Pharmaceutical inhibition of p38α reduces adiposity and enhances the browning of WAT.

<p>(A and B) Representative HE staining of iBAT, iWAT, and eWAT (A), diameter and cross-sectional area (B) of adipocytes in these adipose tissues from SB203580-treated C57BL/6J mice at 2 d postinjection. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (C) BW of C57BL/6J mice before and after 4 wk of SB203580 treatment (control: <i>n</i> = 4, SB203580: <i>n</i> = 5). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D and E) Relative weight of iWAT and eWAT from C57BL/6J mice after 4 wk of SB203580 treatment. These mice were maintained at RT (D, <i>n</i> = 5 per group) or exposed to cold for 2 d before analysis (E, <i>n</i> = 5 per group) as indicated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (F) Relative mRNA levels of UCP-1, PGC1α, ELOVL3, and DIO2 in iWAT from C57BL/6J mice after 4 wk of SB203580 treatment (<i>n</i> = 10 per group). These mice were exposed to cold for 2 d before analysis. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (G) Representative western blots of p-CREB (Ser133) and UCP-1 in iWAT from C57BL/6J mice after 4 wk of SB203580 treatment. These mice were exposed to cold for 2 d before analysis. (H and I) Representative PET/CT images (H) and PET images (I) of C57BL/6J mice after 4 wk of SB203580 treatment. These mice received a daily CL316,243 injection for 8 d before ¹⁸F-FDG injection. White dashed triangles represent the anatomical sites of iWAT. (J) Ex vivo measured ¹⁸F-FDG uptake in iBAT, iWAT, and eWAT to tissue weight ratio by γ counter (<i>n</i> = 3 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K) Relative weight of iWAT from <i>db/db</i> mice after 3 wk of SB203580 treatment (<i>n</i> = 5 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (L and M) Representative HE staining of iWAT (L), diameter and cross-sectional area of adipocytes in iWAT (M) from <i>db/db</i> mice after 3 wk of SB203580 treatment. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. BW, body weight; CREB, cAMP-response element binding protein; CT, computed tomography; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; eWAT, epididymal white adipose tissue; HE staining, hematoxylin-eosin staining; iBAT, interscapular brown adipose tissue; iWAT, inguinal white adipose tissue; NS, not significant; PET, positron emission tomography; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; RT, room temperature; UCP-1, uncoupling protein 1; WAT, white adipose tissue.</p

Loss of p38α in adipose tissues causes minimal effects on BAT.

<p>(A) BT of Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 6 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (B and C) VO₂ (B) and VCO₂ (C) in Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 4 per group). The values were normalized by LM. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D) iBAT weight and relative iBAT weight to BW ratio (iBAT/BW) of Floxed (<i>n</i> = 10) and Fp38αKO (<i>n</i> = 8). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (E-G) Representative HE staining of iBAT (E), diameter and cross-sectional area of adipocytes in iBAT (F), and representative UCP-1 staining of iBAT (G) from Floxed and Fp38αKO mice maintained at RT. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Representative EM images of iBAT from Floxed and Fp38αKO mice maintained at RT at low (top), medium (middle), and high (bottom) magnification, as indicated. (I) Relative mitDNA to nuDNA ratio in unilateral iBAT of Floxed (<i>n</i> = 5) and Fp38αKO (<i>n</i> = 9) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (J) OCR of ADP, Oligomycin, FCCP, and Antimycin A/Rotenone-treated mitochondria derived from iBAT of Floxed and Fp38αKO mice exposed to cold for 2 d (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K) Relative mRNA levels of UCP-1, PGC1α, DIO2, COX8B, and PRDM16 in iBAT from Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 6 per group) or exposed to cold for 2 d (<i>n</i> = 8 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (L and M) Representative western blots of UCP-1 in iBAT from Floxed and Fp38αKO mice maintained at RT (L) or exposed to cold for 2 d (M). (N) BT of Fp38αKO and Floxed mice exposed to cold for 4 h (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (O) Relative mRNA levels of UCP-1 and PGC1α in iBAT from Floxed (<i>n</i> = 7–8) and Fp38αKO (<i>n</i> = 8) mice exposed to cold for 4 h. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (P and Q) Representative western blots (P) and densitometry analysis (Q) of UCP-1 in iBAT from Floxed and Fp38αKO mice exposed to cold for 4 h. The densities of UCP-1 bands were quantitated and normalized to Hsp90 (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (R and S) Relative mRNA levels of PRDM16, DIO2, ELVOL3, COX8B, and CIDEA(R), ATGL, MGL, and HSL (S) in iBAT from Floxed (<i>n</i> = 7–8) and Fp38αKO (<i>n</i> = 6–8) mice exposed to cold for 4 h. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. ADP, adenosine diphosphate; ATGL, adipose triglyceride lipase; BAT, brown adipose tissue; BT, body temperature; BW, body weight; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; EM, electron microscopy; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HE staining, hematoxylin-eosin staining; HSL, hormone-sensitive lipase; iBAT, interscapular brown adipose tissue; IHC, immunohistochemistry; LM, lean mass; MGL, monoglyceride lipase; mitDNA, mitochondrial DNA; NS, not significant; nuDNA, nuclear DNA; OCR, oxygen consumption rate; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; PRDM16, positive regulatory domain containing 16; RT, room temperature; UCP-1, uncoupling protein 1; VCO₂, carbon dioxide production; VO₂, oxygen consumption.</p

The effect of p38α inhibition or deficiency on WAT browning is cell autonomous.

<p>(A-C) Representative HE staining of iWAT (A), diameter and cross-sectional area of adipocytes in iWAT (B), and representative UCP-1 staining of iWAT (C) from C57BL/6J mice after injection of Ad-p38αAF into the fat pad of iWAT. These mice were exposed to cold for 2 d before analysis. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D-F) Relative mRNA levels of UCP-1, PGC1α, DIO2, COX8B, and/or ELVOL3 in iWAT-SVF-derived matured adipocytes treated with SB203580 for 4 or 8 h (D, <i>n</i> = 4 per group), or p38α-specific inhibitor (p38αMAPK-IN-1) for 4 h (E, <i>n</i> = 4 per group), or infected with Lenti-p38αAF (F, <i>n</i> = 3 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (G) Relative mRNA levels of UCP-1, PGC1α, PRDM16, DIO2, ELVOL3, COX8B, and CIDEA in matured adipocytes derived from iWAT-SVF of Floxed and Fp38αKO mice (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Representative fluorescence staining of Mito in matured iWAT-SVF-derived adipocytes infected with Lenti-p38αAF. Bars: 25 μm. (I and J) OCR of Oligomycin, FCCP, and Antimycin A/Rotenone-treated matured adipocytes derived from iWAT-SVF of Floxed and Fp38αKO mice (I) and the AUC of OCR (J) as indicated (<i>n</i> = 5 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K and L) Representative UCP-1 staining of iWAT-SVF-derived matured adipocytes after infection with Lenti-p38αAF (K) or matured adipocytes derived from iWAT-SVF of Floxed and Fp38αKO mice (L). Bars: 10 μm. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Ad-p38αAF, adenovirus expressing p38αAF; AUC, area under the curve; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HE staining, hematoxylin-eosin staining; IHC, immunohistochemistry; iWAT, inguinal white adipose tissue; Lenti-p38αAF, lentivirus expressing p38αAF; MAPK, mitogen-activated protein kinase; Mito, mitochondria; NS, not significant; OCR, oxygen consumption rate; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; PRDM16, positive regulatory domain containing 16; SVF, stromal vascular fraction; UCP-1, uncoupling protein 1; WAT, white adipose tissue.</p

Ablation of p38α in adipose tissues facilitates the browning of WAT.

<p>(A and B) Representative HE staining of iWAT (A), diameter and cross-sectional area of adipocytes in iWAT (B) from Floxed and Fp38αKO mice exposed to cold for 2 d. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (C) Relative mRNA levels of UCP-1, PGC1α, ELOVL3, DIO2, COX8B, and CIDEAin iWAT from Floxed and Fp38αKO mice maintained at RT or exposed to cold for 2 d (RT, <i>n</i> = 4 per group; Cold, <i>n</i> = 8–16 per group). Fold of change was indicated (Floxed versus Fp38αKO). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D) Representative western blots of UCP-1 and TH in iWAT from Floxed and Fp38αKO mice maintained at RT or exposed to cold for 2 d. (E) Representative UCP-1 staining of iWAT from Floxed and Fp38αKO mice exposed to cold for 2 d. Bars: 100 μm. (F) Relative mRNA levels of CD137, TBX1, and TMEM26 in iWAT from Floxed (<i>n</i> = 3) and Fp38αKO (<i>n</i> = 6) mice exposed to cold for 2 d. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (G) Relative mitDNA to nuDNA ratio in unilateral iWAT of Floxed and Fp38αKO mice exposed to cold for 2 d (<i>n</i> = 6 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Representative western blots of p38α, p-p38, p-ATF2, and UCP-1 in iWAT from Floxed and Fp38αKO mice exposed to cold for 2 d. (I) Relative p-ATF2 protein levels in iWAT of Floxed and Fp38αKO mice exposed to cold for 2 d. The densities of p-ATF2 bands were quantitated and normalized to Hsp90 (<i>n</i> = 3 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (J and K) VO₂ (J) and VCO₂ (K) within 48 h after CL316,243 injection in Floxed (<i>n</i> = 5) and Fp38αKO (<i>n</i> = 4) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (L and M) Representative HE staining of iWAT (L), diameter and cross-sectional area of adipocytes in iWAT (M) from Floxed and Fp38αKO mice exposed to cold for 7 d. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (N) Relative mRNA levels of UCP-1, PGC1α, PRDM16, ELOVL3, DIO2, COX8B, and CIDEA in iWAT from Floxed and Fp38αKO mice exposed to cold for 7 d (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (O) Representative UCP-1 staining of iWAT from Floxed and Fp38αKO mice exposed to cold for 7 d. Bars: 100 μm. (P) Relative mRNA levels of UCP-1, COX8B, DIO2, ELOVL3, PGC1α, and PRDM16 in iWAT from 5-wk-old Floxed (<i>n</i> = 6) and Fp38αKO (<i>n</i> = 6) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (Q–S) Total amounts of protein in iBAT (<i>n</i> = 6–9) and iWAT (<i>n</i> = 5–11), and total UCP-1 protein per depot (<i>n</i> = 3) in both iBAT and iWAT of Floxed and Fp38αKO mice maintained at RT or exposed to cold for 2 d. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. ATF2, activating transcription factor 2; ATGL, adipose triglyceride lipase; BAT, brown adipose tissue; BT, body temperature; BW, body weight; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; HE staining, hematoxylin-eosin staining; HSL, hormone-sensitive lipase; iBAT, interscapular brown adipose tissue; IHC, immunohistochemistry; iWAT, inguinal white adipose tissue; MGL, monoglyceride lipase; mitDNA, mitochondrial DNA; ND, not detectable; NS, not significant; nuDNA, nuclear DNA; OCR, oxygen consumption rate; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; PRDM16, positive regulatory domain containing 16; RT, room temperature; TBX1, T-box 1; TH, tyrosine hydroxylase; TMEM26, transmembrane protein 26; UCP-1, uncoupling protein 1; VCO₂, carbon dioxide production; VO₂, oxygen consumption; WAT, white adipose tissue.</p

Adipocyte-specific deletion of p38α leads to a lean phenotype and increased glucose tolerance and insulin sensitivity.

<p>(A-C) Representative western blots of p38α and p-p38 in iBAT (A), iWAT (B), and eWAT (C) from Floxed and Fp38αKO mice as indicated. (D-F) Representative western blots of p38α in other tissues, including liver (D), skeletal muscle (E), and macrophages (F) from Floxed and Fp38αKO mice as indicated. (G) Cumulative gross energy intake and feces of Floxed and Fp38αKO mice for 24 h (<i>n</i> = 4 per group). Mice were maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Growth curve of Floxed (<i>n</i> = 15) and Fp38αKO (<i>n</i> = 8–13) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (I and J) BW, FM, and FM to BW ratio (FM/BW) of Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 11 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K and L) GTT (K, <i>n</i> = 8 per group) and ITT (L, <i>n</i> = 8 per group) in Floxed and Fp38αKO mice. AUCs were calculated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01. AUC, area under curve; BW, body weight; eWAT, epididymal white adipose tissue; FM, fat mass; GTT, glucose tolerance test; iBAT, interscapular brown adipose tissue; ITT, insulin tolearance test; iWAT, inguinal white adipose tissue; NS, not significant; RT, room temperature.</p

Deletion of p38α in adipose tissues prevents diet-induced obesity and improves metabolism.

<p>(A) Representative photographs of Floxed and Fp38αKO mice after HFD feeding and iBAT, iWAT, and liver dissected from these mice. (B) Change of BW of Floxed and Fp38αKO mice upon HFD feeding (<i>n</i> = 6 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (C and D) Weight of iWAT and eWAT (C), relative FW of iWAT and eWAT (D) to BW ratio of Floxed (<i>n</i> = 6–8 per group) and Fp38αKO (<i>n</i> = 6 per group) mice after HFD feeding. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (E and F) GTT (E) and ITT (F) in Floxed and Fp38αKO mice after HFD feeding (<i>n</i> = 8 per group). AUCs were calculated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (G and H) Representative HE staining of iWAT (G), diameter and cross-sectional area of adipocytes in iWAT (H) from HFD-fed Floxed and Fp38αKO mice exposed to cold for 2 d. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (I) Relative mRNA levels of UCP-1, ELOVL3, COX8B, and CIDEA in iWAT from HFD-fed Floxed (<i>n</i> = 6) and Fp38αKO (<i>n</i> = 5–6) mice exposed to cold for 2 d. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (J) Representative UCP-1 staining of iWAT from HFD-fed Floxed and Fp38αKO mice exposed to cold for 2 d. Bars: 100 μm. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. AUC, area under curve; BW, body weight; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; eWAT, epididymal white adipose tissue; FW, fat weight; GTT, glucose tolerance test; HE staining, hematoxylin-eosin staining; HFD, high-fat diet; iBAT, interscapular brown adipose tissue; ITT, insulin tolerance test; iWAT, inguinal white adipose tissue; NS, not significant; UCP-1, uncoupling protein 1.</p