Background: Metabolic dysfunction–associated steatotic liver disease (MASLD) is highly prevalent and closely linked to insulin resistance, endothelial dysfunction, and increased cardiovascular risk. Endothelial nitric oxide synthase (eNOS)–derived nitric oxide (NO) maintains vascular tone, endothelial integrity, and metabolic homeostasis. In the liver, eNOS is expressed in endothelial and parenchymal cells, where it supports hepatic perfusion and lipid metabolism. Hepatic endothelial dysfunction promotes inflammation, steatosis, and insulin resistance.
Purpose: To elucidate the role of eNOS in the pathogenesis of MASLD in mice.
Methods: Global eNOS knockout mice (g-eNOS KO; eNOSΔ/Δ) and WT controls (eNOSflox/flox) were fed either a Western diet (WD; high-sucrose, 45% kcal from fat) or an obesogenic high-fat diet (HFD; 60% kcal from fat) for up to 12 weeks. Body weight and food intake were monitored bi-weekly. Glucose tolerance was assessed by intraperitoneal glucose tolerance tests (IP-GTT) at baseline and every 3 weeks by measuring plasma glucose before and up to 120 min after glucose injection (1 g/kg BW). Liver steatosis, fibrosis, and inflammation were evaluated histologically and by biochemical lipid quantification. Hepatic gene expression for eNOS pathways (eNOS, iNOS, nNOS, Arg1), inflammation (NF-κB), fibrosis (Col1A), and lipid metabolism (Srebf, PPARα) was analyzed by RT-PCR and Western blot.
Results: WT and g-eNOS KO mice fed a 45% kcal Western diet (WD) for 12 weeks gained more weight than chow-fed controls, with only mild, non-significant impairment of glucose tolerance and fasting glucose. Both groups developed moderate hepatic steatosis (WT 2.5 [0.75–3]; g-eNOS KO 2 [1–3]) without further liver pathology. In contrast, a 60% kcal high-fat diet (HFD) caused marked weight gain in both genotypes, exceeding that of WD-fed mice. g-eNOS KO mice showed significantly greater weight gain at 9 weeks, but the difference versus WT was no longer significant at 12 weeks. Fasting glucose levels were comparable; however, g-eNOS KO mice displayed a higher glucose AUC during IP-GTT. After 12 weeks of HFD, both groups developed severe hepatic steatosis (WT 3 [1.5–3]; g-eNOS KO 3 [3–3]) with hepatocyte ballooning but no fibrosis. HFD feeding for 4–12 weeks reduced relative heart, spleen, and kidney weight–to–body weight ratios, more markedly in g-eNOS KO mice, suggesting enhanced visceral fat accumulation and ectopic lipid storage. In WT mice, 12 weeks of HFD did not alter hepatic eNOS but reduced Arg1 expression. Srebf, PPAR-α, and Col1a mRNA were upregulated in WT, consistent with activation of lipogenic and fibrotic pathways. In g-eNOS KO mice, HFD-induced Srebf activation was absent, indicating impaired lipogenic transcription.
Conclusions: Compared to WD, the 60% HFD induced greater weight gain and hepatic steatosis in both WT and g-eNOS KO mice. Global eNOS deficiency further aggravated metabolic alterations and impaired glucose tolerance. The absence of Srebf upregulation in g-eNOS KO mice points to a role of eNOS-derived NO in hepatic lipogenic activation. eNOS thus emerges as a key regulator of metabolic adaptation and liver homeostasis in diet-induced MASLD.
