MAP3K8 (TPL2/COT) affects obesity-induced adipose tissue inflammation without systemic effects in humans and in mice

Abstract

Chronic low-grade inflammation in adipose tissue often accompanies obesity, leading to insulin resistance and increasing the risk for metabolic diseases. MAP3K8 (TPL2/COT) is an important signal transductor and activator of pro-inflammatory pathways that has been linked to obesity-induced adipose tissue inflammation. We used human adipose tissue biopsies to study the relationship of MAP3K8 expression with markers of obesity and expression of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8). Moreover, we evaluated obesity-induced adipose tissue inflammation and insulin resistance in mice lacking MAP3K8 and WT mice on a high-fat diet (HFD) for 16 weeks. Individuals with a BMI >30 displayed a higher mRNA expression of MAP3K8 in adipose tissue compared to individuals with a normal BMI. Additionally, high mRNA expression levels of IL-1β, IL-6 and IL-8, but not TNF -α, in human adipose tissue were associated with higher expression of MAP3K8. Moreover, high plasma SAA and CRP did not associate with increased MAP3K8 expression in adipose tissue. Similarly, no association was found for MAP3K8 expression with plasma insulin or glucose levels. Mice lacking MAP3K8 had similar bodyweight gain as WT mice, yet displayed lower mRNA expression levels of IL-1β, IL-6 and CXCL1 in adipose tissue in response to the HFD as compared to WT animals. However, MAP3K8 deficient mice were not protected against HFD-induced adipose tissue macrophage infiltration or the development of insulin resistance. Together, the data in both human and mouse show that MAP3K8 is involved in local adipose tissue inflammation, specifically for IL-1β and its responsive cytokines IL-6 and IL-8, but does not seem to have systemic effects on insulin resistance.

Figure 1. MAP3K8 in humans is associated with higher BMI and cytokine expression.

MAP3K8 mRNA expression in human subcutaneous adipose tissue, associated with (a) BMI, (b) plasma insulin values, (c) plasma glucose levels, (d) HOMA-IR, (e) adipocyte sell size cell size and (f) crown-like structures. *p<0.05. n = 51, 50, 71, 70 respectively. HOMA-IR  =  Homeostatic Model Assessment for insulin resistance.

Figure 2. MAP3K8 in humans is associated with IL-1β, IL-6 and IL-8 cytokine expression.

Biopsies from subcutaneous adipose tissue were obtained from healthy subjects with varying levels of obesity. Association of MAP3K8 mRNA expression in human subcutaneous adipose tissue with mRNA expression of (a) IL-1ß, (b) IL-6, (c) IL-8, (d) TNF-α, (e) serum amyloid A levels (SAA: Q1≤0.7 mg/L, Q4≥1.6 mg/L), (f) C-reactive protein (CRP: Q1≤0.5 mg/L, Q4≥2.0 mg/L). *p<0.05, **p<0.01.

Figure 3. Obesity and macrophage influx in adipose tissue of HFD-fed WT and MAP3K8-ko animals.

MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a) Bodyweight development upon LFD or HFD feeding. (b) Epididymal white adipose tissue (eWAT) weight after 16 weeks of LFD or HFD. (c) Liver weight after 16 weeks of LFD or HFD. (d) Plasma CXCL1 levels after 16 weeks of LFD or HFD (e) Macrophage influx into the adipose tissue as determined by immunohistochemistry, F4/80 (serotec) staining: 20× magnification or 40× as indicated: (f) Number of crown-like structures per field. (g–i) qPCR analysis for macrophage infiltration markers, (g) CD68, (h) F4/80, (i) MCP-1 in adipose tissue of MAP3K8-ko and WT animals. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. Inflammatory profile of the adipose tissue of HFD-fed WT and MAP3K8-ko animals.

MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a–f) qPCR analysis for cytokines (a) TNF-α, (b) IFNγ, (c) IL-1β, (d) CXCL-1, (e) IL-6 and (f) IL-1Ra. n = 9 mice per group. Relative phosphorylation of NFκB p65 (g) and ERK 1/2 (h) in eWAT of MAP3K8-ko and WT animals after HFD-feeding (i). * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. MAP3K8-ko mice display similar bodyweight and insulin sensitivity compared to WT mice.

MAP3K8-ko and WT mice were fed a LFD or HFD during 16 weeks. (a) Plasma insulin and (b) plasma glucose levels after diet intervention. Insulin (itt) and oral glucose (ogtt) tolerance tests after 16 weeks of diet intervention. (c) itt after 16 weeks of HFD and (d) area under the curve itt. (e) ogtt after 16 weeks of HFD and (f) area under the curve of ogtt. n = 9 mice per group. * p<0.05, ** p<0.01, *** p<0.001.