The Regulation And Mechanism Of Dacinostat On Energy Metabolism In Mice Fed High-fat Diet

Excessive fat deposition will cause the quality of livestock products to decline,and at the same time obesity has become one of the diseases that endanger human health worldwide,and is the main cause of insulin resistance,type II diabetes and non-alcoholic fatty liver disease.In present study,through network pharmacology screening,a potential compound that regulates fat deposition,dacinostat,was discovered.Subsequently,we treated the mice fed with high-fat diets with dacinostat by intraperitoneal injection and conducted the experiments in vitro to evaluate the effect of dacinostat on lipid metabolism and energy homeostasis in mice.The results will be helpful to clarify the role of dacinostat in the energy homeostasis of animals and provide theoretical basis for the regulation of animal fat deposition,obesity,and related diseases.1.Add 10 n M dacinostat to the induction medium II of mouse primary adipocytes for4-6 days.Lipid accumulation,mRNA and protein expression of genes involved in lipogenesis,lipolysis and mitochondrial metabolism were measured.The lipid accumulation in mouse primary white adipocytes showed a significant decrease after treated with dacinostat(P<0.01).Further,dacinostat significantly reduced mRNA expression of lipogenic-related genes,including Cebpb,Pparg,Cd36,Fabp4,Acc1,Fasn and Dgat2.Protein expression of C/EBP?,Ppar?and FAS was also down-regulated(P<0.05).Meanwhile,dacinostat significantly induced the protein expression of ATGL and HSL(P<0.05).On the other hand,dacinostat significantly increased the mRNA expression levels of BAT marker genes(including Prdm16,Ppargc1a,Cox7a1,Ucp1)and beige-related genes(including Cd137,Tmem26,Tbx1),also significantly increased the protein expression levels of UCP1 and PGC1?(P<0.05).In mouse primary brown adipocytes,dacinostat also significantly reduce intracellular lipid accumulation,and significantly reduce the mRNA expression levels of lipogenic-related genes,including Cebpb,Pparg,Cd36,Fasn and Dgat2.Meanwhile,dacinostat significantly increased the protein expression levels of ATGL and HSL.On the other hand,dacinostat significantly increased the mRNA expression levels of the BAT marker genes(including Prdm16,Ppargc1a,Cox7a1,Ucp1)and significantly increased the protein expression levels of UCP1 and PGC1?.In summary,dacinostat may reduce lipid accumulation in adipocytes via inducing browning or beige of mouse primary white adipocytes and activating thermogenesis of mouse primary brown adipocytes to promote lipolysis and inhibit lipogenesis.2.8-week-old male C57BL/6J mice were continuously fed with a high-fat diet for 9weeks,during which they were injected intraperitoneally with 6.6%DMSO and dacinostat(1mg/kg body weight)dissolved in 6.6%DMSO.Then we evaluated growth biological traits,glucose tolerance,insulin sensitivity,serum biochemical indicators,body temperature and cold tolerance in mice.The results showed,dacinostat could significantly reduce the bodyweight gain of mice induced by high-fat diets without affecting food intake and intestinal absorption for triglycerides(P<0.05).Also,dacinostat significantly improved glucose tolerance and insulin sensitivity(P<0.05).Meanwhile,compared with control group,the hepatosomatic index and level of TG in liver were significantly reduced(P<0.01).In addition,dacinostat significantly increased the surface temperature on the position of the scapular BAT and inguinal WAT(P<0.05).The tolerance to cold challenge was also significantly increased(P<0.01).Taken together,dacinostat may increase thermogenesis to increase energy metabolism levels,thereby prevent HFD-induced obesity,improve glucose tolerance and insulin sensitivity,and reduce ectopic lipid deposition in liver in mice.3.The muti-position white adipose tissue(WAT)and scapular brown adipose tissue(BAT)were dissected and extracted.Then we observed the morphology,calculated the weight ratio,measured the mitochondrial DNA content,mRNA and protein expression of mitochondrial-related genes.Acetylated protein expression levels and ChIP-qPCR were also be analyzed.The results showed,dacinostat significantly reduced the proportion of adipose tissue in body(P<0.05)without affecting the weight of main organs(such as heart,spleen,lung and kidney)and main muscles(gastrocnemius(Gas)and anterior tibial(TA)muscles).At the same time,dacinostat significantly increased the number of adipocytes with a cross-sectional area of less than 3,000?m~2 in e WAT(P<0.05)but significantly reduced the number of adipocytes with a cross-sectional area greater than 5000?m~2(P<0.01).Similarly,in iWAT the number of adipocytes with a cross-sectional area of 1000-3000?m~2 increased significantly(P<0.01)but the number of adipocytes with a cross-sectional area greater than7000?m~2 was extremely reduced(P<0.01).At the same time,dacinostat significantly increased the mt DNA/g DNA ratio in BAT(P<0.01)and iWAT and significantly increased the mRNA expression levels of mitochondrial-related genes(including Ucp1,Ppargc1a,Cidea,Cox7a1,Cpt1b),also significantly increased the protein expression levels of UCP1and PGC1?(P<0.05).Moreover,dacinostat also significantly increased the acetylation levels of proteins in BAT and iWAT(P<0.05),especially acetylation at histone H3 lysine 27(H3K27ac).The ChIP-qPCR analysis of H3K27ac showed that dacinostat significantly increased acetylation on Ucp1 transcription start site,Ucp1 enhancers,and the MEF or cAMP response element(CRE)region of Pgc-1?in brown and white adipocytes(P<0.01).In a word,dacinostat may promote Ucp1 and Pgc-1?transcription activation by inducing acetylation on Ucp1 transcription start site,Ucp1 enhancers,and the MEF or cAMP response element(CRE)region of Pgc-1?,and then activate BAT and induce WAT browning,ultimately reducing lipid deposition in adipose tissue.In summary,dacinostat promotes Ucp1 and Pgc-1?transcription activation by inducing acetylation on Ucp1 transcription start site,Ucp1 enhancers,and the MEF or cAMP response element(CRE)region of Pgc-1?,and then activate BAT and induce WAT browning,increased energy expenditure in mice,ultimately help mice prevent HFD-induced obesity.