Molecular Mechanism Of Insulin Resistance Of Endothelia Cells In Metabolic Syndrome

Objective: To evaluate the relationship between insulin resistance and endothelium -dependent vasodilatation in Chinese male obese subjects with normal blood glucose, serum total cholesterol and blood pressure and to explore the non-traditional risk factors in macrovascular disease in patients with metabolic syndrome.Subjests and Methods: 20 euglycemic obese males(OB) and 12 age and sex matched lean controls(lean) were involved in this study. All subjects underwent euglycemic hyperinsulinemia clamp study with an insulin infusion rate of 120mU/m2/min to evaluate the peripheral glucose disposal rate (GDR)in steady-state, brachial artery ultrasound study to assess the endothelium-dependent vasodilation(EDV) and independent vasodilation (EIV). The serum lipids profile and the levels of ROS, MDA,GSH-PX,GSH, hs-CRP,PAI-l and free fatty acids(FFAs).were also measured. Results: The serum concentrations of ROS, MDA, hs-CRP and PAI-1 in OB group were significantly higher than those of the controls (p<0.05). while the serum levels of GSH-PX, GSH were significantly lower. The serum FFAs and triglyceride concentrations were elevated compared with those of the controls. The GDR obtained during euglycemic hyperinsulinemia clamp study were markedly decreased in OB group (P<0.05). The flow-mediateddilation of the brachial artery caused by reactive hyperemia (EDV) were impaired in OB group and the relaxation time were shorter than that of the controls (p<0.05).The EIV was not significantly different between two groups(p>0.05). The significantly negative correlations was observed between the serum levels of ROS,MDA,hs-CRP, PAI-1,FFA and EDV (p<0.05), while there was a significantly positive correlation between the serum GSH-PX,GSH concentrations and EDV (p<0.05). Conclusion: An impaired endothelium-dependent vasodilation was observed in obese subjects although their blood glucose, serum total cholesterol and blood pressure were noamal. The elevated circulating FFAs concentration, enhanced oxidative stress and chronic inflammation may play a role to causing endothelial dysfunction in metabolic syndrome subjects. Aims: Elevated FFAs levels have been shown to impair endothelium dependent vasodilation (EDV) which may related with the high incidence of microvascular disease in insulin resistant patients. Here we test the hypothesis that FFAs induced endothelial dysfunction may caused by inhibited endothelial nitric oxide synthase (eNOS) mRNA expression and activity.Materials and methods: Male S-D rats underwent 6 hr infusion in two groups: Normal Control (NC, n=20): infused with normal sailine; FFA (n=20): infused with FFAs (20%intralipid ± heparin). Following infusion aortas were harvested and cleaned of adhering tissue and blood inside the lumen.The endothelial cells were gleaned by scraper. The endothelial cells were minced and homogenized by ultrasound. To assess free fatty acids(FFAs) and triglyceride(TG) in serum and endothelial homogenate; To assess the serum levels of ROS, MDA, GSH and hsCRP.The analysis of blood nitrite/nitrate (NOx) was performed by high- performance liquid chromatography (HPLC). the expression of eNOS phosphorylation at Ser1177 in aortas from SD rats was examined by immunohistochemistry.The eNOS mRNA expression in vascular homogenate was analyzed by semi-quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR). The eNOS activity in endothelial homogenate was measured by HPLC.Results: The level of serum FFA(FFA:1146.9±223.1umol/L,vs.C:327.3± 106.5umol/L,p<0.01)and triglyceride (2.3±0.5 mmol/L,vs.0.70±0.3mmol/L) were elevated compared with the controls.The level of FFA(298.9±49.5 umol/L,vs.143.1±23.1 umol/L, p<0.01)and triglyceride(1.16±0.26mmol/L,vs.0.81±0.18mmol/L) in endothelial homogenate were also higher. The serum levels of ROS, MDA, hs-CRP in the FFA group were significantly higher than the controls (p<0.05). while the serum levels of GSH were significantly lower than those of the controls.the serum NOx concentration were lower (FFA:12.6±1.9, vs.C:17.2±3.9micro-mol/L, p<0.01). the expression of eNOS phosphorylation at Ser1177 positive immunostaining were found in the intima.the density of eNOS phosphorylation at Ser1177 positive immunostaining was lower in FFA group (P<0.05). The level of eNOS mRNA was decreased by approximately 33% (FFA:1.42,vs.C: 2.12, p < 0.01). The eNOS activity in endothelial homogenate was 0.50±0.62 micro-mol/L in FFA group and 0.96±0.66 micro-mol/L in C group (p < 0.01), decreased by approximately 48%.Conclusions: The present study indicated that elevated FFAs concentration directly and independently reduces endothelial NO production which may secondary to inhibitions of eNOS mRNA expression and activity. The mechanism underline may related with the increased oxidative stress and inflammation caused by elevated FFA concentration.Aims: It is found that FFAs may impair nitric oxide (NO) production by endothelial cells in the previous study. However, the signaling pathways involved has not been characterized. Here we test the hypothesis that decreased NO production caused by elevated circulating free fatty acids levelis related with inhibited IRS-1-Akt-eNOS-NO signaling pathway in endothelial cells and implore wether the enhanced PKC activity, oxidative stress and inflammation play roles druing these process. Materials and methods: Male S-D rats underwent 6 hr infusion in two groups: Control (C, n=30): infused with normal sailine; FFA (n=30): infused with FFAs (20%intralipid + heparin). The euglycemic hyperinsulinemia clamp study was conducted to evaluate the peripheral glucose disposal rate (GDR) during last hours of infusion. At the end of infusion rats were killed and aortas were harvested The endothelial cells were gleaned by scraper. The serine phosphorylation and protein levels of IRS-1, Akt and eNOS in rat aortic endothelial cells were determined by western blotting. The tyrosine phosphorylation and protein levels of p44/42 MAP kinase (ERK-1/2) were detected by same method. The expression of PKCα in aortas was examined by immunohistochemistry. PKC activity inendothelial cells was measured by liquid scintillation counting. The protein levels of PKCα, NFκBp65 and IκBαin rat aortic endothelial cells were detected by western blotting.The plasma FFA levels were determined by an enzymic method. Results: The results showed that in FFA group the plasma FFAs concentration was higher (FFA:1206.4±321.6 micro-mol/L, vs. NC: 356.5±117.9micro-mol /L,p<0.01) and the GIR was decreased (FFAgroup:21.0±1.8mg · min-1 ·kg-1, vs.NC group: 26.3±2.3mg ·min-1·kg-1,P<0.05). The levels of phosphorylation at IRS-1 tyrosine, Akt Ser473 , and eNOS Ser1177 was decreased to 34%, 46% and 36% respectively in FFA groupm, (p<0.05). while the protein levels of IRS-1, Akt and eNOS were not found significant different in both groups (p>0.05). In contrast, there were not significantly different both in tyrosine phosphorylation levels and protein levels of p44/42 MAP kinase in two groups (p>0.05). The total PKC activity of rat aortic endothelial cells was increased in FFA group (P<0.05)PKCα positive immunostaining were found in aortas of rats.the density of PKCα positive immunostaining was higher in FFA group (P<0.05). the total PKC activity of rat aortic endothelial cells was increased in FFA group (P<0.05). The protein levels of PKCαand NFκBp65 were increased by approximately 69% and 47% respectively (p< 0.05), whereas the protein levels of IκBαin FFA group was decreased by approximately 22% (p<0.05). Conclusions: The previous study indicated acute elevation of FFA directly and independently reduces endothelial NO production.The mechanism underline may related, at least partly, down-regulates insulin signaling (IRS-1-Akt-eNOS-NO). whereas p44/42 MAP kinase signaling pathway was not altered in rat aortic endothelial cells. It is suggested that elevated FFA level selectively inhibited insulin signaling by PKC dependent mechanism. The oxidative stress and inflammation caused by FFA may play a roles during this process.Aim:This experiment aims at researching effects of oleic acid on eNOS activity and expression in vitro. To evaluate the possible protective effects on HUV-EC-C mechanisms of PKC inhibitor(GF109203X,GFX), NAD(P)H oxidase inhibitor (Diphenylene iodonium, DI),NF-KB inhibitor(pyrrolidine dithiocarbamate, PDTC) and insulin involved.Methods: The HUV-EC-C was selected to be a model and following aspects were studied: (l)Effects of oleic acid on the endothelial dysfunction: ①The eNOS activity in HUV-EC-C was measured by HPLC. ②The expression of eNOS phosphorylation at Ser1177 in HUV-EC-Cs was examined by immunohistochemistry.(2) Effects of oleic acid on PKC activity and expression in HUV-EC-C:①To investigate the role of PKCα in endothelial dysfunction in HUV-EC-C induced by oleic acid,the translocation of PKCa in a single HUV-EC-C after oleic acid was observed by Laser-Scanning Confocal Microscope.②The PKC activity was measured by liquid scintillation counting.(3)Effects of oleic acid on oxidative stress in HUV-EC-C: the level of ROS in supernatant were with chemistry method by spectro-photometer.(4)Effects of oleic acid on NFκB expression in HUV-EC-C: ①Effects of oleic acid on NFκB expression in HUV-EC-C by immunohistochemistry;②Effects of PDTC on oleic acid-mediated changes in NFκB expression in HUV-EC-C.(5)Effects of oleic acid on insulin signaling in HUV-EC-C: ①Effects of oleic acid on insulin-stimulated eNOS activity by HPLC;②Effects of insulin on oleic acid -mediated changes in eNOS activity and expression in HUV-EC-C (6) Effects of GFX, DI and PDTC on oleicacid-mediated ROS produces in HUV-EC-C. (7) Effects of GFX, DI and PDTC on oleic acid-mediated changes in eNOS activity and expression in HUV-EC-C.Results: Oleic acid inhibited eNOS activity and expression in HUV-EC-C. Oleic acid could induce the translocation of PKCa from plasm to membrane in HUV-EC-C. the density of PKCa positive immunostaining was higher in oleic acid group. Oleic acid could increase totle PKC activity and ROS produce in HUV-EC-C. Oleic acid could induce the translocation of NFκB from plasm to nucleus in HUV-EC-C. however PDTC could inhibit this effects.. Insulin could partly blund the inhibition of eNOS activity and expression by Oleic acid in HUV-EC-C . GFX and DI could discrease oleic acid-mediated ROS produce in HUV-EC-C,however PDTC couldn't inhibit this effects.9.GFX, DI and PDTC could partly correct the inhibition of eNOS activity by Oleic acid in HUV-EC-C.Conclusion :PKC-mediated oxidative stress and inflammation could involved in this mechanism,which oleic acid inhibited eNOS activity and expression in HUV-EC-C.INS, GFX, DI and PDTC could partly correct the inhibition of eNOS activity by oleic acid. Effects of anti-oxidative stress, anti -inflammation and discreased PKC activity may play a roles in this process,which oleic acid inhibited eNOS activity and expression in HUV-EC-C.