Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle

Background

The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.

Methodology/Principal Findings

To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and ¹H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H₂O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.

Conclusions

This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.     

Increased intrahepatic triglyceride is associated with peripheral insulin resistance: in vivo MR imaging and spectroscopy studies

Recent studies have indicated that the mass/content of intramyocellular lipid (IMCL), intrahepatic triglyceride (IHTG), visceral fat (VF), and even deep abdominal subcutaneous fat (SF) may all be correlated with insulin resistance. Since simultaneous measurements of these parameters have not been reported, the relative strength of their associations with insulin action is not known. Therefore, the goals of this study were 1) to simultaneously measure IMCL, IHTG, VF, and abdominal SF in the same nondiabetic individuals using noninvasive (1)H-magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) and 2) to examine how these fat stores are correlated with systemic insulin sensitivity as measured by whole body glucose disposal (R(d)) during euglycemic-hyperinsulinemic clamp studies. Positive correlations were observed among IMCL, IHTG, and VF. There were significant inverse correlations between whole body R(d) and both IMCL and VF. Notably, there was a particularly tight inverse correlation between IHTG and whole body R(d) (r = -0.86, P < 0.001), consistent with an association between liver fat and peripheral insulin sensitivity. This novel finding suggests that hepatic triglyceride accumulation has important systemic consequences that may adversely affect insulin sensitivity in other tissues.     

Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.     

Saturated fatty acid‐induced insulin resistance is associated with mitochondrial dysfunction in skeletal muscle cells

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as obesity and type 2 diabetes mellitus. These high levels of plasma FFA are proposed to play an important role for the development of insulin resistance but the mechanisms involved are still unclear. This study investigated the effects of saturated and unsaturated FFA on insulin sensitivity in parallel with mitochondrial function. C2C12 myotubes were treated for 24 h with 0.1 mM of saturated (palmitic and stearic) and unsaturated (oleic, linoleic, eicosapentaenoic, and docosahexaenoic) FFA. After this period, basal and insulin‐stimulated glucose metabolism and mitochondrial function were evaluated. Saturated palmitic and stearic acids decreased insulin‐induced glycogen synthesis, glucose oxidation, and lactate production. Basal glucose oxidation was also reduced. Palmitic and stearic acids impaired mitochondrial function as demonstrated by decrease of both mitochondrial hyperpolarization and ATP generation. These FFA also decreased Akt activation by insulin. As opposed to saturated FFA, unsaturated FFA did not impair glucose metabolism and mitochondrial function. Primary cultures of rat skeletal muscle cells exhibited similar responses to saturated FFA as compared to C2C12 cells. These results show that in muscle cells saturated FFA‐induced mitochondrial dysfunction associated with impaired insulin‐induced glucose metabolism. J. Cell. Physiol. 222:187–194, 2010. © 2009 Wiley‐Liss, Inc.     

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity

Protein kinase C theta (PKC-theta) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/R), working as "dominant negative," specifically in skeletal muscle. These mice are viable and fertile, however, by the 6-7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin-sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6-7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin-stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over-expression of dominant negative PKC-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.     

Streptozotocin induces G2 arrest in skeletal muscle myoblasts and impairs muscle growth in vivo

Streptozotocin (STZ) is used extensively to induce pancreatic -cell death and ultimately diabetes mellitus in animal models. However, the direct effects of STZ on muscle are largely unknown. To delineate the effects of STZ from the effects of hypoinsulinemia/hyperglycemia, we injected young rats with 1) saline (control), 2) STZ (120 mg/kg) or 3) STZ and insulin (STZ-INS; to maintain euglycemia). STZ rats demonstrated significantly elevated blood glucose throughout the 48-h protocol, while control and STZ-INS rats were euglycemic. Body mass increased in control (13 ± 4 g), decreased by 19 ± 2 g in STZ and remained unchanged in STZ-INS rats (–0.3 ± 2 g). Cross-sectional areas of gastrocnemius muscle fibers were smaller in STZ vs. control (1,480 ± 149 vs. 1,870 ± 40 µm², respectively; P < 0.05) and insulin treatment did not rescue this defect (STZ-INS: 1,476 ± 143 µm²). Western blot analysis revealed a detectable increase in ubiquitinated proteins in the STZ skeletal muscles compared with control and STZ-INS. To further define the effects of STZ on skeletal muscle, independent of hyperglycemia, myoblasts were exposed to varying doses of STZ (0.25–3.0mg/ml) in vitro. Both acute and chronic exposures of STZ significantly impaired proliferative capacity in a dose-dependent manner. Within STZ-treated myoblasts, increased reactive oxygen species was associated with significant G₂/M phase cell-cycle arrest. Taken together, our findings show that the effects of STZ are not -cell specific and reveal that STZ should not be used for studies examining diabetic myopathy. satellite cell; diabetes; diabetic model; type 1 diabetes mellitus; cell cycle; proliferation; hypertrophy     

Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor

Background

Length and intensity of signal transduction via cytokine receptors is precisely regulated. Degradation of certain cytokine receptors is mediated by the ubiquitin ligase SCF(βTrCP). In several instances, Janus kinase (Jak) family members can stabilise their cognate cytokine receptors at the cell surface.

Principal Findings

In this study we show in Hek293 cells that Jak2 binding to the growth hormone receptor prevents endocytosis in a non-catalytic manner. Following receptor activation, the detachment of phosphorylated Jak2 induces down-regulation of the growth hormone receptor by SCF(βTrCP). Using γ2A human fibroblast cells we show that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, strongly suggesting that the modes of endocytosis are identical. Different Jak2 RNA levels in HepG2, IM9 and Hek293 cells indicate the importance of cellular concentration on growth hormone receptor function. Both Jak2 and βTrCP bind to neighbouring linear motifs in the growth hormone receptor tail without the requirement of modifications, indicating that growth hormone sensitivity is regulated by the cellular level of non-committed Jak2.

Conclusions/Significance

As signal transduction of many cytokine receptors depends on Jak2, the study suggests an integrative role of Jak2 in cytokine responses based on its enzyme activity as well as its stabilising properties towards the receptors.     

Hrs is a positive regulator of VEGF and insulin signaling

Both VEGF and insulin are implicated in the pathogenesis of diabetic retinopathy. While it has been established for many years that the number of cell surface receptors impacts upon VEGF and insulin action, little is known about the precise machinery and proteins driving VEGF-R2 and IR degradation. Here, we investigate the role of Hepatocyte growth factor-Regulated tyrosine kinase Substrate (Hrs), a regulator of RTK trafficking, in VEGF and insulin signaling. We report that ectopic expression of Hrs increases VEGF-R2 and IR number and tyrosine phosphorylation, leading to amplification of their downstream signaling. The UIM (Ubiquitin Interacting Motif) domain of Hrs is required for Hrs-induced increases in VEGF-R2, but not in IR. Furthermore, Hrs is tyrosine-phosphorylated in response to VEGF and insulin. We show that the UIM domain is required for Hrs phosphorylation in response to VEGF, but not to insulin. Importantly, Hrs co-localizes with both VEGF-R2 and IR and co-immunoprecipitates with both in a manner independent of the Hrs-UIM domain. Finally, we demonstrate that Hrs inhibits Nedd4-mediated VEGF-R2 degradation and acts additively with Grb10. We conclude that Hrs is a positive regulator of VEGF-R2 and IR signaling and that ectopic expression of Hrs protects both VEGF-R2 and IR from degradation.     

Effects of hormone therapy on insulin signaling proteins in skeletal muscle of cynomolgus monkeys

We have previously shown that hormone therapy (HT) with medroxyprogesterone acetate (MPA) alone or in combination with conjugated equine estrogens (CEE) impairs insulin sensitivity. In the current study, we sought to determine if the effect of MPA on whole body insulin sensitivity is associated with alterations in insulin signaling proteins in skeletal muscle. Ovariectomized cynomolgus monkeys were treated for 2 years with either no hormones (n = 10), CEE (0.625 mg/day human equivalent, n = 11) or CEE + MPA (2.5 mg/day human equivalent, n = 12). At the end of the study, biopsies of rectus femoris muscle were flash frozen in the basal and insulin-stimulated (10 min post-intravenous insulin injection) state. Immunoblotting revealed that CEE + MPA monkeys had significantly less glucose transporter 4 (GLUT4) expression (ANOVA P = 0.001), but there was no significant treatment effect on expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, or the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). There was a tendency for decreased insulin receptor tyrosine phosphorylation with CEE + MPA treatment (ANOVA P = 0.14). These deficiencies in skeletal muscle insulin signaling likely contribute to the unfavorable changes in whole body insulin sensitivity associated with CEE + MPA treatment.     

PTEN,a widely known negative regulator of insulin/PI3K signaling,positively regulates neuronal insulin resistance

Lipid and protein tyrosine phosphatase, phosphatase and tension homologue (PTEN), is a widely known negative regulator of insulin/phosphoinositide 3-kinase signaling. Down-regulation of PTEN is thus widely documented to ameliorate insulin resistance in peripheral tissues such as skeletal muscle and adipose. However, not much is known about its exact role in neuronal insulin signaling and insulin resistance. Moreover, alterations of PTEN in neuronal systems have led to discovery of several unexpected outcomes, including in the neurodegenerative disorder Alzheimer''s disease (AD), which is increasingly being recognized as a brain-specific form of diabetes. In addition, contrary to expectations, its neuron-specific deletion in mice resulted in development of diet-sensitive obesity. The present study shows that PTEN, paradoxically, positively regulates neuronal insulin signaling and glucose uptake. Its down-regulation exacerbates neuronal insulin resistance. The positive role of PTEN in neuronal insulin signaling is likely due to its protein phosphatase actions, which prevents the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), the kinases critically involved in neuronal energy impairment and neurodegeneration. Results suggest that PTEN acting through FAK, the direct protein substrate of PTEN, prevents ERK activation. Our findings provide an explanation for unexpected outcomes reported earlier with PTEN alterations in neuronal systems and also suggest a novel molecular pathway linking neuronal insulin resistance and AD, the two pathophysiological states demonstrated to be closely linked.     

Calorie restriction prevents the development of insulin resistance and impaired insulin signaling in skeletal muscle of ovariectomized rats

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32-70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser(473) phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser(307), SAPK/JNK Thr(183)/Tyr(185), and p38 mitogen-activated protein kinase (MAPK) Thr(180)/Tyr(182) was increased (24-62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX+CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.     

Insulin resistance in the skeletal muscle of women with PCOS involves intrinsic and acquired defects in insulin signaling

Insulin resistance in polycystic ovary syndrome (PCOS) is due to a postbinding defect in signaling that persists in cultured skin fibroblasts and is associated with constitutive serine phosphorylation of the insulin receptor (IR). Cultured skeletal muscle from obese women with PCOS and age- and body mass index-matched control women (n = 10/group) was studied to determine whether signaling defects observed in this tissue in vivo were intrinsic or acquired. Basal and insulin-stimulated glucose transport and GLUT1 abundance were significantly increased in cultured myotubes from women with PCOS. Neither IR beta-subunit abundance and tyrosine autophosphorylation nor insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity differed in the two groups. However, IRS-1 protein abundance was significantly increased in PCOS, resulting in significantly decreased PI 3-kinase activity when normalized for IRS-1. Phosphorylation of IRS-1 on Ser312, a key regulatory site, was significantly increased in PCOS, which may have contributed to this signaling defect. Insulin signaling via IRS-2 was also decreased in myotubes from women with PCOS. In summary, decreased insulin-stimulated glucose uptake in PCOS skeletal muscle in vivo is an acquired defect. Nevertheless, there are intrinsic abnormalities in glucose transport and insulin signaling in myotubes from affected women, including increased phosphorylation of IRS-1 Ser312, that may confer increased susceptibility to insulin resistance-inducing factors in the in vivo environment. These abnormalities differ from those reported in other insulin resistant states consistent with the hypothesis that PCOS is a genetically unique disorder conferring an increased risk for type 2 diabetes.     

Histopathological changes in rat pancreas and skeletal muscle associated with high fat diet induced insulin resistance

The effects of a high fat diet on the development of diabetes mellitus, insulin resistance and secretion have been widely investigated. We investigated the effects of a high fat diet on the pancreas and skeletal muscle of normal rats to explore diet-induced insulin resistance mechanisms. Forty-four male Wistar rats were divided into six groups: a control group fed standard chow, a group fed a 45% fat diet and a group fed a 60% fat diet for 3 weeks to measure acute effects; an additional three groups were fed the same diet regimens for 8 weeks to measure chronic effects. The morphological effects of the two high fat diets were examined by light microscopy. Insulin in pancreatic islets was detected using immunohistochemistry. The homeostasis model assessment of insulin resistance index and insulin staining intensity in islets increased significantly with acute administration of high fat diets, whereas staining intensity decreased with chronic administration of the 45% fat diet. Islet areas increased significantly with chronic administration. High fat diet administration led to islet degeneration, interlobular adipocyte accumulation and vacuolization in the pancreatic tissue, as well as degeneration and lipid droplet accumulation in the skeletal muscle tissue. Vacuolization in the pancreas and lipid droplets in skeletal muscle tissue increased significantly with chronic high fat diet administration. We suggest that the glucolipotoxic effects of high fat diet administration depend on the ratio of saturated to unsaturated fatty acid content in the diet and to the total fat content of the diet.