The Effect Of TNFRIBP-Fc Fusion Protein On Obesity Linked Insulin Resistance

Obesity, with an increasing prevalence, has been become the most common metabolic disorder in the world. Obesity is highly associated with insulin resistance and is the biggest risk factor 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, preceds overt diabetes and signifies a metabolic disorder called syndrome X (concurrence of type 2 diabetes, dyslipidemias, hypertension, and cardiovascular disease).Tumor necrosis factor (TNF-α), produced chiefly by activated monocytes/macrophages and T lymphocytes, is a pleiotropic cytokine with a wide range of biological effects, such as anti-infection, antitumor, immune regulation and inflammation. Recently TNF-αhas been found is synthesised and secreted by adipose tissue and plays a key role in insulin resistance. The general mechanism of TNF-α-induced insulin resistance involves inhibition of insulin receptor signaling as demonstrated in variety of cell types and obese animals. TNF-αinhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1) at least in part by induction of serine phosphorylation of IRS-1. Although it has been previously shown that the lack of TNF-αfunction improve insulin sensitivity in obese animals, no definite conclusion about which TNFR mediates this action of TNF-α.TNFRI blocking peptide (TNFRIBP) was obtained by screening a phage displayed random 12 amino acid peptide library using soluble recombinant human TNFRI in our previous study. TNFRIBP was able to block TNF-αbioactivity by competitively binding to TNFRI. In order to enhance its affinity and prolong its half-life time, we inserted the oligonucleotide sequence of this peptide into the pIg/3C eukaryotic expression vector that contains an upsteam signal peptide sequence necessary for the secretion of chimeras and a downstream human IgG1-Fc fragment sequence, so we got the recombinant vector pIg/3C-TNFRIBP-Fc to secretively express TNFRIBP-Fc fusion protein. The aim of this study to establish stable cell lines to express TNFRIBP-Fc fusion protein by cotransfection CHO-K1 and BHK-21 cells with this recombinant vector and pcDNA3.1 which contains neo gene for selection, gain plenty of purified protein for treatment insulin resistance linked to obesity in vitro and in vivo. This study is expected to lay the foundaton for development of new peptide drug to treat TNF-αrelated diseases.1. Stable express and purify fusion protein TNFRIBP-Fc⑴Establish stable cell lines: CHO-K1 and BHK-21 cells were cotransfected with pIg/3C-TNFRIBP-Fc vector and pcDNA3.1 which contained neo gene for selection, then G418 resistant transformants were selected. After subcloning through three limited-dilution , seven stable cell lines which had high productivity of fusion protein and three stable cell lines which had high productivity of Fc protein were picked out, assessed by ELISA.⑵Purify fusion protein and Fc protein: We cultured these stable cell lines in quantity in the medium without serum and collected the culture supernatants. Fusion protein and Fc protein from supernatants were purified by chromatography on protein A-sepharose CL-4B. Approximately 50μg of fusion protein or 40μg of Fc protein was purified from 100ml of cultural supernatant, quantitied by a sandwich ELISA.⑶Identify molecular weight by SDS-PAGE: The molecule weight of the purified protein and Fc protein were about 37kD and 36kD respectively analyzed by SDS-PAGE.⑷Identify its biological activities: Indirect immunofluorescene showed that fusion protein was able to bind murine TNFRI. TNF-αbioassay suggested that fusion protein, but not Fc protein, suppressed TNF-α-induced cell death in a dose dependent manner. As low as 160ng/ml, the fusion protein completely inhibited the cytotoxicity induced by 100U/ml TNF-α.2. Effect of fusion protein on insulin resistance in vitro⑴Induce 3T3-L1 preadipocytes to differentiate into adipocytes: Briefly, the monolayer cell were maintained in medium A (DMEM with 10% fetal calf serum). Differentiation was induced by incubating the cells with medium A supplemented with 10μg/ml insulin, 1μM dexamethasone, 0.5mM IBMX for 3 days, followed by another 2 days incubation with medium A supplemented with 10μg/ml insulin. The cells were further incubated in medium A for an additional 2 days to complete the adipocyte conversion. Followed this procedure, great than 90% of the cells had the morphological and biochemical properties of adipocytes.⑵High glucose induce insulin resistance in 3T3-L1 adipocytes: 3T3-L1 adipocytes were treated with different concentration glucose for 24 hours, the insulin-stimulated 3H-2-deoxyglucose incorporation test were used to determin insulin sensitivity of 3T3-L1 cells. Results showed that 25mM glucose induced the adipocytes insulin resistance, for decreasing insulin-stimulated increase in glucose uptake by 150 times.⑶The effect of the two forms of endogenous TNF-αon insulin resistance induce by high glucose: 3T3-L1 adipocytes treated with different concentration glucose for 24 hours. Results showed when the cells were induced insulin resistance by 25mM glucose, the TM TNF-αprotein level was declined from 81.4% to 3.56% (p<0.01) and sTNF-αlevel was increased from 63.7 to 113.0 pg/ml (p<0.01), compared to group with 15mM glucose. Insulin resistance could be blocked in part by 160ng/ml anti-TACE antibody or 160ng/ml TNFR1BP-Fc fusion protein, compared to group induced insulin resistance by 25mM glucose, fusion protein and anti-TACE antibody increased insulin-stimulated glucose uptake by 20 and 50 times, respectively.⑷The effect of the two forms of exogenous TNF-αon reponse of 3T3-L1 adipocytes to insulin: 3T3-L1 adipocytes were treated with the two forms of exogenous TNF-αfor 24 hours and detected glucose uptake with insulin stimulation. Compared to the control group without any stimulation, sTNF-αdecreased insulin-stimulated glucose uptake by 96%, but TMTNF-αincreased two times. We alse found that fusion protein or anti-TNF-αantibody can completely block both of sTNF-αand TMTNF-αeffects when they were put together alone.⑸The effect of the two forms of exogenous TNF-αon insulin signaling: 3T3-L1 adipocytes were treated with the two forms of TNF-αfor 24 hours and after that we detected the level of IRS-1 tyrosine phospharylation and serine phopharylation on 270 site (Ser270) to find out its molecular mechanism. We found that sTNF-αstimulated serine phospharylation of IRS-1 and inhibited tyrosine phopharylation, TM TNF-αacted on the contrary: TM TNF-αinhibited IRS-1 Ser270 phospharylation without effect on tyrosine phopharylation.3. Effect of fusion protein on insulin resistance in vivo⑴Establish insulin resistance animal model: Four-week-old male wistar rats weighing between 110 and 130g were fed either with a normal diet (ND) or a high-fat and high-sucrose diet (HFS). Obesity associated insulin resistance was induced by HFS diet for 16 weeks, manifesting as increased body weight by 18%, significant hyperinsulinaemia but euglycemia maintenance and increased HOMAIR index by three times when compared with ND group.⑵Treat animals: After 16 weeks, besides the ND group, the rats with HFS were randomly assigned into four treated groups: fusion protein treatment group, Fc protein treatment group, rabbit anti-TNF-αpolyclonal antibody treatment group and the HFS control group. The rats were given ingtravenoursly with 500μg TNFR1BP-Fc fusion protein or Fc protein or rabbit anti-TNF-αpolyclonal antibody in 0.1 ml normal saline (NS) or just 0.1ml NS treatmeny respectively, twice a week for four weeks. ⑶Inhibitory effect of the fusion protein on body weight, adiposity, serum triglyceride and total chcholesteraemia: At the end of 20 weeks, the mean body weight gain, the weight of epididymal fat pads and the level of serum triglyceride of HFS group was 2 times,1.8 and 3.7 times than that of ND group. Besides that histological analysis of adipose tissue also showed that adipocytes were larger in HFS group than in ND group. The treatment with fusion protein, but not Fc protein for 4 weeks resulted in a significantly decrease weight gain by 26%, fat pads by 30% and the triglyceride level by 50%。Similarity, histological analysis of adipose tissue also shows that adipocytes were larger in HFS fed rats than in control rats. In contrast, the treatment of fusion protein, but not Fc protein, diminished their larger diameter. All these measurements of anti-TNF-αantibody (HFS+Ab) group showed no difference between the ND group. Total cholesterol levels were unchanged in the plasma among the four group.⑷The effect of the fusion protein on glucose metabolism and insulin sensitivity: The blood glucose leveles of all groups showed no difference among them and kept in normal limits, but the serum insulin, C-peptide and HOMAIR were about two folds of ND. The pancreatic islet was compensatory hypertrophy. The Insulin and glucose tolerance experiment explained that the sensitivity of insulin significantly impaired by HFS diet compared to ND group. After treatment with fusion protein, the parameter of insulin, C-peptide and HOMAIR nearly descended to 50%. The compensatory hypertrophy of pancreatic was released too. The Insulin and glucose tolerance experiment directly illustrated that the insulin sensitivity notably increased after post-treatment, but there was no effect of treatment with Fc. The insulin sensitivity of HFS+Ab group showed no difference between the ND group.⑸The effect of the fusion protein on TNFαprotein level by ELISA: We detected the TNFαin serum and in the extract of tissue protein. In HFS group, the content of TNFαin muscle and adipose tissue, obviously upgraded. In fusion protein group, the content of TNFαin serum and adipose tissue downgraded to 50%, however, in muscle downgraded to 80%. In the HFS+Ab group, the content of TNFαin serum and adipose tissue downgraded to 60%, however, in muscle downgraded to 80%.There was no difference in liver tissue of all groups. The levels of TNFαin adipose tissue and muscle tissue were 2.5 and 1.4 folds than that of ND group. ⑹The effect of fusion protein on TNFαprotein in adipose and pancreatic tissue by immunity histochemistry: We compared the expression level of TNFαin adipose and pancreatic tissue by immunity histochemistry. Expression level of TNFαobviously increased compared to ND group and decreased after post-treatment by fusion protein.⑺The effect of fusion protein on IRS-1 tyrosine phosphorylation: We tested tyrosine phosphrylation of IRS-1 by IP-Western blotting. The phosphorylation of IRS-1 was significantly inhibited in fat by 80%, muscle by 68% and liver by 32% for HFS-fed rats compared with that of ND-fed rats. Treatment with fusion protein led to a striking increase of IRS-1 phosphorylation by 54% in fat and by 50% in muscle. Treatment with antibody led to increase of IRS-1 phosphorylation by 75% in fat and by 59% in muscle.4. Cloning, expression and bioactivity appraisement of human sTNFRII and preparation its polyclonal antibody⑴Construct and express the recombiant of human soluble TNF receptorII(sTNFRII) by E.coli: The extracellular domain cDNA of human TNFRII was obtained by RT-PCR from human monocytes. The gene fragment was inserted into pET-28a(+) and positive recombinant was identified by sequencing without mutation. Protein expression was induced by IPTG in E.coli. The expression product was isolated and purified by Ni-NTA agrose. Results showed that the highest expression was achieved after induction for 6 h with IPTG.. SDS-PAGE showed an extra protein band which was around 32kD in size, which occuppied 30% of the total protein in E.coli.⑵Identify its biological activities: Western blotting showed that recombinant sTNFRII was able to bind anti-TNFRII antibody. Indirect immunofluorescene indicated that sTNFRⅡcould inhibit the binding of GFP-sTNF-αto TNFR on L929. TNF-αbioassay suggested that as low as 20μg/ml, sTNFRII completely inhibited the cytotoxicity induced by 100U/ml TNF-α.⑶Prepare sTNFRII polyclonal antibody: we used recombinant sTNFRII as antigen to immune rabbit and gained polyclonal antibody. The titres of the sTNFRII specific ployclonal antibody obtained were 1:32000 and 1:16000.Conclusion:⑴Eukaryotic stable expresses and purifies the fusion protein TNFRIBP-Fc which it is bioactive in murine system;⑵In vitro we identify Fusion protein ameliorates insuin resistant of 3T3-L1 adipocyte induced by high glucose and sTNF-αthrough downregulation sTNF-αexpression level and inhibit their interaction between sTNF-αand TNFRI;⑶TM TNF-αand sTNF-αplay different role in insulin resistance:TM TNF-αcan improve insulin sensitivity but sTNF-αinduced insulin resistance;⑷TNFRIBP-Fc fusion protein improves obesity and insulin resistance induced by high fat and high sucrose diet and protected form the obesity-related reduction in the insulin recetor signaling in muscle and fat tissues, mainly responsible for peripheral glucose uptake with stimulation by insulin.