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Chronic hyperglycemia in type 2 diabetes (T2D) causes pancreatic β-cell dysfunction, which is well known as glucose toxicity (GT), whereas the treatment of T2D improves that dysfunction. However, its underlying molecular mechanism has not yet been clarified. In this study, we aimed to identify and analyze a novel factor whose gene expression was altered by GT (GT-sensitive) and involved in β-cell function. For this purpose, type 2 diabetic db/db mice were treated with empagliflozin to selectively alleviate GT, as we recently reported (Shimo N, et al., 2015). Using the islet RNA from empagliflozin-treated (E) and untreated (U) mice, we performed DNA microarray analysis, and only 43 factors out of 33,779 probes showed more than two times as high signal levels in E as U. Among them, we focused on Tmem163, which is reported to be associated with T2D risk in GWAS and involved in the regulation of intracellular zinc levels. The Tmem163 mRNA expression levels in islets were significantly lower in U than normal control mice, and higher in E than U, which indicated that Tmem163 was GT-sensitive. The tissue distribution analysis by immunohistochemistry and immunoelectron microscopy revealed that Tmem163 was highly localized in pancreatic β-cells and accumulated in the membrane of insulin granule. Next, we found that Tmem163 knockdown significantly reduced glucose-stimulated and KCl-stimulated insulin secretion in MIN6 cells. In vivo, Tmem163 null mice (N) were comparable in body weight and blood glucose levels, but showed decreased glucose tolerance and insulin secretion in intraperitoneal glucose tolerance test, compared with wild type mice. Furthermore, the zinc content of islets in N tended to be lower than a hetero knockout mouse, suggesting a mechanism for β-cell dysfunction. These in vitro and in vivo findings suggest that the decreased expression of the novel GT-sensitive gene Tmem163 in islets causes the insufficient insulin secretion from pancreatic β-cells induced by chronic hyperglycemia. Disclosure N. Shimo: Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. T. Matsuoka: None. D. Kawamori: Research Support; Self; Japan Society for the Promotion of Science, Japan Diabetes Society. Speaker's Bureau; Self; Ajinomoto Co., Inc., Astellas, K.K., Daiichi Sankyo Company, Limited, Dai-nippon Sumitomo Pharmaceutials, MSD K.K., Sanofi K.K., Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Pharmaceuticals, Kowa Pharmaceuticals, Eli Lilly and Company. S. Takebe: None. T. Miyatsuka: Research Support; Self; Eli Lilly and Company, Japan Society for the Promotion of Science, Daiichi Sankyo Company, Limited, Novartis Pharma K.K., Astellas Pharma Inc., MSD K.K., Takeda Pharmaceutical Company Limited. I. Shimomura: Research Support; Self; Appliance Company, Panasonic Corporation. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Covidien Japan Inc.. Research Support; Self; Daiichi Sankyo Company, Limited. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Eli Lilly Japan K.K., Eli Lilly Japan K.K., Kaken Pharmaceutical Co., Ltd.. Speaker's Bureau; Self; Kowa Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., MSD K.K., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K.. Research Support; Self; Novo Nordisk Pharma Ltd.. Speaker's Bureau; Self; Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd.. Research Support; Self; Roche DC Japan K.K., Rohto Pharmaceutical Co., Ltd. Speaker's Bureau; Self; Rohto Pharmaceutical Co., Ltd, Sanwa Kagaku Kenkyusho Co., Ltd.. Research Support; Self; Sanofi K.K.. Speaker's Bureau; Self; Sanofi K.K.. Research Support; Self; Shionogi & Co., Ltd.. Speaker's Bureau; Self; Sumitomo Dainippon Pharma Co., Ltd.. Research Support; Self; Taisho Toyama Pharmaceutical Co., Ltd.. Speaker's Bureau; Self; Taisho Toyama Pharmaceutical Co., Ltd.. Research Support; Self; Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Takeda Pharmaceutical Company Limited. Research Support; Self; Teijin Pharma Limited. Speaker's Bureau; Self; Teijin Pharma Limited.

Among the therapeutic avenues being explored for replacement of the functional islet β-cell mass lost in type 1 diabetes (T1D), reprogramming of adult cell types into new β-cells has been actively pursued. Notably, mouse islet α-cells will transdifferentiate into β-cells under conditions of near β-cell loss, a condition similar to T1D. Moreover, human islet α-cells also appear to poised for reprogramming into insulin-positive cells. Here we have generated transgenic mice conditionally expressing the islet β-cell–enriched Mafa and/or Pdx1 transcription factors to examine their potential to transdifferentiate embryonic pan–islet cell Ngn3-positive progenitors and the later glucagon-positive α-cell population into β-cells. Mafa was found to both potentiate the ability of Pdx1 to induce β-cell formation from Ngn3-positive endocrine precursors and enable Pdx1 to produce β-cells from α-cells. These results provide valuable insight into the fundamental mechanisms influencing islet cell plasticity in vivo.

Pdx1, a β-cell-specific transcription factor, has been shown to play a crucial role in maintaining β-cell function through transactivation of β-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against β-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in β cells (βPdx1) and crossed these mice with Ins2Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of βPdx1; Ins2Akita mice. The βPdx1; Ins2Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that βPdx1; Ins2Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of βPdx1; Ins2Akita mice. These findings suggest that the sustained expression of Pdx1 improves β-cell failure in Ins2Akita mice, at least partially through the preserving expression of β-cell-specific genes as well as improved localization of GLUT2.

Alleviation of hyperglycaemia and hyperlipidemia improves pancreatic β-cell function in type 2 diabetes. However, the underlying molecular mechanisms are still not well clarified. In this study, we aimed to elucidate how the expression alterations of key β-cell factors are altered by the short-term selective alleviation of glucotoxicity or lipotoxicity. We treated db/db mice for one week with empagliflozin and/or bezafibrate to alleviate glucotoxicity and/or liptotoxicity, respectively. The gene expression levels of Pdx1 and Mafa, and their potential targets, insulin 1, Slc2a2, and Glp1r, were higher in the islets of empagliflozin-treated mice, and levels of insulin 2 were higher in mice treated with both reagents, than in untreated mice. Moreover, compared to the pretreatment levels, Mafa and insulin 1 expression increased in empagliflozin-treated mice, and Slc2a2 increased in combination-treated mice. In addition, empagliflozin treatment enhanced β-cell proliferation assessed by Ki-67 immunostaining. Our date clearly demonstrated that the one-week selective alleviation of glucotoxicity led to the better expression levels of the key β-cell factors critical for β-cell function over pretreatment levels, and that the alleviation of lipotoxicity along with glucotoxicity augmented the favorable effects under diabetic conditions.

While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-β cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes.

The transcription factor MafA is a key regulator of insulin gene expression and maturation of islet β cells. Despite its importance, the regulatory mechanism of MafA gene expression is still unclear. To identify the transcriptional regulators of MafA, we examined various transcription factors, which are potentially involved in β cell differentiation. An adenovirus-mediated overexpression study clearly demonstrated that Onecut1 suppresses the promoter activity of MafA through the Foxa2-binding cis-element on the MafA enhancer region (named area A). However, ChIP analysis showed that Foxa2 but not Onecut1 could directly bind to area A. Furthermore, overexpression of Onecut1 inhibited the binding of Foxa2 onto area A upon ChIP analysis. Importantly, insertion of a mutation in the Foxa2-binding site of area A significantly decreased the promoter activity of MafA. These findings suggest that Onecut1 suppresses MafA gene expression through the Foxa2-binding site. In the mouse pancreas, MafA expression was first detected at the latest stage of β cell differentiation and was scarcely observed in Onecut1-positive cells during pancreas development. In addition, Onecut1 expression was significantly increased in the islets of diabetic db/db mice, whereas MafA expression was markedly decreased. The improved glucose levels of db/db mice with insulin injections significantly reduced Onecut1 expression and rescued the reduction of MafA expression. These in vivo experiments also suggest that Onecut1 is a negative regulator of MafA gene expression. This study implicates the novel role of Onecut1 in the control of normal β cell differentiation and its involvement in β cell dysfunction under diabetic conditions by suppressing MafA gene expression.