Aims/Introduction: Heparan sulfate (HS) mediates a variety of molecular recognition events that are essential for differentiation, morphogenesis and homeostasis through various HS forms that result from differential sulfate modification. Recently, we found that HS is localized exclusively around beta-cells in islets of adult mice and is required for insulin secretion. The aim of this study was to examine the contribution of HS sulfate groups to insulin secretion. Materials and Methods: Glucose-induced insulin secretion (GIIS) was examined in mouse pancreatic islets, the mouse pancreatic beta-cell line MIN6 cells and its derivative MIN6T3 cells after removal of sulfate groups by sodium chlorate, a competitive inhibitor of glycosaminoglycan sulfation. Quantitative reverse transcription polymerase chain reaction was used for analyzing messenger ribonucleic acid (mRNA) expression of HS modification enzymes. Expression of HS 3-O-sulfotransferase isoform-1 (Hs3st1) was silenced and GIIS was examined. Results: Impaired insulin secretion by islets, MIN6 cells and MIN6T3 cells was observed after treatment with sodium chlorate. Sodium chlorate-treatment upregulated the mRNA expression of sulfotransferases expressed in MIN6T3 cells. Expression of the Hs3st1 was strongly upregulated by sodium chlorate-treatment, and its silencing by RNA interference reduced GIIS in MIN6T3 cells. Conclusions: Our data suggest that the 3-O-sulfate group of HS that is modified by Hs3st1 plays a significant role(s) in the insulin secretory pathway, selectively through an interaction with factor(s) upstream of membrane depolarization in beta-cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00205.x, 2012)

Aims: We studied whether Notch signalling plays a role in beta cell neogenesis induced from adult exocrine pancreatic cells by treatment with growth factors. Materials and Methods: Beta cell neogenesis was induced in rat acinar cell cultures treated with EGF and LIF as reported recently. Real-time RTPCR, Western Blot and immunocytochemistry were used to analyse Notch signalling components. Notch signalling was stimulated with recombinant Jagged1 and DLL4 or was inhibited with recombinant Notch1 extracellular domain. Results: We found upregulation of mRNA for Notch1 and its target Hes1 in the cultures. The Notch-ligand Jagged1 was expressed at high levels, whereas Jagged2 was very low and DLL1 nearly undetectable. When Notch signalling was activated by Jagged1 or DLL4, a marked inhibition of beta cell neogenesis was seen, namely a reduction in the percentage of insulin-positive cells from 9.4% to 1.1%. Previously, we showed that re-expression of Ngn3 drives beta cell neogenesis in this model. We presently found a strong decrease in Ngn3 protein expression (by immunocytocemistry) after Jagged1 or DLL4 mediated Notch activation. Inhibition of Notch activation by recombinant Notch1 extracellular domain resulted in a significant amplification of beta cell neogenesis, namely increasing the number of beta cells from 9.4% to 25%. Conclusions: We demonstrate a recapitulation of embryonic lateral inhibition in an adult model of beta cell neogenesis. Notch-ligands Jagged1 or DLL4 downregulate Ngn3 and abrogate beta cell neogenesis. Treatment of the cells with rNotch1 to inhibit activation of Notch1 by its endogenous ligands, leads to an amplification of beta cell neogenesis. These findings could have an important impact on therapeutic beta cell regeneration.

Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-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 beta-cell regeneration.

Cyclic ADP-ribose (cADPR) induces the release of Ca2+ from microsomes of pancreatic islets for insulin secretion. It has been demonstrated that cADPR binds to FK506-binding protein 12.6 (FKBP 12.6) on rat islet ryanodine receptor and that the binding of cADPR to FKBP12.6 frees the ryanodine receptor from FKBP12.6, causing it to release Ca2+ [Noguchi, N., Takasawa, S., Nata, K., Tohgo, A., Kato, I., Ikehata, F., Yonekura, H., Okamoto, H., 1997. Cyclic ADP-ribose binds to FK506-binding protein to release Ca2+ from islet microsomes. J. Biol. Chem. 272, 3133–3136.]. In this study, we cloned, characterized the structural organization of the human FKBP12.6, which is highly homologous to human FKBP12, and analyzed the promoters for FKBP12.6 and FKBP12. Human FKBP12.6 gene spanned about 16 kb in length and consisted of four exons and three introns. The positions of exon–intron junction of the FKBP12.6 gene were perfectly matched with those of FKBP12 gene except that FKBP12 has an additional exon, exon V, to code exclusively for 3′-UTR. Fluorescence in situ hybridization revealed that the FKBP12.6 gene was located on chromosome 2 p21–23, which is different from the locus (chromosome 20 p13) of the FKBP12 gene. Reporter gene analyses revealed that the regions of −58∼−24 of FKBP12.6 and −106∼−79 of FKBP12 are important for promoter activities. The promoters contain a consensus transcription factor binding sequence for Sp family in FKBP12.6 and Ets-1 in FKBP12. Electrophoretic mobility shift assays showed that nuclear proteins bind to the promoters. The DNA/protein complex on FKBP12.6 promoter was competed out by Sp1 consensus probe and the complex was supershifted by anti-Sp3 antibodies. On the other hand, the DNA/protein complex on FKBP12 promoter was competed out by Ets-1 consensus probe but not by its mutant probe, indicating that Sp3 and Ets-1 play an essential role in transcription of FKBP12.6 and FKBP12, respectively.

Shin Takasawa et al. (1) challenge the role of inositol 1,4,5-triphosphate (IP3) as an intracellular second messenger that mobilizes Ca21 in pancreatic P cells. They found that cyclic adenosine diphosphate-ribose (cADP-ribose), but not IP3, releases Ca2+ from islet microsomes. It is difficult to reconcile their results with many studies that establish IP3 as an intracellular Ca2+mobilizing second messenger in pancreatic 1 cells (2). Confronted with such provocative results, we performed a series of experiments to compare the Ca2+-mobilizing actions of the two second messengers in 13 cells. We used clonal insulin-secreting RINm5F cells and cells obtained from ob/ob mice, where more than 95% of the islet cells correspond to normal 13 cells. The cells were permeabilized by high-voltage electric discharges, a technique that creates clean holes in the plasma membrane, but leaves intracellular Ca2+-storing organelles in situ and undamaged (3). We found pronounced Ca2+ release when IP3 was added to insulin-secreting RINm5F cells (Fig. 1A) or to pancreatic 13 cells from ob/ob mice (Fig. 1B). In marked contrast to the results in the report by Takasawa et al., there was no Ca2+ release after the addition of cADP-ribose. In experiments with 13 cells, we first added a low dose of caffeine to sensitize the release mechanism that presumably might respond to cADP-ribose. After maximal Ca2+ release by IP3, further Ca2+ was released from 13 cells by the sulfhydryl reagent thimerosal which, as we have shown before, indicates the possible existence of a Ca2+-induced Ca2+ release mechanism in 1 cells (4). With the use of intact 1 cells, we looked for the caffeinesensitive intracellular Ca2+ pool on which cADP-ribose presumably acts. In small clusters of 1 cells that had been loaded with Fura-2, in the absence of extracellular Ca2+, there was marked Ca2+ release from intracellular stores by IP3-forming agonists, whereas caffeine-induced Ca2+ release was absent (5). Detailed studies using caffeine indicate that, in the 1 cell, caffeine increases intracellular free Ca2+ concentration ([Ca2+1i1) by a mechanism unrelated to its intracellular Ca2+-mobilizing action (5). Furthermore, we used the patch-clamp technique to monitor the Ca2+-sensitive K+ conductance in 13 cells for detection of any small release of Ca2+ following the addition of cADP-ribose. This method is more sensitive than fluorimetric methods, and it has been used to record increases in [Ca2+ ]i after intracellular application of 1P3 and guanosine 5 '-0(3-thiotriphosphate) (GTP-y-S) in the pancreatic 1 cell (6). Even so, we were unable to obtain evidence of Ca2+ release from intracellular stores after the addition of cADP-ribose in 14 out of 14 cells, whereas formation of IP3 potently induced release of Ca2+ (Fig. 2). These results raise several questions. First, what might be the reason for the absence of IP3-induced Ca2+ release as reported by Takasawa et al.? Their procedure of purification of microsomes might have adversely affected the IP3-sensitive Ca2+ stores, which can be easily damaged during fractionation (7). Second, why was cADP-ribose-induced Ca2+ release seen in their preparation but not in ours? We do not have a definitive answer to this question. It is possible that cADP-riboseinduced Ca2+ release in the 13 cell is small in magnitude and requires rigorous experimental conditions to be detected. Alternatively, the source of Ca2+ released in the experiments of Takasawa et al. might be cells other than 1 cells. It should be recalled that the islets used by Takasawa et al. contain a large proportion of cells that are not 13 cells. We avoided this potential problem by using a tumor cell line and an almost pure preparation of normal 13 cells as well as by performing experiments on single mouse 13 cells. A possible explanation for our negative results with cADP-ribose could be that our preparation of the compound was inactive. However, precautions were taken to ensure that this was not the case. By using cADP-ribose from different sources, who verified the activity of the substance in other cell systems, we guarded against the possibility that the lack of effect in our experimental system was not simply a result of an inactive batch of the compound. Moreover, cADP-ribose seems to be a stable compound (8). With the aim of taking a more physiological experimental approach (that is, using cells instead of isolated organelles), we deliberately did not exactly duplicate the experiments conducted by Takasawa et al. Hence, there remains a possibility that some experimental factors might have adversely affected the cADP-ribose-sensitive release mechanism in our system. Takasawa et al. also demonstrate that extracts of islets incubated in a high con-

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library [J. Biol. Chem. 263 (1988) 2111], encodes a secretory protein with a growth stimulating effect on pancreatic β cells that ameliorates the diabetes of 90% depancreatized rats [Proc. Natl. Acad. Sci. USA 91 (1994) 3589] and non-obese diabetic mice [Diabetes 51(Suppl. 3) (2002) S478]. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5′-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Iα, REG Iβ, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158–175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5′ HIP/PAP 3′-5′ RS 3′-3′ REG Iα 5′-5′ REG Iβ 3′-3′ REG III 5′. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.

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.Material and methods We examined the occurrence of anti-REG autoantibodies in patients' sera (265 Type 1, 368 Type 2 diabetes patients, and 75 unrelated control subjects) by Western blot analysis, and evaluated inhibitory effects of the sera on REG-stimulated beta-cell proliferation by a 5'-Bromo-2'-deoxyuridine (BrdU) incorporation assay in vitro.Results Anti-REG autoantibodies were found in 24.9% of Type 1, 14.9% of Type 2 and 2.7% of control subjects (P = 0.0004). There were significant differences between the autoantibody positive and negative groups in the duration of disease in the Type 1 subjects (P = 0.0035), and the age of onset in the Type 2 subjects (P = 0.0274). The patient sera containing anti-REG autoantibodies significantly attenuated the BrdU incorporation by REG (35.6 +/- 4.06% of the control), whereas the nondiabetic sera without anti-REG autoantibodies scarcely reduced the incorporation (88.8 +/- 5.10%).Conclusion Anti-REG autoantibodies, which retard beta-cell proliferation in vitro, are found in some diabetic patients. Thus, autoimmunity to REG may be associated with the development/acceleration of diabetes in at least some patients.

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 beta-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 Reg(+/+) mice. We then produced transgenic mice carrying the Reg gene under the control of the rat insulin II promoter (Ins-Reg) to express Reg in beta-cells. Isolated islets from the Ins-Reg transgenic mice showed increased [(3)H]thymidine incorporation. By intercrossing, we produced NOD mice carrying the Ins-Reg transgene and found that development of diabetes in the resultant Ins-Reg transgenic NOD mice was significantly retarded, coinciding with an increase in the pancreatic beta-cell mass. These results indicate that Reg plays an important role in beta-cell growth/regeneration.