Effect Of NEFA On PC MRNA, PEPCK MRNA Expression And Enzyme Activity Of In Vitro Culture Bovine Hepatocytes

Liver is main organ of glyconeogenesis, that supply glucose of 90% for uminant. PC and PEPCK is key enzyme of glyconeogenesis, there enzymatic activity and substrate (energy precursors) control rate of glyconeogenesis. As we all know, TG mainly adipose tissue triglyceride hydrolysis (fat mobilization). Fat mobilization is result that periparturient dairy cows associated with negative energy balance. One hand ,lipolysis can retrieve the lower energy that caused by glyconeogenesis; Other, lipolysis can release large amouts of NEFA into the blood and liver, that can cause fatty liver and ketosis. NEFA level is signal of negative energy balance, dairy cows have any negative energy balance factors would result in increased blood concentration of NEFA. The objective of present studies was to detect the effect of NEFA on PC and PEPCK of mRNA Expression and enzyme activity by vitro culture bovine hepatocytes technique,Real-time RT-PCR and enzyme activity determine. In this study were used to provid experiment and theory foundation for fatty liver.Experimental trough cow primary hepatocyte in vitro, using SYBR-Greenâ… Real-time RT-PCT method to detect that NEFA on PC mRNA and PEPCK mRNA expression; Using Speed detection PEPCK activity and Sigma kit of pc activity. First, as a newborn calf for hepatocyte, using slightly improved access to collagenase digestion of hepatocyte and cell culture, to be experiments when hepatocyte adherent completely. In primary cultured hepatocyte with different concentrations of NEFA that 0mM, 0.5mM, 1mM, 1.5mm and 2mM, respectively. Incubation after 12h: 1) using SYBR-Greenâ… Real-time RT-PCT method determine PC mRNA and PEPCK mRNA expression. The results showed that: With the increased concentration of NEFA, PC mRNA abundance of hepatocyte downward trend, the concentration of the control group and a significant difference or very significant(P<0.01 or 0.05); PEPCK mRNA abundance of hepatocyte downward trend too, and when concentration of NEFA at 1.5mM, PEPCK mRNA abundance decline is significant. Note: PC mRNA and PEPCK mRNA of hepatocyte inhibited by NEFA, and with dose inhibition. 2)Using Speed determine PEPCK activity. The results showed that: With the increased concentration of NEFA, PEPCK activity of hepatocyte showed a downward trend ,even high concentrations of NEFA can completely inhibit PEPCK activity. 3)Using Sigma Kit of PC determine PC activity. The results showed that with the increased concentration of NEFA, PC activity of hepatocyte was decreased, when concentration of NEFA beyond 1.5mM, the experimental group and the control group was significant difference(P<0.05).To sum up, NEFA inhibition PC mRNA, PEPCK mRNA and PC activity, PEPCK activity of hepatocyte. Instructions in the perinatal negative energy balance, Because of fat mobilization, blood NEFA concentrations and liver uptake of NEFA to increased. NEFA can inhibit glyconeogenesis by inhibit PC mRNA , PEPCK mRNA expression and PC activity, PEPCK activity ,thus further exacerbating the negative energy.