Ablation of eNOS does not promote adipose tissue inflammation

Abstract

Adipose tissue (AT) inflammation is a hallmark characteristic of obesity and an important determinant of insulin resistance and cardiovascular disease; therefore, a better understanding of factors regulating AT inflammation is critical. It is well established that reduced vascular endothelial nitric oxide (NO) bioavailability promotes arterial inflammation; however, the role of NO in modulating inflammation in AT remains disputed. In the present study, 10-wk-old C57BL6 wild-type and endothelial nitric oxide synthase (eNOS) knockout male mice were randomized to either a control diet (10% kcal from fat) or a Western diet (44.9% kcal from fat, 17% sucrose, and 1% cholesterol) for 18 wk (n= 7 or 8/group). In wild-type mice, Western diet-induced obesity led to increased visceral white AT expression of inflammatory genes (e.g., MCP1, TNF-α, and CCL5 mRNAs) and markers of macrophage infiltration (e.g., CD68, ITGAM, EMR1, CD11C mRNAs, and Mac-2 protein), as well as reduced markers of mitochondrial content (e.g., OXPHOS complex I and IV protein). Unexpectedly, these effects of Western diet on visceral white AT were not accompanied by decreases in eNOS phosphorylation at Ser-1177 or increases in eNOS phosphorylation at Thr-495. Also counter to expectations, eNOS knockout mice, independent of the diet, were leaner and did not exhibit greater white or brown AT inflammation compared with wild-type mice. Collectively, these findings do not support the hypothesis that reduced NO production from eNOS contributes to obesity-related AT inflammation.

Keywords: Western diet; adipose tissue; endothelial nitric oxide synthase; obesity.

Fig. 1.

Body weight (A), percent body fat (B), epididymal adipose tissue (AT) weight (C), retroperitoneal AT weight (D), interscapular brown AT weight (E), and kilocalories consumed per week (F) in wild-type and eNOS knockout mice fed a control diet vs. Western diet. Values for reported body weight, percent fat, and fat pad weights are from the time of death. Values for kilocalories consumed/week are calculated as an average over the 18-wk feeding period. Data are expressed as means ± SE. D denotes main effect of diet, G denotes main effect of genotype, and D×G denotes diet by genotype interaction. Significant P values (<0.05) are highlighted in bold; n = 7 to 8/group.

Fig. 2.

Representative histology images (×10) of visceral white (i.e., epididymal) AT stained for Mac-2 (A), fold differences in Mac-2-positive stained area (B), and average adipocyte size (C) in wild-type and eNOS knockout mice fed a control diet vs. Western diet. Data are expressed as means ± SE. D denotes main effect of diet, G denotes main effect of genotype, D×G denotes diet by genotype interaction. Significant P values (<0.05) are highlighted in bold; n = 5/group.

Fig. 3.

Visceral white (i.e., epididymal) (A) and brown (i.e., interscapular) (B) AT gene expression in wild-type and eNOS knockout mice fed a control diet vs. Western diet. Data are expressed as means ± SE. D denotes main effect of diet (P < 0.05). No significant main effects of genotype or interactions were found (all P > 0.05); n = 7 to 8/group.

Fig. 4.

Visceral white (i.e., epididymal) AT protein content in wild-type and eNOS knockout mice fed a control diet vs. Western diet. Data are expressed as means ± SE. D denotes main effect of diet, G denotes main effect of genotype, D×G denotes diet by genotype interaction. Significant P values (<0.05) are highlighted in bold; n = 7 to 8/group. For the nitrotyrosine blot, an average of all bands (between 75 kDa and 25 kDa) was calculated.

Fig. 5.

Aorta gene expression in wild-type and eNOS knockout mice fed a control diet vs. Western diet. Data are expressed as means ± SE. G denotes main effect of genotype (P < 0.05). No significant main effects of diet or interactions were found (all P > 0.05); n = 4 to 8/group.