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, Grb2, Nck, Crk, Fyn, Csk, phospholipase Cg, and SHP2. IRS1 also contains more than 30 potential serine/threonine phosphorylation sites in motif
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recognized by various kinases such as casein kinase II, protein kinase C (PKC), protein kinase B/Akt, Jun amino-terminal kinase ( JNK), and mitogen-activated protein (MAP) kinase. The crucial role for these substrate proteins has been documented both in tissue culture model systems and by the use of homologous recombination in mice. In particular, disruption of the IRS1 gene results in growth retardation and mild insulin resistance, whereas IRS2 null mice display peripheral insulin resistance, impaired insulin secretion, and diabetes. Interestingly, the IRS1 null mice do not become diabetic due to pancreatic beta cell compensation. However, the IRS2 mice have a marked reduction in beta cell mass due to a marked inhibition of beta cell development. Although mice carrying an ablated IRS3 gene did not display any detectable phenotype, disruption of IRS4 resulted in modest growth retardation and mild insulin resistance. Both physiological and pathophysiological regulation of IRS protein function occur at multiple levels. Several studies have shown that serine/threonine phosphorylation of IRS proteins can reduce insulinstimulated tyrosine phosphorylation, demonstrating that serine/threonine phosphorylation functions in a feedback-inhibitory mechanism. In this regard, members of the MAP kinase superfamily are capable of catalyzing serine phosphorylations of the IRS proteins. Studies indicate that the stress-induced serine kinase JNK is one such kinase. JNK is stimulated during acute or chronic inflammation and in response to inflammatory cytokines such as TNF-a. JNK phosphorylates IRS1 and IRS2, Shc, and Gab1, and both IRS1 and IRS2 contain JNK-binding motifs. This motif mediates the specific association of JNK with IRS1, catalyzing the phosphorylation of a serine residue on the COOH-terminal side of the phosphotyrosine-binding (PTB) domain (Ser307 in [murine] IRS1, Ser312 in [human] IRS1). Phosphorylation of this residue inhibits the function of the PTB domain, disrupting the association between the insulin receptor and IRS1 and inhibiting tyrosine phosphorylation. This mechanism might explain, at least in part, the insulin resistance that occurs during trauma and obesity. IRS serine/threonine phosphorylation may occur downstream of other kinases activated by inflammatory signals. High doses of salicylates are shown to reverse hyperglycemia, hyperinsulinemia, and dislipidemia in obese rodents by sensitizing the insulinsignaling pathway, including IRS protein tyrosine phosphorylation. The effect of salicylates is attributed to inhibition of IkB kinase-b (IKK-b), resulting in decreased IRS serine/threonine phosphorylation. Importantly, heterozygous disruption of IKK-b protects
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against the development of insulin resistance during high-fat feeding and in obese, leptin-deficient (ob/ob) mice. Although there is no physical interaction between IRS proteins and IKK-b, salicylates increase insulin-stimulated phosphorylation of IRS proteins in the liver, demonstrating that IKK-b inhibits insulin receptor function through the activation of an intermediate kinase(s) or through activation of NFkB. Whereas this type of feedback via serine/threonine phosphorylation accounts for relatively short-term desensitization, the IRS proteins also undergo long-term down-regulation. Prolonged insulin stimulation substantially reduces IRS1 and IRS2 protein levels through a 26S proteosome degradative pathway. Furthermore, insulin stimulates the ubiquitination of IRS2, and reduction of IRS2 by ubiquitin/proteosome-mediated proteolysis in mouse embryo fibroblasts lacking IRS1 dramatically inhibits the activation of Akt and ERK1/2 in response to insulin or IGF-1. The activity of the ubiquitin/proteosome system is also elevated in diabetes, promoting the degradation of the IRS proteins and exacerbating insulin resistance.
PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY Tyrosine phosphorylated IRS proteins engage several effectors that contain SH2 domains. In particular, the SH2 domains of type 1A PI 3-kinase regulatory subunits directly interact with tyrosine phosphorylated IRS proteins, resulting in the targeting and activation of the PI 3-kinase catalytic subunit. Once activated and/or appropriately targeted, this kinase generates PI 3,4,5P3 in the plasma membrane and perhaps a small amount of PI 3,4,5P3 in other intracellular membrane compartments. Generation of PI 3,4,5P3 is essential for insulin-stimulated glucose uptake; glycogen, lipid, and protein synthesis; and changes in gene expression. There are several regulatory (p85a, p85b p55/AS53, p55PIK, and p50) and catalytic (110a and 110b) subunits of this lipid kinase. Surprisingly, genetic ablation of the p85a regulatory subunit results in enhanced insulin sensitivity, probably due to excess levels of the other regulatory subunits compared with the catalytic subunit. PI 3,4,5P3 functions as a signaling intermediate that can target and activate the serine/threonine kinase PIdependent kinase (PDK1). PDK1 contains a pleckstrin homology (PH) domain that has a high affinity and selectivity for PI 3,4,5P3. PI 3,4,5P3 also interacts with the PH domain of Akt. Akt is a substrate for PDK1, and phosphorylation on two carboxyl-terminal serine/threonine residues results in the activation of
the Akt kinase. Similarly, PDK1 can also phosphorylate an activating threonine residue in the atypical PKC isoforms, PKCl and PKCz. The Akt protein kinases exist as three isoforms, all of which are activated by phosphorylation on T308 and S473. On growth factor stimulation, Akt localizes near the plasma membrane, where it becomes phosphorylated on T308 by PDK1. Following phosphorylation and activation, Akt can remain associated with the plasma membrane or intracellular compartments, or it can translocate to the nucleus. Expression of constitutively active Akt stimulates glucose uptake in 3T3L1 adipocytes, whereas Akt inhibition through the use of dominant negative mutants partially inhibits the insulin-stimulated glucose transport. Although Akt1 knockout mice have no significant alteration in whole body glucose homeostasis, Akt2 gene ablation results in moderate insulin resistance and impaired glucose tolerance. These data demonstrate that Akt2 plays an important role in insulin-regulated glucose metabolism but that Akt1 can partially compensate for the loss of Akt2 function. PKCs are a large family of serine/threonine kinases that have also been implicated in several actions of insulin. There are three subgroups of PKCs: the classical cPKCs, the novel nPKCs, and the atypical aPKCs. Although these different family members appear to play overlapping but distinct roles, the aPKCs have been implicated in the stimulation of glucose uptake and protein synthesis by insulin in tissue culture model systems. Although the classical PKCa isoform has been ablated in mice and displays enhanced insulin sensitivity, atypical PKC knockout mice are not yet available.
PI 3-KINASE-INDEPENDENT PATHWAY Although numerous studies have demonstrated that PI 3-kinase activation is necessary, several lines of evidence have suggested that additional signals are required to fully stimulate GLUT4 translocation and glucose uptake. For example, activation of PI 3-kinase by platelet-derived growth factor (PDGF), interleukin-4 (IL-4), or engagement of integrin receptors does not induce GLUT4 translocation in adipocytes despite activation of PI 3-kinase. In addition, two insulin receptor mutants have been identified that are capable of inducing PI 3-kinase activation but not GLUT4 translocation. These data suggest that although the PI 3-kinase pathway is necessary for insulin-stimulated GLUT4 translocation, this pathway is not sufficient and at least one additional signaling pathway is required.
18 A potential clue toward identifying the PI 3-kinaseindependent arm of insulin action emerged from the idea that signal initiation might be segregated into discrete compartments in the plasma membrane. One candidate for such compartments is caveolae, that is, small invaginations of the plasma membrane containing caveolin that are a subset of lipid raft domains. These localized regions are enriched in lipid-modified signaling proteins, glycosylphosphatidylinositol (GPI)anchored proteins, glycolipids, sphingolipids, and cholesterol. Insulin stimulates the tyrosine phosphorylation of caveolin, the major structural protein in caveolae. Investigation of this pathway reveals the phosphorylation of another insulin receptor substrate, the proto-oncogene c-Cbl. The insulin-stimulated phosphorylation of Cbl occurs only in cell lines that respond to insulin by changes in glucose and lipid metabolism and not in other fibroblast lines, despite the presence of Cbl and an active insulin receptor. The phosphorylation of Cbl requires the presence of the adapter protein APS that recruits Cbl to the insulin receptor. APS is a member of the lnk family of adapter proteins, containing both a PH domain and an SH2 domain. Once phosphorylated, the insulin receptor recruits APS via an interaction between the activation loop of the receptor and the SH2 domain of APS. On binding to the receptor, APS undergoes phosphorylation on a single tyrosine residue. This, in turn, recruits Cbl to the receptor–APS complex via an interaction with the SH2 domain of Cbl and leads to the tyrosine phosphorylation of Cbl on three residues. The Cbl-associated protein (CAP) is identified in a two-hybrid screen using Cbl as bait and contains three carboxyl-terminal SH3 domains. CAP is expressed predominantly in insulin-sensitive tissues and in differentiated 3T3-L1 adipocytes but not in preadipocytes. Expression of the CAP gene is increased by the insulin-sensitizing thiazolidinedione (TZD) drugs. TZD activation of PPAR-g directly activates the transcription of CAP through a PPAR-g response element in the CAP promoter. Moreover, TZD-stimulated increases in CAP expression lead to a more robust phosphorylation of Cbl in response to insulin, establishing a potential primary link between TZD-induced insulin sensitization and insulin signal transduction. The CAP protein is recruited with Cbl to the insulin receptor due to an interaction of its third carboxylterminal SH3 domain with Cbl and another direct interaction with APS through its amino- and carboxyl-terminal SH3 domains. The localization and stabilization of this signaling complex in lipid rafts appear to result from its association with the hydrophobic protein flotillin. This interaction is localized to
Insulin Action, Post-Receptor Mechanisms
amino-terminal sequences of CAP that contain homology to the gut peptide sorbin, that is, the sorbin homology (SoHo) domain. Dominant interfering SH3 deletion mutants of CAP that bind to flotillin but not Cbl, or SoHo deletion mutants that bind to Cbl but not flotillin, interfere with the localization of Cbl to lipid rafts. Moreover, these mutants specifically block insulin-stimulated GLUT4 translocation and glucose uptake. The SH2 domain of the small adapter protein CrkII also associates with tyrosine-phosphorylated Cbl and thereby becomes recruited to plasma membrane lipid raft subdomains. In addition to its aminoterminal SH2 domain, CrkII contains two tandem SH3 domains, the first of which constitutively interacts with the proline-rich domain of the guanyl nucleotide exchange factor, C3G. Consistent with this mechanism, insulin also recruits C3G to lipid rafts, a process that is blocked by expression of the dominantinterfering CAP mutant lacking the SH3 domains. C3G can function as a guanine exchange factor (GEF) for several small guanosine triphosphate (GTP)-binding proteins, including Rap1 and R-Ras/ TC21. In screens for small GTP-binding proteins that could potentially regulate insulin-stimulated GLUT4 translocation, the Rho family member TC10 has been identified. Overexpression of this protein has profound effects on insulin-stimulated GLUT4 translocation and glucose uptake. Moreover, C3G activates the exchange of GTP for guanosine diphosphate (GDP) on TC10 both in vitro and in vivo. Unlike other members of the Rho family that undergo geranylgeranylation and interact with guanyl nucleotide dissociation inhibitors, TC10 is subjected to both farnesylation and palmitoylation in a manner analogous to that of H-Ras. These posttranslational modifications are responsible for the targeting of TC10 to lipid raft domains. Moreover, mistargeting of TC10 into non-lipid raft regions of the plasma membrane prevents its activation by insulin and alters the ability of TC10 to modulate insulin-stimulated GLUT4 translocation. In addition, disruption of the lipid raft with cholesterol-extracting drugs or by overexpression of inhibitory forms of caveolin completely blocks TC10 activation as well as the stimulation of glucose transport by insulin. Moreover, treatment of cells with the cholesterol-extracting drug b-cyclodextrin also blocks the stimulation of glucose transport. TC10 also interacts with one of the components of the exocyst complex, Exo70, in a GTP-dependent fashion. Exo70 translocates to the plasma membrane in response to insulin via the activation of TC10,
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where it assembles a multiprotein complex that includes Sec6 and Sec8 as well as other proteins that have not yet been identified. Overexpression of an Exo70 mutant blocks insulin-stimulated glucose uptake but not the trafficking of GLUT4 to the plasma membrane. However, this mutant does block the extracellular exposure of the GLUT4 protein. These data demonstrate that the exocyst may play a critical role in the targeting of the GLUT4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.
CYTOSKELETON AND GLUCOSE TRANSPORT It is well established that the cytoskeleton plays an important role in the stimulation of glucose uptake by insulin. Although the PI 3-kinase pathway can influence actin polymerization, changes occur in actin function that are PI 3-kinase independent. For example, insulin was reported to enhance the localization of the unconventional myosin (Myo1c) to GLUT4 compartments that are associated with actin filaments independent of PI 3-kinase activity. Overexpression of wild-type Myo1c enhances insulin-stimulated GLUT4 translocation, whereas expression of a dominant negative Myo1c mutant inhibits this action. Thus, Myo1c may function in a PI 3-kinase-independent insulinsignaling pathway controlling GLUT4 trafficking. Microtubules have also been implicated in the regulation of GLUT4 translocation. Kinesin KIF5B is partially colocalized with perinuclear GLUT4, and expression of dominant negative kinesin mutants blocks outward GLUT4 vesicle movements. These events are not affected by specific inhibition of PI 3-kinase activity. Thus, insulin signaling appears to target and/or initiate the movement of GLUT4containing membranes on microtubules via conventional kinesin (KIF5B) and subsequently actin through PI 3-kinase-independent mechanisms.
SNARE-MEDIATED GLUT4 VESICLE DOCKING/FUSION AND TRANSPORT ACTIVATION It is well established that vesicle-mediated fusion with target membranes requires a high-affinity interaction between specific pairs of target membrane SNAP receptors (t-SNAREs) and those of vesicle membrane SNAP receptors (v-SNAREs). Considerable progress has been made in identifying the v-SNAREs and
t-SNAREs that facilitate GLUT4 vesicle translocation. VAMP2 or syntaptobrevin 2 is the predominant v-SNARE found in GLUT4 vesicles, whereas syntaxin 4 and SNAP23 function as the major t-SNAREs. Many of the accessory components of GLUT4 vesicles have been identified, and intensive efforts are under way to isolate the component of the various subcellular GLUT4 compartments. Adapter molecules that regulate the interaction between VAMP2 and syntaxin 4 have been identified and include Munc18c, Synip, and tomosyn. As described previously, it is well established that insulin induces a redistribution of the GLUT4 protein from intracellular storage compartments to the plasma membrane. Although it was generally accepted that this was sufficient to account for the increase in glucose uptake, more recent studies have suggested that a second event is required for the acquisition of glucose transport activity, perhaps one dependent on the activation of the p38 MAP kinase.
INSULIN REGULATES GLUCOSE USE AND STORAGE In addition to stimulating uptake, insulin directly regulates the use and storage of glucose. Following its transport into the cell, insulin can activate enzymes responsible for the oxidation of glucose and its storage as glycogen or lipid. The hormone also can inhibit enzymes that promote the breakdown of lipid or glycogen. Intracellular glucose undergoes a regulated phosphorylation step and is directed to a storage pathway involving glycogen synthesis or an oxidative pathway culminating in the rate-limiting enzyme pyruvate dehydrogenase in the mitochondrion. The resulting two carbon intermediates serve as substrates for de novo FFA synthesis and subsequent esterification with glycerol to form triglyceride. Numerous enzymes that control these pathways are regulated by insulin through processes that involve covalent modifications such as phosphorylation, allosteric regulation, or changes in gene expression. The storage of glucose as glycogen allows for its rapid mobilization as a metabolic fuel. Glycogen can be metabolized to produce adenosine triphosphate (ATP) in the absence of oxygen and can produce more ATP per oxygen molecule used than does lipid when used under aerobic conditions. Thus, most tissues rely on glucose as their primary energy source under postprandial conditions and during times of stress or the initial stages of starvation. Insulin regulates glycogen metabolism primarily via the phosphorylation states
20 and activities of the enzymes that catalyze glycogen synthesis or breakdown. Glycogen synthase is inactivated by its phosphorylation at multiple residues. Phosphorylation of glycogen synthase occurs at nine different sites in a hierarchical fashion. These phosphorylations are catalyzed by a number of different kinases, including calmodulin-dependent kinases, casein kinase, cyclic adenosine monophosphate (cAMP)dependent protein kinase (PKA), AMP-activated kinase, and glycogen synthase kinase. Glycogen synthase kinase-3 (GSK3) plays a major role in the phosphorylation and inactivation of glycogen synthase. GSK3 is phosphorylated on Ser9 by the Akt protein kinase, which is activated by insulin via PDK1 as described previously. Phosphorylation at this site reduces the catalytic activity of the kinase. Thus, one mechanism by which insulin can increase the overall activity of glycogen synthase is through the inactivation of GSK3. Phosphorylation of glycogen synthase by GSK3 occurs in a sequential manner, beginning at Ser652 (site 4), followed by Ser648 (site 3c), Ser644 (site 3b), and Ser640 (site 3a). The phosphorylation of glycogen synthase by GSK3 can occur only after the initial phosphorylation of site 5 (Ser656) that is catalyzed by casein kinase II. Glycogen synthase can also be activated via direct dephosphorylation mediated by protein phosphatase 1 (PP1), the major phosphatase thought to stimulate glycogen synthase activation by insulin. PP1 can also dephosphorylate and inactivate glycogen phosphorylase and phosphorylase kinase. Because PP1 has relatively broad substrate specificity in vitro, a number of targeting subunits have been identified that facilitate the recruitment of the phosphatase to specific subcellular locales, including myofibrils, neuronal dendrites, nucleus, and glycogen particles. Four glycogentargeting subunits of PP1 have been described. Although these share certain conserved structural domains for the binding of PP1, glycogen, and PP1 substrates, they vary in their interactions with the enzymatic machinery of glycogen metabolism, tissue distribution, and roles in glycogen metabolism.
GM–Muscle Glycogen Targeting Subunit The expression of PPP1R3a (GM, RGL) is restricted to striated muscle fibers and cardiac muscle. GM is unique in that it possesses phosphorylation sites that have been implicated in the regulation of muscle glycogen synthesis by b-adrenergic stimuli and a large C-terminal region that is required for association with the sarcoplasmic reticulum. Phosphorylation of Ser67, which lies within the PP1-binding
Insulin Action, Post-Receptor Mechanisms
region, results in the dissociation of GM from PP1, and this may contribute to decreases in glycogen synthesis due to increased intramyocellular cAMP. Two mouse models of targeted disruption of the PPP1R3a gene have been reported, and both animal models exhibit greater than 80% decreases in the levels of steady-state muscle glycogen, paralleled by decreased activity of glycogen synthase and increased glycogen phosphorylase. These data demonstrate that GM plays a vital role in the regulation of glycogen metabolism in muscle. Paradoxically, one of the reported GM knockout models develops severe glucose intolerance and insulin resistance and becomes obese with aging, whereas the other does not. This discrepancy may be due to differences in genetic background, suggesting that dramatic decreases in muscle glycogen alone are not sufficient to produce insulin resistance without the contribution of other genetic or environmental factors.
GL PPP1R4 (GL) is a liver-specific glycogen-targeting subunit. This subunit is unique in that it does not bind glycogen synthase yet still appears to play a role in the regulation of hepatic glycogen synthase phosphatase activity. However, GL possesses a unique high-affinity glycogen phosphorylase a binding region at its C terminus. The binding of active phosphorylase by GL is thought to permit the allosteric inhibition of liver glycogen synthase phosphatase by phosphorylase a during periods of net glycogenolysis. Overexpression of GL in primary hepatocytes leads to significant increases in steady-state glycogen. The expression of GL is down-regulated during prolonged fasting and in streptozotocin diabetes and can be restored by refeeding or insulin treatment.
PTG Protein targeting to glycogen (PTG, PPP1R5) is expressed in a variety of tissues that store glycogen. PTG is expressed most highly in adipose, liver, heart, striated muscle, brain, and kidney. PTG is known to bind glycogen synthase, glycogen phosphorylase, and phosphorylase kinase in addition to PP1 and glycogen. The binding of enzymatic substrates to PTG can occur in the absence of glycogen in vitro and is mediated by a single binding region in the molecule. Overexpression of PTG in Chinese hamster ovary cells stably expressing the insulin receptor, primary hepatocytes, 3T3-L1 adipocytes, cultured human muscle cells, or intact rat liver results in dramatic
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increases in steady-state glycogen levels. However, unlike the other targeting subunits, overexpression of PTG seems to lock cells in a glycogenic mode, where they become resistant to the effects of glycogenolytic stimuli and glycogen feedback inhibition. Appropriate regulation of PTG expression is required to maintain the appropriate balance between glycogen synthesis and glycogenolysis. Targeted disruption of one allele of the gene for PTG results in moderate decreases in steady-state glycogen in adipose, heart, and liver, with some minor effects in white fiber striated muscle. Homozygous deletion of PTG results in embryonic lethality. PTG heterozygous mice develop glucose intolerance and mild insulin resistance, hyperinsulinemia, and hyperleptinemia with aging. This phenotype appears to arise from a repartitioning of fuel stores into lipid due to the decreases in whole body glycogen stores, thereby resulting in the accumulation of triglyceride in muscle and an attenuation of insulin signaling. The expression of PTG is downregulated in rat liver due to fasting or streptozotocin diabetes and can recover on refeeding or insulin treatment, much like GL. However, the changes in PTG expression occur much sooner on the commencement of fasting than do the changes in GL expression.
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The biosynthesis and breakdown of lipids are tightly regulated by insulin by modulating the activity of enzymes that catalyze the rate-limiting reactions of these pathways and by controlling expression of the genes encoding these enzymes. The hormone also stimulates the recruitment of fatty acids into tissues by hydrolysis of triacylglycerol derived from circulating very low-density lipoproteins and intestinal chylomicrons, which are by-products of hepatic lipid synthesis or dietary influx. Under normal conditions, lipogenesis occurs predominantly in adipose depots and in the liver. Two coupled processes are involved: de novo fatty acid synthesis from two carbon intermediates and the subsequent esterification of activated fatty acids to glycerol to form triglyceride (the final lipid storage form). The rate-limiting step of lipogenesis is the carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by the enzyme acetyl-CoA carboxylase (ACC). The activity of this enzyme depends on its state of polymerization, which is favored by binding of the allosteric activator citrate. Aggregation of the monomer into the active polymeric form is also stimulated by the phosphorylation at a specific site in response to
21 insulin. Conversely, phosphorylation at alternate sites by cAMP-dependent PKA or by the AMP-activated protein kinase results in the depolymerization and deactivation of ACC. Insulin also promotes the dephosphorylation of these sites. Oxidation of fatty acids occurs primarily in liver, as well as in cardiac and striated muscle, which uses fatty acids as a main fuel in the resting state. Insulin indirectly inhibits fatty acid b-oxidation by increasing malonyl-CoA production (via ACC activation). Malonyl-CoA allosterically inhibits the activity of carnitine palmitoyl transferase I, and this catalyzes the rate-limiting transport of activated fatty acids into the mitochondrion where oxidation takes place. In a similar fashion, feedback inhibition by malonyl-CoA and long-chain fatty acyl CoA esters also affects ACC activity by triggering depolymerization of the active polymer. The integrated coordination of lipogenesis and b-oxidation by insulin also occurs at the level of gene expression, mediated by two main transcription factors. These trans-acting factors include isoforms of the sterol receptor enhancer-binding proteins (SREBPs) and PPAR-g. SREBPs participate with other trans-acting factors, such as the upstream stimulatory factors, to mediate the effects of insulin on the expression of various genes involved in hepatic lipid metabolism, such as that encoding fatty acid synthetase. SREBP2 appears to be involved in hepatic cholesterol metabolism because its overexpression results in increased synthesis of hydroxymethylglutaryl (HMG)-CoA reductase and farnesyl pyrophosphate synthase. The role of SREBP1 in fatty acid/triglyceride metabolism seems to be restricted mainly to the liver, although it likely plays an important role in adipocyte cholesterol metabolism. SREBP null mice show no changes in adipose mass, or in the expression of acetyl-CoA carboxylase or fatty acid synthetase, although the up-regulation of hepatic lipogenic genes during fasting/refeeding is prevented. PPAR-g is a nuclear hormone receptor present mainly in adipose tissue, but it is also expressed at low levels in liver. PPAR ligands include prostaglandins, fatty acids, and the synthetically derived thiazolidinediones. PPAR-g expression is induced by insulin, and its promoter is itself trans-activated by SREBP1. PPAR-g expression is critical for adipocyte differentiation and is required for the trans-activation of adipocyte gene products involved in lipid metabolism, including acetyl-CoA synthetase, adipocyte fatty acid-binding protein, fatty acid transport protein, the glycerolneogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and lipoprotein lipase (LPL).
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This nuclear receptor also increases insulin sensitivity in cells and in vivo, explaining the antidiabetic effects of the PPAR-g-activating drugs. The circulating levels of insulin required to inhibit lipolysis are far below those required to stimulate glucose uptake into muscle. This physiological property results in an initial inhibition of fatty acid release into the circulation from adipose depots, and increased uptake of dietary-derived lipids by the peripheral tissues occurs following ingestion of a meal. Lipolysis in the fat cells is catalyzed by the enzyme hormone-sensitive lipase (HSL) in a mechanism that requires the translocation of the enzyme from the cytoplasm to the periphery of the lipid droplet. Upon phosphorylation by PKA, the enzyme undergoes catalytic activation and is translocated to the lipid droplet by a mechanism involving the PKA substrate perilipin. The phosphorylation of perilipin results in a dispersion of the normal perilipin ‘‘coat’’ around the lipid droplet, thereby potentially increasing the accessible surface area of the droplet to HSL activity. Insulin prevents the phosphorylation of HSL primarily by attenuating cAMP signaling via activation of cyclic nucleotide phosphodiesterase 3B, which catalyzes the conversion of cAMP to 50 -AMP. Insulin also increases the dephosphorylation of HSL and inhibits the phosphorylation of perilipin. Regulation of HSL localization also involves the docking protein lipotransin, and HSL is capable of interacting with lipotransin after phosphorylation by PKA, thereby allowing for its docking at the outer surface of the lipid droplet. Lipotransin possesses an intrinsic ATPase activity that allows for a dissociation of the complex after docking, permitting HSL to proceed with the catalysis. Insulin may freeze the HSL–lipotransin complex at the docking site, thereby preventing the dissociation step that is required for HSL to catalyze triglyceride hydrolysis. The release of dietary- or hepatic-derived fatty acids from circulating lipoprotein complexes is catalyzed
Insulin Action, Post-Receptor Mechanisms
by LPL, which catalyzes the rate-limiting step in the hydrolysis of lipoprotein-associated triglycerides to yield FFAs and 2-monoacylglycerol. Insulin appears to increase the expression and release of LPL from fat cells. Insulin stimulation of LPL activity occurs by both wortmannin-sensitive PI 3-kinase and rapamycin-sensitive p70 S6-kinase-signaling pathways.
See Also the Following Articles Insulin and Insulin-like Growth Factors, Evolution of Insulin Secretion: Functional and Biochemical Aspects Insulin Secretion, Physiology
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Further Reading Boss, A., Guilherme, A., Robida, S. I., Nicoloro, S. M., Zhou, Q. L., Jiang, Z. Y., Pomerleau, D. P., and Czech, M. P. (2002). Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature 420, 821–824. Brady, M. J., and Saltiel, A. R. (2001). The role of protein phosphatase-1 in insulin action. Recent Prog. Horm. Res. 56, 157–173. Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. Science 296, 1655–1657. Inoue, M., Chang, L., Hwang, J., Chiang, S. H., and Saltiel, A. R. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422, 629–633. Mauvais-Jarvis, F., Kulkarni, R. N., and Kahn, C. R. (2002). Knockout models are useful tools to dissect the pathophysiology and genetics of insulin resistance. Clin. Endocrinol. 57, 1–9. Mora, S., and Pessin, J. E. (2002). An adipocentric view of signaling and intracellular trafficking. Diab. Metab. Res. Rev. 18, 345–356. Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M. (2002). Transcriptional regulation of adipogenesis. Genes Dev. 14, 1293–1307. Saltiel, A. R., and Pessin, J. E. (2002). Insulin signaling pathways in time and space. Trends Cell. Biol. 12, 65–71. Unger, R. H., and Orci, L. (2001). Diseases of liporegulation: New perspective on obesity and related disorders. FASEB J. 15, 312–321. White, M. F. (2002). IRS proteins and the common path to diabetes. Am. J. Physiol. Endocrinol. Metab. 283, E413–E422.