Dataset Information

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

ABSTRACT: Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of β-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo "autophagometer."

SUBMITTER: Seiliez I

PROVIDER: S-EPMC4835979 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

Seiliez Iban I Belghit Ikram I Gao Yujie Y Skiba-Cassy Sandrine S Dias Karine K Cluzeaud Marianne M Rémond Didier D Hafnaoui Nordine N Salin Bénédicte B Camougrand Nadine N Panserat Stéphane S

Autophagy 20160101 2

Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP c ...[more]

PMID: 26902586

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Project description:Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo.Abbreviations: ANOVA: analysis of variance; Atg: autophagy related; Bcl2l11/Bim: BCL2 like 11; d.p.f.: days post-fertilization; Cryaa: crystallin, alpha a: DMSO: dimethyl sulfoxide; Elavl3: ELAV like neuron-specific RNA binding protein 3; ER: estrogen receptor; ERT2: modified ligand-binding domain of human ESR1/estrogen receptor α; Gal4: galactose-responsive transcription factor 4; GFP: green fluorescent protein; h.p.f.: hours post-fertilization; HSP: heat-shock protein; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; SD: standard deviation; SEM: standard error of the mean; UAS: upstream activating sequence; Ubb: ubiquitin b.

Dataset Information

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

Publications

Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay.

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