Hepatic INSIG2 Is Inhibited By IRI And Contributes To Post-transplantation Hyperlipidemia And Hepatic Steatosis Via Akt-Srebp Pathway

Background:Liver transplantation（LT）is the most effective treatment for end-stage liver disease.With the improvement of the long-term survival rate of liver transplant recipients and the increase of donor organs in China,however,chronic diseases in liver transplant recipients have acquired much more attention from the transplant community,e.g.post-transplant hyperlipidemia,cardiovascular disease,and chronic kidney disease,etc.Ischemia-reperfusion injury（IRI）is an important factor which restricts the prognosis of LT recipients.IRI mediates inflammation and oxidative stress by promoting the largely enhanced production of reactive oxygen species in hepatic cells.Under the condition of oxidative stress,liver IRI can induce the remodeling of lipid homeostasis,such as alterations of fatty acids de novo synthesized by hepatic cells and increases of the storage of neutral lipids in hepatic cells,further promotes post-transplantation hyperlipidemia（PTHL）and hepatic steatosis.What’s more,insulin induced gene 2（INSIG2）can response to the insulin and regulate hepatic lipogenesis.However,the mechanism by which liver IRI causes morbid hepatic lipogenesis is still unclear.Therefore,we aim to study the roles of INSIG-SREBP pathway in hepatic lipogenesis after liver IRI.Aim:Based on the clinical observation that recipients after LT are susceptible to hyperlipidemia,this study focuses the expression of INSIG2 induced by LT and liver IRI and aims to explore the mechanism by which INSIG2 contributes to morbid hepatic lipogenesis after LT.Methods:PART 1:Based on the LT model and the liver IRI models,the blood lipid indexes of the animal models with HFD were measured by the automatic biochemical analyzer.The hepatic triglyceride（TG）was stained with oil red O and compared in a semi-quantitative manner by the software Image J.The insulin resistance of mice was detected by measuring fasting blood glucose,fasting insulin concentration and oral glucose tolerance tests.PART 2:Through PCR array,the genes with significant differences caused by LT in rats were screened out（including INSIG2）,and then verified by q RT-PCR and western blotting.Primary hepatocytes of mice were extracted.The hypoxia/reoxygenation（H/R）model of primary hepatocytes was established to simulate liver IRI using hypoxia incubator（O₂ concentration can be as low as 1.0%）,and then the expression of CREBZF and INSIG2 were determined that whether they could be influenced by liver IRI.PART 3:The dual-luciferase reporter assay was used to verify the interaction between CREBZF and INSIG2.The expression of genes related to lipogenesis in IRI liver tissues were examined by western blot.When Insig2 transcription in Hep G2 cells was silenced by Si RNAs and/or Akt was inhibited by the Akt-1/2 inhibitor,the expression of genes related to lipogenesis were examined by western blot and the concentrations of TG and cholesterol in the cells were tested by kits.PART 4:Finally,the transcriptome sequencing data of donor liver tissues before and after LT in the GEO database were compared and analyzed.INSIG2 expression levels in preoperative donor’s liver tissues from hyperlipidemia recipients and non-hyperlipidemia recipients after LT were determined by immunohistochemistry.Results:The LT and the liver IRI model can cause increases of blood lipid indexes and hepatic TG.We prove that with high-fat diet,mice in the IRI and LT group show significant increases in fasting blood glucose levels and decreased insulin secretion capacity.A total of 11 genes in the PCR array including INSIG2 show significant changes after LT and IRI.IRI can inhibit the expression of INSIG2 and promote the increase of CREBZF expression in hepatic tissues and hepatocytes from 3 different perspectives.It is proved that CREBZF co-actives ATF4 to inhibit the transcription activities of the INSIG2.Akt-Srebp signaling is significantly activated in the liver with IRI and in primary hepatocytes with H/R,while the expression of Srebp in Hep G2 cells after H/R is significantly inhibited while treated with Akti-1/2 inhibitors.By silencing the expression of INSIG2 in Hep G2 cells,the Akt-Srebp is activated,the expression of lipogenesis-related genes increases,and the content of intracellular TG and cholesterol show insignificant increase.Akti-1/2 inhibitor treatment can partially reverse the activated lipogenesis genes and increases of intracellular TG and cholesterol concentrations,which are caused by the inhibition of INSIG2 expression.We use the GEO database to retrieve the transcriptome sequencing data of 40 pairs of donor liver tissues before and after the LT.The expression of INSIG2 show significantly inhibition after transplantation.And 56 cases of pre-LT donor liver tissues are tested by immunohistochemistry,and the INSIG2 immunohistochemical score of the recipients with PTHL is significantly lower than that of the none-PTHL.Conclusions:1.LT/liver IRI causes hepatic lipogenesis disorders;2.The expression of donor liver INSIG2 in PTHL recipients is significantly lower than non-PTHL recipients;3.IRI inhibits the expression of INSIG2 through CREBZF activation,then promotes lipogenesis through SREBP.