亮氨酸对黄牛皮下脂肪细胞棕色化的影响

Effect of Leucine on Browning of Subcutaneous Adipocytes in Yellow Cattle

旨在分离黄牛皮下前体脂肪细胞,探究亮氨酸对白色脂肪细胞棕色化以及脂质代谢的影响。本研究采取4头3岁的健康雄性黄牛((351.7±42.5)kg)皮下脂肪组织分离前体脂肪细胞,混合后于细胞分化D 6-D 9添加不同浓度亮氨酸(0、0.5、1、2、4、8 mmol·L^(-1))(n=6)。RT-PCR检测棕色化标志基因,筛选亮氨酸诱导脂肪细胞棕色化的浓度,后续试验分为对照组(CON)和最佳浓度亮氨酸添加组(Leu)(n=6)。RT-PCR检测线粒体生物合成关键基因表达、mtDNA拷贝数以及脂质代谢相关基因的表达,Western Blot检测棕色化关键蛋白以及信号通路蛋白表达。Mito-tracker Green染色和油红O染色分别检测细胞内线粒体含量和脂滴形态的变化。结果表明,成功分离得到黄牛皮下前体脂肪细胞并诱导为成熟脂肪细胞。4 mmol·L^(-1)亮氨酸显著提高脂肪细胞中棕色化标志因子UCP1、PRDM 16和TMEM 26的mRNA相对表达量,线粒体生物合成关键基因SIRT1、TFAM和NRF 1/2的mRNA相对表达量,上调关键蛋白UCP1、CD137和SIRT1的表达量(P<0.01)。亮氨酸显著提高细胞中mtDNA拷贝数和线粒体含量(P<0.01),降低细胞内ATP含量(P<0.05),促使细胞脂滴由单房大脂滴向多房小脂滴转变,并降低细胞内甘油三酯的含量(P<0.01)。亮氨酸抑制脂肪合成基因ACC、FAS表达,促进脂肪分解基因ATGL、HSL和脂肪酸β氧化基因PPARα、CPT1、ACOX表达(P<0.01)。添加亮氨酸极显著上调p-AMPKα/AMPKα和PGC1α的蛋白表达量(P<0.01)。综上所述,亮氨酸能促进黄牛脂肪细胞棕色化和线粒体生物合成,促进脂肪分解抑制脂肪合成,减少细胞内脂质沉积,并上调p-AMPKα/AMPKα和PGC1α的蛋白表达量。

This experiment was conducted to isolate subcutaneous preadipocytes from yellow cattle and study the effect of leucine on browning white adipocytes and lipid metabolism.Preadipocytes were isolated from subcutaneous adipose tissue of 43-year-old healthy male yellow cattle((351.7±42.5)kg),mixed and added with different concentrations of leucine(0,0.5,1,2,4 and 8 mmol·L^(-1))at the differentiation D 6-D 9(n=6).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 to set the control group(CON)and optimal concentration leucine addition group(Leu)(n=6).RT-PCR was used to detect the key mitochondrial biosynthesis genes expression,mtDNA copy number and genes expression 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 showed that subcutaneous preadipocytes were isolated and differentiated into mature adipocytes successfully.4 mmol·L^(-1) leucine significantly increased the relative mRNA expression of browning marker factors UCP1,PRDM 16 and TMEM 26 and key genes of mitochondrial biosynthesis SIRT1,TFAM and NRF 1/2,and increased the protein expression of UCP1,CD137 and SIRT1 in adipocytes(P<0.01).Leucine significantly increased the mtDNA copy number and mitochondrial content(P<0.01),decreased the intracellular ATP content(P<0.05).Leucine induced the transformation of lipid droplets from large single-compartment to small multi-compartment and decreased intracellular triglyceride content(P<0.01).Leucine decreased the expression of lipid synthesis genes ACC and FAS,while promoting the expression of lipolysis genes ATGL and HSL and fatty acidβ-oxidation genes PPARα,CPT 1 and ACOX1(P<0.01).Leucine also significantly upregulated protein expression levels of p-AMPKα/AMPKαand PGC1α(P<0.01).In conclusion,leucine promotes browning and mitochondrial biosynthesis in preadipocytes,promotes lipolysis and inhibits lipid synthesis,reduces lipid deposition,and upregulates the protein expression of p-AMPKα/AMPKαand PGC1α.