The E3 Ubiquitin Ligase Peli1 Ablation Alleviates Diet-Induced Imbalanced Hepatic Energy Expenditure And Insulin Resistance

BackgroundMetabolic syndrome is a group of syndromes including abdominal obesity,hyperglycemia,hypertension and dyslipidemia,and the core features of the pathogenesis are the chronic low inflammation and insulin resistance.The incidence of global metabolic syndrome is increasing every year.Insulin resistance is a common risk factor for metabolic diseases including diabetes,atherosclerosis and other cardiovascular disorders.The pathogenesis of obesity-related insulin resistance has not been thoroughly elucidated,and the effective prevention and treatment methods are absent.TLRs/IL-1R signaling pathways play key roles in innate immune response involving in immune surveillance and host defense.Under the long-term of obesity,elevated levels of free fatty acids,inflammation and activated stress signaling such as JNK and IKKs caused by excessive expansion of adipose tissue contribute to the increased serine phosphorylation of IRS1 and decreased tyrosine phosphorylation of IRS1,and eventually lead to the impaired insulin signaling pathway.In particular,activation of JNK can directly induce serine phosphorylation of IRS1 and promote insulin resistance.Meanwhile,obesity-related intestinal microflora imbalance increases the levels of LPS(lipopolysaccharide,endotoxin)and then activates a variety of signaling pathways through TLRs/IL-1R signaling pathways,promoting in a series of inflammatory factors such as IL-1?,IL-6,TNF?,IL-12p40,IFN?,and resulting the injured insulin signaling pathway in liver,skeletal muscle and other insulin targeting tissues.As a key regulatory molecule in TLRs/IL-1R signaling pathways,E3 ubiquitin ligase Peli1 plays an important role in the regulation of inflammatory response,cell injury and apoptosis.Peli has the FHA domain and the RING domain at its N-terminus and C-terminus,respectively.The most well known part of RING domain is the E3 ubiquitin ligase activity.It is reported that Peli1 knock-out could inhibit LPS or Poly(I:C)-induced the expression of TNF-?,IL-6 in mice,which is related to the decreased ubiquitin of RIP 1 and transcriptional activity of NF-?B.Peli1 can promote the degradation of TRAF3 by promoting the ubiquitination of cIAP and improve the activity of transcription factor AP-1 in the central nervous system.Besides,the negative regulatory factor Smad6 can interact with Peli1 and inhibit IL-1?-induced the activation of related signaling pathways.According to our previous studies in the mouse models of cardiac hypertrophy and ischemia-reperfusion injury,the increased expression of Peli1 and activation of its E3 ubiquitin ligase could enhance pathological remodeling of ventricular chambers,while inhibited Peli1 expression could alleviate inflammatory infiltrates of myocardium which may be related to the decreased ubiquitination of TRAF6 and transcriptional activity of NF-?B.A large number of studies have shown that insulin resistance or type 2 diabetes patients show lipid metabolic disorders and mitochondrial dysfunction.The reduction of mitochondrial oxidative function,mitochondrial biosynthesis,and especially the expression of genes related to mitochondrial oxidative metabolism,such as CPT1,PGC1?,HNF4?,etc.resulting in decreased oxidizing ability and the accumulation of intra-cellular lipid.HNF4?,a liver-enriched nuclear hormone receptor,influences the expression of a series of genes involved in bile acid,lipid,glucose and drugs metabolism.Knockout of HNF4? significantly increases the susceptibility of fatty liver and hyperlipidemia in mice.In addition,the mutations of HNF4? in human can lead to Maturity onset diabetes of the young(MODY).Recent studies have shown that HNF4a can bind to the DR1 domain of the CPT1A promoter and mediate the transcriptional activation of CPT1? together with PGC1?,which promotes the mitochondrial fatty acids oxidation in the liver,and plays an important role in lipid metabolism.It is also reported that increased ?-oxidation of fatty acids can inhibit obesity and improve insulin resistance,which is an important target for intervention of metabolic syndrome.It remains unknown whether Peli1 knock-out could plays a role in obesity-associated lipid metabolic disorders and insulin resistance through regulating HNF4? expression and TLRs/IL-1R signaling pathways.In other words,the molecular mechanisms involved in Peli1 mediated obesity and metabolic syndrome need to be explored further.Objective1.Clarify whether Peli1 knock-out has a protective effect on hepatic lipid metabolic disorders induced by high fat diets,and the molecular mechanism involved.2.Elucidate whether Peli1 knock-out can improve HFD-induced insulin resistance.3.To clarify whether Peli1 knockout has a regulatory role in liver inflammation and the underline molecular mechanisms.Methods1.Models1.1.Animal modelsTo elucidate the effect of Peli1-deficiency on HFD-induced obesity and insulin resistance,we fed mice with high fat diet for 16 weeks.6 weeks C57/BL6 male mice were randomly divided into four groups:wild type+control group(WT-Chow),wild type+high fat diet group(WT-HFD),Peli1 knockout+control diet group(Peli1-/--Chow),Peli1 knockout+ high fat diet group(Peli1-/--HFD).Monitor the effect of Peli1 knock-out on HFD-induced obesity,insulin resistance and metabolic inflammation.1.2.Cell modelExcessive fatty acids of obesity individuals can produce inflammatory mediators and/or activate pattern recognition receptors to activate inflammatory pathways,resulting in metabolic inflammation.In order to elucidate the molecular mechanism of Peli1 in regulating HFD-induced insulin resistance,we stimulated the primary hepatocytes,Kupffer cells and embryonic fibroblasts cells with the free fatty acid(500 ?M)and poly(I:C)(100 ?g/ml)to induce insulin resistance in vitro,detected the levels of inflammatory cytokines and investigated the molecular mechanisms of Peli1 involved in regulating FFA and poly(I:C)induced insulin resistance and inflammatory cytokines secretion.2.Observation targets and MethodsTo evaluate the obesity degree,we recorded body weight weekly,calculated LW/BW,white fat weight/body weight,and detected serological indicators.H/E staining was used to evaluate hepatic lipid deposition,inflammatory infiltration of adipose tissue and adipocyte sizeThe physiological metabolism and behavior of mice were monitored by TSE PhenoMaster System.qRT-PCR was used to calculate the mRNA levels of HNF4?and other lipid metabolic genes.The levels of HNF4? protein in the total protein,cytoplasmic protein and nuclear protein were detected by Western blot.The tyrosine phosphorylation of IRS 1 and the ubiquitination level of HNF4? in the nuclear protein were measured by immunoprecipitation.The insulin sensibility was evaluated by glucose tolerance test and insulin tolerance test.The expression of Peli1,phosphorylation of IRS 1 and phosphorylation of AKT were detected by Western blot.The level of Traf3,cIAP2 protein and phosphorylation of MAPKs were detected by Western blot.The ubiquitination level of Traf3 and cIAP2 were detected by immunoprecipitation.The levels of IRF3 protein and phosphorylation in cytoplasmic protein and nuclear protein were detected by Western blot.The nuclear translocation of NF-?B was detected by EMSA,and the mRNA level of inflammatory cytokines was detected by qRT-PCR.ResultsPart I:Peli1 knockout protected mice from HFD-induced liver lipid metabolic disorders1)Peli1 knockout inhibited the expansion of systemic adipose tissue.The weight of mice was recorded weekly.After 16 weeks,the body weight of WT-Chow group had no significant difference from Peli1-/--Chow group,and the body weight of WT-HFD group was significantly higher than that of WT-Chow group,while Peli1 knock-out could significantly improve this.The weight and the cross-sectional area of the epididymal fat and subcutaneous fat significantly increased compared with the normal diet group after 16 weeks.Meanwhile,the area of brown fat cells and the lipid droplets in the cytoplasm were significantly increased in HFD-fed group compared with the normal diet group.These indicators in Peli1-/--HFD group were significantly improved compared with WT-HFD group.The levels of triglyceride and free fatty acid in serum were significantly increased in HFD-fed group compared with the normal diet group,which were improved in Peli1 knock-out group.2)Peli1 knockout improved the lipid accumulation in liver.The morphology of the mouse liver showed the enlarged,grayish and greasy liver of WT-HFD group,which was improved in Peli1 knock-out group.The LW/BW ratio was also decreased in Peli1 knock-out mice.H&E staining and oil-red O staining showed that the WT-Chow group had distinct cell borders and few lipid droplets in the cytoplasm.Meanwhile,the size of hepatocytes was increased,and there were increased lipid droplets in the cytoplasm in WT-HFD group.Hepatic lipid deposition was significantly improved in Peli1-/--HFD group compared with that in WT-HFD group.3)Peli1 knockout improved systemic metabolism.TSE PhenoMaster System showed the rate of heat generation,oxygen consumption and carbon dioxide production were significantly reduced in HFD-fed mice after 16 weeks,which were reversed in Peli1 knock-out group.However,there was no significant difference in food intake,water consumption and exercise among groups,suggesting that the metabolic rate of Peli1 knock-out mice was higher than that of wild-type mice.In addition,both in day and night,respiratory quotient was significantly decreased in 16-week HFD-fed mice,which was even lower in Peli1-/--HFD group during the day.Moreover,Peli1 knockout could effectively up-regulate the lipid oxidation rate in daytime.Heat generation was mainly in the internal during daytime,suggesting that Peli1 knockout could increase the lipids consumption.4)Peli1 mediated the post-translational modification of HNF-4?.The results of RT-PCR showed that Peli1 had no significant effect on the mRNA levels of lipid metabolism-related genes in skeletal muscle and brown fat,while the genes related to hepatic lipid metabolism were significantly increased after Peli1 knockout,such as carnitine palmitoyltransferase 1?(CPT1A),carnitine palmitoyltransferase(CPT2),long-chain acyl-CoA dehydrogenase(LCAD),mediumchain acyl-CoA dehydrogenase(MACD),peroxisome proliferator activated receptor-coactivator 1?(PGC1?),showing that Peli1 knock out can effectively promote hepatic fat utilization,increase heat generation and fatty acid ?-oxidation.The mRNA level of HNF4? in the livers of HFD-fed group was significantly decreased,which was significantly reversed by Peli1 knockout.The total protein,cytoplasmic protein and nuclear protein were extracted from liver tissues of 16 weeks HFD-fed mice.The HNF4? protein level in the high fat diet group was significantly lower than that in the normal diet group,and the protein expression of HNF4? was abnormally increased in the liver of the Peli1 knock-out groups.FFA or poly(I:C)significantly induce the decreased protein level of HNF4?in the nucleus,which was reversed by Peli1 deletion.Primary hepatocyte pre-treated with proteasome inhibitor(MG 132 or ALLN)or lysosomal inhibitor(chloroquine and ammonium chloride)and stimulated with FFA(4 hr)or poly(I:C)(8 hr)showed that the proteasome inhibitors could reverse decreased HNF4? protein levels caused by the FFA or poly(I:C)stimulation,while the lysosome inhibitors had no significant effect.Stimulated primary hepatocytes with FFA(30min)or poly(I:C)(1hr)significantly increased the levels of total ubiquitination and K48 site ubiquitination,which were significantly reversed by Peli1 deletion.In 293 cells,we also found that Peli1 was essential for ubiquitination of HNF4?.Part ?:Peli1 knockout improved HFD-induced insulin resistance.1)In vivo.The fasting blood glucose and insulin levels in the HFD-fed group were significantly higher than those in the normal diet group,which was reversed in Peli1 knock-out mice.Glucose tolerance test(GTT)and insulin tolerance test(ITT)showed that Peli1 knockout could protect mice from HFD-induced glucose tolerance and insulin resistance.The area under the curve of each group was consistent with the above results in the glucose tolerance test and the insulin tolerance test.The level of p-AKT(ser473)in the WT-HFD group was significantly decreased compared with that in the WT-Chow group,and the levels of p-IRS1(Ser307)and total serine phosphorylation of IRS 1 were significantly increased in the WT-HFD group,which was reversed in Peli1 knock-out mice.It was indicated that Peli1 knockout could effectively improve HFD-induced insulin resistance.2)In vitro.Western blot showed that stimulated primary hepatocytes with 500 ?M FFA for 4 hours to establish the models of insulin resistance.Compared with BSA,FFA could significantly increase the level of p-IRS1(ser307)and decrease the levels of p-tyr-IRS1,p-AKT(Thr308)and p-AKT(ser473),which were reversed by Peli1 deletion.Meanwhile,stimulated primary hepatocytes with 100 ?g/ml poly(I:C)for 8 hr to establish the model of insulin resistance,which was also reversed by Peli1 deficiency.Part ?:Peli1 could regulate FFA-induced activation of Traf3/MAPKs pathway in primary hepatocytes.1)FFA could significantly induce the levels of p-JNK and p-p38 in primary hepatocytes,which was reversed by Peli1 deficiency.However,poly(I:C)did not significantly induce the activation of MAPKs in primary hepatocytes.2)Compared with the control group,the protein level of Traf3 in FFA-treated group was significantly decreased,which was reversed by Peli1 deficiency.In addition,Peli1 deletion significantly inhibited FFA-induced K48 site ubiquitination of Traf3,as well as the ubiquitination of cIAP2.3)Poly(I:C)did not induce the decreased protein level of Traf3.Immunoprecipitation showed that poly(I:C)induced K63 site ubiquitination of Traf3.In addition,poly(I:C)did not affected the ubiquitination levels of cIAP2.4)LPS or poly(I:C)could significantly increase the levels of total IRF3 and p-IRF3 in the nucleus and reduce the cytoplasmic p-IRF3 in MEFs.Likewise,results in primary hepatocytes were consistent with the above results.In addition,Peli1 deletion could inhibit FFA,LPS or poly(I:C)-induced NF-?B nuclear translocation in primary hepatocytes.5)FFA could induce increased levels of pro-inflammatory cytokines,such as IL-1?,IL-6,TNF?,IL-12p40,which were reversed by Peli1 deletion,and the effect of poly(I:C)was weak.ConclusionIn summary,Peli1 knockout significantly improves HFD-induced obesity and insulin resistance.Peli1 interacts with HNF4? and negatively regulates the level of HNF4? at levels of transcription and post-translational modification.On the one hand,Peli1 mediates ubiquitination and degradation of HNF4?,and regulate the expression of lipid metabolism genes.On the other hand,Peli1 promotes MAPKs activation by regulating ubiquitination of c-IAP and TFAF3.Thus,Peli1 plays a key role in diet-induced metabolic regulation.The above studies provide a vital theory for developing effective biological target points and new drugs that can delay the progression of metabolic syndrome.