Neutrophil infiltration regulates clock-gene expression to organize daily hepatic metabolism.

Liver metabolism follows diurnal fluctuations through the modulation of molecular clock genes. Disruption of this molecular clock can result in metabolic disease but its potential regulation by immune cells remains unexplored. Here, we demonstrated that in steady state, neutrophils infiltrated the mouse liver following a circadian pattern and regulated hepatocyte clock-genes by neutrophil elastase (NE) secretion. NE signals through c-Jun NH2-terminal kinase (JNK) inhibiting fibroblast growth factor 21 (FGF21) and activating Bmal1 expression in the hepatocyte. Interestingly, mice with neutropenia, defective neutrophil infiltration or lacking elastase were protected against steatosis correlating with lower JNK activation, reduced Bmal1 and increased FGF21 expression, together with decreased lipogenesis in the liver. Lastly, using a cohort of human samples we found a direct correlation between JNK activation, NE levels and Bmal1 expression in the liver. This study demonstrates that neutrophils contribute to the maintenance of daily hepatic homeostasis through the regulation of the NE/JNK/Bmal1 axis.BGT and MC were fellows of the FPI: Severo Ochoa CNIC program (SVP-2013–067639) and (BES-2017–079711) respectively. IN was funded by EFSD/Lilly grants (2017 and 2019), the CNIC IPP FP7 Marie Curie Programme (PCOFUND-2012–600396), EFSD Rising Star award (2019), JDC-2018-Incorporación (MIN/JDC1802). T-L was a Juan de la Cierva fellow (JCI2011–11623). C.F has a Sara Borrell contract (CD19/00078). RJD is an Investigator of the Howard Hughes Medical Institute. This work was funded by the following grants to GS: funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n˚ ERC 260464, EFSD/Lilly European Diabetes Research Programme Dr Sabio, 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (Investigadores-BBVA-2017) IN[17] _BBM_BAS_0066, MINECO-FEDER SAF2016-79126-R and PID2019-104399RB-I00 , EUIN201785875, Comunidad de Madrid IMMUNOTHERCAN-CM S2010/BMD-2326 and B2017/BMD-3733 and Fundación AECC AECC PROYE19047SABI and AECC: INVES20026LEIV to ML. MM was funded by ISCIII and FEDER PI16/01548 and Junta de Castilla y León GRS 1362/A/16 and INT/M/17/17 and JL-T by Junta de Castilla y León GRS 1356/A/16 and GRS 1587/A/17. The study was additionally funded by MEIC grants to ML (MINECO-FEDER-SAF2015-74112-JIN) AT-L (MINECO-FEDERSAF2014-61233-JIN), RJD: Grant DK R01 DK107220 from the National Institutes of Health. AH: (SAF2015-65607-R). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015–0505).S

MKK6 controls T3-mediated browning of white adipose tissue

El aumento de la capacidad termogénica del tejido adiposo para mejorar el gasto de energía del organismo se considera una estrategia terapéutica prometedora para combatir la obesidad. Aquí nosotros informe que la expresión del activador MAPK p38 MKK6 está elevada en el tejido adiposo blanco de individuos obesos. Usando animales knockout y shRNA, mostramos que la eliminación de Mkk6 aumenta el gasto de energía y la capacidad termogénica del tejido adiposo blanco, protegiendo a los ratones contra la obesidad inducida por la dieta y el desarrollo de la diabetes. La eliminación de Mkk6 aumenta la expresión de UCP1 estimulada por T3 en los adipocitos, lo que aumenta su capacidad termogénica. De manera mecánica, demostramos que, en el tejido adiposo blanco, p38 se activa mediante una ruta alternativa que involucra AMPK, TAK y TAB. Nuestros resultados identifican MKK6 en los adipocitos como un posible objetivo terapéutico para reducir la obesidad.Increasing the thermogenic capacity of adipose tissue to enhance organismal energy expenditure is considered a promising therapeutic strategy to combat obesity. Here, we report that expression of the p38 MAPK activator MKK6 is elevated in white adipose tissue of obese individuals. Using knockout animals and shRNA, we show that Mkk6 deletion increases energy expenditure and thermogenic capacity of white adipose tissue, protecting mice against diet-induced obesity and the development of diabetes. Deletion of Mkk6 increases T3-stimulated UCP1 expression in adipocytes, thereby increasing their thermogenic capacity. Mechanistically, we demonstrate that, in white adipose tissue, p38 is activated by an alternative pathway involving AMPK, TAK, and TAB. Our results identify MKK6 in adipocytes as a potential therapeutic target to reduce obesity.• Guadalupe Sabio Buzo y Rebeca Acin Pérez pertenecen a Programa Ramón y Cajal • Elisa Manieri pertenece a Caixa • Ministerio de Economía y Competitividad. Proyecto FPI BES-2014-069332, para Valle Montalvo Romeral • Ministerio de Economía y Competitividad. Proyecto FPI BES-2011-043428, para Edgar Bernardo • Ministerio de Economía y Competitividad y FEDER SAF2016-79126-R y Comunidad de Madrid S2010 / BMD-2326, para Guadalupe Sabio Buzo • ISCIII y FEDER, PI10 / 01692 e I3SNS-INT12 / 049, para Miguel Marcos Martín • Junta de Castilla y León GRS 681 / A / 11, para Lourdes Hernández Cosido • Ministerio de Economía y Competitividad. BFU2015-70664-R, Xunta de Galicia 2015-CP080 y PIE13 / 00024, y ERC281408, para Rubén Nogueiras Pozo • Unión Europea. Becas europeas UE0 / MCA1108 y UE0 / MCA1201; y la Comunidad de Madrid CAM / API1009, para Rubén Nogueiras Pozo • Junta de Extremadura y FEDER BR15164, para Francisco Centeno Velázquez • Ministerio de Economía y Competitividad. . BFU2013-46109-R, para Clara V. Álvarez Villamarín • European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. ERC 260464peerReviewe

p38γ is essential for cell cycle progression and liver tumorigenesis

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)–cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

p38α blocks brown adipose tissue thermogenesis through p38δ inhibition

<div><p>Adipose tissue has emerged as an important regulator of whole-body metabolism, and its capacity to dissipate energy in the form of heat has acquired a special relevance in recent years as potential treatment for obesity. In this context, the p38MAPK pathway has arisen as a key player in the thermogenic program because it is required for the activation of brown adipose tissue (BAT) thermogenesis and participates also in the transformation of white adipose tissue (WAT) into BAT-like depot called beige/brite tissue. Here, using mice that are deficient in p38α specifically in adipose tissue (p38α^Fab-KO), we unexpectedly found that lack of p38α protected against high-fat diet (HFD)-induced obesity. We also showed that p38α^Fab-KO mice presented higher energy expenditure due to increased BAT thermogenesis. Mechanistically, we found that lack of p38α resulted in the activation of the related protein kinase family member p38δ. Our results showed that p38δ is activated in BAT by cold exposure, and lack of this kinase specifically in adipose tissue (p38δ ^Fab-KO) resulted in overweight together with reduced energy expenditure and lower body and skin surface temperature in the BAT region. These observations indicate that p38α probably blocks BAT thermogenesis through p38δ inhibition. Consistent with the results obtained in animals, p38α was reduced in visceral and subcutaneous adipose tissue of subjects with obesity and was inversely correlated with body mass index (BMI). Altogether, we have elucidated a mechanism implicated in physiological BAT activation that has potential clinical implications for the treatment of obesity and related diseases such as diabetes.</p></div

p38α^Fab-KO mice are protected against diet-induced obesity and diabetes.

<p>(A) Body weight time course in Fab-Cre and p38α^Fab-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 p38α^Fab-KO mice (mean ± SEM; Fab-Cre HFD <i>n =</i> 10 mice; p38α^Fab-KO HFD <i>n =</i> 11 mice; Fab-Cre ND <i>n =</i> 9 mice; p38α^Fab-KO ND <i>n =</i> 8 mice). (B) NMR analysis of fat mass in p38α^Fab-KO and Fab-Cre mice after 8 weeks of HFD (mean ± SEM; Fab-Cre <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 8 mice). (C) Representative haematoxylin–eosin and oil red O staining of liver sections (Fab-Cre <i>n =</i> 6 mice; p38α^Fab-KO <i>n =</i> 6 mice; and 3 pictures from each mouse). Scale bar: 50 μm. (D) Fasting and fed blood glucose in Fab-Cre and p38α^Fab-KO mice fed the HFD (8 weeks) (mean ± SEM; Fab-Cre <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 11 mice). (E) GTT and ITT in Fab-Cre and p38α^Fab-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 <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 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 <i>n =</i> 6 mice; p38α^Fab-KO <i>n =</i> 6 mice; and 3 pictures from each mouse). Scale bar: 50 μm. *<i>p</i> < 0.05, ***<i>p</i> < 0.001 Fab-Cre versus p38α^Fab-KO. ‘&&’ indicates <i>p</i> < 0.01, ‘&&&’ indicates <i>p</i> < 0.001 Fab-Cre ND versus Fab-Cre HFD (2-way ANOVA coupled with Bonferroni’s post-tests or <i>t</i> test or Welch’s test when variances were different). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004455#pbio.2004455.s015" target="_blank">S1 Data</a>. 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.</p

p38s regulate respiratory capacity of brown adipocytes.

<p>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 <i>Gapdh</i> mRNA (mean ± SEM; WT <i>n =</i> 5 wells; p38δ^−/− <i>n =</i> 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 p38α^Fab-KO mice (mean ± SEM; Fab-Cre <i>n =</i> 3 wells; p38α^Fab-KO <i>n =</i> 5 wells) and of (C) WT or p38δ^−/− mice (mean ± SEM; WT <i>n =</i> 3 wells; p38δ^−/− <i>n =</i> 4 wells). (D–E) OCR to NE (1 μM) and ISO (1 μM) in differentiated brown adipocytes from Fab-Cre and p38α^Fab-KO mice (mean ± SEM; Fab-Cre <i>n =</i> 7 or p38α^Fab-KO <i>n =</i> 7 wells treated with NE; and Fab-Cre <i>n =</i> 8 or p38α^Fab-KO <i>n =</i> 8 wells treated with ISO) (panel D) or from WT or p38δ^−/− mice (mean ± SEM; WT <i>n =</i> 22 or p38δ^−/− <i>n =</i> 12 wells treated with NE; and WT <i>n =</i> 12 or p38δ^−/− <i>n =</i> 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 p38α^Fab-KO mice was abolished by pretreatment with BIRB796 (10 μM) for 1 hour (mean ± SEM; Fab-Cre <i>n =</i> 6 or p38α^Fab-KO <i>n =</i> 7 wells treated with NE; and Fab-Cre <i>n =</i> 7 or p38α^Fab-KO <i>n =</i> 8 wells treated with ISO). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004455#pbio.2004455.s015" target="_blank">S1 Data</a>. 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.</p

p38α^Fab-KO mice are protected against diet-induced obesity and diabetes.

<p>(A) Body weight time course in Fab-Cre and p38α^Fab-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 p38α^Fab-KO mice (mean ± SEM; Fab-Cre HFD <i>n =</i> 10 mice; p38α^Fab-KO HFD <i>n =</i> 11 mice; Fab-Cre ND <i>n =</i> 9 mice; p38α^Fab-KO ND <i>n =</i> 8 mice). (B) NMR analysis of fat mass in p38α^Fab-KO and Fab-Cre mice after 8 weeks of HFD (mean ± SEM; Fab-Cre <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 8 mice). (C) Representative haematoxylin–eosin and oil red O staining of liver sections (Fab-Cre <i>n =</i> 6 mice; p38α^Fab-KO <i>n =</i> 6 mice; and 3 pictures from each mouse). Scale bar: 50 μm. (D) Fasting and fed blood glucose in Fab-Cre and p38α^Fab-KO mice fed the HFD (8 weeks) (mean ± SEM; Fab-Cre <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 11 mice). (E) GTT and ITT in Fab-Cre and p38α^Fab-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 <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 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 <i>n =</i> 6 mice; p38α^Fab-KO <i>n =</i> 6 mice; and 3 pictures from each mouse). Scale bar: 50 μm. *<i>p</i> < 0.05, ***<i>p</i> < 0.001 Fab-Cre versus p38α^Fab-KO. ‘&&’ indicates <i>p</i> < 0.01, ‘&&&’ indicates <i>p</i> < 0.001 Fab-Cre ND versus Fab-Cre HFD (2-way ANOVA coupled with Bonferroni’s post-tests or <i>t</i> test or Welch’s test when variances were different). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004455#pbio.2004455.s015" target="_blank">S1 Data</a>. 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.</p

p38α^Fab-KO mice have higher energy expenditure and increased BAT thermogenesis.

<p>Fab-Cre and p38α^Fab-KO mice were fed an HFD for 8 weeks. (A) Analysis of eWAT expansion in HFD-fed Fab-Cre and p38α^Fab-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 p38α^Fab-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 <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 8 mice). (C) Body (mean ± SEM; Fab-Cre <i>n =</i> 20 mice; p38α^Fab-KO <i>n =</i> 18 mice) and skin temperature of surrounding interscapular BAT (mean ± SEM; Fab-Cre <i>n =</i> 10 mice; p38α^Fab-KO <i>n =</i> 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 p38α^Fab-KO mice are indicated: **<i>p</i> < 0.01 (<i>t</i> test or Welch’s test when variances were different). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004455#pbio.2004455.s015" target="_blank">S1 Data</a>. 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.</p