Objectives-Obesity causes inflammation and insulin resistance in the vasculature as well as in tissues involved in glucose metabolism such as liver, muscle, and adipose tissue. To investigate the relative susceptibility of vascular tissue to these effects, we determined the time course over which inflammation and insulin resistance develops in various tissues of mice with diet-induced obesity (DIO) and compared these tissue-based responses to changes in circulating inflammatory markers. Methods and Results-Adult male C57BL/6 mice were fed either a control low-fat diet (LF; 10% saturated fat) or a high-fat diet (HF, 60% saturated fat) for durations ranging between 1 to 14 weeks. Cellular inflammation and insulin resistance were assessed by measuring phospho-IB␣ and insulin-induced phosphorylation of Akt, respectively, in extracts of thoracic aorta, liver, skeletal muscle, and visceral fat. As expected, HF feeding induced rapid increases of body weight, fat mass, and fasting insulin levels compared to controls, each of which achieved statistical significance within 4 weeks. Whereas plasma markers of inflammation became elevated relatively late in the course of DIO (eg, serum amyloid A [SAA], by Week 14), levels of phospho-IB␣ in aortic lysates were elevated by 2-fold within the first week. The early onset of vascular inflammation was accompanied by biochemical evidence of both endothelial dysfunction (reduced nitric oxide production; induction of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1) and insulin resistance (impaired insulin-induced phosphorylation of Akt and eNOS). Although inflammation and insulin resistance were also detected in skeletal muscle and liver of HF-fed animals, these responses were observed much later (between 4 and 8 weeks of HF feeding), and they were not detected in visceral adipose tissue until 14 weeks. Conclusions-During obesity induced by HF feeding, inflammation and insulin resistance develop in the vasculature well before these responses are detected in muscle, liver, or adipose tissue. This observation suggests that the vasculature is more susceptible than other tissues to the deleterious effects of nutrient overload.

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.

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-We investigated whether NADPH oxidase-dependent production of superoxide contributes to activation of NF-B in endothelial cells by the saturated free fatty acid palmitate. Methods and Results-After incubation of human endothelial cells with palmitate at a concentration known to induce cellular inflammation (100 mol/L), we measured superoxide levels by using electron spin resonance spectroscopy and the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). Palmitate exposure induced a Ͼ2-fold increase in superoxide levels, an effect associated with activation of NF-B signaling as measured by phospho-IB␣, NF-B activity, IL-6, and ICAM expression. Reduction in superoxide levels by each of 3 different interventions-pretreatment with superoxide dismutase (SOD), diphenylene iodinium (DPI), or knockdown of NADPH oxidase 4 (NOX4) by siRNA-attenuated palmitate-mediated NF-B signaling. Inhibition of toll like receptor-4 (TLR4) signaling also suppressed palmitate-mediated superoxide production and associated inflammation, whereas palmitatemediated superoxide production was not affected by overexpression of a phosphorylation mutant IB␣ (NF-B super repressor) that blocks cellular inflammation downstream of IKK␤/NF-B. Finally, high-fat feeding increased expression of NOX4 and an upstream activator, bone morphogenic protein (BMP4), in thoracic aortic tissue from C57BL/6 mice, but not in TLR4 Ϫ/Ϫ mice, compared to low-fat fed controls. S aturated FFAs such as palmitate readily induce endothelial inflammation, including increased IKK␤-NF-B signaling, via a mechanism that involves activation of Toll-like receptors (TLR) that are key components of the innate immune system. Among the consequences of TLR4-induced activation of NF-B is impaired vascular insulin signaling and reduced nitric oxide production. 1 Based on these and other observations, elevated circulating concentrations of saturated free fatty acids (FFA) are implicated in the mechanism underlying obesity-associated inflammation and insulin resistance in endothelial cells, but the mechanism underlying this link has yet to be established. Conclusions-TheseOne potential mechanism whereby exposure to saturated FFA induces cellular inflammation is via reactive oxygen species (ROS) such as superoxide (O 2 ⅐Ϫ ) 2 that can be generated by both mitochondrial electron transport and by cytosolic enzymes such as the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. These enzymes transfer electrons from NADPH across cell membranes and are a major source of cytoplasmic ROS. The electron acceptor for this reaction is oxygen, producing superoxide radicals. Of 7 NOX homologues that have been identified in nonphagocytic cells, NOX4 is the major species expressed in endothelial cells, with NOX1, NOX2, and NOX5 being expressed at much lower levels. In vascular tissues of db/db mice, a genetic model of severe obesity and diabetes attributable to a mutation in the leptin receptor, expression of NOX1, NOX4, and p22 phox (a smaller subun...

Objective-Diet-induced obesity (DIO) in mice causes vascular inflammation and insulin resistance that are accompanied by decreased endothelial-derived NO production. We sought to determine whether reduced NO-cGMP signaling contributes to the deleterious effects of DIO on the vasculature and, if so, whether these effects can be blocked by increased vascular NO-cGMP signaling. Methods and Results-By using an established endothelial cell culture model of insulin resistance, exposure to palmitate, 100 mol/L, for 3 hours induced both cellular inflammation (activation of IKK␤-nuclear factor-B) and impaired insulin signaling via the insulin receptor substrate-phosphatidylinositol 3-kinase pathway. Sensitivity to palmitate-induced endothelial inflammation and insulin resistance was increased when NO signaling was reduced using an endothelial NO synthase inhibitor, whereas endothelial responses to palmitate were blocked by pretreatment with either an NO donor or a cGMP analogue. To investigate whether endogenous NO-cGMP signaling protects against vascular responses to nutrient excess in vivo, adult male mice lacking endothelial NO synthase were studied. As predicted, both vascular inflammation (phosphorylated IB␣ and intercellular adhesion molecule levels) and insulin resistance (phosphorylated Akt [pAkt] and phosphorylated eNOS [peNOS] levels) were increased in endothelial NO synthase Ϫ/Ϫ (eNOS) mice, reminiscent of the effect of DIO in wild-type controls. Next, we asked whether the vascular response to DIO in wild-type mice can be reversed by a pharmacological increase of cGMP signaling. C57BL6 mice were either fed a high-fat diet or remained on a low-fat diet for 8 weeks. During the final 2 weeks of the study, mice on each diet received either placebo or the phosphodiesterase-5 inhibitor sildenafil, 10 mg/kg per day orally. In high-fat diet-fed mice, vascular inflammation and insulin resistance were completely prevented by sildenafil administration at a dose that had no effect in mice fed the low-fat diet. Conclusion-Reduced