1. Improvement Of TmTNF-α In Insulin Resistance Of Adipocytes And Its Underlying Mechanism. 2. Construction Of Conserved Sequence Deleted TNF-α Mutant And The Trimerization Of Remodeled TNFRBP.

Obesity is highly associated with insulin resistance and is one of the biggest risk factors for non-insulin-dependent diabetes mellitus. Insulin resistance or the decrease in insulin sensitivity, defined as a smaller than expected biological response to a given dose of insulin, shows a decrease in glucose uptake by insulin-acted peripheral tissue, especially muscle and adipose tissue and an increase in hepatic gluconeogenesis. Dyslipidemias, hypertension, and cardiovascular disease are often concurrent with type 2 diabetes.Tumor necrosis factor-α(TNF-α) is a pleiotropic cytokine with a wide range of biological effects. It exists as a 26-kDa transmembrane form (tmTNF-α) that is cleaved by the TNF-αconverting enzyme (TACE) to release a 17-kDa soluble molecule (sTNF-α). It has been confirmed that sTNF-αis one of the most important molecules which links obesity and insulin resistance and plays an important pathogenic role. However, our preliminary work has confirmed that the two forms of TNF-αhad opposite effects on insulin resistance. In this study, we aim to determine the impact of tmTNF-αon insulin resistance of adipocytes and further to investigate its molecular mechanisms and signaling pathways, which provides a novel clue on the intervention of obesity and its various complications.The main results are as follows:1. tmTNF-αincreased insulin sensitivity of 3T3-L1 adipocytes via directly improving insulin signaling3T3-L1 adipocytes were treated with the two forms of exogenous TNF-αand the insulin signaling was detected. sTNF-αinhibited the tyrosine phosphorylation of IRS-1 and the phosphorylation of the downstream molecule AKT; while tmTNF-αinduced the tyrosine phosphorylation of IRS-1 and the phosphorylation of AKT. It brought about an increase in insulin sensitivity of 3T3-L1 adipocytes, leading to a two-fold elevation of insulin-stimulated glucose uptake compared with control cells.2. tmTNF-αincreased insulin sensitivity of 3T3-L1 adipocytes by downregulating the expression of IL-6 and MCP-1(1) We used realtime PCR and ELISA respectively to suggest that tmTNF-α, whose role is contrary to sTNF-α, significantly inhibited the mRNA and protein expression of insulin resistant molecules such as IL-6 and MCP-1;(2) sTNF-αenhanced the lipolysis of the adipocytes,which increased FFA in the supernatant; while tmTNF-αhad no statistical difference compared with the control group ,which showed tmTNF-αdidn't enhance the lipolysis of the adipocytes;(3) tmTNF-αdownregulated the expression of insulin resistant molecules via the inhibition of the degradation of IκB-αto prevent the activation of NF-κB.3. tmTNF-αincreased insulin sensitivity of 3T3-L1 adipocytes by upregulating the expression of adiponectin(1) tmTNF-αremarkably improved the mRNA expression of adiponectin; while sTNF-αinhibited its expression;(2) tmTNF-αremarkably improved the expression of PPAR-γ; GW9662,an inhibitor of PPAR-γ,could decrease the mRNA expression of adiponectin tmTNF-αinduced; when the adipocytes were treated with GW9662 alone , it showed no observed effect.(3) Additionally, GW9662 could partly block the glucose uptake and the phosphorylation of AKT which is an insulin signaling molecule; while there was no observed effect when treated with GW9662 alone.Conclusions: tmTNF-αand sTNF-αyield different effects on insulin resistance of adipocytes. (1) tmTNF-αcould elevate insulin sensitivity of 3T3-L1 adipocytes via directly improving insulin signaling transduction; (2) tmTNF-αdownregulated the expression of insulin resistant molecules such as IL-6 and MCP-1 via preventing the activation of NF-κB; (3) tmTNF-αupregulated the expression of insulin-sensitive molecule adiponectin via increasing the expression of PPAR-γto increase the insulin sensitivity of adipocytes. Part 2: Construction of conserved sequence deleted TNF-αmutant and the trimerization of remodeled TNFRBPTumor necrosis factor superfamily(TNF-SF) participate in the comprehensive physiological and pathological process, mediating various biological effects, such as inflammation, apoptosis, immune regulation. The common feature of these membrane proteins is the tendency on the trimer and they share highly conserved amino acid sequence in their primary structures. Therefore, we hypothesized that highly conserved sequence of the members of the TNF superfamily are likely to be common features for their trimer formation.Because 119-130 locates just in the highly conserved sequence of TNF-αand in the connecting parts of TNF-αmonomers, in this study, we constructed pcDNA3.0-TNF-α△119-122 mutant recombinant plasmid by overlapping PCR, which provided an important clue and experimental basis for studying the relationship of the structure and function of TNF-α. However, the effect of TNF-αdepends on the combination with TNFR of the target cell. We screened a phage displayed random 12 amino acid peptide library using soluble recombinant human TNFRI in our previous study. TNFRBP is able to block TNF-αbioactivity by competitively binding to TNFRI. According to the protein molecule simulation, we simulated the spacial comformation and characteristics of TNF-α, screening the key amino acid and the best virtual model for the trimer formation of TNF-αin vitro. TNFRBP was remodeled by linking the conserved sequences, Ile58 Tyr59 Ser60 and Gly122 Val123 Phe124 selected from TNF-α, to the N and C terminal. It is expected that the remodeled TNFRBP can increase the competitiveness with the wild type ligand and overcome the defection of easy degradation to elevate its biological effects, which have important theoretical significance and practical value for the fundamental research and clinical application. The main results are as follows:1. Construction, cloning and identification of pcDNA3.0-TNF-α△119-122 (1) Construction and cloning of mutant recombinant plasmid: Using the plasmid pcDNA3.0-wtTNF-αas a template, synthesizing fragments for the missing tmTNF-α(△119-122) by overlapping PCR. After digestion with endonucleases(BamHI and XhoI), the TNF-αfragment was inserted into pcDNA3.0 plasmid by T4 DNA ligase. And then the connected products were transformed into E. coli DH5αand the positive clones were screened by Ampicillin resistance. (2) Identification of the positive clones: Firstly the positive clones were confirmed by colony PCR, followed identification with endonucleases digestion, and further DNA sequence analysis also proved that the recombinant missed TNF-α△119-122, suggesting expected mutant pcDNA3.0-TNF-α△119-122 recombinant plasmid was successfully constructed.2. The trimer formation of the remodeled TNFRBP by Chemical cross-linking With sTNF-αprotein as the positive control, we detected the trimer formation of remodeled TNFRBP by Chemical cross-linking. After Chemical cross-linking, sTNF-αcould form monomer, dimmer and trimer at the same time, while there was only monomer existence, and no dimers, trimer formation of TNFRBP. It demonstrated that the remodeled TNFRBP couldn't form trimer in vitro.3. Blockage of sTNF-α-mediated cytotoxicity by the primary TNFRBP and the remodeled TNFRBP We confirmed that the primary TNFRBP had evident antagonistic effect on the cytotoxicity of sTNF-αand the antagonist rate was increased with increasing concentration within the scope of certain, while there was no obvious inhibitory effect of the remodeled TNFRBP on the cytotoxicity of sTNF-α.Conclusion: (1) The pcDNA3.0-TNF-α△119-122 mutant recombinant plasmid was successfully constructed, which layed the foundation for the further detection on its biological function to verify the relationship between the site 119-122 and the trimerization of TNF-α; (2) The remodeled TNFRBP couldn't form trimer in vitro and it deprived of the TNF-blocking effect of the primary TNFRBP. This study provided a clue for the further study on the relationship of structure and function of TNF-α.