Overexpression of endothelial nitric oxide synthase prevents diet-induced obesity and regulates adipocyte phenotype

Abstract

Rationale: Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high-fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied.

Objective: The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance.

Methods and results: In db/db mice and in high-fat diet-fed wild-type C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS transgenic mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with wild-type mice, high-fat diet-fed eNOS transgenic mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS transgenic mice showed higher levels of PPAR-α and PPAR-γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption.

Conclusions: These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high-fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.

Figure 1. Nutrient excess alters tissue eNOS levels

Tissue levels of eNOS from mice fed a low fat (LFD) or high fat diet (HFD) for 6 or 12 weeks; age-matched db/db mice were included as an additional model of T2D: (A) Representative Western blots of eNOS from epididymal adipose tissue and aorta. (B,C) Quantification of eNOS expression from panel A. n = 3–4 per group;**p<0.01 vs. 6 week LFD. (D,E) Levels of eNOS in adipose tissue and aorta from wild-type (WT), littermate eNOS-TG hemizygous, and eNOS-TG homozygous mice. n = 3 per group; **p<0.01 vs. indicated groups.

Figure 2. eNOS prevents diet-induced obesity

Weight gain, adiposity, and indirect calorimetry measurements from WT and eNOS-TG mice fed a low fat (LFD) or high fat diet (HFD): (A) Body weights during 6 weeks of LF feeding, n = 22–26 per group; (B) body weights during 6 weeks of HF feeding, n = 26 per group; (C) summarized weight gain over the course of 6 weeks and 12 weeks of HF feeding, n = 28–29 per group for 6 week group, n = 4–7 per group for 12 week group; (D) food intake, n = 4 per group; (E) representative DexaScan images of mice fed a LFD or HFD for 6 weeks; (F) body fat percentage, n = 8–12 per group; (G) lean mass percentage, n = 8–12 per group; and (H) tibia length for mice fed a LFD or HFD for 6 weeks, n = 8–12 per group; (I) average oxygen consumption (VO₂); (J) average carbon dioxide production (VCO₂); (K) respiratory exchange ratio (RER); and (L) ambulatory counts. n = 4 per group; *p<0.05, **p<0.01, and ***p<0.001 vs. indicated groups; ^#p<0.05 vs. WT HFD.

Figure 3. Effect of eNOS overexpression on indices of insulin resistance

After 6 weeks of a low fat (LFD) or high fat diet (HFD), glucose tolerance and insulin sensitivity were examined in WT and eNOS-TG mice: (A) Non-fasting and fasting glucose levels; white bars, WT LFD; blue bars, eNOS-TG LFD; white hatched bars, WT HFD; blue hatched bars, eNOS-TG HFD; (B) HbA1c; (C–E) glucose tolerance tests; and (F–H) insulin tolerance tests. n = 14 per group; *p<0.05 vs WT LFD or otherwise indicated groups.

Figure 4. eNOS overexpression decreases diet-induced adipocyte hypertrophy

Adipocyte size measurements from WT and eNOS-TG mice fed a LFD or HFD for 6 weeks: (A) Representative hematoxylin and eosin-stained images of adipose tissue from the epididymal fat pad (×20 magnification; scale bar = 100 μm); (B) Mean adipocyte area; (C) Distribution of adipocyte sizes from mice fed a LFD (upper panel) and a HFD (lower panel). n = 5 per group, *p<0.05 vs. WT LFD; ^#p<0.05 vs. WT HFD.

Figure 5. Metabolomic analyses of adipose tissues from high fat-fed mice

Metabolomic analyses of epididymal adipose tissue metabolites from WT and eNOS-TG mice fed HFD for 6 weeks: (A) Univariate analysis: t-tests of compounds from adipose tissues. All metabolites above the dotted line were found to be significantly different between WT and eNOS-TG mice (p<0.05). A table of these metabolites can be found in the data supplement (Online Table II); (B) Multivariate analysis: partial least squares-discriminant analysis (PLS-DA); (C) Hierarchial clustering: Heatmap and dendogram using the the most significantly different metabolites. (D) The significant metabolites were subjected to pathway impact analysis using Metaboanalyst MetPA and the Mus musculus pathway library. Fisher’s exact test was used for overrepresentation analysis, and relative betweenness centrality was used for pathway topology analysis. n = 14 animals: 7 WT HFD, 7 eNOS-TG HFD.

Figure 6. Overexpression of eNOS regulates intermediary metabolism in adipose tissue

Metabolite analysis from adipose tissues of WT and eNOS-TG mice fed HFD for 6 weeks: (A) z-score plots of significantly changed metabolites; (B) Correlation analysis was assessed using the Spearman rank correlation test, and the metabolites that correlated with citrulline were then examined. (C) Super- and sub-pathway distribution of adipose tissue metabolites found to be significantly different between WT and eNOS-TG mice. n=14 animals: 7 WT HFD and 7 eNOS-TG HFD.

Figure 7. Mitochondria are increased in the adipose tissue of eNOS-TG mice

Measurements of mitochondria in epididymal adipose tissue and skeletal muscle from WT and eNOS-TG mice: (A) Representative Western blots of adipose tissue eNOS, PGC1α, ALDH2, COX4I1, and Sirt3; GAPDH was used as a loading control. (B) Representative Western blots of skeletal muscle eNOS, PGC1α, VDAC, COX4I1, and Sirt3. GAPDH was used as a loading control. (C) Quantification of protein expression from panel A. (D) Quantification of protein expression from panel B. n=3 per group; *p<0.05 vs. WT; White bars, WT; blue bars, TG. (E) Immunofluorescence images of adipose tissue sections from HF-fed WT (panel i) and eNOS-TG (panel ii) mice; the sections were stained with MitoID-Red as a qualitative index of mitochondrial mass. Scale bar=200 μM (F) Representative photomicrograph of adipocytes isolated from HF-fed WT and eNOS-TG mice (600,000 adipocytes per well). (G) mRNA analysis of Ppara and Pparg. White bars, WT LFD; blue bars, eNOS-TG LFD; white hatched bars, WT HFD; blue hatched bars, eNOS-TG HFD; n=6 per group; *p<0.05 vs. indicated groups.

Figure 8. eNOS overexpression increases adipose tissue mitochondrial energetics

Extracellular flux (XF) analysis of adipose tissue explants from WT and eNOS-TG mice fed a HFD for 14 wks: (A) Representative photomicrographs of adipose tissue explants used for XF analysis; (B) Oxygen consumption rates (OCR) of adipose tissue explants: After three baseline measurements, antimycin A and rotenone (AA/Rot) were injected to identify the mitochondria-dependent OCR. (C) Mitochondrial OCR calculated from measurements in panel B. (D) Extracellular acidification rate (ECAR) measured from adipose explants; ECAR is a surrogate measure of glycolytic rate. n = 3–4 per group, *p<0.05 vs WT.