Effects Of Early Feeding Patterns On Nrg4 Expression In Adipose Tissue And Relationship With Hepatic Lipid Metabolism

Non-alcoholic fatty liver disease(NAFLD)is characterized as diffuse hepatocellular steatosis in the absence of significant alcohol consumption and other obvious definitive liver damage factors.It is closely associated with obesity,and the prevalence of NAFLD in obese children and adolescents is up to 50%.The onset of paediatric NAFLD is asymptomatic.Therefore,in order to develop strategies to effectively prevent NAFLD in adulthood,it is important to better understand the mechanisms by which obesity increases susceptibility to NAFLD in childrenIn 1998,Lucas defined the "Nutritional Programming" as metabolic programming effects of early life nutrition on adult diseases,which means that nutritional status in critical or sensitive period has programming effects on body and organ function.Early life overnutrition is closely related to obesity and NAFLD occurrence in adulthood Liver is the main site of lipid metabolism,and excessive triglyceride(TG)accumulation in NAFLD is primarily caused by the imbalance of hepatic lipid homeostasis between the acquisition(uptake and synthesis)and removal(export and oxidation)of TG/fatty acid.Several rate-limiting enzymes and transcription factors participate in hepatic lipid metabolism.The activities of these enzyme systems are also regulated by dietary.Early life overnutrition displayed increased enzymes activation involved in lipid synthesis in the livers and increased the risk of NAFLD in adulthood However,it is not clear how early nutrition environment mediates programmed changes in hepatic lipid metabolism,and there are no definite targets for prevention and treatment of NAFLDAdipose tissue is the largest endocrine organ in the whole body.It participates in the pathological process of metabolic diseases caused by obesity through the crosstalk with liver,brain and pancreas by adipocytokines,enzymes,transcription factors.Nrg4 is an adipocytokine,belonging to the family of Neuregulin(NRG).Nrg4,an endocrine link between brown adipose tissue(BAT)and liver,negatively regulates hepatic de novo lipogenesis(DNL)and decreases the occurence of NAFLD.Nrg4 is abundant in BAT,followed by subcutaneous adipose tissue(SAT),with the least visceral adipose tissue(VAT),which is consistent with the browning ability of adipose tissue.While cold,drugs and nutrients can induce the Browning change of WAT in different ways,up-regulation the expression of heat-producing genes such as Ucp1 and PGC1,thus increasing the heat production and energy consumption.It is suggested that Nrg4 expression may be increased by inducing brown-like changes in WAT,and then the hepatic lipid metabolism disorder caused by obesity may be reducedLong-chain polyunsaturated fatty acids(ω3-PUFAs),as natural ligands for the peroxisome proliferator-activated receptor γ(PPARy),is a key molecule in the regulation of preadipocyte differentiation,which affects the differentiation and the ultimate differentiation of adipocyte.Studies suggest that after ω3-PUFA diet,the rats could resume normal heat metabolism and reduce adipose tissue accumulation Therefore,while exploring the regulatory pathways of Nrg4 in adipocytes,new members and new mechanisms,involved in the regulation of the differentiation of preadipocytes into browning adipocytes from the point of view of nutritional intervention,will help to open a new field of Nrg4 treatment of NAFLDTherefore,we assumed:1)Early life overnutrition environment could reduce heat production,inhibited adipose tissue Nrg4 expression in rats and increased the occurrence of high fat-induced NAFLD;ω3-PUFA dietary intervention could induce WAT browning,increase heat production and Nrg4 expression,and decrease the risk of NAFLD occurrence;2)ω3 PUFA(EPA)regulated the Nrg4 expression in adipocytes with time and dose stimulation;3)ω3-PUFA(EPA)regulated the browning of adipocytes through PPARy pathway,and promoted Nrg4 expression and secretion in adipocytes;4)Exogenous Nrg4 inhibited gene expression related to lipid synthesis and reduced lipid accumulation in hepatic steatosis cells.In order to verify these hypotheses,we conducted a series of studies,which are reported as follows:Part ⅠEffects of Early Feeding patterns on Nrg4 Expression in Adipose Tissue and Relationship with NAFLDObjectiveTo observe the effects of early feeding patterns on Nrg4 expression in adipose tissues and hepatic lipid metabolism,and to clarify the relationship with NAFLD.Materials and Methods1.Establishment the neonatal overfeeding rat model,Male Sprague-Dawley rat pup litters were assigned randomly to normal litters(NL)or small litters(SL)on postnatal day 3.After weaning,the NL rats were given standard chow and high fat diet(NL-HF),while the SL rats were given standard chow,high fat diet(SL-HF)and ω3-PUFA diet enriched with polyunsaturated fatty acids(SL-FO)until postnatal week 13(W13).2.Monitor the body weight,food intake and body temperature of rats regularly.The changes in glucose and lipid metabolism,liver and adipose tissue weight,liver and adipose tissue morphology were observed at W3 and W13.At the end of the study,liver B-ultrasound was performed,and VO2 consumption,VCO2 production and heat production were monitored by a combined indirect calorimetry system(TSE systems)in W13.3.Real-time qPCR was used to detect mRNA expression of Nrg4,Ucpl,PPARy,PRDM16 and PGC1α in adipose tissue and enzymes related to lipid metabolism in liver.The hepatic TG content was detected with TG test kits.Results1.At the end of the experiment,the body and liver weight of SL rats were higher than those in NL rats(P<0.05).The body weight and liver weight of NL-HF group and SL-HF group were higher than those in the control group,and those of SL-HF rats were highest among groups(P<0.05);in contrast,the gains of body as well as liver weight were lower in SL-FO rats compared with SL rats at W13(P<0.05).2.The body temperature,O2 consumption,CO2 production,and heat production in SL rats were all significantly lower than those in NL rats(P<0.05);the body temperature,O2 consumption,CO2 production and heat production of NL-HF group and SL-HF group were also significantly lower than those of control group(P<0.05);body temperature,O2 consumption,CO2 output and heat production in SL-FO rats were all higher than those in SL rats(P<0.05).3.The liver TG content in the SL group was higher than that in the NL group(P<0.05),and the B-ultrasound showed a slight fatty liver change in SL rats.High fat diet elevated TG content in liver of the NL-HF and SL-HF rats compared with the control group(P<0.05)and presented the fatty liver in rats,and B-ultrasound showed moderate fatty liver in the SL-HF rats.There was no significant difference in hepatic TG content between the NL and SL-FO groups(P>0.05)and no abnormal changes in liver B-ultrasoun.4.Hepatic ACC,SCD1 and FASN and nuclear transcription factor SREBP-1c mRNA expression in SL rats was higher than NL rats at W3 and persisted until W13;high-fat diet accelerated the increase of lipogenesis-related genes expression in SL rats(P<0.05).In SL-FO rats,the expression of above lipid synthesis related genes in the liver was significantly decreased(P<0.05).There was no significant difference in the mRNA expression of genes related to fatty acid uptake,oxidation and export mRNA expression level between NL and SL groups at adulthood(P>0.05)5.Nrg4 was expressed in various tissues of the body,showed as BAT>SAT>VAT>lung,heart,liver or other peripheral organs.Nrg4 expression levels in BAT,SAT and VAT of rats in SL group were significantly lower than those in NL group(P<0.05).High-fat diet reduced Nrg4 expression level in SAT and VAT(P<0.05),but not affected Nrg4 expression level in BAT(P>0.05).Nrg4 expression levels in BAT,SAT and VAT of rats in SL-FO rats increased significantly compared to SL rats(P<0.05)6.The mRNA expression of Ucpl in BAT and VAT of SL,NL-HF and SL-HF rats and PGC1α in BAT,SAT and VAT of rats in SL,NL-HF and SL-HF rats were significantly lower than those in NL rats(P<0.05).The mRNA expression levels of Ucpl in BAT and VAT and PGC1α in BAT,SAT and VAT in SL-FO rats increased significantly compared with those in SL rats(P<0.05)ConclusionThere is a significant depot difference in Nrg4 expression of adipose tissues.Both of lactation and after weaning were the critical period for the regulation of Nrg4 expression in adipose tissue.Early life overnutrition inhibited Nrg4 expression in BAT and WAT and lasted to adulthood,ω3-PUFA diet after weaning induced WAT browning,restored the Nrg4 expression in BAT and WAT,attenuated hepatic lipid synthesis,and finally reduced the risk of NAFLD occurrencePart ⅡThe molecular pathway of the regulation of Nrg4 by eicosapentaennoic acid in adipocytes from different depotsObjectiveTo investigate the regulation and molecular mechanism of Eicosapentaenoic Acid(EPA)on Nrg4 expression in adipocytes at different stages of differentiation and from different sourcesMaterials and Methods1.Culture the Human visveral Preadipocyte(HPA)and induce into mature adipocytes.At various differentiation stages,the HPA cells were treated with EPA in different concentrations and to determine the optimal time and concentration of EPA acting on the expression of Nrg42.The primary preadipocyte were extracted from BAT and SAT of C57BL/6 mice at 3-4 weeks and induced into mature B AC and WAC.At various differentiation stages,the BAC or WAC were treated with EPA in different concentrations and to determine the optimal time and concentration of EPA acting on the expression of Nrg43.Observe the effect of PPARy agonist(Rosiglitazone,Rosiglitazone,Rosi)and PPARy antagonist(GW9662)on the expression and secretion of Nrg4 in HPA adipocytes4.Oil red O staining was used to observe the lipid accumulation in adipocytes Real-time qPCR was used to detect the expression of Nrg4,Ucp1,PPARγ,PGC1α and PRDM16 mRNA in different adipocytes,and the content of Nrg4 protein in the culture supernatant of HPA adipocytes was detected by ELISAResults1.Oil red O staining showed that with the differentiation and maturation of HPA,BAC and WAC,the lipid droplets in the cells increased gradually,and the Nrg4 mRNA expression was significantly up-regulated,and reached its peak on the 12th and 8th day of differentiation of HPA and primary adipocyte,respectively2.EPA intervention had significant time and dose effects.Although late intervention increased Nrg4 mRNA expression,the required EPA concentration(BAC,WAC,HPA cells respectively 20,50,200 μM)is significantly higher than that required for the whole differentiation process(respectively 10,50,10μM)3.Rosiglitazone upregulated the Nrg4 mRNA expression and the content of Nrg4 in cell culture supernatants in HPA at the late stage or throughout the differentiation(P<0.05),while GW9662 inhibited the increase of Nrg4 mRNA expression and Nrg4 content in the culture supernatant of HPA induced by EPA(P<0.05)4.Both EPA and Rosi intervention significantly reduced lipid accumulation in adipocytes,and up-regulated Ucpl and PGC1α mRNA expression(P<0.05),while GW9662 inhibited the up-regulation of Ucpl and PGC1α mRNA expression induced by EPAConclusionNrg4 expression was markedly induced during adipocyte differentiation.EPA raised Nrg4 mRNA expression in HPA,BAC and WAC,and presented the obvious time and dose effect.Long-term exposure to the physiological dose of EPA is more conducive to inducing brown-like changes in adipose cells and up-regulating Nrg4 expression.Activation or inhibition of PPARy significantly affected the expression and secretion levels of Nrg4 in adipocytes,suggesting that PPARγ might be an important pathway for EPA to regulate brown-like changes in adipocytes and promote Nrg4 expression and secretionPart ⅢThe Protective Effect of Nrg4 on Hepatic Lipid MetabolismObjectiveTo investigate the regulatory effect of Nrg4 on lipid metabolism related gene expression in hepatocytesMaterials and MethodsOverexpress ErbB4 in HepG2 cells by adenovirus,induce HepG2 cells to be steatosis by sodium oleate(OA),and and finally the cells treated with recombinant Nrg4 protein(10-200ng/ml)for 48 hours.Observe the effects of Nrg4 intervention on lipid accumulation,TG content,and lipid metabolism-related gene expression in HepG2 cells.Real-time qPCR was used to detect mRNA expression levels of lipid metabolism related genes.Enzyme assay kit was used to detect intracellular TG content Observe the hepatocellular lipid accumulation by Oil red O staining and detect the TG content with TG test kits.Results1.OA induced HepG2 cells with ErbB4 overexpression,with increased lipid droplets and TG content in the cells compared with the control group(P<0.05),and finally established the fatty liver cell model2.After OA-induced,the mRNA expression of lipid synthesis(ACC,SCD1,FASN)and their nuclear transcription factor(SREBP-1c)were significantly increased compared with the control group(P<0.05),while the gene expression of lipid-input(LPL,L-FABP),lipid export(MTP),lipid oxidation(CPT1)and their nuclear transcription factor(PPARα)were significantly lower compared to the control group(P<0.05)2.Nrg4 protein(≥100ng/ml)treated on HepG2 cells with ErbB4 overexpression for 48h,significantly inhibited the up-regulation of intracellular lipid droplets,TG content,and lipogenesis-related genes induced by OA induction(P<0.05),but with no significant effect on the expression of lipid input,oxidation,and export related genesConclusionNrg4 inhibited the up-regulated expression of OA-induced lipid synthesis genes ACC,SCD1,FASN and their nuclear transcription factor SREBP-1c mRNA by ErbB4 pathway,and finally reduced lipid accumulation in cells.