Owen L. Woodman

Allosteric Ligands of the Glucagon-Like Peptide 1 Receptor (GLP-1R) Differentially Modulate Endogenous and Exogenous Peptide Responses in a Pathway-Selective Manner: Implications for Drug Screening

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 Ca2+ 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.

Modulation of the Glucagon-Like Peptide-1 Receptor Signaling by Naturally Occurring and Synthetic Flavonoids

The glucagon-like peptide 1 receptor (GLP-1R) is a promising target for the treatment of type II diabetes mellitus because of its role in metabolic homeostasis. In recent years, difficulties with peptide therapies have driven the search for small-molecule compounds to modulate the activity of this receptor. We recently identified quercetin, a naturally occurring flavonoid, as a probe-dependent, pathway-selective allosteric modulator of GLP-1R-mediated signaling. Using Chinese hamster ovary cells expressing the human GLP-1R, we have now extended this work to identify the structural requirements of flavonoids to modify GLP-1R binding and signaling (cAMP formation and intracellular Ca2+ mobilization) of each of the GLP-1R endogenous agonists, as well as the clinically used exogenous peptide mimetic exendin-4. This study identified a chemical series of hydroxyl flavonols with the ability to selectively augment calcium (Ca2+) signaling in a peptide agonist-specific manner, with effects only on truncated GLP-1 peptides [GLP-1(7–36)NH2 and GLP-1(7–37)] and exendin-4, but not on oxyntomodulin or full-length GLP-1 peptides [GLP-1(1–36)NH2 and GLP-1(1–37)]. In addition, the 3-hydroxyl group on the flavone backbone (i.e., a flavonol) was essential for this activity, however insufficient on its own, to produce the allosteric effects. In contrast to hydroxyl flavonols, catechin had no effect on peptide-mediated Ca2+ signaling but negatively modulated peptide-mediated cAMP formation in a probe-dependent manner. These data represent a detailed examination of the action of different flavonoids on peptide agonists at the GLP-1R and may aid in the development of future small molecule compounds targeted at this receptor.

Vascular and anti-oxidant actions of flavonols and flavones

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.

Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes.

BACKGROUND AND PURPOSE To 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.

Vasorelaxant and antioxidant activity of flavonols and flavones: Structure-activity relationships

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 Ca2+-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 Ca2+, 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.

Isoflavones, Equol and Cardiovascular Disease: Pharmacological and Therapeutic Insights

Isoflavones are an important class of phytoestrogens that are found at extrememly high levels in soy. Up until recently, daidzein and genistein were considered to be the most important and hence most studied isoflavones, however more recently attention has shifted to isoflavone metabolies. Equol represents the main active product of daidzein metabolism, produced via specific microflora in the gut. It has a longer half life and greater biological activity, including superior antioxidant activity. Yet, whilst the majority of animals produce equol following soy consumption, as much as 30-50 % of the adult human population cannot. This inability to produce equol in as much as half the population is thought to provide some explanation for the failure of soy to reveal any substantial health benefits in clinical studies. This article will comprehensively review literature investigating the potential cardiovascular benefits of daidzein and its metabolites, paying particular attention to equol. It will focus on the relative vasorelaxant activity, effects on nitric oxide synthase (NOS), antioxidant activity and potential for the treatment and prevention of hypertension and stroke. Findings obtained in both animal and human studies will be reviewed with the hope of gaining an insight into the experimental and clinical importance of equol to the cardiovascular benefits of soy.

Vasorelaxant and antioxidant activity of the isoflavone metabolite equol in carotid and cerebral arteries

BACKGROUND AND PURPOSE Equol is the main active intestinal metabolite of the isoflavone daidzein and is postulated to be responsible for the cardiovascular benefits of soy. Cerebral vascular effects of equol are unknown. We compared the vasorelaxant and antioxidant effects of equol and daidzein in carotid and basilar artery of normal and hypertensive rats. EXPERIMENTAL APPROACH Relaxant responses to equol and daidzein were measured in the isolated carotid artery and in the basilar artery in vivo. Effects of nitric oxide synthase (NOS) inhibition, high extracellular K(+), endothelial removal and gender on responses to equol were investigated in carotid arteries. Antioxidant activity was assessed as the reduction of NADPH-induced superoxide levels. Hypertension was induced using angiotensin II (0.7 mg/kg per day for 14 days). KEY RESULTS In normotensive rats, equol displayed vasorelaxant activity similar to daidzein. The relaxant effect of equol was independent of an intact endothelium, NOS activity, K(+) channels and gender. In the basilar artery, where superoxide levels are higher, equol exerted weak antioxidant effects, whereas effects of daidzein were insignificant. During hypertension, equol-induced vasorelaxation was preserved, whereas relaxant responses to daidzein were impaired. CONCLUSIONS AND IMPLICATIONS Equol possesses substantial vasodilator and weak antioxidant activity in cerebral arteries, with similar activity to daidzein, whereas in hypertension the vasorelaxant response to equol, but not daidzein, is preserved. However, daidzein possesses comparable direct vascular effects with equol, without the need for intestinal conversion to equol. Nevertheless, equol may represent a more useful therapeutic agent during cerebral vascular disease.

Coronary vasoconstriction induced by leukotrienes in the anaesthetized dog

In the anaesthetized dog LTC4, LTD4 (0.3-10 micrograms, injected into the coronary artery) and the thromboxane-mimetic U46619 (5-10 micrograms) decreased coronary blood flow. LTE4 (1-10 micrograms), however, did not affect coronary blood flow. The vasoconstrictor responses to LTC4, LTD4 or U46619 were not altered by the cyclo-oxygenase inhibitor indomethacin (5 mg/kg i.v.). LTC4 and LTD4 did not stimulate the release of any prostaglandin-like substance into the pericardial fluid. It is concluded that LTC4 and LTD4 are able to produce coronary vasoconstriction in vivo independent of the production of any cyclo-oxygenase metabolites of arachidonic acid.

Daidzein and 17β-Estradiol Enhance Nitric Oxide Synthase Activity Associated with an Increase in Calmodulin and a Decrease in Caveolin-1

Isoflavones, such as daidzein, are proposed to possess vasculoprotective properties, perhaps through a mechanism similar to estrogen. Our experiments aimed to test the hypothesis that daidzein and 17β-estradiol enhance endothelium-dependent relaxation through an increase in NO synthesis due to an increase in activity or expression of endothelial nitric oxide synthase (eNOS). Male rats were treated with daidzein (0.2 mg/kg per day sc), 17β-estradiol (0.1 mg/kg per day sc), or vehicle for 7 days and reactivity of isolated aortic rings was then determined. ACh-induced relaxation was significantly enhanced in aortic rings from rats treated with daidzein or 17β-estradiol but the relaxant responses to the endothelium-independent dilators sodium nitroprusside or isoprenaline were not different. Nitrite production and the level of cGMP were significantly greater in aortae from daidzein and 17β-estradiol compared with vehicle-treated rats. Daidzein and 17β-estradiol did not alter eNOS protein in endothelium-intact aortae but reduced expression of caveolin-1 and increased expression of calmodulin, changes that would account for an increase in eNOS activity. There were no differences between groups in the expression of calmodulin and caveolin-1 in arteries when the endothelium was removed. Daidzein or 17β-estradiol treatment selectively enhances endothelium-dependent relaxation in male rats through an increase in eNOS activity. The increase in eNOS activity is associated with a decreased expression of caveolin-1 and an increased expression of calmodulin in endothelial cells.

High-fructose diet elevates myocardial superoxide generation in mice in the absence of cardiac hypertrophy

OBJECTIVE Dietary fructose intake has increased considerably in recent decades and this has been paralleled by an increase in the incidence of insulin resistance, especially in children and adolescents. The impact of a high-fructose diet on the myocardium is not fully understood. The aims of this study were to characterize the murine metabolic and cardiac phenotypes associated with a high-fructose diet and to determine whether this diet imparts differential effects with age. METHODS Juvenile (4 wk) and adult (14 wk) C57Bl/6 mice were fed a 60% fructose diet or isoenergetic control (starch) diet for 6 wk. RESULTS At completion of the dietary intervention (at ages 10 and 20 wk), fructose-fed mice were normotensive; hyperinsulinemia and cardiac hypertrophy were not evident. Interestingly, fructose-fed mice exhibited lower blood glucose levels (10 wk: 4.81+/-0.28 versus 5.42+/-0.31 mmol/L; 20 wk: 4.88+/-0.30 versus 5.96+/-0.42 mmol/L, P<0.05) compared with controls. Nicotinamide adenosine dinucleotide phosphate-driven myocardial superoxide production was significantly increased in fructose-fed mice at both ages (by approximately 29% of control at 10 wk of age and 16% at 20 wk, P<0.01). No increase in aortic superoxide production was observed. Fructose feeding did not alter gene expression of the antioxidant thioredoxin-2, suggesting an imbalance between myocardial reactive oxygen species generation and antioxidant induction. CONCLUSION These findings indicate that increased myocardial superoxide production may represent an early and primary cardiac pathologic response to the metabolic challenge of excess dietary fructose in juveniles and adults that can be detected in the absence of cardiac hypertrophy and hypertension.