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

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

Structure of cucurbitacins.

<p>Structure of cucurbitacins.</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

Serum lipid levels in experimental mice.

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

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

Effect of cucurbitacin E on body fat content

<p>(A) Comparative weekly body weight gain trends in SD, HFD, HFD and CuE (L) 0.25mg/kg, HFD and CuE (H) 0.5mg/kg and HFD+Orlistat mice for 10 weeks (n = 12–15). (B) Proportion of perigonadal, (C) perirenal (D), mesenteric (E) and subcutaneous fat pads weights. (F) Proportion of total body fat content.(G) Visceral fat index. n = 10–12 in each group, results represent means ± SEMs. *P < 0.05, mice treated with CuE or Orlistat vs HFD-MetS mice.</p