Growth factor induced activation of phosphoinositide 3-kinase and protein kinase B (PKB) leads to increased activity of the mammalian target of rapamycin (mTOR). This subsequently leads to increased phosphorylation of eIF4E binding protein-1 (4EBP1) and activation of p70 ribosomal S6 protein kinase (p70(S6K)), both of which are important steps in the stimulation of protein translation. The stimulation of translation is attenuated in cells deprived of amino acids and this is associated with the attenuation of 4EBP1 phosphorylation and p70(S6K) activation. It has been suggested that PKB regulates mTOR function by phosphorylation although direct phosphorylation of mTOR by PKB has not been demonstrated previously. In the present work, we have found that PKB directly phosphorylates mTOR and, using phosphospecific antibodies, we have shown this phosphorylation occurs at Ser(2448). Insulin also induces phosphorylation on Ser(2448) and this effect is blocked by wortmannin but not rapamycin, consistent with the effect being mediated by PKB. Amino-acid starvation rapidly attenuated the reactivity of the Ser(2448) phosphospecific antibody with mTOR and this could not be restored by either insulin stimulation of cells or incubation with PKB in vitro. Our findings demonstrate that mTOR is a direct target for PKB and support the conclusion that regulation of phosphorylation of Ser(2448) is a point of convergence for the counteracting regulatory effects of growth factors and amino acid levels.

Objective: Reduced circulating omentin levels have been reported in obesity and type 2 diabetes, but data were mostly derived from univariate analyses in small study samples. This study aimed to investigate the relationship between omentin, abnormal glucose tolerance and related metabolic factors in a large population-based cross-sectional study. Design and methods: Serum omentin was measured by ELISA in 1092 participants of the German KORA F4 survey (2006)(2007)(2008). Associations between omentin serum levels, glucose tolerance (assessed with an oral glucose tolerance test) and diabetesrelated factors were estimated using logistic and linear regression models respectively. Results: Serum levels of omentin were not related to categories of glucose tolerance. However, serum omentin was positively associated with whole-body insulin sensitivity index (ISI (composite)) and HDL cholesterol and showed inverse associations with 2-h post-load glucose, fasting insulin, homeostasis model assessment-estimated insulin resistance, BMI and triglycerides (all P%0.03 after adjustment for age, sex and lifestyle factors). Further adjustment for BMI and/or serum lipids attenuated the associations with parameters of glucose metabolism, whereas adjustment for serum adiponectin virtually abolished all aforementioned associations. In contrast, adjustment for omentin had no effect on the positive association between adiponectin levels and ISI (composite). Conclusions: The data from this large population-based cohort show that circulating omentin levels are associated with insulin sensitivity. Our observations further suggest that omentin acts via upregulation of adiponectin, which in turn affects lipid metabolism and thereby also indirectly enhances insulin sensitivity, but mechanistic studies are required to corroborate this hypothesis.

Circulating WISP1 levels and WISP1 expression in VAT are increased in obesity independent of glycaemic status. Furthermore, WISP1 impaired insulin signalling in muscle and liver cells.

Testosterone (T) levels are decreased in obese men, but the underlying causes are incompletely understood. Our objective was to explore the relation between low (free) T levels and male obesity, by evaluating metabolic parameters, subcutaneous adipose tissue (SAT) aromatase expression, and parameters of the hypothalamic–pituitary–gonadal axis. We recruited 57 morbidly obese men [33 had type 2 diabetes (DM2)] and 25 normal-weight men undergoing abdominal surgery. Fourteen obese men also attended a follow-up, 2 years after gastric bypass surgery (GBS). Circulating T levels were quantified by LC–MS/MS, whereas free T levels were measured using serum equilibrium dialysis and sex hormone-binding globulin, luteinizing hormone, and follicle-stimulating hormone by immunoassay. SAT biopsies were used to determine adipocyte cell size and aromatase expression by real-time PCR. Total and free T levels were decreased in obese males versus controls, with a further decrease in obese men with DM2 versus obese men without DM2. There were no differences in aromatase expression among the study groups, and sex steroids did not correlate with aromatase expression. Pearson analysis revealed an inverse association between (free) T and SAT cell size, triglycerides, and HOMA-IR. Multivariate analysis confirmed the inverse association between (free) T and SAT cell size (β = −0.321, P = 0.037 and β = −0.441, P = 0.011, respectively), independent of age, triglycerides, HOMA-IR, obesity, or diabetes. T levels were normalized 2 years after GBS. These data suggest that SAT cell size rather than SAT aromatase expression or parameters of the hypothalamic–pituitary–gonadal axis is related to low T in male obesity, which points to adipose cell size-related metabolic changes as a major trigger in decreased T levels.

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.

WNT1 inducible signaling pathway protein 1 (WISP-1/CCN4) is a novel adipokine, which is upregulated in obesity, and induces a pro-inflammatory response in macrophages in-vitro. Preclinical observations suggested WISP-1/CCN4 as a potential candidate for novel obesity therapy targeting adipose tissue inflammation. Whether circulating levels of WISP-1/CCN4 in humans are altered in obesity and/or type 2 diabetes (T2DM) and in the postprandial state, however, is unknown. This study assessed circulating WISP-1/CCN4 levels in a) paired liquid meal tests and hyperinsulinemic- euglycemic clamps (cohort I, n = 26), b) healthy individuals (cohort II, n = 207) and c) individuals with different stages of obesity and glucose tolerance (cohort III, n = 253). Circulating plasma and serum WISP-1/CCN4 concentrations were measured using a commercially available ELISA. WISP-1/CCN4 levels were not influenced by changes in insulin and/or glucose during the tests. In healthy individuals, WISP-1/CCN4 was detectable in 13% of plasma samples with the intraclass correlation coefficient of 0.93 (95% CI: 0.84-0.96) and in 58.1% of the serum samples in cohort III. Circulating WISP-1/CCN4 positively correlated with body mass index, body fat percentage, leptin and triglyceride levels, hip circumference and fatty liver index. No differences in WISP-1/CCN4 levels between individuals with normal glucose tolerance, impaired glucose tolerance and T2DM were found. The circulating concentrations of WISP-1/CCN4 showed no acute regulation in postprandial state and correlated with anthropometrical obesity markers and lipid profiles. In healthy individuals, WISP-1/CCN4 levels are more often below the detection limit. Thus, serum WISP-1/CCN4 levels may be used as a suitable biomarker of obesity.

Secretory products from epicardial adipose tissue (EAT) from patients with type 2 diabetes (T2D) impair cardiomyocyte function. These changes associate with alterations in miRNA expression, including the induction of miR-208a. Recent studies suggest that activation of the cardiac-specific renin-angiotensin system (RAS) may affect cardiac energy metabolism via induction of miR-208a. This study investigated whether cardiomyocyte dysfunction induced by conditioned media (CM) from EAT-T2D involves activation of the RAS/miR-208a pathway. Therefore, primary adult rat cardiomyocytes were incubated with CM generated from EAT biopsies from patients with T2D and without T2D (ND). Exposing cardiomyocytes to CM-EAT-T2D reduced sarcomere shortening and increased miR-208a expression versus cells exposed to CM-EAT-ND or control medium. The angiotensin II receptor type 1 (AGTR1) antagonist losartan reversed these effects. Accordingly, incubation with angiotensin II (Ang II) reduced sarcomere shortening, and lowered palmitate-induced mitochondrial respiration and carnitine palmitoyltransferase 1c (CPT1c) expression in cardiomyocytes. Locked-nucleic-acid-mediated inhibition of miR-208a function reversed the detrimental effects induced by Ang II. Interestingly, Ang II levels in CM-EAT-T2D were increased by 2.6-fold after culture with cardiomyocytes. The paracrine activation of the cardiac-specific RAS by CM-EAT-T2D was corroborated by increases in the expression of AGTR1 and renin, as well as a reduction in angiotensin-converting enzyme 2 levels. Collectively, these data show that secretory products from EAT-T2D impair cardiomyocyte contractile function and mitochondrial β-oxidation via activation of the cardiac-specific RAS system and induction of miR-208a, and suggest that alterations in the secretory profile of EAT may contribute to the development of diabetes-related heart disease.

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