Kwan Yi Chu1, Liam O'Reilly, Georg Ramm, Trevor J Biden. 1. Diabetes and Metabolism Division, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW, 2010, Australia, k.chu@garvan.org.au.
Abstract
AIMS/HYPOTHESIS: Defective beta cell function during lipid oversupply and type 2 diabetes is associated with dysregulation of lysosomal function and autophagy. Whether this dysregulation represents augmentation or inhibition is unclear because of technical limitations in assaying autophagy. The current aim was to determine the effects of high-fat feeding on true autophagic flux in beta cells in vivo in mice, and to establish the relationship between autophagy, endoplasmic reticulum (ER) stress and apoptosis. METHODS: Green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) mice were fed chow or high-fat diets for 8-10 weeks and injected with 100 mg kg(-1) day(-1) chloroquine for 5 days, prior to being killed, to block clearance of autophagic markers. Pancreases and livers were fixed and GFP-LC3 aggregates or autophagosomes were detected by fluorescence or electron microscopy, respectively. Independently, islets isolated from chow or high-fat-fed mice were treated for 2 h with chloroquine ex vivo, and immunoblotting was performed for markers of autophagy (LC3 lipidation - LC3II and p62/SQSTM1), ER stress (C/EBP homology protein [CHOP], phosphorylated eukaryotic initiation factor 2α [p-eIFα] and inositol requiring enzyme 1α [p-IRE1α]) and apoptosis (cleaved caspase-3). RESULTS: Numbers of autophagosomes and GFP puncta were increased in beta cells by combined high-fat feeding and chloroquine injection, indicative of enhanced autophagic flux. By contrast, GFP puncta were attenuated in liver under the same conditions. Relative to chow-fed controls, islets isolated from fat-fed mice exhibited higher LC3II levels when treated ex vivo with chloroquine. The combination of high-fat feeding and acute chloroquine treatment induced CHOP, p-eIF2α and caspase-3, but not either treatment alone. CONCLUSIONS/ INTERPRETATION: We provide the first in vivo demonstrations that high-fat feeding increases autophagic flux in pancreatic beta cells, and that this serves to protect against induction of terminal ER stress. We also highlight an approach for monitoring dietary alterations in autophagic flux using ex vivo manipulation of isolated islets.
AIMS/HYPOTHESIS: Defective beta cell function during lipid oversupply and type 2 diabetes is associated with dysregulation of lysosomal function and autophagy. Whether this dysregulation represents augmentation or inhibition is unclear because of technical limitations in assaying autophagy. The current aim was to determine the effects of high-fat feeding on true autophagic flux in beta cells in vivo in mice, and to establish the relationship between autophagy, endoplasmic reticulum (ER) stress and apoptosis. METHODS: Green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) mice were fed chow or high-fat diets for 8-10 weeks and injected with 100 mg kg(-1) day(-1) chloroquine for 5 days, prior to being killed, to block clearance of autophagic markers. Pancreases and livers were fixed and GFP-LC3 aggregates or autophagosomes were detected by fluorescence or electron microscopy, respectively. Independently, islets isolated from chow or high-fat-fed mice were treated for 2 h with chloroquine ex vivo, and immunoblotting was performed for markers of autophagy (LC3 lipidation - LC3II and p62/SQSTM1), ER stress (C/EBP homology protein [CHOP], phosphorylated eukaryotic initiation factor 2α [p-eIFα] and inositol requiring enzyme 1α [p-IRE1α]) and apoptosis (cleaved caspase-3). RESULTS: Numbers of autophagosomes and GFP puncta were increased in beta cells by combined high-fat feeding and chloroquine injection, indicative of enhanced autophagic flux. By contrast, GFP puncta were attenuated in liver under the same conditions. Relative to chow-fed controls, islets isolated from fat-fed mice exhibited higher LC3II levels when treated ex vivo with chloroquine. The combination of high-fat feeding and acute chloroquine treatment induced CHOP, p-eIF2α and caspase-3, but not either treatment alone. CONCLUSIONS/ INTERPRETATION: We provide the first in vivo demonstrations that high-fat feeding increases autophagic flux in pancreatic beta cells, and that this serves to protect against induction of terminal ER stress. We also highlight an approach for monitoring dietary alterations in autophagic flux using ex vivo manipulation of isolated islets.
Authors: Eri Iwai-Kanai; Hua Yuan; Chengqun Huang; M Richard Sayen; Cynthia N Perry-Garza; Lucy Kim; Roberta A Gottlieb Journal: Autophagy Date: 2008-01-18 Impact factor: 16.016
Authors: M Masini; M Bugliani; R Lupi; S del Guerra; U Boggi; F Filipponi; L Marselli; P Masiello; P Marchetti Journal: Diabetologia Date: 2009-04-15 Impact factor: 10.122