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

Structure of cucurbitacins.

<p>Structure of cucurbitacins.</p

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

Serum lipid levels in experimental mice.

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

Effect of CuE on adipose tissue morphology and function.

<p>(A) H and E stained tissue of perigonadal fat from (I) SD (II) HFD (III) HFD+CuE (L) (IV) HFD+CuE (H); and (V) HFD+Orlistat mice. (B-D) Lipogenic gene expression of SREBP, FASN and ACACA genes was measured in visceral fat tissue, by quantitative PCR. (E) Serum adiponectin concentration. (F) Serum leptin levels were measured by ELISA. (G-I) Macrophage infiltration and recruitment gene expression of CD11b, MCP-1 and CCR2 genes was measured in visceral fat tissue, by quantitative PCR. *P < 0.05 HFD-MetS mice treated with CuE or Orlistat vs HFD mice model of MetS. Results are mean ± S.E. (n = 12–15). (J) Serum TNF-alpha concentration.</p

Determination of the effect of CuE on JAK-STAT signaling.

<p>(A) Total protein from abdominal fat of all mice groups was separated on 7.5% SDS-PAGE gels, and immunoblotted with a phospho-tyrosine1007/1008 antibody. The same blots were stripped and reprobed with a polyclonal JAK-2 protein antibody. The levels of tyrosine phosphorylation of JAK-2 in the immunoblots were quantified using densitometry and normalized to the JAK-2 protein. (B) Phospho STAT5A tyrosine 694 antibody was used for immunoblotting STAT5A phosphorylation. (C) PPAR-gamma antibody was used to measure expression of PPAR-gamma. The data are presented as mean ± SEMs, n = 5–6, *P < 0.05 mice treated with CuE or Orlistat vs HFD-MetS mice.</p

Effect of cucurbitacins on adipogenesis.

<p>(A) Oil red O staining in 3T3-L1 preadipocytes differentiated into adipocytes and treated with different concentrations of cucurbitacins. GM, growth media, DM, differentiation media. (B) Quantification of cellular TG content. Cellular triglyceride content is relative to cells treated with DM alone (deemed 100%). n = 4–5 independent experiments, results represent mean ± SEMs. *P < 0.05, cucurbitacins vs. cells treated with DM alone. (C) Glucose uptake in adipocytes. Differentiated 3T3-L1 adipocytes were incubated with 20ng/ml TNF-alpha and CuE for 24 hours followed by stimulation with 10nM insulin for 1 hour. Results are mean ± SEMs of five experiments, *P < 0.05. cucurbitacins vs. cells treated with TNF-alpha alone.</p

CuE treatment improved insulin resistance in mice.

<p>(A) Measurement of blood glucose levels in mice when challenged with intraperitoneal glucose tolerance test (IP-GTT). Area under the curve (AUC) quantification for GTT. (B) Measurement of blood insulin levels during IP-GTT. Area under the curve (AUC) quantification for glucose stimulated insulin secretion (GSIS). Results represent mean ± SEMs. n = 10–12 in each group, *P < 0.05, mice treated with CuE or Orlistat vs HFD mice model of MetS.</p