Resistin Its Binding Peptides 3T3-L1 Cell Differentiation Between Studies

Diabetes mellitus, a worldwide prevailing disease, is a principal cause of morbidity and mortality in human populations with serious complications. Only a minority of patients suffers from type 1 diabetes. Type 2 diabetes is the most common form of this disease; it arises when the body no longer response properly to its own insulin. Driving the epidemic of type 2 diabetes is the continuing increase in obesity. But the mechanisms that increased adipose tissue in obese patients promotes insulin resistance are still poorly understood. Adipocytes can secret a variety of polypeptides, such as free fatty acids (FFA), tumor necrosis factor a (TNFa), Leptin, adiponectin etc, which may affect insulin action in target organs. But those signal molecules are only part contributors of insulin resistance. Resistin, a novel cysteine-rich protein secreted by adipocyte, was recently identitfied as a member of the diverse family of adipocyte-derived polypeptides that may link obesity with type 2 diabetes mellitus.Resistin expression in adipose tissue or its serum concentration was upregulated in both diet-induced and genetic obese mice and human patients, and was decreased by treatment with rosiglitazone, an anti-diabetic drug that can enhance insulin sensitivity. Resistin treatment impaired glucose tolerance, whereas immunoneutralization of the resistinprotein in mice increased insulin sensitivity. Moreover, resistin is expressed mainly in adipose tissues, and was found to be significantly upregulated during the differentiation of 3T3-L1 preadipocytes. We speculate that resistin my promote preadipocyte differentiation. Surprisingly, adverse effect was reported concerning both resistin and resistin-like molecule a. In our studies, rat resistin gene was inserted into pDual GC and pEGFP-N2 expression vectors for examinating the effects of resistin overexpression in 3T3-L1 cells before and after differentiation which was induced by 3-isobutyl-1-methyxanthine (MIX), insulin, and dexamethasone (DEX), and for subcellular localization inspection respectively. Smaller conserved fragments between rat and mouse resistin gene were inserted into short interference RNA (siRNA) expression vectors, for examination of the effects of targeted resistin inhibition on differentiation of rat resistin-overexpression 3T3-L1 cells. Prior to stimulation, the resistin transfected 3T3-L1 cells contained many more small lipid droplets than non-transfected 3T3-L1 cells. Following stimulation, differentiation in the resistin transfected 3T3-L1 cells was dramatically promoted, especially in the early stages. Stimulation of differentiation was also observed in non-transfected 3T3-L1 cells grown in resistin protein containing conditioned medium. The expression of adipocyte differentiation- associated markers such as C/EBP a , RXR a and LPL was upregulated in resistin overexpressing cells, whereasexpression of Pref-1, an inhibitor of preadipocyte differentiation, was downregulated. In addition, expression of two of the three tested siRNAs inhibited the adipoconversion process, providing further evidence that resistin promotes the differentiation of preadipocytes to adipocytes.Then, how does resistin exert its function? It is presumed that resistin is transported by bloodstream and binds to its receptor on the surface of target cells. In an attempt to identify resistin binding partners, we constructed a multiple tissue cDNA phage display library and screened for resistin binding activity. Three cDNA fragments with the same llbp 5' end sequence were selected. No open reading frame (ORF) was found, because all reading frames were interrupted by one or two TGA stop codons, suggesting that readthrough has occurred within the phage vecter. A pair of reverse complementary oligodeoxynucleotides including the identity sequence with a TGA stop codon and 6 x His coding sequence was inseret into the left and right arms of T7Select phage display vector and readthrough was confirmed by plaque lift assay using an anti-6 xHis antibody. To investigate co-localization...