Effect Of Insulin, Glucose And Fatty Acid On Resistin MRNA Expression Of Bovine Adipocyte In Vitro

The insulin-resistance may occur in fat cow which decreases sensitivity to insulin, and influences adjustment of insulin on glucose and fat metabolism. Cytokines secreted by adipocyte may be relative to the disorder of fat metabolism and the insulin-resistance. The adipocyte culture in vitro is as an ideal model to study the impact of adipocyte cytokines on fat metabolism.Resistin has been known as a link between insulin-resistance and obesity. Resistin can cause insulin-resistance in liver, and its mRNA level increases in adipose tissue during lactation period. Up to now, there is little information on the effect of resistin on fat metabolism of dairy cow in periparturient period. The objective of present studies was to detect abundance of resistn mRNA of adipoctes in different differentiation time and to evaluate the effect of different concentration of insulin, glucose and nonesterified fatty acids on resistin mRNA of adipocytes in vitro by RT-PCR in order to understand the relations among insulin- resistance, fat and resistin.Firstly, serial culture of primary adipocyte was carried out to observe cellular dynamic morphological changes, draw growth curve by lencocyte count, survey fat content by extracting oil red O from the stained cells and determine degree of adipocyte differentiation. The cellular differentiated markers were detected by reverse transcription-polymerase chain reaction (RT-PCR). The cellular survival rate after freezed was counted by trypen-blue-stain. The results are as follow: the cells by serial subcultivation remained a character of accumulating fat. The growth curve and fat change curve coincided between different eras. The cellular differentiated markers, such as lpoproteinesterase (LPL), leptin (LP), 3-glycerol phosphate dehydrogenase (GPDH) were detected. The rate of cellular survival arrived 87%. The results show that the adipocyte after serial subcultivation could be used as a model to research adipocyte factor, but degree of preadipocyte differentiation was not determined by investigating LPL mRNA, LP mRNA and GPDH mRNA.Secondly, a 151bp sequence of bovine resistin cDNA was cloned using total RNA of adipose tissue and a pair of primers. After sequencing analysis, the homology compared with sequence of Genebank is 98%. Total RNA was extracted from adipocyte of primary culture for 4d, 6d, 8d, 10d, 12d, 14d, 16d . AS an inner-reference of β-actin, abundance of resistin mRNA was analyzed quantitively. Abundance of resistin mRNA of adipocyte decreased from 4d to 12d after primary culture, and then rose again .The cells has been mature adipocyte refewrenced red oil O. It is concluded that expression of resistin mRNA occurred in both preadipocyte and adipocyte, and decreases during cell differentiation. It elevates with gradual increasing adipogenesis of the cultured adipocyte.Thirdly, different concentrations of insulin, glucose and nonesterified fatty acids were...