The farnesoid X receptor (FXR; NR1H4) is a nuclear hormone receptor that functions as the bile acid receptor. In addition to the critical role FXR plays in bile acid metabolism and transport, it regulates a variety of genes important in lipoprotein metabolism. We demonstrate that FXR also plays a role in carbohydrate metabolism via regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression. Treatment of either H4IIE or MH1C1 rat hepatoma cell lines as well as primary rat or human hepatocytes with FXR agonists led to stimulation of PEPCK mRNA expression to levels comparable to those obtained with glucocorticoid receptor agonists. We examined the physiological significance of FXR agonist-induced enhancement of PEPCK expression in primary rat hepatocytes. In addition to inducing PEPCK expression in primary hepatocytes, FXR agonists stimulated glucose output to levels comparable to those observed with a glucocorticoid receptor agonist. Consistent with these observations, treatment of C57BL6 mice with GW4064 significantly increased hepatic PEPCK expression. Activation of FXR initiated a cascade involving induction of peroxisome proliferator-activated receptor alpha and TRB3 expression that is consistent with stimulation of PEPCK gene expression via interference with a pathway that may involve Akt-dependent phosphorylation of Forkhead/winged helix transcription factor (FOXO1). The FXR-peroxisome proliferator-activated receptor alpha-TRB3 pathway was conserved in rat hepatoma cell lines, mice, as well as primary human hepatocytes. Thus, in addition to its role in the regulation of lipid metabolism, FXR regulates carbohydrate metabolism.
OBJECTIVEThe clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor.RESEARCH DESIGN AND METHODSScreening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo.RESULTSNovel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment.CONCLUSIONSThese studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.
Ontazolast is a potent inhibitor (IC50 = 1 nm) of calcium ionophore A23187-stimulated leukotriene B4 (LTB4) biosynthesis in human peripheral blood leukocytes. The compound is practically insoluble in water (0.14 microgram/mL) and previous studies in animals have demonstrated extensive presystemic drug clearance through hepatic first-pass metabolism. Bioavailability of a suspension formulation in rats was less than 1%, but increased to approximately 9% when administered as a 20% soybean oil-in-water emulsion. The emulsion formulation and three additional lipid-based formulations were administered by gavage to conscious, minimally restrained rats in a novel, double-cannulated model to determine the effects of formulation on systemic blood absorption and mesenteric lymph transport of ontazolast. The bioavailability of ontazolast was significantly and substantially enhanced by all of the lipid-based formulations. While these formulations also significantly increased the amount of ontazolast transported by the lymph, the total amounts transported were insufficient to account for the improvement in bioavailability, which may be due to the elimination or reduction of the barriers of poor aqueous solubility and slow dissolution to absorption of ontazolast from the gastrointestinal tract, or the effects of lipid on the gastrointestinal membrane permeability, transit time, or metabolism of ontazolast. Semisolid SEDDS formulations, composed of Peceol and Gelucire 44/14, produced bioavailability similar to the emulsion formulation. The total amount of ontazolast transported by the lymph varied directly with the amount of concurrent triglyceride transport and appeared to be favored by formulations that prolong gastric emptying time or promote rapid absorption of ontazolast from the gastrointestinal tract.
G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a key drug target class. Recently, allosteric drugs that can cobind with and modulate the activity of the endogenous ligand(s) for the receptor have become a major focus of the pharmaceutical and biotechnology industry for the development of novel GPCR therapeutic agents. This class of drugs has distinct properties compared with drugs targeting the endogenous (orthosteric) ligand-binding site that include the ability to sculpt cellular signaling and to respond differently in the presence of discrete orthosteric ligands, a behavior termed "probe dependence." Here, using cell signaling assays combined with ex vivo and in vivo studies of insulin secretion, we demonstrate that allosteric ligands can cause marked potentiation of previously "inert" metabolic products of neurotransmitters and peptide hormones, a novel consequence of the phenomenon of probe dependence. Indeed, at the muscarinic M 2 receptor and glucagon-like peptide 1 (GLP-1) receptor, allosteric potentiation of the metabolites, choline and GLP-1(9 -36)NH 2 , respectively, was ϳ100-fold and up to 200-fold greater than that seen with the physiological signaling molecules acetylcholine and GLP-1(7-36)NH 2 . Modulation of GLP-1(9 -36)NH 2 was also demonstrated in ex vivo and in vivo assays of insulin secretion.This work opens up new avenues for allosteric drug discovery by directly targeting modulation of metabolites, but it also identifies a behavior that could contribute to unexpected clinical outcomes if interaction of allosteric drugs with metabolites is not part of their preclinical assessment.
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