Naoya Noguchi

CD38 is critical for social behaviour by regulating oxytocin secretion

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

Cyclic ADP-ribose Binds to FK506-binding Protein 12.6 to Release Ca2+ from Islet Microsomes

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

Structure, chromosomal localization and expression of mouse genes encoding type III Reg, RegIIIα, RegIIIβ, RegIIIγ

Abstract Reg (regenerating gene), first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet β-cells. Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, Reg family, which consists of three subtypes (type I, II, III) based on the primary structures of the encoded proteins of the genes. In mouse, type I and type II Reg genes (i.e. RegI and RegII gene) have so far been isolated. In the present study, the complete nucleotide (nt) sequences of the cDNAs and genes encoding murine type III Reg (regenerating gene product), RegIIIα, RegIIIβ and RegIIIγ were determined. RegIIIα, RegIIIβ and RegIIIγ encode 175-, 175- and 174-amino acid (aa) proteins, respectively, with 60–70% homology. All three genes are composed of six exons and five introns spanning approx. 3 kb, and exhibit distinctive structural features unique for members of the Reg gene family. All the mouse Reg genes, RegIIIα, RegIIIβ, RegIIIγ, RegI and RegII, are assigned to the adjacent site of chromosome 6C by fluorescence in situ hybridization (FISH). RegIIIα, RegIIIβ and RegIIIγ were expressed weakly in pancreas, strongly in intestinal tract, but not in hyperplastic islets, whereas both RegI and RegII were expressed in hyperplastic islets. These results suggest that genes of the mouse Reg family are derived from a common ancestor gene by several gene duplications, and have obtained divergency in expression and function in the process of genetic evolution.

Cyclin D1 activation through ATF-2 in Reg-induced pancreatic β-cell regeneration

Regenerating gene product (Reg) is induced in pancreatic β‐cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces β‐cell regeneration was unknown. In the present study, we found that Reg increased phospho‐ATF‐2, which binds to −57 to −52 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 β‐cell regeneration.

Important role of heparan sulfate in postnatal islet growth and insulin secretion.

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet beta-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in beta-cells. These mice exhibited abnormal islet morphology with reduced beta-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.

Human REG family genes are tandemly ordered in a 95-kilobase region of chromosome 2p12

Reg, first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet β‐cells. Recently, it has been revealed that Reg and Reg‐related genes constitute a multigene family, the Reg family. In human, the four REG family genes, i.e., REG Iα, REG Iβ, REG‐related sequence (RS) and HIPIPAP, have so far been isolated. In this study, we analyzed YAC clones containing the four genes and performed two‐color FISH to determine the map order of the genes. The human REG family genes are tandemly ordered in the 95‐kbp DNA region of chromosome 2p12 as follows: 2cen‐HIPIPAP‐RS‐REG Iα‐REG Iβ‐ptel.

Autoantibodies to REG, a beta-cell regeneration factor, in diabetic patients

Background  Regenerating gene (Reg) product, Reg, acts as an autocrine/paracrine growth factor for beta‐cell regeneration. The presence of autoimmunity against REG may affect the operative of the regenerative mechanisms in beta cells of Type 1 and Type 2 diabetes patients. We screened sera from Type 1 and Type 2 diabetes subjects for anti‐REG autoantibodies, searched for correlations in the general characteristics of the subjects with the presence of anti‐REG autoimmunity, and tested the attenuation of REG‐induced beta‐cell proliferation by the autoanitibodies.

FKBP12.6 disruption impairs glucose-induced insulin secretion

Cyclic ADP-ribose (cADPR), accumulated in pancreatic beta-cells in response to elevated ATP levels after glucose stimulation, mobilizes Ca(2+) from the endoplasmic reticulum through the ryanodine receptor (RyR) and thereby induces insulin secretion. We have recently demonstrated in an in vitro study that cADPR activates RyR through binding to FK506-binding protein 12.6 (FKBP12.6), an accessory protein of RyR. Here we generated FKBP12.6-deficient (FKBP12.6(-/-)) mice by homologous recombination. FKBP12.6(-/-) mice showed glucose intolerance coupled to insufficient insulin secretion upon a glucose challenge. Insulin secretion in response to glucose was markedly impaired in FKBP12.6(-/-) islets, while sulfonylurea- or KCl-induced insulin secretion was unaffected. No difference was found in the glucose oxidation rate between FKBP12.6(-/-) and wild-type islets. These results indicate that FKBP12.6 plays a role in glucose-induced insulin secretion downstream of ATP production, independently of ATP-sensitive K(+) channels, in pancreatic beta-cells.

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca(2+) mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca(2+) concentrations in pancreatic islets, resulting from Ca(2+) mobilization from RyRs as well as Ca(2+) influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [(3)H]ryanodine binding. Intracellular Ca(2+) release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.

Upregulation of REG Iα accelerates tumor progression in pancreatic cancer with diabetes

Diabetes is now generally accepted as a crucial event in the process of pancreatic cancer (PaC). However, molecular mechanisms underlying the relationship between diabetes and PaC are not fully understood. Regenerating gene (REG) Iα is a growth factor affecting pancreatic islet beta cells, and it has been shown to be involved in the carcinogenesis in gastrointestinal tract. It is rational to speculate that REG Iα plays a potential role in the pathogenesis of PaC with diabetes. The aim of this study was to evaluate the REG Iα protein expression profile in PaC with and without diabetes, and define the contribution of REG Iα on PaC development. We found that REG Iα protein preferentially expressed in cancerous tissues of PaC patients with diabetes by Western blot. REG Iα positive cancer cells in PaC with diabetes (n = 38) was significantly higher than that in subjects without diabetes (n = 42, p < 0.05) by immunohistochemical analysis. Furthermore, we found that overexpression of REG Iα protein in PaC cell lines resulted in accelerated cell proliferation and consequently tumor growth, both in vitro and in vivo. The findings suggest that REG Iα may act as one of the tumor promoter and contribute to the aggressive nature of PaC, especially in the subpopulation with diabetes. This study would shed new insights for understanding the molecular mechanisms underlying the link between diabetes and PaC.