Role For Erythropoietin On Insulin Signaling Of Palmitate Treated HepG2 Cells

Objective To investigate the role for erythropoietin (EPO) on hepatic insulin signaling of palmitate (PA) induced HepG2 cells and high fat-diet induced C57BL/6mice.Methods HepG2 cells were intervened with palmitic acid (250 μ mmol/L), EPO (10U) and resveratrol (Rsv,10μ mmol/L), activator of SIRT1 (Nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1), respectively. Cells were randomly assigned into:Control, insulin (Ins, 100nM), palmitic acid (PA), PA+EPO (10U) and PA+resveratrol (10 μ M), respectively. Then HepG2 cells under both normal and palmitic acid were transfected with SIRT1 RNAi mixed with lipofacfectamine2000 and were divided into following four groups:Control, EPO, EPO+Scramble, EPO +siSIRT1. Independent samples t- test was used for data analysis. The expression of insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase/ protein kinase B(PI3K/AKT) and SIRT1 were detected by Western blot. The expression of SIRT1 mRNA level was determined by qRT-PCR. Male C57BL/6 mice were fed with high-fat diet for 12 weeks and then treated with EPO (2000 IU/kg) or vehicle saline for three weeks. Protein levels of hepatic insulin signaling were determined using western blot; Oil O Red were used to evaluate the lipid content of livers.Results Compared with control group, levels of pIRS-2(Ser731) increased and pAkt(Ser473),SIRT1 decreased significantly in palmitate induced HepG2 cells (1.25±0.06 vs 1.00±0.03、0.77±0.02 vs 1.00±0.03、0.74±0.03 vs 1.00±0.03,t=6.048、 -13.686、-10.649). After EPO treatment, levels of pAkt(Ser473) and SIRT1 under insulin resistance conditions were significantly enhanced while pIRS-2(Ser731) were significantly decreased (1.60±0.14 vs 1.00±0.08、1.39±0.03 vs 1.00±0.03,0.70±0.03 vs 1.00±0.11,t=6.330、25.868、-4.853, all P<0.05). However, when SIRT1 expression was inhibited by small interfering RNA (SiRNA), neither PI3Kp85 nor pAKT(Ser473) levels were altered in response to EPO under both normal and insulin resistant conditions. In animal experiments, SIRT1、PI3Kp85、pAKT(Ser473) decreased significantly after 12-weeks of high-fat diet (0.63±0.13 vs 1.00±0.05,0.64±0.03 vs 1.00 ±0.02,0.29±0.01 vs 1.00±0.04, respectively, pIRS-2(Ser731) significantly increased (2.65±0.29 vs 1.00±0.05). EPO intervention could significantly increase SIRT1、PI3Kp85. pAKT(Ser473) levels (0.97±0.04 vs 0.63±0.13,0.96±0.02 vs 0.64±0.03、0.68±0.01 vs 0.29±0.01, F=19.100,287.482,504.096, respectively, all P<0.05)and decrease pIRS-2(Ser731) levels (1.02±0.03 vs 2.65±0.29, F=92.198, all P<0.05).Conclusions EPO positively regulates impaired PI3K/Akt signaling caused by PA through SIRT1 in HepG2 cells as well as hepatic insulin signaling induced by high fat-diet in C57BL/6 mice.Background and aimsPigment epithelium-derived factor (PEDF) was recently found to be closely associated with metabolic syndrome and insulin resistance. However, there is a lack of evidence quantitating PEDF levels in different glucose tolerance state and whether metformin treatment could influence it remains unknown. We aimed to investigate serum PEDF levels in subjects with different glucose tolerance and the effect of metformin treatment on PEDF levels in type 2 diabetic patients.Materials and methodsCirculating PEDF levels and metabolic profiles were assessed in 517 Chinese participants with normal glucose tolerance (NGT), impaired glucose regulation (IGR) and type 2 diabetes mellitus (T2DM). PEDF levels after 24-weeks metformin treatment in 63 patients with T2DM were also analyzed. Serum estradiol levels were determined in females. The study protocol was approved by the Ethics Committee of Drum Tower Hospital and written informed consent was obtained from all study participants.ResultsSerum PEDF level was significantly higher in subjects with IGR [9.51 (7.6-11.4) ug/ml] and T2DM [9.7 (7.9-11.7) ug/ml], compared with the controls [8.5 (7.1-10.7) ug/ml, p=0.010] and metformin treatment significantly decreased PEDF levels in T2DM patients with weight loss (p=0.027). We also found that men exhibited remarkably higher serum PEDF levels [9.58 (7.92-11.92) ug/ml] than women [8.88 (7.35-10.77) ug/ml, p=0.003] and this difference still existed even after adjusting for BMI, WC and WHR (p=0.009). Spearman correlation analysis demonstrated that PEDF levels were positively and significantly correlated with risk factors regarding metabolic syndrome and visceral obesity such as body mass index, waist circumference, waist-to-hip ratio, fasting and 2h glucose (p<0.001). Multiple stepwise regression analysis revealed that PEDF was associated with alanine aminotransferase (p<0.001), triglycerides (p=0.002) and diastolic blood pressure (p=0.002) after adjustment for age and gender. Subjects with a higher tertile of PEDF concentration exhibited a higher prevalence of overweight or obesity than those in the lower tertile (p<0.001). Furthermore, we found that serum estradiol level was negatively associated with PEDF (r=-0.216, p=0.013) and was an independent determinant of PEDF in post-menopause females.ConclusionPEDF concentration is significantly elevated in patients with IGR and could be reduced after metformin treatment in T2DM patients with weight loss. Decreased estradiol level was an independent risk factor for the elevated PEDF and may contribute to the occurrence of T2DM in post-menopause females.