Autoamplification of tumor necrosis factor-alpha: a potential mechanism for the maintenance of elevated tumor necrosis factor-alpha in male but not female obese mice

Abstract

Although tumor necrosis factor-alpha (TNF-alpha) 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-alpha remain elusive. We hypothesized that autoamplification of TNF-alpha contributes to the maintenance of elevated TNF-alpha in obesity. Treatment of 3T3-L1 adipocytes with TNF-alpha, or injection of TNF-alpha into C57BL/6J mice, up-regulated TNF-alpha mRNA in adipocytes and in adipose tissues, respectively. Ob/ob male but not female mice lacking TNF-alpha receptors showed significantly lower levels of adipose TNF-alpha mRNA when compared with TNF-alpha receptor-expressing ob/ob mice. Thus, the lack of endogenous TNF-alpha signaling reduced adipose TNF-alpha mRNA in ob/ob male mice. Additionally, hyperinsulinemia potentiated this TNF-alpha-mediated autoamplification response in adipose tissues and in adipocytes in a synergistic and dose-dependent manner. Studies in which TNF-alpha was injected into lean mice lacking individual TNF-alpha receptors indicated that TNF-alpha autoamplification in adipose tissues was mediated primarily via the p55 TNF-alpha receptor whereas the p75 TNF-alpha receptor appeared to augment this response. Finally, TNF-alpha autoamplification in adipocytes occurred via the protein kinase C signaling pathway and the transcription factor nuclear factor-kappaB. Thus, TNF-alpha can positively autoregulate its own biosynthesis in adipose tissue, contributing to the maintenance of elevated TNF-alpha in obesity.

Figure 1

Autoamplification 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.

Figure 2

Autoamplification of TNF-α mRNA in adipose tissue of wild-type and TNF-α receptor-deficient lean mice. Lean wild-type mice or TNFR-deficient lean mice were injected intraperitoneally with 4 μg of murine recombinant TNF-α or saline. Three hours later, adipose tissues were collected and analyzed for TNF-α mRNA as described in Materials and Methods. N = 4 ± SD. ***P < 0.001; ns, not significant.

Figure 3

Effect of TNF-α on the cellular localization of TNF-α mRNA in wild-type and TNFR-deficient lean mice. In situ hybridization was performed on paraffin sections of adipose tissues from wild-type saline-treated mice (A) and TNF-α-treated wild-type (B), p55^−/−/p75^−/− (C), p55^−/− (D), and p75^−/− (E) mice. a, adipocytes. Arrowheads indicate positive signals for TNF-α (B and E). Each picture is a representative of 12 slides. Original magnifications, ×400.

Figure 4

A: 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.

Figure 5

TNF-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.

Figure 6

A: 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.

Figure 7

Effect 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.