CBF1 is clinically prognostic and serves as a target to block cellular invasion and chemoresistance of EMT-like glioblastoma cells.

BACKGROUND: Glioblastoma is the most common and most lethal primary brain cancer. CBF1 (also known as Recombination signal Binding Protein for immunoglobulin kappa J, RBPJ) is the cardinal transcriptional regulator of the Notch signalling network and has been shown to promote cancer stem-like cells (CSCs) in glioblastoma. Recent studies suggest that some of the malignant properties of CSCs are mediated through the activation of pro-invasive programme of epithelial-to-mesenchymal transition (EMT). Little is known whether CBF1 is involved in the EMT-like phenotype of glioma cells. METHODS: In a collection of GBM neurosphere lines, we genetically inhibited CBF1 and investigated the consequences on EMT-related properties, including in vitro invasiveness by Boyden chambers assay, chemoresistance using a clinical drug library screen and glycolytic metabolism assessing live-cell extracellular acidification rate. We also compared CBF1 expression in cells exposed to low and high oxygen tension. In silico analysis in large-scale Western and Eastern patient cohorts investigated the clinical prognostic value of CBF1 expression in low- and high-grade glioma as well as medulloblastoma. RESULTS: Mean CBF1 expression is significantly increased in isocitrate dehydrogenase 1 (IDH1) R132H mutant glioblastoma and serves as prognostic marker for prolonged overall survival in brain tumours, particularly after therapy with temozolomide. Hypoxic regions of glioblastoma have higher CBF1 activation and exposure to low oxygen can induce its expression in glioma cells in vitro. CBF1 inhibition blocks EMT activators such as zinc finger E-box-binding homeobox 1 (ZEB1) and significantly reduces cellular invasion and resistance to clinically approved anticancer drugs. Moreover, we indicate that CBF1 inhibition can impede cellular glycolysis. CONCLUSIONS: Mean CBF1 activation in bulk tumour samples serves as a clinical predictive biomarker in brain cancers but its intratumoral and intertumoral expression is highly heterogeneous. Microenvironmental changes such as hypoxia can stimulate the activation of CBF1 in glioblastoma. CBF1 blockade can suppress glioblastoma invasion in vitro in particular in cells undergone EMT such as those found in the hypoxic niche. Targeting CBF1 can be an effective anti-EMT therapy to impede invasive properties and chemosensitivity in those cells.

Low serum omentin levels in the elderly population with Type 2 diabetes and polyneuropathy.

AIMS: To investigate the hypothesis that high serum levels of omentin, an adipokine with anti-inflammatory, insulin-sensitizing and cardioprotective properties, may be related to a lower risk of diabetic sensorimotor polyneuropathy. METHODS: The association between serum omentin level and polyneuropathy was estimated in people aged 61-82 years with Type 2 diabetes (47 with and 168 without polyneuropathy) from the population-based KORA F4 study. The presence of clinical diabetic sensorimotor polyneuropathy was defined as bilateral impairment of foot vibration perception and/or foot pressure sensation. Omentin levels were determined by enzyme-linked immunosorbent assay. RESULTS: Serum omentin level was inversely associated with polyneuropathy after adjustment for age, sex, height, waist circumference, hypertension, total cholesterol, smoking, alcohol intake and physical activity [odds ratio 0.45 (95% CI 0.21-0.98); P = 0.043]. Although omentin was positively correlated with adiponectin (r = 0.55, P < 0.0001) and inversely with tumour necrosis factor-α (r = -0.30, P = 0.019), additional adjustment for adiponectin and tumour necrosis factor-α had little impact on the association. CONCLUSIONS: Serum levels of omentin are reduced in people with Type 2 diabetes and diabetic sensorimotor polyneuropathy, independently of established risk factors of polyneuropathy. This association is only partially explained by biomarkers of subclinical inflammation.

Knockdown of PRAS40 inhibits insulin action via proteasome-mediated degradation of IRS1 in primary human skeletal muscle cells.

AIMS/HYPOTHESIS: The proline-rich Akt substrate of 40 kDa (PRAS40) is a component of the mammalian target of rapamycin complex 1 (mTORC1) and among the most prominent Akt substrates in skeletal muscle. Yet the cellular functions of PRAS40 are incompletely defined. This study assessed the function of PRAS40 in insulin action in primary human skeletal muscle cells (hSkMC). METHODS: Insulin action was examined in hSkMC following small interfering RNA-mediated silencing of PRAS40 (also known as AKT1S1) under normal conditions and following chemokine-induced insulin resistance. RESULTS: PRAS40 knockdown (PRAS40-KD) in hSkMC decreased insulin-mediated phosphorylation of Akt by 50% (p < 0.05) as well as of the Akt substrates glycogen synthase kinase 3 (40%) and tuberous sclerosis complex 2 (32%) (both p < 0.05). Furthermore, insulin-stimulated glucose uptake was reduced by 20% in PRAS40-KD myotubes (p < 0.05). Exposing PRAS40-KD myotubes to chemokines caused no additional deterioration of insulin action. PRAS40-KD further reduced insulin-mediated phosphorylation of the mTORC1-regulated proteins p70S6 kinase (p70S6K) (47%), S6 (43%), and eukaryotic elongation 4E-binding protein 1 (100%), as well as protein levels of growth factor receptor bound protein 10 (35%) (all p < 0.05). The inhibition of insulin action in PRAS40-KD myotubes was associated with a reduction in IRS1 protein levels (60%) (p < 0.05), and was reversed by pharmacological proteasome inhibition. Accordingly, expression of the genes encoding E3-ligases F-box protein 32 (also known as atrogin-1) and muscle RING-finger protein-1 and activity of the proteasome was elevated in PRAS40-KD myotubes. CONCLUSIONS/INTERPRETATION: Inhibition of insulin action in PRAS40-KD myotubes was found to associate with IRS1 degradation promoted by increased proteasome activity rather than hyperactivation of the p70S6K-negative-feedback loop. These findings identify PRAS40 as a modulator of insulin action.

Anti-inflammatory cytokines and risk of type 2 diabetes.

Proinflammatory processes have been investigated extensively in the development of type 2 diabetes, but our knowledge on anti-inflammatory proteins is rather limited. This article summarizes studies that investigated associations between circulating levels of anti-inflammatory cytokines and incident type 2 diabetes preferably in prospective epidemiological studies. Adiponectin is the only known anti-inflammatory protein whose circulating levels are decreased before type 2 diabetes. In contrast, concentrations of interleukin-1 receptor antagonist (IL-1RA), transforming growth factor-ß1 (TGF-ß1) and growth differentiation factor-15 (GDF-15) are increased and indicate the presence of a compensatory, but eventually futile, counter-regulation of proinflammatory stimuli. Importantly, a proof-of-principle study using recombinant IL-1RA to improve metabolic control in patients with type 2 diabetes demonstrated that a more pronounced upregulation of this protein than that found in the natural course of diabetes development may have clinical relevance. Other interesting candidates like omentin (which shows similar associations with metabolic parameters as adiponectin), interleukin-10 (IL-10) and secreted frizzled-related protein-5 (Sfrp5) are currently less well studied with sometimes conflicting results regarding their association with type 2 diabetes. Thus, further research is required to better understand the causal role of proinflammatory cytokines, hypoadiponectinaemia and the upregulation of anti-inflammatory proteins before the onset of type 2 diabetes.

Targeting of mitochondrial reactive oxygen species production does not avert lipid-induced insulin resistance in muscle tissue from mice.

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.

Novel insights into glucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options?

At pharmacological concentrations, glucocorticoids (GCs) display potent anti-inflammatory effects, and are therefore frequently prescribed by physicians to treat a wide variety of diseases. Despite excellent efficacy, GC therapy is hampered by their notorious metabolic side effect profile. Chronic exposure to increased levels of circulating GCs is associated with central adiposity, dyslipidaemia, skeletal muscle wasting, insulin resistance, glucose intolerance and overt diabetes. Remarkably, many of these side-effects of GC treatment resemble the various components of the metabolic syndrome (MetS), in which indeed subtle disturbances in the hypothalamic-pituitary-adrenal (HPA) axis and/or increased tissue sensitivity to GCs have been reported. Recent developments have led to renewed interest in the mechanisms of GC's diabetogenic effects. First, 'selective dissociating glucocorticoid receptor (GR) ligands', which aim to segregate GC's anti-inflammatory and metabolic actions, are currently being developed. Second, at present, selective 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibitors, which may reduce local GC concentrations by inhibiting cortisone to cortisol conversion, are evaluated in clinical trials as a novel treatment modality for the MetS. In this review, we provide an update of the current knowledge on the mechanisms that underlie GC-induced dysmetabolic effects. In particular, recent progress in research into the role of GCs in the pathogenesis of insulin resistance and beta-cell dysfunction will be discussed.

Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.

Myocardial insulin action and the contribution of insulin resistance to the pathogenesis of diabetic cardiomyopathy.

Heart disease is the leading cause of death in patients with insulin resistance and type 2 diabetes (DM2). Even in the absence of coronary artery disease and hypertension, functional and structural abnormalities exist in patients with well-controlled and uncomplicated DM2. These derangements are collectively designated by the term diabetic cardiomyopathy (DCM). Changes in myocardial energy metabolism, due to altered substrate supply and utilization, largely underlie the development of DCM. Insulin is an important regulator of myocardial substrate metabolism, but also exerts regulatory effects on intracellular Ca2+ handling and cell survival. The current paper reviews the multiple functional and molecular effects of insulin on the heart, all of which ultimately seem to be cardioprotective both under normal conditions and under ischemia. In particular, the dismal consequences of myocardial insulin resistance contributing to the development of DCM will be discussed.

Dynamics of insulin signalling in liver during hyperinsulinemic euglycaemic clamp conditions in vivo and the effects of high-fat feeding in male mice.

Insulin is an important regulator of hepatic carbohydrate, lipid, and protein metabolism, and the regulation of these processes by insulin is disturbed under conditions of insulin resistance and type 2 diabetes. Despite these alterations, the impact of insulin resistance on insulin signalling in the liver is not well defined. Variations in time and dose of insulin stimulation as well as plasma glucose levels may underlie this. The present study aimed at determining the dynamics of activation of hepatic insulin signalling in vivo at insulin concentrations resembling those achieved after a meal, and addressing the effects of high-fat feeding. An unexpected finding of this study was the biphasic activation pattern of the IRS-PI3K-PKB/Akt pathway. Our findings indicate that the first burst of activation contributes to regulation of glucose metabolism. The physiological function of the second peak is still unknown, but may involve regulation of protein synthesis. Finally, high-fat feeding caused hepatic insulin resistance, as illustrated by a reduced suppression of hepatic glucose production. A sustained increased phosphorylation of the serine/threonine kinases p70S6kinase and Jun N-terminal kinase in the absence of insulin may underlie the abrogated phosphorylation of the IRS proteins and their downstream targets.

Hyperosmotic stress activates the insulin receptor in CHO cells.

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.

Modulation of insulin-stimulated glycogen synthesis by Src Homology Phosphatase 2.

We have examined the requirement of the protein tyrosine phosphatase Src Homology Phosphatase 2 (SHP2) for insulin-stimulated glycogen synthesis. To this end, 3T3L1 fibroblasts were stably transfected with either wild type or a catalytically inactive C463A-mutant of SHP2, and analysed for insulin-induced glycogen synthesis, tyrosine phosphorylation of the insulin receptor and IRS-1, and activation of phosphatidylinositol 3'-kinase (PI 3'-kinase). Glycogen synthesis was stimulated 9.1+/-0.9-fold by insulin in untransfected cells. In cells expressing the dominant-negative C463A-SHP2 mutant, the stimulation of glycogen synthesis by insulin was strongly enhanced (18.7+/-2.7-fold stimulation), while this response was impaired in cells overexpressing wild-type SHP2 (6.6+/-1.1-fold stimulation). When exploring the early post-receptor signalling pathways that contribute to glycogen synthesis, we found that insulin stimulated the tyrosine phosphorylation of IRS-1, and the activation of IRS-1-associated PI 3'-kinase more strongly in C463A-SHP2 expressing 3T3L1-cells (18.1+/-4.7-fold) than in parental 3T3L1 cells (6.8+/-0.5-fold). In 3T3L1 cells overexpressing wild-type SHP2, the insulin stimulation of IRS-1 tyrosine phosphorylation and the activation of PI 3'-kinase (4.5+/-1.0-fold) were impaired. An enhanced activity of SHP2 leads to negative modulation of insulin signalling by reducing the tyrosine phosphorylation of IRS-1 and the concomitant activation of PI 3'-kinase. This results in an impaired ability of insulin to stimulate glycogen synthesis.

The insulin sensitizing effect of topiramate involves KATP channel activation in the central nervous system.

BACKGROUND AND PURPOSE: Topiramate improves insulin sensitivity, in addition to its antiepileptic action. However, the underlying mechanism is unknown. Therefore, the present study was aimed at investigating the mechanism of the insulin-sensitizing effect of topiramate both in vivo and in vitro. EXPERIMENTAL APPROACH: Male C57Bl/6J mice were fed a run-in high-fat diet for 6 weeks, before receiving topiramate or vehicle mixed in high-fat diet for an additional 6 weeks. Insulin sensitivity was assessed by hyperinsulinaemic-euglycaemic clamp. The extent to which the insulin sensitizing effects of topiramate were mediated through the CNS were determined by concomitant i.c.v. infusion of vehicle or tolbutamide, an inhibitor of ATP-sensitive potassium channels in neurons. The direct effects of topiramate on insulin signalling and glucose uptake were assessed in vivo and in cultured muscle cells. KEY RESULTS: In hyperinsulinaemic-euglycaemic clamp conditions, therapeutic plasma concentrations of topiramate (∼4 µg·mL(-1) ) improved insulin sensitivity (glucose infusion rate + 58%). Using 2-deoxy-D-[(3) H]glucose, we established that topiramate improved the insulin-mediated glucose uptake by heart (+92%), muscle (+116%) and adipose tissue (+586%). Upon i.c.v. tolbutamide, the insulin-sensitizing effect of topiramate was completely abrogated. Topiramate did not directly affect glucose uptake or insulin signalling neither in vivo nor in cultured muscle cells. CONCLUSION AND IMPLICATIONS: In conclusion, topiramate stimulates insulin-mediated glucose uptake in vivo through the CNS. These observations illustrate the possibility of pharmacological modulation of peripheral insulin resistance through a target in the CNS.

The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the alpha2-adrenergic receptors in beta cells.

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.

Loss of 50% of excess weight using a very low energy diet improves insulin-stimulated glucose disposal and skeletal muscle insulin signalling in obese insulin-treated type 2 diabetic patients.

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.

Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification.

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.

Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice.

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.

Cardiac dysfunction induced by high-fat diet is associated with altered myocardial insulin signalling in rats.

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.

Molecular mechanisms of contraction-regulated cardiac glucose transport.

Insulin and contraction are the most important regulators of glucose utilization in cardiac muscle. In contrast with insulin, the intracellular signalling elements of contraction have remained unexplored. In the present studies, adult rat ventricular cardiomyocytes were electrically stimulated to perform rhythmic contractions to permit the determination of potential sites of convergence of contraction and insulin signalling to glucose transport. The participation of phosphoinositide 3-kinase (PI-3K) in Ca(2+)- and contraction-stimulated 3-O-methylglucose transport was suggested by the great sensitivity of this process towards the PI-3K inhibitors wortmannin and LY294002 and by the presence of PI-3K activity in anti-phosphotyrosine immunoprecipitates from contracted cells. Initial signalling events of insulin action, including receptor kinase activation, the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 and the recruitment of PI-3K to IRS-1 and IRS-2, were found not to be involved in contraction-mediated signalling. However, immunoprecipitation of p85alpha revealed a markedly enhanced tyrosine phosphorylation of an unknown co-precipitated 200 kDa protein in response to both stimuli. It is concluded that contraction-regulated cardiac glucose transport involves the activation of PI-3K in response to upstream signalling pathways different from that of insulin.

Diversification of cardiac insulin signaling involves the p85 alpha/beta subunits of phosphatidylinositol 3-kinase.

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.

Replacement of the conserved tyrosine 1210 by phenylalanine in the insulin receptor affects insulin-induced dephosphorylation of focal adhesion kinase but leaves other responses intact.

The families of tyrosine and serine/threonine kinases exhibit shared clusters of conserved amino acid residues. Some conserved residues are confined to the family of tyrosine kinases (TKs), like Tyr at position 1210 in the insulin receptor. Nearly all TKs have at this position Tyr, whereas Ser/Thr kinases generally have Phe at this site. The three-dimensional structure of the insulin receptor TK domain shows Tyr1210 to be located in the cleft, below bound ATP, in a region which potentially contributes to substrate binding. We have examined whether this specific Tyr residue contributes to the generation of TK-specific responses, such as Tyr phosphorylation of Shc, activation of Ras and Erk1,2, and stimulation of DNA synthesis. In addition, we have examined the contribution of Tyr1210 to insulin receptor-specific responses as Tyr phosphorylation of IRS1, stimulation of glycogen synthesis, and dephosphorylation of focal adhesion kinase (FAK). Wild-type and a mutant insulin receptor, in which Tyr1210 was replaced by Phe, were stably expressed in CHO cells, and clones expressing similar numbers of insulin receptors were selected. It was found that replacement of Tyr1210 by Phe resulted in a receptor which was nearly inactive in inducing dephosphorylation of FAK. The mutant receptor was able to induce RasGTP formation, glycogen synthesis, and activation of phosphatidylinositol 3-kinase, though the magnitude of stimulation of some responses was decreased. These findings indicate that Tyr1210 is not essential for the induction of tyrosine kinase-specific responses, such as activation of the Shc/Ras/Erk1,2 pathway and mitogenicity. On the other hand, the abrogation of insulin-induced FAK dephosphorylation indicates that Tyr1210 is involved in coupling of the activated receptor to some downstream targets. Thus, Tyr1210 may fine tune the signal generated by the activated insulin receptor.