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• W1605764573 abstract "Although tumor necrosis factor-α (TNF-α) is elevated in adipose tissue in obesity and may contribute to the cardiovascular and metabolic risks associated with this condition, the mechanisms leading to elevated TNF-α remain elusive. We hypothesized that autoamplification of TNF-α contributes to the maintenance of elevated TNF-α in obesity. Treatment of 3T3-L1 adipocytes with TNF-α, or injection of TNF-α into C57BL/6J mice, up-regulated TNF-α mRNA in adipocytes and in adipose tissues, respectively. Ob/ob male but not female mice lacking TNF-α receptors showed significantly lower levels of adipose TNF-α mRNA when compared with TNF-α receptor-expressing ob/ob mice. Thus, the lack of endogenous TNF-α signaling reduced adipose TNF-α mRNA in ob/ob male mice. Additionally, hyperinsulinemia potentiated this TNF-α-mediated autoamplification response in adipose tissues and in adipocytes in a synergistic and dose-dependent manner. Studies in which TNF-α was injected into lean mice lacking individual TNF-α receptors indicated that TNF-α autoamplification in adipose tissues was mediated primarily via the p55 TNF-α receptor whereas the p75 TNF-α receptor appeared to augment this response. Finally, TNF-α autoamplification in adipocytes occurred via the protein kinase C signaling pathway and the transcription factor nuclear factor-κB. Thus, TNF-α can positively autoregulate its own biosynthesis in adipose tissue, contributing to the maintenance of elevated TNF-α in obesity. Although tumor necrosis factor-α (TNF-α) is elevated in adipose tissue in obesity and may contribute to the cardiovascular and metabolic risks associated with this condition, the mechanisms leading to elevated TNF-α remain elusive. We hypothesized that autoamplification of TNF-α contributes to the maintenance of elevated TNF-α in obesity. Treatment of 3T3-L1 adipocytes with TNF-α, or injection of TNF-α into C57BL/6J mice, up-regulated TNF-α mRNA in adipocytes and in adipose tissues, respectively. Ob/ob male but not female mice lacking TNF-α receptors showed significantly lower levels of adipose TNF-α mRNA when compared with TNF-α receptor-expressing ob/ob mice. Thus, the lack of endogenous TNF-α signaling reduced adipose TNF-α mRNA in ob/ob male mice. Additionally, hyperinsulinemia potentiated this TNF-α-mediated autoamplification response in adipose tissues and in adipocytes in a synergistic and dose-dependent manner. Studies in which TNF-α was injected into lean mice lacking individual TNF-α receptors indicated that TNF-α autoamplification in adipose tissues was mediated primarily via the p55 TNF-α receptor whereas the p75 TNF-α receptor appeared to augment this response. Finally, TNF-α autoamplification in adipocytes occurred via the protein kinase C signaling pathway and the transcription factor nuclear factor-κB. Thus, TNF-α can positively autoregulate its own biosynthesis in adipose tissue, contributing to the maintenance of elevated TNF-α in obesity. Tumor necrosis factor-α (TNF-α) is a multifunctional cytokine involved in the pathogenesis of multiple disease states including inflammation, obesity, and insulin resistance.1Beutler B Cerami A The biology of cachectin/TNF-a primary mediator of the host response.Annu Rev Immunol. 1989; 7: 625-655Crossref PubMed Scopus (1494) Google Scholar, 2Barbara JA van Ostade X Lopez A Tumor necrosis factor-alpha (TNF-alpha): the good, the bad and potentially very effective.Immunol Cell Biol. 1996; 74: 434-443Crossref PubMed Scopus (102) Google Scholar, 3Sethi JK Hotamisligil GS The role of TNFa in adipocyte metabolism.Cell Dev Biol. 1999; 10: 19-29Crossref Scopus (360) Google Scholar The TNF-α gene encodes a 26-kd protein primarily produced as a type II transmembrane protein, which is cleaved by the metalloprotease TNF-α converting enzyme to generate a soluble 51-kd trimeric TNF-α.4Kriegler M Perez C DeFay K Albert I Lu S A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF.Cell. 1988; 53: 45-53Abstract Full Text PDF PubMed Scopus (930) Google Scholar, 5Black RA Rauch CT Kozlosky CJ Peschon JJ Slack JL Wolfson MF Castner BJ Stocking KL Reddy P Srinivasan S Nelson N Boiani N Schooley KA Gerhart M Davis R Fitzner JN Johnson RS Paxton RJ March CJ Cerretti DP A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.Nature. 1997; 385: 729-733Crossref PubMed Scopus (2705) Google Scholar, 6Moss ML Jin SL Milla ME Bickett DM Burkhart W Carter HL Chen WJ Clay WC Didsbury JR Hassler D Hoffman CR Kost TA Lambert MH Leesnitzer MA McCauley P McGeehan G Mitchell J Moyer M Pahel G Rocque W Overton LK Schoenen F Seaton T Su JL Becherer JD Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.Nature. 1997; 385: 733-736Crossref PubMed Scopus (1481) Google Scholar Both forms of TNF-α are biologically active and elicit their actions via two distinct membrane receptors, a 55-kd isoform (p55) and a 75-kd isoform (p75) receptor.7Tartaglia LA Goeddel DV Two TNF receptors.Immunol Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1002) Google Scholar, 8Smith CA Farrah T Goodwin RG The TNF receptor superfamily of cellular and viral-proteins—activation, costimulation, and death.Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1837) Google Scholar TNF-α expression is increased in adipose tissue in obesity, and there is evidence linking elevated TNF-α to the development of insulin resistance.9Hotamisligil GS Shargill NS Spiegelman BM Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6121) Google Scholar, 10Hotamisligil GS Arner P Caro JF Atkinson RL Spiegelman BM Increased adipose tissue expression of tumor necrosis factor-a in human obesity and insulin resistance.J Clin Invest. 1995; 95: 2409-2415Crossref PubMed Scopus (2960) Google Scholar Additionally, we and others have shown that TNF-α contributes to the elevated expression of prothrombotic and inflammatory genes [eg, plasminogen activator inhibitor 1 (PAI-1), transforming growth factor-β, and monocyte chemoattractant protein 1 (MCP-1)] associated with obesity.11Samad F Uysal KT Wiesbrock SM Pandey M Hotamisligil GS Loskutoff DJ Tumor necrosis factor a is a key component in the obesity-linked elevation of plasminogen activator inhibitor-1.Proc Natl Acad Sci USA. 1999; 96: 6902-6907Crossref PubMed Scopus (193) Google Scholar, 12Samad F Yamamoto K Pandey M Loskutoff D Elevated expression of transforming growth factor-b in adipose tissue from obese mice.Mol Med. 1997; 3: 37-48Crossref PubMed Google Scholar, 13Sartipy P Loskutoff DJ Monocyte chemoattractant protein 1 in obesity and insulin resistance.Proc Natl Acad Sci USA. 2003; 100: 7265-7270Crossref PubMed Scopus (908) Google Scholar Although, these studies suggest that TNF-α may promote many of the obesity-linked pathologies, the physiological component of obesity that triggers the production of TNF-α remains elusive. Triglycerides and/or free fatty acids may be inducers of TNF-α expression because feeding rats a high-fat diet results in a significant increase in TNF-α mRNA and protein in fat pads, whereas mice lacking the fatty acid binding protein aP2 do not express TNF-α in adipose tissue.14Morin CL Eckel RH Marcel T Pagliassotti MJ High fat diets elevate tissue-derived tumor necrosis factor-a activity.Endocrinology. 1997; 138: 4665-4671Crossref PubMed Scopus (65) Google Scholar, 15Hotamisligil GS Johnson RS Distel RJ Ellis R Papaioannou VE Spiegelman BM Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein.Science. 1996; 274: 1377-1379Crossref PubMed Scopus (651) Google Scholar In this study, we hypothesized that in the obese adipose tissue, TNF-α can positively autoregulate its own biosynthesis. This hypothesis is supported by the fact that the TNF-α promotor contains binding sites for the nuclear factor (NF)-κB transcription factor,16Collart MA Baeuerle P Vassalli P Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kB-like motifs and of constitutive and inducible forms of NF-kB.Mol Cell Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar known to be involved in TNF-α expression and also known to be activated by TNF-α itself in various cell types.17Wajant H Henkler F Scheurich P The TNF-receptor-associated factor family—scaffold molecules for cytokine receptors, kinases and their regulators.Cell Signal. 2001; 13: 389-400Crossref PubMed Scopus (308) Google Scholar Thus, according to our hypothesis, weight gain leads to an initial but modest increase in TNF-α (eg, induced by free fatty acids, hypoxia, or other mechanisms). This initial increase in TNF-α is then further augmented via an autoregulatory TNF-α amplification loop in the local milieu of the obese adipose tissue, and this autocrine process may contribute to the maintenance of elevated TNF-α in obesity. The studies described in this report support this hypothesis and further show that TNF-α autoamplification in the adipose tissue occurs predominantly through the p55 TNF-α receptor and involves the protein kinase C signaling pathway and the transcription factor NF-κB. Furthermore, hyperinsulinemia, which is often associated with obesity, appears to potentiate TNF-α autoamplification in adipose tissue and adipocytes in a synergistic and dose-responsive manner. Finally, gender seems to be a determining factor in the expression and regulation of TNF-α mRNA in adipose tissue of obese mice. All animal studies were reviewed and approved by our Institutional Animal Care and Use Committee and the Animal Research Committee, in accordance with Public Health Policy regarding the use and care of laboratory animals. Adult obese mice (C57BL/ob/ob; 20 to 24 weeks of age) and wild-type C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Age-matched ob/ob mice deficient in either one (p55 or p75) or both TNFRs (p55 and p75) were generated by crossing and back-crossing lean mice deficient in these receptors to ob/ob mice as described,11Samad F Uysal KT Wiesbrock SM Pandey M Hotamisligil GS Loskutoff DJ Tumor necrosis factor a is a key component in the obesity-linked elevation of plasminogen activator inhibitor-1.Proc Natl Acad Sci USA. 1999; 96: 6902-6907Crossref PubMed Scopus (193) Google Scholar, 18Uysal KT Wiesbrock SM Marino MW Hotamisligil GS Protection from obesity-induced insulin resistance in mice lacking TNF-a function.Nature. 1997; 389: 610-614Crossref PubMed Scopus (1902) Google Scholar and genotyped using polymerase chain reaction (PCR)-based assays.18Uysal KT Wiesbrock SM Marino MW Hotamisligil GS Protection from obesity-induced insulin resistance in mice lacking TNF-a function.Nature. 1997; 389: 610-614Crossref PubMed Scopus (1902) Google Scholar In some experiments, lean mice lacking p55 TNFR, p75 TNFR, or both, and wild-type controls were injected intraperitoneally with recombinant murine TNF-α (4 μg per mouse in 100 μl of sterile saline; Genzyme Diagnostics, Cambridge, MA). Control animals were injected with 100 μl of saline. Three hours later, adipose tissues were removed and processed for in situ hybridization (below) or the preparation of total RNA. For in vivo insulin experiments, mice were injected intraperitoneally with 5 U of regular human insulin (Himulin R; Eli Lilly, Indianapolis, IN), and the controls were injected with an equivalent volume of saline. At various times thereafter, adipose tissues were removed and processed for the preparation of total RNA. Total RNA was isolated using the Ultraspec RNA isolation system according to the manufacturer's directions (Biotecx Laboratories, Inc., Houston, TX). 3T3-L1 mouse embryo fibroblasts were obtained from the American Type Culture Collection (Rockville, MD). The culturing of these cells (in six-well plates) and their differentiation from preadipocytes to mature adipocytes was performed as described previously.19Green H Kehinde O An established preadipose cell line and its differentiation in culture: II. Factors affecting the adipose conversion.Cell. 1975; 5: 19-27Abstract Full Text PDF PubMed Scopus (1089) Google Scholar TNF-α treatment was performed after a 24-hour pretreatment in serum-free medium containing 0.2% bovine serum albumin. Cells were harvested 3 hours later and TNF-α mRNA expression was determined by real-time reverse transcriptase (RT)-PCR. In some experiments, the cells were pretreated with an inhibitor of protein kinase C (PKC) (GF109203X; Calbiochem, La Jolla, CA) or inhibitors of the transcription factor NF-κB (HNE or SN50; Calbiochem) for 1 hour before administration of TNF-α. Total RNA was isolated 3 hours after TNF-α treatment using the Ultraspec RNA isolation system according to the manufacturer's directions (Biotecx Laboratories, Inc.), and the relative level of TNF-α mRNA was determined using real-time RT-PCR. The concentration of TNF-α mRNA was determined by real-time RT-PCR (I Cycler; Bio-Rad Laboratories, Hercules, CA) and the use of a standard curve prepared from a linearized synthetic plasmid containing upstream and downstream primer sets for TNF-α and β-actin (internal control), respectively.11Samad F Uysal KT Wiesbrock SM Pandey M Hotamisligil GS Loskutoff DJ Tumor necrosis factor a is a key component in the obesity-linked elevation of plasminogen activator inhibitor-1.Proc Natl Acad Sci USA. 1999; 96: 6902-6907Crossref PubMed Scopus (193) Google Scholar, 20Pandey M Tuncman G Hotamisligil GS Samad F Divergent roles for p55 and p75 TNF-a receptors in the induction of plasminogen activator inhibitor-1.Am J Pathol. 2003; 162: 933-941Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Standard RNA was prepared by in vitro transcription of the synthetic linearized plasmid using the Riboprobe Gemini II In Vitro Transcription System (Promega, Madison, WI) as previously described.11Samad F Uysal KT Wiesbrock SM Pandey M Hotamisligil GS Loskutoff DJ Tumor necrosis factor a is a key component in the obesity-linked elevation of plasminogen activator inhibitor-1.Proc Natl Acad Sci USA. 1999; 96: 6902-6907Crossref PubMed Scopus (193) Google Scholar, 20Pandey M Tuncman G Hotamisligil GS Samad F Divergent roles for p55 and p75 TNF-a receptors in the induction of plasminogen activator inhibitor-1.Am J Pathol. 2003; 162: 933-941Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar cDNA was prepared from either 1 μg of total RNA extracted from tissues or cells and from various concentrations (107 to 103 molecules) of the standard RNA as previously described.11Samad F Uysal KT Wiesbrock SM Pandey M Hotamisligil GS Loskutoff DJ Tumor necrosis factor a is a key component in the obesity-linked elevation of plasminogen activator inhibitor-1.Proc Natl Acad Sci USA. 1999; 96: 6902-6907Crossref PubMed Scopus (193) Google Scholar Real-time PCR amplifications were performed using 2.5 μl of cDNA, primers at a concentration of 150 nmol/L, and the SYBR green PCR master mix (Perkin-Elmer, Emeryville, CA) in a total volume of 25 μl, under cycling conditions used previously.20Pandey M Tuncman G Hotamisligil GS Samad F Divergent roles for p55 and p75 TNF-a receptors in the induction of plasminogen activator inhibitor-1.Am J Pathol. 2003; 162: 933-941Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar The concentrations of TNF-α and β-actin mRNA were determined using the standard curve constructed with the amplification data obtained from the various concentrations of the standard synthetic plasmid. TNF-α mRNA levels were then normalized to β-actin mRNA and expressed as per μg of total RNA.11Samad F Uysal KT Wiesbrock SM Pandey M Hotamisligil GS Loskutoff DJ Tumor necrosis factor a is a key component in the obesity-linked elevation of plasminogen activator inhibitor-1.Proc Natl Acad Sci USA. 1999; 96: 6902-6907Crossref PubMed Scopus (193) Google Scholar In situ hybridization was performed as described by using 35S-labeled anti-sense or sense TNF-α riboprobes.21Samad F Yamamoto K Loskutoff DJ Distribution and regulation of plasminogen activator inhibitor-1 in murine adipose tissue in vivo: induction by tumor necrosis factor-a and lipopolysaccharide.J Clin Invest. 1996; 97: 37-46Crossref PubMed Scopus (286) Google Scholar Slides were exposed in the dark at 4°C for 4 to 8 weeks. After slides were developed, they were counterstained with hematoxylin and eosin. Plasma TNF-α levels were determined using the Cytoscreen Mouse TNF-α Immunoassay Kit from Biosource International (Camarillo, CA) according to the manufacturer's instructions. The results are expressed as the mean ± SD. Statistical comparisons of results were performed using two-way analysis of variance (see Figure 2, Figure 4, Figure 5, Figure 6) or one-way analysis of variance (see Figure 1, Figure 7) using Prism 3.02 software (GraphPad, San Diego, CA). When the results passed the analysis of variance test, we performed Bonferroni's multiple comparison posttest to calculate the relevant P values (Figures 2, 4, 5, and 7) or tested whether there was a significant linear trend (Figure 1, Figure 6). In all cases, significance levels were set at *P < 0.05, **P < 0.01, and ***P < 0.001.Figure 4A: Expression of TNF-α mRNA in adipose tissues. Total RNA was extracted from adipose tissues of lean and obese (ob/ob), male and female mice. TNF-α mRNA expression was determined using real-time RT-PCR analysis. N = 3 ± SD. *P < 0.05; ns, not significant. B: Expression of TNF-α mRNA in adipose tissues from ob/ob and TNF-α receptor-deficient ob/ob mice. Total RNA was extracted from the adipose tissues of male and female ob/ob mice and ob/ob mice lacking both TNFRs (p55−/−/p75−/−). TNF-α mRNA was determined using real-time RT-PCR. N = 3 for each group, error bars represent ± SD. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5TNF-α plasma levels in wild-type and TNF-α receptor-deficient obese mice. Plasma was collected from male and female ob/ob mice and ob/ob mice lacking both TNFRs (p55−/−/p75−/−). Plasma TNF-α levels were determined using an ELISA assay as described in Materials and Methods. N = 4 for each group and error bars represent ± SD. ***P < 0.001; **P < 0.01; ns, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6A: Effect of insulin and/or TNF-α on TNF-α mRNA expression in 3T3-L1 adipocytes. 3T3-L1 adipocytes were either treated with insulin, TNF-α, or TNF-α (3 ng/ml) plus increasing doses of insulin. Total RNA was isolated 3 hours after treatments, and TNF-α mRNA expression was determined using real-time RT-PCR. N = 3 ± SD. **P < 0.01 for linear trend. B: Effect of insulin treatment on TNF-α mRNA expression in adipose tissues. Male ob/ob and lean mice were injected intraperitoneally with saline or 5 U of regular human insulin. Mice were sacrificed, and adipose tissues removed. Total RNA was extracted and analyzed for TNF-α mRNA expression by real-time RT-PCR. Each time point on the graph represents the mean ± SD of three animals. **P < 0.01; ns, not significant for linear trend.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Autoamplification of TNF-α mRNA in 3T3-L1 adipocytes. Total RNA was isolated from untreated 3T3-L1 adipocytes and adipocytes treated with recombinant mouse TNF-α for 3 hours. TNF-α expression was determined using real-time RT-PCR. N = 3 ± SD. ***P < 0.001 for linear trend.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effect of inhibitors of TNF-α signaling on TNF-α mRNA expression in adipocytes. 3T3-L1 adipocytes were pretreated for 1 hour with DMSO, or with the indicated inhibitors as described in Materials and Methods. Cells were then treated with 8 ng/ml of mouse recombinant TNF-α for 3 hours, and TNF-α mRNA expression was determined using real-time RT-PCR. For A and B, n = 3 ± SD. ***P < 0.001; *P < 0.05; ns, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because TNF-α signaling has been shown to result in activation of the NF-κB transcription factor and the TNF-α promotor contains NF-κB sites,16Collart MA Baeuerle P Vassalli P Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kB-like motifs and of constitutive and inducible forms of NF-kB.Mol Cell Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar, 17Wajant H Henkler F Scheurich P The TNF-receptor-associated factor family—scaffold molecules for cytokine receptors, kinases and their regulators.Cell Signal. 2001; 13: 389-400Crossref PubMed Scopus (308) Google Scholar we investigated the effect of TNF-α treatment on TNF-α mRNA expression in 3T3-L1 adipocytes. In vitro cultured 3T3-L1 adipocytes were treated with increasing amounts of recombinant murine TNF-α (1, 3, 6, 8, or 10 ng/ml; endotoxin-free). After 3 hours of TNF-α treatment, total RNA was isolated, and TNF-α mRNA levels were determined using quantitative real-time RT-PCR. TNF-α treatment of 3T3-L1 adipocytes resulted in a dose-dependent increase in TNF-α mRNA expression, with a maximum induction of 10-fold with 10 ng/ml of TNF-α compared to untreated control cells (Figure 1). These results suggest the existence of an autoamplification loop for the induction and maintenance of elevated TNF-α expression in adipocytes. We next investigated the autoamplification of TNF-α expression in vivo in mouse adipose tissue, and the role of p55 and p75 TNF receptors in mediating this response. Injection of recombinant murine TNF-α intraperitoneally into lean male wild-type mice resulted in a 10-fold increase (Figure 2; P < 0.001) in TNF-α mRNA in adipose tissue whereas this response was not observed in TNF-α-treated male p55−/−/p75−/− or p55−/− mice (Figure 2). However, TNF-α-treated male p75−/− mice did show a fourfold increase in adipose tissue TNF-α mRNA (Figure 2), although this did not reach statistical significance. The observation that TNF-α induction in p75−/− mice is less than half of the induction observed in wild-type mice, together with the observation that p55 deficiency leads to a complete lack of induction, indicates that p75 alone (p55−/−) cannot elicit a response and suggests that it can potentiate the response mediated by p55. These results were also confirmed by in situ hybridization (Figure 3). For example, no signal for TNF-α was detected in adipose tissue from untreated wild-type mice (Figure 3A). However, after TNF-α treatment, a strong hybridization signal was apparent in adipose tissue in the wild-type (Figure 3B) and p75−/− mice (Figure 3E), but not in p55−/−/p75−/− (Figure 3C), or p55−/− (Figure 3D) mice. Tissues hybridized with a sense probe (negative control) did not show any signal (not shown). Similar to the quantitative real-time RT-PCR data (Figure 2), the intensity of the hybridization signals in TNF-α-treated lean p75−/− mice (Figure 3E) was weaker than that observed in TNF-α-treated wild-type mice (Figure 3B). In both cases, the positive signals appeared to be associated with multiple cell types including cells that morphologically resembled adipocytes (Figure 3, B and E). The participation of other cell types such as macrophages also may be involved. It was previously shown that TNF-α mRNA levels are increased in adipose tissue obtained from obese mice compared to lean mice.9Hotamisligil GS Shargill NS Spiegelman BM Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6121) Google Scholar Interestingly, in the present study we observed significant gender-based differences in TNF-α mRNA levels in adipose tissue from obese ob/ob mice (Figure 4A), which was not apparent in previous studies because essentially all of those studies were conducted in male mice. Although adipose tissue TNF-α mRNA levels measured in lean mice did not differ significantly between sexes, adipose tissue TNF-α mRNA were threefold higher in male obese mice when compared to females (Figure 4A; P < 0.05). As a result, relative differences in adipose tissue TNF-α mRNA levels between obese and lean mice were statistically significant in males (sixfold; P < 0.05) but not in females. Plasma levels of TNF-α protein appeared to be slightly higher in ob/ob male mice compared with ob/ob females; however, this increase was not significant (Figure 5). It is probable that the majority of TNF-α produced is used locally in the tissues and/or bound to receptors and does not get into the circulation, therefore masking the gender-based differences observed for TNF-α mRNA expression. Plasma TNF-α levels were undetectable in male and female lean mice. The potential contribution of TNF-α autoamplification to elevated adipose tissue TNF-α mRNA expression in obesity was examined using ob/ob mice that expressed or lacked either one or both of the TNFRs. When compared with TNFR-expressing male ob/ob mice, TNF-α receptor-deficient male ob/ob mice had significantly reduced levels (85% reduction; P < 0.001) of TNF-α mRNA in adipose tissue (Figure 4B), suggesting that the lack of endogenous TNF-α signaling markedly reduced the levels of TNF-α mRNA in the adipose tissues of these mice. Male ob/ob mice deficient for p55 alone showed a 67% decrease (P < 0.01) in adipose tissue TNF-α mRNA levels. Although a decrease in adipose tissue TNF-α mRNA levels was also observed in p75-deficient male ob/ob mice, this decrease was not significant. In contrast to what we observed in male mice, TNF receptor deficiency did not affect adipose tissue TNF-α mRNA levels in female ob/ob mice (Figure 4B), suggesting that TNF-α autoamplification does not significantly contribute to the elevated levels of TNF-α in adipose tissues of female obese mice. Next, we investigated whether gender-based differences observed in adipose tissue TNF-α mRNA in obese mice was also reflected in circulating plasma TNF-α levels. We also analyzed how receptor deficiency in obese mice affects plasma TNF-α levels. Although TNF-α mRNA was elevated in adipose tissue in male compared to female obese mice (Figure 4), circulating plasma TNF-α levels were identical in normal male and female obese mice (Figure 5). Interestingly, the lack of both TNF-α receptors resulted in an increase in plasma TNF-α levels (Figure 5), with the increase being more pronounced in males compared to females (sixfold versus fourfold). These results potentially suggest that the majority of TNF-α is bound to its receptors in tissues and the lack of receptors allows for its accumulation in the circulation. The increased levels of plasma TNF-α in TNFR-deficient male obese mice compared to female mice also suggest that male mice have higher levels of TNF-α protein and a larger proportion of TNF-α is normally bound to its receptors in male mice compared with females. Unlike the increase in plasma TNF-α observed in mice lacking both TNFRs, plasma TNF-α levels in p55-deficient ob/ob mice does not differ significantly from levels measured in normal ob/ob mice, and no sex-based differences were observed in these mice (Figure 5). These results appear to suggest that the majority of TNF-α may actually be bound to the p75 TNFR in both male and female mice. Obese mice lacking the p75 receptor showed only a 58% increase in plasma TNF-α level compared to normal obese mice (30 pg/ml versus 19 pg/ml), but this was not statistically significant. The overall plasma TNF-α levels in the p75−/− mice were similar in both male and female mice. Because obesity is associated with hyperinsulinemia and increased TNF-α in adipose tissue,9Hotamisligil GS Shargill NS Spiegelman BM Adipose expression of tumor necrosis factor-a: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6121) Google Scholar we investigated whether increased insulin would potentiate TNF-α autoamplification. In vitro-cultured 3T3-L1 adipocytes were treated with a suboptimal dose (3 ng/ml) of TNF-α in the absence or presence of increasing amounts of insulin (10, 100, and 1000 nmol/L). After 3 hours of treatment, total RNA was prepared from the cells and changes in TNF-α mRNA expression were determined using real-time quantitative RT-PCR. Compared to untreated control, insulin treatment alone, even at the maximum dose of 1000 nmol/L, resulted in a statistically significant, although only modest, increase in TNF-α mRNA expression (Figure 6A). In contrast, a suboptimal dose of TNF-α (3 ng/ml) resulted in a dramatic increase in TNF-α mRNA expression. The effects of TNF-α on TNF-α mRNA were potentiated by insulin in a synergistic and dose-dependent manner (Figure 6A). Experiments were performed to determine whether this synergistic effect occurred in vivo. Insulin (5 U) was injected into both lean wild-type and obese ob/ob mice, and total RNA was prepared from adipo" @default.
• W1605764573 created "2016-06-24" @default.
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• W1605764573 date "2006-02-01" @default.
• W1605764573 modified "2023-10-16" @default.
• W1605764573 title "Autoamplification of Tumor Necrosis Factor-α" @default.
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