The glucagon-like peptide-1 (GLP-1) receptor is a key regulator of insulin secretion and a major therapeutic target for treatment of diabetes. However, GLP-1 receptor function is complex, with multiple endogenous peptides that can interact with the receptor, including full-length (1-37) and truncated (7-37) forms of GLP-1 that can each exist in an amidated form and the related peptide oxyntomodulin. We have investigated two GLP-1 receptor allosteric modulators, Novo Nordisk compound 2 (6,7-dichloro2-methylsulfonyl-3-tert-butylaminoquinoxaline) and quercetin, and their ability to modify binding and signaling (cAMP formation, intracellular Ca 2ϩ mobilization, and extracellular signal-regulated kinase 1/2 phosphorylation) of each of the naturally occurring endogenous peptide agonists, as well as the clinically used peptide mimetic exendin-4. We identified and quantified stimulus bias across multiple endogenous peptides, with response profiles for truncated GLP-1 peptides distinct from those of either the full-length GLP-1 peptides or oxyntomodulin, the first demonstration of such behavior at the GLP-1 receptor. Compound 2 selectively augmented cAMP signaling but did so in a peptide-agonist dependent manner having greatest effect on oxyntomodulin, weaker effect on truncated GLP-1 peptides, and negligible effect on other peptide responses; these effects were principally driven by parallel changes in peptide agonist affinity. In contrast, quercetin selectively modulated calcium signaling but with effects only on truncated GLP-1 peptides or exendin and not oxyntomodulin or full-length peptides. These data have significant implications for how GLP-1 receptor targeted drugs are screened and developed, whereas the allosterically driven, agonist-selective, stimulus bias highlights the potential for distinct clinical efficacy depending on the properties of individual drugs.

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The mechanism of the relaxant action and the structure-activity relation of flavonols (fisetin, quercetin, and 3,3',4'-trihydroxyflavone) and flavones (apigenin, chrysin, and luteolin) were examined in rat isolated thoracic aorta. The control responses to flavonols and flavones were compared with responses observed after the removal of the endothelium or in the presence of the L-type Ca2+ channel blocker, nifedipine (10(-7) M). The effects of flavonoids on contraction caused by the influx of extracellular Ca2+ and agonist-induced release of intracellular Ca2+ also were investigated. The flavones exhibited endothelium-independent vasorelaxation, whereas the removal of the endothelium significantly decreased the sensitivity of the relaxant responses to the flavonols without affecting the maximal relaxation. In the presence of nifedipine, the responses to apigenin, luteolin, and quercetin were significantly inhibited, but relaxation to chrysin, fisetin, and 3,3',4'-trihydroxyflavone was unaffected. All flavonols and flavones caused concentration-dependent inhibition of the contractile responses to exogenous application of Ca2+ and the release of intracellular Ca2+ stimulated by phenylephrine. Of the six flavonoids examined, 3,3',4'-trihydroxyflavone was the most potent when causing vasorelaxation or inhibition of contraction caused by the influx or release of Ca2+. In conclusion, these studies provide evidence that the hydroxyl substitution in the carbon 3 position that characterizes the flavonols is important in stimulating endothelium-dependent vasorelaxation, and the absence of hydroxyl substitution on the A phenolic ring enhances the relaxant action.

BACKGROUND AND PURPOSETo investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats. EXPERIMENTAL APPROACHWire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels. KEY RESULTSSuperoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate-and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer 473 Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling. CONCLUSION AND IMPLICATIONSThe contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer 473 Akt and/or eNOS uncoupling. AbbreviationsEDHF, endothelium-derived hyperpolarizing factor; eNOS, endothelial nitric oxide

1. Flavonols and flavones are plant-derived polyphenolic compounds that are commonly consumed in the diet. Epidemiological studies indicating that high dietary intake of flavonols reduces the risk of mortality due to coronary heart disease have provoked interest in the mechanism of this cardioprotective effect. 2. We have investigated the structure-activity relationships of a range of flavonols and flavones with regard to their vascular relaxant and anti-oxidant activity. In rat isolated thoracic aorta, the synthetic flavonol 3',4'-dihydroxyflavonol (DiOHF) was found to be a significantly more potent vasorelaxant than the naturally occurring compounds chrysin, apigenin, luteolin, quercetin and fisetin. Similarly, DiOHF was significantly more potent than those compounds in the inhibition of calcium-induced contraction of the rat aorta. 3. 3',4'-Dihydroxyflavonol was also found to significantly inhibit superoxide radical generation in a cell-free system in the presence of xanthine/xanthine oxidase or by rat isolated aorta in the presence of NADPH. In the presence of oxidant stress generated by pyrogallol or xanthine/xanthine oxidase, endothelium-dependent relaxation of rat aortic rings was impaired. 3',4'-Dihydroxyflavonol was able to significantly improve endothelium-dependent relaxation in the presence of those oxygen radical generators. 4. In addition, DiOHF was found to significantly improve dilatation in the rat hindquarters vasculature after exposure to ischaemia and reperfusion. 3',4'-Dihydroxyflavonol was found to be equally effective whether applied before ischaemia or during ischaemia just before reperfusion. 5. In conclusion, DiOHF is an effective vasodilator and anti-oxidant that is able to prevent vascular reperfusion injury. We suggest that DiOHF may be useful as an adjunct to thrombolytic therapy in the management of reperfusion injury.

Non-alcoholic steatohepatitis (NASH) develops from non-alcoholic fatty liver disease (NAFLD). Currently, around 25% of the population is estimated to have NAFLD, and 25% of NAFLD patients are estimated to have NASH. NASH is typically characterized by liver steatosis inflammation, and fibrosis driven by metabolic disruptions such as obesity, diabetes, and dyslipidemia. NASH patients with significant fibrosis have increased risk of developing cirrhosis and liver failure. Currently, NASH is the second leading cause for liver transplant in the United States. More importantly, the risk of developing hepatocellular carcinoma from NASH has also been highlighted in recent studies. Patients may have NAFLD for years before progressing into NASH. Although the pathogenesis of NASH is not completely understood, the current “multiple-hits” hypothesis suggests that in addition to fat accumulation, elevated oxidative and ER stress may also drive liver inflammation and fibrosis. The development of clinically relevant animal models and pharmacological treatments for NASH have been hampered by the limited understanding of the disease mechanism and a lack of sensitive, non-invasive diagnostic tools. Currently, most pre-clinical animal models are divided into three main groups which includes: genetic models, diet-induced, and toxin + diet-induced animal models. Although dietary models mimic the natural course of NASH in humans, the models often only induce mild liver injury. Many genetic and toxin + diet-induced models rapidly induce the development of metabolic disruption and serious liver injury, but not without their own shortcomings. This review provides an overview of the “multiple-hits” hypothesis and an evaluation of the currently existing animal models of NASH. This review also provides an update on the available interventions for managing NASH as well as pharmacological agents that are currently undergoing clinical trials for the treatment of NASH.

This data indicates that the antihypertrophic actions of ANP are accompanied by reduced levels of superoxide, suggesting an antioxidant action contributes to the antihypertrophic actions of ANP.

We investigated the structure-activity relationships regarding vascular and antioxidant activity of a range of synthetic flavonols and flavones with 3 or fewer hydroxyl (OH) or methoxyl substitutions. The relaxant responses and ability of the flavones/flavonols to inhibit phenylephrine (PE)- and Ca-induced contraction was determined in rat isolated thoracic aorta. The ability of these compounds to reduce the level of superoxide and preserve endothelium-dependent relaxation in the presence of oxidative stress was also examined. Four compounds impaired contraction to PE or Ca, in the potency order 3'-hydroxyflavonol>3',4'-dihydroxyflavonol>7,4'-dihydroxyflavonol>3',4'-dihydroxyflavone. Flavonol, 3',4'-dimethoxyflavonol, and flavone were significantly less active. The flavonoids caused concentration-dependent reductions in superoxide produced by rat aorta in the presence of NADPH. The most active compounds, 3',4'-dihydroxyflavonol and 7,4'-dihydroxyflavonol, preserved endothelium-dependent relaxation in the presence of oxidative stress caused by pyrogallol or xanthine/xanthine oxidase. The results indicate that the catechol group is not critical for vascular relaxant or antioxidant activity, but rather, the important determinants for higher vascular and antioxidant activity of these compounds are the presence of a C3 OH group and the total number of OH substituents, respectively. These results have allowed the identification of the structural characteristics that promote vascular and antioxidant activity of flavonols, which may lead to the development of agents useful in treatment of cardiovascular disease.