Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity

Abstract

Aims: Obesity is a risk factor for diabetes and cardiovascular diseases, with the incidence of these disorders becoming epidemic. Pathogenic responses to obesity have been ascribed to adipose tissue (AT) dysfunction that promotes bioactive mediator secretion from visceral AT and the initiation of pro-inflammatory events that induce oxidative stress and tissue dysfunction. Current understanding supports that suppressing pro-inflammatory and oxidative events promotes improved metabolic and cardiovascular function. In this regard, electrophilic nitro-fatty acids display pleiotropic anti-inflammatory signalling actions.

Methods and results: It was hypothesized that high-fat diet (HFD)-induced inflammatory and metabolic responses, manifested by loss of glucose tolerance and vascular dysfunction, would be attenuated by systemic administration of nitrooctadecenoic acid (OA-NO2). Male C57BL/6j mice subjected to a HFD for 20 weeks displayed increased adiposity, fasting glucose, and insulin levels, which led to glucose intolerance and pulmonary hypertension, characterized by increased right ventricular (RV) end-systolic pressure (RVESP) and pulmonary vascular resistance (PVR). This was associated with increased lung xanthine oxidoreductase (XO) activity, macrophage infiltration, and enhanced expression of pro-inflammatory cytokines. Left ventricular (LV) end-diastolic pressure remained unaltered, indicating that the HFD produces pulmonary vascular remodelling, rather than LV dysfunction and pulmonary venous hypertension. Administration of OA-NO2 for the final 6.5 weeks of HFD improved glucose tolerance and significantly attenuated HFD-induced RVESP, PVR, RV hypertrophy, lung XO activity, oxidative stress, and pro-inflammatory pulmonary cytokine levels.

Conclusions: These observations support that the pleiotropic signalling actions of electrophilic fatty acids represent a therapeutic strategy for limiting the complex pathogenic responses instigated by obesity.

Keywords: Inflammation; Nitro-fatty acid signalling; Obesity; Pulmonary hypertension; Xanthine Oxidase.

Figure 1

HFD induces weight gain and adiposity. (A) Mice subjected to HFD or NC were weighed weekly. (B and C) Fat tissue was determined by DEXA and abdominal fat pads were weighed at sacrifice. There was no statistical significance between HFD and HFD mice treated with OA-NO₂ for the last 6.5 weeks. Data represent mean ± SEM; ★P < 0.001 to NC; %P < 0.05 to NC; no significance between HFD and HFD + OA-NO₂ (n ≥ 6 per group).

Figure 2

OA-NO₂ improves glucose tolerance. (A and B) Fasting blood glucose (A) and plasma insulin (B) levels were measured at 20 weeks. (C) GTT was performed with an ip injection of 1.5 g/kg glucose in 0.9% NaCl at Week 18.5. Data represent mean ± SEM; (A and B) One-way ANOVA; (C) two-way ANOVA; ★P < 0.0001 to NC; ^P < 0.0001 to HFD; #P < 0.01 to HFD; ★★★P < 0.0001 to NC and HFD + OA-NO₂; ★★P < 0.01 to NC and HFD + OA-NO₂; %P < 0.05 to NC; no significance between NC and HFD + OA-NO₂ at any time point (n ≥ 6 per group).

Figure 3

OA-NO₂ limits HFD-induced inflammation, normalizes adipokine levels, and prevents generation of protein carbonyls. (A) Pro-inflammatory cytokines (MCP-1 and IL-6), macrophage markers (F4/80 and CD68), and adipokines (adiponectin and leptin) were determined by qPCR in abdominal fat. (B) Circulating leptin and (C) adiponectin levels were measured by radioimmunoassay and Luminex singleplex, respectively. Plasma adiponectin levels were normalized to fat mass (g) determined by DEXA in Fig. 1B. (D) Plasma MIP-1α, IL-6, and TNF-α were measured by Luminex multiplex. (E) Relative protein carbonyls were determined by DNP hydrazone product detection. Data represent mean ± SEM; ★P < 0.001 to NC; ^P < 0.001 to HFD; #P < 0.01 to HFD; &P < 0.05 to HFD; +P < 0.01 to NC; %P < 0.05 to NC; no significance between NC and HFD + OA-NO₂ at any time point unless noted (n ≥ 6 per group).

Figure 4

OA-NO₂ prevents obesity-induced PAH. (A) Representative tracings of PV loops in NC controls (double dashed), HFD (dashed), and HFD + OA-NO₂ groups for the last 6.5 of 20-week feeding (solid). (B–E) Haemodynamic indices were determined at 20 week in NC controls, HFD, and HFD + OA-NO₂-treated mice for RVESP (B), PVR (C), RV CI (D), and Tau (E). Data represent mean ± SEM; ★P < 0.01 to NC; #P < 0.05 to HFD; no significance between NC and HFD + OA-NO₂ (n ≥ 6 per group).

Figure 5

OA-NO₂ reduces pulmonary vascular remodelling and inflammation. (A) Representative confocal images of vascular remodelling stained with α-SMA and (B) average SM thickness of pulmonary arterioles were analysed (α-SMA, red; 4′,6-diamidino-2-phenylindole (DAPI), blue; and XO, green). (C) Lung inflammatory cytokine expression was determined by qPCR using Taqman assays. Data represent mean ± SEM; ★P < 0.001 to NC; #P < 0.01 to HFD; &P < 0.05 to HFD; no significance between NC and HFD + OA-NO₂ (n ≥ 6 per group).

Figure 6

OA-NO₂-mediated reduction in XO activity rescues HFD-induced PAH. (A) Lung XO activity and (B) representative PV loops for HFD (dashed) and HFD + febux (solid) (B). (C–F) Febuxostat effects on lung XO activity (C), RVESP (D), RV CI (E), and Tau (F). Data represent mean ± SEM; (A) one-way ANOVA, ★P < 0.001 to NC; ^P < 0.001 to HFD; (C–F) t-test, #P < 0.05 to HFD (n ≥ 6 per group).