Effect Of Leucine On Browning And Mitochondrial Biosynthesis In Subcutaneous And Intramuscular Adipocytes Of Yellow Cattle

Fat is an important form of nutrient deposition in beef cattle fattening and feeding.The location and quantity of fat deposition affect the cattle carcass quality and meat quality of cattle,as well as production costs and breeding profits.Improving intramuscular fat deposition and reducing subcutaneous fat deposition are targets for cost-effective production.The transformation of white adipocytes into beige adipocytes under certain stimulation is called "white adipocyte browning".Beige adipocytes can maintain the temperature of young animals through non-shiveringly thermogenesis and promote the dissipation of chemical energy as heat,reducing fat deposition and improving meat quality as well as brown adipocytes.Leucine,as an important functional amino acid,not only promotes protein synthesis but also plays a certain regulatory role in lipid metabolism.However,the regulatory mechanism of Leu on white adipocyte browning is still unclear.Therefore,this study isolated subcutaneous and intramuscular primary preadipocytes from yellow cattle to investigate the regulation effects of leucine on the browning and mitochondrial biogenesis of white adipocytes in cattle.This study has important implications for exploring the nutritional metabolism regulation of fat and the nutrient deposition of beef cattle.Trial 1.Isolation and identification of subcutaneous and intramuscular preadipocytes in yellow cattle.After obtaining subcutaneous adipose tissue and longissimus dorsi muscle tissue from cattle,preadipocytes were isolated and cultured,then growth curve determination and immunohistochemical identification were performed.Oil red O staining was performed during the differentiation process of preadipocytes,and the relative m RNA expression of the key differentiating genes PPARγ,C/EBPα,ACC,FABP4 and ADIPOQ was detected by RT-PCR.The results were as follows:(1)When subcutaneous and intramuscular preadipocytes were isolated and cultured for 1d,some cells adhered to the wall and presented a short spindle shape,which gradually transformed into a fiber shape.The cells entered the logarithmic growth phase at D3 and the plateau phase at D7,and the growth curve was "S" shaped.The preadipocyte marker protein PREF-1 was positively expressed fluorescently and located at the cell membrane.As the time of adipocyte differentiation increased,the content of intracellular lipid droplets significantly increased(P<0.05).After 9 days of differentiation,large round lipid droplets were formed and stained red with oil red O.The relative m RNA expression of PPARγ,C/EBPα,and FABP4 increased initially and then decreased,reaching their peak on D6 in adipocyte differentiation(P<0.05).The relative m RNA expression of ACC significantly increased on D6-9 in adipocyte differentiation(P<0.05).The relative m RNA expression level of ADIPOQ gradually increased and reached its peak on D9 during adipocyte differentiation(P<0.05).(2)During the 3-4 days of culture,the growth rate of intramuscular adipocytes was significantly higher than that of subcutaneous adipocytes(P<0.05).At the end of differentiation,the lipid content in subcutaneous adipocytes was higher than that in intramuscular adipocytes(P<0.05).During differentiation,the relative m RNA expression levels of PPARγ,C/EBPα and FABP4 were higher in subcutaneous preadipocytes than in intramuscular preadipocytes(P<0.05).These results indicated that the well-activated subcutaneous and intramuscular preadipocytes were successfully isolated from yellow cattle,and they could differentiate into mature adipocytes after the 9 d differentiation period,the lipid accumulation capacity of subcutaneous preadipocytes was higher than that of intramuscular preadipocytes.Trial 2.Effect of leucine on browning and mitochondrial biosynthesis in subcutaneous and intramuscular adipocytes of yellow cattleAt D6-9 of differentiation,additional leucine at 0,0.5,1,2,4 and 8 m M was added to the maintenance medium.RT-PCR was used to detect the expression level of key browning genes to select leucine concentrations that induce browning,and follow-up experiments were executed choosing the control group(CON)and optimal concentration leucine addition group(Leu).RT-PCR was used to detect the key mitochondrial biosynthesis genes,mt DNA copy number and genes related to lipid metabolism.The expression of key proteins of browning as well as pathway proteins were detected by Western Blot.Intracellular mitochondrial content and lipid droplet morphology were detected by Mito-tracker Green staining and Oil Red O staining,respectively.The results were as follows:(1)4 m M and 2 m M leucine promoted the relative m RNA expression of UCP1,PRDM16,TMEM26,Cidea,and Cited1 in subcutaneous and intramuscular adipocytes,respectively(P<0.05),with the highest fold upregulation of the relative m RNA expression of UCP1.4 m M and 2 m M leucine separately promoted the protein expression levels of UCP1,CD137,and SIRT1 in subcutaneous and intramuscular adipocytes(P<0.05).Therefore,4 m M and 2 m M leucine were chosen to treat subcutaneous and intramuscular adipocytes separately.(2)After 4 m M and 2 m M leucine treatment of subcutaneous and intramuscular adipocytes,respectively,intracellular lipid droplets shifted from single-compartment large lipid droplets to multi-compartment small lipid droplets,and intracellular lipid content was significantly reduced(P<0.01).4 m M and 2 m M leucine addition significantly increased the relative m RNA expression of key genes for mitochondrial biosynthesis(P<0.05),significantly increased the mitochondrial content and mt DNA copy number in cells(P<0.01)and decreased intracellular ATP content(P<0.05).Leucine promoted the relative m RNA expression of key lipolysis enzymes ATGL and HSL and key fatty acid β-oxidation enzymes PPARα,ACOX1 and CPT1(P<0.01)and inhibited the relative m RNA expression of key lipid synthesis enzymes ACC and FAS(P<0.01).(3)After 4 m M and 2 m M leucine treatment of subcutaneous and intramuscular adipocytes,respectively,the relative m RNA expressions of AMPKα1 and PGC1α were significantly increased in both adipocytes(P<0.01),the expressions of p-AMPKα/AMPKα and PGC1αproteins were significantly increased(P<0.01).These results indicated that Leucine-induced browning of subcutaneous and intramuscular adipocytes differed in optimal concentration.leucine promoted browning and mitochondrial biosynthesis in subcutaneous and intramuscular adipocytes,promoted lipolysis and inhibited lipid synthesis,reduced intracellular lipid deposition,and upregulated the protein expression of p-AMPKα/AMPKα and PGC1α.Trial 3.Signaling pathway of leucine on browning and mitochondrial biosynthesis in subcutaneous adipocytes of yellow cattleTrials 1 and 2 have demonstrated that subcutaneous adipocytes have better lipid accumulation capacity than intramuscular adipocytes and that leucine can induce subcutaneous adipocyte browning.Additionally,subcutaneous adipose tissue is a key site of research on the browning phenomenon and the reduction of subcutaneous fat deposition is also a demand in practical production.Furthermore,browning of intramuscular adipocytes could lead to lower meat quality in cattle,so it is necessary to explore the mechanism of leucine on subcutaneous adipocytes browning and mitochondrial biogenesis in cattle.The study divided the samples into a control group(CON group),a leucine group(Leu group),an AMPK inhibitor group(CC group),and a leucine and AMPK inhibitor treated group(Leu+CC group),and measured the relative m RNA expression level of browning markers and genes related to mitochondrial biogenesis,the protein expression level of browning markers and the AMPKα/PGC1α signaling pathway,the number of intracellular mitochondria,and the content of intracellular lipid and lipid droplets.The results were as follows:(1)Compared with the CON group,the CC group significantly reduced the relative m RNA expression of PGC1α and protein expression of p-AMPKα/AMPKα and PGC1α(P<0.05).(2)Compared with the CON group,the CC group significantly decreased the relative m RNA expression of browning marker UCP1,PRDM16,CD137 and mitochondrial biosynthesis key gene TFAM(P<0.05),significantly decreased the protein expression of UCP1 and p-AMPKα/AMPKα.Compared with the Leu group,the Leu+CC group significantly decreased intracellular mitochondrial content(P<0.05),and significantly increased the lipid content in adipocytes(P<0.05).These results indicated that leucine promoted browning and mitochondrial biosynthesis via AMPKα/PGC1α signaling pathway.In conclusion,this study successfully isolated subcutaneous and intramuscular preadipocytes from yellow cattle,which could be induced into mature adipocytes,and the lipid accumulation capacity of subcutaneous preadipocytes was higher than that of intramuscular preadipocytes.4 m M and 2 m M leucine separately induced browning of adipocytes and promoted their mitochondrial biosynthesis,while inhibiting lipid synthesis to promote lipolysis and reduce lipid deposition.In addition,leucine affected browning and mitochondrial biosynthesis in subcutaneous adipocytes of cattle through AMPKα/PGC1α signaling pathway.