The Mechanism Of Conditioned Medium From Contractive Skeletal Muscle Cells Reversing Insulin Resistance And Dysfunction Of Endothelial Cells

Objective:Obesity is an important risk factor for type 2 diabetes.Vascular complications are the leading cause of the death of type 2 diabetes mellitus.Obese adipose tissue rapidly expends,leading adipocytes hypertrophy and preceding the rate of angiogenesis.These are the main causes of adipose tissue hypoxia.Meanwhile,adipose tissue infiltrated with macrophages.These lead to insulin resistance and chronic low inflammation.The adipose tissue secrets elevated levels of TNF-α,MCP-1 and other proinflammatory cytokines,as well as free fatty acids.However,anti-inflammatory factors such as adiponectin released from adipose tissue were reduced.These adipokines in turn affected insulin function in liver and skeletal muscle,leading to systemic insulin resistance and eventually type 2 diabetes while b cells function failuure.It can also induce endothelium insulin resistance and dysfunction,leading to cardiovascular disease.Endothelium dysfunction is the pathophysiology of cardiovascular disease.Improved endothelium function may prevent cardiovascular disease.On the other hand,physical exercise can increase endothelium-dependent vasodilatation to improve insulin resistance and endothelium function.Exercise also stimulates skeletal muscle to secrete a variety of cytokines and specific myokines,which may play important roles in the benefit of exercise.But the related mechanism is not clear.In this study,electric pulse stimulation was used to stimulate muscle cells contraction.Then,the conditioned medium from muscle cells was collected and the effect of conditioned medium on insulin resistance and endothelial cells dysfunction were studied.Content:Part 1.The effect of conditioned medium from contracting muscle cells(CM-EPS)on inflammation and apoptosis of endothelial cells.Inflammation and apoptosis of endothelial cells.were induced with conditioned medium from hypoxic adipocyte(CM-H).Then endothelial cells were co-cultured with CM-EPS.Macrophage migration,the mRNA expressions of E-selectin,ICAM-1,MCP-1 and IL-6,the phosphorylation of IKKα/β and NF-κB,SOCS3 protein expression and the apoptosis of endothelial cells were detected.Part 2.The effect of conditioned medium from contracting muscle cells(CM-EPS)on insulin resistance and endothelial cells dysfunction.Insulin resistance and endothelial cells dysfunction were induced with conditioned medium from hypoxic adipocyte(CM-H).Then endothelial cells were co-cultured with CM-EPS.The phosphorylation of Akt and eNOS and endothelial cell NO levels were detected.Phosphorylation of AMPK and eNOS were detected under CM-EPS treatment.The endothelial cells dysfunction was induced by CM-H following addition of CM_EPS with or without compound C prior treatment.Phosphorylation of AMPK and eNOS were measured.Methods:3T3-L1 adipocytes were incubated under normoxia or hypoxia condition.The supernatant was collected as adipocyte conditioned medium(CM-N and CM-H).C2C12 skeletal muscle cells were stimulated with electric pulse for 12 hours.The supernatant was collected as conditioned medium from skeletal muscle cells(CM-EPS).HUVECs were co-cultured with CM-N or CM-H with CM-EPS,Macrophages migration were detected with transwell system,mRNA expressions of E-selectin,ICAM-1,MCP-1 and IL-6 were measured by real-time PCR.The phosphorylation of IKKα/β,NF-κB,Akt,eNOS and SOCS3 protein levels were measured by Western blot.The NO concentration was measured by ELISA.Endothelial cell apoptosis was detected by flow cytometry.The phosphorylation of AMPK and eNOS were detected by Western blot.After endothelial cells cultured with CM-N and CM-H for 16 h,endothelial cells were stimulated with CM-EPS,then We also used Compound C to inhibit AMPK.Western blot was used to detect AMPK and eNOS phosphorylation of endothelial cells.Results:Part 1.The mRNA expressions of E-selectin,ICAM-1,MCP-1 and IL-6 in HUVECs were reduced upon CN-H treatment.The phosphorylations of IKKα/β and NF-κB,SOCS3 protein levels and endothelial cell apoptosis were significantly reduced upon CM-H treatment.Part 2.CM-EPS reverses the effect of CM-H on phosphorylation of Akt and eNOS and increases the NO production in HUVECs.CM-EPS stimulated the phosphorylations of AMPK and eNOS in HUVECs,reaching maximum at 1h which were inhibited by AMPK inhibitor Compound C.Conclusion:1.CM-EPS significantly reduced the macrophages migration towards to CM-H-treated HUVECs.CM-EPS significantly reduced the mRNA expressions of E-selectin,ICAM-1 and MCP-1 in HUVECs.2.CM-EPS significantly reduced the mRNA level of IL-6,phosphorylation of IKKα/β and NF-κB,the protein level of SOCS3 and the apoptosis of CM-H-treated HUVECs.3.CM-EPS significantly improved CM-H-induced HUVECs insulin resistance and dysfunction.4.CM-EPS improves endothelial function via phosphorylation of AMPK.In summary,CM-EPS reversed endothelial cells inflammation,apoptosis,insulin resistance and dysfunction caused by CM-H.