Mechanism Of Exogenous Valine Supplementation On Fat Deposition In Mice

When the energy intake of moving objects is greater than the consumption,fat will accumulate excessively,leading to obesity,accompanied by diabetes,hypertension,hyperlipidemia,fatty liver,cardiovascular disease and other metabolic diseases.A low protein diet can reduce weight without limiting calorie intake.Branched chain amino acids(BCAAs),including valine(Val),leucine(Leu),and isoleucine(Ile),are not only essential amino acids for animal growth and development,but also have important regulatory roles in lipid metabolism and fat deposition.Previous studies have shown that there is a strong correlation between valine metabolism and fat metabolism,which can participate in regulating the fatty acid composition and lipid metabolism of skeletal muscle.However,it is unclear how exogenous valine regulates fat deposition in mouse liver and adipose tissue.Therefore,this study takes KM male mice as the research object,and explores the impact of exogenous valine on their fat deposition by adding valine(w/v)to drinking water,providing a new idea for in-depth understanding of the regulatory mechanism of animal fat deposition,and also providing a reference for the addition of valine in animal feed.The specific results are as follows:(1)Valine and growth and development in male miceAfter a week of adaptive feeding,healthy KM male rats aged 3 weeks were randomly divided into three groups(n=12),namely,the control group(Control),the 0.30% valine group(0.30% valine),and the 0.45% valine group(0.45% valine).Valine was added to drinking water(w/v)for 7 weeks.The results showed that there was no significant difference in food intake among mice(P>0.05),but the body weight of mice treated with valine increased significantly(P>0.05),and that of mice treated with 0.45% valine increased significantly.Valine treatment did not affect the organ indexes of the main organs such as heart,liver,and kidney in mice(P>0.05);However,it significantly affected the spleen index(both significantly increased between 0.45% Valine group and Control group and 0.30% Valine group)(P<0.01).The white fat index and beige fat index in the0.30% Valine group and 0.45% Valine group were significantly higher than those in the Control group(P<0.05);There was no significant difference in brown fat index among the groups,but there was a trend of increase after valine treatment.Adding valine increased the fasting blood glucose(P<0.05),increased gluconeogenesis(P<0.01),and decreased insulin sensitivity(P<0.05 or P<0.01)in mice.Adding valine can induce abnormal glucose metabolism and insulin resistance in mice.Valine treatment changes the contour of liver lobules and the arrangement of hepatocyte cords in a dose dependent manner,increasing liver lipid droplet deposition(HE and oil red O).White adipocytes were significantly enlarged and decreased(P<0.05),while brown adipocytes were significantly decreased and increased(P<0.05).It can be seen that valine can promote fat deposition in mice,induce abnormal glucose metabolism,and produce insulin resistance.(2)Adipose tissue transcriptome sequencingWhite and brown adipose tissue samples were taken from the Control group and0.45% Valine group,and three samples from each group were subjected to transcriptome sequencing.The difference analysis in white adipose tissue obtained 740 differential genes(DEGs)(534 upregulated and 206 downregulated)(screening criteria: | log2 Fold change | ≥ 1,Pvalue<0.05,the same below).The GO functional enrichment analysis of DEGs showed that these DEGs were significantly enriched in multiple biological processes such as transmembrane transport complexes,protein maturation,reproductive process regulation,hormone metabolism,heparin binding,serine hydrolase activity,and steroid binding;The results of KEGG pathway analysis showed that DEGs were involved in signal pathways related to bile secretion,diurnal entrainment,mineral absorption,and cholesterol metabolism.Differential analysis in brown adipose tissue yielded 980 differential genes(DEGs)(517 upregulated and 465 downregulated).The GO function enrichment analysis of DEGs showed that these DEGs were significantly enriched in biological processes such as inflammatory response,adipocyte differentiation,lipid metabolism,polysaccharide metabolism,autophagy,and circadian rhythm;KEGG pathway analysis showed that DEGs were involved in MAPK signaling pathway,type I diabetes,T cell receptor signaling pathway,circadian rhythm and other related signaling pathways.There are 57 differential genes(DEGs)in white fat and brown fat.Based on the transcriptome analysis results,nine DEGs were screened for RT-q PCR validation(white fat: Atp1a1,Apod,Hp,Nceh1,Cacna1 h,Pawr,Sfrp1,Ccn2,Per1;brown fat: C3,Hp,Agl,Per1,Prkaa2,Fabp5,Nr1d1,Zc3h12 a,Per2),and the expression trend of nine DEGs in white fat was basically consistent with that of RNS-seq;The expression trend of seven DEGs in brown fat was basically consistent with that of RNS-seq,while Fabp5 and Zc3h12 a did not change.Transcriptome sequencing results showed that valine treatment altered multiple pathways and genes in white and brown fat in mice,thereby affecting lipid metabolism.(3)Expression of genes and proteins related to valine and fat depositionRT-q PCR showed that valine treatment significantly or extremely significantly increased the fatty synthesis gene C/EBP in the liver α And SCD1 expression,reducing fat β Oxidative gene PPAR α And CPT-1 expression(P<0.05 or P<0.01);Significant or extremely significant increase in white fat synthesis genes SREBP-1c,C/EBP α、Expression of FAS,SCD1,and Acaca(P<0.05 or P<0.01);Significantly reduces beige PPAR α、 Expression of CPT-1 and SCD1(P<0.05 or P<0.01);Significantly increased the expression of SCD1 in brown fat(P<0.01).ELISA detection showed that the liver AMPK concentration in 0.45% Valine group was significantly higher than that in Control group,while P-AMPK/AMPK was significantly lower than that in Control group(P<0.05);The concentrations of SCD1 and AMPK in white fat in 0.45 Valine group were significantly higher than those in Control group,while P-AMPK and P-AMPK/AMPK were significantly lower than those in Control group(P<0.05);The concentration of SCD1 in brown fat in 0.45 Valine group was significantly higher than that in Control group(P<0.05).In summary,exogenous addition of valine can promote weight gain in mice in a low dose dependent manner,alter the microstructure of the liver,and induce glucose metabolism abnormalities and insulin resistance in mice.Exogenous valine treatment can increase the expression of fat synthesis genes in mice and inhibit β Expression of oxidation genes;Increase the expression of SCD1 protein while inhibiting the phosphorylation of AMPK to a certain extent,thereby promoting fat deposition in mice.