Effects Of Leucine On Mitochondrial Function In Primary Skeletal Muscle Cell Treated With Palmitate

Objective: To investigate the effects of leucine on mitochondrial biogenesis and mitochondrial functions in primarily cultured skeletal muscle cell treated with palmitate.Methods: CCK-8 was used to detect cell viability of primarily cultured skeletal muscle cells treated with leucine or palmitate at different intervention time and doses.Western blot was used to detect the protein expression level of pSer 473 AKT after skeletal muscle cells were treated with leucine at different doses.The skeletal muscle cells were treated without leucine and palmitate(Con),0.25 mmol/L leucine(Leu),0.25mmol/L palmitate(PA)and 0.25 mmol/L leucine with 0.25 mmol/L palmitate(PL)for24 hours,respectively.Western blot was used to detect the protein expression level of pSer 473 AKT,mTOR and BCAA catabolic enzymes(BCATm,BCKDK and BCKDC).Real-time PCR were carried out to assess the mtDNA content and the mRNA levels of mitochondrial biogenesis related genes,such as PGC-1?,NRF-1,TFAM and SIRT-1 in skeletal muscle cells.The mitochondrial mass of skeletal muscle cells were detected by Mitotracker Green FM staining.The mitochondrial ROS production of skeletal muscle cells were detected by MitoSOX red staining.The ATP production of mitochondria in skeletal muscle cells were measured by colorimetry.Results:1.Determination of intervention doses and methods: The cell viability of skeletal muscle cells were significantly decreased when cells were treated by palmitate higher than 0.4 mmol/L for 24 hours.Different doses of leucine had no significant effect on the cell viability of skeletal muscle cells.The expression of insulin-stimulated pSer473 AKT was significantly increased in skeletal muscle cells when the dose of leucine was 0.25 mmol/L.The cell viability of skeletal muscle cells were significantlydecreased when cells were treated by 0.25 mmol/L palmitate for 24 hours then0.25 mmol/L leucine for 24 hours.The cell viability of skeletal muscle cells were significantly decreased when cells were treated by 0.4 mmol/L palmitate together with0.25 mmol/L leucine for 24 and 48 hours,respectively.Therefore,the dose and method of intervention in this study were determined to be 0.25 mmol/L palmitate together with 0.25 mmol/L leucine for 24 hours.2.Insulin sensitivity of skeletal muscle cells:The expression of insulin-stimulated pSer 473 AKT was significantly decreased in skeletal muscle cells of PA compared with that of Con.The expression of insulin-stimulated pSer473 AKT was significantly increased in skeletal muscle cells of PL compared with that of PA.However,there was no significant difference in mTOR expression level.3.Protein expression of relative enzyme of BCAAs catabolism: The expression of BCATm was significantly increased in skeletal muscle cells of PL compared with that of PA.There was no significant difference in BCKDH expression level.However,the expression of BCKDK,a kinase that regulates the activity of BCKDH by phosphorating it,was significantly decreased in skeletal muscle cells of PL compared with that of PA.These results indicating that leucine could increase the catabolism of BCAA in skeletal muscle cells.4.Mitochondria biogenesis related genes in skeletal muscle cells: The mRNA levels of PGC-1? and TFAM were significantly increased and the mRNA levels of SIRT-1 were significantly decreased in skeletal muscle cells of PL compared with that of PA.The mitochondrial mass in skeletal muscle cells of PA was significantly decreased with that of Con.The mitochondrial mass in skeletal muscle cells of PL was increased significantly compared with that of PA.However,the content of mtDNA in skeletal muscle cells did not change significantly.5.Mitochondrial function in skeletal muscle cells: The content of mitochondrial ROS was significantly increased in skeletal muscle cells of PA compared with that of Con.The mitochondrial ATP synthesis was significantly increased and the mitochondrial ROS was significantly decreased in skeletal muscle cells of PL compared with that of PA.Conclusion: Taken together,these results indicate that under the conditions of this experiment,leucine can significantly enhance the insulin sensitivity of skeletal muscle cells and increase the catabolism of BCAA in skeletal muscle cells.Meanwhile,leucine can promote the mitochondria biogenesis,reduce the mitochondrial oxidative stress and enhance the mitochondrial function of skeletal muscle cells treated with palmitate.