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.

The Cbfa1/Runx2 (referred to herein as Cbfa1) transcription factor has been shown to be essential for osteoblast differentiation and bone formation during embryogenesis. PTH given intermittently is a proven bone anabolic agent. Here, we investigated whether PTH regulates the expression and/or activity of Cbfa1 in osteoblastic cells and in a rat metatarsal organ culture assay. PTH was found to regulate Cbfa1 mRNA in the rat osteosarcoma cell line UMR106 in a concentration-dependent manner. The effect of PTH was mimicked by forskolin, an activator of adenylate cyclase leading to the protein kinase A pathway. PTH administered intermittently for 5 d in vivo was found to stimulate Cbfa1 protein in the rat proximal tibiae metaphysis. To demonstrate PTH regulation of Cbfa1 activity, a construct containing six tandem Cbfa1 binding elements fused to luciferase was shown to be rapidly stimulated in response to PTH. This stimulation preceded the effects on mRNA regulation and resulted from a protein kinase A-mediated increase in Cbfa1 activity. Finally, using a neonate rat metatarsal organ culture system, we demonstrated dose-dependent anabolic responsiveness to PTH and to Cbfa1 overexpression from an adenoviral construct. We further showed that Cbfa1 antisense oligonucleotides that blocked adenoviral Cbfa1-induced anabolic effects in this organ culture model also abolished the PTH-mediated anabolic increase. These findings suggest a requirement for Cbfa1 in mediating the anabolic effects of PTH. Thus, regulation of Cbfa1 expression or activity is an important mechanism by which PTH controls osteoblast function.

An in vivo adenoviral gene delivery system was utilized to assess the effect of overexpressing protein kinase C (PKC)-on rat skeletal muscle glucose transport activity. Female lean Zucker rats were injected with adenoviral/human PKC-(hPKC-) and adenoviral/LacZ in opposing tibialis anterior muscles. One week subsequent to adenoviral/gene delivery rats were subjected to hind limb perfusion. The hPKC-protein was expressed at the same level (fast-twitch white) or at ϳ80% of the level (fast-twitch red) of endogenous PKC-, thus approximately doubling the amount of PKC-in tibialis anterior. Basal glucose transport activity was elevated ϳ3.4-and 2-fold, respectively, in fast-twitch white and red hPKC-muscle relative to control. Submaximal insulin-stimulated glucose transport activity, corrected for basal transport, was ϳ90 and 40% over control values, respectively, in fast-twitch white and red hPKCmuscle. The enhancement of glucose transport activity in muscle expressing hPKC-occurred in the absence of any change in GLUT1 or GLUT4 protein levels, suggesting a redistribution of existing transporters to the cell surface. These results demonstrate that an adenoviral vector can be used to deliver expressible hPKC-to adult rat skeletal muscle in vivo and also affirm a role for PKC-in the regulation of glucose transport activity.

To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.