ectopic ucp1 overexpression in white adipose tissue improves … · 2015. 10. 15. · ectopic ucp1...

Anne-Laure Poher,1 Christelle Veyrat-Durebex,2 Jordi Altirriba,1 Xavier Montet,3

Didier J. Colin,4 Aurélie Caillon,1 Jacqueline Lyautey,1 andFrançoise Rohner-Jeanrenaud1

Ectopic UCP1 Overexpression in WhiteAdipose Tissue Improves InsulinSensitivity in Lou/C Rats, a Modelof Obesity ResistanceDiabetes 2015;64:3700–3712 | DOI: 10.2337/db15-0210

Brown adipose tissue (BAT), characterized by the pres-ence of uncoupling protein 1 (UCP1), has been describedas metabolically active in humans. Lou/C rats, originat-ing from the Wistar strain, are resistant to obesity. Wepreviously demonstrated that Lou/C animals expressUCP1 in beige adipocytes in inguinal white adipose tissue(iWAT), suggesting a role of this protein in processessuch as the control of body weight and the observedimproved insulin sensitivity. A b3 adrenergic agonist wasadministered for 2 weeks in Wistar and Lou/C rats toactivate UCP1 and delineate its metabolic impact. Thetreatment brought about decreases in fat mass andimprovements in insulin sensitivity in both groups. InBAT, UCP1 expression increased similarly in response tothe treatment in the two groups. However, the interventioninduced the appearance of beige cells in iWAT, associatedwith a marked increase in UCP1 expression, in Lou/Crats only. This increase was correlated with a markedlyenhanced glucose uptake measured during euglycemic-hyperinsulinemic clamps, suggesting a role of beige cellsin this process. Activation of UCP1 in ectopic tissues, suchas beige cells in iWAT, may be an interesting therapeuticapproach to prevent body weight gain, decrease fat mass,and improve insulin sensitivity.

Obesity has reached epidemic proportions worldwide andhas become a major global public health problem in recentdecades (1). It represents a considerable risk factor for thedevelopment of several comorbidities, among which is type2 diabetes (1). Globally, the key component of the obesity

epidemic is long-term dysregulation of energy balance. In viewof the relative lack of drugs suppressing appetite, approachesto increase energy expenditure are viewed as potential newtherapeutic options to treat obesity and metabolic complica-tions. Along this line, brown adipose tissue (BAT), now knownto be present in adult humans (2–4), is the focus of majorinterest, due to its role in inducing thermogenesis (5). Themitochondria of brown adipocytes are characterized by thepresence of uncoupling protein 1 (UCP1), which uncouplesoxidative phosphorylation from ATP synthesis, resulting inheat production (6). This process consumes substantialamounts of free fatty acids (FFAs) and glucose (7).

In rodents, brown adipocytes are found in discrete areas,such as interscapular, cervical, peri-renal, and intercostaldepots (8), which are referred to as “classical” BAT depots. Inwhite adipose tissue (WAT), brown-like cells, called beige orbrite cells, express UCP1 (9). The existence of a specific pre-cursor, different from classical white or brown adipocytes,arising from smooth muscle cells that would differentiateinto beige adipocytes in WAT has been proposed (10). More-over, some studies suggest that, under specific conditions,most or all of white adipocytes transdifferentiate into beigeadipocytes (11).

Interestingly, whatever their developmental origin,white, beige, and brown adipocytes seem to greatly differin their function. As mentioned above, BAT is the effectororgan of nonshivering thermogenesis that, by utilizinglarge quantities of glucose and lipids from the circulation,can promote negative energy balance. Moreover, as recentlyreviewed by Peirce and Vidal-Puig (12), the role of BAT

1Laboratory of Metabolism, Division of Endocrinology, Diabetology, Hypertensionand Nutrition, Department of Internal Medicine Specialties, Faculty of Medicine,University of Geneva, Geneva, Switzerland2Department of Cell Physiology and Metabolism, Faculty of Medicine, Universityof Geneva, Geneva, Switzerland3Division of Radiology, Faculty of Medicine, University of Geneva, Geneva, Switzerland4MicroPET/SPECT/CT Imaging Laboratory, Centre for BioMedical Imaging, UniversityHospital of Geneva, Geneva, Switzerland

Corresponding author: Anne-Laure Poher, [email protected].

Received 13 February 2015 and accepted 20 July 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0210/-/DC1.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

3700 Diabetes Volume 64, November 2015

METABOLISM

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activation might be broader than solely promoting negativeenergy balance. Indeed, such activation was described toexert anti–type 2 diabetic effects (13,14), associated withimprovements of dyslipidemia (rev. in 15). These effectsare partly interrelated but can also be dissociated andexerted by different UCP1-expressing adipocyte types.

The Lou/C rat is a model of age- and diet-inducedobesity resistance, which also exhibits a lower body fatmass, increased leptin sensitivity, and improved insulinsensitivity compared with Wistar animals (16–18). Wealso demonstrated that the most striking differencesbetween Lou/C and Wistar rats were the presence, ininguinal WAT (iWAT) of the Lou/C group, of Ucp1 (16,17)and of a marked Adrb3 overexpression (17).

In the current study, we used the Lou/C rat as a modelto investigate the impact of UCP1 activation on glucosemetabolism. To this end, various groups of Wistar and Lou/Crats were subcutaneously infused for 2 weeks with a b3adrenoreceptor agonist (CL-316243), and UCP1 in BAT andWAT depots, as well as the insulin-stimulated glucose utili-zation rate, of different tissues was determined.

RESEARCH DESIGN AND METHODS

Animals and DietsTwo month-old male Lou/C and Wistar rats were pur-chased from Harlan UK Limited (Oxon, U.K.) and CharlesRiver (L’Arbresle, France), respectively. They were housedin pairs under controlled conditions (22°C; light on 7:00 A.M.–7:00 P.M.) and were allowed free access to water anddiet (RMI; Hersteller, Essex, U.K.). Osmotic minipumps(Alzet, Cupertino, CA) delivering a b3 agonist (CL-316243,1 mg/kg/day; Tocris, Bristol, U.K.,) or NaCl 0.9% for 14 dayswere placed subcutaneously at the age of 12 weeks. One weekbefore and during the treatment, body weight and food in-take were measured daily (9:00 A.M.). A first colony was sub-mitted to a glucose tolerance test (GTT) at day 9 and a bodyfat composition analysis by MRI (Echo-MRI) at day 14. Asecond colony was submitted to a computed tomography(CT) scan on day 12 and a euglycemic-hyperinsulinemic clamp(EHC) on day 14. A third colony underwent a positron emis-sion tomography (PET) scan coupled to a micro-CT on day14. A fourth colony was housed in pairs at 22°C or at30°C in a climatic chamber (Meditest H 1300L; Froilabo,Meysieu, France).

Except for EHC experiments, all rats were killed between9:00 A.M. and 1:00 P.M., using isoflurane (Halocarbon Labora-tories, River Edge, NJ) anesthesia and rapid decapitation.Trunk blood was collected to measure the concentration ofvarious hormones and metabolites. Tissues were freezeclamped and stored at –80°C for determination of geneand protein expressions. The procedures were approved bythe ethics committee of our university and were in accor-dance with the Swiss guidelines for animal experimentation.

GTTWistar and Lou/C rats were food deprived for 4 h (from9:00 to 1:00 P.M.). A glucose load of 1.5 g/kg i.p. was

administered. Blood samples were collected by tail nickingand were used for further analyses of plasma glucose andinsulin levels.

EHCRats were overnight fasted and anesthetized with pentobar-bital (50 mg/kg i.p.; Abbott Laboratories, Chicago, IL). Theglucose infusion rate (GIR) required to maintain euglycemiaunder insulin-stimulated conditions (18 mU/kg/min; ActrapidHM, Novo Nordisk, Bagsvaerd, Denmark) was determinedas previously described (19,20). At the end of the EHC, thein vivo insulin-stimulated glucose utilization indexof individual tissues was measured, using 2-deoxy-D-[1,2-3H]glucose (30 mCi/rat, NET 328A; PerkinElmer,Schwerzenbach, Switzerland) (20). Rats were killed bydecapitation and tissues stored at –80°C. The 2-deoxy-D-[1,2-3H]glucose–specific activity was measured in deproteinizedblood samples. Determination of tissue concentration of2-deoxy-D-[1,2-3H]glucose-6-phosphate allowed for the cal-culation of the in vivo glucose utilization index of individ-ual tissues and was expressed in nanograms of glucose permilligram of tissue per minute (21,22).

Plasma MeasurementsPlasma glucose levels were measured by the glucoseoxidase method (Glu; Roche Diagnostics GmbH, Rotkreuz,Switzerland). FFA and triglyceride (TG) levels were deter-mined using a Wako Chemicals GmbH (Neuss, Germany)and a Biomérieux (Marcy l’Etoile, France) kit, respectively.Plasma leptin (Linco Research, Inc., St Charles, MO) levelswere measured using a double antibody radioimmuno-assay kit. Plasma insulin levels were determined usingan ELISA commercial kit (10-1250; Mercodia, Uppsala,Sweden).

Tissue Processing and RT-PCRRNA was reverse transcribed (M-MLV-RT; Invitrogen,Basel, Switzerland) and quantitative PCR (qPCR) wasperformed using the SYBR green PCR Master Mix (AppliedBiosystems, Warrington, U.K.) on a Stepone Plus machine(Applied Biosystems). Primers (Supplementary Table 1)were designed with the PrimerExpress software (AppliedBiosystems). Results were normalized to the expressionlevels of the housekeeping gene, ribosomal protein S29(Rps29).

Mitochondrial DNA Copy NumberQuantification of mitochondrial DNA (mtDNA) copy num-ber was achieved by qPCR. Briefly, DNA was extracted fromiWAT using the DNeasy Blood and Tissue kit (Qiagen,Düsseldorf, Germany). Nuclear and mtDNA copy numberswere assessed by real-time PCR using primers targetedtoward the Cox1 gene (for mtDNA) and nuclear RNAseP(for nuclear DNA).

Western BlottingFrozen tissues were homogenized in ice-cold RIPA buffer. Pro-tein levels were quantified using a BCA protein assay (Pierce,

diabetes.diabetesjournals.org Poher and Associates 3701

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Lausanne, Switzerland). Ten and 50 mg protein, respectively,was used for BAT andWAT SDS-PAGE. UCP1 antibody (Abcam,Cambridge, U.K.) was used at a concentration of 1/10,000for BAT and 1/1,000 for WAT before the secondary antibody(anti-rabbit 1/5,000) was added. Housekeeping proteinsERM (Santa Cruz Biotechnology, Santa Cruz, CA) and actin(Millipore, Billerica, MA) were used at 1/2,000 and1/100,000, with anti-goat (1/10,000) or anti-mouse (1/5,000)antibody, respectively. Detection was performed with an en-hanced chemiluminescence detection system (AmershamBiosciences, Amersham, U.K.). Signals were quantified usingthe PXi and the Genetools software from Syngene UK.

Body CompositionAn EchoMRI-700 quantitative nuclear magnetic resonanceanalyzer (Echo Medical Systems, Houston, TX) was used tomeasure body composition (total fat and lean body mass).Rats were also scanned on a multidetector CT scanner(Discovery 750 HD; GE Healthcare, Milwaukee, WI) todetermine the volume of the various fat depots.

Light MicroscopyTissues were fixed in formalin (10%) after dissection.They were washed in PBS, dehydrated, cleared, and finallyembedded in paraffin blocks, which were cut at 7 mm andthen stained with hematoxylin-eosin (Sigma-Aldrich, Buchs,Switzerland). For immunohistochemistry, the same UCP1antibody as that used for Western blots was used at a con-centration of 1/100 before the secondary anti-rabbit Cy3antibody (1/250) (Jackson ImmunoResearch).

[18F]-fluorodeoxyglucose Glucose UptakeMeasurement in TissuesRats were overnight fasted and anesthetized usingisoflurane. Glycemia was measured before the experi-ment. Animals received a 3.5 mg/kg i.p. injection offurosemide (Lasix; Sanofi, Paris, France) to empty theirbladder. [18F]-fluorodeoxyglucose ([18F]-FDG) was injectedvia the pudendal vein (20 MBq/rat). Glucose uptake was mea-sured with a microPET/CT (Triumph; TriFoil, Chatsworth, CA).A micro-CT analysis was simultaneously performed to localizethe [18F]-FDG emission in different structures. Data were an-alyzed by the Osirix software (Pixmeo, Geneva, Switzerland).

Data AnalysesResults are expressed as mean6 SEM. Comparison betweenthe four groups was performed by GraphPad Prism (SanDiego, CA), using the two-way ANOVA analysis followedby a Tukey post hoc test. Correlation between the Ucp1mRNA expression and the glucose uptake index in WATwas calculated using the Pearson correlation coefficient.Statistical significance was established at P , 0.05.

RESULTS

As previously reported (16,17), Lou/C had a lower bodyweight than Wistar rats. The 2-week CL-316243 treat-ment had no impact on body weight gain in Wistar andLou/C animals (Fig. 1A). Total food intake of Lou/C was

lower than that of Wistar rats, but it was unaffected bythe treatment in both groups (Fig. 1A). Interestingly, foodefficiency was similar in Wistar and Lou/C rats whethertreated or not (Fig. 1A). Analysis of body composition byMRI (EchoMRI) further showed that the b3 agonist treat-ment decreased the total fat mass and consequently in-creased the lean body mass in both Wistar and Lou/Canimals (Fig. 1B). In addition, CT scan analysis showeda significant treatment-induced decrease in the mass of allfat depots (interscapular BAT, subcutaneous WAT, andintra-abdominal WAT) in the two animal groups, exceptfor subcutaneous WAT of Lou/C rats (Fig. 1C). This was inkeeping with a significant or with a trend toward a signif-icant decrease in leptinemia in b3 agonist–treated Wistarand Lou/C rats, respectively (Table 1). Qualitatively sim-ilar results were obtained for plasma glucose, insulin, andTG levels, while plasma FFA concentrations were unaf-fected by the treatment in both groups (Table 1).

To evaluate the impact of the b3 agonist treatment onglucose metabolism, a GTT was performed at day 9 of thestudy (Fig. 2A). The treatment had no impact on basalglycemia in both groups (data not shown). As indicated bythe changes in glycemia from baseline values, as well asthe areas under the curve (AUCs) during the whole tests,glucose tolerance of Wistar and Lou/C rats was improvedby the treatment (Fig. 2A). With regard to insulin, thetreatment effect on the AUC during the GTT almostreached statistical significance (P = 0.057), this effect be-ing clearly more marked in the Lou/C than in the Wistargroup (Fig. 2B).

Peripheral insulin sensitivity was then evaluated byperforming EHC. Basal and steady-state (clamp) values ofglycemia and insulinemia are provided in SupplementaryTable 2. As previously reported (16,17), the GIR of Lou/Crats was higher than that of Wistar animals, indicatingimproved insulin sensitivity (Fig. 2C). In both the Wistarand the Lou/C group, the b3 agonist treatment increasedthe GIR (Fig. 2C). As clearly depicted by the dynamic GIRchanges, the values reached in Wistar-treated rats weresimilar to those of the untreated Lou/C group, while theb3 agonist treatment further increased the GIR in Lou/Canimals (Fig. 2C). Glucose uptake by individual insulin-sensitive tissues was then assessed, using the 2-deoxyglucosetechnique (20–22). The treatment had no impact on skeletalmuscle glucose uptake in both groups (Fig. 2D). It resulted insimilar two- to threefold increases in glucose uptake in BATof Wistar and Lou/C rats (Fig. 2D). Interestingly, glucoseuptake by different WAT depots was not significantlymodified in Wistar but was markedly increased in Lou/Canimals. This was observed in abdominal WAT (epididymalWAT [eWAT] and retroperitoneal WAT) and in the iWATfat depot (Fig. 2D).

To try understanding the mechanisms underlying the b3agonist treatment–induced increases in BAT and WATglucose uptake, and in view of the well-known effect ofCL-316243 on UCP1 (23), we measured the expression of thisprotein in these tissues. Considering BAT, hematoxylin-eosin

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staining revealed less lipid inclusions and therefore acti-vated tissue in the Wistar and the Lou/C treated groups(Fig. 3A). Accordingly, Ucp1 mRNA expression was in-creased by fourfold by the treatment in the two groups(Fig. 3B). These results were confirmed by Western blotanalysis (Fig. 3C). The treatment also had similar effects

in Wistar and Lou/C rats regarding the expression of theAdrb3, which was markedly inhibited (Fig. 3B).

Subsequently, BAT activity was evaluated by PET scananalysis, using [18F]-FDG. Figure 3D shows micro-CT, PETscan, and merged micro-CT and PET scan images of onerepresentative animal per group. PET scan allowed for the

Figure 1—CL-316243 decreases fat mass in both Wistar and Lou/C rats. A: Total body weight gain of Wistar and Lou/C rats measured after14 days of CL-316243 treatment 1 mg/kg/day (CL) or saline solution (NaCl), total food intake measured after 9 days, and food efficiencycalculated from Dbody weight gain divided by total food intake at 9 days. B: Determination of the percent lean and fat mass repartitionusing MRI. C: Determination of the volume of various fat depots by CT scan. Results are means 6 SEM of 8 experiments per group.Statistical significance assessed by two-way ANOVA: #strain effect; @treatment effect; $interaction between strain and treatment.*Significant difference by post hoc pairwise comparison between different conditions. Single, double, and triple symbols indicate P < 0.05,P < 0.01, and P < 0.001, respectively.

Table 1—CL-316243 improves metabolic parameters

Wistar Lou/C

NaCl CL-316243 NaCl CL-316243

Glycemia (mmol/L) 7.6 6 0.3 6.6 6 0.2†† 6.2 6 0.2 5.5 6 0.2‡

Insulin (ng/mL) 2.06 6 0.50 0.71 6 0.15†† 0.54 6 0.09 0.40 6 0.04

Leptin (ng/mL) 3.80 6 0.19 2.37 6 0.22††† 1.44 6 0.21 0.98 6 0.12

TG (mg/mL) 1.49 6 0.13 0.91 6 0.09††† 0.88 6 0.08 0.54 6 0.03‡

FFA (mmol/L) 0.25 6 0.02 0.23 6 0.01 0.42 6 0.05 0.32 6 0.03

Results are means 6 SEM of 8 experiments per group. Plasma was collected from rats receiving NaCl or CL-316243 treatment for 14days starting at the age of 12 weeks. Statistical analyses were performed using two-way ANOVA (Tukey posttest) with ††,0.01,†††,0.001, Wistar NaCl vs. Wistar CL; ‡,0.05 Lou/C NaCl vs. Lou/C CL.


Figure 2—Improvement in insulin sensitivity in Lou/C rats is due to an increase in glucose uptake by WAT. A: DGlycemia during a GTT (1.5g/kg i.p.) after 4 h of fasting; glucose AUC at 120 min. B: Plasma insulin levels during the GTT; insulin AUC after 60 min. C: GIR during (rightpanel) and at the end (left panel) of EHC in Wistar NaCl, Wistar treated with CL-316243 (CL), Lou/C NaCl, and Lou/C treated withCL-316243 rats. D: Tissue-specific insulin-stimulated glucose uptake. eWAT, epididymal WAT; rpWAT, retroperitoneal WAT; Qr, quadricepsred; Qw, quadriceps white. Results are means 6 SEM of 6–9 experiments. Statistical significance assessed by two-way ANOVA: #straineffect; @treatment effect; $interaction between strain and treatment. *Significant difference by post hoc pairwise comparison betweendifferent conditions. Single, double, and triple symbols indicate P < 0.05, P < 0.01, and P < 0.001, respectively. For the dynamic changesof Dglucose and insulinemia during the GTT (A and B, left panels), as well as of GIR as a function of time (C, right panel): *Wistar NaCl vs.Lou/C NaCl; †Wistar NaCl vs. Wistar CL-316243; ‡Lou/C NaCl vs. Lou/C CL-316243; §Wistar CL vs. Lou/C CL-316243. gluc, glucose.


detection of three main BAT depots: at the basis of thebrain (cervical BAT), in the interscapular region, and alongthe vertebral column. As clearly revealed after quantifica-tion of the signals in all the animals studied, the b3 agonisttreatment increased glucose uptake to a similar extent ineach of these three depots in Wistar and Lou/C rats,whereas the signals were absent under standard conditions(Fig. 3D).

In line with our previous observation of measurableUCP1 expression in iWAT of Lou/C, but not of Wistarrats (16,17), we looked at the potential presence ofUCP1-positive beige adipocytes in this tissue after theb3 agonist treatment. Examination of hematoxylin-eosinstaining revealed the presence of multilocular adipocytesin the b3 agonist–treated Lou/C group only (Fig. 4A).Immunofluorescence analysis further showed that these

Figure 3—UCP1 is equally overexpressed in BAT in both Wistar (W) and Lou/C (L) rats by CL-316243. A: Representative hematoxylin-eosinstaining of BAT (7 mm). Scale bars = 50 mm. B: Ucp1 and b3 adrenoreceptor (Adrb3) mRNA expression in BAT. C: Protein expression byWestern blot with relative quantification in BAT. D: Representative sagittal cuts of [18F]-FDG uptake measured by microPET. microCT wasperformed to localize glucose uptake in the tissues; quantification of glucose uptake in interscapular BAT (iBAT) (29), cervical BAT (cBAT),and paravertebral BAT (pvBAT) in control and CL-316243–treated Wistar and Lou/C rats. Results are means 6 SEM of 4–8 experiments.Statistical significance assessed by two-way ANOVA: @, treatment effect. *Significant difference by post hoc pairwise comparison betweendifferent conditions. Double and triple symbols indicate P < 0.01 and P < 0.001, respectively. CL, CL-316243.


Figure 4—UCP1 is overexpressed in the subcutaneous iWAT depot in Lou/C rats by CL-316243. A: Representative hematoxylin-eosinstaining in iWAT (7 mm). Scale bars = 50 mm. iWAT sections were stained with an anti-UCP1 antibody. Hematoxylin staining was used torecognize structures. The sections were examined by fluorescence microscopy. Red indicates UCP1. B: Ucp1, Adrb3, and Pgc1a mRNAexpression in iWAT. C: UCP1 protein expression measured by Western blot with relative quantification in iWAT. D: Representative axial cutsof [18F]-FDG uptake measured by microPET. microCT was performed to localize glucose uptake in the tissues; quantification of glucoseuptake in iWAT of control and CL-316243 (CL)-treated Wistar and Lou/C rats. SUV, standard uptake value. E: Mitochondrial DNA copynumber in iWAT. DNA copies of mitochondrial complex I (COX1, mtDNA) were normalized to the DNA levels of nuclear RNAseP (nDNA).


multilocular adipocytes expressed UCP1, demonstratingthe presence of beige cells in the iWAT of Lou/C treatedrats (Fig. 4A). In keeping with these results, Ucp1 mRNAexpression was increased a hundred fold in iWAT of Lou/Crats in response to the treatment. It was also induced iniWAT of Wistar treated animals, reaching values measuredin the untreated Lou/C group (Fig. 4B). Western blot anal-ysis clearly showed that UCP1 protein was expressed in theiWAT of the Lou/C treated group only (Fig. 4C). In linewith the results obtained for Ucp1, the expression of Pgc1a(Fig. 4B), of Tbx1 (Supplementary Fig. 1), and of variousmarkers of mitochondrial biogenesis (Supplementary Fig. 1),as well as mitochondrial DNA (Fig. 4E), were markedlyenhanced by the treatment in Lou/C rats (600% increase inthe mtDNA copy number). Next, we measured the activityof iWAT by PET scan coupled with micro-CT and observeda higher glucose uptake in treated Lou/C rats comparedwith the three other groups (Fig. 4D). To substantiatethe existence of the previously proposed link between glu-cose uptake/insulin sensitivity and Ucp1 expression in ad-ipose tissue (24), we looked at the correlation betweenthese two parameters, considering all the animals studied.As shown on Fig. 4F, the data fitted a hyperbolic regressionwith a positive and significant correlation, suggesting thatUCP1-positive beige cells in iWAT may be involved in theimprovement in insulin sensitivity in this tissue. Withregard to the expression of the Adrb3, it was markedlyoverexpressed in iWAT of Lou/C compared with Wistarrats, in which it was barely detectable. The b3 agonisttreatment downregulated the expression of the Adrb3 inthe Lou/C but not in the Wistar group (Fig. 4B).

Given that the insulin-induced glucose uptake wasincreased by the b3 agonist, not only in iWAT, but also inthe eWAT, we examined the potential presence of beigeadipocytes in this depot. Multilocularization of adipocyteswas observed in Lou/C-treated rats only (SupplementaryFig. 2A), in keeping with the induction of a marked Ucp1gene expression (by 160-fold) (Supplementary Fig. 2B). ThemRNA expression of the Adrb3 was low in both Wistar andLou/C rats, with no intergroup difference and no impact ofthe b3 agonist treatment (Supplementary Fig. 2B).

It is well-known that thermoneutrality in rodents isreached at ;28–30°C (24°C for rats and 30°C for mice)and that, although it is the usual temperature used inanimal quarters, 22°C represents a cold stimulus increas-ing UCP1 expression and activity (25). It was thereforepossible that the presence of UCP1-positive beige cells inWAT observed in the Lou/C group without any treatmentwas a consequence of a better temperature sensing thanin Wistar rats. To address this issue, we maintained

groups of Wistar and Lou/C rats at 22°C or at 30°C for4 weeks. At 30°C, both food intake and body weight gainwere reduced in Wistar, as well as in Lou/C rats (Fig. 5A).This resulted in similar food efficiencies in response to thetreatment under the two experimental conditions, withlower values in the Lou/C than in the Wistar groups (Fig.5B). Regarding BAT Ucp1 expression, it was lower in Lou/Cthan in Wistar animals, and it was reduced by theexposure at 30°C in the Wistar group (Fig. 5D). Suchconditions also brought about a decrease in BAT Adrb3expression in Wistar and Lou/C rats (Fig. 5D). In iWAT,Ucp1 expression was doubled in Lou/C compared withWistar at 22°C, although this did not reach statisticalsignificance, and it remained at the same values at 30°C(Fig. 5E). In this tissue, expression of the Adrb3 washigher in Lou/C than in Wistar rats, with no effect ofthe temperature (Fig. 5E). Similar observations weremade for the GIR measured during EHC (Fig. 5C), indi-cating that the improved insulin sensitivity of the Lou/Cgroup is maintained at 30°C and is therefore not linked tocold exposure, even of minor magnitude.

Finally, the expression of genes encoding for enzymesknown to be involved in lipid metabolism was measuredin BAT and iWAT. Acetyl CoA carboxylase represents therate-limiting step in fatty acid (FA) synthesis. Carnitinepalmitoyl transferase-1a is the rate-limiting step of theFA oxidation pathway, mediating FA transport from thecytosol to the mitochondria. Medium- and large-chainacyl-CoA dehydrogenases (MCAD and LCAD) are involvedin FA oxidation. PEPCK allows for the in situ productionof glycerol-3-phosphate (glyceroneogenesis) and thereforefor the formation of TG. Hormone-sensitive lipase (HSL)is one of the main enzymes involved in lipolysis. In iWAT,Acc1 expression was twofold higher in Lou/C than inWistar rats in the absence of b3 agonist treatment. Fur-thermore, it was more than doubled in the Lou/C groupin response to the treatment, while it remained unchangedin Wistar rats (Fig. 6A). As expected from its inhibitoryregulation by malonyl CoA, Cpt1a expression was lower inLou/C than in Wistar animals, with a trend toward a fur-ther decrease in the b3 agonist–treated group (Fig. 6A).On the contrary, the b3 agonist treatment resulted in anenhancement of Hsl, Lcad, and Mcad expression in Lou/Crats, without any change in the Wistar group (Fig. 6A).Similar results were obtained for the expression of Pepck(Fig. 6A).

In BAT, Acc1 expression was similar in Wistar and Lou/Cuntreated rats and it was reduced by the treatment inthe Lou/C group only (Fig. 6B). The expression of Cpt1awas under the detection limit, as a likely consequence of

F: Nonlinear regression with hyperbolic correlation between 2-DG glucose uptake and Ucp1 expression in iWAT. Results are means6 SEMof 8 experiments per group. Statistical significance assessed by two-way ANOVA: #strain effect; @treatment effect; $interaction betweenstrain and treatment. *Significant difference by post hoc pairwise comparison between different conditions. Single and triple symbolsindicate P < 0.05 and P < 0.001, respectively. gluc, glucose.


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Figure 5—Differences between Wistar and Lou/C rats are not due to a difference in thermoneutrality. A: DBody weight (BW) gain of Wistarand Lou/C rats measured between days 4 and 30 of 22°C (control) and 30°C exposure. Total food intake (FI) measured between days 4 and30. B: Food efficiency calculated from Dbody weight gain divided by total food intake between days 4 and 30. C: GIR measured at the endof EHCs in Wistar and Lou/C rats housed at 22°C and at 30°C. D: Ucp1 and Adrb3 mRNA expression in BAT. E: Ucp1 and Adrb3 mRNAexpression in iWAT. Results are means 6 SEM of 8–12 experiments. Statistical significance assessed by two-way ANOVA: #strain effect;@treatment effect; $interaction between strain and treatment. *Significant difference by post hoc pairwise comparison between differentconditions. Double and triple symbols indicate P < 0.01 and P < 0.001, respectively.


elevated Acc1 expression in this tissue. There was nodifference in Hsl expression in Wistar and Lou/C ratswhether treated or not (data not shown). In controlanimals, Pepck was less expressed in the Lou/C comparedwith the Wistar group but it was increased by the treat-ment in Lou/C rats to reach the expression level mea-sured in Wistar rats (Fig. 6B).

DISCUSSION

This study was undertaken to determine the impact ofrecruiting UCP1-expressing adipocytes in a model of obesity

resistance characterized by the presence of beige cells in iWAT(16,17). For this purpose, Lou/C and Wistar rats were treatedfor 2 weeks with a b3 agonist (CL-316243). The treatmenthad no effect on food intake and body weight gain, while itdecreased the fat mass as well as the weight of interscapularBAT and of various WAT depots in the two groups.

In terms of its effects on UCP1 expression, differenttypes of results were obtained in BAT and WAT whencomparing Wistar and Lou/C rats. In BAT, we and otherspreviously reported that Ucp1 mRNA and/or protein levelswere lower in Lou/C than in Wistar rats at 22°C or at 25°C

Figure 6—The higher glucose uptake in iWAT is explained by an increase in FA synthesis in CL-treated Lou/C rats. A: Acc1, Cpt1a, Hsl,Pepck, Lcad, and Mcad mRNA expression in iWAT. B: Acc1 and Pepck mRNA expression in BAT. Results are means 6 SEM of 8experiments per group. Statistical significance assessed by two-way ANOVA: #strain effect; @treatment effect. *Significant differenceby post hoc pairwise comparison between different conditions. Single, double, and triple symbols indicate P < 0.05, P < 0.01, and P <0.001, respectively. AU, arbitrary units; CL, CL, CL-316243; ctrl, control.


(17,26), whereas, in the current study, this was observedfor UCP1 protein at 22°C but not for the Ucp1 gene at 22°Cor 30°C. Regarding the effects of the b3 agonist treatment,it increased Ucp1 and decreased Adrb3 expression in BAT ofboth groups to a similar extent. These results, confirmed bythe measurement of UCP1 protein, closely correlated withthe observation of similar increases in BAT glucose uptakein Wistar and Lou/C rats, as measured during EHC and by[18F]-FDG–PET scan analysis.

UCP1-expressing beige cells are known to be present invarious WAT depots (27,28). Chronic cold exposure wasshown to recruit beige adipocytes in WAT, resulting inWAT “browning” (29,30). Interestingly, resistance to diet-induced obesity in rats and mice has been suggested todepend on the induction of beige adipocyte recruitmentin WAT (16,31). Moreover, such increased recruitment ofbeige cells has been shown to compensate for decreasedBAT thermogenesis, and the specific loss of beige adipo-cytes due to adipose tissue–specific deletion of a transcrip-tional factor involved in Ucp1 expression (32), PRDM16,was shown to cause obesity (32,33). Beige adipocytes arealso strikingly involved in the regulation of glucose metab-olism, as demonstrated by the beneficial effects on glucosetolerance and insulin sensitivity observed in transgenicmice overexpressing PRDM16 in iWAT (32).

In the current study, we showed that the b3 agonisttreatment affected the Lou/C group only, markedly increas-ing UCP1 gene and protein expression, mitochondrial bio-genesis, and the insulin-stimulated glucose uptake measuredduring EHC (subcutaneous and intra-abdominal fat depots)or using [18F]-FDG–PET scan analysis (subcutaneous fat de-pot) as previously reported (34). Furthermore, in iWAT fromLou/C- treated rats, a hyperbolic correlation between glucoseuptake and Ucp1 mRNA expression could be observed. Al-though this does not allow conclusions about cause-effectrelationships, these data suggest that the amount of beigeadipocytes in WAT depots can determine the overall tissueglucose utilization rate. Such conclusion is supported bythe appearance of multilocular beige cells in iWAT ofCL-316243–treated Lou/C rats, as determined by histologicalmeans. Whether these beige cells result from activation ofexisting adipocytes or from transdifferentiation of whiteinto brown cells is an important issue that will need to beaddressed in future work.

As schematized by Fig. 7, the molecular mechanismsunderlying the b3 agonist–induced increase in glucosemetabolism in iWAT of Lou/C rats seem to involve induc-tions of increased FA synthesis (de novo lipogenesis[DNL]) and lipolysis (increased Acc1 and Hsl, respec-tively), as well as of enhanced FA b-oxidation (increasedMcad and Lcad). Additionally, the marked increase in theexpression of Pepck in iWAT of Lou/C treated rats sug-gests the presence of increased glyceroneogenesis. Simi-lar data of gene expression responsible for enzymesinvolved in lipid metabolism in adipose tissues werereported in normal mice treated with CL-316243 for 7days (35).

With regard to the overall insulin-stimulated glucoseutilization, as reflected by the GIR during EHC (under ourexperimental conditions of suppressed hepatic glucoseproduction), we observed that it was similarly increasedby the treatment in Wistar and Lou/C rats. Given thatskeletal muscle glucose utilization was unaltered by thetreatment in the two groups, only BAT and WAT glucoseuptake could be held responsible for the treatment-induced increase in GIR. By considering the total massof BAT and WAT, as well as the mean glucose uptake inthese tissues, it was possible to approximate the percentcontribution of each of these two tissues to the increasedGIR. Thus, in Wistar rats, both BAT (;30%) and WAT(;60%) contributed to the GIR enhancement in responseto the treatment. In contrast, in Lou/C rats, the treatmentdoubled BAT glucose uptake but decreased the BAT massby twofold. Therefore, in these animals, the stimulatoryeffect of the treatment on GIR could only be attributedto increased glucose uptake in WAT depots containingbeige cells. It should, however, be mentioned that thesecalculations may underestimate BAT glucose uptake, asthe reduction in tissue weight may reflect loss of lipidsdue to increased thermogenic activity induced by the b3agonist treatment. These data are in keeping with previouslyreported results showing that chronic stimulation of the

Figure 7—Schematic representation of a beige adipocyte in iWATof b3 agonist–treated Lou/C rats, showing the occurrence of futilecycling between lipolysis and lipogenesis. Higher glucose uptake isconverted to malonyl-CoA by acetyl CoA carboxylase 1 (ACC1),which is markedly increased. Together with hormone-sensitivelipase (HSL), this increases the amount of FA within the cell. TheseFA are used by the mitochondria, as suggested by the increase inthe expression of enzymes involved in b-oxidation (large-chainacyl-CoA dehydrogenase [LCAD] and medium chain acyl-CoA de-hydrogenase [MCAD]), despite a slight decrease in carnitine palmitoyltransferase-1 (CPT1) (likely due to an increase in the productionof malonyl-CoA). Enhanced FA utilization induces the productionof acetyl-CoA, as well as of a proton gradient between the twomitochondrial membranes. Such a gradient is dissipated by UCP1,increasing heat production and decreasing ATP synthesis. Part ofmitochondrial acetyl-CoA can be transformed to oxaloacetate in thecytosol (after its transformation to citrate), where it can be convertedto glycerol-3-phosphate (G3P) via PEPCK (glyceroneogenesis), ulti-mately increasing lipogenesis. Yellow arrows represent lipogenic,whereas green ones represent lipolytic pathways.


sympathetic nervous system stimulates glucose uptakein WAT and BAT without any effect in skeletal muscles(36). The predominant role of beige cells in WAT glucoseuptake is further supported by the previous observationthat the increased glucose utilization under sympatheticstimulation depends on UCP1 activation (37,38).

Altogether, the results of the current study extendother data showing that chronic cold exposure or b3agonist treatment exerts beneficial effects on glucosemetabolism in mice with high-fat diet–induced obesity(rev. in 12) and normalizes hyperglycemia and hyper-triglyceridemia in mouse models of diabetes and dysli-pidemia (39–41). They also extend previous data (42),supporting the notion of interdependency between glu-cose uptake, lipid metabolism, and thermogenesis. Asrecently further discussed (43), a particularly importantrole for the futile cycling between FA synthesis andoxidation linked to thermogenesis and glucose disposalis played by DNL. This may be particularly relevant inbeige cells of WAT depots, in keeping with the obser-vation that WAT is required for the full thermogenicresponse to CL-316243 (44). To go a step further in theunderstanding of the mechanisms involved in theeffects of b3 agonists, several data indicate that DNLis required for the synthesis of several signaling lipidswith systemic effects (“lipokines”) supporting metabolichomeostasis. The presence of these molecules in ourdifferent experimental groups could therefore be of in-terest and will be investigated in the future.

To conclude, induction of DNL coupled with a simulta-neous increase in FA oxidation as occurs during browningof WAT (CL-316243–treated normal mice [35]; CL-316243–treated Lou/C rats [present study]) appears to be importantfor the regulation of glucose homeostasis and may thereforebe of clinical relevance for the treatment of obesity and type2 diabetes in humans.

Acknowledgments. The authors thank Professor Claes Wollheim andProfessor Jean-Paul Giacobino for critical comments on the manuscript.Funding. This study was supported by grants from the Swiss National Foun-dation (Bern, Switzerland), Fonds De La Recherche Scientifique (31003A_134919and 310030_160290), and the 278373 DIABAT Collaborative Project from theEuropean Community (Diabat/FP7_HEALTH_2011_TWO_STAGE). A.-L.P. was re-cipient of a grant from Fondation d’Endocrinologie (Geneva, Switzerland).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. A.-L.P. carried out the animal, qPCR, Western blot,and immunofluorescence studies and drafted the manuscript. A.-L.P., C.V.-D., andF.R.-J. conceived the study, participated in its design and coordination, and draftedthe manuscript. C.V.-D., J.A., A.C., and J.L. helped with the animal treatments. J.A.carried out biostatistical analysis and drafted the manuscript. X.M. performed CTscan and its analysis. D.J.C. performed [18F]-FDG. J.L. participated in qPCR experi-ments. All authors read and approved the final manuscript. F.R.-J. is the guarantorof this work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.Prior Presentation. Parts of this study were presented in abstract form atthe 49th Annual Meeting of the European Association for the Study of Diabetes,Barcelona, Spain, 23–27 September 2013; the 2013 Congress of Société

Francophone du Diabète, Montpellier, France, 26–29 March 2013; the 1st AnnualMeeting of DIABAT, Berlin, Germany, 6 October 2012; the 2nd Annual Meeting ofDIABAT, Zurich, Switzerland, 18 October 2013; the 3rd Annual Meeting of DIABAT,Nice, France, 9 October 2014; and the 21st Meeting of the Association G2L2,Geneva, Switzerland, 12 April 2013.

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