Effects Of Honokiol And High-fat Intervention On White Fat Browning In Mice And Its Mechanisms

The study was aimed to investigate the effects of honokiol(HON)and high fat intervention on the browning of white fat and its molecular mechanism in mice.According to the condition of body weight,60 C57BL/6 mice aged 6 weeks and in good status were randomly divided into 5 groups,with 6 replicates,each replicate has 2 mice,after 3 days of adaptive feeding.The five groups were fed normal chow diet(ND),high fat diet(HFD),and HFD supplementing 0.02%,0.04% and 0.08% HON for 8 weeks.Growth performance,adipose tissue weight and morphology,levels of blood lipid,the expression of browning of epididymal white adipose tissue and its related protein and mRNA,the microbiota and the metabolites of cecum was determined.The results showed that:(1)Effects of HON on growth performance of fed mice with high fat dietThe final body weight and feed conversion rate of HFD-fed mice increased significantly(P<0.05),while the total intake(TFI)decreased significantly(P<0.05)compared with ND-fed mice.HON supplementing could significantly inhibit the weight gain of HFD-fed mice(P<0.05),HON with low and high-dose groups significantly decreased FCR(P<0.05),while had no significant effect of middle-dose(P>0.05).HFD+0.08%HON group significantly decreased TFI of mice(P<0.05)compare to other groups supplementing HON(P>0.05).Those results indicated that the inhibition of weight gain in high fat fed mice by HON was achieved by reducing feed conversion rate,not by reducing feed intake and energy intake.(2)Effects of HON on adipose tissue weight,tissue Morphology and Blood Lipid level in High-fat-fed miceIn the HFD-fed group,the weight of WAT(epididymal,inguinal,mesenteric,perirenal),levels of serum triglyceride(TG),cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)was significantly increased(P <0.05),and the level of high-density lipoprotein cholesterol(HDL-C)was significantly lower(P<0.05)compared to ND-fed group.Compared with the HFD group,the addition of the low dose of HON could significantly lower epididymal,inguinal,mesenteric WAT weight(P<0.05),the middle dose HON could inhibit the increase in the weight of the WAT,but the effect was not significant(P>0.05),The addition of high-dose of HON could not only significantly reduce epididymal,inguinal,mesenteric,perirenal WAT weight,but also reduce the weight of BAT(P<0.05).What's more,the addition of HON could significantly lower the levels of TG,TC,LDL-C in the mice(P<0.05).HE staining of epididymal adipose tissue was found that the diameter of the adipose tissue cells in HFD-fed group increased significantly compared with ND-fed group(P<0.05).Compared with HFD group,supplementing HON could significantly reduce the diameter of the adipose tissue cells of the mice caused by high-fat feeding(P<0.05).These results suggested that HON inhibited the weight gain of high fat-fed mice by increasing the mobilization and utilization of adipose tissue and thus reducing the weight of WAT.(3)Effects of HON on browning and its related mRNA and protein expression of epididymal WAT in mice fed with high fatTMT-labeled quantitative proteomics was used to sequence the epididymal WAT of ND,HFD,HFD+0.08%HON of mice and finally identify the differential protein,steroid O-acyltransferase 1(SOAT1),which is closely related to fatty acid synthesis.The differential protein was verified by RT-PCR and Western-blot,and it was found that the mRNA and protein expression of SOAT1 in epididymal WAT of HFD-fed group was significantly higher than that in ND-fed group(P<0.05).In comparison to HFD group,supplementing HON could significantly decrease the mRNA and protein expression of SOAT1(P<0.05).In addition,compared to ND-fed group of mice,HFD-fed group significantly inhibited the expression of epididymal uncoupling protein 1(UCP1),phosphorylated AMP-activated protein kinase(P-AMPK)/AMP-activated protein kinase(AMPK)and its downstream protein phosphorylation acetyl coenzyme A carboxylase(P-ACC)/acetyl coenzyme A carboxylase(ACC)and carnitine palmItoyl transferase 1A(CPT1A)protein and the mRNA expression of UCP1(P<0.05),and significantly increased the expression of CCAAT/ enhancer binding protein(C/EBP?),sterol regulatory element-binding protein 1C(SREBP-1C)mRNA,and fatty acid synthetase(FAS),ACC mRNA(P<0.05),which are closely related to SOAT1 and fatty acid synthesis.Compared with HFD group,the addition of HON significantly increased UCP1,P-AMPK/AMPK and its downstream P-ACC/ACC,CPT1 A protein expression,and mRNA expression of UCP1,PRDM16(P<0.05),significantly decreased the protein expression of C/EBP? protein and FAS,SREBP-1C mRNA expression(P<0.05).Those results revealed that supplementing HON could promote the browning of WAT in mice fed with high fat diet,it was also an important reason that adding HON could inhibit the weight gain and decrease the feed conversion rate of mice.Moreover,AMPK-ACC-CPT1 A and C/EBP?-SOAT1-SREBP-1C pathway may be an important molecular mechanism of WAT browning.(4)Effects of HON on short chain fatty acids and microbial Indexes of cecum contents in mice fed with High fatHFD-fed group significantly reduced the level of cecum butyrate(P<0.05),but had no significant effect on the acetate,propionate and isovalerate compared to ND-fed group of mice(P>0.05).Compared with the HFD group,the addition of low,high-dose HON could significantly increase the level of butyrate(P<0.05),and the high dose of HON could also markedly increase the level of acetate,propionate and total SCFAs(P<0.05).What's more,the results of 16 S RNA sequencing showed that HFD-fed group remarkably decreased abundance of Akkermancia,increased abundance of Oscillospira(P<0.05),but had no significant effect on the abundance of Bacteroides(P>0.05)compared with ND group.Adding HON dramatically elevated the abundance of Akkermansia,and decreased the abundance of Oscillospira(P<0.05),and remarkably elevated the abundance of Bacteroides that mainly produced short-chain fatty acids with the addition of high-dose HON(P<0.05)compared with HFD group.Correlation analysis revealed that Akkermansia and total short chain fatty acids were negatively correlated with body weight and positively correlated with UCP1 protein expression.Those results indicated that HON could be beneficial to the organism by regulating the composition of gut microbiota and the level of metabolite,and the browning of WAT might be an important way to play a role.In conclusion,HON can promote the browning of WAT and improve the imbalance of lipid metabolism and energy metabolism induced by high fat feeding in mice.AMPK-ACC-CPT1 A and C/EBP?-SOAT1-SREBP-1C pathway is an important molecular mechanism for the induction of browning,and gut microbiota and metabolites are important factors affecting browning of WAT.