Novel Spatiotemporal Analysis for Exploring a-Cell Differentiation

Abstract

Aims: In vivo studies have demonstrated that pancreatic α-cells have potency to transdifferentiate into insulin-producing β cells, which could lead to potential therapy to cure diabetes. In addition, inappropriate glucagon secretion from α cells has been shown to be involved in the pathophysiology of diabetes. Although such findings highlight the need for better understanding of α-cell biology, it remains to be elucidated where α cells originate and what regulates α cell fate: α cell neogenesis, maturation and dedifferentiation. To address this issue, we generated a novel reporter mouse “Gcg-Timer.” Methods: We have previously generated Neurog3-Timer mouse (Miyatsuka et al. Diabetes 2009), in which newborn endocrine cells can be sorted by FACS separately from differentiated cells. However, we failed to obtain spatiotemporal information of newborn cells due to the low fluorescent intensity of DsRed-E5. To overcome this difficulty, we used a new reporter construct eGFP-IRES-mRFP and inserted it into the proglucagon gene. In this Gcg-Timer mouse, green fluorescence can be detected first, because mRFP expression is controlled by IRES and therefore lower than eGFP expression. As a result, newly-generated α cells exhibit green fluorescence and can be distinguished from more differentiated α cells exhibiting both green and red fluorescence. Results: Both microscopic analysis and flow cytometry revealed that green-dominant α cells were observed in Gcg-Timer mice at embryonic and neonatal stages until postnatal day 3 (P3), but not at P14 and adult stages. Intriguingly, red dominant cells, which were negative for glucagon staining, were detected at P14, indicating that α-to-non-α transition occurs at this stage. Conclusions: Gcg-Timer mice enabled us to label not only newly-generated α cells but also dedifferentiated α cells in a time-dependent manner. The α-cell neogenesis occurs specifically during embryonic and early neonatal stages under physiological condition. Disclosure M. Himuro: 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.. L. Suzuki: None. M. Miura: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Sanofi K.K.. Research Support; Self; Kowa Pharmaceuticals America, Inc.. T. Katahira: None. M. Takahashi: None. Y. Nishida: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited, AstraZeneca. S. Oshima: None. R. Okamoto: None. H. Watada: Advisory Panel; Self; AstraZeneca. Consultant; Self; Astellas Pharma US, Inc., AstraZeneca, Boehringer Ingelheim GmbH, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Novartis AG, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Development Center Asia, Pte. Ltd.. Research Support; Self; Abbott, Astellas Pharma US, Inc., AstraZeneca, Bayer AG, Benefit One Health Care Co., Ltd., Boehringer Ingelheim GmbH, Bristol-Myers Squibb Company, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Johnson & Johnson Diabetes Institute, LLC., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nitto Boseki Co., Ltd., Novartis AG, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Development Center Asia, Pte. Ltd., Terumo Medical Corporation.