Modulation der Insulinsignalübertragung durch Phosphorylierung von Serin-357 imInsulin-Rezeptor-Substrat-1

The activation of the protein kinase C (PKC) family of serine/threonine kinases contributes to the modulation of insulin signaling and the PKC-dependent phosphorylation of IRS-1 has been implicated in the development of insulin resistance. Here we demonstrate Ser-357 of rat IRS-1 as a novel PKC-delta-dependent phosphorylation site in skeletal muscle cells upon stimulation with insulin and phorbol ester using phospho-Ser-357 antibodies and active and kinase dead mutant of PKC-delta. Phosphorylation of this site was simulated using IRS-1 Glu357 and shown to reduce insulin-induced tyrosine phosphorylation of IRS-1, to decrease activation of Akt and subsequently to diminish phosphorylation of GSK-3. When the phosphorylation was prevented by mutation of Ser-357 to alanine, these effects of insulin were enhanced. When the adjacent Ser-358, present in mouse and rat IRS-1, was mutated to alanine, which is homologous to the human sequence, the insulin-induced phosphorylation of GSK-3 or tyrosine phosphorylation of IRS-1 was not increased. Moreover, both, active PKC-delta and phosphorylation of Ser-357 were shown to be necessary for the attenuation of insulinstimulated Akt phosphorylation. The phosphorylation of Ser-357 could lead to increased association of PKC-delta to IRS-1 upon insulin stimulation, which was demonstrated with IRS-1 Glu357. Together, these data suggest that phosphorylation of Ser-357 mediates at least in part the adverse effects of PKC-delta activation on insulin action.Die Isoenzyme der Proteinkinase C (PKC), einer Familie von Serin-/Threoninkinasen, sind an der Modulation der Insulinsignalübertragung beteiligt. Die PKC-abhängige Phosphorylierung von Insulinrezeptor-Substrat (IRS)-1 wird im Zusammenhang mit der Entwicklung der peripheren Insulinresistenz diskutiert. In vorliegender Arbeit konnte erstmals gezeigt werden, dass Serin-357 von IRS-1 (Ratte) ein PKC-delta-abhängige Phosphorylierungsstelle in Skelettmuskelzellen ist. Der Beweis wurde mit einem selbst hergestellten Phospho-Ser-357-spezifischen Antikörper in Insulin oder Phorbolester stimulierten Zellen unter Verwendung einer Kinase-inaktiven PKC-delta geführt. Weiterhin konnte durch Simulierung einer Phosphorylierung an Ser-357 durch eine IRS-1 Glu-357-Mutante unter anderem gezeigt werden, dass es hierdurch zu einer Reduktion der Tyrosinphosphorylierung von IRS-1 kommt, sowie die Aktivierung von Akt vermindert ist und in der Folge auch die Phosphorylierung von GSK-3 abgeschwächt ist. Demgegenüber werden diese Insulin-vermittelten Effekte verstärkt wenn die Phosphorylierung durch eine Punktmutation von Ser-357 zu Alanin verhindert wird. Eine potentielle funktionelle Rolle des direkt benachbarten Ser-358 konnte durch eine Mutation dieses Restes zu Alanin ausgeschlossen werden. Außerdem konnte gezeigt werden, dass sowohl die aktive PKC-delta als auch die Phosphorylierung von Ser-357 für die Abschwächung der insulinabhängigen Phosphorylierung von Akt notwendig sind. Eine erhöhte Assoziation von PKC-delta und IRS-1 nach Insulinstimulation konnte ebenfalls durch Simulierung der Ser-357 Phosphorylierung mit der IRS-1 Glu-357-Mutanten gezeigt werden. Zusammenfassend zeigen diese Daten, dass die Phosphorylierung von Serin 357 in IRS-1, mindestens zum Teil, die negative Wirkung der PKC-delta-Aktivierung auf die Insulinsignalübertragung vermittelt

Gendering flood early warning systems:the case of Pakistan

After the devastating 2010 flood in Pakistan, an early warning system (EWS) for river floods has been established in the Lai basin passing through the twin cities of Islamabad and Rawalpindi. Inequalities in society are amplified at the time of disasters, and EWS that are people-centred proved more effective in communicating risk and saving people. This article undertakes a gender analysis of Pakistan's EWS for each of the four pillars of people-centred EWS in order to highlight gendered and classed vulnerabilities to flood. Focus group discussions and key informant interviews were conducted with members of relevant institutions and communities in four neighbourhoods across the length of the Lai basin for understanding how gendered vulnerability impacts the acquisition of the risk messages, how congruent are the risk messages in EWS with gendered risk perception, and to what extent formal EWS enable or hinder behavioural responses to the risk messages. The EWS in the capital of Pakistan comes up short on all the criteria for a people-centred gender-sensitive EWS. Technocratic approach, lack of citizens' involvement and communication gap between the official jargonistic early warning messages and communities at risk are the major obstacles. Despite the establishment of ad hoc cells for addressing gender issues, gender is hardly operationalised and does not go beyond a token recognition

Developmental androgen excess programs sympathetic tone and adipose tissue dysfunction and predisposes to a cardiometabolic syndrome in female mice.

International audienceAmong women, the polycystic ovarian syndrome (PCOS) is considered a form of metabolic syndrome with reproductive abnormalities. Women with PCOS show increased sympathetic tone, visceral adiposity with enlarged adipocytes, hypoadiponectinemia, insulin resistance, glucose intolerance, increased inactive osteocalcin, and hypertension. Excess fetal exposure to androgens has been hypothesized to play a role in the pathogenesis of PCOS. Previously, we showed that neonatal exposure to the androgen testosterone (NT) programs leptin resistance in adult female mice. Here, we studied the impact of NT on lean and adipose tissues, sympathetic tone in cardiometabolic tissues, and the development of metabolic dysfunction in mice. Neonatally androgenized adult female mice (NTF) displayed masculinization of lean tissues with increased cardiac and skeletal muscle as well as kidney masses. NTF mice showed increased and dysfunctional white adipose tissue with increased sympathetic tone in both visceral and subcutaneous fat as well as increased number of enlarged and insulin-resistant adipocytes that displayed altered expression of developmental genes and hypoadiponectinemia. NTF exhibited dysfunctional brown adipose tissue with increased mass and decreased energy expenditure. They also displayed decreased undercarboxylated and active osteocalcin and were predisposed to obesity during chronic androgen excess. NTF showed increased renal sympathetic tone associated with increased blood pressure, and they developed glucose intolerance and insulin resistance. Thus, developmental exposure to testosterone in female mice programs features of cardiometabolic dysfunction, as can be observed in women with PCOS, including increased sympathetic tone, visceral adiposity, insulin resistance, prediabetes, and hypertension

Cucurbitacin E reduces obesity and related metabolic dysfunction in mice by targeting JAK-STAT5 signaling pathway

<div><p>Several members of cucurbitaceae family have been reported to regulate growth of cancer by interfering with STAT3 signaling. In the present study, we investigated the unique role and molecular mechanism of cucurbitacins (Cucs) in reducing symptoms of metabolic syndrome in mice. Cucurbitacin E (CuE) was found to reduce adipogenesis in murine adipocytes. CuE treatment diminished hypertrophy of adipocytes, visceral obesity and lipogenesis gene expression in diet induced mice model of metabolic syndrome (MetS). CuE also ameliorated adipose tissue dysfunction by reducing hyperleptinemia and TNF-alpha levels and enhancing hypoadiponectinemia. Results show that CuE mediated these effects by attenuating Jenus kinase- Signal transducer and activator of transcription 5 (JAK- STAT5) signaling in visceral fat tissue. As a result, CuE treatment also reduced PPAR gamma expression. Glucose uptake enhanced in adipocytes after stimulation with CuE and insulin resistance diminished in mice treated with CuE, as reflected by reduced glucose intolerance and glucose stimulated insulin secretion. CuE restored insulin sensitivity indirectly by inhibiting JAK phosphorylation and improving AMPK activity. Consequently, insulin signaling was up-regulated in mice muscle. As CuE positively regulated adipose tissue function and suppressed visceral obesity, dyslipedemia, hyperglycemia and insulin resistance in mice model of MetS, we suggest that CuE can be used as novel approach to treat metabolic diseases.</p></div

Structure of cucurbitacins.

<p>Structure of cucurbitacins.</p

Determination of the effect of CuE on insulin signaling.

<p>(A) Total protein from skeletal muscle of all mice groups was separated on 7.5% SDS-PAGE gels, and immunoblotted with either phospho IRS-1 serine 307 or phospho AKT serine 473 or phospho-AMPK-Thr 172 or phospho JAK-tyrosine1007/1008 antibody. (B) The levels of phosphorylation in the immunoblots were quantified using densitometry and normalized to their respective total proteins expression. The data are presented as mean ± SEMs, n = 5–6, *P < 0.05 mice treated with CuE or Orlistat vs HFD-MetS mice.</p

Serum lipid levels in experimental mice.

<p>Serum lipid levels in experimental mice.</p