Knockdown Of ETAR Inhibits P66Shc-mediated Hepatic Mitochondrial Fragmentation And Related Metabolic Disorders

Aim: Emerging evidence from animal studies and clinical trials indicates that serum endothelin-1(ET-1)levels are significantly increased under diabetic conditions,and the systemic inhibition of ET-1 signaling by endothelin receptor antagonists can improve some pathological features of diabetes and its complications.It is demonstrated that endothelin type A receptor(ETAR)plays an important role in the pathology of ET-1-mediated diabetes.Metabolic disorders in the liver can lead to systemic metabolic disorders,which promotes the development and progression of diabetes and its complications.However,the effects as well as the mechanistic targets of hepatic ET1/ETAR signaling inhibition on the pathology of metabolic diseases remain unclear.In addition,it has been shown that mitochondrial fragmentation participates in the regulation of the pathological process of diabetes-related metabolic disorders.This study aims to clarify whether and how hepatic knockdown of ETAR improves liver mitochondrial fragmentation and related metabolic disorders in diabetic mice induced by high-fat food(HFD).Methods: In this study,diabetic mice were induced by HFD.Liver-selective knockdown of ETAR was achieved by tail vein injection of adeno-associated virus 8(AAV 8).Serum ET-1 level was detected by ELISA;the levels of proteins in signaling pathways were measured by Western blot;the metabolic parameters and mitochondrial functions were assayed by biochemical technology.The pathological changes of liver tissue were analyzed by HE staining;mitochondrial fragmentation was observed by transmission electron microscope analysis and Mito-Tracker Red CMXRos staining;mitochondrial translocation of p66 Shc was detected by immunofluorescence.Mito Sox staining was used to evaluate mitochondrial ROS production;the m RNA levels of inflammation-related factors were determined by Real-time PCR.Finally,the interaction between PKCδ and p66 Shc was analyzed by co-immunoprecipitation(Co-IP).In addition,L02 cells were treated with ET-1 to further verify the effects of knockdown of ETAR in vitro.Results: Firstly,liver-specific knockdown of ETAR improved blood glucose,triglycerides levels,as well as ameliorating insulin resistance and liver injuries in HFD-induced diabetic mice,indicating that hepatic knockdown of ETAR could effectively improve the systemic and liver glucose and lipid metabolism disorders in diabetic mice.Our findings further showed that the potential mechanism underlying the improvement of hepatic ETAR knockdown on metabolic disorders was associated with the inhibition of p66Shc-mediated hepatic mitochondrial fragmentation and subsequent oxidative stress-related damage in diabetic mice.The results revealed that hepatic ETAR knockdown restored the mitochondrial membrane potential ΔΨm and ATP production,meanwhile alleviating the mitochondrial fragmentation and subsequent oxidative stress and inflammation in diabetic mice.However,overexpression of p66 Shc in L02 cells treated with ET-1 eliminated the beneficial effects of ETAR knockdown on mitochondrial function and fragmentation.PKCδ is an important regulatory protein involved in mitochondrial function,insulin resistance and metabolic disorders,therefore,we explored that the potential role of PKCδ in linking hepatic ETAR knockdown and p66Shc-mediated mitochondrial dysfunction.The results showed that hepatic ETAR knockdown significantly restrained the phosphorylation of PKCδ and its plasma membrane translocation,as well as its interaction with p66 Shc in diabetic mice.These results suggested that hepatic ETAR knockdown inactivated p66 Shc by inhibiting PKCδ in HFD-induced diabetic mice,thus improving above-mentioned mitochondria fragmentation,dysfunction and related metabolic disorders.Furthmore,PKCδ knockdown also resulted in inactivation of p66 Shc in L02 cells treated with ET-1.Moreover,these ETAR knockdown-mediated actions were confirmed in ET1-treated L02 cells.Conclusion: In this study,hepatic knockdown of ETAR significantly inhibited p66Shc-mediated mitochondrial fragmentation and dysfunction,and subsequent oxidative stress-related damage,thereby effectively improving liver glucose and lipid metabolism in HFD-fed mice,and indicated that hepatic knockdown of ETAR may be a promising therapeutic strategy for metabolic diseases.