2185-P: Estimation of Autophagy Flux by a Novel Fluorescent Reporter in Organs Involved in Glucose Metabolism Including Pancreatic ß Cells

Autophagy is a bulk degradation system which plays an important role to maintain cell homeostasis. We have shown that deficiency of autophagy in pancreatic β cells leads to their failure and deterioration of glucose tolerance. On the other hand, there has been controversy over induction of autophagic flux in β cells following different physiological contexts, as it has been difficult to estimate autophagy flux by using conventional methods such as estimation of LC3-lipidation. Recently, application of a novel autophagy flux, GFP-LC3-RFP-LC3ΔG, is reported, which enables us to evaluate autophagy flux by calculating ratio between the fluorescence of GFP and RFP, as GFP-LC3 is degraded specifically in autolysosomes, whereas RFP-LC3ΔG remains intact in cytosol as an internal control. To improve its usefulness, we modified it to construct pHluorin-LC3-mCherry. First, we expressed this probe in MIN6 cells and confirmed decrease of pHluorin/mCherry ratio following induction of autophagic flux in starvation or administration of specific mTOR inhibitor, Torin-1. Next, we generated knock-in mouse in which the probe is expressed under the control of CAG promotor in ROSA26 locus. Its expression was confirmed based on Western blot and fluorescent microscopy in almost all the tissues that we examined. Then we stimulated the mice with starvation and detected that pHluorin/mCherry ratio decreases after starvation in several organs, including liver and muscle which are deeply involved in glucose homeostasis. Finally, we investigated whether autophagic flux is changed after starvation in pancreatic beta cells and successfully demonstrated that the flux is increased following prolonged starvation. In addition, we also illustrated upregulation of autophagic flux in isolated islets after glucose and amino acid starvation. These results show dynamic change of autophagic flux after starvation in many tissues related to glucose homeostasis including βcells. Disclosure S. Aoyama: None. Y. Nishida: None. R. Yazawa: None. I. Tanida: None. T. Miyatsuka: None. H. Watada: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kissei Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Pfizer Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker9s Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Kowa Pharmaceutical Europe Co. Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Sanwa Chemical Industry Co. Ltd., Takeda Pharmaceutical Company Limited.