Chronic NOS inhibition accelerates NAFLD progression in an obese rat model

Abstract

The progression in nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis is a serious health concern, but the underlying mechanisms remain unclear. We hypothesized that chronic inhibition of nitric oxide (NO) synthase (NOS) via N(ω)-nitro-L-arginine methyl ester (L-NAME) would intensify liver injury in a rat model of obesity, insulin resistance, and NAFLD. Obese Otsuka Long-Evans Tokushima fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats received control or L-NAME (65-70 mg·kg(-1)·day(-1))-containing drinking water for 4 wk. L-NAME treatment significantly (P < 0.05) reduced serum NO metabolites and food intake in both groups. Remarkably, despite no increase in body weight, L-NAME treatment increased hepatic triacylglycerol content (+40%, P < 0.05) vs. control OLETF rats. This increase was associated with impaired (P < 0.05) hepatic mitochondrial state 3 respiration. Interestingly, the opposite effect was found in LETO rats, where L-NAME increased (P < 0.05) hepatic mitochondrial state 3 respiration. In addition, L-NAME induced a shift toward proinflammatory M1 macrophage polarity, as indicated by elevated hepatic CD11c (P < 0.05) and IL-1β (P = 0.07) mRNA in OLETF rats and reduced expression of the anti-inflammatory M2 markers CD163 and CD206 (P < 0.05) in LETO rats. Markers of total macrophage content (CD68 and F4/80) mRNA were unaffected by L-NAME in either group. In conclusion, systemic NOS inhibition in the obese OLETF rats reduced hepatic mitochondrial respiration, increased hepatic triacylglycerol accumulation, and increased hepatic inflammation. These findings suggest an important role for proper NO metabolism in the hepatic adaptation to obesity.

Keywords: Nω-nitro-l-arginine methyl ester; liver; nitric oxide; obese Otsuka Long-Evans Tokushima fatty rat.

Fig. 1.

Effects of N^ω-nitro-l-arginine methyl ester (l-NAME) on hepatic steatosis (A and B) and mRNA expression of inflammatory M1 (C and D) and M2 (E and F) markers, total macrophage (Mφ) markers (G and H), and markers of cell adhesion [monocyte chemotactic protein 1 (MCP-1; I) and ICAM (J)]. OLETF, Otsuka Long-Evans Tokushima fatty; LETO, Long-Evans Tokushima Otsuka; TAG, triacylglycerol. Values are means ± SE; n = 7–10/group. Different letter superscripts denote significant differences (P < 0.05).

Fig. 2.

Effect of l-NAME treatment on hepatic nitric oxide (NO) synthase (NOS) activity, total hepatic nitrate and nitrite (NO_X), NOS isoform expression, and kinases known to regulate endothelial NOS (eNOS) activation. A: representative Western blot images. AMPK, α-AMP-activated protein kinase. B: activity of Ca²⁺-dependent and -independent NOS in liver homogenate. AU, arbitrary units; ND, not determined. C–I: effects of l-NAME treatment on hepatic NO_X (C); protein expression of inducible NOS (iNOS; D), total eNOS (E), S1177-phosphorylated eNOS (F), T172-phosphorylated AMPK (F), and S473-phosphorylated Akt (H); and mRNA expression of hypoxia-inducible factor 1α (HIF-1α; I). Values are means ± SE; n = 10/group. Different letter superscripts denote significant differences (P < 0.05).

Fig. 3.

Effects of l-NAME treatment on hepatic mitochondrial function (A and B), content (C), and biogenesis (D and E). PGC-1α, peroxisome proliferator-activated receptor-γ coactivator 1α; TFAM, transcription factor A mitochondrial. Values are means ± SE; n = 5–10/group. Different letter superscripts denote significant differences (P < 0.05).

Fig. 4.

Effects of l-NAME treatment on total hepatic SOD activity (A) and mRNA expression of SOD1 (B) and SOD2 (C). Values are means ± SE; n = 10/group. Different letter superscripts denote significant differences (P < 0.05).

Fig. 5.

Increased hepatic steatosis in l-NAME-treated OLETF rats is independent of markers of lipid export [microsomal triglyceride transfer protein (MTTP; B)], fatty acid uptake (C), de novo lipogenesis [acetyl-CoA carboxylase (ACC; D), S79-phosphorylated ACC (p-ACC; E), and fatty acid synthase (FAS; F)], or red gastrocnemius (RG) skeletal muscle S473 phosphorylation-specific Akt (p-Akt; G). Values are means ± SE; n = 10/group. Different letter superscripts denote significant differences (P < 0.05).