No abstract

Intracellular Ca2؉ mobilization occurs in a variety of cellular processes and is mediated by two major systems, the inositol 1,4,5-trisphosphate (IP 3 ) and cyclic ADP-ribose (cADPR) systems. cADPR has been proposed to be a second messenger for insulin secretion induced by glucose in pancreatic ␤-cells (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370 -373). Here we show that the cADPR signal system for insulin secretion is replaced by the IP 3 system in diabetic ␤-cells such as ob/ob mouse islets and RINm5F cells. We measured the cADPR content in these ␤-cells by radioimmunoassay and found that the increase of the cADPR content by glucose did not occur in ob/ob mouse islets and RINm5F cells, whereas the increased cADPR level by glucose was observed in normal rat and mouse islets. Microsomes of these diabetic ␤-cells released Ca 2؉ in response to IP 3 but not to cADPR. In the diabetic ␤-cells, CD38 (ADP-ribosyl cyclase/cADPR hydrolase) and type 2 ryanodine receptor mRNAs were scarcely detected and, in contrast, an increased expression of IP 3 receptor mRNAs was observed. The diabetic ␤-cells secreted insulin rather by carbamylcholine than by glucose.

The regeneration of pancreatic islet ␤ cells is important for the prevention and cure of diabetes mellitus. We have demonstrated that the administration of poly(ADP-ribose) synthetase͞polymerase (PARP) inhibitors such as nicotinamide to 90% depancreatized rats induces islet regeneration. From the regenerating islet-derived cDNA library, we have isolated Reg (regenerating gene) and demonstrated that Reg protein induces ␤-cell replication via the Reg receptor and ameliorates experimental diabetes. However, the mechanism by which Reg gene is activated in ␤ cells has been elusive. In this study, we found that the combined addition of IL-6 and dexamethasone induced the expression of Reg gene in ␤ cells and that PARP inhibitors enhanced the expression. Reporter gene assays revealed that the ؊81 Ϸ ؊70 region (TGCCCCTCCCAT) of the Reg gene promoter is a cis-element for the expression of Reg gene. Gel mobility shift assays showed that the active transcriptional DNA͞protein complex was formed by the stimulation with IL-6 and dexamethasone. Surprisingly, PARP bound to the cis-element and was involved in the active transcriptional DNA͞protein complex. The DNA͞protein complex formation was inhibited depending on the autopoly(ADP-ribosyl)ation of PARP in the complex. Thus, PARP inhibitors enhance the DNA͞ protein complex formation for Reg gene transcription and stabilize the complex by inhibiting the autopoly(ADP-ribosyl)ation of PARP.P ancreatic ␤ cells of the islets of Langerhans are the only cells that produce insulin in humans as well as in almost all animals, but they have a limited capacity for regeneration, which is a predisposing factor for the development of diabetes mellitus. Strategies for influencing the replication and growth of the ␤-cell mass are therefore important for the prevention and͞or treatment of diabetes (1). We have established a model for islet regeneration in 90% depancreatized rats treated with poly(ADP-ribose) synthetase͞ polymerase (PARP) inhibitors such as nicotinamide and 3-aminobenzamide: Regenerating islets in the pancreatic remnants of PARP inhibitor-treated rats were markedly enlarged and consisted largely of insulin-producing ␤ cells, preventing the development of diabetes that would otherwise have been caused by the 90% pancreatectomy (2). In screening the regenerating islet-derived cDNA library, we found a novel gene and named it Reg (regenerating gene) (3-5). The rat Reg cDNA encoded a 165-amino acid protein with a 21-amino acid signal peptide. We also isolated the human REG cDNA, which encoded a 166-amino acid protein with a 68% amino acid sequence identity to the rat Reg protein (3). Rat and human Reg proteins stimulated the replication of pancreatic ␤ cells and increased the ␤-cell mass in 90% depancreatized rats and in nonobese diabetic mice, resulting in the amelioration of diabetes (6, 7). We have recently identified a Reg protein receptor that mediates a growth signal of Reg protein for ␤-cell regeneration (8). The expression of the Reg receptor, however, was not increased in reg...

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets. We have demonstrated in vitro and in vivo that the exogenous addition of rat and human Reg gene products, Reg/REG proteins, induced ␤-cell replication via the Reg receptor and thereby ameliorated experimental diabetes. In the present study, we produced Reg knockout mice by homologous recombination. The Reg gene disruption resulted in a null mutation. Knockout mice developed normally. Islets from the Reg knockout mice appeared morphologically indistinguishable from those of normal controls. However, [3 H]thymidine incorporation in isolated islets from Reg knockout mice was decreased. When hyperplastic islets were induced by the injection of goldthioglucose, the average islet size in Reg knockout mice was significantly smaller than that of control

The regeneration of pancreatic islet beta cells is important for the prevention and cure of diabetes mellitus. We have demonstrated that the administration of poly(ADP-ribose) synthetase/polymerase (PARP) inhibitors such as nicotinamide to 90% depancreatized rats induces islet regeneration. From the regenerating islet-derived cDNA library, we have isolated Reg (regenerating gene) and demonstrated that Reg protein induces beta-cell replication via the Reg receptor and ameliorates experimental diabetes. However, the mechanism by which Reg gene is activated in beta cells has been elusive. In this study, we found that the combined addition of IL-6 and dexamethasone induced the expression of Reg gene in beta cells and that PARP inhibitors enhanced the expression. Reporter gene assays revealed that the -81 approximately -70 region (TGCCCCTCCCAT) of the Reg gene promoter is a cis-element for the expression of Reg gene. Gel mobility shift assays showed that the active transcriptional DNA/protein complex was formed by the stimulation with IL-6 and dexamethasone. Surprisingly, PARP bound to the cis-element and was involved in the active transcriptional DNA/protein complex. The DNA/protein complex formation was inhibited depending on the autopoly(ADP-ribosyl)ation of PARP in the complex. Thus, PARP inhibitors enhance the DNA/protein complex formation for Reg gene transcription and stabilize the complex by inhibiting the autopoly(ADP-ribosyl)ation of PARP.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca 2 ϩ mobilization for insulin secretion by glucose in pancreatic ␤ cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD ϩ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 ( P Յ 0.05). Insulin secretion from pancreatic islets by glucose is significantly inhibited by the addition of the NIDDM sera with anti-CD38 antibodies ( P Յ 0.04-0.0001), and the inhibition of insulin secretion is abolished by the addition of recombinant CD38 ( P Յ 0.02). The increase of cADPR levels in pancreatic islets by glucose was also inhibited by the addition of the sera ( P Յ 0.05). These results strongly suggest that the presence of anti-CD38 autoantibodies in NIDDM patients can be one of the major causes of impaired glucose-induced insulin secretion in NIDDM.

Regenerating gene product (Reg) is induced in pancreatic b-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces b-cell regeneration was unknown.In the present study, we found that Reg increased phospho-ATF-2, which binds to À57 to À52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce b-cell regeneration.

No abstract