ABSTRACT

We recently showed that activation of G protein-coupled receptor 119 (GPR119) (also termed glucose dependent insulinotropic receptor) improves glucose homeostasis via direct cAMP-mediated enhancement of glucose-dependent insulin release in pancreatic beta-cells. Here we show that GPR119 also stimulates incretin hormone release and thus may regulate glucose homeostasis by this additional mechanism. GPR119 mRNA was found to be expressed at significant levels in intestinal subregions that produce glucose-dependent insulinotropic peptide and glucagon-like peptide (GLP)-1. Furthermore, in situ hybridization studies indicated that most GLP-1-producing cells coexpress GPR119 mRNA. In GLUTag cells, a well-established model of intestinal L-cell function, the potent GPR119 agonist AR231453 stimulated cAMP accumulation and GLP-1 release. When administered in mice, AR231453 increased active GLP-1 levels within 2 min after oral glucose delivery and substantially enhanced total glucose-dependent insulinotropic peptide levels. Blockade of GLP-1 receptor signaling with exendin(9-39) reduced the ability of AR231453 to improve glucose tolerance in mice. Conversely, combined administration of AR231453 and the DPP-4 inhibitor sitagliptin to wild-type mice significantly amplified both plasma GLP-1 levels and oral glucose tolerance, relative to either agent alone. In mice lacking GPR119, no such enhancement was seen. Thus, GPR119 regulates glucose tolerance by acting on intestinal endocrine cells as well as pancreatic beta-cells. These data also suggest that combined stimulation of incretin hormone release and protection against incretin hormone degradation may be an effective antidiabetic strategy.

Pancreatic ␤-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor have shown significant promise as antidiabetic agents by virtue of their ability to amplify glucose-dependent insulin release and preserve pancreatic ␤-cell mass. These effects are mediated via stimulation of cAMP through ␤-cell GLP-1 receptors. We report that the G␣ s -coupled receptor GPR119 is largely restricted to insulin-producing ␤-cells of pancreatic islets. Additionally, we show here that GPR119 functions as a glucose-dependent insulinotropic receptor. Unlike receptors for GLP-1 and other peptides that mediate enhanced glucose-dependent insulin release, GPR119 was suitable for the development of potent, orally active, small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in both HIT-T15 cells and rodent islets. In both cases, loss of GPR119 rendered AR231453 inactive. AR231453 also enhanced glucose-dependent insulin release in vivo and improved oral glucose tolerance in wild-type mice but not in GPR119-deficient mice. Diabetic KK/A y mice were also highly responsive to AR231453. Orally active GPR119 agonists may offer significant promise as novel antihyperglycemic agents acting in a glucose-dependent fashion. (Endocrinology

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotidebinding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.CED4 family proteins constitute a unique family of caspaseactivating molecules. The founding member of this family, CED4, was discovered in the nematode Caenorhabditis elegans in screens for genes that are essential for developmental programmed cell death (1). CED4 contains an N-terminal CARD 1 followed by an NB domain, the later containing classical Walker A and B box motifs recognized as important in binding nucleotide triphosphates. CED4 functions as an activator of the caspase, CED3, in vitro and in vivo (2, 3). The NB domain of CED4 oligomerizes in an ATP-dependent manner (4, 5), whereas the CARD binds a complementary N-terminal CARD found in the zymogen proform of CED3 (6). Protease activation is thought to result from the induced proximity of CED3 zymogens bound to oligomerized CED4, where the weak intrinsic protease activity of the proenzymes is sufficient for trans-proteolysis of closely juxtaposed pro-caspases (4, 7). Proteolytic cleavage of pro-CED3 then produces the large and small subunits of the heterotetrameric, autonomously active enzyme.The closest homologue of CED4 identified in humans and other mammals thus far is Apaf1 (apoptosis protease-activating factor-1) (8). Similar to CED4, the Apaf1 protein contains a CARD, followed by an NB domain that shares significant amino acid sequence identity with the NB domains of CED4 and a family of ATPases associated with pathogen resistance (R genes) in plants (3, 5, 9), thus constituting the NB-ARC (Apaf-1/R gene/CED4) domain family (also known as NACHT domain). Unlike CED4, however, the NB-ARC domain of Apaf1 is followed by multiple WD repeats. These WD domains participate in auto-repression of Apaf1, locking it into an inactive, unoligomerized state ...