Lipolysis Of HIF-1α On Adipose Tissue Of Cows With Ketosis

Ketosis is a common nutritional metabolic disease in cows during the perinatal period.When dairy cows enter the early lactation stage,milk production increases rapidly and the prevalence of ketosis in dairy cows is at its highest.This period is characterized by lower energy intake than body requirements,leading to negative energy balance（NEB）in dairy cows.The increase of NEB duration and milk yield intensity will further aggravate the negative energy balance.In order to maintain the energy balance and meet the energy demand,the adipose tissue of dairy cows begins to mobilize fat to make up the gap between energy intake and output,leading to the increase of plasma NEFA concentration,and the metabolic dysfunction of adipose tissue may occur during this process.In recent years,some studies have reported the relationship between adipose tissue dysfunction and adipose tissue hypoxia.The hypoxia response of tissue cells is mainly mediated by hypoxia inducible factor（HIF）,which is composed of two dimeric subunits,αsubunit which is the most sensitive to oxygen concentration andβsubunit which is widely present in all tissues.Under normal conditions,the alpha subunit of HIF-1 is hydroxylated by proline hydroxylase（PHDs）,which allows ubiquitin ligase（VHL）to inhibit HIF-1αactivity and then proteolysis via the proteasome pathway.Under anoxic conditions,the hydroxylation step is inhibited,leading to the stable presence and increased expression of HIF-1a.According to previous reports,HIF-1αsignaling pathway is highly expressed in adipose tissue of cows with ketosis,and HIF-1αis involved in regulating the expression of lipase and vascular endothelial factor（VEGF）,which plays an important role in the regulation of fat metabolism.Therefore,we proposed a scientific hypothesis on the effect of HIF-1αon adipose tissue metabolism of cows with ketosis,and further explored the mechanism and influence of HIF-1αon the regulation of adipose tissue lipid metabolism,hypoxia and insulin signaling pathway of cows with ketosis,providing a new direction and a new path for the study of cows with ketosis.In this study,the effects of HIF-1αon hypoxia and lipolysis in adipose tissue of cows with ketosis were investigated from two aspects:in vivo and in vitro.Adipose tissue samples from healthy cows and 12 Holstein cows with clinical ketosis were selected from blood ketone body test.Hypoxia-related proteins,lipase and insulin signaling proteins in adipose tissue samples were detected by Western Blotting.Primary bovine adipose cells were collected and cultured in vitro.Bovine adipose cells were treated with 200μmol/L cobalt chloride（CoCl₂）to induce hypoxia,and the adipose cells were transfected with HIF-1αoverexpression and HIF-1αinhibitor.After treatment,cultured bovine adipose cells were collected in vitro.Applied Western Hypoxia-related factors（HIF-1α,GLUT4,FLT-1,PHD2 and VEGF）,key enzymes of lipid metabolism（ATGL,PLIN1,PKA,p-HSL and HSL）,and key molecules of insulin signaling pathway（Akt,p-Akt and PI3K）were detected by Blotting.The in vivo results showed that the expression levels of hypoxia-related proteins HIF-1α（P<0.01）,GLUT4（P<0.05）,FLT-1（P<0.01）,PHD2（P<0.01）and VEGF（P<0.01）in adipose tissue of ketodiseased cows were significantly increased compared with those of healthy cows.Compared with the control group,the key enzyme p-HSL/HSL（P<0.05）,ATGL（P<0.01）,PLIN1（P<0.01）and PKA（P<0.01）were increased.The protein expressions of insulin signal p-Akt/Akt（P<0.01）and PI3K（P<0.01）in ketosis adipose were significantly increased.The results showed that the hypoxy-related molecules HIF-1α（P<0.01）,GLUT4 P<0.01),PHD2（P<0.01）,VEGF（P<0.05）and FLT-1（P<0.05）protein expression level was significantly increased,the above results were consistent with the in vivo experimental results,and the cow adipocyte hypoxia model was successfully constructed.To investigate the effect of HIF-1αon CoCl₂induced hypoxia,we detected HIF-1αoverexpression and the expression of hypoxy-related proteins after interference.The results showed that HIF-1αoverexpression（Ad-HIF1α）further aggravated CoCl₂induced hypoxia in adipocytes.Interference with HIF-1α（Si-HIF1α）alleviated CoCl₂induced adipocyte hypoxia.After HIF-1αoverexpression adenovirus was transfected into adipocytes or HIF-1αinhibitors were added,the results showed that HIF-1αoverexpression（Ad-HIF1α）further increased the lipolysis of adipocytes and enhanced the expression of key molecules of insulin signaling pathway compared with the control group.Interference with HIF-1α（Si-HIF1α）inhibited the expression of key molecules in adipocyte lipolysis and insulin signaling pathway.In conclusion,the high expression of HIF-1αin adipose tissue of cows with ketosis enhances the lipolysis effect of adipose tissue.Under hypoxia condition,increased HIF-1αexpression in bovine adipose cells up-regulated insulin signaling pathway,and inhibition of HIF-1αdown-regulated insulin signaling pathway and lipolysis intensity.These results indicate that HIF-1αplays an important role in maintaining oxygen balance and lipid metabolism homeostasis in adipose tissue of cows with ketosis.