| Obesity is considered as a chronic low-grade inflammatory state that results in insulin resistance, and also linked to a wide array of other pathophysiological sequelae including hypertension, hyperlipidemia, atherosclerosis, and type2diabetes. Disruption in the delicate balance between anti-inflammatory and pro-inflammatory adipokines secreted by adipose tissue could be involved in the induction and maintenance of obesity-related chronic inflammatory state, and contribute to the development of obesity-linked insulin resistance. Tumor necrosis factor (TNF-a) is one of adipokines secreted by adipose tissue, and exists in two bioactive forms, a26-kDa transmembrane form (tmTNF-α) and a17-kDa soluble form (sTNF-α). sTNF-α has been widely recognized as a key factor linking obesity and insulin resistance, facilitating insulin resistance through a variety of molecular mechanisms. However, so far the impact of tmTNF-a on insulin resistance remains poorly understood. The aim of the present study was to investigate the impact of tmTNF-a on obesity-induced insulin resistance and its molecular mechanisms, and to provide new clues for the intervention and treatment of obesity and insulin resistance. The main results are as follows:Part1Exogenous tmTNF-α increases insulin sensitivity of adipocytes via forward signaling mediated by TNFR23T3-L1preadipocytes were induced to differentiate into mature adipocytes, and then directly stimulated with the two forms of TNF-α for24h. The insulin signaling, production of pro-and anti-inflammatory adipokines and their molecular pathways were detected.Ⅰ. Exogenous tmTNF-α increases insulin sensitivity of3T3-L1adipocytes and human primary adipocytes1. tmTNF-α enhanced the insulin-stimulated glucose uptake in adipocytes:[H]-2-DG uptake experiments showed that, in contrast to the inhibitory effect of sTNF-α, tmTNF-α improved cellular glucose uptake in response to insulin in adipocytes, and this insulin-sensitizing effect of tmTNF-a could be blocked by pretreatment with an TNF-α antibody.2. tmTNF-α directly improved insulin signaling in adipocytes:Western analysis showed that, in contrast to the effect of sTNF-a, tmTNF-a significantly increased insulin-stimulated tyrosine phosphorylation of IRS-1and phosphorylation of Akt, which both are key components in insuin signaling.3. Exogenous tmTNF-a promoted the expression and membrane translocation of GLUT4in adipocytes: Western blot analysis showed that, in contrast to the effect of sTNF-a, tmTNF-a evidently promoted the expression of GLUT4, and its translocation from the cytosol to the membrane fraction in response to insulin.These results above indicated that tmTNF-a increased insulin sensitivity of adipocytes via directly improved insulin signaling.Ⅱ. Exogenous tmTNF-a downregulated pro-inflammatory adipokines via inhibition of NF-κB pathway1. Exogenous tmTNF-a inhibited NF-κB activation:We previously demonstrated that, in contrast to the effect of sTNF-a, tmTNF-a could inhibit the gene transcription and protein synthesis of pro-inflammatory adipokines IL-6and MCP-1. Since IL-6and MCP-1are NF-κB target genes, we used Western blot to assess the impact of the two forms of TNF-a on NF-κB activation. The results showed that, in contrast to the promotive effect of sTNF-a, tmTNF-a markedly inhibited the degradation of IκB-α and the nuclear translocation of NF-κB p65from the cytoplasm, indicating that NF-κB activation was suppressed by tmTNF-α.2. Exogenous tmTNF-α inhibited NF-κB pathway via A20:We used Real-time RT-PCR to measure mRNA expression of A20, a negative feedback regulator of NF-κB signaling pathway, and verified that A20gene expression could be induced by both forms of TNF-a. Silencing A20expression by siRNA promoted the degradation of IκB-a induced by sTNF-a, but completely eliminated the inhibitory effect of tmTNF-a on IκB-α degradation. siA20further improved mRNA transcription of IL-6and MCP-1upregulated by sTNF-a, while blocked diminished gene transcription of these two adipokines downregulated by tmTNF-α.These results above suggested that tmTNF-α inhibited NF-κB pathway via A20to downregulate the production of pro-inflammatory adipokines IL-6and MCP-1, and consequently, indirectly increased insulin sensitivity of adipocytes. Ⅲ. Exogenous tmTNF-α promoted the expression of insulin-sensitizing adipokine adiponectin via PPAR-γ1. Exogenous tmTNF-α promoted the production of adiponectin:ELISA analysis confirmed that, in contrast to the effect of sTNF-α, tmTNF-α promoted the synthesis and secretion of anti-inflammatory adipokine adiponectin.2. Exogenous tmTNF-α promoted adiponectin expression and increased insulin sensitivity via PPAR-γ:We previously verified that tmTNF-α remarkably improved the expression of PPAR-y, a transcription factor of adiponectin, and treatment with the specific PPAR-y antagonist GW9662blocked tmTNF-α-mediated upregulation of adiponectin mRNA expression. We used glucose uptake experiments to further confirm that treatment with GW9662totally prohibited the insulin-stimulated glucose uptake enhanced by tmTNF-α.These results above hinted that tmTNF-α promoted adiponectin expression via PPAR-y, contributing to the enhancement of insulin sensitivity.Ⅳ. Exogenous tmTNF-α exerts the insulin-sensitizing effect via TNFR23T3-L1adipocytes were pretreatment with TNFR1and TNFR2neutralizing antibodies respectively, and then stimulated with the two forms of TNF-α for24h. The results showed that:1. Exogenous tmTNF-a increased insulin sensitivity via TNFR2:TNFR1blockage abolished sTNF-α-induced inhibition of the insulin-stimulated glucose uptake, while TNFR2blockage totally canceled tmTNF-α-induced promotion of the insulin-stimulated glucose uptake and the tyrosine phosphorylation of IRS-1.2. Exogenous tmTNF-a downregulated inflammatory response via TNFR2: TNFR1blockage completely abolished sTNF-α-induced promotion of IL-6mRNA expression, while TNFR2blockage totally canceled tmTNF-a-induced inhibition of IL-6gene transcription and IκB-α degradation.3. Exogenous tmTNF-α upregulated anti-inflammatory response via TNFR2: TNFR1blockage largely reversed sTNF-α-induced inhibition of adiponectin mRNA expression, while TNFR2blockage totally canceled tmTNF-α-induced upregulation of the levels of adiponectin mRNA and PPAR-γ protein. These results above indicated that tmTNF-a increased insulin sensitivity of adipocytes via TNFR2.Part2The impact of tmTNF-a antibody on high fat diet-induced obese miceⅠ. Specificity identification of tmTNF-a antibody in vitroWe previously prepared a rabbit anti-mouse tmTNF-a specific polyclonal antibody. ELISA analysis revealed that this antibody could bind specifically to tmTNF-a antigen peptide without cross-reaction to sTNF-a. Western blot showed that this antibody only interacted with26kD tmTNF-a, but not with17kD sTNF-a. FACS confirmed that tmTNF-a antibody could increase the surface expression of tmTNF-a on3T3-L1adipocytes.Ⅱ. Determination of the therapeutic dose of tmTNF-a antibody in vivoIn order to determine a therapeutic dose of tmTNF-a antibody in vivo, we injected intraperitoneally different doses of tmTNF-a antibody into C57BL/6mice, and detected the expression of tmTNF-a in epididymal adipose tissue and skeletal muscle tissue24h after injection. IHC analysis showed that400μg tmTNF-a antibody could effectively increase the expression of tmTNF-a both in adipose tissue and skeletal muscle. Western blot analysis declared that600μg tmTNF-a antibody could remarkably increase the expression of tmTNF-a in these two tissues. So we used600μg tmTNF-a antibody in the next studies.Ⅲ. The impact of tmTNF-a antibody on tmTNF-a enzymolysis in high fat diet-induced obese miceAfter being fed with high fat diet (HFD) for2weeks, C57BL/6mice were injected intraperitoneally with either600μg purified rabbit anti-mouse tmTNF-a antibody or same amount of rabbit IgG or PBS, three times a week. The treatment lasted12weeks. Mice fed a normal diet were used as a chow control.1. The impact of tmTNF-a antibody on tmTNF-a expression in adipose tissue of HFD-induced obese mice:IHC and Western blot analyses showed that HFD induced a slight increase in tmTNF-a expression in adipose tissue of HFD-induced obese mice, while tmTNF-a antibody caused a significant increase in tmTNF-a expression in adipose tissue.2. The impact of tmTNF-a antibody on tmTNF-a expression in skeletal muscle tissue of HFD-induced obese mice:IHC and Western blot analyses showed that HFD increased tmTNF-a expression in skeletal muscle tissue of HFD-induced obese mice, while tmTNF-a antibody caused a further increase in tmTNF-a expression in skeletal muscle tissue.3. The impact of tmTNF-a antibody on tmTNF-a expression in liver tissue of HFD-induced obese mice:IHC and Western blot analyses showed that HFD increased tmTNF-a expression in liver tissue of HFD-induced obese mice, while tmTNF-a antibody caused a further increase in tmTNF-a expression in liver tissue.4. The impact of tmTNF-a antibody on plasma sTNF-a level in HFD-induced obese mice:CBA analysis revealed that obesity induced a remarkable increase in plasma sTNF-a level, while tmTNF-a antibody completely inhibited sTNF-a secretion induced by obesity.These results above suggested that tmTNF-a antibody could significantly block the proteolytic cleavage of tmTNF-a, contributing to increased expression of tmTNF-a in adipose tissue, skeletal muscle and liver, which are crucial organs for glucose metabolism, and therefore, the impact of tmTNF-a on glucose and lipid metabolism and insulin resistance in obese mice could be observed.Ⅳ. The impact of tmTNF-a antibody on lipid metabolism in high fat diet-induced obese mice1. The impact of tmTNF-a antibody on body weight in high fat diet-induced obese mice:Body weight curve showed that body weight of HFD-fed mice was higher than chow control from2weeks after HFD feeding, and tmTNF-a antibody did not impact body-weight gain induced by HFD. After12weeks of antibody treatment, there was no difference in body weight between antibody treatment group and HFD control groups.2. The impact of tmTNF-a antibody on fat mass in high fat diet-induced obese mice:The weight of the epididymal fat pads showed that, HFD caused increased fat mass in mice, while tmTNF-a antibody resulted in a further increase in fat mass. HE staining of adipose tissue revealed that, the increase in fat cell size seen in HFD-fed mice was accompanied by the increased infiltration of inflammatory cells, meanwhile, larger adipocytes but reduced infiltration of inflammatory cells were seen in tmTNF-a antibody treatment group than those in HFD control groups.3. The impact of tmTNF-a antibody on liver in high fat diet-induced obese mice: Increased liver weights and elevated ALT levels were confirmed in HFD-fed mice. HE staining also displayed hepatic steatosis, as many single large droplets had displaced the nucleus and ballooning degeneration causing conspicuous swelling of the hepatocytes and cytoplasmic vacuolation, which could be stained with Oil Red O. The application of tmTNF-a antibody could significantly reduce liver weights and prevented the development of hepatic steatosis. ALT levels have returned to normal after antibody treatment.4. The impact of tmTNF-a antibody on blood lipids in high fat diet-induced obese mice:Plasma total cholesterol (TC), triglyceride (TG), HDL and LDL levels were evidently increased in HFD-fed obese mice. tmTNF-a antibody treatment drastically lowered the levels of TG and LDL, and further increased HDL levels, whereas had no effect on hypercholesterolemia.Ⅴ. The impact of tmTNF-a antibody on glucose metabolism in high fat diet-induced obese mice1. The impact of tmTNF-a antibody on fasting plasma glucose and insulin in high fat diet-induced obese mice:HFD-fed obese mice had normal fasting blood glucose values. In contrast, fasting insulin levels in HFD-fed mice were significantly increased. The application of tmTNF-a antibody improved obesity-induced hyperinsulinemia, but further elevated fasting blood glucose levels.2. The impact of tmTNF-a antibody on glucose tolerance and insulin sensitivity in high fat diet-induced obese mice:Impaired glucose tolerance and insulin sensitivity were determined in HFD-fed obese mice. Although tmTNF-a antibody could not improve impaired glucose tolerance, it could partially improve insulin sensitivity, indicating that tmTNF-a antibody-induced hyperglycemia is mainly related to inadequate insulin production.3. The impact of tmTNF-a antibody on pancreas in high fat diet-induced obese mice:A compensatory hypertrophy of pancreatic islets with increased insulin-positive cells was observed in HFD-fed obese mice. tmTNF-a antibody treatment markedly suppressed obesity-induced hyperplasia and hypertrophy of pancreatic islets. IHC experiments confirmed that tmTNF-a antibody could enhance the expression of tmTNF-a in pancreatic islets, suggesting that this antibody might induce apoptosis of pancreatic islet cells. TUNEL analysis demonstrated that tmTNF-a antibody led to increased apoptosis in pancreatic cells, which could be the reason for inadequate compensatory hypertrophy.4. The impact of tmTNF-a antibody on GLUT4expression in various tissues in high fat diet-induced obese mice:Western blot analysis showed that, the expression of GLUT4in membrane protein fraction of adipose tissue and skeletal muscle was evidently decreased in HFD-fed mice. tmTNF-a antibody markedly increased GLUT4expression in membrane protein fraction of adipose tissue and skeletal muscle, and induced a slight GLUT4expression in membrane fraction of liver tissue.Ⅵ. The impact of tmTNF-a antibody on plasma pro-and anti-inflammatory adipokines in high fat diet-induced obese miceWe used CBA method to measure the plasma contents of pro-inflammatory adipokines IL-6, MCP-1and anti-inflammatory adipokine adiponectin in all mice. The results showed that, plasma IL-6and MCP-1concentrations were significantly increased, while plasma adiponection levels were remarkably decreased in HFD-fed obese mice. tmTNF-a antibody treatment restored the levels of these pro-and anti-inflammatory adipokines to normal.Part3Switch characteristic of tmTNF-a in adipose tissue of obese patientsⅠ. TNF-α expression in abdominal subcutaneous adipose tissue of normal controls and obese patientsWe collected abdominal subcutaneous adipose tissues of normal controls (BMI<25kg/m2) and obese patients (BMI>30kg/m2) from the operating room of Tongji hospital. Western blot analysis showed that the expression of TNF-α in total protein from adipose tissue lysate of obese patients was significantly higher than those of normal controls. We used statistical software to analyze the correlation between BMI and TNF-α expression in abdominal subcutaneous adipose tissue of21(12normal and9obese) individuals. The results showed that BMI is positively correlated with TNF-a expression in adipose tissue.Ⅱ. tmTNF-a expression in membrane fraction and sTNF-a content in tissue homogenate from abdominal subcutaneous adipose tissue of normal controls and obese patientsWestern blot and ELISA results demonstrated that tmTNF-a expression in membrane fraction and sTNF-a content in tissue homogenate from adipose tissue of obese patients were significantly higher than those of normal controls.Ⅲ. TACE expression and activity in abdominal subcutaneous adipose tissue of normal controls and obese patientsWe used fluorescent substrate assay and Western Blot to detect TACE activity and its expression in abdominal subcutaneous adipose tissue of human subjects, respectively. The results showed that TACE expression and activity in adipose tissue of obese subjects were significantly higher than those of normal controls, indicating that elevated TACE expression and activity are responsible for increased tmTNF-a switch.IV. Timp-3expression in abdominal subcutaneous adipose tissue of normal controls and obese patientsWe used Western Blot to explore the expression of timp-3, an inhibitor of TACE activity, in abdominal subcutaneous adipose tissue of human subjects. The results displayed that timp-3expression in adipose tissue of obese subjects were significantly lower than those of normal controls, indicating that decreased timp-3expression might be the reason for increased TACE activity.Conclusions:tmTNF-a could improve insulin signaling transduction and the expression and membrane translocation of GLUT4to increase glucose uptake in adipocytes via TNFR2. It inhibits NF-kB activation via A20to reduce the expression of insulin-resistant moleculars IL-6and MCP-1, whilst enhances the expression of insulin-sensitive molecular adiponectin via PPAR-y, both contributing to increased insulin sensitivity in adipocytes. Furthermore, tmTNF-a antibody could effectively inhibit the ectodomain shedding of tmTNF-a in vivo, consequently, increase tmTNF-a expression in adipose tissue, skeletal muscle, liver and pancreas. tmTNF-a antibody treatment improved obesity-induced abnormal lipid metabolism and chronic inflammatory state, and partially alleviated obesity-induced insulin resistance. However, this antibody could induce apoptosis in pancreatic cells, resulting in inadequate compensatory insulin secretion and hyperglycemia. In line with animal model, tmTNF-a expression in membrane fraction and sTNF-a content in tissue homogenate from abdominal subcutaneous adipose tissue of obese patients were significantly increased, indicating an enhanced switch of tmTNF-α. Meantime, TACE expression and activity were increased, and the expression of timp-3, an inhibitor of TACE activity, were markedly decreased in adipose tissue of obese subjects, which are the reasons for enhanced switch of tmTNF-a. |
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