Skeletal Muscle Glucose Metabolism and Insulin Resistance

Skeletal Muscle Glucose Metabolism and Insulin Resistance L Norton and R DeFronzo, University of Texas Health Sciences Center at San Antonio, San Antonio, TX, USA ã 2014 Elsevier Inc. All rights reserved.

Glossary Insulin A peptide hormone secreted from the b cells of the pancreas that is essential for the regulation of carbohydrate, fat, and protein metabolism in multiple tissues in the body, including the skeletal muscle, liver, and fat. Insulin stimulates the uptake of glucose in skeletal muscle, promotes glucose storage in the form of glycogen in the muscle and liver, and promotes the storage of fat in fat cells or adipocytes. An absence of insulin is the cause of type 1 diabetes and an inability to respond to insulin is a hallmark feature of type 2 diabetes mellitus. Insulin resistance A physiological condition in which cells of the body fail to respond appropriately to normal levels of circulating insulin. As a result, insulin fails to stimulate the uptake of glucose into insulin-sensitive tissues (i.e., the muscle, fat, and liver) resulting in an increase in the circulating levels of glucose, or hyperglycemia. Skeletal muscle A form of striated muscle that is under voluntary control and comprised of elongated multinucleated muscle cells, or myocytes, which often are referred to as muscle fibers or myofibers. Skeletal muscle myofibers contain myofibrils, which are made up of actin

Abbreviations AMPK AS160 ATP DAG FFA FOXO GLUT GS GSK HK IGT Insr IR IRS Km mRNA

Adenosine monophosphate kinase Rab GTPase-activating protein AS160 Adenosine triphosphate Diacylglycerol Free fatty acid Forkhead transcription factor Glucose transporter Glycogen synthase Glycogen synthase kinase Hexokinase Impaired glucose tolerance Insulin receptor gene Insulin receptor Insulin receptor substrate Michaelis constant Messenger ribonucleic acid

Normal Whole-Body Glucose Homeostasis

and myosin filaments and which form the basic contractile unit of skeletal muscle. Skeletal muscles are attached to the skeleton by strong elastic tendons and control virtually all body movement and regulate posture. The skeletal muscle has a high demand for energy in the form of glucose and is responsible for most of the glucose uptake from the blood following the ingestion of a meal, and, therefore, it plays a critical role in the maintenance of blood sugar levels (glycemia) in humans. Type 2 diabetes mellitus A metabolic disorder characterized by high blood sugar levels (hyperglycemia) in the presence of insulin resistance. This is in contrast to type 1 diabetes that is caused by a destruction of b cells in the pancreas leading to an absolute deficiency of insulin in the body. Type 2 diabetes makes up around 90% of all diabetes cases and is caused primarily by obesity, although  20% of all patients with type 2 diabetes are lean. There are  300 million people with type 2 diabetes across the globe and the disease is associated with serious long-term complications, including heart disease, strokes, blindness, and kidney failure.

NAD NADH PI3K p70S6K PDC PDH PDPK PDK PDP PIP3 PKB/AKT PKC PP1 SH2 SNAP TSC2

Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide (reduced) Phosphatidylinositol 3-kinase p70 ribosomal protein S6 kinase Pyruvate dehydrogenase complex Pyruvate dehydrogenase 3-Phospoinositide-dependent protein kinase Pyruvate dehydrogenase kinase Pyruvate dehydrogenase phosphatase Phosphatidylinositol (3,4,5)-triphosphate Protein kinase B Protein kinase C Protein phosphatase 1 SRC homology-2 protein N-ethylmaleimide-sensitive attachment protein Tuberous sclerosis complex-2

A discussion of skeletal muscle glucose metabolism and insulin resistance naturally begins with an overview of the mechanisms involved in the maintenance of normal whole-body glucose homeostasis in the basal or postabsorptive state (10– 12 h overnight fast), following ingestion of a typical mixed

meal. In the postabsorptive state, the great majority of total body glucose disposal takes place in insulin-independent tissues and 50% of all glucose utilization occurs in the brain, which is insulin-independent and becomes saturated at a plasma glucose concentration of about 40 mg dl 1 (2.2 mM). Another 25% of glucose disposal occurs in the splanchnic area (liver plus gastrointestinal tissues) and this also is insulin-independent.

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The remaining 25% of glucose utilization in the postabsorptive state takes place in insulin-dependent tissues – primarily the skeletal muscle – and, to a lesser extent, adipose tissue. This basal glucose utilization, 2.0 mg kg 1 min, is precisely matched by the rate of endogenous glucose production. Approximately 85% of endogenous glucose production is derived from the liver, and the remaining 15% is produced by the kidney. Glycogenolysis (the breakdown of glycogen to glucose) and gluconeogenesis (the formation of new glucose from glucose precursors) contribute equally to the basal rate of hepatic glucose production in humans. Following glucose ingestion, the increase in plasma glucose concentration stimulates insulin release, and the combination of hyperinsulinemia and hyperglycemia (i) stimulates glucose uptake by splanchnic (liver and gut) and peripheral (primarily muscle) tissues and (ii) suppresses endogenous (primarily hepatic) glucose production. As mentioned earlier, the majority (80–85%) of glucose uptake by peripheral tissues occurs in muscle, with a small amount (4–5%) being metabolized by adipocytes. Although fat tissue is responsible for only a small amount of total body glucose disposal, it plays an important role in the maintenance of total body glucose homeostasis by regulating the release of free fatty acids (FFA) from stored triglycerides and through the production of adipocytokines that influence insulin sensitivity in the muscle and liver. Insulin is a potent antilipolytic hormone and even small increments in the plasma insulin concentration markedly inhibit lipolysis, leading to a decline in the plasma FFA levels. The decline in plasma FFA concentration augments muscle glucose uptake and contributes to the inhibition of hepatic glucose production. Therefore, changes in the plasma FFA concentration in response to increased plasma levels of insulin and glucose play an important role in the maintenance of normal glucose homeostasis. The role of FFA in cellular mechanisms of insulin resistance will be discussed in more detail in the succeeding text.

In addition to insulin, glucagon also plays a central role in the regulation of whole-body glucose homeostasis. The role of the glucagon primarily is to promote the breakdown of stored liver glycogen to glucose. Under postabsorptive conditions, approximately half of total hepatic glucose production is dependent upon the maintenance of normal basal glucagon levels and inhibition of basal glucagon secretion with somatostatin causes a reduction in hepatic glucose production and plasma glucose concentration. After a glucose-containing meal, glucagon secretion is inhibited by hyperinsulinemia, and the resultant hypoglucagonemia contributes to the suppression of hepatic glucose production and maintenance of normal postprandial glucose tolerance.

Normal Glucose Metabolism in Human Skeletal Muscle The skeletal muscle plays a key role in maintaining glucose homeostasis and its major function in this regard is to take up glucose from the blood for oxidation and generation of energy in the form of adenosine triphosphate (ATP) or for storage in the form of glycogen. Skeletal muscle cells are able to take up glucose in an insulin-independent and insulin-dependent manner, the latter making up by far the majority of glucose uptake in humans. Physical exercise has an insulin-like effect on the skeletal muscle and is one of the major mechanisms whereby glucose can enter the muscle cell in an insulinindependent manner, but during the postprandial stage, insulin-mediated glucose uptake predominates. Binding of insulin to the insulin receptor (IR) activates a cascade of intracellular events (known collectively as insulin signaling) that leads to the eventual uptake of glucose from the blood into the muscle cell (Figure 1). In the presence of insulin, the IR phosphorylates insulin receptor substrate (IRS) proteins, which are linked to the activation of the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B (PKB) pathway and the

Glucose transporter (GLUT-4)

Insulin receptor P P

P P

PTP 1B SHC

Grb2

CbI/CAP complex IRS1/2/3/4

G-6-P

PI-3 K

SOS/Ras

PTEN SHIP2 UGPglucose

PDK MEK

aPKC

Hexokinase

Akt

Oxidative glucose metabolism

Glycogen synthase

MAP kinase GSK3

Gene expression growth regulation

p70S6k PP1

Signal transduciton

Glycogen

Glucose utilization Glycogen/lipid/protein synthesis

Figure 1 The insulin signaling pathway and the regulation of glucose uptake. See text for more details.

Obesity / Metabolism / Nutrition | Skeletal Muscle Glucose Metabolism and Insulin Resistance

translocation of the glucose transporter type 4 (GLUT4) to the plasma membrane. This key central pathway is responsible for the large majority of glucose transport into insulin-sensitive tissues. Skeletal muscle insulin resistance – which is the reduced ability of the muscle cell to respond appropriately to circulating insulin and stimulate glucose uptake – is primarily the result of disruption of various stages of the insulin signaling pathway, which will be discussed in the second part of this article. But to understand the mechanisms of insulin resistance, we must first understand the insulin signaling pathway in detail.

The Insulin Signaling Pathway The IR is the first point of contact between circulating insulin and the insulin signaling response. For insulin to exert its effects, it must first bind to and then activate the IR by phosphorylating key tyrosine residues on the b chain. This results in the translocation of IRS-1 protein to the plasma membrane, where it interacts with the IR and itself undergoes tyrosine phosphorylation. This leads to the activation of PI3K and AKT/PKB, eventually resulting in glucose transport into the cell via the GLUT4 transporter. The importance of the IR to glucose metabolism in multiple tissues has been unequivocally demonstrated through the use of transgenic rodent models; targeted disruption of the insulin receptor gene (Insr) in mice results in a marked rise in plasma glucose (hyperglycemia) and insulin (hyperinsulinemia), as well as ketoacidosis, growth retardation, and, eventually, death shortly after birth. Skeletal muscle-specific IR knockout mice demonstrate impaired insulin signaling and decreased insulin-dependent glucose transport. Following its activation, IR tyrosine kinase phosphorylates specific intracellular proteins, of which at least nine have been identified. In muscle, insulin-IRS-1 serves as the major docking protein that interacts with the IR tyrosine kinase and undergoes tyrosine phosphorylation in regions containing specific amino acid sequence motifs that, when phosphorylated, serve as recognition sites for proteins containing src homology 2 (SH2) domains. Mutation of these specific tyrosines severely impairs the ability of insulin to stimulate muscle glycogen synthesis, glucose oxidation, and other acute metabolic and growthpromoting effects of insulin. Perhaps the best studied SH2 protein is the regulatory subunit of PI3K. In the muscle, the phosphorylated tyrosine residues of IRS-1 mediate an association with the 85 kDa regulatory subunit of PI3K, leading to activation of the enzyme. PI3K is comprised of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. The latter catalyzes the 3-prime phosphorylation of phosphatidylinositol (PI), PI-4phosphate, and PI-4,5-diphosphate, which are able to bind and activate additional proteins via their pleckstrin homology domains, resulting in the stimulation of glucose transport. The most critical of these proteins is the 3-phospoinositidedependent protein kinase 1 (PDPK1), which is partially responsible for the activation of AKT/PKB. AKT/PKB requires phosphorylation at two sites for its full activation, Thr308 and Ser473; PDPK1 enhances the activity of AKT/PKB by phosphorylation of Thr308, and recent evidence suggests that the mammalian target of rapamycin complex 2 is responsible for AKT/PKB activation at the ser473 site. The positive actions of PI3K can be negatively regulated at the level of phosphatidylinositol (3,4,5)-trisphosphate (PIP3)

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by phospholipid phosphatases, such as phosphatase and tensin homologue that dephosphorylates and inactivates PIP3. The activation of PI3K by phosphorylated IRS-1 also leads to the activation of glycogen synthase (GS), via a process that involves activation of AKT/PKB and subsequent inhibition of additional kinases, such as glycogen synthase kinase (GSK)-3, and activation of protein phosphatase 1 (PP1). Inhibitors of PI3K impair glucose transport and block the activation of GS and the expression of hexokinase (HK)-II, which is a critical enzyme that phosphorylates glucose that enters the cell and enables its oxidation and/or storage. The action of insulin to increase protein synthesis and inhibit protein degradation also is mediated by PI3K. Other proteins with SH2 domains, including the adapter protein Grb2 and Shc, also interact with IRS-1 and become phosphorylated following exposure to insulin. Grb2 and Shc link IRS-1/IRS-2 to the mitogenactivated protein (MAP) signaling pathway, which plays an important role in the generation of transcription factors and promotes cell growth, proliferation, and differentiation. Inhibition of the MAP kinase pathway prevents the stimulation of cell growth by insulin but has no effect on the metabolic actions of the hormone. The AKT/PKB protein mediates most of the PI3K-dependent metabolic actions of insulin, primarily through the direct or indirect phosphorylation of multiple protein substrates including PKs, signaling proteins, and transcription factors. One of the major roles of the AKT/PKB pathway is to stimulate glucose uptake into the cell and upregulate glycogen synthesis. AKT/ PKB regulates glucose uptake by phosphorylating and inhibiting the Rab GTPase-activating protein AS160 (AS160), which is an intermediate step between activation of insulin signaling and GLUT4 translocation. The activation of AS160 triggers the activation of Rab small GTPases that are important for the cytoskeletal reorganization that is required for the translocation of GLUT4 to the plasma membrane. Phosphorylation of GSK by AKT/PKB decreases its activity towards GS and thereby increases glycogen synthesis. AKT/PKB also activates the mTOR pathway via phosphorylation and inhibition of tuberin, or tuberous sclerosis complex-2 (TSC2), which is in complex with hamartin (TSC1). The mTOR pathway regulates protein synthesis by phosphorylating the proteins p70 ribosomal protein S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (short). Finally, AKT/PKB regulates the expression of gluconeogenic and lipogenic enzymes, in part by controlling the activity of the forkhead (FOX) class of transcription factors – a family of around 100 members, several of which may be critical for insulin action. One of these transcription factors, forkhead transcription factor (FOXO)1, activates gluconeogenic genes in the liver and inhibits adipogenesis, actions which are inhibited by insulin through AKT-/ PKB-mediated phosphorylation of FOXO1, which inhibits its transcriptional activity. FOXO1 may also have an important role in the regulation of carbohydrate and fat oxidation via actions on the pyruvate dehydrogenase complex (PDC).

Insulin Signaling and Glucose Transport Activation of the insulin signaling pathway through AKT/PKB leads to the translocation of intracellular GLUTs to the plasma membrane where they facilitate glucose uptake into the target

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tissue. While there are 12 major GLUTs (divided into three major classes) with distinct tissue distributions, the most abundant in insulin-sensitive tissues is GLUT4. GLUT4 has a glucose Michaelis constant (Km) of 5 mM, which is close to the plasma glucose concentration of a healthy human. In the skeletal muscle and adipose tissue, the concentration of GLUT4 at the plasma membrane increases markedly in response to insulin. In basal unstimulated muscle and adipose tissue, GLUT4 is mostly sequestered in the cytoplasm and constitutively cycles to and from the plasma membrane through slow endocytosis and fast exocytosis, and it is thought that insulin stimulates the exocytic arm and reduces the endocytic arm of this cycling. Under these conditions, GLUT4-containing vesicles slowly fuse with the plasma membrane and internalize GLUT4 transporters present on the cell membrane, returning these transporters to an intracellular cytoplasmic pool. The absolute amount of GLUT4 present on the cell surface in the absence of insulin signaling is, therefore, determined by this exocytosis–endocytosis cycling. Approximately 5% of the total GLUT4 pool is present on the cell surface under basal, noninsulin-stimulated conditions. Insulin rapidly stimulates glucose transport primarily by enhancing the exocytosis arm of this GLUT4 cycling such that within a few minutes,  50% of GLUT4 transporters relocate to the plasma membrane. Although the precise mechanisms are still unclear, it is thought that the regulation of GLUT4 cycling involves multiple proteins involved in vesicle trafficking, and insulin signaling facilitates the fusion of GLUT4 containing vesicles with the plasma membrane. AKT/PKB can target components of the vesicle – plasma membrane fusion machinery, which comprises the vesicle-associated membrane protein-2, the N-ethylmaleimide-sensitive attachment protein-23 (SNAP23), and the membrane-associated SNAP protein syntaxin-4 as well as synip, tomosyn, and munc18 that bind syntaxin4 to modulate the insulin-dependent gain in membraneassociated GLUT4.

Atypical Protein Kinase C and Glucose Uptake A number of protein kinase C (PKC) isoforms have been linked to the regulation of the insulin signaling pathway, both in a negative and a positive fashion. The PKC family is made up of a number of PKs consisting of  10 isozymes. They are commonly further subdivided into conventional (classical), novel, and atypical forms. Conventional PKCs consist of a, b, and g isoforms and require calcium, diacylglycerol (DAG), and a phospholipid for activation. The novel PKCs (nPKC) consist of d, e, Z, and y isoforms and require DAG but not calcium for their activation. In contrast, the atypical PKCs (aPKC) include z and l\i isoforms, but these do not require either calcium or DAG for activation and are similar to AKT/PKB, which require PIP3 and PDPK1/PDPK2 phosphorylation. In addition to the phosphorylation and activation of AKT/PKB, it has been demonstrated that an additional target of PDPK1 is PKC-z and -l\i, and there is now evidence to suggest that these aPKC isoforms serve as molecular switches that participate in turning on glucose transport responses during insulin action. The aPKCs exist in cells in a folded state and, upon activation by acidic lipids (e.g., PIP3), unfold and become active through a number of complementary mechanisms, including PDPK1 phosphorylation, autophosphorylation, and relief of autoinhibition,

suggesting that mechanism exists to activate aPKCindependent of phosphorylation and PDPK1. Following unfolding, aPKCs may become exposed to protease activity and are then converted to short-lived, constitutively active M-type kinases. Defects in insulin-mediated aPKC activation have consistently been reported in the muscle and adipocyte of T2DM patients and in a number of animal models of insulin resistance. Stable expression of kinase-inactive aPKC in myotubes inhibits insulin-mediated GLUT4 translocation, whereas overexpression of constitutively active aPKC mimics the action of insulin on GLUT4 translocation. Furthermore, in embryonic stem cells that lack PKC-l, which are then differentiated to adipocytes, insulin fails to stimulate glucose transport. Together, these studies strongly suggest that aPKCs play an important role in glucose uptake/GLUT4 translocation in the skeletal muscle and adipose tissue. There is currently no evidence to suggest that the conventional or nPKC isoforms play a similar role in insulin-stimulated glucose transport/GLUT4 translocation. Indeed, as mentioned earlier, they appear to be more relevant in states of insulin resistance.

Glucose Phosphorylation Once glucose has successfully entered the cell, isoenzymes of HK catalyze the conversion of glucose to glucose 6-phosphate (G6P) – essentially trapping glucose within the cell. HKI, HKII, and HKIII are single-chain peptides that have a number of properties in common, including a high affinity for glucose and inhibition by G6P. HKII is expressed in insulin-sensitive tissues (adipose and muscle tissue), whereas HKI is expressed in brain and erythrocytes. HKIV, also known as glucokinase, can be further subdivided into HKIVB, which is believed to be the glucose sensor in the b cell, and HKIVL, which is important for hepatic glucose metabolism. Glucose, once phosphorylated, has essentially three fates in the cell: it can be partially and anaerobically metabolized to lactate and released into the circulation, oxidized in the mitochondria to generate energy in the form of ATP, or it can be stored as energy in the form of glucogen.

Glycogen Metabolism: Nonoxidative Glucose Disposal During rest, storage of glucose as glycogen is enhanced by insulin stimulation of glucose uptake. Following phosphorylation to G6P by HKII, glucose may enter glycolysis to produce ATP or be converted to glucose-1-phosphate for glycogen synthesis. GS is one of the key enzymes controlling the rate of muscle glycogen synthesis and utilizes UDP-glucose to add glucose molecules by a1 – four linkages – the rate-limiting step in glycogen synthesis. The activity of GS is controlled by covalent modification (phosphorylation/dephosphorylation), allosteric activation, and enzyme translocation (Figure 2). The enzyme is phosphorylated on up to nine residues by several kinases, resulting in enzyme deactivation and decreased sensitivity to allosteric activators. These kinases include cAMP-dependent protein kinase A (PKA), calmodulin-dependent kinases, adenosine monophosphate kinase (AMPK), and GSK3. Conversely, the dephosphorylation and activation of GS are controlled

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Glycogen synthesis P Glycogen synthase a Pi

H2O

Glycogen synthase b

Protein phosphorylase-1

+

Insulin

P Pi Phosphorylase kinase

H2O Phosphorylase kinase P Pi Glycogen phosphorylase b

H2O Glycogen phosphorylase a

Glycogen degradation

Principles of biochemistry, 4/e ã2006 Pearson prentice hall, Inc.

Figure 2 The regulation of glycogen metabolism by insulin. See text for more details.

primarily by PP1. These enzymes themselves are also closely regulated: GSK3 can be negatively regulated by phosphorylation by AKT/PKB following stimulation by insulin and positively regulated by cAMP following adrenergic stimulation. Similarly, PP1 can be upregulated by insulin via complex mechanisms leading to dephosphorylation and activation of GS. The relative contribution of GSK3 and PP1 to GS activity is not clear, but recent reports on transgenic mice have questioned the ability of insulin to activate GS via PP1. Alternative mechanisms exist to regulate GS activity in addition to phosphorylation/dephosphorylation. For example, binding of G6P to GS unfolds the enzyme resulting in allosteric activation and causes conformational changes in GS that favor dephosphorylation of the enzyme. Furthermore, translocation of GS to glycogen particles in response to stimuli (i.e., insulin) is an additional mechanism whereby GS activity is regulated. During exercise, muscles rely on glycolytic pathways to provide ATP for continued contraction. Glycogen breakdown is controlled by the enzyme glycogen phosphorylase, which in turn is activated by a number of stimuli related to muscle contraction (e.g., calcium), thereby allowing glycogen breakdown to change in parallel with the energy demands during exercise. Repletion of glycogen stores involves an increase in GS activity after exercise. However, despite net glycogen breakdown during exercise, mechanisms exist to activate GS and therefore increase glycogen synthesis during exercise in an insulin-independent manner. An increase in GS activity in exercise appears to be dependent on the mode, duration, and intensity of the exercise as some studies have demonstrated a reduction in GS activity during high-intensity exercise. The insulin independence of GS activation during exercise suggests that exercise and insulin utilize different signaling pathways to activate GS. These mechanisms may be related to glycogen levels themselves, phosphatase (PP1) and kinase (GSK3, PKA, and AMPK) activation and allosteric factors (G6P).

Carbohydrate Oxidation: Oxidative Glucose Disposal The pyruvate dehydrogenase (PDH) enzyme is part of the multienzyme PDC, which catalyzes the physiologically irreversible decarboxylation of pyruvate to acetyl-CoA and is often referred to as a ‘gatekeeper’ in the oxidation of carbohydrate (Figure 3). PDC links the degradation of intracellular glycogen and extracellular glucose via glycolysis, as well as the oxidation of extracellular pyruvate and lactate, to the energy requirements of the cell. When the glucose supply is high, the combination of acetyl-CoA with oxaloacetate provides a precursor for malonyl-CoA production. Malonyl-CoA can limit the mitochondrial uptake (and therefore oxidation) of FFA via inhibition of carnitine palmitoyltransferase I. On the other hand, when glucose availability is low or FFA supply and oxidation is sufficient to meet the cellular energy demands, PDC activity is suppressed, limiting the conversion of pyruvate to acetyl-CoA. This response to glucose scarcity may be crucial for glucose conservation. Flux through the PDC is tightly regulated to maintain glucose homeostasis during both the fed and fasting states. This regulation is achieved via a combination of three major mechanisms: (1) reversible phosphorylation/dephosphorylation, (2) modifications of the activities of the regulatory components by the redox state and acetyl-CoA/ CoA ratio, and (3) transcriptional regulation of the regulatory components. The PDC complex contains two specific regulatory enzymes, pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). These two enzymes catalyze a phosphorylation/dephosphorylation cycle involving specific serine residues on the PDH enzyme. The phosphorylation of the PDH completely inactivates the PDC, and therefore, the activity of PDC reflects the balance between the activities of PDK (which phosphorylates and inactivates PDH) and PDP (which dephosphorylates and activates PDH). The regulation

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High (Ca2+)

PDP 1

Pyruvate

PDP 2 (Multiple target sites)

Low (O)

PDK 1

High (Acetyl-CoA) High (NADH)

PDK 2

ATP

PDK 3

Starvation and Nutrient deprivation

PDK 4

De

NAD+

Ac ti os vati ph on or yla tio n

ph

Inhibition Phosphorylation

Pyruvate dehydrogenase

t en an n m o i r t Pe hibi In

Arsinate and organic arsenical Acetyl coenzyme A

CO and NADH

Figure 3 Carbohydrate oxidation and the pyruvate dehydrogenase complex (PDC). See text for more details.

of these enzymes is also tightly regulated. The PDP enzymes (PDP1 and PDP2) are variably expressed in tissues, but the dominant PDP1 enzyme in the skeletal muscle requires magnesium and is stimulated by calcium. Short-term regulation of PDK includes its inhibition by pyruvate and its activation by acetyl-CoA and nicotinamide adenine dinucleotide (reduced) (NADH) – products of the PDC reaction and FFA oxidation. To date, four isoforms of PDK have been identified (PDK1-4) and each exhibits tissue-specific regulation. PDK1 is found in the heart, pancreas, and skeletal muscle; PDK2 is ubiquitously expressed in the fed state; PDK3 has a limited tissue distribution and PDK4 is expressed at high levels in the heart, skeletal muscle, kidney, liver, and pancreas. The relative catalytic activity of the PDK isoforms towards PDH varies such that PDK2 and PDK4 exhibit the highest activity. In addition, each isoform is differentially affected by short-term regulatory metabolites. For example, PDK2 is most sensitive to inhibition to pyruvate, whereas PDK4 is relatively insensitive to pyruvate but instead is more sensitive to an increase NADH/NAD ratio.

Cellular Mechanisms of Insulin Resistance in the Skeletal Muscle Insulin resistance – defined as a state of reduced responsiveness to normal circulating levels of insulin – plays a major role in the development and progression of T2DM, and it is a hallmark feature of the disease. Insulin resistance is one of the earliest detectable defects in patients at high risk of developing T2DM, illustrated by the finding that normal glucose-tolerant offspring of parents with T2DM frequently display insulin resistance from an early age. Insulin resistance has traditionally been viewed in terms of the effects of insulin on glucose metabolism and, as such, many early studies focused on the possible reduction in early insulin signaling events including abnormalities in the structure and number of IRs and defects in the phosphorylation of early effectors of the insulin signaling cascade. However, it is now widely recognized that insulin

resistance involves impairments in the enzymes that regulate glycogen synthesis as well as fat and carbohydrate metabolism and mitochondria function, and it is likely that multiple signaling and metabolic defects combine in the skeletal muscle to manifest insulin resistance. In the second half of this article, we will discuss the cellular mechanisms involved in insulin resistance and begin with an overview of the defects along the insulin signaling pathway and then progress to consider mechanisms further downstream of these pathways, including glycogen synthesis, substrate oxidation, and mitochondria function.

Insulin Resistance and IR Number and Affinity Both receptor and postreceptor defects contribute to insulin resistance in individuals with T2DM. Some studies have demonstrated a modest 20–30% reduction in insulin binding to monocytes and adipocytes from T2DM patients, but this has not been a consistent finding. The decrease in insulin binding is due to a reduction in the number of IRs without change in IR affinity. However, caution should be employed in interpreting these studies, since the muscle and liver, not adipocytes, are the major tissues responsible for the regulation of glucose homeostasis in vivo and insulin binding to solubilized receptors obtained from the skeletal muscle, and the liver has been shown to be normal in obese and lean diabetic individuals. Moreover, a decrease in IR number cannot be demonstrated in over half of T2DM subjects, and it has been difficult to demonstrate a correlation between reduced insulin binding and the severity of insulin resistance. A variety of defects in IR internalization and processing have been described in syndromes of severe insulin resistance and diabetes. However, the IR gene has been sequenced in T2DM patients from diverse ethnic populations and, with very rare exceptions, physiologically significant mutations in the IR gene have not been observed. This excludes a structural gene abnormality in the IR as a cause of common T2DM.

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Insulin Resistance and IR Tyrosine Kinase Activity IR tyrosine kinase activity has been examined in the skeletal muscle, adipocytes, and hepatocytes from normal-weight and obese diabetic subjects. Most, but not all, investigators have found a reduction in tyrosine kinase activity that cannot be explained by alterations in IR number or IR binding affinity (see in the succeeding text). However, restoration of normoglycemia by weight loss has been shown to correct the defect in IR tyrosine kinase activity, suggesting that the defect in tyrosine kinase is acquired secondary to some combination of hyperglycemia, distributed intracellular glucose metabolism, hyperinsulinemia, and insulin resistance – all of which improved after weight loss. Exposure of cultured fibroblasts to high glucose concentration also inhibits IR tyrosine kinase activity. Since IR tyrosine kinase activity assays are performed in vitro, the results of these assays could provide misleading information with regard to IR function in vivo. To circumvent this problem, investigators have employed the euglycemic hyperinsulinemic clamp with muscle biopsies and antiphosphotyrosine immunoblot analysis to provide a ‘snapshot’ of the insulin-stimulated tyrosine phosphorylation state of the receptor in vivo. In insulinresistant obese nondiabetic and T2DM subjects, a substantial decrease in IR tyrosine phosphorylation has been demonstrated. However, when insulin-stimulated IR tyrosine phosphorylation was examined in normal glucose-tolerant, insulin-resistant individuals (offspring of two diabetic parents) at high risk of developing T2DM, a normal increase in tyrosine phosphorylation of the IR was observed. These findings are consistent with the concept that impaired IR tyrosine kinase activity in T2DM patients is acquired secondary to hyperglycemia or some other metabolic disturbance.

Insulin Signaling Defects in Insulin Resistance In insulin-resistant obese nondiabetic subjects, the ability of insulin to activate IR and IRS-1 tyrosine phosphorylation in the muscle is modestly reduced, while in T2DM individuals, insulin-stimulated IR and IRS-1 tyrosine phosphorylation are severely impaired. Association of the p85 subunit of PI3K with IRS-1 and activation of PI3K also are greatly attenuated in obese nondiabetic and T2DM subjects compared to lean healthy controls. The decrease in insulin-stimulated association of the p85 regulatory subunit of PI3K with IRS-1 is closely correlated with the reduction in insulin-stimulated muscle GS activity and in vivo insulin-stimulated glucose disposal. Impaired regulation of PI3K gene expression by insulin also has been demonstrated in the skeletal muscle and adipose tissue of patients with T2DM. In animal models of diabetes, an 80–90% decrease in insulin-stimulated IRS-1 phosphorylation and PI3K activity has been reported. In insulin-resistant, normal glucose-tolerant offspring of T2DM parents, IRS-1 tyrosine phosphorylation, and the association of p85 protein/PI3K activity with IRS-1 are markedly decreased despite normal tyrosine phosphorylation of the IR; these insulin signaling defects are correlated closely with the severity of insulin resistance, measured with the euglycemic insulin clamp technique. In summary, impaired association of PI3K with IRS-1 and its subsequent activation are characteristic abnormalities in patients with T2DM, and these defects are correlated closely

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with in vivo muscle insulin resistance. A common mutation in the IRS-1 gene (Gly 972 Arg) has been associated with type 2 diabetes, insulin resistance, and obesity, but the physiological significance of this mutation remains to be established. Insulin resistance of the PI3K signaling pathway contrasts with an intact stimulation of the MAP kinase pathway by insulin in insulin-resistant T2DM and obese nondiabetic individuals. Physiological hyperinsulinemia increases MEK1 activity and ERK1/2 phosphorylation and activity similarly in lean healthy subjects and in insulin-resistant obese nondiabetic and T2DM patients. Intact stimulation of the MAP kinase pathway by insulin in the presence of insulin resistance in the PI3K pathway may play an important role in the development of atherosclerosis. If the metabolic PI3K pathway is impaired, plasma glucose levels rise, resulting in increased insulin secretion and hyperinsulinemia. Because IR function is normal or only modestly impaired, especially early in the natural history of T2DM, this leads to excessive stimulation of the MAP kinase (mitogenic) pathway in vascular tissues, with resultant proliferation of vascular smooth muscle cells, increased collagen formation, and increased production of growth factors and inflammatory cytokines.

Insulin Resistance and Glucose Transport As described in the first part of this article, activation of the insulin signal transduction system in insulin target tissues stimulates glucose transport via a mechanism that involves translocation of an intracellular pool of GLUTs (associated with low-density microsomes) to the plasma membrane and their subsequent activation after insertion into the cell membrane. In adipocytes and muscle of patients with T2DM glucose transport activity is severely impaired. In adipocytes from human and rodent models of T2DM, GLUT4 messenger ribonucleic acid (mRNA) and protein content are markedly reduced, and the ability of insulin to elicit a normal translocation response and to activate the GLUT4 transporter after insertion into the cell membrane is decreased. In contrast to adipocytes, muscle tissue from lean and obese T2DM subjects exhibits normal levels of GLUT4 mRNA expression and normal levels of GLUT4 protein, thus demonstrating that transcriptional and translational regulation of GLUT4 is not impaired in insulin resistance. In contrast to the normal expression of GLUT4 protein and mRNA in muscle of T2DM subjects, every study that has examined adipose tissue has reported reduced basal and insulin-stimulated GLUT4 mRNA levels and decreased GLUT4 transporter number in all subcellular fractions. These observations also demonstrate that GLUT4 expression in humans is subject to tissue-specific regulation. Using complex triple-tracer techniques, the in vivo dose–response curve for the action of insulin on glucose transport in the forearm skeletal muscle has been examined in T2DM subjects, and insulin-stimulated inward muscle glucose transport has been shown to be severely impaired. Impaired in vivo muscle glucose transport in T2DM patients also has been demonstrated using MRI and PET imaging techniques. Since the number of GLUT4 transporters in the muscle of diabetic subjects is normal, impaired GLUT4 translocation and decreased intrinsic activity of the GLUT are responsible for the defect in muscle glucose transport. Finally, it should be noted that large

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Obesity / Metabolism / Nutrition | Skeletal Muscle Glucose Metabolism and Insulin Resistance

populations of T2DM patients have been screened for mutations in the GLUT4 gene, but such mutations are very uncommon and, when detected, have been of questionable physiological significance.

nPKCs and Insulin Resistance A number of the nPKC isoforms (nPKCs) have been associated with negative regulation of the insulin signaling pathway. This is in contrast to the positive effects of the aPKCs on GLUT4 translocation and glucose uptake (see in the succeeding text). The nPKCs include d, e, Z, and y isoforms and require DAG but not calcium for their activation and as such have consistently been linked to insulin resistance associated with increased lipid availability in the skeletal muscle. Lipid infusion in rats and humans impairs insulin-stimulated glucose disposal in the muscle and is associated with concomitant activation of PKC-d and -y. It has been suggested that one mechanism whereby PKC-d and PKC-y reduce insulin sensitivity is via serine phosphorylation of the IRS-1 and this has recently been demonstrated in vitro. This serine phosphorylation may reduce the ability of IRS to activate PI3K and thus contribute to a reduction in insulin-stimulated glucose uptake and glycogen synthesis. An additional nPKC, PKC-e, has also been shown to be upregulated in the skeletal muscle of the diabetic Psammomys obesus rat.

Insulin Resistance and Glucose Phosphorylation In the skeletal muscle, HKII transcription is regulated by insulin, whereas HKI mRNA and protein levels are not affected by insulin. In response to physiological euglycemic hyperinsulinemia of 2–4 h duration, HKII cytosolic activity, protein content, and mRNA levels increase by 50–200% in healthy nondiabetic subjects, and this is associated with the translocation of HKII from the cytosol to the mitochondria. In forearm muscle, insulin-stimulated glucose transport (measured with the triple-tracer technique) is markedly impaired in lean T2DM patients. However, the rate of intracellular glucose phosphorylation is impaired to an even greater extent, resulting in an increase in the free glucose concentration within the intracellular space that is accessible to glucose. These observations indicate that in T2DM individuals, while both glucose transport and glucose phosphorylation are severely resistant to the action of insulin, impaired glucose phosphorylation (HKII) appears to be the rate-limiting step for insulin action. Studies using 31P-NMR in combination with 1-14C-glucose also have demonstrated that both insulin-stimulated muscle glucose transport and glucose phosphorylation are impaired in T2DM subjects, but results from this study suggest that the defect in transport exceeds the defect in phosphorylation. Because of methodological differences, the results of the triple tracer and MRI studies cannot easily be reconciled. Nonetheless, these studies are consistent in demonstrating that abnormalities in both muscle glucose phosphorylation and glucose transport are well established and occur early in the natural history of T2DM and cannot be explained by glucose toxicity. In healthy nondiabetic subjects, a physiological increase in the plasma insulin concentration for as little as 2–4 h increases muscle HKII activity, gene transcription, and translation. In lean patients with T2DM, the ability of insulin to augment

HKII activity and mRNA levels is markedly reduced compared to controls. Decreased basal muscle HKII activity and mRNA levels and impaired insulin-stimulated HKII activity in T2DM subjects have been reported by other investigators. A decrease in insulin-stimulated muscle HKII activity also has been described in subjects with impaired glucose tolerance (IGT). Several groups have looked for point mutations in the HKII gene in individuals with T2DM and, although several nucleotide substitutions have been found, none are close to the glucose and ATP binding sites and none have been associated with insulin resistance. Thus, an abnormality in the HKII gene is unlikely to explain the inherited insulin resistance in common variety T2DM.

Insulin Resistance and Glycogen Synthesis Following phosphorylation by HKII, glucose can either be converted to glycogen or enter the glycolytic pathway. Of the glucose that enters the glycolytic pathway, 90% is oxidized and the remaining 10% is released as lactate. At low physiological plasma insulin concentrations, glycogen synthesis and glucose oxidation contribute approximately equally to glucose disposal. However, with increasing plasma insulin concentrations, glycogen synthesis predominates. Impaired insulin-stimulated glycogen synthesis is a characteristic finding in all insulin-resistant states including obesity, IGT, diabetes, and diabesity in all ethnic groups and accounts for the majority of the defect in insulin-mediated whole-body glucose disposal. Impaired glycogen synthesis also has been documented in the normal glucose-tolerant offspring of two diabetic parents, in the first-degree relatives of T2DM individuals, and in the normoglycemic twin of a monozygotic twin pair in which the other twin has T2DM. GS is the key insulin-regulated enzyme that controls the rate of muscle glycogen synthesis. Insulin activates GS by stimulating a cascade of phosphorylation/dephosphorylation reactions, which ultimately lead to the activation of PP1 (also called GS phosphatase). The regulatory subunit of PP1 has two serine phosphorylation sites. Phosphorylation of site 2 by PKA inactivates PP1, while phosphorylation of site 1 by insulin activates PP1, leading to the stimulation of GS. Phosphorylation of site 1 of PP1 by insulin in muscle is catalyzed by insulin-stimulated protein kinase-1 (ISPK-1). Because of their central role in muscle glycogen formation, the three enzymes – GS, PP1, and ISPK-1 – have been extensively studied in the individuals with T2DM. GS exists in an active (dephosphorylated) and an inactive (phosphorylated) form. Under basal conditions, total GS activity in T2DM subjects is reduced and the ability of insulin to activate GS is severely impaired. The ability of insulin to stimulate GS also is diminished in the normal glucose-tolerant, insulin-resistant relatives of individuals with T2DM. In insulin-resistant nondiabetic and diabetic Pima Indians, activation of muscle PP1 (GS phosphatase) by insulin is severely reduced. Since PP1 dephosphorylates GS, leading to its activation, the defect in PP1 plays an important role in the muscle insulin resistance of T2DM. The effect of insulin on GS gene transcription and translation in vivo has been studied extensively. Most studies have demonstrated that insulin does not increase GS mRNA or protein expression in human muscle. However, GS mRNA and protein levels are decreased in muscle

Obesity / Metabolism / Nutrition | Skeletal Muscle Glucose Metabolism and Insulin Resistance

of patients with T2DM, explaining in part the decreased GS activity. The major abnormality in GS regulation in T2DM is its lack of dephosphorylation and activation by insulin, as a result of IR signaling abnormalities. The GS gene has been the subject of intensive investigation, and DNA sequencing has revealed either no mutations or very rare nucleotide substitutions that cannot adequately explain the defect in insulin-stimulated GS activity. The genes encoding the catalytic subunits of PP1 and ISPK-1 have been examined in Pima Indians and Danes with type 2 diabetes. Several silent single nucleotide polymorphisms were found in the PP1 and ISPK-1 genes in the Danish population, but the mRNA levels of both genes were normal in the skeletal muscle. No structural gene abnormalities in the catalytic subunit of PP1 were detected in Pima Indians. Thus, neither mutations in the PP1 and ISPK-1 genes nor abnormalities in their translation can explain the impaired enzymatic activities of GS and PP1 that have been observed in vivo. Similarly, there is no evidence that an alteration in glycogen phosphorylase plays any role in the abnormality in glycogen formation in T2DM. In summary, GS activity is severely impaired in individuals with T2DM, but the molecular etiology of the defect remains to be determined.

Insulin Resistance and Substrate Utilization Carbohydrate and fat are the primary fuel sources for mitochondrial ATP production in human skeletal muscle. An interaction between the oxidations of these two fuels has been suggested as a potential mechanism underlying insulin resistance in the skeletal muscle, and this hypothesis prevails today but remains controversial. Randle and colleagues proposed the concept of the ‘glucose–fatty acid cycle’ in the 1960s while studying rat heart and diaphragm muscle. The main feature of this hypothesis is that increased fat oxidation in muscle would inhibit both PDH and phosphofructokinase (PFK) by accumulation of acetyl-CoA and citrate, respectively. Inhibition of these enzymes would lead to reduced flux through the glycolytic pathway and thus an increase in G6P concentrations, inhibiting HK and resulting in reduced glucose uptake and oxidation. In T2DM individuals, the glycolytic/ glucose oxidative pathway has been show to be impaired, but while a single study has suggested that PFK activity is modestly reduced in muscle biopsies from T2DM subjects, most evidence indicates that the activity of PFK is normal. Insulin has no effect on muscle PFK activity, mRNA levels, or protein content in either nondiabetic or diabetic individuals. However, PDH is a key insulin-regulated enzyme whose activity in muscle is acutely stimulated by insulin. In T2DM patients, insulin-stimulated PDH activity is decreased in human adipocytes and in the skeletal muscle, and insulin resistance is associated with increased PDK activity and subsequent reductions in PDC flux. PDK activity is increased in several oxidative tissues in vivo in response to nutritional interventions that increase lipid supply and oxidation. For example, PDK expression and/or activity increases with starvation, insulin resistance induced by high-fat feeding, and streptozotocin-induced diabetes, and this has been linked to an increase in circulating and intracellular FFA and related metabolites. However, recent studies have shown that PDK4 expression is downregulated by insulin independently of FFA levels, and because the states of insulin resistance mentioned

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earlier are characterized by insulin deficiency, it is possible that an increase in PDK4 is a consequence of the inability of insulin to suppress PDK4 expression (i.e., insulin resistance). Recent studies have also implicated FOXO1 in the regulation of PDK4 by insulin. FOXO1 is expressed in insulin-sensitive tissues, including the skeletal muscle, and, as discussed earlier, is phosphorylated by AKT/PKB through the insulin signaling pathway. Following phosphorylation by AKT/PKB, FOXO1 translocates from the nucleus to the cytosol resulting in reduced transcriptional activity. Therefore, a reduction in insulin signaling due to insulin resistance would activate FOXO1 and lead to an increase in PDK4 expression. Taken together, these pathways may provide a possible link between insulin signaling and the control of carbohydrate and fat oxidation and thereby provide an integrative model of insulin resistance.

Mitochondria Dysfunction and Insulin Resistance Studies in humans using molecular, biochemical, and magnetic resonance spectroscopic techniques have documented a defect in mitochondrial oxidative phosphorylation in various insulinresistant states. Most of these studies have been performed in the skeletal muscle because of its accessibility. In vivo measurement of oxidative phosphorylation with 31P nuclear magnetic resonance (NMR) has also demonstrated impaired ATP synthesis in various insulin-resistant states. Insulin-resistant offspring of subjects with T2DM have a 30–40% decrease in the resting metabolic flux through the TCA (tricarboxylic acid cycle) and oxidative phosphorylation in the skeletal muscle, measured with magnetic resonance spectroscopy. A similar impairment in resting metabolic flux through oxidative phosphorylation has been reported in subjects with T2DM. Further, unlike lean, insulinsensitive individuals, subjects with diabetes and normal glucosetolerant, insulin-resistant offspring of two parents with diabetes fail to increase mitochondrial oxidative phosphorylation flux following insulin stimulation despite a significant increase in glucose disposal in the skeletal muscle. Moreover, subjects with T2DM have decreased exercise tolerance and impaired recovery of intracellular phosphocreatine concentration following exercise, indicating that the mitochondrial defect in oxidative phosphorylation may contribute to impaired exercise capacity in insulin-resistant individuals. Insulin resistance also is a characteristic feature in the normal aging process and is associated with a decrease in mitochondrial ATP synthesis rate and an increase in intramyocellular fat content. Lastly, the experimental induction of insulin resistance in the skeletal muscle, caused by a physiological increase in plasma FFA concentration in lean, healthy, insulin-sensitive individuals, also is associated with a decrease in oxidative phosphorylation flux in the skeletal muscle. Collectively, these results indicate that, regardless of etiology, insulin resistance in the skeletal muscle is associated with decreased mitochondrial oxidative phosphorylation in the skeletal muscle.

Summary In summary, postbinding defects in insulin action primarily are responsible for the insulin resistance in T2DM. Diminished insulin binding, when present, is modest and secondary to downregulation of the IR by chronic hyperinsulinemia.

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Obesity / Metabolism / Nutrition | Skeletal Muscle Glucose Metabolism and Insulin Resistance

In patients with T2DM and overt fasting hyperglycemia, a number of postbinding defects have been demonstrated, including reduced IR tyrosine kinase activity, insulin signal transduction abnormalities, decreased glucose transport, diminished glucose phosphorylation, and impaired GS activity. The glycolytic/glucose oxidative pathway is largely intact and, when defects are observed, they appear to be acquired secondary to enhanced FFA/lipid oxidation. From the quantitative standpoint, impaired glycogen synthesis represents the major pathway responsible for the insulin resistance in T2DM and is present long before the onset of overt diabetes, that is, in normal glucose-tolerant, insulin-resistant prediabetic subjects and in individuals with IGT. The impairment in GS activation appears to result from a defect in the ability of insulin to phosphorylate IRS-1, causing a reduced association of the p85 subunit of PI3K with IRS-1 and decreased activation of the enzyme PI3K.

Further Reading General DeFronzo, R.A., 1998. Lilly lecture. The triumvirate: beta cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37, 667–687. DeFronzo, R.A., 1997. Pathogenesis of type 2 diabetes mellitus: metabolic and molecular implications for identifying diabetes genes. Diabetes 5, 117–269. DeFronzo, R.A., 2009. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes 58, 773–795. DeFronzo, R.A., Ferrannini, E., 2010. Regulation of intermediatory metabolism during fasting and feeding. In: Jameson, J.L., DeGroot, L.J. (Eds.), Endocrinology. Saunders Elsevier, Philadelphia, PA, pp. 673–698.

Insulin Signaling Alessi, D.R., James, S.R., Downes, C.P., Holmes, A.B., Gaffney, P.R., Reese, C.B., et al., 1997. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr. Biol. 7, 261–269. Bell, G.l., Kayano, T., Buse, J.B., Burant, C.F., Takeda, J., Lin, D., et al., 1990. Molecular biology of mammalian glucose transporters. Diabetes Care 13, 198–200. Bruning, J.C., Michael, M.D., Winnay, J.N., Hayashi, T., Horsch, D., Accili, D., et al., 1998. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol. Cell 2, 559–569. Dugani, C.B., Klip, A., 2005. Glucose transporter 4: cycling, compartments and controversies. EMBO Rep. 6, 1137–1142. Frame, S., Cohen, P., 2001. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359, 1–16. Greenberg, C.C., Jurczak, M.J., Danos, A.M., Brady, M.J., 2006. Glycogen branches out: new perspectives on the role of glycogen metabolism in the integration of metabolic pathways. Am. J. Physiol. Endocrinol. Metab. 291, E1–E8. Joshi, R.L., Lamothe, B., Cordonnier, N., Mesbah, K., Monthioux, E., Jami, J., et al., 1996. Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J. 15, 1542–1547. Joost, H.G., Bell, G.I., Best, J.D., Birnbaum, M.J., Charron, M.J., Chen, Y.T., et al., 2002. Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators. Am. J. Physiol. Endocrinol. Metab. 282, E974–E976. Kane, S., Sano, H., Liu, S.C., Asara, J.M., Lane, W.S., Garner, C.C., et al., 2002. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J. Biol. Chem. 277, 22115–22118. Katzen, H.M., Soderman, D.D., Wiley, C.E., 1970. Multiple forms of hexokinase. Activities associated with subcellular particulate and soluble fractions of normal and streptozotocin diabetic rat tissues. J. Biol. Chem. 245, 4081–4096. Maehama, T., Dixon, J.E., 1999. PTEN: a tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol. 9, 125–128. Mothe-Satney, I., Peraldi, P., Rocchi, S., Sawka-Verhelle, D., Tartare-Deckert, S., Giudicelli, J., 2001. Surfing the insulin signaling web. Eur. J. Clin. Invest. 31, 966–977.

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Mechanisms of Skeletal Muscle Insulin Resistance Bandyopadhyay, G., Standaert, M.L., Galloway, L., Moscat, J., Farese, R.V., 1997. Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138, 4721–4731. Beeson, M., Sajan, M.P., Dizon, M., Grebenev, D., Gomez-Daspet, J., Miura, A., et al., 2003. Activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5(PO4)3 is defective in muscle in type 2 diabetes and impaired glucose tolerance: amelioration by rosiglitazone and exercise. Diabetes 52, 1926–1934. Comi, R.J., Grunberger, G., Gorden, P., 1987. Relationship of insulin binding and insulin-stimulated tyrosine kinase activity is altered in type II diabetes. J. Clin. Invest. 79, 453–462. DeFronzo, R.A., Tobin, J.D., Andres, R., 1979. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am. J. Physiol. 237, E214–E223. Dey, D., Basu, D., Roy, S.S., Bandyopadhyay, A., Bhattacharya, S., 2006. Involvement of novel PKC isoforms in FFA induced defects in insulin signaling. Mol. Cell. Endocrinol. 246, 60–64. Dresner, A., Laurent, D., Marcucci, M., Griffin, M.E., Dufour, S., Cline, G.W., et al., 1999. Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity. J. Clin. Invest. 103, 253–259. Farese, R.V., 2002. Function and dysfunction of aPKC isoforms for glucose transport in insulin-sensitive and insulin-resistant states. Am. J. Physiol. Endocrinol. Metab. 283, E1–E11. Freidenberg, G.R., Henry, R.R., Klein, H.H., Reichart, D.R., Olefsky, J.M., 1987. Decreased kinase activity of insulin receptors from adipocytes of non-insulindependent diabetic subjects. J. Clin. Invest. 79, 240–250. Ginsberg, H., Kimmerling, G., Olefsky, J.M., Reaven, G.M., 1975. Demonstration of insulin resistance in untreated adult-onset diabetic subjects with fasting hyperglycemia. J. Clin. Invest. 55, 454–461. Hashiramoto, M., Osawa, H., Ando, M., Murakami, A., Nishimiya, T., Nakano, M., et al., 2005. A nonsense mutation in the Arg345 of the insulin receptor gene in a Japanese type A insulin-resistant patient. Endocr. J. 52, 499–504. Himsworth, H.P., Kerr, R.B., 1939. Insulin-sensitive and insulin-insensitive types of diabetes mellitus. Clin. Sci. 4, 120–152. Ikeda, Y., Olsen, G.S., Ziv, E., Hansen, L.L., Busch, A.K., Hansen, B.F., et al., 2001. Cellular mechanism of nutritionally induced insulin resistance in Psammomys obesus: overexpression of protein kinase Cepsilon in skeletal muscle precedes the onset of hyperinsulinemia and hyperglycemia. Diabetes 50, 584–592. Kahn, B.B., Shulman, G.I., DeFronzo, R.A., Cushman, S.W., Rossetti, L., 1991. Normalization of blood glucose in diabetic rats with phlorizin treatment reverses

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