| Purpose: Obesity is a risk factor of atherosclerotic.The synthesis and secretion of saturated fatty acids and pro-inflammatory cytokines such as TNF-? and MCP-1 increases in obese adipose tissue,whereas anti-inflammatory cytokines such as adiponectin decreases.These are easy to induce chronic inflammation,liver steatosis and cardiovascular diseases.Obese adipose tissue is hypoxic.We previous reported that the inflammatory cytokines from adipocytes cultured under hyoxic condition induce insulin resistance in muscle cells and hepatocytes.However,whether these factors have effect on hepatocellular inflammation signals and vascular endothelial cells,as well as induces endothelial dysfunction remain unclear.Liraglutide,glucagon like peptide-1(GLP-1)analogue,is an anti-diabetic drug.Recent animal and clinical studies found that it might have cardiovascular protective effect,but the mechanism remains incompletely clear.Therefore,we explore the effects of the factors released from hypoxic adipocytes and saturated fatty acids on hepatocellular inflammation signlas and endothelial function.We aslo explore the molecular mechanism of liraglutide-improved endothelial dysfunction.Methods: In the first part,the adipocytes were cultured in normoxic or hypoxic condition for 16 h,respectively.The supernatants were collect as conditioned medium.The hepatocytes were incubated with conditioned medium for 16 h.The phosphorylation of JNK,NF-?B and IKK?/? were examined by Western blot.In the second part,the adipocytes were cultured in normoxic or hypoxic condition for 16 h,respectively.The supernatants were collect as conditioned medium.The HUVECs were incubated with conditioned medium for 16 h.The phosphorylation of JNK,NF-?B,IKK?/? and e NOS were examined by Western blot.The m RNA expressions of IL-6,E-selectin,VCAM-1 and ICAM-1 were detected by real-time PCR.The macrophages migration towards HUVECs was detected by transwell assay.The NO level was measured with a Nitric Oxide Colorimetric Assay Kit.The apoptosis of endothelial cells was detected by flow cytometry.In the third part,the HUVECs were incubated with palmitate(PA)for 16 h.The phosphorylation or protein of JNK,IKK?/?,e NOS and SOCS3 were examined by Western blot.The m RNA expression of IL-6,E-selectin,VCAM-1 and ICAM-1 were detected by real-time PCR.The macrophages migration towards HUVECs was detected by transwell assay.The NO level was measured with a Nitric Oxide Colorimetric Assay Kit.In the fourth part,the HUVECs were incubated with PA for 16 h following treatment with 100 n M liraglutide for 30 min.The phosphorylation or protein levels of JNK,IKK?/?,e NOS,PKC,AMPK and SOCS3 were examined by Western blot,respectively.The m RNA expression of IL-6,E-selectin,VCAM-1 and ICAM-1 were detected by real-time PCR.The macrophages migration towards HUVECs was detected by transwell assay.The NO level was measured with a Nitric Oxide Colorimetric Assay Kit.We also used the inhibitor of AMPK,c PKC and n PKC to further explore the molecular mechanism of liraglutide on improving endothelial dysfunction.Results: In the first part of this study,results show that the conditioned medium from hypoxia adipocytes significantly elevated the phosphorylation of JNK,NF-?B and IKK?/?.In the second part of this study,results show that the conditioned medium from hypoxia adipocytes significantly elevated not only the phosphorylation of JNK,NF-?B and IKK?/?,but also the m RNA expression of IL-6,E-selectin,VCAM-1 and ICAM-1 of HUVECs.It promoted macrophages migration towards HUVECs.It decreased the phosphorylation of e NOS and the level of NO.It promoted endothelial cells apoptosis.In the third part of this study,results show that PA significantly elevated not only the phosphorylation or protein of JNK,IKK?/? and SOCS3,but also the m RNA expression of IL-6,E-selectin,VCAM-1 and ICAM-1 of HUVECs.PA promoted macrophages migration towards HUVECs.PA decreased the phosphorylation of e NOS and the level of NO.In the fourth part of this study,results show that liraglutide obviously inhibited the phosphorylation of JNK,IKK?/? and protein level of SOCS3 induced by PA.It reversed the m RNA expressions of IL-6,E-selectin,VCAM-1 and ICAM-1 induced by PA.It inhibited macrophages migration towards HUVECs.Liraglutide significantly increased phosphorylation of e NOS,AMPK and the release of NO.However,liraglutide had no effect on PKC phosphorylation.In addition,inhibition of AMPK by Compound C(CC)significantly decreased e NOS phosphorylation and NO release.Inhibition of c PKC and n PKC by G?6983 had no effect on the phosphorylation of e NOS.Lastly,the effect of leptin and liraglutide together on e NOS phosphorylation was significantly higher than each stimulus alone under PA treatment.Conclusions: 1.The conditioned medium from hypoxia adipocytes significantly elevated the expression of inflammatory signaling molecules in hepatocytes.2.The conditioned medium from hypoxia adipocytes significantly elevated the phosphorylation of inflammatory signaling molecules,the m RNA expression of adhesion molecule in HUVECs and promoted macrophages migration towards HUVECs.3.The conditioned medium from hypoxia adipocytes promoted cell apoptosis in HUVECs.4.The conditioned medium from hypoxia adipocytes impares endothelial function.5.PA obviously elevated the phosphorylation of inflammatory signaling molecules,the m RNA expression of adhesion molecule and promoted macrophages migration towards HUVECs.6.PA impares endothelial function.7.Liraglutide significantly decreased the phosphorylation of the inflammatory signaling molecules and the m RNA expression of adhesion molecule induced by PA on HUVECs.8.Liraglutide significantly inhibited the microscope migration towards HUVECs induced by PA.9.Liraglutide improved the endothelial dysfunction in an AMPK-depented manner.10.c PKC and n PKC were not involved in liraglutide-improved endothelial dysfunction.11.Liraglutide has additive effect with leptin on improved endothelial dysfunction under PA treatment. |
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