Reduced hepatic eNOS phosphorylation is associated with NAFLD and type 2 diabetes progression and is prevented by daily exercise in hyperphagic OLETF rats

Abstract

We tested the hypothesis that nonalcoholic fatty liver disease (NAFLD) is associated with reduced hepatic endothelial nitric oxide synthase (eNOS) activation status via S1177 phosphorylation (p-eNOS) and is prevented by daily voluntary wheel running (VWR). Hyperphagic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an established model of obesity, type 2 diabetes (T2D) and NAFLD, and normophagic controls [Long-Evans Tokushima Otsuka (LETO)] were studied at 8, 20, and 40 wk of age. Basal hepatic eNOS phosphorylation (p-eNOS/eNOS) was similar between LETO and OLETFs with early hepatic steatosis (8 wk of age) and advanced steatosis, hyperinsulinemia, and hyperglycemia (20 wk of age). In contrast, hepatic p-eNOS/eNOS was significantly lower (P < 0.05) in OLETF rats with T2D advancement and the transition to more advanced NAFLD with inflammation and fibrosis [increased tumor necrosis factor-α (TNF-α), CD68, and CD163 mRNA expression; 40 wk of age]. Reduced hepatic eNOS activation status in 40-wk OLETF rats was significantly correlated with reduced p-Akt/Akt (r = 0.73, P < 0.05), reduced serum insulin (r = 0.59, P < 0.05), and elevated serum glucose (r = -0.78, P < 0.05), suggesting a link between impaired glycemic control and altered hepatic nitric oxide metabolism. VWR by OLETF rats, in conjunction with NAFLD and T2D prevention, normalized p-eNOS/eNOS and p-Akt/Akt to LETO levels. Basal activation of hepatic eNOS and Akt are maintained until advanced NAFLD and T2D development in obese OLETF rats. The prevention of this reduction by VWR may result from maintained insulin sensitivity and glycemic control.

Keywords: Akt; OLETF; eNOS; exercise; hepatic; type 2 diabetes.

Fig. 1.

Representative Oil-Red O staining for neutral lipid staining in sedentary Otsuka Long-Evans Tokushima Fatty (OLETF-sed) and sedentary Long-Evans Tokushima Otsuka (LETO-sed) rats at 8, 20, and 40 wk of age and voluntary wheel running (VWR) OLETF (OLETF-ex) rats at 40 wk of age.

Fig. 2.

Basal activation status of hepatic endothelial nitric oxide synthase (eNOS) and Akt. Representative Western blots are depicted in A, with means ± SE (n = 6–8/group) shown for total eNOS protein content (B), S1177 phosphorylated (p)-eNOS/eNOS (C), total Akt (D), and S473 p-Akt/Akt (E). ^a,b,c Significant difference (P < 0.05) within group across age. *Significant difference (P < 0.05) between groups within age. AU, arbitrary units.

Fig. 3.

Effects of VWR on hepatic eNOS and Akt phosphorylation in 40-wk OLETF rats. Representative Western blots are depicted in A, with means ± SE (n = 6–8/group) shown for total eNOS (B), p-eNOS/eNOS (C), total Akt (D), and p-Akt/Akt (E). ^a,b Significant difference (P < 0.05) within group across age.

Fig. 4.

Total hepatic nitric oxide content (NO_X; A), α-smooth muscle actin (α-SMA) protein content (B), inducible nitric oxide synthase (iNOS) mRNA expression (C), tumor necrosis factor-α (TNF-α) mRNA expression (D), CD163 mRNA expression (E), and CD68 mRNA expression (F) in 40-wk OLETF and LETO rats. Values are means ± SE (n = 4–8/group). ^a,bSignificant difference (P < 0.05) within group across age.

Fig. 5.

Hepatic protein abundance of caveolin-1 (Cav-1; A), guanosine triphosphate cyclohydrolase-1 (GTPCH-1; B), adenosine monophosphate activated protein kinase (AMPK; C), and p-AMPK (D) in 40-wk OLETF and LETO rats. Values are means ± SE (n = 7–8/group). ^a,bSignificant difference (P < 0.05) within group across age.

Fig. 6.

Significant (P < 0.05) correlations of p-eNOS/eNOS with p-Akt/Akt (r = 0.73; A), serum glucose (r = −0.78; B), serum insulin (r = 0.59; C), and α-SMA protein content (r = −0.51; D) in 40-wk-old LETO-sed, OLETF-sed, and OLETF-ex rats.