Recent studies using magnetic resonance spectroscopy have shown that decreased insulin-stimulated muscle glycogen synthesis due to a defect in insulin-stimulated glucose transport activity is a major factor in the pathogenesis of type 2 diabetes. The molecular mechanism underlying defective insulin-stimulated glucose transport activity can be attributed to increases in intramyocellular lipid metabolites such as fatty acyl CoAs and diacylglycerol, which in turn activate a serine/threonine kinase cascade, thus leading to defects in insulin signaling through Ser/Thr phosphorylation of insulin receptor substrate (IRS)-1. A similar mechanism is also observed in hepatic insulin resistance associated with nonalcoholic fatty liver, which is a common feature of type 2 diabetes, where increases in hepatocellular diacylglycerol content activate protein kinase C-epsilon, leading to reduced insulin-stimulated tyrosine phosphorylation of IRS-2. More recently, magnetic resonance spectroscopy studies in healthy lean elderly subjects and healthy lean insulin-resistant offspring of parents with type 2 diabetes have demonstrated that reduced mitochondrial function may predispose these individuals to intramyocellular lipid accumulation and insulin resistance. Further analysis has found that the reduction in mitochondrial function in the insulin-resistant offspring can be mostly attributed to reductions in mitochondrial density. By elucidating the cellular and molecular mechanisms responsible for insulin resistance, these studies provide potential new targets for the treatment and prevention of type 2 diabetes.

Insulin resistance is characteristic of obesity, type 2 diabetes, and components of the cardiometabolic syndrome, including hypertension and dyslipidemia, that collectively contribute to a substantial risk for cardiovascular disease. Metabolic actions of insulin in classic insulin target tissues (eg, skeletal muscle, fat, and liver), as well as actions in nonclassic targets (eg, cardiovascular tissue), help to explain why insulin resistance and metabolic dysregulation are central in the pathogenesis of the cardiometabolic syndrome and cardiovascular disease. Glucose and lipid metabolism are largely dependent on mitochondria to generate energy in cells. Thereby, when nutrient oxidation is inefficient, the ratio of ATP production/oxygen consumption is low, leading to an increased production of superoxide anions. Reactive oxygen species formation may have maladaptive consequences that increase the rate of mutagenesis and stimulate proinflammatory processes. In addition to reactive oxygen species formation, genetic factors, aging, and reduced mitochondrial biogenesis all contribute to mitochondrial dysfunction. These factors also contribute to insulin resistance in classic and nonclassic insulin target tissues. Insulin resistance emanating from mitochondrial dysfunction may contribute to metabolic and cardiovascular abnormalities and subsequent increases in cardiovascular disease. Furthermore, interventions that improve mitochondrial function also improve insulin resistance. Collectively, these observations suggest that mitochondrial dysfunction may be a central cause of insulin resistance and associated complications. In this review, we discuss mechanisms of mitochondrial dysfunction related to the pathophysiology of insulin resistance in classic insulin-responsive tissue, as well as cardiovascular tissue.

Insulin resistance is a major hallmark in the development of type 2 diabetes, which is characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in muscle. The nuclear hormone receptor coactivator PGC-1 (peroxisome proliferator-activated (PPAR)-gamma coactivator-1) has been implicated in the onset of type 2 diabetes. Hepatic PGC-1 expression is elevated in mouse models of this disease, where it promotes constitutive activation of gluconeogenesis and fatty acid oxidation through its association with the nuclear hormone receptors HNF-4 and PPAR-alpha, respectively. Here we show that PGC-1-deficient mice, generated by adenoviral delivery of PGC-1 RNA interference (RNAi) to the liver, experience fasting hypoglycemia. Hepatic insulin sensitivity was enhanced in PGC-1-deficient mice, reflecting in part the reduced expression of the mammalian tribbles homolog TRB-3, a fasting-inducible inhibitor of the serine-threonine kinase Akt/PKB (ref. 6). We show here that, in the liver, TRB-3 is a target for PPAR-alpha. Knockdown of hepatic TRB-3 expression improved glucose tolerance, whereas hepatic overexpression of TRB-3 reversed the insulin-sensitive phenotype of PGC-1-deficient mice. These results indicate a link between nuclear hormone receptor and insulin signaling pathways, and suggest a potential role for TRB-3 inhibitors in the treatment of type 2 diabetes.

Most lifestyle-related chronic diseases are characterized by low-grade systemic inflammation and insulin resistance. Excessive tumor necrosis factor-alpha (TNF-alpha) concentrations have been implicated in the development of insulin resistance, but direct evidence in humans is lacking. Here, we demonstrate that TNF-alpha infusion in healthy humans induces insulin resistance in skeletal muscle, without effect on endogenous glucose production, as estimated by a combined euglycemic insulin clamp and stable isotope tracer method. TNF-alpha directly impairs glucose uptake and metabolism by altering insulin signal transduction. TNF-alpha infusion increases phosphorylation of p70 S6 kinase, extracellular signal-regulated kinase-1/2, and c-Jun NH(2)-terminal kinase, concomitant with increased serine and reduced tyrosine phosphorylation of insulin receptor substrate-1. These signaling effects are associated with impaired phosphorylation of Akt substrate 160, the most proximal step identified in the canonical insulin signaling cascade regulating GLUT4 translocation and glucose uptake. Thus, excessive concentrations of TNF-alpha negatively regulate insulin signaling and whole-body glucose uptake in humans. Our results provide a molecular link between low-grade systemic inflammation and the metabolic syndrome.

Inherited defects in signaling pathways downstream of the insulin receptor have long been suggested to contribute to human type 2 diabetes mellitus. Here we describe a mutation in the gene encoding the protein kinase AKT2/PKBbeta in a family that shows autosomal dominant inheritance of severe insulin resistance and diabetes mellitus. Expression of the mutant kinase in cultured cells disrupted insulin signaling to metabolic end points and inhibited the function of coexpressed, wild-type AKT. These findings demonstrate the central importance of AKT signaling to insulin sensitivity in humans.

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.

An epidemic surge in the incidence of obesity has occurred worldwide over the past two decades. This alarming trend has been triggered by lifestyle habits that encourage overconsumption of energy-rich foods while also discouraging regular physical activity. These environmental influences create a chronic energy imbalance that leads to persistent weight gain in the form of body fat and a host of other abnormalities in metabolic homeostasis. As adiposity increases, so does the risk of developing comorbidities such as diabetes, hypertension, and cardiovascular disease. The intimate association between obesity and systemic metabolic dysregulation has inspired a new area of biochemistry research in which scientists are seeking to understand the molecular mechanisms that link chronic lipid oversupply to tissue dysfunction and disease development. The purpose of this chapter is to review recent findings in this area, placing emphasis on lipid-induced functional impairments in the major peripheral organs that control energy flux: adipose tissue, the liver, skeletal muscle, and the pancreas.

Malonyl-CoA is an allosteric inhibitor of carnitine palmitoyltransferase (CPT) I, the enzyme that controls the transfer of long-chain fatty acyl (LCFA)-CoAs into the mitochondria where they are oxidized. In rat skeletal muscle, the formation of malonyl-CoA is regulated acutely (in minutes) by changes in the activity of the beta-isoform of acetyl-CoA carboxylase (ACCbeta). This can occur by at least two mechanisms: one involving cytosolic citrate, an allosteric activator of ACCbeta and a precursor of its substrate cytosolic acetyl-CoA, and the other involving changes in ACCbeta phosphorylation. Increases in cytosolic citrate leading to an increase in the concentration of malonyl-CoA occur when muscle is presented with insulin and glucose, or when it is made inactive by denervation, in keeping with a diminished need for fatty acid oxidation in these situations. Conversely, during exercise, when the need of the muscle cell for fatty acid oxidation is increased, decreases in the ATP/AMP and/or creatine phosphate-to-creatine ratios activate an isoform of an AMP-activated protein kinase (AMPK), which phosphorylates ACCbeta and inhibits both its basal activity and activation by citrate. The central role of cytosolic citrate links this malonyl-CoA regulatory mechanism to the glucose-fatty acid cycle concept of Randle et al. (P. J. Randle, P. B. Garland. C. N. Hales, and E. A. Newsholme. Lancet 1: 785-789, 1963) and to a mechanism by which glucose might autoregulate its own use. A similar citrate-mediated malonyl-CoA regulatory mechanism appears to exist in other tissues, including the pancreatic beta-cell, the heart, and probably the central nervous system. It is our hypothesis that by altering the cytosolic concentrations of LCFA-CoA and diacylglycerol, and secondarily the activity of one or more protein kinase C isoforms, changes in malonyl-CoA provide a link between fuel metabolism and signal transduction in these cells. It is also our hypothesis that dysregulation of the malonyl-CoA regulatory mechanism, if it leads to sustained increases in the concentrations of malonyl-CoA and cytosolic LCFA-CoA, could play a key role in the pathogenesis of insulin resistance in muscle. That it may contribute to abnormalities associated with the insulin resistance syndrome in other tissues and the development of obesity has also been suggested. Studies are clearly needed to test these hypotheses and to explore the notion that exercise and some pharmacological agents that increase insulin sensitivity act via effects on malonyl-CoA and/or cytosolic LCFA-CoA.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a sugar attachment to serine or threonine hydroxyl moieties on nuclear and cytoplasmic proteins. In many ways, O-GlcNAcylation is similar to phosphorylation because both post-translational modifications cycle rapidly in response to internal or environmental cues. O-GlcNAcylated proteins are involved in transcription, translation, cytoskeletal assembly, signal transduction, and many other cellular functions. O-GlcNAc signaling is intertwined with cellular metabolism; indeed, the donor sugar for O-GlcNAcylation (UDP-GlcNAc) is synthesized from glucose, glutamine, and UTP via the hexosamine biosynthetic pathway. Emerging research indicates that O-GlcNAc signaling and its crosstalk with phosphorylation are altered in metabolic diseases, such as diabetes and cancer.

To understand better the defects in the proximal steps of insulin signaling during type 2 diabetes, we used differentiated human skeletal muscle cells in primary culture. When compared with cells from control subjects, myotubes established from patients with type 2 diabetes presented the same defects as those previously evidenced in vivo in muscle biopsies, including defective stimulation of phosphatidylinositol (PI) 3-kinase activity, decreased association of PI 3-kinase with insulin receptor substrate (IRS)-1 and reduced IRS-1 tyrosine phosphorylation during insulin stimulation. In contrast to IRS-1, the signaling through IRS-2 was not altered. Investigating the causes of the reduced tyrosine phosphorylation of IRS-1, we found a more than twofold increase in the basal phosphorylation of IRS-1 on serine 636 in myotubes from patients with diabetes. Concomitantly, there was a higher basal mitogen-activated protein kinase (MAPK) activity in these cells, and inhibition of the MAPKs with PD98059 strongly reduced the level of serine 636 phosphorylation. These results suggest that IRS-1 phosphorylation on serine 636 might be involved in the reduced phosphorylation of IRS-1 on tyrosine and in the subsequent alteration of insulin-induced PI 3-kinase activation. Moreover, increased MAPK activity seems to play a role in the phosphorylation of IRS-1 on serine residue in human muscle cells.

Nuclear, cytoplasmic, and mitochondrial proteins are extensively modified by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. This sugar modification regulates fundamental cellular processes in response to diverse nutritional and hormonal cues. The enzymes O-GlcNAc transferase (OGT) and O-linked β-N-acetylglucosaminase (O-GlcNAcase) mediate the addition and removal of O-GlcNAc, respectively. Aberrant O-GlcNAcylation has been implicated in a plethora of human diseases, including diabetes, cancer, aging, cardiovascular disease, and neurodegenerative disease. Because metabolic dysregulation is a vital component of these diseases, unraveling the roles of O-GlcNAc in metabolism is of emerging importance. Here, we review the current understanding of the functions of O-GlcNAc in cell signaling and gene transcription involved in metabolism, and focus on its relevance to diabetes, cancer, circadian rhythm, and mitochondrial function.

Insulin resistance condition is associated to the development of several syndromes, such as obesity, type 2 diabetes mellitus and metabolic syndrome. Although the factors linking insulin resistance to these syndromes are not precisely defined yet, evidence suggests that the elevated plasma free fatty acid (FFA) level plays an important role in the development of skeletal muscle insulin resistance. Accordantly, in vivo and in vitro exposure of skeletal muscle and myocytes to physiological concentrations of saturated fatty acids is associated with insulin resistance condition. Several mechanisms have been postulated to account for fatty acids-induced muscle insulin resistance, including Randle cycle, oxidative stress, inflammation and mitochondrial dysfunction. Here we reviewed experimental evidence supporting the involvement of each of these propositions in the development of skeletal muscle insulin resistance induced by saturated fatty acids and propose an integrative model placing mitochondrial dysfunction as an important and common factor to the other mechanisms.

The storage of triglyceride (TG) droplets in nonadipose tissues is called ectopic fat storage. Ectopic fat is associated with insulin resistance and type 2 diabetes mellitus (T2DM). Not the triglycerides per se but the accumulation of intermediates of lipid metabolism in organs, such as the liver, skeletal muscle, and heart seem to disrupt metabolic processes and impair organ function. We describe the mechanisms of ectopic fat depositions in the liver, skeletal muscle, and in and around the heart and the consequences for each organs function. In addition, we systematically reviewed the literature for the effects of diet-induced weight loss and exercise on ectopic fat depositions.

This study was conducted to investigate the possible involvement of protein kinase C (PKC) and serine/threonine phosphorylation of the insulin receptor in insulin resistance and/or obesity. Insulin receptor tyrosine kinase activity was depressed in muscle from obese insulin-resistant patients compared with lean insulin-responsive control subjects. Alkaline phosphatase treatment resulted in a significant 48% increase in in vitro insulin-stimulated receptor tyrosine kinase activity in obese but not lean muscle. To investigate the involvement of PKC in skeletal muscle insulin resistance and/or obesity, membrane-associated PKC activity and the protein content of various PKC isoforms were measured in human skeletal muscle from lean, insulin-responsive, and obese insulin-resistant patients. Membrane-associated PKC activity was not changed; however, PKC-beta protein content, assayed by Western blot analysis, was significantly higher, whereas PKC-theta, -eta, and -mu were significantly lower in muscle from obese patients compared with muscle from lean control subjects. Incubation of muscle strips with insulin significantly increased membrane-associated PKC activity in muscle from obese but not lean subjects. PKC-delta, -beta, and -theta were translocated from the cytosol to the membrane fraction in response to insulin treatment. These results suggest that in skeletal muscle from insulin-resistant obese patients, insulin receptor tyrosine kinase activity was reduced because of hyperphosphorylation on serine/threonine residues. Membrane-associated PKC-beta protein was elevated under basal conditions, and membrane-associated total PKC activity was increased under insulin-stimulated conditions in muscle from obese insulin-resistant patients. Thus, we postulate that the decreased tyrosine kinase activity of the insulin receptor may be caused by serine/threonine phosphorylation by PKC.

Insulin resistance is a major risk factor for developing type 2 diabetes caused by the inability of insulin-target tissues to respond properly to insulin, and contributes to the morbidity of obesity. Insulin action involves a series of signaling cascades initiated by insulin binding to its receptor, eliciting receptor autophosphorylation and activation of the receptor tyrosine kinase, resulting in tyrosine phosphorylation of insulin receptor substrates (IRSs). Phosphorylation of IRSs leads to activation of phosphatidylinositol 3-kinase (PI3K) and, subsequently, to activation of Akt and its downstream mediator AS160, all of which are important steps for stimulating glucose transport induced by insulin. Although the mechanisms underlying insulin resistance are not completely understood in skeletal muscle, it is thought to result, at least in part, from impaired insulin-dependent PI3K activation and downstream signaling. This review focuses on the molecular basis of skeletal muscle insulin resistance in obesity and type 2 diabetes. In addition, the effects of insulin-sensitizing agent treatment and lifestyle intervention of human insulin-resistant subjects on insulin signaling cascade are discussed. Furthermore, the role of Rho-kinase, a newly identified regulator of insulin action in insulin control of metabolism, is addressed.

Resistance to the biological action of insulin in its target tissues is a cardinal feature of non-insulin-dependent diabetes mellitus. Protein-tyrosine phosphatases (PTPases) have been postulated to play a key role in the regulation of the insulin action pathway, especially in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo. To evaluate whether changes in the activity and/or abundance of candidate skeletal muscle PTPases is associated with severe resistance to insulin in an animal model, we measured PTPase enzyme activity and PTPase protein level by immunoblotting in subcellular fractions of skeletal muscle in lean (+/?), insulin-resistant obese (fa/fa), and diabetic (ZDF/Drt-fa/fa) Zucker rats. Using a phosphotyrosylmyelin basic protein substrate, the solubilized-particulate fraction PTPase activity was increased by 65% and 74% (P < .05) and in vitro dephosphorylation of a recombinant rat insulin receptor kinase domain was increased by 104% and 114% in obese and diabetic animals, respectively (P < .01). These changes in PTPase activity were associated with an increase in specific immunoreactivity of leukocyte common antigen-related PTPase ([LAR] by 42% and 50%), PTPase 1B (by 61% and 69%), and the SHZ domain containing PTPase (SH-PTP2) (by 44% and 48%) in the solubilized-particulate fraction of obese and diabetic animals, respectively (P < .05). In diabetic muscle, increased SH-PTP2 abundance was also associated with a shift of SH-PTP2 to a plasma membrane component, which may have important consequences for the activation of this enzyme in the insulin-resistant state. These results provide evidence that specific PTPases play a role in the insulin resistance of this genetic model of obesity and non-insulin-dependent diabetes.

Ceramides (Cer) are implicated in obesity-associated skeletal muscle and perhaps adipocyte insulin resistance. We examined whether the sphingolipid content of human subcutaneous adipose tissue and plasma varies by obesity and sex as well as the relationship between ceramide content and metabolic indices. Abdominal subcutaneous adipose biopsies were performed on 12 lean adults (males = 6), 12 obese adults (males = 6) for measurement of sphingolipid content and activity of the main ceramide metabolism enzymes. Blood was sampled for glucose, insulin (to calculate homeostasis model assessment-estimated insulin resistance (HOMA(IR))) adiponectin, and interleukin-6 (IL-6) concentrations. Compared to lean controls, total ceramide content (pg/adipocyte) was increased by 31% (P < 0.05) and 34% (P < 0.05) in obese females and males, respectively. In adipocytes from obese adults sphingosine, sphinganine, sphingosine-1-phosphate, C14-Cer, C16-Cer, and C24-Cer were all increased. C18:1-Cer was increased in obese males and C24:1-Cer in obese females. For women only, there was a negative correlation between C16-Cer ceramide and plasma adiponectin (r = -0.77, P = 0.003) and a positive correlation between total ceramide content and HOMA(IR) (r = 0.74, P = 0.006). For men only there were significant (at least P < 0.05), positive correlations between adipocyte Cer-containing saturated fatty acid and plasma IL-6 concentration. We conclude that the sexual dimorphism in adipose tissue behavior in humans extends to adipose tissue sphingolipid content its association with adiponectin, IL-6 and insulin resistance.

Insulin resistance is present in liver and muscle of subjects with type 2 diabetes and obesity. Recent studies suggest that such insulin resistance could be related to abnormalities in lipid-mediated signal transduction; however, the nature of these abnormalities is unclear. To examine this question further, tissue levels of diacylglycerol (DAG), malonyl-CoA, and triglyceride (TG) were determined in liver and soleus muscle of obese insulin-resistant KKAy mice and lean C57 BL control mice. In addition, the effects of treatment with pioglitazone, an antidiabetic agent that acts by increasing insulin sensitivity in muscle, liver, and other tissues, were assessed. The KKAy mice were hyperglycemic (407 vs. 138 mg/dl), hypertriglyceridemic (337 vs. 109 mg/dl), hyperinsulinemic (631 vs. 15 mU/ml), and weighed more (42 vs. 35 g) than the control mice. They also had 1.5- to 2.0-fold higher levels of malonyl-CoA in both liver and muscle, higher DAG (twofold) and TG (1.3-fold) levels in muscle, and higher TG (threefold), but not DAG, levels. Treatment of the KKAy mice with pioglitazone for 4 days decreased plasma glucose, TGs, and insulin by approximately 50% and restored hepatic and muscle malonyl-CoA levels to control values. In contrast, pioglitazone increased hepatic and muscle DAG levels two- or threefold. It has no effect on muscle or hepatic TG content, and it slightly increased hepatic TGs in the control group. The results indicate that abnormalities in tissue lipids occur in both liver and muscle of the KKAy mouse and that they are differentially altered when insulin sensitivity is enhanced by treatment with pioglitazone.(ABSTRACT TRUNCATED AT 250 WORDS)