Satoko Yamada

Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

Background Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings The expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. Conclusions Sweet taste receptor is expressed in β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms.

Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic β cells

The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium‐evoked insulin secretion. In rat β cells transfected with green fluorescent protein‐tagged PKC‐α (PKC‐α–EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca2+]c), which was accompanied by transient translocation of PKC‐α–EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC‐θ–EGFP, the C1 domain of PKC‐γ and PKC‐ɛ–GFP. A high concentration of glucose induced repetitive elevation of [Ca2+]c and repetitive translocation of PKC‐α–EGFP. Diazoxide completely blocked both elevation of [Ca2+]c and translocation of PKC‐α–EGFP. We then studied the role of conventional PKC in calcium‐evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Gö‐6976, an inhibitor of conventional PKC, and PKC‐α pseudosubstrate fused to antennapedia peptide (Antp‐PKC19–31) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö‐6976 and Antp‐PKC19–31. However, when islets were stimulated for 60 min with high glucose, both Gö‐6976 and Antp‐PKC19–31 reduced glucose‐induced insulin secretion. Similar results were obtained by transfection of dominant‐negative PKC‐α using adenovirus vector. Taken together, PKC‐α is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization‐induced insulin secretion per se, but it also augments glucose‐induced secretion.

Differentiation of adult hepatic stem-like cells into pancreatic endocrine cells

To apply cell transplantation for treatment of diabetes mellitus, a sufficient number of β-cell sources are required. In the present study, we examined whether an epithelial cell line obtained from normal adult rat liver, namely hepatic stem-like (HSL) cells, which can be converted to both hepatocytes and billiary epithelial cells, could be a potential β-cell source. The growth speed of HSL cells was rapid and these cells were easily expanded in vitro. Bipotential hepatic stem cells, HSL cells, also expressed PGP9.5, which is expressed in neurons, β-cells, and progenitor cells of the pancreatic endocrine cells as well. Sodium butyrate induced morphological changes in HSL cells and converted them into flattened cells with large cytoplasm. When HSL cells were incubated with a combination of 5 mM sodium butyrate and 1 nM betacellulin, most of the cells were converted into morphologically neuron-like cells. RT-PCR analysis revealed that a series of transcriptional factors involved in differentiation of pancreatic endocrine cells was induced by the treatment with sodium butyrate and betacellulin. mRNAs for insulin, pancreatic polypeptide, and somatostatin were also observed. Immunoreactive pancreatic polypeptide, somatostatin, and insulin were detected in sodium butyrate and betacellulin-treated HSL cells. In conclusion, HSL cells obtained from adult normal liver also have the potential to differentiate into pancreatic endocrine cells in vitro. HSL cells may be one of the potential β-cell sources for cell transplant therapy for insulin-dependent diabetes.

Conophylline Suppresses Pancreatic Stellate Cells and Improves Islet Fibrosis in Goto-Kakizaki Rats

Activin A is a differentiation factor for β-cells and is effective to promote β-cell neogenesis. Activin A is also an autocrine activator of pancreatic stellate cells, which play a critical role in fibrogenesis of the pancreas. Conophylline (CnP) is a natural compound, which reproduces the effect of activin on β-cell differentiation and promotes β-cell neogenesis when administered in vivo. However, its effect on stellate cells is not known. We therefore investigated the effect of CnP on stellate cells both in vitro and in vivo. Unlike activin A, CnP inhibited activation of cultured stellate cells and reduced the production of collagen. We then analyzed the involvement of stellate cells in islet fibrosis in Goto-Kakizaki (GK) rats, a model of type 2 diabetes mellitus. In pancreatic sections obtained from 6-wk-old GK rats, CD68-positive macrophages and glial fibrillary acidic protein- and α-smooth muscle actin-positive stellate cells infiltrated into islets. Later, the number of macrophages was increased, and the α-smooth muscle actin staining of stellate cells became stronger, indicating the involvement of stellate cells in islet fibrosis in GK rats. When CnP was administered orally for 4 wk, starting from 6 wk of age, invasion of stellate cells and macrophages was markedly reduced and islet fibrosis was significantly improved. The insulin content was twice as high in CnP-treated rats. These results indicate that CnP exerts antifibrotic actions both in vitro and in vivo and improves islet fibrosis in Goto-Kakizaki rats.

Diminution of early insulin response to glucose in subjects with normal but minimally elevated fasting plasma glucose. Evidence for early beta-cell dysfunction

Aim Systematic analysis of β‐cell function in Japanese health examinees.

Size-related and size-unrelated functional heterogeneity among pancreatic islets

Functional heterogeneity of pancreatic islets was systematically analyzed for the first time using freshly isolated single rat pancreatic islets. First, 60 islets were sequentially exposed to 3, 9.4, 15.6, and 24.1 mM glucose for 30 min each in incubation experiments: 36 (60%) responded in a concentration-dependent and 19 (32%) in an all-or-none manner, and 5 (8%) islets did not respond to high glucose. As a group, the larger the islet, the higher the beta cell glucose sensitivity. However, glucose-stimulated elevation of [Ca2+]i in the beta cell. insulin/glucagon ratio in the islet, and expression of glucose transporter 2, glucokinase, and pancreatic duodenal homeobox factor-1 in the beta cell were not significantly related to islet size. Second, 50 islets were stimulated with 16.7 mM glucose in perifusion. A biphasic insulin release was found in 39 (78%), and no or little first phase response in 11 (22%) islets, irrespective of the islet size. Nevertheless, when the response was plotted as a group, it was clearly biphasic. Islet size, insulin content and the amount of insulin release were positively correlated with each other. In conclusion, there are size-related and size-unrelated functional diversity among pancreatic islets. The reason for such heterogeneity remained to be determined.

A Betacellulin Mutant Promotes Differentiation of Pancreatic Acinar AR42J Cells into Insulin-Producing Cells with Low Affinity of Binding to ErbB1

Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion.

Extracellular matrix modulates insulin production during differentiation of AR42J cells: Functional role of Pax6 transcription factor

Extracellular matrix (ECM) modulates differentiation of pancreatic β‐cells during development. However, the mechanism by which ECM proteins modulate differentiation is not totally clear. We investigated the effect of ECM proteins on differentiation β‐cells in vitro. We investigated the effect of basement membrane ECM on differentiation of AR42J cells and rat ductal cells. First, we examined the effect of reconstituted basement membrane, Matrigel on differentiation of AR42J cells induced by activin and betacellulin. Matrigel augmented insulin production and increased the expression of GLUT2, SUR1, and glucokinase. Among various transcription factors investigated, Matrigel markedly upregulated the expression of Pax6. When Pax6 was overexpressed in cells treated with activin and betacellulin, the expression of insulin was upregulated. Conversely, knockdown of Pax6 significantly reduced the insulin expression in cells cultured on Matrigel. The effects of Matrigel on insulin‐production and induction of Pax6 were reproduced partially by laminin‐1, a major component of Matrigel, and inhibited by anti‐integrin‐β1 antibody. Matrigel also enhanced the activation of p38 mitogen‐activated kinase induced by activin and betacellulin, which was inhibited by anti‐β1 antibody. Finally, the effect of Matrigel on differentiation was reproduced in rat cultured ductal cells, and Matrigel also increased the expression of Pax6. These results indicate that basement membrane ECM augments differentiation of pancreatic progenitor cells to insulin‐secreting cells by upregulating the expression of Pax6. J. Cell. Biochem. 112: 318–329, 2011.

Reversal of streptozotocin-induced hyperglycemia by continuous supply of betacellulin in mice

Previous studies have shown the efficacy of betacellulin (BTC) to promote β-cell regeneration. Because of its short half-life, however, the effect of BTC may have been underestimated. This study was conducted to assess the effect of continuous administration of BTC on β-cell regeneration. Adenovirus vectors encoding proBTC (Ad-proBTC) and mature BTC (Ad-mBTC) were prepared, and the efficacy of secretion of BTC was compared in AML12 hepatocytes. When AML12 cells were infected with Ad-proBTC or Ad-mBTC, cells infected with Ad-mBTC secreted considerably larger amount of BTC. We then infused Ad-mBTC into the mouse tail vein. Expression of BTC was detected in the liver for at least 21 days, and serum BTC was maintained at approximately 1 ng/ml for 7 days. When Ad-mBTC was infused immediately after administration of STZ (170 mg/kg), elevation of the plasma glucose induced by STZ was markedly inhibited, and the plasma glucose concentration remained at less than 200 mg/dl for 21 days. The insulin content and the β-cell mass were significantly increased in Ad-mBTC-infused mice. These results indicate that continuous administration of BTC is quite effective in promoting regeneration of β-cells.

P-170 Evaluation of the questionnaire of members of the Japan Association for Diabetes Education and Care

We distributed a questionnaire to the members in July 2007. The members included doctors, nurses, pharmacists and people with diabetes. We surveyed the problems they felt in their daily life and whether there had been changes before and after they become a member of our association. We sent out questionnaires by post to all members who attended the headquarters of our association. We sent 4,000 questionnaires and received back 1967. The collection rate was therefore 49.175%. The results are as follows: 1) The ratio of Type 1 diabetes was 34% of the whole and Type 2 was 61%. 2) Research on how long it took to begin seeing a doctor regularly after people had been diagnosed with diabetes showed 58% of people with Type 1 diabetes began to see a doctor within 1 month, but 17% of them began after more than 6 years. As for people with Type 2 diabetes, 28% began under 1 month, while 30% began after more than 6 years. 3) Asked about suspension of treatment, showed the total of people who had suspended treatment was 10%. Of these, 68% with Type 2 diabetes had once, 25% of them had twice, while 45% with Type 1 diabetes had once and 40% of them had twice. 4) Research on whether the members were satisfied or not with the contents of our monthly journal “Sakae”. 78% of the members read it every month certainly and 20% of them read it sometimes. 35% the members are fully satisfied with the contents, and 52% of them are satisfied to some extent. 5). Asked whether there were changes in diet after becoming a member of our association as multiple answer questions. 32% became conscious of their intake of calories, with 28% not taking too much salt, sugar, and fat, 17% not eating snacks. There was no change for 31% people. 6) Asked whether there were changes in exercise after becoming the member of our association as multiple answer questions, 35% had tried walking more, whereas 39% had not changed their level of exercise. 7) Asked whether there had been a change in their knowledge about diabetes after becoming the member of our association as a multiple answer question, 67% felt their knowledge had progressed, 25% had begun gathering information about diabetes, while 15% did not consider their knowledge had changed. There are a relatively large number of people with Type 1 diabetes in our association, and even some of these people take a certain period to begin seeing a doctor. As for people with Type 2 diabetes, there is a bipolarization between before 1 month and more than 6 years. Nearly 80% of our members are satisfied with our monthly journal “Sakae”, and they learned about diet, exercise, knowledge about diabetes from this magazine. About 70% of members had changed their diet, 60% changed their exercise, and nearly 80% feel a change in their level of knowledge about diabetes. Our association contributes to the improvement of daily life of people with diabetes to some extent, but needs to make considerably more effort to provide people with more information about diabetes.