Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Chronic antagonism of the mineralocorticoid receptor ameliorates hypertension and end organ damage in a rodent model of salt-sensitive hypertension.

We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.

Chronic Inhibition of Renal Outer Medullary Potassium Channel Not Only Prevented but Also Reversed Development of Hypertension and End-Organ Damage in Dahl Salt-Sensitive Rats.

The renal outer medullary potassium (ROMK) channel mediates potassium recycling and facilitates sodium reabsorption through the Na+/K+/2Cl- cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Evidence from the phenotype of humans and rodents with functional ROMK deficiency supports the contention that selective ROMK inhibitors (ROMKi) will represent a novel diuretic with potential of therapeutic benefit for hypertension. ROMKi have recently been synthesized by Merck & Co, Inc. The present studies were designed to examine the effects of ROMKi B on systemic hemodynamics, renal function and structure, and vascular function in Dahl salt-sensitive rats. Four experimental groups-control, high-salt diet alone; ROMKi B 3 mg·kg-1·d-1; ROMKi B 10 mg·kg-1·d-1; and hydrochlorothiazide 25 mg·kg-1·d-1-were included in prophylactic (from week 1 to week 9 on high-salt diet) and therapeutic studies (from week 5 to week 9 on high-salt diet), respectively. ROMKi B produced sustained blood pressure reduction and improved renal and vascular function and histological alterations induced by a high-salt diet. ROMKi B was superior to hydrochlorothiazide at reducing blood pressure. Furthermore, ROMKi B provided beneficial effects on both the plasma lipid profile and bone mineral density. Chronic ROMK inhibition not only prevented but also reversed the development of hypertension and end-organ damage in Dahl salt-sensitive rats. Our findings suggest a potential utility of ROMKi B as a novel antihypertensive agent, particularly for the treatment of the salt-sensitive hypertension patient population.

Effects of carvedilol on structural and functional outcomes and plasma biomarkers in the mouse transverse aortic constriction heart failure model.

INTRODUCTION: Despite the widespread use of the mouse transverse aortic constriction heart failure model, there are no reports on the characterization of the standard-of-care agent carvedilol in this model. METHODS: Left ventricular pressure overload was produced in mice by transverse aortic constriction between the innominate and left common carotid arteries. Carvedilol was administered at multiple dose levels (3, 10 and 30 mg/kg/day per os; yielding end-study mean plasma concentrations of 0.002, 0.015 and 0.044 µM, respectively) in a therapeutic design protocol with treatment initiated after the manifestation of left ventricular remodeling at 3 weeks post transverse aortic constriction and continued for 10 weeks. RESULTS: Carvedilol treatment in transverse aortic constriction mice significantly decreased heart rate and left ventricular dP/dt (max) at all dose levels consistent with ß-adrenoceptor blockade. The middle dose of carvedilol significantly decreased left ventricular weight, whereas the higher dose decreased total heart, left and right ventricular weight and wet lung weight compared to untreated transverse aortic constriction mice. The higher dose of carvedilol significantly increased cardiac performance as measured by ejection fraction and fractional shortening and decreased left ventricular end systolic volume consistent with the beneficial effect on cardiac function. End-study plasma sST-2 and Gal-3 levels did not differ among sham, transverse aortic constriction control and transverse aortic constriction carvedilol groups. Plasma brain natriuretic peptide concentrations were elevated significantly in transverse aortic constriction control animals (~150%) compared to shams in association with changes in ejection fraction and heart weight and tended to decrease (~30%, p = 0.10-0.12) with the mid- and high-dose carvedilol treatment. CONCLUSION: A comparison of carvedilol hemodynamic and structural effects in the mouse transverse aortic constriction model versus clinical use indicates a strong agreement in effect profiles preclinical versus clinical, providing important translational validation for this widely used animal model. The present plasma brain natriuretic peptide biomarker findings support the measurement of plasma natriuretic peptides in the mouse transverse aortic constriction model to extend the translational utility of the model.

Glucagon like receptor 1/ glucagon dual agonist acutely enhanced hepatic lipid clearance and suppressed de novo lipogenesis in mice.

Lipid lowering properties of glucagon have been reported. Blocking glucagon signaling leads to rise in plasma LDL levels. Here, we demonstrate the lipid lowering effects of acute dosing with Glp1r/Gcgr dual agonist (DualAG). All the experiments were performed in 25 week-old male diet-induced (60% kCal fat) obese mice. After 2 hrs of fasting, mice were injected subcutaneously with vehicle, liraglutide (25nmol/kg) and DualAG (25nmol/kg). De novo cholesterol and palmitate synthesis was measured by deuterium incorporation method using D2O. 13C18-oleate infusion was used for measuring fatty acid esterification. Simultaneous activation of Glp1r and Gcgr resulted in decrease in plasma triglyceride and cholesterol levels. DualAG enhanced hepatic LDLr protein levels, along with causing decrease in content of plasma ApoB48 and ApoB100. VLDL secretion, de novo palmitate synthesis and fatty acid esterification decreased with acute DualAG treatment. On the other hand, ketone levels were elevated with DualAG treatment, indicating increased fatty acid oxidation. Lipid relevant changes were absent in liraglutide treated group. In an acute treatment, DualAG demonstrated significant impact on lipid homeostasis, specifically on hepatic uptake, VLDL secretion and de novo synthesis. These effects collectively reveal that lipid lowering abilities of DualAG are primarily through glucagon signaling and are liver centric.

GPR40 partial agonists and AgoPAMs: Differentiating effects on glucose and hormonal secretions in the rodent.

GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.

Discovery of Benzimidazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Discovery of Triazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Genetic deletion and pharmacological inhibition of phosphodiesterase 10A protects mice from diet-induced obesity and insulin resistance.

Phosphodiesterase 10A (PDE10A) is a novel therapeutic target for the treatment of schizophrenia. Here we report a novel role of PDE10A in the regulation of caloric intake and energy homeostasis. PDE10A-deficient mice are resistant to diet-induced obesity (DIO) and associated metabolic disturbances. Inhibition of weight gain is due to hypophagia after mice are fed a highly palatable diet rich in fats and sugar but not a standard diet. PDE10A deficiency produces a decrease in caloric intake without affecting meal frequency, daytime versus nighttime feeding behavior, or locomotor activity. We tested THPP-6, a small molecule PDE10A inhibitor, in DIO mice. THPP-6 treatment resulted in decreased food intake, body weight loss, and reduced adiposity at doses that produced antipsychotic efficacy in behavioral models. We show that PDE10A inhibition increased whole-body energy expenditure in DIO mice fed a Western-style diet, achieving weight loss and reducing adiposity beyond the extent seen with food restriction alone. Therefore, chronic THPP-6 treatment conferred improved insulin sensitivity and reversed hyperinsulinemia. These data demonstrate that PDE10A inhibition represents a novel antipsychotic target that may have additional metabolic benefits over current medications for schizophrenia by suppressing food intake, alleviating weight gain, and reducing the risk for the development of diabetes.

Effects of anacetrapib on plasma lipids, apolipoproteins and PCSK9 in healthy, lean rhesus macaques.

Inhibition of cholesteryl ester transfer protein (CETP) has been vigorously pursued as a potential therapy to treat patients who are at an elevated risk for coronary artery disease. Anacetrapib, a novel CETP inhibitor, has been shown clinically to raise HDL cholesterol and reduce LDL cholesterol when provided as monotherapy or when co-administered with a statin. Preclinically, the effects of anacetrapib on the functionality and composition of HDL have been extensively studied. In contrast, the effects of anacetrapib on other parameters related to lipoprotein metabolism and cardiovascular risk have been difficult to explore. The aim of the present investigation was to evaluate the effects of anacetrapib in rhesus macaques and to compare these to effects reported in dyslipidemic humans. Our results from two separate studies show that administration of anacetrapib (150 mg/kg q.d. for 10 days) to rhesus macaques results in alterations in CETP activity (reduced by more than 70%) and HDL cholesterol (increased by more than 110%) which are similar to those reported in dyslipidemic humans. Levels of LDL cholesterol were reduced by more than 60%, an effect slightly greater than what has been observed clinically. Treatment with anacetrapib in this model was also found to lead to statistically significant reductions in plasma PCSK9 and to reduce cholesterol excursion in the combined chylomicron and remnant lipoprotein fraction isolated from plasma by fast protein liquid chromatography. Collectively, these data suggest that rhesus macaques may be a useful translational model to study the mechanistic effects of CETP inhibition.

Diacylglycerol acyltransferase-1 (DGAT1) inhibition perturbs postprandial gut hormone release.

Diacylglycerol acyltransferase-1 (DGAT1) is a potential therapeutic target for treatment of obesity and related metabolic diseases. However, the degree of DGAT1 inhibition required for metabolic benefits is unclear. Here we show that partial DGAT1 deficiency in mice suppressed postprandial triglyceridemia, led to elevations in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) only following meals with very high lipid content, and did not protect from diet-induced obesity. Maximal DGAT1 inhibition led to enhanced GLP-1 and PYY secretion following meals with physiologically relevant lipid content. Finally, combination of DGAT1 inhibition with dipeptidyl-peptidase-4 (DPP-4) inhibition led to further enhancements in active GLP-1 in mice and dogs. The current study suggests that targeting DGAT1 to enhance postprandial gut hormone secretion requires maximal inhibition, and suggests combination with DPP-4i as a potential strategy to develop DGAT1 inhibitors for treatment of metabolic diseases.

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release--potential insight into mechanism of action.

OBJECTIVE: Investigation was conducted to understand the mechanism of action of diacylglycerol acyltransferase 1 (DGAT1) using small molecules DGAT1 inhibitors, compounds K and L. DESIGN AND METHODS: Biochemical and stable-label tracer approaches were applied to interrogate the functional activities of compounds K and L on TG synthesis and changes of carbon flow. Energy homeostasis and gut peptide release upon DGAT1 inhibition was conducted in mouse and dog models. RESULTS: Compounds K and L, dose-dependently inhibits post-prandial TG excursion in mouse and dog models. Weight loss studies in WT and Dgat1(-/-) mice, confirmed that the effects of compound K on body weight loss is mechanism-based. Compounds K and L altered incretin peptide release following oral fat challenge. Immunohistochemical studies with intestinal tissues demonstrate lack of detectable DGAT1 immunoreactivity in enteroendocrine cells. Furthermore, (13) C-fatty acid tracing studies indicate that compound K inhibition of DGAT1 increased the production of phosphatidyl choline (PC). CONCLUSION: Treatment with DGAT1 inhibitors improves lipid metabolism and body weight. DGAT1 inhibition leads to enhanced PC production via alternative carbon channeling. Immunohistological studies suggest that DGAT1 inhibitor's effects on plasma gut peptide levels are likely via an indirect mechanism. Overall these data indicate a translational potential towards the clinic.

Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure.

The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK(-/-) pups recapitulated features identified in the ROMK null mice. The ROMK(+/-) rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK(+/+) littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation.

Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms.

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. However, the underlying mechanisms for these effects are unclear. It was suggested that SCFAs may regulate gut hormones via their endogenous receptors Free fatty acid receptors 2 (FFAR2) and 3 (FFAR3), but direct evidence is lacking. We examined the effects of SCFA administration in mice, and show that butyrate, propionate, and acetate all protected against diet-induced obesity and insulin resistance. Butyrate and propionate, but not acetate, induce gut hormones and reduce food intake. As FFAR3 is the common receptor activated by butyrate and propionate, we examined these effects in FFAR3-deficient mice. The effects of butyrate and propionate on body weight and food intake are independent of FFAR3. In addition, FFAR3 plays a minor role in butyrate stimulation of Glucagon-like peptide-1, and is not required for butyrate- and propionate-dependent induction of Glucose-dependent insulinotropic peptide. Finally, FFAR3-deficient mice show normal body weight and glucose homeostasis. Stimulation of gut hormones and food intake inhibition by butyrate and propionate may represent a novel mechanism by which gut microbiota regulates host metabolism. These effects are largely intact in FFAR3-deficient mice, indicating additional mediators are required for these beneficial effects.