Impact of Cold-Deacclimation on Brown Adipose Tissue Mitochondrial Energetics, Structure and Mitophagy

Abstract

Brown adipose tissue (BAT) generates heat through non-shivering thermogenesis (NST) to help animals adapt to cold environments. Adaptation to cold induces several molecular processes in BAT, including increased uncoupling protein 1 (UCP1) expression, mitochondrial number, and mitochondrial activity. However, the mechanisms governing the deactivation of NST during cold- deacclimation in BAT remain unclear. Our study examines the changes in mitochondrial content, structure, and energetics in C57BL6/J (WT) mice and mito-QC reporter mice at several time intervals (3 hours, 12 hours, 24 hours, and 48 hours) after moving from a cold-adapted state (7 days at 4°C) to a thermoneutral environment (30°C). We hypothesized that during cold-deacclimation BAT mitochondrial content decreases, mitophagy increases, cristae structure changes, cristae surface area decreases, and oxidative activity decreases. Investigations included mito-QC mice, which express fusion protein (mCherry-GFP) in the mitochondrial outer membrane; during mitophagy the GFP fluorescence diminishes while mCherry fluorescence is preserved. Findings revealed an increase in mitophagy in BAT at 48 hours of cold-deacclimation (p=0.05). However, no changes were observed in key autophagy and apoptotic proteins Parkin, LC3I/II or Beclin in BAT during cold-deacclimation. Fission and fusion markers FIS1 and OPA1 remained unchanged, but FUNDC1 and MFN1 decreased at 48 hours, suggesting mitochondrial fragmentation (p<0.05). Quantitative analysis of transmission electron micrographs demonstrated a decrease in mitochondrial/cytoplasmic area by 48 hours (p<0.001), indicating reduced mitochondrial content. Lipid droplet/cytoplasmic area increased by approximately 50% at 48 hours (p<0.01), consistent with reduced lipolysis during adaptation to thermoneutrality. High-resolution respirometry of permeabilized BAT explants revealed decreased complex I-supported uncoupling at 24 hours (p<0.001) and 48 hours (p<0.01). Complex II-mediated uncoupled respiration decreased at 48 hours (p<0.05), while complex III-derived uncoupled respiration decreased at 3 hours (p<0.05), 24 hours (p<0.05), and 48 hours (p<0.001). Isolated BAT mitochondria exhibited reduced Complex II (p<0.05) and III-mediated uncoupled respiration (p<0.01) at 48 hours. Both total protein content per milligram of tissue (p=0.0001) and total mitochondrial protein per milligram of tissue (p<0.05) decreased over time. However, total UCP1 protein content in BAT remained unchanged over the 48-hour period. Findings overall are consistent with the conclusion that cold-deacclimation decreases mitochondrial content and oxidative activity with increased mitophagy in BAT. Future research directions include investigating details of metabolic changes in BAT during cold-deacclimation using metabolomics.