Effects Of Sarco/Endo Plasmic Reticulum Ca²⁺-ATPase2 On ER Stress And Glucose Metabolism Under Intermittent Hypoxia In Hepatocytes

Objective The objective of this study was to explore the changes of hepatic gluconeogenesis after exposed to obstructive sleep apnea-hypopnea syndrome,（OSAHS）characteristic intermittent hypoxia（IH）,and to investigate alterations of Sarco（endo）plasmic reticulum Ca²⁺-ATPase 2（SERCA2）and phosphorylation of endoplasmic reticulum stress（ER stress）associated proteins induced by IH.Furthermore,the essential role of SERCA2/ER stress in the development of glucose metabolism during IH was to be determined by overexpressing SERCA2,for the further exploration of the mechanisms underlying abnormal glucose metabolism in OSAHS.Methods To construct the IH model of hepatocytes,cells were incubated in normal air as control group（21%O₂,5%CO₂,37℃）and intermittent hypoxia group（21%O₂ 5min,1%O₂ 5min,5%CO₂,37℃,6 circles/h）over 24h,respectively.For gluconeogenesis assessment,after 24h IH/room air exposure,cells were washed with warm PBS twice to remove glucose,and incubated in glucose-free,phenol-free Dulbecco’s Modified Eagle’s Medium（DMEM）without glutamine,supplemented with gluconeogenic substrates（sodium pyruvate and lactate）for another 6h prior to harvesting.After completion of the exposure periods,cells were washed twice with 2 mL phosphate buffered saline（PBS）and lysed with RIPA lysis buffer.Protein quantity was determined using the BCA protein assay and used for data normalization.Glucose production was assessed using Glucose Oxidase Activity Assay Kit.Glucose concentration was normalized to cellular protein concentration,western blot was performed following incubation of hepatocytes with room air/IH exposure over 24 hours;SERCA2,p-PERK,p-eIF2α,p-Akt（Ser473）and glucose-6-phosphate（G6Pase）expression were measured.By lentiviral transfection to overexpress SERCA2,p-PERK,p-eIF2α,p-Akt（Ser473）and G6Pase expression were examined again,as well as gluconeogenesis.Results Comparing to control group after culture,we found that hepatocytes gluconeogenesis in IH group significantly increased,this change was accompanied by decreased phosphorylation of Akt（S473）and increased G6Pase.Expression of SERCA2was reduced in IH group,while ER stress associated proteins like p-PERK,p-eIF2αwere markedly increased when hepatocytes were exposed to IH.Furthermore,overexpression of SERCA2 alleviated ER stress,improved Akt（ser473）phosphorylation level,and significantly reduced the gluconeogenesis and G6Pase to normal level.Conclusion Intermittent hypoxia induced glucose metabolism dysfunction in hepatocytes.SERCA2 protein significantly decreased in IH group.SERCA2overexpression in hepatocytes alliveated ER stress,suggesting that SERCA2 may be a regulator of ER stress.The current findings further indicated that SERCA2 induced ER stress may play some role in OSAHS associated glucose metabolic dysfunction.