Proteome imbalance of mitochondrial electron transport chain in brown adipocytes leads to metabolic benefits
Adaptive thermogenesis of brown adipose tissue (BAT) is critical for thermoregulation and contributes to total energy expenditure. However, whether BAT has non-thermogenic functions is largely unknown. Here, we describe that mice with a BAT-specific Liver kinase b1 deletion (Lkb1BKO mice) exhibited impaired mitochondrial respiration and thermogenesis in BAT, but reduced adiposity and liver triglyceride accumulation under high-fat-diet feeding at room temperature. Importantly, these metabolic benefits were also present in Lkb1BKO mice at thermoneutrality, where BAT thermogenesis was not required. Mechanistically, decreased mRNA levels of mtDNA-encoded electron transport chain (ETC) subunits and ETC proteome imbalance led to impaired mitochondrial respiration in BAT of Lkb1BKO mice. Furthermore, reducing mtDNA gene expression directly in BAT by removing mitochondrial transcription factor A (Tfam) in BAT also showed ETC proteome imbalance and the tradeoff between BAT thermogenesis and systemic metabolism at both room temperature and thermoneutrality. Collectively, our data demonstrates that ETC proteome imbalance in BAT regulates systemic metabolism independently of BAT thermogenic capacity.
Sample Processing Protocol
Mouse models used: Two genotypes of mice: Mice with either WT or brown adipose tissue(BAT)-specific Liver kinase b1 deletion (Lkb1BKO). Purified BAT mitochondria from 10-12-week old male mice housed at RT or thermoneutrality (n=3 for each genotype/condition) were resuspended in 8 M urea, 50 mM Tris, 5 mM CaCl2, 100 mM NaCl, and protease inhibitors. Mitochondria Isolation: Freshly dissected BAT tissue from about 8-week-old male and female mice was homogenized in a Dounce homogenizer with 5ml ice-cold mitochondria isolation buffer (210mM Mannitol, 70mM Sucrose, 1mM EGTA, 5mM HEPES pH7.5, 0.5% BSA). The homogenates were filtered through cheesecloth to remove residual particulates and intact mitochondria were isolated by differential centrifugation using a previously described protocol (Methods Enzymol 1979;55:65-78). The mitochondrial pellet was resuspended in 25L of isolation buffer and protein was quantitated using the Bradford assay. Mitochondria were lysed by probe sonication on ice, and proteins reduced by the addition of 5 mM DTT for 30 min at 37C, followed cysteine alkylation by the addition of 15 mM iodoacetamide at RT for 45 min in the dark. The reaction was then quenched by the addition of 15 mM DTT for 15 minutes at RT. Proteins were first digested by the addition of endoproteinase LysC (Wako LC) at a 1:50 substrate:enzyme and incubated for 2h at RT. Next, samples were further digested by the addition of trypsin (Promega) at 1:100 substrate:enzyme, and incubated overnight at 37C. Protein digests were then acidified by the addition of 0.5% triflororacetic acid, and samples desalted on C18 stage tips (Rainin). Peptides were resuspended in 4% formic acid and 3% acetonitrile, and approximately 1μg of digested mitochondria proteins was loaded onto a 75μm ID column packed with 25cm of Reprosil C18 1.9μm, 120Å particles (Dr. Maisch). Peptides were eluted into a Q-Exactive Plus (Thermo Fisher) mass spectrometer by gradient elution delivered by an Easy1200 nLC system (Thermo Fisher). The gradient was from 4.5% to 31% acetonitrile over 165 minutes.
Data Processing Protocol
All data were searched against the Mus musculus uniprot database (downloaded July 22, 2016). Peptide and protein identification searches, as well as label-free quantitation, were performed using the MaxQuant data analysis algorithm, and all peptide and protein identifications were filtered to a 1% false-discovery rate (Cox et al., 2014; Cox and Mann, 2008). MSstats analysis was used for data normalization, and calucluation of log2 fold changes and statistical significance.
Masand R, Paulo E, Wu D, Wang Y, Swaney DL, Jimenez-Morales D, Krogan NJ, Wang B. Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits. Cell Metab. 2018 27(3):616-629.e4 PubMed: 29514069