| High concentration of blood non-esterified fatty acids(NEFA)is a high incidence of perinatal dairy cows,which is caused by the decrease in dry matter intake(DMI)and the restoration of lactation in perinatal cows due to uterine compression,resulting in negative energy balance(NEB).In order to compensate for the negative energy balance,a large amount of NEFA is mobilized into the blood through body fat mobilization,resulting in a large increase in NEFA in the blood of perinatal cows,forming high concentration of blood NEFA.Skeletal muscle,as the largest organ of the body,involve in more than 80% of glucose metabolism,and its role in maintaining energy balance in perinatal cows has not been reported.To investigate the effect of high NEFA to mitochondria and tricarboxylic acid cycle of bovine skeletal muscle cells,in this study,we used CCK-8 method,oil red O staining,transmission electron microscopy,flow cytometry,BODIPY 493/503 fluorescence staining technology,q RT-PCR to detect the effect of NEFA to the skeletal muscle cell.1.Effects of different concentrations of NEFA and treatment duration on viability and morphology of bovine skeletal muscle cellsThe results of stimulating bovine skeletal muscle cells with gradient concentration of NEFA showed that after 2 h of stimulation with 1.2 mmol/L or 1.5 mmol/L NEFA,the survival rate of bovine skeletal muscle cells was significantly increased(P<0.01);After 2 h+1.8 mmol/L treatment,the cells became shrinkage and shedding,and the cell survival rate significantly decreased(P<0.01);Stimulating cells with 1.5 mmol/L NEFA showed no significant decrease in cell survival rate among different treatment durations,indicating that 1.5 mmol/L NEFA had no inhibitory effect on cell survival and was the optimal treatment concentration.In addition,transmission electron microscopy observed that 2 h+1.5 mmol/L NEFA treatment caused structural loss,volume reduction,darkening of matrix color,and disordered arrangement of mitochondrial cristae in a large number of mitochondria in bovine skeletal muscle cells,and some mitochondria showed vacuolar morphology,indicating that NEFA caused damage to the mitochondrial ultrastructure of bovine skeletal muscle cells.2.Acute effect of high NEFA on bovine skeletal muscle cellsThe results showed that after 1.5 mmol/L NEFA stressed skeletal cells for 2 h,the proportion of cells with mitochondrial membrane potential decreased significantly(P<0.01),the activity of CAT in cells decreased significantly(P<0.01),the activity of SOD decreased significantly(P<0.05),and the concentration of MDA in culture medium increased significantly(P<0.05).The results of oil red O staining and BODIPY493/503 lipid droplet staining showed that small lipid droplets appeared around the nucleus of skeletal muscle cells after NEFA treatment.In addition,under the NEFA treatment group,the key factors for intracellular mitochondrial generation and function,NRF1 and PGC-1α were observed.The mRNA abundance of MFN2,COI,CO II,CO III,and CO IV was significantly lower than that of the control group(P<0.01);The expression level of the fatty acid transmitochondrial transport related gene CPT1 B was significantly increased(P<0.01),and the expression of the triglyceride hydrolase PNPLA2 and the long chain fatty acid elongation related enzyme ELOVL6 was significantly downregulated(P<0.05).The above results indicate that NEFA can affect the generation and fusion of mitochondria in skeletal muscle cells,causing abnormalities in important metabolic processes such as fatty acid transport,triglyceride breakdown,and long-chain fatty acid elongation.In summary,treatment with 1.5mmol/L NEFA for 2 h resulted in acute effects on skeletal muscle cells.3.Effects of NEFA on mitochondrial function and metabolism in bovine skeletal muscle cellsThe NEFA treatment time was extended to 12 h to explore the effect of high concentration of NEFA stimulation on bovine skeletal muscle cells.The NEFA treatment time was extended to 12 h to explore the effect of high concentration of NEFA stimulation on bovine skeletal muscle cells.The results showed that compared with 0h+1.5 mmol/L,12 h+1.5 mmol/L NEFA treated skeletal muscle cells caused the decrease of mitochondrial membrane potential more cells(P<0.01),and the number and volume of lipid droplets in the cells increased.The mRNA expression levels of PGC-1α,a gene regulating mitochondrial gene replication and transcription,and MFN2,a fusion-related gene regulating mitochondria,were significantly lower than those of 0h control group(P<0.01),and the mRNA expression levels of NRF1 were significantly decreased(P<0.05).The mRNA abundance of CO IV gene related to mitochondrial aerobic metabolism was significantly increased(P<0.05).The mRNA expression levels of PNPLA2,ELOVL1 and ELOVL6,and GLUT4,the key enzyme of glucose transport,were significantly decreased(P<0.01),while the mRNA expression levels of CPT1 B were significantly increased(P<0.01).In conclusion,mitochondrial function of skeletal muscle cells treated with 1.5 mmol/L NEFA for 12 h was disordered compared with that treated with 0 h.The results showed that compared with 0 h+1.5 mmol/L,12 h+1.5mmol/L NEFA treated skeletal muscle cells caused the decrease of mitochondrial membrane potential more cells(P<0.01),and the number and volume of lipid droplets in the cells increased.The mRNA expression levels of PGC-1α,a gene regulating mitochondrial gene replication and transcription,and MFN2,a fusion-related gene regulating mitochondria,were significantly lower than those of 0 h control group(P<0.01),and the mRNA expression levels of NRF1 were significantly decreased(P<0.05).The mRNA abundance of CO IV gene related to mitochondrial aerobic metabolism was significantly increased(P<0.05).The mRNA expression levels of PNPLA2,ELOVL1 and ELOVL6,and GLUT4,the key enzyme of glucose transport,were significantly decreased(P<0.01),while the mRNA expression levels of CPT1 B were significantly increased(P<0.01).In conclusion,mitochondrial function of skeletal muscle cells treated with 1.5mmol /L NEFA for 12 h was disordered compared with that treated with 0 h.Compared with 2 h treatment group,the proportion of mitochondrial membrane potential decline decreased in 12 h treatment group,and mRNA abundance of genes related to mitochondrial generation and fusion and aerobic metabolism increased,suggesting that skeletal muscle cells may initially adapt to high NEFA environment after 12 h of high NEFA stress.However,with the increase of time of 1.5 mmol/L NEFA stress,the effect of fatty acid transport,production and glucose membrane translocation in cells was significantly enhanced.In conclusion,high concentration of NEFA can cause fatty acid metabolism disorders in bovine skeletal muscle cells,resulting in mitochondrial morphology and function disorders and cell metabolism abnormalities. |
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