The Role Of Lipin1 In Autophagy In Diabetic Peripheral Neuropathy

BackgroundDiabetic peripheral neuropathy(DPN)is a chronic complication of diabetes that is difficult and ineffective to treat.At present,the mechanisms underlying the pathogenesis of DPN remain unclear,which require to be further investigated.The typical neuropathological changes of DPN include axonal injury,focal segmental deletion of myelin,and neuronal damage.Hyperglycemia,advanced glycation end products and other stimuli,as noxious stimulus signals,can damage sensory neurons and axons.Under the harmful stimulation of hyperglycemia,Schwann cells would proliferate and migrate again,resulting in axonal degeneration and demyelination.Autophagy plays an important role in neurodegenerative diseases.Recent studies have revealed autophagy is involved in the development of DPN due to long-term hypoxia,lack of nerve factors,and oxidative stress.However,the relationship between autophagy and peripheral nerve injury,especially in DPN,has been poorly studied.Qu et al.reported that high glucose led to the decrease of Schwann cells autophagy and the aggravation of Schwann cells injury.However,Towns R,et al.reported that excessive activation of autophagy in diabetic mice neurons may lead to neuronal loss,DPN and neuropathic pain.Therefore,the changes of autophagy in diabetes mellitus are complicated and the relationships between autophagy and peripheral nerve need further investigations.Lipin 1 is an enzyme produced by the expression of LIPIN that is closely related to glycolipid metabolism.Lipin 1 converts phosphatidic acid(PA)to DAG,which is a messenger that activates PKC/PKD signaling pathway and participates in the formation and maturation of autophagosome.Therefore,Lipin 1-DAG-PKD signaling pathway maybe involved in the autophagy regulation.In Lipin 1-deficient mice,a disorder of peripheral nerve myelination,including demyelination and axonal degeneration,has been observed.Based on the above,we proposed:Lipin 1 can reduce autophagy disorders and alleviate DPN,thus providing novel perspectives to the treatment of DPN.Part I The effects and mechanisms of Lipinl on autophagy of sciatic nerve in DPN ratsObjective To investigate the effects and mechanisms of Lipin 1 on autophagy of sciatic nerve in DPN rats.Methods1.Diabetic(DM)rat model was induced by intraperitoneal injection of streptozotocin(STZ).Blood glucose and body weight of rats were monitored regularly.2.The motor nerve conduction velocity(MNCV)of sciatic nerve and paw mechanical withdrawal threshold(PMWT)were measured 8-week after DM model was established.Ultrastructural changes of sciatic nerve were observed under electron microscope.The expression of Lipin 1 in sciatic nerve was analyzed by q-PCR.The diacylglycerol(DAG)levels were measured by enzyme-linked immunosorbent assay(ELISA).Autophagy-related protein levels were detected by Western Blotting.3.DPN rats were divided into:Lipin 1 overexpression group(ADV-Lipin1)and Adenovirus empty shell group(ADV-Lipin1-Con).NC rats were also divided into:Lipin]knock-down group(ADV-Lipin1-RNAi)and Adenovirus empty shell group(ADV-Lipin1-RNAi-Con).Different adenoviruses(ADV)were injected via intrathecal.4.After 2 weeks of ADV injection,MNCV and PMWT were measured.Ultrastructural changes of sciatic nerve were observed under electron microscope.The expression of Lipin 1,the DAG and autophagy-related protein levels were detected.Results1.The fasting blood glucose(FBG)of DM group was more than 16.7mmol/l.Compared with NC group,the FBG of DM group was significantly higher while body weight was significantly lower.2.MNCV and PMWT in DPN group were lower than those in NC group.The electron microscope showed that the demyelination of the sciatic nerve was occurred as well as the lamella was obviously loose in DPN.rats.Compared with NC rats,Lipin 1 expression and DAG levels in sciatic nerve of DPN rats decreased.In the sciatic nerve of DPN rats,autophagy was excessively activated.3.After ADV injection,MNCV and PMWT in ADV-Lipin1 group increased as compared to the ADV-Lipin1-Con group.Meanwhile,the expression of Lipin 1 and DAG levels in sciatic nerve in ADV-Lipin1 group increased significantly,the myelin sheath separation of sciatic nerve was improved significantly and the lamella was dense.The autophagy reaction in the sciatic nerve of ADV-Lipin1 group was improved.4.After ADV injection,the MNCV and PMWT of rats decreased in ADV-Lipin1RNAi group as compared to the ADV-Lipinl-RNAi-Con group.Moreover,the expression of Lipin 1 and DAG levels in the sciatic nerve of rats significantly decreased in ADV-Lipin1-RNAi group.The sciatic nerve showed mild swelling and the lamella was looser in ADV-Lipin1-RNAi group as compared to the ADV-Lipin1-RNAi-Con rats.Autophagy reaction in the sciatic nerve of ADV-Lipin1-RNAi group was over activated.Conclusion1.Lipin 1 deficiency caused disorder of lipid metabolism and excessive enhancement of autophagy reaction in rat sciatic nerve,resulting in the demyelination of sciatic nerve,denaturation and injury of neuronal cell bodies,and impairment of normal nerve function.2.The loss of Lipin 1 could lead to the occurrence and development of DPN by regulating autophagy.Part II The effects and mechanisms of Lipin1 on cell activity,apoptosis and autophagy in rat Schwann cells(RSC96 cells)ObjectiveTo investigate the effects and mechanisms of Lipin1 on cell activity,apoptosis and autophagy in rat Schwann cells(RSC96 cells)in vitro.Methods1.RSC96 cells were incubated with different concentrations of glucose for 24h,48h and 72h,and the same concentration of mannitol was used as control.The activity and apoptosis rate of RSC96 cells were measured.Normal glucose(NG)and high glucose(HG)were used to interfere with RSC96 cells.Lipin1 expression,DAG levels and autophagy related protein levels were measured.The number of autophagosomes or autophagy lysosomes in RSC96 cells was observed under electron microscope.2.Lipin1 overexpression lentivirus was transfected into RSC96 cells(LV-Lipin1)and empty shell lentivirus was used as control(LV-Lipin1-Con).After successful transfection,RSC96 cells were intervened with HG,then cell activity,apoptosis rate and DAG levels were measured.The expression of Lipin1 and autophagy related proteins were detected by Western-blot.The number of autophagosomes or autophagy lysosomes was observed under electron microscope.3.Lipin1 knock-down lentivirus was transfected into RSC96 cells(LV-Lipin1RNAi)and empty shell lentivirus was used as control(LV-Lipin1-RNAi-Con).After successful transfection,RSC96 cells were intervened with NG,then cell activity,apoptosis rate and DAG levels were measured.Meanwhile,the expression of Lipin 1 and autophagy related proteins were detected.The number of autophagosomes or autophagy lysosomes was observed under electron microscope.Results1.HG resulted in decreased expression of Lipin 1 in RSC96 cells,decreased cell viability and increased apoptosis rate.The number of autophagosome or autophagy lysosome increased significantly,and the autophagy reaction was over activated.2.Compared with LV-Lipinl-Con group,overexpression of Lipin1 significantly increased cell viability and decreased apoptosis rate decreased in RSC96 cell.The number of autophagosome or autophagy lysosome decreased,and the autophagic reaction was inhibited.3.Silencing Lipin1 significantly deceased the cell viability and increased the apoptosis in RSC96 cells compared with LV-Lipin1-RNAi-Con group.The number of autophagosome or autophagy lysosome increased,and the autophagy reaction was over activated.Conclusion1.Lipin1 deficiency resulted in lipid metabolism disorder,excessive enhancement of autophagy reaction,decreased cell activity and increased apoptosis in Schwann cells.2.Lipin1 could affect the normal physiological function of Schwann cells by regulating autophagy.