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Aims/hypothesis: High-fat, high-sucrose diet (HF)-induced reactive oxygen species (ROS) levels are implicated in skeletal muscle insulin resistance and mitochondrial dysfunction. Here we investigated whether mitochondrial ROS sequestering can circumvent HF-induced oxidative stress; we also determined the impact of any reduced oxidative stress on muscle insulin sensitivity and mitochondrial function., Methods: The Skulachev ion (plastoquinonyl decyltriphenylphosphonium) (SkQ), a mitochondria-specific antioxidant, was used to target ROS production in C2C12 muscle cells as well as in HF-fed (16 weeks old) male C57Bl/6 mice, compared with mice on low-fat chow diet (LF) or HF alone. Oxidative stress was measured as protein carbonylation levels. Glucose tolerance tests, glucose uptake assays and insulin-stimulated signalling were determined to assess muscle insulin sensitivity. Mitochondrial function was determined by high-resolution respirometry., Results: SkQ treatment reduced oxidative stress in muscle cells (-23% p < 0.05), but did not improve insulin sensitivity and glucose uptake under insulin-resistant conditions. In HF mice, oxidative stress was elevated (56% vs LF p < 0.05), an effect completely blunted by SkQ. However, HF and HF+SkQ mice displayed impaired glucose tolerance (AUC HF up 33%, p < 0.001; HF+SkQ up 22%; p < 0.01 vs LF) and disrupted skeletal muscle insulin signalling. ROS sequestering did not improve mitochondrial function., Conclusions/interpretation: SkQ treatment reduced muscle mitochondrial ROS production and prevented HF-induced oxidative stress. Nonetheless, whole-body glucose tolerance, insulin-stimulated glucose uptake, muscle insulin signalling and mitochondrial function were not improved. These results suggest that HF-induced oxidative stress is not a prerequisite for the development of muscle insulin resistance.

Context: Low testosterone accompanied by elevated estradiol associates with the development of metabolic dysfunction in men., Objective: The aim of the study was to explore the hypothesis that alterations in sex steroid levels induce metabolic dysfunction through adipokines., Design: Circulating levels of sex steroids and 28 adipokines were determined in a cross-sectional study of morbidly obese men and aged-matched controls, as well as in a randomized clinical trial with healthy young men in which obesity-related alterations in sex steroid levels were mimicked by treatment with an aromatase inhibitor plus estradiol patches., Results: Morbidly obese men had lower testosterone levels than normal-weight controls. Estradiol levels were increased in morbidly obese men (without DM2) as compared to normal-weight controls. Circulating levels of multiple proinflammatory cytokines, including IL-1Ra, IL-5, IL-6, IL-10, leptin, monocyte chemoattractant protein 1 (MCP1), and macrophage inflammatory protein 1α, positively associated with estradiol and negatively with testosterone. The associations with estradiol, but not with testosterone, remained significant after adjusting for adipocyte cell size. In a separate clinical trial, the direct adverse effects of lowering testosterone and raising estradiol on MCP1 were substantiated in vivo., Conclusions: Initial alterations in sex steroid levels may contribute to metabolic dysfunction through adverse effects on adipokine levels in obese men. The direct adverse effects on MCP1, a chemokine highly linked to the development of metabolic dysfunction, were substantiated in a trial mimicking obesity-related alterations of sex steroid levels in healthy young males.

Context: Reduction of 50% excess body weight, using a very low-calorie diet (VLCD; 450 kcal/d) improves insulin sensitivity in obese type 2 diabetes mellitus patients., Objective: The objective of the study was to evaluate whether adding exercise to the VLCD has additional benefits., Design: This was a randomized intervention study., Setting: The study was conducted at a clinical research center in an academic medical center., Subjects: Twenty-seven obese [body mass index 37.2 ± 0.9 kg/m(2) (mean ± sem)] insulin-treated type 2 diabetes mellitus patients., Intervention: Patients followed a 16-wk VLCD. Thirteen of them simultaneously participated in an exercise program (E) consisting of 1-h, in-hospital training and four 30-min training sessions on a cycloergometer weekly., Outcome Measures: Insulin resistance was measured by a hyperinsulinemic euglycemic clamp. Insulin signaling, mitochondrial DNA (mtDNA) content, and intramyocellular lipid content was measured in skeletal muscle biopsies., Results: Baseline characteristics were identical in both groups. Substantial weight loss occurred (-23.7 ± 1.7 kg VLCD-only vs. -27.2 ± 1.9 kg VLCD+E, P = NS within groups). The exercise group lost more fat mass. Insulin-stimulated glucose disposal increased similarly in both study groups [15.0 ± 0.9 to 39.2 ± 4.7 μmol/min(-1) · kg lean body mass (LBM(-1)) VLCD-only vs. 17.0 ± 1.0 to 37.5 ± 3.5 μmol/min(-1) · kg LBM(-1) in VLCD+E], as did phosphorylation of the phosphatidylinositol 3-kinase-protein kinase B/AKT insulin signaling pathway. In contrast, skeletal muscle mtDNA content increased only in the VLCD+E group (1211 ± 185 to 2288 ± 358, arbitrary units, P = 0.016 vs. 1397 ± 240 to 1196 ± 179, P = NS, VLCD-only group). Maximum aerobic capacity also only increased significantly in the VLCD+E group (+6.6 ± 1.7 ml/min(-1) · kg LBM(-1) vs. +0.7 ± 1.5 ml/min(-1) · kg LBM(-1) VLCD-only, P = 0.017)., Conclusion: Addition of exercise to a 16-wk VLCD induces more fat loss. Exercise augments maximum aerobic capacity and skeletal muscle mtDNA content. These changes are, however, not reflected in a higher insulin-stimulated glucose disposal rate.

The stress protein heat shock protein 60 (Hsp60) induces secretion of proinflammatory mediators from murine adipocytes. This study aimed to study Hsp60 as a mediator of adipose tissue inflammation and skeletal muscle cell (SkMC) insulin sensitivity and to quantify plasma Hsp60 concentrations in lean and obese individuals. Regulation of Hsp60 release and Hsp60-induced cytokine secretion and signaling was measured in human adipocytes and SkMCs. Adipocytes exhibited higher Hsp60 release than preadipocytes and SkMCs, which was further stimulated by cytokines and Toll-like receptor (TLR)-4 activation. Hsp60 activated extracellular signal-related kinase (ERK)-1/2, Jun NH(2)-terminal kinase (JNK), p38, nuclear factor (NF)-κB, and impaired insulin-stimulated Akt phosphorylation in adipocytes. Furthermore, Hsp60 stimulated adipocytes to secrete tumor necrosis factor-α, interleukin (IL)-6, and IL-8. In SkMCs, Hsp60 activated ERK1/2, JNK, and NF-κB and inhibits insulin signaling and insulin-stimulated glucose uptake. SkMCs released IL-6, IL-8, and monocyte chemoattractant protein-1 on Hsp60 stimulation. Plasma Hsp60 was higher in obese males than in lean males and correlated positively with BMI, blood pressure, leptin, and homeostasis model assessment-insulin resistance. In summary, Hsp60 is released by human adipocytes, increased in plasma of obese humans, and induces insulin resistance. This is accompanied by activation of proinflammatory signaling in human adipocytes and SkMCs. Thus, Hsp60 might be a factor underlying adipose tissue inflammation and obesity-associated metabolic disorders.

Insulin signaling in the central nervous system (CNS) is required for the inhibitory effect of insulin on glucose production. Our aim was to determine whether the CNS is also involved in the stimulatory effect of circulating insulin on the tissue-specific retention of fatty acid (FA) from plasma. In wild-type mice, hyperinsulinemic-euglycemic clamp conditions stimulated the retention of both plasma triglyceride-derived FA and plasma albumin-bound FA in the various white adipose tissues (WAT) but not in other tissues, including brown adipose tissue (BAT). Intracerebroventricular (ICV) administration of insulin induced a similar pattern of tissue-specific FA partitioning. This effect of ICV insulin administration was not associated with activation of the insulin signaling pathway in adipose tissue. ICV administration of tolbutamide, a K(ATP) channel blocker, considerably reduced (during hyperinsulinemic-euglycemic clamp conditions) and even completely blocked (during ICV administration of insulin) WAT-specific retention of FA from plasma. This central effect of insulin was absent in CD36-deficient mice, indicating that CD36 is the predominant FA transporter in insulin-stimulated FA retention by WAT. In diet-induced insulin-resistant mice, these stimulating effects of insulin (circulating or ICV administered) on FA retention in WAT were lost. In conclusion, in insulin-sensitive mice, circulating insulin stimulates tissue-specific partitioning of plasma-derived FA in WAT in part through activation of K(ATP) channels in the CNS. Apparently, circulating insulin stimulates fatty acid uptake in WAT but not in BAT, directly and indirectly through the CNS.

Treatment with the dopamine receptor D2 (DRD2) agonist bromocriptine improves metabolic features in obese patients with type 2 diabetes by a still unknown mechanism. In the present study, we investigated the acute effect of bromocriptine and its underlying mechanism(s) on insulin secretion both in vivo and in vitro. For this purpose, C57Bl6/J mice were subjected to an intraperitoneal glucose tolerance test (ipGTT) and a hyperglycemic (HG) clamp 60min after a single injection of bromocriptine or placebo. The effects of bromocriptine on glucose-stimulated insulin secretion (GSIS), cell membrane potential and intracellular cAMP levels were also determined in INS-1E beta cells. We report here that bromocriptine increased glucose levels during ipGTT in vivo, an effect associated with a dose-dependent decrease in GSIS. During the HG clamp, bromocriptine reduced both first-phase and second-phase insulin response. This inhibitory effect was also observed in INS-1E beta cells, in which therapeutic concentrations of bromocriptine (0.5-50nM) decreased GSIS. Mechanistically, neither cellular energy state nor cell membrane depolarization was affected by bromocriptine whereas intracellular cAMP levels were significantly reduced, suggesting involvement of G-protein-coupled receptors. Surprisingly, the DRD2 antagonist domperidone did not counteract the effect of bromocriptine on GSIS, whereas yohimbine, an antagonist of the alpha2-adrenergic receptors, completely abolished bromocriptine-induced inhibition of GSIS. In conclusion, acute administration of bromocriptine inhibits GSIS by a DRD2-independent mechanism involving direct activation of the pancreatic alpha2-adrenergic receptors. We suggest that treatment with bromocriptine promotes beta cells rest, thereby preventing long-lasting hypersecretion of insulin and subsequent beta cell failure., (Copyright 2010 Elsevier Inc. All rights reserved.)

Insulin stimulates cardiac long-chain fatty acid (LCFA) and glucose uptake via translocation of human homolog of rat fatty acid translocase (CD36) and GLUT4 respectively, from intracellular membrane compartments to the sarcolemma, a process dependent on the activation of phosphatidylinositol-3 kinase. To identify downstream kinases of insulin signaling involved in translocation of CD36 and GLUT4 in the heart, we tested i) which cardiac protein kinase C (PKC) isoforms (alpha, delta, epsilon or zeta) are activated by insulin, and ii) whether PKC isoform-specific inhibition affects insulin-stimulated substrate uptake in the heart. Insulin-stimulated LCFA and glucose uptake were completely blunted by inhibition of PKC-zeta, but not by inhibition of conventional or novel PKCs. Concomitantly, translocation of CD36 and GLUT4 to the sarcolemma was completely blunted upon inhibition of PKC-zeta. However, insulin, in contrast to the diacylglycerol-analog phorbol-12-myristate-13-acetate (PMA), did not induce membrane-attachment of the conventional and novel PKCs-alpha, -delta, and -epsilon. PKC-zeta was already entirely membrane-bound in non-stimulated cells, and neither insulin nor PMA treatment had any effect on the subcellular localization of PKC-zeta. Furthermore, insulin treatment did not change phosphorylation of PKC-alpha, -delta, and -zeta or enzymatic activity of PKC-zeta towards a PKC-zeta substrate peptide. It is concluded that PKC-zeta, but not any other PKC isoform, is necessary for insulin-induced translocation of GLUT4 and CD36. However, PKC-zeta is already fully active under basal conditions and not further activated by insulin, indicating that its role in insulin-stimulated uptake of both glucose and LCFA is permissive rather than regulatory.

Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and beta-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.

In postmortem brains of patients with major depression, the expression of corticotrophin-releasing factor (CRF) is enhanced and that of brain-derived neurotrophic factor (BDNF) decreased. In mice over-expressing neuronal CRF (an animal model for depression) the expression of urocortin 1 (Ucn1) in the non-preganglionic Edinger-Westphal nucleus (npEW) is strongly down-regulated. Therefore, we hypothesized that an altered activity of Ucn1 neurons in the npEW would contribute to the pathogenesis of major depression. To test this hypothesis we measured Ucn1 mRNA and BDNF mRNA levels in the npEW of seven male and four female, drug-free suicide victims with major depression, and compared the data with those obtained from 10 male and seven female individuals without neurological and psychiatric disorders (controls). We show that compared with controls, the Ucn1-mRNA level in npEW neurons is about 9.12 times higher in male but unchanged in female suicide victims. Furthermore, BDNF mRNA expression in microdissections of npEW was 3.36 times lower in male suicide victims, but 5.27 times higher in female victims, compared with controls. Our data also show that male suicide victims had almost 11.47 times more Ucn1 and 4.26 times less BDNF mRNA in the npEW than female suicide victims. We discuss the significance of these data for npEW Ucn1 and BDNF, and propose that altered expressions of Ucn1 and BDNF in the npEW contribute to the pathogenesis of major depression and/or suicidality in a gender-specific manner.

Aims/hypothesis: Both energy restriction (ER) per se and weight loss improve glucose metabolism in obese insulin-treated type 2 diabetic patients. Short-term ER decreases basal endogenous glucose production (EGP) but not glucose disposal. In contrast the blood glucose-lowering mechanism of long-term ER with substantial weight loss has not been fully elucidated. The aim of this study was to investigate the effect of loss of 50% of excess weight [50% excess weight reduction (EWR)] on EGP, whole-body insulin sensitivity and the disturbed myocellular insulin-signalling pathway in ten obese insulin-treated type 2 diabetic patients., Methods: A euglycaemic-hyperinsulinaemic clamp with stable isotopes ([6,6-(2)H2]glucose and [2H5]glycerol) combined with skeletal muscle biopsies was performed during a very low energy diet (VLED; 1,883 kJ/day) on day 2 and again after 50% EWR. Oral blood glucose-lowering agents and insulin were discontinued 3 weeks prior to the VLED and at the start of the VLED, respectively., Results: Loss of 50% EWR (20.3+/-2.2 kg from day 2 to day of 50% EWR) normalised basal EGP and improved insulin sensitivity, especially insulin-stimulated glucose disposal (18.8+/-2.0 to 39.1+/-2.8 micromol kg fat-free mass(-1) min(-1), p=0.001). The latter was accompanied by improved insulin signalling at the level of the recently discovered protein kinase B/Akt substrates AS160 and PRAS40 along with a decrease in intramyocellular lipid (IMCL) content., Conclusions/interpretation: Considerable weight loss in obese, insulin-treated type 2 diabetic patients normalises basal EGP and improves insulin sensitivity resulting from an improvement in insulin signal transduction in skeletal muscle. The decrease in IMCL might contribute to this effect.

Aims/hypothesis: Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD)., Methods: Rats were exposed to an HFD or a low-fat diet (LFD). In vivo cardiac function was monitored by echocardiography. Substrate uptake and utilisation were determined in isolated cardiomyocytes., Results: Feeding an HFD for 8 weeks induced left ventricular dilation in the systolic phase and decreased fractional shortening and the ejection fraction. Insulin-stimulated glucose uptake and proline-rich Akt substrate 40 phosphorylation were 41% (p < 0.001) and 45% (p < 0.05) lower, respectively, in cardiomyocytes from rats on the HFD. However, long-chain fatty acid (LCFA) uptake was 1.4-fold increased (p < 0.001) and LCFA esterification into triacylglycerols and phospholipids was increased 1.4- and 1.5-fold, respectively (both p < 0.05), in cardiomyocytes from HFD compared with LFD hearts. In the presence of the CD36 inhibitor sulfo-N-succinimidyloleate, LCFA uptake and esterification were similar in LFD and HFD cardiomyocytes. In HFD hearts CD36 was relocated to the sarcolemma, and basal phosphorylation of a mediator of CD36-trafficking, i.e. protein kinase B (PKB/Akt), was increased., Conclusions/interpretation: Feeding rats an HFD induced cardiac contractile dysfunction, which was accompanied by the relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The permanent presence of CD36 at the sarcolemma resulted in enhanced rates of LCFA uptake and myocardial triacylglycerol accumulation, and may contribute to the development of insulin resistance and diabetic cardiomyopathy.

Aims/hypothesis: Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice., Materials and Methods: Chronic hepatic steatosis and hepatic insulin resistance were induced by high-fat feeding of male C57BL/6Jico mice for 6 weeks. In addition, acute hepatic steatosis in the absence of insulin resistance was induced by pharmacological blockade of beta-oxidation using tetradecylglycidic acid (TDGA). mTOR signalling was examined in liver homogenates., Results: High-fat feeding caused obesity (p<0.001), hepatic steatosis (p<0.05) and hepatic insulin resistance (p<0.05). The phosphorylation of mTOR and its downstream targets p70S6 kinase and S6 ribosomal protein was two-fold higher in mice on a high-fat diet than in mice fed standard chow (all p<0.05) and associated with enhanced rates of protein synthesis. Acute induction of hepatic steatosis with TDGA had no effect on mTOR activity. The increased activity of the mTOR pathway in livers from mice on a high-fat diet could not be ascribed to diet-induced alterations in known modulators of mTOR activity such as circulating plasma leucine levels, phosphorylation of protein kinase B and AMP-activated protein kinase, and changes in mitochondrial function., Conclusions/interpretation: High-fat diet induces increase of the mTOR nutrient sensing pathway in association with hepatic insulin resistance, but not with hepatic lipid accumulation as such.

Introduction: Long-chain fatty acids and glucose are the predominant substrates for cardiac metabolic energy production. While in the healthy heart there is a distinctive and very finely tuned balance between the utilization of these metabolic substrates, in chronic cardiac disease this balance is upset to the use of primarily glucose (e.g., cardiac hypertrophy and failure) or primarily fatty acids (e.g., diabetic cardiomyopathy). Cardiac substrate preference is regulated not only at the level of mitochondrial oxidation (Randle cycle) but also at the level of sarcolemmal uptake of substrates., Molecular Mechanism of Cardiac Substrate Uptake: The latter occurs by translocation of specific substrate transporters, namely fatty acid translocase/CD36 and plasma membrane fatty acid-binding protein (FABPpm) to regulate fatty acid transport, and GLUT4 to regulate glucose transport, from intracellular storage pools to the sarcolemma. Both insulin and cardiac muscle contractions increase the cellular uptake of fatty acids and glucose simultaneously by these mechanisms. Although the signal transduction pathways involved in eliciting substrate transporter trafficking have only partly been disclosed, recent studies indicate the feasibility of selective recruitment of either CD36 or GLUT4 to the sarcolemma, thereby increasing the uptake of a single class of substrates and thus altering the substrate preference of cardiac muscle cells., Concluding Remarks: As a result, selective modulation of the sarcolemmal localization of fatty acid- and/or glucose transporters holds promise as a therapeutic tool to rectify a disruption of the cardiac substrate balance occurring in chronic cardiac disease.

Aims/hypothesis: Diabetic cardiomyopathy (DCM) is common in type 2 diabetes. In DCM, insulin resistance may alter cardiac substrate supply and utilisation leading to changes in myocardial metabolism and cardiac function. In rats, exposure to excessive alimentary fat, inducing a type 2 diabetic phenotype, may result in myocardial insulin resistance and cardiac functional changes resembling DCM., Materials and Methods: Rats received high-fat (HFD) or low-fat (LFD) diets for 7 weeks. Prior to killing, insulin or saline was injected i.p. Contractile function and insulin signalling were assessed in papillary muscles and ventricular lysates, respectively., Results: Fasting and post-load blood glucose levels were increased in HFD- vs LFD-rats (all p < 0.02). Mean heart weight, but not body weight, was increased in HFD-rats (p < 0.01). HFD-hearts showed structural changes and triglyceride accumulation. HFD-muscles developed higher baseline and maximum forces, but showed impaired recovery from higher workloads. Insulin-associated modulation of Ca2+-induced force augmentation was abolished in HFD-muscles. HFD reduced insulin-stimulated IRS1-associated phosphatidylinositol 3'-kinase activity and phosphorylation of protein kinase B, glycogen synthase kinase-3beta, endothelial nitric oxide synthase, and forkhead transcription factors by 40-60% (all p < 0.05). Insulin-mediated phosphorylation of phospholamban, a critical regulator of myocardial contractility, was decreased in HFD-hearts (p < 0.05)., Conclusions/interpretation: HFD induced a hypertrophy-like cardiac phenotype, characterised by a higher basal contractile force, an impaired recovery from increased workloads and decreased insulin-mediated protection against Ca2+ overload. Cardiac dysfunction was associated with myocardial insulin resistance and phospholamban hypophosphorylation. Our data suggest that myocardial insulin resistance, resulting from exposure to excessive alimentary fat, may contribute to the pathogenesis of diabetes-related heart disease.

Synaptosomal-associated protein of 25 kDa (SNAP-25) regulates various membrane fusion processes including exocytosis by endocrine and neural cells. To increase our understanding of the occurrence and regulation of SNAP-25 isoforms, we identified and characterized SNAP-25a and SNAP-25b mRNAs in the pituitary gland and brain of the amphibian Xenopus laevis. The proteins are strongly conserved and are resistant to botulinum neurotoxin A but not to botulinum neurotoxin E, as shown by Western blotting. The spatial distribution of the two SNAP-25 isoforms was assessed with in situ hybridization. Both SNAP-25a mRNA and SNAP-25b mRNA reside in cells in the pituitary distal lobe and, particularly, in the endocrine melanotrope cells in the pituitary intermediate lobe. The melanotrope cells are involved in the background adaptation process of the skin by releasing alpha-melanophore-stimulating hormone. Quantitation of the respective in situ hybridization signals in the Xenopus pars intermedia indicated a differential response, SNAP-25b mRNA being more highly expressed in black-adapted animals than SNAP-25a mRNA, and more than in white-adapted toads. This differential upregulation was also studied by real-time reverse transcriptase polymerase chain reaction, showing that in the intermediate pituitary lobe, both isoforms are physiologically controlled by the background light intensity stimulus, but with different intensities; in black-adapted animals SNAP-25b mRNA is upregulated by 3.33 times compared with white-adapted animals, but SNAP-25a only by 1.96 times. As to neural tissue, in situ hybridization showed that both isoforms coexist throughout the brain, sometimes with similar strengths, but in various areas either SNAP-25a mRNA or SNAP-25b mRNA expression is prevalent. It is speculated that each of the SNAP-25 isoforms in the Xenopus pituitary and brain has a distinct function in cellular fusion processes including secretion, and that their occurrence and regulation depend on the type of secreted neurotransmitter/hormone and/or the activity state of the cell.

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.

Ventricular cardiomyocytes and cardiac tissue of lean and genetically obese (fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85 alpha and p85 beta for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these mechanisms for cardiac insulin resistance. Western blot analysis of cardiomyocyte lysates revealed expression of p85 alpha and p85 beta but no detectable amounts of the splice variants of p85 alpha. Essentially no p85 alpha subunit of PI 3-kinase was found to be associated with insulin receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in p85 beta associated with IRS-1, leading to a three- to fourfold increase in p85 beta-associated PI 3-kinase activity. This response was significantly reduced in obese animals. Comparable results were obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 +/- 0.7-fold over basal) of p85 alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85 beta could be detected in GLUT-4-containing vesicles. Recruitment of the p110 catalytic subunit of PI 3-kinase and a twofold increase in enzyme activity in GLUT-4-containing vesicles by insulin was observed only in lean rats. We conclude that, in the heart, p85 alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85 beta represents the main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation. Furthermore, multiple defects of PI 3-kinase activation, involving both the p85 alpha and the p85 beta adaptor subunits, may contribute to cardiac insulin resistance.

The role of the mammalian target of rapamycin (mTOR) was investigated in insulin responsive cell lines. mTOR was expressed at high levels in insulin responsive cell types and in 3T3-L1 cells mTOR expression levels increased dramatically as cells differentiated from fibroblasts into insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically localized to microsomal membranes. Protein kinase activity directed towards mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was due to an autokinase activity present in mTOR. The mTOR autokinase and the protein kinase activity of the p110 alpha isoform of PI 3-kinase shared several notable similarities; (a) both were maximally active in the presence of Mn2+ but also showed significant activity in the presence of Mg2+ (b) neither were inhibited by the presence of non-ionic detergent and (c) both were inhibited by wortmannin and LY294002, known inhibitors of the PI 3-kinase lipid kinase activity. These data taken together indicate the autokinase activity lay in the PI 3-kinase homology domain. In summary mTOR is a membrane anchored protein kinase that is active in conditions encountered in vivo and the fact it is highly expressed in insulin responsive cell types is consistent with a role in insulin signalling.