The Mechanism Of Retinol Binding Protein4Affects The Adipogenesis Of Porcine Preadipocytes

Adipogenesis is now considered to be a dynamic and plastic process, and involves the coordination of a tightly regulated transcriptional program via numerous paracrine and endocrine factors, which regulate the differentiation of adipocytes and maintain the phenotype of mature adipocytes. It is now well recognized that adipose tissue is one of endocrinal organs and secretes various adipokines, which are likely to represent major contributions to adipose tissue biology. This dysregulation of adipose-secreted factors may promote insulin resistance and obesity-linked metabolic disorders. Moreover, adipokines also effect the fat accumulation and muscle development in livestock. So, it is critically important to more fully understand the molecular mechanisms of adipokines in the adipogenesis and meat quality improvement in animal husbandry.Retinol-binding protein4(RBP4), one of the members of the lipocalin superfamily, is a specific transport protein for retinoid (vitamin A). RBP4is responsible to transport retinoid from liver to extrahepatic tissues and assists in retinoid metabolism and actions. Until2005, it has been reported that RBP4is a novel adipocyte-secreted hormone that regulates the glucose absorption in adipocytes and provokes insulin resistant states associated with obesity. The new function of RBP4promotes to explore the relation between pathological and physiological mechanism, adipokines and inflammatory response. Therefore, it is possible that RBP4will be a new therapeutic target. However, the mechanisms underlying RBP4affects preadipocyte differentiation are still not well understood.Previous studies have shown that RBP4expression is significant difference in the adipose tissue of different developmental stages in the same porcine breed, with a putative important role in the differentiation of adipocytes. To investigate the RBP4biological function, its expression was firstly examined in the adipogenesis. Then RBP4expression and interference vector were constructed to increase or decrease its level in porcine preadipocytes. The changes in morphology and gene expression levels were examined by Oil Red O staining, real-time qPCR and Western blot analysis to evaluate the effect of RBP4on the differentiation of porcine preadipocytes. Next, insulin-induced and the specific inhibitor of insulin signaling (LY294002) was used to clarify the interaction between RBP4and insulin signaling. Finally, frozen section, immunofluorescence and Western blot were adopted to detect Ad-RBP4affects abdominal fat tissue in mice, which provide more evidence for experiment in vitro.The specific results are as follows:1. The expression of RBP4was increased in the porcine adipogenesis. RBP4was highly expressed in adipoctyes compared to preadipocytes and was predominantly localized in the cytoplasm of preadipocytes and adipocytes.2. RBP4recombinant adenovirus vector was constructed correctly, virus titer was8.6×109pfu/ml. RBP4expression was remarkably increased in porcine preadipocytes in Ad-RBP4. The results showed that adipogenic differentiation of porcine preadipocytes was inhibited by the overexpression of RBP4. It reflected that mRNA and protein expression levels of adipocyte marker genes were decreased and mRNA of lipolysis genes were enhanced.3. Porcine RBP4lentivirus interfering vector was constructed. Virus titer was6.5×107pfu/ml, then infected the porcine preadipocytes, RBP4mRNA and protein expression was remarkably reduced by approximate60%in porcine preadipocytes. The data confirmed that preadipocytes differentiation was promoted and the expression levels of PPARy, SREBP-lc and aP2were increased in adipocytes by knockdown of RBP4.4. RBP4had a suppressive effect as well on the differentiation of porcine preadipocytes by decreasing the activation of insulin signaling pathway. However, the activation of insulin signaling mediated by the knockdown of RBP4in porcine preadipocytes was recovered in the suppression of LY294002. RBP4interacted physically with AKT in mature porcine adipocytes. Taken together, RBP4mainly changed the activity of AKT, which involved in regulating the insulin signaling.5. Ad-RBP4was injected in mice in order to promote RBP4ectopic expression. Compared with control, the rate of abdominal fat deposition was slower and their adipocytes size was smaller in Ad-RBP4injection group. Besides, RBP4could induce the insulin resistance in abdominal fat tissue.The work presented in this thesis establishes RBP4as a potent negative regulator of adipogenesis of porcine adipocytes. In addition, the underlying cellular mechanism of RBP4induces insulin resistance in adipocytes is that RBP4inhibits the phosphorylation of AK.T at threonine (308) and blocks the insulin signaling pathway.