The Regulatory Mechanism Of MiR-181a On Glucose And Lipid Metabolic Homeostasis In Bovine Hepatocytes In Vitro

Because of the dry matter intake decreased and the energy requirement increased, transition dairy cows often in a state of negative energy balance(NEB). Meanwhile, the NEB will cause the fat mobilization, resulting in dramatically increasing of nonestesterified fatty acids in plasma(NEFAs). However, excessive NEFAs release by adipose tissue are stored in the liver and associated with impaired hepatocytes organelles and signal transduction, this can induce energy unbalance. Mi RNAs represent endogenous, noncoding 20-24 nucleotide RNAs that are widespread exist in all multicellular organisms. The mature mi RNAs regulate target gene expression at posttranscriptional level. Abnormal expression of mi RNAs has been observed in the insulintarget tissues, including adipose tissue, liver and muscle of rat models with type 2 diabetes. Mounting evidences have shown that mi RNAs are critically involved in the regulation of metabolism homeostasis, but in dairy cows, very little is known about the regulation of mi RNAs under normal and disease conditions. The objective of this study is to evaluate the roles of mi R-181 a in the regulation of glucose and lipid homeostasis in bovine hepatocytes in vitro.Detecting the concentration of plasma and liver mi R-181 a in clinical ketosis cows, the results showed that the expression of mi R-181 a in ketosis cows was significantly higher than in healthy cows. Bovine hepatocytes were treated with different concentration of NEFAs(0, 0.6, 1.2, 2.4 mmol/L). The results showed that mi R-181 a expression was increased after treatment in a dose-dependent, and significantly higher in the 1.2, 2.4 mmol/L treatment groups than in the control group. In addition, in the 1.2, 2.4 mmol/L treatment groups, the insulin sensitivity was significantly decreased and lipid deposition was significantly increased. These results suggest that the expression of mi R-181 a is related to the metabolism of glucose and lipid in bovine hepatocytes.Overexpression of mi R-181 a in bovine hepatocytes significantly impaired insulin signaling pathway and decreased insulin-stimulated glucose uptake and increased the key gluconeogenic genes PEPCK and G-6-P. At the same time, SREBP-1c, the key genes in lipid synthesis, were significantly increased and PPARα, the key genes of lipid oxidation, were significantly decreased in the mi R-181 a overexpression group than the negative group. However, inhibition of mi R-181 a in bovine hepatocytes significantly enhanced insulin signaling pathway and increased insulin-stimulated glucose uptake, and the SREBP-1c were significantly decreased and the PPARαwere significantly increased. Notably, under high concentration of NEFAs treatment, inhibition of mi R-181 a significantly upregulated the insulin signaling pathway and insulin-stimulated glucose uptake. Meanwhile, high concentration of NEFAs induced lipid deposition was also decreased by inhibition of mi R-181 a in bovine hepatocytes. Taken together, these results indicated that mi R-181 a overexpression can impaired the homeostasis of glucose and lipid metabolism and inhibition of endogenous mi R-181 a is helpful to maintain glucose and lipid homeostasis in bovine hepatocytes.Putative target of mi R-181 a were searched using prediction program, Target Scan. SIRT1 was selected as an ideal candidate. As expected, mi R-181 a mimics transfection significantly decreased the SIRT1 protein and m RNA levels in bovine hepatocytes. In contrast, the transfection of bovine hepatocytes with the inhibitors of mi R-181 a increased the expression of SIRT1. These observations clearly indicate that SIRT1 is a direct target gene of mi R-181 a in bovine hepatocytes. To further confirm the role of SIRT1 in the regulation of energy balance, EX527, a selective inhibitor of SIRT1, was used. As expected, EX427 significantly decreased SIRT1 expression and dramatically impaired energy balance in bovine hepatocytes. The results were similar to overexpression of mi R-181 a in bovine hepatocytes. When bovine hepatocytes were treated with SIRT1 agonist Resveratrol under high concentration of NEFAs treatment conditions, the homeostasis of glucose and lipid metabolism were recovered from abnormal.Summing up the above results, we conclude that overexpression of mi R-181 a in bovine hepatocytes impaire glucose and lipid homeostasis. SIRT1 is a direct target of mi R-181 a. In addition, inhibition of mi R-181 a or activation of SIRT1 with Res recovere glucose and lipid homeostasis in bovine hepatocytes, even under high concentration of NEFAs treatment conditions.