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Effect of Insig-1/SCAP/SREBP-1c Signal on Lipid Metabolism in Hepatocytes under Endoplasmic Reticulum Stress

Author: FangDianLiang
Tutor: ShenZuo
School: Chongqing Medical University
Course: Internal Medicine
Keywords: ERS SREBP-1c Insig-1 SCAP
CLC: R575.5
Type: PhD thesis
Year: 2013
Downloads: 183
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Abstract


Background and objective Non-alcoholic fatty liver disease(NAFLD) is one of the most common chronic liver diseases, which hasbecome an important public health problem. In developed countries, itaffects20~30%adults. Excessive accumulation of lipids (especially fattyacids and triglycerides) in hepatocyte is thought to be the key step.Whatever stressor that disrupts fatty acid intake, β-oxidation and synthesisof lipoprotein could induce abnormal lipid deposition. Increasedendogenous synthesis of hepatic lipid contributes to the development ofNAFLD. Endoplasmic reticulum stress refers to adaptation to insultsintracellularly or extracellularly. It suggests that hepatic steatosis andsteatohepatitis induced by ERS gets great attention. It indicates that ERSdisrupts the balance of lipid metabolism and promotes lipid deposition inliver. Sterol regulatory element binding protein-1c(SREBP-1c),which is thekey transcription factor, upregulates the expression of lipogenic genes,including Fatty Acid Synthase (FAS), Acetyl-CoA Carboxylase (ACC1)and Stearoyl-CoA Desaturase1(SCD1), leading to lipid accumulation. SREBP cleavage-activating protein (SCAP) escorts SREBP from ER toGolgi. Insulin induced gene (Insig) retains the SCAP/SREBP complex inthe ER and blocks the proteolytic activation of SREBP. Merely reportsabout effect of Insig-1/SCAP/SREBP-1c signal pathway in lipidmetabolism were found. Our study is going to explore the effect ofInsig-1/SCAP/SREBP-1c pathway on lipid metabolism under ERS inducedby Thapsigargin in L02and HepG2cells. The clarification andunderstanding of the molecular mechanism of Insig-1/SCAP/SREBP-1csignal pathway in lipid metabolism under ERS could provide new ideas andmethods for preventing NAFLD.Methods1Establishment of the model of steasosis induced by ERS and its possiblemechanism1.1Establishment and verification of the model of steasosis induced byERSThe appropriate concentration of Thapsigargin was determined bydetecting the viability of L02and HepG2cells under differentconcentrations of Tg through Cell counting kit (CCK-8).The GRP-78protein expression levels were determined by Western blot. Ultra-structureof L02and HepG2cells were observed under endoplasmic reticulum stress.1.2Measurement of hepatic lipid under ERSThe concentration of triglycerides and the lipid deposition were assessed through intracellular triglyceride measurement and oil red Ostaining respectively after the induction of1μM Tg for24and48h in L02and HepG2cells1.3Mechanism of steasosis induced by ERSThe mRNA and protein expression of Insig-1、SCAP、SREBP-1c、LXR、FAS and ACC1were detected by Real-Time PCR and Western blot.2The Insig-1/SCAP/SREBP-1c signal pathway involved in lipidmetabolism under ERS2.1Effect of over-expression of Insig-1on lipid metabolism under ERSpCMV6-Insig-1-DDK plasmid containing DDK label protein wasconstructed and transfected into L02and HepG2cells. The proteinexpression of SREBP-1c、 nSREBP-1c and FAS were determined byWestern blot.2.2Effect of inhibition of SREBP-1c on hepatic lipid metabolism underERSThe miRNA targeting SREBP-1c was constructed and transfected toL02and HepG2cells. Triglyceride content and FAS and ACC1proteinexpression were measured respectively after SREBP-1c silencing.Results1Establishment of the model of steasosis induced by ERS and its possiblemechanism1.1Establishment and verification of the hepatic steasosis induced by ERS Cell viability was evaluated through cck-8.There was no seriousdamage after Tg treatment for24h.The inhibition ratio were9.25%、9.95%、11.12%、13.22%and6.17%、6.76%、9.98%、14.81%respectively underthe concentrations of0.5μM、1μM、2μM and4μM Tg for48h.Celldisplayed severely morphologic changes when Tg treatment for72h.According to the references, we chose1μM as the best concentrationfor inducing ERS after24、48h.Compared to the control group, the GRP-78protein increased significantly after Tg treatment in a time-dependentmanner (P<0.05).Tg exposure led to ER dilatation dramatically.1.2Measurement of hepatic lipid under ERSThere was no significant difference about triglyceride content after Tgtreatment for24h (P>0.05), while the lipid droplet deposited obviouslyand triglyceride content increased markedly after Tg treatment for48h inL02and HepG2cells (P<0.05).1.3Mechanism of hepatic steasosis induced by ERSCompared to the control group, the SCAP and SREBP-1c expressionof mRNA and protein increased markedly under ERS in L02and HepG2cells (P<0.05), while the nSREBP-1c increased even more significantly.The protein expression of FAS and ACC1increased as well (P<0.05), whilethere was no significant difference in protein expression of LXR (P>0.05).Tg treatment upregulated Insig-1mRNA in L02cells whiledown-regulated in HepG2cells. Tg treatment decreased Insig-1protein levels significantly in both L02and HepG2cells (P<0.05).2.1Effects of over-expression of Insig-1on lipid metabolism under ERSCompared to the control group, the DDK label protein was clearlydetected after pCMV6-Insig-1-DDK transfection. Insig-1over-expressionreduced intracellular triglyceride content obviously under ERS in L02andHepG2cells (P<0.05).There was no significant difference in SREBP-1cprecursor protein expression between groups (P>0.05).In contrast, thenSREBP-1c and FAS protein decreased significantly (P<0.05).2.2Inhibition of SREBP-1c on hepatic lipid metabolism under ERSSREBP-1c targeted miRNA expression vectors were constructedsuccessfully. SREBP-1c-1miRNA suppressed SREBP-1c proteinexpression dramatically (P<0.05).Compared to the control group,expression of FAS and ACC1proteins were suppressed after SREBP-1cknockdown accordingly (P<0.05).The triglyceride content reducedobviously and the intracellular lipid droplets decreased significantly in L02and HepG2cells.Conclusion1Endoplasmic reticulum stress promotes liver lipid deposition in L02and HepG2cells.2ERS is involved in the activation of SREBP-1c of at transcriptionaland post-transcriptional level. Over-expression of SREBP-1c、FASand ACC1coupled with reduction of Insig-1are closely linked to dys-regulation of lipid metabolism induced by endoplasmicreticulum stress.3The Insig-1/SCAP/SREBP-1c signal pathway contributes to fataccumulation in hepatocytes under endoplasmic reticulum stress.

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Liver metabolic disorders
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