Combined Hyperglycemia and Hyperinsulinemia-induced Insulin Resistance in Adipocytes is associated with Dual Signaling Defects mediated by PKC-ζ

A hyperglycemic and hyperinsulinemic environment characteristic of type 2 diabetes causes insulin resistance. In adipocytes, defects in both insulin sensitivity and maximum response of glucose transport have been demonstrated. To investigate the molecular mechanisms, freshly isolated rat adipocytes were incubated in control (5.6 mM glucose, no insulin) and high glucose (20 mM)/high insulin (100 nM) (HG/HI) for 18 h to induce insulin resistance. Insulin resistant adipocytes manifested decreased sensitivity of glucose uptake associated with defects in IRS-1 Tyr phosphorylation, association of p85 subunit of phosphatidylinositol-3-kinase, AktSer473 and Thr308 phosphorylation accompanied by impaired glucose transporter 4 translocation. In contrast, PKC-ζ activity was augmented by chronic HG/HI. Inhibition of PKC-ζ with a specific cell permeable peptide reversed the signalling defects and insulin sensitivity of glucose uptake. Transfection of dominant-negative kinase-inactive PKC-ζ blocked insulin resistance, while constitutively-active PKC-ζ recapitulated the defects. The HG/HI incubation was associated with stimulation of IRS-1Ser318 and AktThr34 phosphorylation, targets of PKC-ζ. Transfection of IRS-1S318A and AktT34A each partially corrected, while combined transfection of both completely normalized insulin signaling. In vivo hyperglycemia/hyperinsulinemia in rats, for 48h similarly resulted in activation of PKC-ζ and increased phosphorylation of IRS-1 Ser318 and AktThr 34. These data indicate that impairment of insulin signaling by chronic HG/HI is mediated by dual defects at IRS-1 and Akt mediated by PKC-ζ.</p

Curcumin Prevents High Fat Diet Induced Insulin Resistance and Obesity via Attenuating Lipogenesis in Liver and Inflammatory Pathway in Adipocytes

Background: Mechanisms underlying the attenuation of body weight gain and insulin resistance in response to high fat diet (HFD) by the curry compound curcumin need to be further explored. Although the attenuation of the inflammatory pathway is an accepted mechanism, a recent study suggested that curcumin stimulates Wnt signaling pathway and hence suppresses adipogenic differentiation. This is in contrast with the known repressive effect of curcumin on Wnt signaling in other cell lineages. Methodology and Principal Findings: We conducted the examination on low fat diet, or HFD fed C57BL/6J mice with or without curcumin intervention for 28 weeks. Curcumin significantly attenuated the effect of HFD on glucose disposal, body weight/fat gain, as well as the development of insulin resistance. No stimulatory effect on Wnt activation was observed in the mature fat tissue. In addition, curcumin did not stimulate Wnt signaling in vitro in primary rat adipocytes. Furthermore, curcumin inhibited lipogenic gene expression in the liver and blocked the effects of HFD on macrophage infiltration and the inflammatory pathway in the adipose tissue. Conclusions and Significance: We conclude that the beneficial effect of curcumin during HFD consumption is mediated by attenuating lipogenic gene expression in the liver and the inflammatory response in the adipose tissue, in the absence o

The oxidative burst in tomato plants induced by race-specific elicitors of Cladosporium fulvum

grantor: University of TorontoOne of the intriguing features of plant disease resistance is an initial oxidative burst. The present study, using the 'Cladosporium fulvum '-tomato pathosystem, explored the oxidative burst in both leaf tissues and in cell cultures following treatment with race specific elicitors obtained from the fungus. The oxidative burst was detected by 2',7'dichlorofluorescin diacetate (DCFH-DA), a probe for detection of H2O2. A variety of tests, including the use of catalase, showed that the probe measured both intracellular and extracellular H2O2. Ile oxidative burst occurred within 1-2 hr in leaf tissues and within a few minutes in cell cultures, respectively, after elicitor treatment. In cell cultures, the oxidative burst was abolished by inhibitors of NADPH oxidase, suggesting that this enzyme is responsible for the generation of O2-, a precursor of H2O2. As well, O2- was detected 'in planta' by the Mn2+/diaminobenzidine technique. Prevention of increased cytosolic Ca2+ appeared to block the increased H2O2 generation. Studies with other inhibitors suggested that protein kinase(s) and phospholipase C were involved in the oxidative burst, supporting the possibility that the signaling mechanism leading to the oxidative burst in tomato cells is similar to that observed in neutrophils. Activities of antioxidant enzymes, e.g., superoxide dismutase, catalase, and glutathione 'S'-transferase, were not changed in leaves undergoing an oxidative burst. Total peroxidase activity, including ascorbate peroxidase, increased in elicitor-treated leaves but the activity of ascorbate peroxidase decreased by 4 hr. Elicitor-induced necrosis in leaves was significantly delayed, but not completely inhibited, by catalase or by scavengers of active oxygen species (AOS). An oxidative burst was still induced at elicitor dilutions which caused no visible necrosis and only high concentrations (ca 1M) of H2O2 could mimic the visible leaf necrosis induced by elicitor. Growth of 'C. fulvum' germ tubes was inhibited in vitro by moderate levels of H2O2, suggesting a possible role for H2O2 in restricting colonization. A model is proposed which describes the relationship between AOS, generated from an oxidative burst, and the fate of both host cells and invading fungal cells.Ph.D

The oxidative burst in tomato plants induced by race-specific elicitors of Cladosporium fulvum

grantor: University of TorontoOne of the intriguing features of plant disease resistance is an initial oxidative burst. The present study, using the 'Cladosporium fulvum '-tomato pathosystem, explored the oxidative burst in both leaf tissues and in cell cultures following treatment with race specific elicitors obtained from the fungus. The oxidative burst was detected by 2',7'dichlorofluorescin diacetate (DCFH-DA), a probe for detection of H2O2. A variety of tests, including the use of catalase, showed that the probe measured both intracellular and extracellular H2O2. Ile oxidative burst occurred within 1-2 hr in leaf tissues and within a few minutes in cell cultures, respectively, after elicitor treatment. In cell cultures, the oxidative burst was abolished by inhibitors of NADPH oxidase, suggesting that this enzyme is responsible for the generation of O2-, a precursor of H2O2. As well, O2- was detected 'in planta' by the Mn2+/diaminobenzidine technique. Prevention of increased cytosolic Ca2+ appeared to block the increased H2O2 generation. Studies with other inhibitors suggested that protein kinase(s) and phospholipase C were involved in the oxidative burst, supporting the possibility that the signaling mechanism leading to the oxidative burst in tomato cells is similar to that observed in neutrophils. Activities of antioxidant enzymes, e.g., superoxide dismutase, catalase, and glutathione 'S'-transferase, were not changed in leaves undergoing an oxidative burst. Total peroxidase activity, including ascorbate peroxidase, increased in elicitor-treated leaves but the activity of ascorbate peroxidase decreased by 4 hr. Elicitor-induced necrosis in leaves was significantly delayed, but not completely inhibited, by catalase or by scavengers of active oxygen species (AOS). An oxidative burst was still induced at elicitor dilutions which caused no visible necrosis and only high concentrations (ca 1M) of H2O2 could mimic the visible leaf necrosis induced by elicitor. Growth of 'C. fulvum' germ tubes was inhibited in vitro by moderate levels of H2O2, suggesting a possible role for H2O2 in restricting colonization. A model is proposed which describes the relationship between AOS, generated from an oxidative burst, and the fate of both host cells and invading fungal cells.Ph.D

The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism

Adaptor proteins link surface receptors to intracellular signaling pathways and potentially control the way cells respond to nutrient availability. Mice deficient in p66Shc, the most recently evolved isoform of the Shc1 adaptor proteins and a mediator of receptor tyrosine kinase signaling, display resistance to diabetes and obesity. Using quantitative mass spectrometry, we found that p66Shc inhibited glucose metabolism. Depletion of p66Shc enhanced glycolysis and increased the allocation of glucose-derived carbon into anabolic metabolism, characteristics of a metabolic shift called the Warburg effect. This change in metabolism was mediated by the mammalian target of rapamycin (mTOR) because inhibition of mTOR with rapamycin reversed the glycolytic phenotype caused by p66Shc deficiency. Thus, unlike the other isoforms of Shc1, p66Shc appears to antagonize insulin and mTOR signaling, which limits glucose uptake and metabolism. Our results identify a critical inhibitory role for p66Shc in anabolic metabolism.National Institutes of Health (U.S.)United States. Dept. of DefenseW. M. Keck FoundationLAM FoundationNational Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award)National Institutes of Health (U.S.) (K99 Award