The Effects And Mechanism Of Nobiletin On Lipid Metabolism In Liver-specific Knockout Of Bmal1 Mice Fed With High-Fat Diet

Objectives:Disruption of liver circadian rhythms is closely related to metabolic syndrome.Liver plays a central role in lipid homeostasis.Mice with liver-specific knockout core clock gene exhibited metabolic disorders including dyslipidemia.Small-molecule modulators enhancing clock system are emerging as powerful tools to ameliorate metabolic disorders.Nobiletin(NOB),a naturally occurring small-molecule compound enriched in citrus peels,displays potential lipid lowering and circadian-enhancing properties in diet-induced obese mice and db/db mutant mice.However,the requirements of specific clock gene for the beneficial effects of NOB on metabolism are not well understood.This study intends to establish a high-fat diet(HFD)-induced liver-specific knockout of Bmal1(Bmal1LKO)mice model with NOB intervention,and investigate the effects of NOB on lipid metabolism in mice with specific organ rhythm disruption.Methods:1.Liver tissue and serum samples of wild type(WT)and Bmal1LKO mice fed normal chow diet were collected at 7 time points,then liver and serum triglyceride(TG),serum cholesterol(TC),serum glucose,liver clock and clock-controlled related metabolic genes were detected to determine the time of NOB gavage.2.Mice with Bmal1LKO fed a high-fat diet ad libitum 8 weeks.Throughout the last 4 weeks,the mice were treated with either vehicle(0.5%Sodium carboxymethyl cellulose)or NOB(200 mg/kg body weight)via oral gavage every day,in the time window of ZT4-ZT5.Liver and serum lipids,serum lipidome,liver and adipose clock genes and lipid metabolic genes were measured after mice were sacrificed at ZT4-ZT5.Results:1.In the normal chow diet,the contents of TG and TC in liver and serum of Bmal1LKO mice lost the diurnal variation,and the expression pattern of lipid synthesis genes was not affected,but the oscillations of clock genes and genes that regulated cholesterol synthesis(Hmgcr)and conversion into bile acid(Cyp7a1,Cyp8b1)were lost.Bmal1LKO mice were fed with HFD exhibited spendthrift metabolic phenotype including lower body weight,lower fat tissue,lower liver fat and TG,lower serum glucose in comparison with Bmal1flox/flox mice with the same quantity of food intake.2.Serum lipidomics analysis showed that among the 25 types of lipids detected,NOB increased 12 types of lipids(BG,BMP,PE-O,etc.)in Bmal1flox/flox mice and Bmal1LKO mice,and decreased 7 types of lipids(TAG,PS,SL,etc.).NOB increased only Bmal1flox/flox mice but did not change the PI,PE,PA and LPE lipids in Bmal1LKO mice.Notably,NOB decreased serum LPC in Bmal1flox/flox mice but increased in Bmal1LKO mice.Serum lipidomics suggests that NOB affects serum lipid composition through liver Bmal1 and corresponds to liver and serum TC.3.Compared with vehicle NOB pronouncedly decreased fat tissue weight and serum free fatty acid in both Bmal1flox/flox and Bmal1LKO mice.NOB decreased live TG in accompany with the decreasing mRNA levels of DNL genes(Srebp1c,Acaca,Fasn,Scd1)in both genotypes.There were no significant interactions between genotypes and NOB treatment.NOB increased serum VLDL in Bmal1LKO mice,which is consistent with that NOB increased liver Shp mRNA level while decreased Mttp mRNA level,the key genes that facilitate VLDL assembly and secretion.NOB decreased serum TG in Bmal1flox/flox mice but on effect was observed in Bmal1LKO.NOB decreased the weight of white adipose tissue in Bmal1flox/flox and Bmal1LKO mice,corresponding to decreased expression of lipid synthesis genes and increased expression of lipid hydrolysis genes,which were not affected by liver Bmal1.4.NOB decreased liver and serum TC level in Bmal1flox/flox mice in consistency with the decreasing Hmgcr and increasing Cyp7a1,Cyp8b1,Gata4 and Abcg5 mRNA levels in liver,which meant that NOB led to less cholesterol synthesis and more conversion into bile acid and export into intestine.On the contrary,in Bmal1LKO mice NOB increased Hmgcr mRNA level and had no effects on the genes related to bile acid synthesis and cholesterol excretion,which might contribute to the elevation of liver and serum TC in NOB-treated Bmal1LKO mice.Conclusions:1.In HFD-fed mice,NOB inhibited liver de novo lipogenesis(DNL)and decreased liver TG independently of liver Bmal1.2.The effects of NOB on serum lipid metabolites was changed by the absence of liver Bmal1,which was specifically shown as decrease in serum LPC in Bmal1flox/flox mice but increase in Bmal1LKO mice,suggesting that the beneficial effects of NOB on liver lipid metabolism was reversed by the absence of liver Bmal1.3.The specific depletion of liver Bmal1 reverse the beneficial effects of NOB on hepatic cholesterol homeostasis metabolism,and mRNA expression levels of related genes suggest that these effects are achieved by altering hepatic cholesterol synthesis(Hmgcr),transformation(Cyp7a1Cyp8b1)and excretion(Gata4,Abcg5)as well as VLDL assembly and secretion(Shp,Mttp)processes.