Chronic cold exposure induces autophagy to promote fatty acid oxidation, mitochondrial turnover, and thermogenesis in brown adipose tissue

Figures

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  Figure 1. Chronic cold exposure increases autophagy in BAT and primary brown adipocytes (A) Representative immunoblots and densitometry analysis of MAP1LC3BII, SQSTM1, and MAP1LC3BII:MAP1LC3BI ratio in BAT of mice subjected to 72 h of cold exposure. (B) Immunoblots and densitometry analysis of PRKAA and MTOR pathway in BAT during cold exposure. (C) mRNA expression of autophagy genes in the BAT of mice exposed to cold. Values are means ± SEM for 5 mice in each group. (D) Autophagy flux analysis in primary brown adipocytes. Cells were treated with or without NE for 72 h, and bafilomycin (Baf) was added 6 h before harvest. (E) Confocal microscopic images and quantification of red puncta in brown adipocyte cell line transfected with RFP-eGFP-MAP1LC3B plasmid and incubated with or without 1 μM NE for 72 h. Scale bar, 20 μm. Quantification of images (at least 10 transfected cells per sample in 3 different fields) was done using ImageJ software. (F) mRNA expression of autophagy genes in primary brown adipocytes treated with 1 μM NE for 72 h. Statistical significance shows as ∗p < 0.05.

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  Figure 2. Chronic cold exposure induces lipophagy and β-oxidation in BAT (A–D) Metabolomic profiling of (A) short-, (B) medium-, and (C) long-chain acylcarnitines species and (D) level of TCA cycle intermediates in BAT. (E) Immunoblot and densitometry analysis of lipases and β-oxidation markers in BAT. Values are means ± SEM for 5 mice in each group. Statistical significance shows as ∗p < 0.05.

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  Figure 3. Chronic cold exposure induces mitophagy and mitochondrial turnover in BAT (A) Representative electron microscopic images showing BAT of mice housed at TN (30°C) or in cold (4°C) for 72 h. White box shows an autophagosome containing mitochondria. Scale bar, 0.2 μm. (B) Immunoblots and densitometry of autophagy proteins in mitochondrial fraction of BAT. (C) Confocal microscopic images and quantification of red puncta in brown adipocyte cell line transfected with mito-RFP-EGFP plasmid and treated with or without 1 μM NE for 72 h. Scale bar, 20 μm. Quantification of images (at least 10 transfected cells per sample in 3 different fields) was done using ImageJ software. (D) Immunoblots and densitometry of mitochondrial proteins in BAT. (E) Confocal microscopic images showing brown adipocyte cell line transfected with pMitoTimer plasmid and treated with or without 1 μM NE for 72 h. Bafilomycin (Baf) was added 6 h before harvest. Scale bar, 20 μm. (F) Seahorse analysis of oxygen consumption rate (OCR) for primary brown adipocytes treated with 1 μM NE for 72 h. For the NE-treated group, 1 μM NE was added to the assay media 1 h before OCR measurement. (G) Representative immunoblots and…

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  Figure 4. Autophagy in BAT is required for thermogenesis in chronic cold exposure (A) Representative blots and densitometry of autophagy proteins in BAT of control and Atg5cKO mice housed at TN (30°C) or in cold (4°C) for 72 h. (B) Body temperature of control and Atg5cKO mice housed at TN or in cold for 72 h. (C) Electron microscopic images showing damaged mitochondria in cold-exposed Atg5cKO mice. Scale bars: 2 μm (10,000×) and 0.5 μm (25,000×). (D) Immunoblots and densitometry showing increased level of protein carbonyls in cold-exposed Atg5cKO mice. (E) Representative immunoblots and densitometry showing mitochondrial proteins. (F) Immunoblots showing autophagy proteins in mitochondrial fraction of BAT. (G) Representative immunoblots and densitometry of proteins involved in lipolysis and PRKAA pathway in BAT. (H) Seahorse analysis of OCR in primary brown adipocytes transfected with control or Atg5 siRNA for 48 h before analysis. Statistical significance shows as ∗p < 0.05 and ∗∗∗∗p < 0.0001. See also Figure S1.