OBJECTIVEProinflammatory activation of Kupffer cells is implicated in the effect of high-fat feeding to cause liver insulin resistance. We sought to determine whether reduced endothelial nitric oxide (NO) signaling contributes to the effect of high-fat feeding to increase hepatic inflammatory signaling and if so, whether this effect 1) involves activation of Kupffer cells and 2) is ameliorated by increased NO signaling.RESEARCH DESIGN AND METHODSEffect of NO/cGMP signaling on hepatic inflammation and on isolated Kupffer cells was examined in C57BL/6 mice, eNos−/− mice, and Vasp−/− mice fed a low-fat or high-fat diet.RESULTSWe show that high-fat feeding induces proinflammatory activation of Kupffer cells in wild-type mice coincident with reduced liver endothelial nitric oxide synthase activity and NO content while, conversely, enhancement of signaling downstream of endogenous NO by phosphodiesterase-5 inhibition protects against high fat–induced inflammation in Kupffer cells. Furthermore, proinflammatory activation of Kupffer cells is evident in eNos−/− mice even on a low-fat diet. Targeted deletion of vasodilator-stimulated phosphoprotein (VASP), a key downstream target of endothelially derived NO, similarly predisposes to hepatic and Kupffer cell inflammation and abrogates the protective effect of NO signaling in both macrophages and hepatocytes studied in a cell culture model.CONCLUSIONSThese results collectively imply a physiological role for endothelial NO to limit obesity-associated inflammation and insulin resistance in hepatocytes and support a model in which Kupffer cell activation during high-fat feeding is dependent on reduced NO signaling. Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

Obesity and adiponectin depletion have been associated with the occurrence of nonalcoholic fatty liver disease (NAFLD). The goal of this study was to identify the relationship between weight gain, adiponectin signaling, and development of nonalcoholic steatohepatitis (NASH) in an obese, diabetic mouse model. Leptin-receptor deficient (Leprdb/db) and C57BL/6 mice were administered a diet high in unsaturated fat (HF) (61%) or normal chow for 5 or 10 weeks. Liver histology was evaluated using steatosis, inflammation, and ballooning scores. Serum, adipose tissue, and liver were analyzed for changes in metabolic parameters, messenger RNA (mRNA), and protein levels. Leprdb/db HF mice developed marked obesity, hepatic steatosis, and more than 50% progressed to NASH at each timepoint. Serum adiponectin level demonstrated a strong inverse relationship with body mass (r = −0.82; P < 0.0001) and adiponectin level was an independent predictor of NASH (13.6 μg/mL; P < 0.05; area under the receiver operating curve (AUROC) = 0.84). White adipose tissue of NASH mice was characterized by increased expression of genes linked to oxidative stress, macrophage infiltration, reduced adiponectin, and impaired lipid metabolism. HF lepr db/db NASH mice exhibited diminished hepatic adiponectin signaling evidenced by reduced levels of adiponectin receptor-2, inactivation of adenosine monophosphate activated protein kinase (AMPK), and decreased expression of genes involved in mitochondrial biogenesis and β-oxidation (Cox4, Nrf1, Pgc1α, Pgc1β and Tfam). In contrast, recombinant adiponectin administration up-regulated the expression of mitochondrial genes in AML-12 hepatocytes, with or without lipid-loading. Conclusion Leprdb/db mice fed a diet high in unsaturated fat develop weight gain and NASH through adiponectin depletion, which is associated with adipose tissue inflammation and hepatic mitochondrial dysfunction. We propose that this murine model of NASH may provide novel insights into the mechanism for development of human NASH.

MM, Kowdley KV. Iron overload results in hepatic oxidative stress, immune cell activation, and hepatocellular ballooning injury, leading to nonalcoholic steatohepatitis in genetically obese mice. Am J Physiol Gastrointest Liver Physiol 310: G117-G127, 2016. First published November 12, 2015; doi:10.1152/ajpgi.00246.2015.-The aim of this study was to determine the effect of iron overload in the development of nonalcoholic steatohepatitis (NASH) in a genetically obese mouse model (Lepr db/db ). Leptin receptor-deficient mice were fed a normal or an iron-supplemented chow for 8 wk and switched to normal chow for 8 wk. All dietary iron (DI)-fed mice developed hepatic iron overload predominantly in the reticuloendothelial system. Hepatocellular ballooning injury was observed in the livers of 85% of DI mice, relative to 20% of chow-fed Lepr db/db . Hepatic malonyldialdehyde levels and mRNA levels of antioxidant genes (Nrf2, Gpx1, and Hmox1) were significantly increased in the DI mice. Hepatic mRNA levels of mitochondrial biogenesis regulators Pgc1␣, Tfam, Cox4, and Nrf1 were diminished in the DI mice. In addition, gene expression levels of cytokines (Il6, Tnf␣) and several innate and adaptive immune cell markers such as Tlr4, Inos, CD11c, CD4, CD8, and Ifn␥ were significantly increased in livers of the DI group. Strikingly, Nlrp3, a component of the inflammasome and Il18, a cytokine elicited by inflammasome activation, were significantly upregulated in the livers of DI mice. In addition, RAW 264.7 macrophages loaded with exogenous iron showed significantly higher levels of inflammatory markers (Inos, Tnf␣, Mcp1, Tlr4). Thus dietary iron excess leads to hepatic oxidative stress, inflammasome activation, induction of inflammatory and immune mediators, hepatocellular ballooning injury, and therefore NASH in this model. Taken together, these studies indicate a multifactorial role for iron overload in the pathogenesis of NASH in the setting of obesity and metabolic syndrome. iron excess; hepatocellular ballooning; inflammasome; immune cell activation; reticuloendothelial system NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) is the most prevalent chronic liver disease in the world and is strongly associated with obesity and the attendant metabolic syndrome (17

Rationale Obesity is characterized by chronic inflammation of adipose tissue, which contributes to insulin resistance and diabetes. Although nitric oxide (NO) signaling has anti-inflammatory effects in the vasculature, whether reduced NO contributes to adipose tissue inflammation is unknown. We sought to determine whether 1) obesity induced by high-fat (HF) diet reduces endothelial nitric oxide signaling in adipose tissue, 2) reduced endothelial nitric oxide synthase (eNOS) signaling is sufficient to induce adipose tissue inflammation independent of diet, and 3) increased cGMP signaling can block adipose tissue inflammation induced by HF feeding. Methods and results Relative to mice fed a low-fat diet, HF diet markedly reduced phospho-eNOS and phospho-VASP, markers of vascular NO signaling. Expression of pro-inflammatory cytokines was increased in adipose tissue of eNOS−/− mice. Conversely, enhancement of signaling downstream of NO by phosphodiesterase 5 (PDE-5) inhibition using sildenafil attenuated HF-induced pro-inflammatory cytokine expression and the recruitment of macrophages into adipose tissue. Finally, we implicate a role for Vasodilator- stimulated phosphoprotein (VASP), a downstream mediator of NO-cGMP signaling in mediating eNOS-induced anti-inflammatory effects since VASP−/− mice recapitulated the pro-inflammatory phenotype displayed by eNOS−/− mice. Conclusions These results imply a physiological role for endothelial NO to limit obesity-associated inflammation in adipose tissue and hence identifies the NO-cGMP-VASP pathway as a potential therapeutic target in the treatment of diabetes.

Objective To determine the relationship of serum vitamin D deficiency to histologic features of NAFLD, and associated demographic, clinical, laboratory, and transcriptomic data in the well characterized NASH CRN cohort. Methods Serum vitamin D 25(OH)D (VD) was quantified by liquid chromatography-tandem mass spectrometry in 190 adults (>18 yrs) with biopsy-proven NAFLD. Subjects were categorized according to their level of VD as either sufficient (>30ng/ml), insufficient (≥20≤30ng/ml), or deficient (VDD; <20 ng/ml). Multivariable logistic regression was used to investigate the association of VDD and the presence of definite nonalcoholic steatohepatitis (NASH) and individual histological features of NAFLD after adjusting for age, sex, race, BMI, ALT, and diabetes status. Hepatic transcriptomic data was compared between VDD and non-VDD subjects. Results VDD was present in 55% of subjects and was independently associated with definitive NASH (OR 3.15, 95% CI 1.62–6.15, p=0.001), increased lobular inflammation (OR=1.98, 95%CI, 1.08–3.61, p=0.026), more ballooning (OR=2.38, 95%CI, 1.32–4.30, p=0.004), and the presence of fibrosis (OR=2.32, 95%CI, 1.13–4.77, p=0.022). There was a significant inverse relationship between lower levels of serum resistin and increased VD level category (p=0.013). The KRT10, SEMA3B, SNORD3C, ARSD, and IGKV4-1 genes were differentially expressed (FDR<0.05) between VDD and non-VDD subjects. Gene ontology and pathway analysis suggest activation of the MAPK and NF-kB pathways in VDD NAFLD subjects. Conclusions VDD is prevalent among U.S. adult NAFLD patients and is independently associated with a definitive diagnosis of NASH and increased histological severity. Novel associations in pro-inflammatory pathways were identified that suggest the mechanism for VDD in the pathogenesis of NASH and support dietary and/or lifestyle modifications to increase vitamin D levels in these patients.