The Signaling Mechanism Of NEFAs Inducing Insulin Resistance Through Mitochondrial Dysfunction In Hepatocytes Of Dairy Cows

Fatty liver is a metabolic disease in cows transition and the pathological basis is negative energy balance(NEB). The cows perform high concentrations blood NEFAs and BHBA. The loaded NEFAs re-esterified back into triglyceride(TG) in liver resulting in fatty liver. It has been shown that mitochondrial dysfunction and insulin resistance were found in obese mice and patients with fatty. The insulin sensitivity index of NEB cows, ketosis and fatty liver cows was lower than healthy cows. It suggest that the insulin signaling pathway was damaged in fatty liver cows. Moreover, mitochondrial dysfunction is closely related to the development of insulin resistance. Therefore, we speculated that mitochondrial dysfunction occurred in fatty liver cows, and related with the development of insulin resistance. In this study, we carry out experiment in vivo to determine the state of mitochondrial dysfunction and insulin resistance in fatty liver dairy cows. In addition, calf hepatocytes were cultured in vitro toreveal the mechanism of NEFAs-induced insulin resistance, and effects of mitochondrial dysfunction on insulin resistance.Perinatal cows were devided into healthy group and fatty liver group, and collected the liver samples. TG content was significantly higher than the healthy group. Moreover, HE staining showed serious liver steatosis in fatty liver group. The phosphorylation levels of AKT, GSK3β and IRS2 were significantly decreased and the expression of mitochondrial function related key moleculars PGC-1α, NRF-1, Mfn-2, NDAFA9, SDHA, UQCRC2, COXIV and ATPB were significantly decreased. These results indicate fatty live cows exist insulin resistance and mitochondrial dysfunction.Cultured the calf hepatocytes in vitro and treated with different concentrations of NEFAs. And transfected with adenovirus overexpression PGC-1αand Mfn-2 respectively. Firstly, calf hepatocytes were incubated with 2.4 m M NEFAs, and treated with multiple time points. The result showed that the expression levels of PGC-1α and SDHA was lowest at 9h in treatment with NEFAs. Then, calf hepatocytes were incubated with different concentrations of NEFAs, The result show that the phosphorylation levels of AKT, GSK3β and IRS2 were significantly decreased in the NEFAs treatment groups. The protein expression of PGC-1α, Mfn-2, COI-IV were significant lower in the NEFAs groups than in the control group. The ROS content was significantly higher in the NEFAs treatment groups, while ATP production was significantly lower in the NEFAs treatment groups.PGC-1α was overexpressed in calf hepatocytes, compared with the control groupthe phosphorylation levels of IRS2, GSK3β and AKT and the expression levels of PGC-1α, Mfn-2, COI, COII and COIV were significantly higher. The ROS content was significantly lower, while ATP production and membrane potential results were significantly higher. Mfn-2 was overexpressed in calf hepatocytes, the phosphorylation levels of IRS2, GSK3β and AKT and protein expression levels of Mfn-2, COI, COII and COIV were significantly higher. The ROS content was significantly lower, while ATP production and membrane potential results were significantly higher. Our data showed that fatty liver dairy cows display mitochondrial dysfunction and insulin resistance. High level of non-esterified fatty acid(NEFA) impairs the mitochondrial function and insulin signal pathway of cow hepatocytes in vitro. Furthermore, we showed that these detrimental effects may occur through the modulation of PGC-1α/Mfn-2 and their downstream targets. These findings establish mitochondrial dysfunction as an essential component of organelle insulin resistance that is a metabolic pathologyaccompanied the development of fatty liver.In conclusion, These results suggest that fatty liver dairy cowsdisplay high concentrations blood NEFAs, mitochondrial dysfunction and insulin resistance. Moreover, mitochondrial dysfunction mediates NEFAs-induced insulin resistance.In addition to, overexpression of PGC-1α/Mfn-2ameliorated NEFAs-induced mitochondrial dysfunction and improved insulin resistance in cow hepatocytes.