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 b-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."

Dietary methionine deficiency affects oxidative status, mitochondrial integrity and mitophagy in the liver of rainbow trout (Oncorhynchus mykiss)

“…Livers sampled 2 and 16 h after the last meal were homogenized and analysed according to the previously detailed protocol 24 and using the following antibodies: anti-phospho- ribosomal protein S6 kinase 1 (P- RPS6K1, Thr389, #9205, Cell Signaling Technology); anti- RPS6K1 (#9202, Cell Signaling Technology); phospho-EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1 (P- EIF4EBP1, Thr37/Thr46, #9459, Cell Signaling Technology); anti- EIF4EBP1 (#9452, Cell Signaling Technology); anti-phospho eIF2α (Ser51, #9721, Cell Signaling Technology); anti-carboxyl terminal eIF2α (#9722, Cell Signaling Technology); anti-phospho AMP-activated protein kinase (P-AMPK, Thr172, #2532, Cell Signaling Technology); anti-AMPK (#2532, Cell Signaling Technology); anti-β-tubulin (TUBB)(#2146, Cell Signaling Technology); anti-LC3B (#2775, Cell Signaling Technology). All these antibodies have already been validated in rainbow trout 24 , 28 .…”

Early feeding of rainbow trout (Oncorhynchus mykiss) with methionine deficient diet over a two-week period: consequences for liver mitochondria in juveniles

“…previously detailed (Belghit et al, 2014) using the following antibodies: anti-phospho-ribosomal protein S6 (S6 Ser235/Ser236, 4856, Cell Signaling Technologies); anti-carboxyl terminal S6 (2217, Cell Signaling Technologies); anti-phospho-eukaryotic translation initiation factor 2α (eIF2α Ser51, 9721, Cell Signaling Technology); anti-carboxyl terminal eIF2α (9722, Cell Signaling Technology); anti-β-tubulin (2146, Cell Signaling Technology); anti-translocase of inner mitochondrial membrane 23 (TIM23, 611222, BD Transduction Laboratories TM ), anti-mitofusin2 (MFN2, ab56889, Abcam); anti-parkin RBR E3 ubiquitin protein ligase (PARKIN, ab15954, Abcam); anti-phospho-ubiquitin (Ser65) (Ser65, ABS1513-I, EMD Millipore); anti-total ubiquitin (MAB1510, EMD Millipore); and anti-cleaved poly (ADP-ribose) polymerase (PARP, 95425, Cell Signaling Technology). All of these antibodies (except anti-cleaved PARP) have already been validated in rainbow trout (Belghit et al, 2014;Seiliez et al, 2016;Séité et al, 2018). For cleaved PARP, the amino sequence of the corresponding protein was monitored in the SIGENAE database (http://www.sigenae.org) to check for the conservation of the antigen sequence with the corresponding sequence from mammals, ensuring a good specificity of the mammalian antibody used in the analysis of our samples.…”

“…Reverse transcription-quantitative PCR (RT-qPCR) analysis was performed on whole fry sampled 16 h after the last meal during the first sampling (N=6 per diet) and on the liver of fish sampled 16 h after the feeding trial (second sampling, N=6 per condition) The protocol conditions for sample preparation and RT-qPCR were as previously described (Fontagné-Dicharry et al, 2015;Seiliez et al, 2016). The primers used for qPCR assays were also as described in previous studies (Seiliez et al, 2016;Séité et al, 2018;Séité et al, 2019). For the expression analysis, relative quantification of target gene expression was done using the ΔCT method (Pfaffl et al, 2002).…”

The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis

“…The Oil Red O (Sigma, O1391) staining was done by described previously methods with optimal cutting temperature of frozen samples and consequent staining. 40…”