Fig 1.
p38α in human visceral fat inversely correlated with BMI and directly correlated with UCP1 in human visceral fat and sWAT.
(A) mRNA levels of Mapk14 (p38α) in visceral fat from lean individuals and individuals with obesity—mRNA expression was normalised to the amount of Gapdh mRNA. (B) Correlation between mRNA levels of Mapk14 (p38α) and BMI (r2 = −0,365; p = 0.001) or (C) Ucp1 in visceral fat (r2 = 0.316; p = 0.007). The mRNA levels of Mapk14 (p38α) and Ucp1 were determined by qRT-PCR (n = 71). (D) mRNA levels of Mapk14 (p38α) in sWAT from lean individuals and individuals with obesity. mRNA expression was normalised to the amount of Gapdh mRNA. (E) Correlation between mRNA levels of Mapk14 (p38α) and Ucp1 in sWAT (r2 = 0.320; p < 0.0001). Graph correlating mRNA Mapk14 and log mRNA Ucp1 is also shown. The mRNA levels of Mapk14 (p38α) and Ucp1 were determined by qRT-PCR (n = 168). See also S1 Data. Linear relationships between variables were tested using Pearson’s correlation coefficient. BMI, body mass index; qRT-PCR, quantitative real-time polymerase chain reaction; UCP1, uncoupling protein 1.
Fig 2.
p38αFab-KO mice are protected against diet-induced obesity and diabetes.
(A) Body weight time course in Fab-Cre and p38αFab-KO male (8–10-wk-old) mice fed an HFD over 8 weeks. Data are presented as the increase above initial weight (left panel) or as total weight comparing mice fed an HDF with mice fed an ND (right panel). HFD-induced weight gain was significantly higher in Fab-Cre than p38αFab-KO mice (mean ± SEM; Fab-Cre HFD n = 10 mice; p38αFab-KO HFD n = 11 mice; Fab-Cre ND n = 9 mice; p38αFab-KO ND n = 8 mice). (B) NMR analysis of fat mass in p38αFab-KO and Fab-Cre mice after 8 weeks of HFD (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 8 mice). (C) Representative haematoxylin–eosin and oil red O staining of liver sections (Fab-Cre n = 6 mice; p38αFab-KO n = 6 mice; and 3 pictures from each mouse). Scale bar: 50 μm. (D) Fasting and fed blood glucose in Fab-Cre and p38αFab-KO mice fed the HFD (8 weeks) (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 11 mice). (E) GTT and ITT in Fab-Cre and p38αFab-KO mice fed the HFD for 8 weeks. Mice were fasted overnight (for GTT) or 1 hour (for ITT), and blood glucose concentration was measured in mice given intraperitoneal injections of glucose (1 g/kg of total body weight) or insulin (0.75 U/kg of total body weight) (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 11 mice). (F) Immunohistochemistry of eWAT sections using anti-GLUT4 (green), anti-Cav-1 (red) antibodies, and the nuclear dye DAPI (blue). Location of GLUT4 was analysed in mice treated without or with insulin (1.5 IU/kg) for 15 minutes after overnight fasting. Scale bar: 20 μm. (G) Representative haematoxylin–eosin BAT and eWAT sections (Fab-Cre n = 6 mice; p38αFab-KO n = 6 mice; and 3 pictures from each mouse). Scale bar: 50 μm. *p < 0.05, ***p < 0.001 Fab-Cre versus p38αFab-KO. ‘&&’ indicates p < 0.01, ‘&&&’ indicates p < 0.001 Fab-Cre ND versus Fab-Cre HFD (2-way ANOVA coupled with Bonferroni’s post-tests or t test or Welch’s test when variances were different). See also S1 Data. BAT, brown adipose tissue; Cav-1, caveolin-1; eWAT, epididymal fat; GLUT4, glucose transporter type 4; GTT, glucose tolerance test; HFD, high-fat diet; ITT, insulin tolerance test; ND, normal-chow diet; WAT, white adipose tissue.
Fig 3.
p38αFab-KO mice have higher energy expenditure and increased BAT thermogenesis.
Fab-Cre and p38αFab-KO mice were fed an HFD for 8 weeks. (A) Analysis of eWAT expansion in HFD-fed Fab-Cre and p38αFab-KO mice. Animals were treated with BrdU in the drinking water during the first week of a 6-week HFD. Cartoon explaining the protocol is shown in the left panel. BrdU incorporation into the nuclei was detected by immunofluorescence in eWAT sections (right panel). Cell outlines were stained with anti-perilipin antibody (green) and nuclei, with DAPI (blue). Scale bar: 20 μm. A cell in detail is shown in a bigger magnification for each genotype. Quantification of positive BrdU nuclei is showed in the middle panel. (B) Comparison of energy balance between HFD-fed Fab-Cre and p38αFab-KO mice. HFD-fed mice were examined in a metabolic cage over a 2-day period to measure FI, respiratory exchange, and EE. FI and EE (left) over 2 days were corrected by lean mass. EE expressed as ANCOVA analysis (middle panel) and hour by hour over 48-h period (right panel) are also shown (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 8 mice). (C) Body (mean ± SEM; Fab-Cre n = 20 mice; p38αFab-KO n = 18 mice) and skin temperature of surrounding interscapular BAT (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 7 mice). Lower panels show representative infrared thermal images. (D) Immunoblot analysis of UCP1 levels and Creb and AMPK phosphorylation in lysates from BAT. Quantification is shown in the lower panel. (E) Immunohistochemistry staining of UCP1 after 8 weeks of HFD in BAT. Scale bar: 50 μm. Statistically significant differences between Fab-Cre and p38αFab-KO mice are indicated: **p < 0.01 (t test or Welch’s test when variances were different). See also S1 Data. AMPK, 5' adenosine monophosphate-activated protein kinase; BAT, brown adipose tissue; BrdU, bromodeoxyuridine; Creb, cAMP response element-binding; EE, energy expenditure; eWAT, epididymal fat; FI, food intake; HFD, high-fat diet; IR temperature, infrared temperature; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Fig 4.
p38α controls BAT thermogenesis.
UCP1-Cre and p38αUCP1-KO mice were fed with HFD for 8 weeks. (A) Body weight at the end of the treatment (mean ± SEM; UCP1-Cre n = 12 mice; p38αUCP1-KO n = 14 mice). (B) Weight of BAT, eWAT, sWAT, iWAT, pWAT, and liver relativised to tibia length (mean ± SEM; UCP1-Cre n = 12 mice; p38αUCP1-KO n = 14 mice). (C) Body temperature of HFD-fed UCP1-Cre and p38αUCP1-KO mice (mean ± SEM; UCP1-Cre n = 12 mice; p38αUCP1-KO n = 14 mice). (D) Fasting and fed blood glucose in UCP1-Cre and p38αUCP1-KO mice fed an HFD (8 weeks) (mean ± SEM; UCP1-Cre n = 12 mice; p38αUCP1-KO n = 14 mice). (E) GTT and (F) ITT in UCP1-Cre and p38αUCP1-KO mice fed HFD for 8 weeks. Mice were fasted overnight (for GTT) or 1 hour (for ITT), and blood glucose concentration was measured in mice given intraperitoneal injections of glucose (1 g/kg of total body weight) or insulin (0.75 U/kg of total body weight) (mean ± SEM; UCP1-Cre n = 12 mice; p38αUCP1-KO n = 14 mice). *p < 0.05; **p < 0.01; ***p < 0.001 UCP1-Cre versus p38αUCP1-KO (2-way ANOVA coupled with Bonferroni’s post-tests or t test or Welch’s test when variances were different). See also S1 Data. BAT, brown adipose tissue; eWAT, epididymal fat; GTT, glucose tolerance test; HFD, high-fat diet; ITT, insulin tolerance test; iWAT, inguinal fat; pWAT, perirenal fat; sWAT, subcutaneous fat; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Fig 5.
p38αFab-KO mice have increased BAT thermogenesis under thermoneutrality conditions.
Fab-Cre and p38αFab-KO mice were fed an HFD for 8 weeks and housed at 30 °C. (A) Body weight time course in Fab-Cre and p38αFab-KO male (8–10-wk-old) mice fed a HFD over 8 weeks. Data are presented as the increase above initial weight (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 6 mice). (B) Weight of liver, BAT, eWAT, sWAT, iWAT, and pWAT (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 6 mice). (C) Body and skin temperature of surrounding interscapular BAT from HFD-fed Fab-Cre and p38αFab-KO mice (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 6 mice). Lower panels show representative infrared thermal images. (D) Immunoblot analysis of UCP1 protein levels in lysates from BAT. Quantification is shown in the lower panel. (E) Fasting and fed blood glucose in Fab-Cre and p38αFab-KO mice fed the HFD at 30 °C (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 6 mice). (F) GTT in HFD-fed Fab-Cre and p38αFab-KO at 30 °C. Blood glucose concentration was measured in mice given intraperitoneal injections of glucose (1 g/kg of total body weight) (mean ± SEM; Fab-Cre n = 10 mice; p38αFab-KO n = 6 mice). Statistically significant differences between Fab-Cre and p38αFab-KO mice are indicated: *p < 0.05; **p < 0.01; ***p < 0.001 (t test or Welch’s test when variances were different). See also S1 Data. BAT, brown adipose tissue; eWAT, epididymal fat; GTT, glucose tolerance test; HFD, high-fat diet; IR temperature, infrared temperature; iWAT, inguinal fat; pWAT, perirenal fat; sWAT, subcutaneous fat; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Fig 6.
Activation of p38δ is responsible for BAT activation.
(A) Immunoblot analysis of BAT lysate from Fab-Cre and p38αFab-KO mice fed an HFD for 8 weeks. Left: 23 °C; right: comparison of 23 °C versus 30 °C. (B) qRT-PCR analysis of mRNA expression of p38β (Mapk11), p38γ (Mapk12), and p38δ (Mapk13) in BAT Fab-Cre and p38αFab-KO mice fed an HFD for 8 weeks. mRNA was normalised to level of Gapdh mRNA (mean ± SEM, Fab-Cre n = 15 mice; p38αFab-KO n = 9 mice) (C) Body weight in Fab-Cre and p38δFab-KO male (8–10-wk-old) mice fed an ND over 8 weeks (mean ± SEM; Fab-Cre n = 6 mice; p38δFab-KO n = 6 mice). (D) Body, fat, and lean mass in p38δFab-KO and Fab-Cre mice after 8 weeks of ND measured by NMR (mean ± SEM; Fab-Cre n = 6 mice; p38δFab-KO n = 5 mice). (E) Comparison of energy balance between ND-fed Fab-Cre and p38δFab-KO mice. ND-fed mice were examined in a metabolic cage over a 3-day period to measure FI and EE. FI (upper left panel; mean ± SEM; Fab-Cre n = 12 mice; p38δFab-KO n = 10 mice) and EE (upper right panel; mean ± SEM; Fab-Cre n = 6 mice; p38δFab-KO n = 6 mice) over 2 days were corrected by lean mass. EE expressed as ANCOVA analysis (lower left panel; mean ± SEM; Fab-Cre n = 9 mice; p38δFab-KO n = 12 mice) and hour by hour over a 48-hour period (lower right panel; mean ± SEM; Fab-Cre n = 12 mice; p38δFab-KO n = 12 mice) are also shown. (F) Body temperature of ND-fed Fab-Cre and p38δFab-KO mice (mean ± SEM; Fab-Cre n = 9 mice; p38δFab-KO n = 11 mice). Skin temperature surrounding interscapular BAT in ND-fed Fab-Cre and p38δFab-KO. Right panels show representative infrared thermal images (mean ± SEM; Fab-Cre n = 10 mice; p38δFab-KO n = 12 mice). (G) Adipocytes differentiated from interscapular BAT were stimulated with 100 nM T3 for 48 hours. Immunoprecipitation from cell lysates of p38δ were evaluated by immunoblot with antibodies against phospho-p38 and p38δ. Adipocytes differentiated from sWAT were stimulated with 1 μM NE for 1 hour, and p38 phosphorylation was analysed by immunoblot. (H) Control mice (C57BL/6) were exposed to cold (4 °C) for the indicated time, and phosphorylation of the different p38s in BAT was evaluated by immunoblot (n = 5 for each group; representative blot presented). (I) Body temperature of ND-fed Fab-Cre and p38δFab-KO mice exposed to cold (4 °C) for 1 hour (mean ± SEM; Fab-Cre n = 10 mice; p38δFab-KO n = 8 mice). Skin temperature surrounding interscapular BAT in ND-fed Fab-Cre and p38δFab-KO after 1 hour of cold exposure. Right panels show representative infrared thermal images (mean ± SEM; Fab-Cre n = 9 mice; p38δFab-KO n = 8 mice). *p < 0.05; **p < 0.01; ***p < 0.001 (t test). See also S1 Data. BAT, brown adipose tissue; Creb, cAMP response element-binding; EE, energy expenditure; FI, food intake; HFD, high-fat diet; IR temperature, infrared temperature; ND, normal-chow diet; NE, norepinephrine; NMR, nuclear magnetic resonance; qRT-PCR, quantitative real-time polymerase chain reaction; sWAT, subcutaneous fat.
Fig 7.
p38s regulate respiratory capacity of brown adipocytes.
Primary adipocytes isolated from intercapsular BAT were differentiated in vitro. (A) qRT-PCR analysis of browning genes mRNA expression from primary adipocytes isolated from WT or p38δ−/− mice. mRNA expression was normalised to the amount of Gapdh mRNA (mean ± SEM; WT n = 5 wells; p38δ−/− n = 5 wells). (B) Analysis of mitochondrial DNA content with respect to nuclear DNA by RT-PCR in adipocytes isolated from BAT of Fab-cre or p38αFab-KO mice (mean ± SEM; Fab-Cre n = 3 wells; p38αFab-KO n = 5 wells) and of (C) WT or p38δ−/− mice (mean ± SEM; WT n = 3 wells; p38δ−/− n = 4 wells). (D–E) OCR to NE (1 μM) and ISO (1 μM) in differentiated brown adipocytes from Fab-Cre and p38αFab-KO mice (mean ± SEM; Fab-Cre n = 7 or p38αFab-KO n = 7 wells treated with NE; and Fab-Cre n = 8 or p38αFab-KO n = 8 wells treated with ISO) (panel D) or from WT or p38δ−/− mice (mean ± SEM; WT n = 22 or p38δ−/− n = 12 wells treated with NE; and WT n = 12 or p38δ−/− n = 12 wells treated with ISO) (panel E) analysed by Seahorse assay. Nonmitochondrial respiration was subtracted from OCR values, and all values were normalised to protein content. Upper panels show OCR over time upon different drugs injections: oligomycin (1 μM), FCCP (1 μM), and antimycin A (1 μM) with rotenone (1 μM). Lower panels show basal and NE/ISO-induced OCR. (F) OCR induced by NE and ISO in differentiated brown adipocytes from Fab-Cre and p38αFab-KO mice was abolished by pretreatment with BIRB796 (10 μM) for 1 hour (mean ± SEM; Fab-Cre n = 6 or p38αFab-KO n = 7 wells treated with NE; and Fab-Cre n = 7 or p38αFab-KO n = 8 wells treated with ISO). See also S1 Data. BAT, brown adipose tissue; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; ISO, isoproterenol; NE, norepinephrine; OCR, oxygen consumption rate; qRT-PCR, quantitative real-time polymerase chain reaction; WT, wild-type.
Fig 8.
Regulation of browning and BAT activation by p38 pathway.
Graphical abstract summarising the role of p38 isoforms in adipose tissue. In eWAT, p38α activates browning through the phosphorylation of Creb and ATF2 increasing UCP1 expression. In iWAT and BAT, p38α activation inhibits p38γ and p38δ and in consequence reduces browning and BAT activation, respectively, by down-regulation of UCP1. ATF2, activating transcription factor 2; BAT, brown adipose tissue; Creb, cAMP response element-binding; eWAT, epididymal fat; iWAT, inguinal fat; UCP1, uncoupling protein 1.