| China is the country with the largest pork production and consumption in the world.In2019,China’s pork consumption reached 44.866 million tons,which ranked first in the world.In 2020,China’s meat and eggs production reached 75.19 million tons,in which pork output reached the top,42.25 million tons.With the rapid development of economy and the improvement of living conditions,consumers were more inclined to choose high-quality pork with good color,high tender degree and high lean percentage.Fat deposition was one of the important factors affecting pork quality,and the fat content of pork was directly related to pork flavor and taste.At the same time,excessive fat deposition increased the carcass fat percentage,affect the reproductive performance and reduce the feed conversion ratio.Therefore,the reasonable deposition of pig fat was not only one of the important factors to promote the sustainable development of pig farming in China,but also the research focus in the field of animal nutrition.Branched-chain amino acids(BCAA)were a class of neutral amino acids with similar molecular structure,including leucine,valine and isoleucine,which contained branched aliphatic hydrocarbon chains on theα-carbon chain.BCAA were essential amino acids that could not be synthesized by domestic animals,and played a very important role in regulating glucose and lipid metabolism,protein metabolism and energy supply.Studies showed that BCAA increased daily gain and feed utilization,decreased body fat percentage and improved production performance.Besides,BCAA affected the m RNA N~6-methyladenine(m~6A)level of the partial lipid metabolism genes.But the effect and mechanism of BCAA on fat deposition was still unclear.m~6A was the most common RNA methylation modification in eukaryotic cells,and regulated a variety of biological processes such as adipocyte differentiation and tumgenesis.Therefore,we hypothesized BCAA regulated fat deposition via m~6A.Our study used mice as animal model and 3T3-L1 cells as cell model,to explore the effect of BCAA on fat deposition.The mechanism of BCAA was further explored from the perspective of m RNA m~6A,which laid a theoretical foundation for the application of BCAA in animal production.The main results were as follows:1.Effects of BCAA on fat deposition and metabolism in miceMale 4-week-old C57BL/6J mice were divided into HF group and HF+BCAA group,with11 mice in each group.HF group was with high fat diet,HF+BCAA group was with high fat diet and 5%BCAA in water,feeding for 10 weeks.The results showed there was no significant difference in food intake and water intake between HF group and HF+BCAA group.Compared with HF group,the body weight and fat mass in HF+BCAA group were significantly reduced(P<0.05).The weight of subcutaneous inguinal WAT was reduced by about 50%,and visceral epididymal WAT decreased about 65%.Mice in HF+BCAA group had lower blood glucose under fasting condition(P<0.05),better glucose tolerance(P<0.05)and insulin sensitivity(P<0.05)than that in HF group.These results suggest that BCAA significantly inhibits fat deposition in mice,improves glucose metabolism.2.Effect of BCAA on adipocyte differentiation and polyesterIn this study,3T3-L1 cells were treated by gradient BCAA(0,10,20,30 and 40 m M).The results showed that BCAA inhibited adipocyte polyester in a dose-dependent manner and inhibited the expression of PPARγ,C/EBPαand FABP4(P<0.05).Compared with the control group,BCAA significantly increased the m RNA m~6A level of white adipose tissue and 3T3-L1cells,and decreased the expression of FTO protein.OE-FTO treatment increased FTO expression in BCAA group and partially restored lipid deposition in BCAA group.These results indicate that BCAA inhibit adipocyte polyester and differentiation by decreasing FTO expression and increasing m~6A m RNA level.3.BCAA inhibits adipocyte cycle(1)BCAA inhibits the clonal proliferation of adipocytes.In order to explore the mechanism of BCAA inhibiting fat deposition,we first examined the effects of BCAA on different differentiation stages of precursor adipocytes.3T3-L1 cells were treated with 30 m M BCAA at D0-D2,D0-D4 and D2-D4 stages.The results showed that BCAA inhibited more fat deposition in D0-D2 than in D2-D4.The main biological event at the stage is mitotic clonal expansion of the precursor adipocytes.Thus,we utilized flow cytometry to further analyze the effect of BCAA on cell cycle at the MCE stage.The results showed that the relative number of G0-G1 phase cells in BCAA group increased in a concentration-dependent manner,indicating that BCAA retarded cell cycle progression.RT-q PCR and western blot analysis showed that BCCA significantly inhibited the expression of CCNA2 and CDK2(two key MCE regulatory factors).These results suggest that BCAA inhibits CCNA2 and CDK2 to blocks cell cycle progression,further inhibits fat deposition.(2)BCAA inhibits CCNA2 and CDK2 expression in an m~6A dependent manner by regulating FTO expression.Previous study showed that BCAA regulated lipid metabolism by affecting m~6A levels of lipid metabolism genes.Therefore,we hypothesized whether BCAA affected the expression of CCNA2 and CDK2 in an m~6A-dependent way.The results showed that BCAA increased m~6A levels of CDK2 and CCNA2 m RNA(P<0.05),and accelerated the degradation of CDK2 and CCNA2 m RNA(P<0.05).Considering that YTHDF2,one of the m~6A binding proteins,recognized m RNA containing m~6A modification and accelerate its degradation,we used si YTHDF2 to inhibit the protein expression of YTHDF2,and found that si YTHDF2 significantly increased the m RNA expression levels of CCNA2 and CDK2(P<0.05).Notably,inhibition of YTHDF2 restored the expression of CDK2 and CCNA2 m RNA in BCAA-induced cells,and partially restored lipid deposition.These indicated that BCAA increased CCNA2 and CDK2 m RNA m~6A level,and inhibited CCNA2 and CDK2 expression via YTHDF2.In consideration of the result that BCAA inhibited fat deposition by inhibiting the expression of m~6A demethylase FTO,we speculated that BCAA inhibits CCNA2 and CDK2 in an m~6A dependent manner via regulating the expression of FTO.Our results showed that CCNA2 and CDK2 m RNA and protein expression were significantly decreased after inhibiting endogenous FTO expression in 3T3-L1 cells(P<0.05).CCNA2 and CDK2 protein expression levels were significantly increased in FTO overexpressed cells(P<0.05).In addition,inhibition of YTHDF2 partially restored CDK2 and CCNA2 m RNA expression in si FTO group cells.The above indicated that BCAA increased CCNA2 and CDK2 m RNA m~6A levels by restraining FTO expression,and inhibited CCNA2 and CDK2 expressions via YTHDF2.(3)BCAA regulates NADPH metabolism,to inhibit FTO expression and improve m~6A level.To explore the mechanism how BCAA regulated FTO expression and m~6A level,the study examined the effects of BCAA on NADPH levels in 3T3-L1 cells,WAT and serum.The results showed that BCAA significantly reduced the content of NADPH in 3T3-L1 cells,WAT and serum(P<0.05),decreased the ratio of NADPH/NADP~+in WAT(P<0.05).To further confirm that NADPH mediated the inhibitory effect of BCAA on FTO,NADPH was added to the medium of BCAA-treated 3T3-L1 cells.The results manifested the decreasing of FTO expression induced by BCAA was effectively compensated,and the increase of m~6A level induced by BCAA was effectively inhibited(P<0.05).These results suggest that BCAA regulates FTO protein expression and m~6A level through NADPH.To further explore the mechanism of BCAA-induced NADPH reduction,we examined the expression of major proteins(G6PD and NADK)that determined NADPH level,and found that BCAA significantly inhibited the expression of G6PD in 3T3-L1 cells and WAT.The results showed that adding G6PD activator KCL significantly compensated the NADPH level,remarkably increased the expression of FTO and significantly decreased the m RNA m~6A level in BCAA-treated 3T3-L1 cells.These results suggest that BCAA affects m~6A level by inhibiting G6PD expression to inhibit FTO expression.In conclusion,BCAA reduces the expression of G6PD and NADPH,then downregulates the expression of FTO,increased the m~6A levels of CCNA2 and CDK2,down-regulates the expression of CCNA2 and CDK2 via YTHDF2,blocks the cell cycle at the MCE stage to inhibits the adipocyte differentiation and polyester. |
