Endoplasmic Reticulum Stress-mediated Autophagy And Apoptosis Alleviate Dietary Fat-induced Triglyceride Accumulation In The Intestine Of Yellow Catfish

Lipids is one of important macronutrients for formulated feed.High dietary lipid leads to fat deposition in the intestine,but the underlying mechanism is unknown.We used yellow catfish intestine and their isolated intestinal epithelial cells to test the hypothesis that endoplasmic reticulum(ER)stress,autophagy,and apoptosis mediate fat-induced changes in lipid metabolism.Results are as follows:1 High-fat diet induced endoplasmic reticulum stress,autophagy,apoptosis and lipid deposition in the intestine of Pelteobagrus fulvidracoMale and female yellow catfish(3.79 ± 0.16 g)were fed diets containing lipid at 6.98%(low fat diet,LFD),11.3%(middle fat diet,MFD),or 15.4%(high fat diet,HFD)for 8 wk.TG content,enzymatic activity and qPCR assays were performed on the intestine.Compared to LFD and MFD,HFD increased TG content and activities of lipogenic enzymes(6PGD,ME,ICDH and FAS),up-regulated mRNA levels of genes involved in lipogenesis(6pgd,acca,fas and srebp 1c),lipolysis(cpt1,accb,hsla and hslb),FA transport proteins i-fabp,ER stress(grp78,ire1,xbp1,traf2 and atf 6),autophagy(atg1b,lc3 a,lc3b and atg3)and apoptosis(baxa,caspase 3a and caspase 3b).Thus,HFD up-regulated lipogenesis,lipolysis and FA transport,induced ER stress,activated autophagy and apoptosis in the intestine.2 Endoplasmic reticulum stress-mediated autophagy and apoptosis alleviate fatty acid-induced triglyceride accumulation of the intestinal epithelial cells of yellow catfishIn order to investigate the mechanism of dietary fat affecting intestinal fat deposition in Pelteobagrus fulvidraco,isolated intestinal epithelial cells were pretreated with inhibitors(3-methyladenine(3-MA),4-phenylbutyric acid(4-PBA)or Ac-DVED-CHO(AC))for 1 hour and cultured in control solution or in different concentrations of fatty acids for 24 hours.Preincubation with 4-PBA,utilizing immunoblotting,QPCR,determination of lipogenesis enzyme activitis,triglycerides and fatty acids,detection of the effect of endoplasmic reticulum stress on fatty acid-induced triglycerides deposition in intestinal epithelial cells.Preincubation with 4-PBA,QPCR,low cytometry,laser confocal microscopy combine with acridine orange(AO),Annexin V-FITC/PI staining and caspase 3 enzyme activity assay were used to detect the interaction between endoplasmic reticulum stress and autophagy and apoptosis.Preincubation with 3-MA,utilizing QPCR,cytometry,laser confocal microscopy combine with acridine orange(AO),MDC,Lyso Trscker and BODIPY493/503 staining,transmission electron microscopy,immunoblotting,QPCR,determination of triglycerides,CPT1 enzyme activity assay,HSL and ATGL ELISA kits to measure the autophagy level and lipophagy induced by autophagy-mediated fatty acids.Preincubation with Ac-DVED-CHO,utilizing QPCR,cytometry,laser confocal microscopy combine with Annexin V-FITC/PI staining,immunoblotting and determination of triglycerides,caspase 3 enzyme activity and CPT1 enzyme activity assay,we determined apoptosis levels and apoptosis mediates fatty acid-induced ?-oxidation.The results showed that fatty acids increased endoplasmic reticulum stress level.Endoplasmic reticulum stress mediated fatty acid-induced triglycerides deposition level,autophagy activation level and apoptotic activation level.Fatty acids increased autophagy levels,and autophagy mediated fatty acid-induced lipophagy levels.Fatty acids increased the apoptotic level,and apoptotic mediated fatty acids induced fat decomposition.In conclusion,endoplasmic reticulum stress mediates fatty acid-induced fat deposition,autophagy and apoptosis,and autophagy and apoptosis mediate fatty acid-induced lipophagy and ?-oxidation to reduce fat deposition.Fatty acid up-regulated lipogenesis,lipolysis and fatty acid transport,induced ER stress,activated autophagy and apoptosis;ER stress,autophagy and apoptosis played important regulatory roles in fat-induced changes in lipid metabolism in the intestinal epithelial cells of yellow catfish.