Kenneth J. McCreath,1 Sandra Espada,1 Beatriz G. Gálvez,1 Marina Benito,2,3

Antonio de Molina,4 Pilar Sepúlveda,5 and Ana M. Cervera1,5

Targeted Disruption of theSUCNR1 Metabolic ReceptorLeads to Dichotomous Effects onObesityDiabetes 2015;64:1154–1167 | DOI: 10.2337/db14-0346

A number of metabolites have signaling properties byacting through G-protein–coupled receptors. Succinate,a Krebs cycle intermediate, increases after dysregu-lated energy metabolism and can bind to its cognatereceptor succinate receptor 1 (Sucnr1, or GPR91) to ac-tivate downstream signaling pathways. We show thatSucnr1 is highly expressed in the white adipose tissue(WAT) compartment of mice and regulates adiposemass and glucose homeostasis. Sucnr12/2 mice weregenerated, and weight gain was monitored under basaland nutritional stress (high-fat diet [HFD]) conditions.On chow diet, Sucnr12/2 mice had increased energyexpenditure, were lean with a smaller WAT compart-ment, and had improved glucose buffering. Lipolysismeasurements revealed that Sucnr12/2 mice were re-leased from succinate-induced inhibition of lipolysis,demonstrating a function of Sucnr1 in adipose tissue.Sucnr1 deletion also protected mice from obesity onHFD, but only during the initial period; at later stages,body weight of HFD-fed Sucnr12/2 mice was almostcomparable with wild-type (WT) mice, but WAT contentwas greater. Also, these mice became progressivelyhyperglycemic and failed to secrete insulin, althoughpancreas architecture was similar to WT mice. Thesefindings suggest that Sucnr1 is a sensor for dietaryenergy and raise the interesting possibility that proto-cols to modulate Sucnr1 might have therapeutic utilityin the setting of obesity.

G-protein–coupled receptors (GPCRs) constitute the majorand most diverse group of membrane receptors ineukaryotes (1). Found in the plasma membrane, thesereceptors are tasked with the recognition and transmis-sion of messages from the external environment. An ever-increasing number of GPCRs are now being identified asreceptors for metabolites or energy substrates (2),expanding the repertoire of biological targets to diseasesassociated with the metabolic syndrome, including hyper-tension and type 2 diabetes (3). Metabolites such as lac-tate (4), ketone bodies (5), and a-ketoglutarate (6),known primarily to serve functions distinct to signaling,have been shown to act as ligands for GPCRs. Represen-tative of this new class of receptor is Gpr91, a receptor forthe Krebs cycle intermediate succinate, now termedSucnr1 for succinate receptor (7). The Krebs, or citricacid, cycle occurs at the junction between glycolysis andoxidative phosphorylation (8) and is a key component ofaerobic metabolism by providing reducing equivalents forthe electron transport chain. Interestingly, succinate hasbeen known for many years to increase in tissues duringhypoxia (9–11) and may act as a surrogate marker forreduced oxygenation. Accordingly, Sucnr1 may be acti-vated in times of hypoxic stress such as ischemic injuryin liver (12) or retina (13) or during cardiomyocyte crisis(14). Moreover, dysregulated metabolism, akin to diabetesor the metabolic syndrome, also has the potential to in-crease peripheral blood concentrations of succinate to

1Department of Cardiovascular Development and Repair, Fundación CentroNacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain2Advanced Imaging Unit, Department of Atherothrombosis, Imaging, and Epidemi-ology, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III,Madrid, Spain3CIBER de Enfermedades Respiratorias, Madrid, Spain4Comparative Medicine Unit, Fundación Centro Nacional de Investigaciones Cardio-vasculares Carlos III, Madrid, Spain5Regenerative Medicine and Heart Transplantation Unit, Instituto de InvestigaciónSanitaria La Fe, Valencia, Spain

Corresponding author: Ana M. Cervera, acervera@iislafe.es or amcerveraz@gmail.com.

Received 28 February 2014 and accepted 14 October 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0346/-/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.

1154 Diabetes Volume 64, April 2015

METABOLISM

levels where activation of the Sucnr1 receptor will occur(15,16). Indeed, the low levels (;2–80 mmol/L) of succi-nate found in blood plasma (15–18), in comparison withthat required for activation (in the region of 180 mmol/L[2]), would suggest that Sucnr1 functions more as a sensorof metabolic (or oxidative) damage (19) rather thana physiological mediator of signaling.

Mounting evidence points to an important role forSucnr1 during periods of metabolic dysfunction, such asdiabetes-related hypertension, oxygen-induced retinopa-thy, and the metabolic syndrome (19). Interestingly,obesity is a key component of many metabolic-relateddiseases (20) and can lead to diabetes, hyperlipidemia,and cardiovascular disease (20,21). The effect of Sucnr1deficiency on body composition and metabolism has notpreviously been studied. We reasoned that Sucnr1 couldbe a potential modulator of metabolism since a recentstudy indicated that this receptor participates in thebreakdown (lipolysis) of stored triglycerol (TG) in fat(22). To test this hypothesis, we analyzed the expressionof Sucnr1 in mouse adipose tissue and observed highexpression in the white adipocyte compartment. To gaininsight into the (patho)physiological role of Sucnr1, wecreated mice deficient for this gene using a knockout(KO)-first approach. We show that deficiency of theSucnr1 gene has metabolic consequences for weight gainand glucose homeostasis. Sucnr1 KO mice have a modestlean phenotype under standard diet (SD) conditions, cor-relating with a reduction in the size of white adiposetissue (WAT) depots, increased energy expenditure, andan improved glucose clearance rate. Challenge with high-fat diet (HFD) also resulted in a significant decrease inbody weight in Sucnr1 KO mice, but only during initialperiods. At later periods, body weight of Sucnr1 KO micewas indistinguishable from wild-type (WT) littermates,but WAT content was increased. This appeared to coincidewith a hyperglycemic profile and a reduction in insulinsecretion from the pancreas. Collectively, our resultspoint to a potentially important role for Sucnr1 in meta-bolic disease related with obesity.

RESEARCH DESIGN AND METHODS

Generation of Sucnr1-Mutant Mice, Feeding Regimes,and Indirect CalorimetryTo create Sucnr1-mutant mice, we obtained two Sucnr1-targeted embryonic stem (ES) cell clones from the EuropeanMouse Mutagenesis Consortium (EUCOMM), designated asKO-first, conditional-ready ES cells (23). This flexible systemallows both for gene-trap (GT) reporter constructs and alsofor conditional and null alleles to be generated by exposureto the site-specific recombinases Cre and flippase (www.knockoutmouse.org). ES cells were injected into C57BL/6blastocysts, and chimeric males were bred to C57BL/6females to produce germ line transmission. Heterozygousanimals were intercrossed, and genotyping of F2 progenydetected homozygous mice at the expected Mendelian ratio.Genotyping was performed after weaning by PCR assay

with genomic DNA extracted from tail biopsies using theREDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, SaintLouis, MO). Genotyping primers were primer 1, 59-ACTATAAGCATCACTTCACCACTTCC-39; primer 2, 59-CAACGGGTTCTTCTGTTAGTCC-39; and primer 3, 59-AAGAACAAATGGTAGAACAGTCACGG-39. A PCR product of ;500nucleotides (nt) was obtained from WT DNA, whereasmutant DNA produced an amplification product of;320 nt in size. These KO-first mice were designated GTmice. In order to remove the critical exon of the Sucnr1 gene,together with the neomycin cassette, GT mice were crossedwith Sox2-Cre mice (supplied by Dr. Miguel Torres, CentroNacional de Investigaciones Cardiovasculares [CNIC]) to pro-duce whole-body KO mice. Genotyping was performedas before using the primer 4, 59-ACTTCCAGTTCAACATCAGCCGCTACA-39; primer 5, 59-GTGATTCATCTGTATTATTAGATGAGC-39; and primer 6, 59-CAGCACAACCATCAGAGAAACAATGGACTC-39. A PCR product of ;650 nt indi-cated that recombination had occurred to produce a KOlocus, and a product of ;300 nt identified a WT locus. Allexperiments were carried out using these KO mice.

Mice were housed at the CNIC animal facility underpathogen-free conditions. Male mice were used for allstudies. Mice were given ad libitum access to food andwater unless otherwise noted in experimental methods.Environmental conditions were controlled to 12/12-hlight/dark cycles. Animal studies were approved by thelocal ethics committee. All animal procedures conformedto European Union Directive 86/609/EEC and recommen-dation 2007/526/EC regarding the protection of animalsused for experimental and other scientific purposes,enacted under Spanish law 1201/2005. Male Sucnr1 KOmice and WT mice (5–6 weeks old) obtained from hetero-zygous mating were fed an SD of rodent chow, containing13.4% fat (16% energy; Diet 5K67, LabDiet, Saint Louis,MO) or an HFD containing 34.9% fat (60.9% energy;Diet 58Y1, TestDiet, Saint Louis, MO). Body weightwas recorded weekly. Separate cohorts of animals onSD and HFD were used for metabolic cage analysis usinga 16-chamber indirect calorimetry system (PhenoMaster;TSE Systems, Bad Homburg, Germany). Mice were indi-vidually housed in the chambers for 5 days; the first2 days were used for acclimatization, and data were ana-lyzed from the last 3 days. Oxygen consumption rate,carbon dioxide production rate, feeding, and total loco-motive activity were measured concurrently for eachmouse.

MRI and Spectroscopy AnalysisImaging was performed in the Advanced Imaging Unit atCNIC. Mice on SD or HFD were preanesthetized byinhalation of isoflurane/oxygen (2–4%) and monitored byelectrocardiogram and breathing rhythm (Model 1025; SAInstruments, Inc., NY). Animals were positioned supine ina customized bed with a built-in nose cone supplying inha-latory anesthesia (1–2%) and kept at 35–37°C by a warmairflow. Images were acquired with an Agilent/Varian 7

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Tesla system equipped with a DD2 console and an activeshielded 205/120 gradient insert coil with 130 mT/m max-imum strength and a volume coil (Rapid Biomedical GmbH,Rimpar, Germany) for transmit/receive. For MRI, data werecollected with a two- and three-dimensional (3D) spin-echosequence. T1 weighted two-dimensional images (repetitiontime 380 ms, echo time 13 ms, number of averages 4) wereacquired to visualize the fat qualitatively in high resolution.3D T1 weighted spin-echo acquisition was also acquiredto calculate the body fat–to–total body weight ratio using100 ms repetition time, 8 ms echo time, 1 average. In bothsequences, the field of view was 90 3 45 3 45 mm3 andthe reconstruction matrix was 256 3 256 3 256. ForMRS, the 13C and 1H surface coil was placed over theepididymal fat. 13C spectra were acquired with a repeti-tion time of 1,000 ms, 512 accumulations, and a spectralwidth of 18 kHz. The 1H spectra were acquired with 1,000ms of repetition time, 256 scans, and a spectral width of10 kHz. Images were processed using OsiriX (Pixmeo,Geneva, Switzerland). Quantification of fat compositionfrom 3D images was estimated applying a semiautomaticimage thresholding segmentation plug-in included inthe software and referenced to the total (body) volumescalculated by the same approach. 1H and 13C spectrawere processed with MestReNova (Mestrelab Research,Santiago de Compostela, Spain). An exponential line broad-ening (20 Hz for 13C spectrum and 3 Hz for 1H spectrum)was applied before Fourier transformation. Individualcontributions of the signals from carbonylic, olefinic,methylene, and methyl moieties were quantified by inte-gration after automatic baseline correction. The same pro-cess was applied in 1H spectrum. The areas under eachpeak were used to calculate the fractional contributionof saturated (130 ppm), monounsaturated (130 and128 ppm), and doubly (poly) unsaturated (128 ppm) fattyacids, taking as a reference the area under the carbonylicpeak (24). The areas under the peak in the 1H spectrum,total lipids (1.3 ppm) and water peak (4.7 ppm), were usedto calculate the percentage of total lipids with respect towater content (25).

Ex Vivo Lipolysis AssayMouse epididymal adipose tissue was dissected, weighed,divided into equal pieces, and suspended in 400 mL Krebs-Ringer bicarbonate buffer (Sigma-Aldrich) containing 1%fatty acid-free BSA (Sigma-Aldrich). The tissue sampleswere treated with either PBS or 25 nmol/L isoproterenolor pretreated with 100 mmol/L succinate (for 10 min) or100 ng/mL pertusis toxin for 4 h before isoproterenoltreatment. Samples were incubated on a shaker at 37°C,and glycerol release was measured using a Glycerol Deter-mination kit (Cayman Chemical Company, Ann Arbor, MI).

In Vivo Lipolysis AssayAfter an overnight fast, mice were injected intraperitoneallywith succinate (1.2 mg/kg) or the adenosine A1 receptor ago-nist (R)-N6-(2-phenylisopropyl)-adenosine (PIA; 0.15 nmol/g).

Blood was collected before and 15 min after injection.Serum nonesterified fatty acid (NEFA) was measuredusing the NEFA-HR(2) kit from Wako Chemicals(Richmond, VA).

Tissue Collection and Blood AnalysisAt the end of the study, mice were killed following anovernight fast. Blood samples were collected by cardiacpuncture, and serum was separated by centrifugation.Total serum cholesterol, triglycerides, free fatty acids,hepatic aspartate aminotransferase (AST), and alanineaminotransferase (ALT) were determined using an auto-mated analyzer (Dimension RxL Max Integrated ChemistrySystem; Siemens, Munich, Germany) and commerciallyavailable kits from Siemens (total cholesterol, triglycerides,ALT, and AST) and Wako Chemicals (free fatty acids).Diabetes-related biomarkers in serum were analyzed usingthe Bio-Plex Pro Mouse Diabetes standard 8-plex andadiponectin kits (Bio-Rad Laboratories Inc., Berkeley, CA).Selected tissues were collected and weighed. Tissueswere fixed in neutral buffered formalin solution andprocessed for histological analysis or snap frozen inliquid nitrogen and stored at 280°C for RNA and pro-tein extraction.

Histological AnalysisTissues were fixed in 10% buffered formalin (VWR In-ternational, Radnor, PA), embedded in paraffin, and sec-tioned at 5 mm. For morphometric analysis, sections werestained with hematoxylin and eosin according to standardprotocols. Adipocyte size (from WAT) and islet cell area andcell number (from pancreas) were determined with ImageJsoftware (National Institutes of Health). Sections from sixanimals per group were used, and at least 500 adipocytesper animal were measured from multiple fields. Islet cellarea was quantified from at least four sections per pancreasand expressed as the percentage of total pancreas area. Isletcell density was assessed by counting the number of nucleiin a 1,720 mm2 area from the same sections. Additionalsections were subjected to heat-induced epitope retrievalin citrate-buffer, pH 6, followed by blocking of endogenousperoxidase activity with hydrogen peroxide prior to theimmunostaining. Sections were incubated overnight withantibodies specific for Sucnr1 (Novus Biologicals, Littleton,CO), insulin (Cell Signaling Technology Inc., Danvers, MA),glucagon (Abcam plc, Cambridge, U.K.), Ki67 (Master Diag-nostica, Granada, Spain), or caspase-3 (R&D Systems Inc.,Minneapolis, MN). For Sucnr1 immunodetection, a tyramidesignal amplification kit (Molecular Probes; Thermo FisherScientific, Waltham, MA) was used. For insulin/glucagondouble staining, we used fluorescence-labeled secondaryantibodies. Insulin, Ki67, and caspase-3 were detectedusing an automatic immunostainer (Dako AutostainerPlus; Dako, Glostrup, Denmark) with a horseradish-peroxidase-labeled polymer anti-rabbit as secondaryantibody (EnVision-HRP; Dako) and the chromogenicsubstrate DAB (Dako), followed by counterstainingwith Mayer’s hematoxylin.

1156 SUCNR1 Effects on Obesity Diabetes Volume 64, April 2015

Western Blot AnalysisFor examination of phospho-Akt in WAT, skeletal muscle,and liver samples, fasted mice were injected intraperito-neally with saline or insulin (1.5 units/kg body mass).Extracts were prepared at 10 min postinjection as de-scribed (26). Briefly, tissues were homogenized in 500 mLice-cold lysis buffer (1% Triton X-100, 150 mmol/L NaCl,10 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA, 1% NonidetP-40, and complete mini protease inhibitor cocktail; Roche,Mannheim, Germany) with a mechanical homogenizer, anddebris was cleared by centrifugation at 10,000g at 4°C for10 min. The supernatant was collected and the quantity ofprotein was measured with the BCA kit (Thermo FisherScientific). Protein extracts (40 mg of protein) were exam-ined by protein immunoblot analysis by probing with anti-bodies to Akt and phospho-Ser473 Akt (Cell SignalingTechnology Inc.). Immunocomplexes were detected withthe enhanced chemiluminescence reagent (Amersham, GEHealthcare Bio-Sciences AB, Uppsala, Sweden).

Glucose Tolerance Test and Insulin Tolerance TestFor the intraperitoneal glucose tolerance test (IPGTT), micewere fasted overnight (16–18 h). Conscious mice werethen injected intraperitoneally with glucose (1.5 g/kgbody weight). Blood glucose was monitored in samplesobtained from tail punctures before and after glucoseadministration using a handheld glucometer (AscenciaElite; Bayer AG, Leverkusen, Germany). To measure in-sulin levels in response to glucose, blood was collectedat 0 and 10 min postinjection through the submaxilarvein, and the resulting serum was used to measure in-sulin by ELISA (Millipore Corporation, Billerica, MA).For the intraperitoneal insulin tolerance test (IPITT),mice were fasted for 2 h and injected with insulin(0.75 units/kg).

Flow Cytometry Analysis, Stromal Vascular FractionIsolation, and Preadipocyte DifferentiationThe stromal vascular fraction (SVF) was isolated fromWAT by incubating the tissue for 1 h at 37°C in Krebsbuffer (1 g tissue/3 mL solution) containing 2 mg/mLcollagenase A (Roche Diagnostics, Basel, Switzerland) and20 mg/mL fatty acid–free BSA (Sigma-Aldrich). Digestedtissue was centrifuged at 250g, and the SVF pellet andadipocyte fractions were collected for RNA extraction.For preadipocyte differentiation, the same procedure wasfollowed using subcutaneous WAT. Once the SVF fractionwas collected, SVF cells were differentiated to adipocytes exvivo using a modification of a published protocol (27).Cells were plated (50,000 cells/cm2) and maintained inculture medium (DMEM/F12, 8% FBS, 2 mmol/L L-glutamine,50 units/mL penicillin, and 50 mg/mL streptomycin) for5 days, and differentiation was induced by incubating withculture medium supplemented with 5 mg/mL insulin, 1mg/mL dexamethasone, 25 mg/mL isobutylmethylxanthine,and 1 mmol/L rosiglitazone. After 2 days, cells were incu-bated for an additional 2 days in culture medium containinginsulin and rosiglitazone only and were then maintained in

culture medium supplemented with only insulin for theremaining duration of differentiation (a further 4 days).For flow cytometry analysis of adipocytes, the same en-zyme dissociation procedure was followed for epididymalWAT digestion. Then adipocytes were recovered by cen-trifugation (250g, 10 min, 4°C), collected in 1 mL of ice-cold PBS containing 0.25 mL of Draq5 (BioStatus Limited,Leicestershire, U.K.) to stain the cells, and analyzed ona LSR Fortessa flow cytometer (Becton Dickinson, SanJose, CA). Data were analyzed with FlowJo 1.6 software.

Triglyceride MeasurementHepatic and WAT triglyceride content was measured usingtissue from mice fasted overnight as previously described(28). Briefly, total lipids were extracted from samples(50 mg) by using an 8:1 mixture of chloroform and methanol(4 h). The extracts were mixed with 1 N sulfuric acid andcentrifuged (10 min). Triglyceride content was measuredwith a kit purchased from bioMérieux Inc. (Marcy l’Etoile,France). Oil Red O staining and measurement of intracel-lular triglyceride levels were performed as previously de-scribed (27). Fixed cells or frozen liver sections (10 mm)were stained with Oil Red O (Sigma-Aldrich) for 15–20 minat room temperature. For quantification, cell monolayerswere washed extensively with water to remove unbounddye, and 1 mL of isopropyl alcohol was added to theculture dish. After 5 min, the absorbance (510 nm) ofthe extract was measured by spectrophotometry.

Total Pancreatic Insulin ContentTotal pancreatic insulin content was measured as de-scribed (29). Pancreata were harvested from fed mice,Polytron homogenized (30 s) on ice in 5 mL cold 2 Nacetic acid, boiled for 5 min, transferred to ice, and centri-fuged at 15,000g and 4°C for 15 min. Supernatantextracts were then assayed for protein content and insulincontent (ELISA, Millipore Corporation).

RNA Isolation and Gene Expression AnalysisTotal RNA extraction, cDNA synthesis, and quantifica-tion were performed by standard methods. The expres-sion of mRNA was examined by quantitative reversetranscription–polymerase chain reaction (qRT-PCR) us-ing a 7900 Fast Real Time thermocycler and Fast SYBRGreen assays (Applied Biosystems, Thermo Fisher Scien-tific). Three technical replicates were performed per sam-ple, and relative expression was normalized againstmouse Srp14. Primer sequences are given in Supplemen-tary Table 1.

Statistical AnalysisCalculations were performed using Biogazelle qBase+ andGraphPad Prism 5.0 software (San Diego, CA) and reported asmean 6 SEM. MRI and MRS data were reported as mean 6standard deviation. Statistical comparisons were made usingrepeated-measures ANOVA with Bonferroni posttests forcomparisons between groups at individual time points andStudent t test for independent samples, as appropriate. Sig-nificance was established as a value of P , 0.05.

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RESULTS

Sucnr1 Is Highly Expressed in WATGene transcription analysis of C57BL/6 WT mouse tissuesrevealed high Sucnr1 expression in kidney and liver, withlittle or no expression in lung tissue (Fig. 1A). Theseresults are consistent with previous work demonstratinghigh Sucnr1 expression in kidney medulla (7) and also he-patic stellate cells (12). Interestingly, significant expressionwas also detected in WAT from epididymal fat (Fig. 1A),ratifying the recent grouping of this gene to an adiposecluster in the adult mouse (22). Analysis of fractionatedWAT demonstrated Sucnr1 expression in the mature adi-pocyte but not the stromal cell fraction, which containspreadipocytes (Fig. 1B). In contrast to WAT, Sucnr1 ex-pression was minimal from brown adipose tissue (BAT)deposits (Fig. 1C).

Generation of Sucnr1GT/GT and Sucnr12/2 MiceTo investigate the in vivo function of Sucnr1, we generatedSucnr1-mutant mice by using KO-first targeted ES cell clonesobtained from EUCOMM. The targeting vector, directed tointron 1 of the Sucnr1 gene (Fig. 2A), contained a spliceacceptor (SA) module from the engrailed-2 (En2) gene, fol-lowed by an internal ribosome entry site, which directstranslation of a fusion reporter protein with b-galactosidase(LacZ), which, in turn, is dependent on transcription of theendogenous Sucnr1 promoter. A separate transcription unitcontained a promoter-driven neomycin gene for antibioticselection. These two modules were flanked by flippase rec-ognition target sites, permitting flippase-mediated recombi-nation. Additionally, exon 2 of the gene, which was includedin the targeting vector, was flanked by LoxP sites, allowingconditional ablation of the exon following Cre-mediated re-combination (30). A third LoxP site bordered the neomycincassette (Fig. 2A), ensuring its removal with exon 2. Thus,depending on mating regimes, either whole-body KO miceor conditional KOs can be produced (23). In this work, wedescribe only results from the GT and whole-body KO mice.

Two targeted ES cells clones were injected into mouseblastocysts, and germ line transmission was confirmed

after mating. All mice were on a C57BL/6 background.Mouse genotypes were evaluated by PCR using genomicDNA from tail biopsies, with primers designed to detectcorrectly targeted events (Fig. 2B, primers 1, 2, and 3).Homozygous offspring from heterozygous Sucnr1GT/+

male and female intercrosses were viable and exhibitednormal development. Whole-body KO mice, distinctfrom GT mice by the complete removal of exon 2, wereproduced following a crossing regimen with Sox2-Cremice. Removal of exon 2 and the neomycin module wasconfirmed by PCR using primers 4, 5, and 6, giving theexpected amplification products for WT and KO (Fig. 2C).Because intronic insertions of GT vectors may allow nor-mal mRNA splicing to some degree, we analyzed endoge-nous Sucnr1 levels in GT mice to determine whetherresidual transcripts were expressed. Using primers e1and e2, which bridge exons 1–2 (Fig. 2A), PCR amplifica-tion of cDNA from different tissues of WT mice yieldeda single product with the expected size of ;190 nt(Fig. 2D). Unexpectedly, amplification of cDNA fromSucnr1GT/GT mice with the same primer pair yieldeda larger single product of 300 nt, with no detectable WTSucnr1 expression (Fig. 2D, left panel) suggesting that theSA is not skipped. Introducing an internal ribosome entrysite–specific reverse PCR primer (p7), together with primere1, resulted in the amplification of an intermediate-sizedfragment (220 nt), consistent with the proper functioningof the SA module (Fig. 2D, middle panel). PCR across exons1–2 in Sucnr1KO/KO (Sucnr1 KO) mice failed to amplifya reaction product, consistent with the loss of exon 2(Fig. 2D, right panel). To determine the origin of the un-anticipated 300 nt product in Sucnr1GT/GT mice, and ex-clude the possibility of PCR artifacts, reactions wereperformed on mRNA from different tissues of mice, andthe products were sequenced. Results from sequencing de-termined precisely in all cases that the fusion transcriptgenerated by the proper joining of exon 1 to the SA se-quence had caused activation of a cryptic splice donor sitelocated 115 nt downstream in the En2 sequence, whichspliced to the SA of Sucnr1-exon 2 (Fig. 2E). This splicing

Figure 1—Sucnr1 tissue distribution. A: Relative expression of Sucnr1 in mouse tissues, determined by qRT-PCR. Expression levels (log10)were relative to that in lung tissue arbitrarily chosen as 1 (n = 3 mice). B: Relative expression of Sucnr1 in the SVF and adipocytes isolatedfrom epididymal adipose tissue, examined by qRT-PCR. Adiponectin and macrophage scavenger receptor 1 served as positive controls foradipocyte and stromal fractions, respectively (n = 4 mice). C: Relative expression of Sucnr1 in WAT and BAT compartments, examined byqRT-PCR (n = 3 mice). Data represent mean 6 SEM. ADIP, adipocytes; Adp, adiponectin; H, heart; K, kidney; Li, liver; Lu, lung; Msr1,macrophage scavenger receptor 1.

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resulted in a frameshift with premature stop codons withinexon 2, predicting termination of a truncated Sucnr1 trans-lation product. Curiously, this cryptic splice element in theEn2 gene has been previously described in the literature(31,32), and it seems rather puzzling that a large-scale ge-netic KO platform would use the En2-SA unit with a docu-mented alternative splicing phenomenon. Because of thisincongruity, only KO mice were characterized further.

In accord with mRNA expression, strong and homog-enous b-galactosidase staining was observed throughoutWAT from KO mice but not WT littermates (Fig. 2F, toppanels), indicating robust b-galactosidase activity in this tis-sue driven by the endogenous Sucnr1 promoter. Subse-quently, immunofluorescence analysis of paraffin-embeddedWAT sections revealed that Sucnr1 is present in the plasmamembrane of WT adipocytes but is not detected in equiva-lent samples of KO mice (Fig. 2F, bottom panels). Collec-tively, these results identify the white adipocyte as asignificant reservoir of Sucnr1 expression and suggest thatit might have a functional role in adipocyte metabolism.

Sucnr1 Deficiency Results in Release From Succinate-Induced Inhibition of LipolysisHydrolysis of fat stored as TG in adipose tissue, termedlipolysis, is tightly controlled by hormones and metabo-lites that are secreted according to nutritional status (33).Enzymatic breakdown of TG occurs through a series ofsteps (34) to release one molecule of glycerol and threemolecules of fatty acid. Released fatty acids enter thecirculation and can be taken up and oxidized by peripheraltissues. Interestingly, succinate has been shown to inhibitlipolysis in isolated adipose tissue, presumably throughengagement with Sucnr1. To investigate whether lipolysisis perturbed in Sucnr1 KO mice, we dissected WAT fromepididymal fat regions and measured lipolytic activity inexplant tissue. Glycerol release, as a metric of lipolysis,was similar in WT and Sucnr1 KO mice, both under basaland b-adrenergic agonist (isoproterenol)-stimulated con-ditions (Fig. 3A). In accord with previous findings (22),addition of succinate (100 mmol/L) induced an inhibitionof isoproterenol-stimulated glycerol release in WAT ex-plants from WT mice, and pretreatment of adipocyteswith pertussis toxin blocked the antilipolytic effect ofsuccinate (Fig. 3B). As anticipated, inhibition of lipolysis bysuccinate was absent in equivalent WAT from Sucnr1 KOmice (Fig. 3C). Moreover, intraperitoneal injection ofsuccinate (1.2 g/kg) provoked a significant decrease inserum levels of NEFA in WT mice, whereas the decreasein Sucnr1 KO mice was not significant (Fig. 3D). Further,intraperitoneal injection of the adenosine A1 receptor ag-onist PIA could inhibit lipolysis both in WT and Sucnr1KO mice (Fig. 3E), demonstrating that the response togeneral GPCR-mediated antilipolysis was not perturbedin Sucnr1 KO mice. Taken together, these results establishthat succinate exerts its antilipolytic actions throughSucnr1 coupled to Gi-type G proteins.

Disruption of Sucnr1 Results in a Moderately LeanPhenotype With Reduced WAT CompartmentsGiven that expression of Sucnr1 in WAT alluded to aninvolvement in energy homeostasis, we assessed weightgain in WT and Sucnr1 KO mice on an SD. Total bodyweight did not differ at weaning (13.16 1.1 g for male WTvs. 13.2 6 0.6 g for male KO; n = 8), and both genotypesdisplayed similar weight gains, although a discernibletrend for decreased weight gain in Sucnr1 KO mice was

Figure 2—Generation of Sucnr1-deficient mice. A: Diagram of theEUCOMM targeting vector including the LoxP-flanked exon andSucnr1 WT locus. Primers for genotyping correct targeting eventsare shown by large arrows (1–7); exon-spanning primers are shownby small arrows. Black boxes represent exons. B and C: PCR anal-ysis of GT, KO, and WT alleles. D: Amplification of exons frommRNA produced from liver, kidney, and WAT of WT, GT, and KOmice. Larger product amplified with primers e1+e2 was extractedfor sequencing analysis. E: Schematic representation of alternativesplicing around the KO-first cassette. Correct splicing is denoted bya solid line; the alternative transcript is denoted by a broken line (notto scale). F: Representative b-galactosidase staining of intact adi-pose tissue from WT and KO mice (2003 magnification) demon-strating high LacZ expression in KO mice, which was absent in WTmice. WAT was counterstained with nuclear fast red; immunohisto-chemical staining of WAT (2003 magnification) with an antibody toSUCNR1 confirms its expression in WT but not KO tissue. ACT,actin; FRT, flippase recognition target; IR, internal ribosome entrysite; NEO, neomycin.

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apparent from 10 weeks of diet (Fig. 3F), which was sig-nificant when measured at 50 weeks (41.6 6 1.5 g for WTvs. 35.5 6 1.9 g for Sucnr1 KO mice; P , 0.05) (Fig. 3G).Analysis of body composition was performed by dissec-tion of organs and fat pads, nuclear MRI (NMRI), andMRS. After 18–20 weeks on the diet, mice were killed andorgan weights were measured. No differences betweengenotypes were found in the weights of heart, spleen, liver,and BAT at the end of the study (not shown); however,evident differences were found in the WAT depots. Accord-ingly, retroperitoneal, subcutaneous, and epididymal fatmass was significantly reduced in Sucnr1 KO mice relative

to WT littermates (Fig. 3H). Further, compared with totalbody weight, cumulative fat content was significantly re-duced in Sucnr1 KO animals (Fig. 3I). Visual inspection ofNMRI images of WT and Sucnr1 KO animals suggesteda greater percentage of abdominal fat mass in WT animalsrelative to Sucnr1 KO littermates (Fig. 3J), and quantifica-tion analysis of axial slices demonstrated a trend for reduc-tion (Fig. 3J). In the same experimental setting, 13C-NMRspectra of glycerides were recorded over the entire abdom-inal region (Supplementary Fig. 1A). Quantification of thespectra (24) showed that the fractional contribution ofsaturated fatty acids to the total pool of glycerides was

Figure 3—Lipolysis, body weight measurement, fat distribution, and adipocyte content of Sucnr1-deficient mice A: Measurement oflipolysis in WAT explants from Sucnr1-deficient mice (KO) and WT littermates. Lipolysis was determined by measuring glycerol releasedfrom adipocytes in the absence (–) or presence (+) of 25 nmol/L isoproterenol for 4 h (n = 4 mice per group). Results are expressed as mgglycerol per liter released per mg tissue. B: WAT explants of WT mice were pretreated or not for 4 h with pertussis toxin (100 ng/mL21) priorto the addition of succinate (100 mmol/L), followed 10 min later by isoproterenol (25 nmol/L). Lipolysis was determined by glycerol releaseas in A. A representative experiment from four is shown. C: Lipolysis was measured in WAT explants from Sucnr1-deficient and WTlittermates. Explants were incubated with isoproterenol (25 nmol/L) plus/minus succinate (100 mmol/L), and glycerol release was deter-mined after incubation for 3 h. Results show the percentage of activity with succinate (n = 4 mice per group). D: Fasted mice were injectedintraperitoneally with succinate (1.2 g/kg). Serum NEFA was measured before and 15 min after injection (n = 6 mice per group). E: Fastedmice were injected intraperitoneally with PIA (0.15 nmol/g). Serum NEFA was measured before and 15 min after injection (n = 6 mice pergroup). F: Body weight changes in male Sucnr1-deficient mice (KO) and WT littermates fed an SD for 16 weeks (n = 8–12 mice per group).G: Body weights of male Sucnr1-deficient mice (KO) and WT littermates fed an SD for 50 weeks (n = 8–12 mice per group). H: WAT weightmeasurements after 20 weeks on diet (n = 8–12 per group). I: Total fat weight taken as a percentage of body weight after 20 weeks on diet(n = 8–12 per group). J: NMRI analysis of body fat content and quantification of several measurements (n = 7 mice per group). Results wereexpressed as proportion of fat volumes vs. total mouse volume (fat volume/total volume). K: Lipid quantification. 1H spectra of abdominalcompartments from WT and Sucnr1 KO mice (n = 7 mice per group). Areas under the curve were used to calculate the portion of lipids withrespect to water content. L: Representative hematoxylin and eosin–stained sections (2003magnification) of paraffin-embedded epididymalWAT from mice after 18 weeks of diet and average adipocyte size (n = 8 mice per group). M: Flow cytometry analysis of epididymaladipocytes (n = 6 mice per group). N: Triglyceride (TG) content in epididymal fat from SD-fed mice (n = 6 mice per group). O: Relativeexpression of adipogenesis genes in epididymal WAT after 18 weeks on SD, determined by qRT-PCR analysis: Pparg, Cebpa, and Fabp4(n = 6 mice per group). P: Representative images of Oil Red O–stained adipocytes differentiated from stromal vascular cells collected fromWT and Sucnr1-deleted mice and absorbance measurement (510 nm) of Oil Red O staining (n = 4 mice per group). Q: Relative expression ofadipogenesis genes in differentiated adipocytes from SD-fed mice, determined by qRT-PCR analysis; Pparg, Pref1, and Srebp1c (n = 4mice per group). Data represent mean 6 SEM. Body weight analysis was assessed by repeated-measures ANOVA. *P < 0.05; **P < 0.01;***P < 0.001. epi, epididymal; FSC-A, forward scatter-A; iso, isoproterenol; OD, optical density; PTX, pertusis toxin; ret, retroperitoneal;sub, subcutaneous; succ, succinate.

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significantly increased in Sucnr1 KO animals (0.583 60.292 vs. 0.481 6 0.189; P , 0.05 for Sucnr1 KO vs.WT, respectively), whereas monounsaturated and polyun-saturated fatty acids were comparable between genotypes.Finally, 1H-MRS were also acquired from the abdominalregion to assess lipid content (25). As depicted in repre-sentative spectra of mice, the ratio of water to fat contentwas altered in Sucnr1 KO mice relative to control litter-mates (Supplementary Fig. 1B), and area under the curvemeasurements revealed a significant decrease in fat contentin Sucnr1 KO mice (Fig. 3K). Collectively, these results areconsistent with the fat weight analysis and strongly suggestthat Sucnr1 KO mice have a reduced WAT content.

In agreement with the decreased size of the differentanatomical WAT compartments in SD-fed Sucnr1 KO mice,histological analysis of hematoxylin and eosin–stainedWAT established a significant decrease in adipocyte sizerelative to WT mice (Fig. 3L). Further, flow cytometry anal-ysis of disaggregated adipocytes from epididymal WATrevealed a significant decrease in adipocyte size, measuredas forward scatter (Fig. 3M). These changes were mirroredby a reduction in the concentration of adipose triglycerides(Fig. 3N). As the decrease in adipose mass in Sucnr1 KOmice can result from a reduction in adipocyte size and/ordecreased differentiation, we examined the expression of

genes related to adipocyte differentiation. Although expres-sion levels of the adipogenic transcription factors, peroxi-some proliferator-activated receptor g (Pparg) and CCAAT/enhancer-binding protein a (Cebpa), together with the latedifferentiation marker fatty acid binding protein 4 (Fabp4),tended to be slightly lower in WAT taken from Sucnr1 KOmice, this decrease was not significant (Fig. 3O). Moreover,the differentiation ability of preadipocytes extracted fromthe SVF of adult mice was comparable between bothgroups, as measured by Oil Red O staining of neutral lipids(Fig. 3P) and by expression of the adipocyte marker genesPparg, protein delta homolog-1 (Pref1), and SREBP-1c(Srebp1c), although a nonsignificant increase in Ppargand Srebp1c expression was observed in adipocytes fromSucnr1 KO mice (Fig. 3Q). These results, taken togetherwith the findings of low levels of Sucnr1 expression in SVF,a reduction in adipose tissue mass and adipocyte size, anddecreased lipid content, collectively suggest that Sucnr1 de-letion does not alter adipogenesis but rather results indecreased lipid accumulation and smaller adipocytes.

To further evaluate the metabolic changes caused bySucnr1 deletion, we measured energy expenditure by in-direct calorimetry. Interestingly, the rate of VO2 con-sumption in the Sucnr1 KO group was consistentlyhigher than WT counterparts (Fig. 4A). The respiratory

Figure 4—Indirect calorimetry and UCP expression. Age-matched mice after 20 weeks of SD were individually placed in metabolic cages(PhenoMaster) and allowed to acclimatize for 48 h before readings were taken (n = 7–8 mice per group). A: Mean whole-body oxygenconsumption rate. B: RER (VCO2/VO2). C: Mean food intake (g). D: Mean energy expenditure (kcal$h21$kg21). E: Mean RER over dark andlight cycles. F: Mean food intake over dark and light cycles. G: Mean energy expenditure over dark and light cycles. H: Total ambulatoryactivity (sum of horizontal and vertical counts) over dark and light cycles. Measurements were recorded on a cycle of dark (8:00 P.M. to 8:00A.M.) and light (8:00 A.M. to 8:00 P.M.). I: Relative expression of UCP1, UCP2, and UCP3 in tissues (n = 5–7 per group). Data represent mean6SEM. *P < 0.05; **P < 0.01; ***P < 0.001. D, dark; EE, energy expenditure; L, light; SM, skeletal muscle.

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exchange ratio (RER; VO2/VCO2) of Sucnr1 KO mice wasalso significantly higher than that of WT mice during thedark cycle (Fig. 4B and E), indicating that perhaps less tissuefat is available for oxidation in these mice. Alternatively,the increased RER values may suggest an increase inwhole-body utilization of carbohydrates and proteinover lipids. Surprisingly, food intake was increased inSucnr1 KO mice compared with WT (Fig. 4C and F),and energy expenditure was greater during the dark cycle(Fig. 4D and G), with no change in locomotor activitybetween the two genotypes (Fig. 4H). This increase infood intake together with the reduction in fat mass couldbe due to evident increases in energy expenditure. Thoughwe were unable to find differences in BAT expansion be-tween WT and Sucnr1 KO mice, dysregulated energy ex-penditure often results in overexpression of uncouplingproteins (UCPs) to dissipate energy. However, analysis ofUCP expression revealed comparable levels of UCP2 andUCP3 in skeletal muscle and liver (Fig. 4I). Interestingly,although changes did not reach significance, there wasa trend for lower UCP1 expression in BAT tissue of Sucnr1KO mice, together with elevated levels of UCP1 and UCP2expression in WAT tissue (Fig. 4I), suggesting perhapscompensatory mechanisms. Collectively, these results sug-gest that deletion of Sucnr1 results in substantial altera-tions in energy expenditure and disposition.

HFD Feeding Leads to Increased Fat Deposition inSucnr1 KO Mice and Hepatic SteatosisWe next challenged mice with HFD to provoke obesity.HFD feeding led to increased weight in both genotypes;however, Sucnr1 KO mice gained weight at a slower ratethan WT littermates, which was significant after only;3 weeks of feeding (25.7 6 0.3 vs. 28.6 6 0.5 g; P =0.0006). Variance in body weight widened until week 14when differences became less pronounced and weightcurves were superimposable (Fig. 5A). Predictably, ani-mals on HFD were substantially heavier than SD-fed ani-mals at the end of the study (compare Fig. 5A and Fig.3F), and this correlated with an increase in the weight ofWAT and BAT compartments and also liver. Surprisingly,although Sucnr1 KO mice on HFD were generally leanerthan WT counterparts, the weight of WAT stores at theend of the study was significantly increased (Fig. 5B),whereas adipocyte size was comparable (SupplementaryFig. 2). This finding was in contrast to that observed un-der SD, where Sucnr1 KO mice had significantly less WAT(Fig. 3H and I). In a second cohort of HFD-fed mice usedfor NMRI analysis, body weights of Sucnr1 KO mice after20 weeks on diet were found to be significantly lower thanequivalent WT mice (Fig. 5C), in keeping with the overallobservations. Although no differences were found in theweight of different anatomical WAT compartments be-tween groups in this cohort (Fig. 5D), the proportion offat as a percentage of total body weight was significantlyhigher in Sucnr1 KO animals, as determined by measure-ment of combined fat depots (Fig. 5E). Inspection of

NMRI images indicated comparable abdominal fat volumein both groups (Fig. 5F), and quantitative assessment offat from axial slices revealed no differences (Fig. 5F). Amore accurate quantification was carried out using1H-MRS to determine lipid content. In this case, area underthe curve measurements revealed a significant increase infat content in Sucnr1 KO mice (Fig. 5G), in keeping withthe findings from the first cohort.

Results from indirect calorimetry at 8 weeks on HFDrevealed no significant differences in VO2 consumption orRER (Fig. 5H) in Sucnr1 KO mice compared with WTcontrol, although ingestion of the HFD reduced RER val-ues in both groups relative to SD. Again, total food con-sumption was modestly increased in Sucnr1 KO mice,although this did not reach significance (Fig. 5H). Further-more, locomotor activity was not significantly differentbetween groups (data not shown). These results indicatedthat the initial slower rate of weight gain in Sucnr1 KOmice under HFD was not attributable to alterations inenergy expenditure, food intake, or activity.

Changes in adipose mass were reflected in alterationsof adipokine levels in serum of animals. Compared withWT littermates, the reduced quantity of adipose tissue inSucnr1 KO mice under SD was paralleled by decreasedcirculating levels of the obesity indicator leptin (Fig. 5I).This finding is in keeping with the increased feeding ofSucnr1 KO animals on SD (Fig. 4C and F) and the satietyeffect of this hormone (35). In contrast, HFD feedinginduced a .10-fold increase in serum leptin, which wasnot significantly different between Sucnr1 KO and WTmice (Fig. 5I). In addition, a small but significant decreasein serum levels of the insulin-sensitizing hormone adipo-nectin was observed for Sucnr1 KO mice under both di-etary regimes (Fig. 5I). However, serum levels of theadipokines resistin and plasminogen activator inhibitor-1, although increased in HFD, were not statistically dif-ferent between genotypes (Fig. 5I). Obesity in humansand animal models is often associated with increased cir-culating lipids and ectopic lipid accumulation in nonadi-pose tissues including liver (36). Consistent with thisphenotype, both fasted serum levels of NEFAs and cho-lesterol were elevated in WT mice and Sucnr1 KO mice fedHFD for 18 weeks, whereas TG concentrations remainedin the normal range (Fig. 5J). In accord with the measuredlipolytic rates in explants (Fig. 3A), no differences inplasma parameters were observed between genotypes. Al-though no morphologic alterations were found in liverbetween groups fed an SD, hepatic steatosis was inducedin both genotypes under HFD, evidenced by hematoxylinand eosin staining of abundant vacuolar vesicles in liversections, which were confirmed as lipid droplets by OilRed O staining (Fig. 5K). Additionally, hepatic triglyceridecontent in both genotypes was significantly increased un-der HFD, consistent with the fatty changes to liver (Fig. 5L);however, no significant differences in liver TG content werefound between Sucnr1 KO and WT mice under either diet.Further, HFD levels of AST and ALT were found to be

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significantly increased in Sucnr1 KO mice compared withWT mice, suggesting increased hepatocyte damage (Fig. 5M);the origin of these differences is not known. Consistent withthe increased lipid load in liver, expression analysis revealedchanges in relevant genes (Supplementary Fig. 3), includingthe lipogeneic transcription factor sterol regulatory bindingprotein 1c (Srebp1c), and its target stearyol CoA desaturase-1(Scd1), which were induced under HFD in both genotypes;however, other genes involved in triglyceride synthesis, fatty

acid synthase (Fas) and stearyol CoA desaturase (Acc),were unaltered in HFD-fed mice. Similarly, expression ofthe fatty acid transporter CD36 was upregulated underHFD in both genotypes, although expression was signifi-cantly reduced in Sucnr1 KO mice compared with WTlittermates on SD. Additionally, a modest but significantincrease in the expression of a second fatty acid trans-porter, Fabp4, was found in Sucnr1 KO but not WT miceunder HFD diet, whereas expression of the fatty acid

Figure 5—Analysis of mice under HFD. A: Body weight changes in male Sucnr1-deficient mice (KO) and WT littermates fed HFD for 16weeks (n = 8–12 mice per group). B: Organ weight measurements after 18 weeks on diet (n = 8–12 per group). C: Body weight changes inmale Sucnr1-deficient mice (KO) and WT littermates fed HFD for 20 weeks (n = 8 mice per group). D: WAT weight measurements after 20weeks on diet (n = 8 per group). E: Total fat weight taken as a percentage of body weight after 20 weeks on diet (n = 8 per group). F: NMRIanalysis of body fat content and quantification of several measurements (n = 7 mice per group). Results were expressed as proportion of fatvolumes vs. total mouse volume (fat volume/total volume). G: Lipid quantification. 1H spectra of abdominal compartments from WT andSucnr1 KO mice (n = 7 mice per group). Areas under the curve were used to calculate the portion of lipids with respect to water content. H:Indirect calorimetry of age-matched mice after 8 weeks of HFD was performed as in Fig. 3. Mean whole-body oxygen consumption, meanRER over dark and light cycles, and mean food intake over dark and light cycles. I: Serum levels of adipokines measured in fasted mice atend of study (n = 8–12 mice per group). J: Serum levels of NEFAs, triglycerides, and total cholesterol from Sucnr1-deficient mice (KO) andWT littermates after 18 weeks on diets (n = 8–12 mice per group). K: Representative hematoxylin and eosin–stained and Oil Red O sectionsof paraffin-embedded liver taken from WT and KO mice after 18 weeks on diets (1003 magnification). L: Hepatic triglyceride (TG) contentafter 16 h fasting (n = 8–12 mice per group). M: Serum measurement of ALT and AST from Sucnr1-deficient mice (KO) and WT littermatesafter 18 weeks on HFD (n = 8–12 mice per group). Body weight analysis was assessed by repeated-measures ANOVA. Data representmean 6 SEM. *P < 0.05; **P < 0.01; ***P < 0.001. CHOt, total cholesterol; epi, epididymal; ret, retroperitoneal; sub, subcutaneous.

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transport protein 2 (Fatp2) was downregulated in Sucnr1KO mice under HFD. In contrast to these changes, nodifferences were found between genotypes for the expres-sion of genes related to fatty acid oxidation, althoughcarnitine palmitoyltransferase 1a and 1b (Cpt1a andCpt1b) were downregulated under HFD in both geno-types. Collectively, these data indicate that although theabsence of Sucnr1 results in subtle changes to liver func-tion metrics compared with WT mice, HFD appeared tocause comparable steatosis.

Sucnr1 KO Mice Are Hyperglycemic Under HFD WithReduced Insulin SecretionAlthough the metabolic phenotype of Sucnr1 KO miceappeared modest, the evident differences in adiposity andweight gain prompted us to evaluate glucose and insulinhomeostasis. These parameters were measured in miceafter 16 weeks on diet. Compared with SD, HFD led to anincrease in both fasted and fed levels of serum glucose inSucnr1 KO mice and WT littermates (Fig. 6A). Serumglucose was comparable between genotypes in SD; how-ever, despite their similar final body weights in HFD,Sucnr1 KO mice had significantly higher levels of fastingblood glucose, whereas fed glucose was unaltered (Fig. 6A,right panel). Nevertheless, Sucnr1 KO mice showed sim-ilar levels of fasting insulin as those from WT mice inHFD, which were elevated compared with those in SD(Fig. 6B). To directly assess whether glucose homeostasiswas dysregulated in Sucnr1 KO mice, we performed glu-cose tolerance tests and insulin tolerance tests. Interest-ingly, in the SD group, intraperitoneal challenge with1.5 g/kg glucose resulted in faster clearance in Sucnr1KO mice, as glucose concentrations remained significantlylower compared with WT mice (Fig. 6C, left panel). This isin keeping with the general leanness of KO animals andhigher RER values (Fig. 4B and E), and suggested im-proved insulin sensitivity that promotes carbohydrate uti-lization. As anticipated, glucose clearance deteriorated inthe HFD group (Fig. 6D, left panel). However, in markedcontrast to the findings under SD, Sucnr1 KO mice fed anHFD developed a clear impairment in glucose eliminationcompared with WT mice, with manifest hyperglycemia120 min after glucose injection. Notably, this decreasedability to lower blood glucose was not apparent in Sucnr1KO mice at 11 weeks on HFD (Supplementary Fig. 4),suggesting a progressive glucose intolerance concurringwith the increased WAT weight. Abnormal glucose ho-meostasis could result from either a defect in insulin sen-sitivity of the peripheral tissues or a defect in insulinsecretion. Insulin tolerance tests showed that althoughboth genotypes exhibited decreased insulin sensitivity un-der HFD, Sucnr1 KO mice exhibited comparable insulinsensitivity to WT littermates, as demonstrated by a quickdecline in plasma glucose after an acute insulin injection(Fig. 6C and D, right panels). Furthermore, insulin wasshown to induce phosphorylation of the major insulin-signaling protein Akt (on Ser473) in liver, WAT, and skeletal

muscle to similar intensities in Sucnr1 KO and WT mice onHFD (Fig. 6E). These results pointed to a defect in insulinsecretion as the origin of the hyperglycemia in HFD-fed KOmice. To consider this, we repeated the glucose tolerancetest and measured circulating insulin levels at baseline and10 min after a glucose load (1.5 g/kg). Whereas WT miceshowed a characteristic increase in insulin levels at t = 10 min,Sucnr1 KO mice failed to secrete insulin, although GLP-1levels were similar in both genotypes (Fig. 6F). Collec-tively, these results imply that while Sucnr1 KO micehave significantly enhanced glucose tolerance under SD,the HFD causes a sufficient deficiency in insulin secretionto cause glucose intolerance.

Guided by these findings, we examined islet anatomyand endocrine cell size in pancreatic tissue sections ofHFD-fed mice (Fig. 6G). As expected, morphometric anal-ysis revealed that high-fat feeding increased islet cell size,and this increase was comparable in both genotypes(Fig. 6H). Moreover, b-cell number was also similarly in-creased in Sucnr1 KO and WT mice under HFD, comparedwith SD (Fig. 6I). Also, total (acid-extracted) pancreaticinsulin concentration was similar between genotypes onHFD (Fig. 6J). Immunohistochemistry staining with anantibody to insulin showed that the morphologic featuresof WT and KO islets were comparable (Fig. 6K, left pan-els), and detailed analysis of the islets revealed a normalcell architecture shared between genotypes, with positiveimmunostaining for glucagon (a-cells) in the periphery,while the area of anti-insulin staining was observedmostly in the central region (Fig. 6K, right panels). Exam-ination of b-cell proliferation with an antibody to Ki67indicated a similar and low (,1%) frequency of Ki67-positive cells in both WT and KO mice, and very fewapoptotic (caspase-3 positive) b-cells were detected in ei-ther genotype (Fig. 6L). Thus our results are consistentwith an insulin secretory defect in Sucnr1 KO mice.

DISCUSSION

Impaired cellular metabolism is a defining hallmark ofmany disease states and can affect homeostatic signalingprocesses. As the Krebs cycle is the central metabolicpathway in aerobic organisms, it is particularly significantthat succinate, an intermediate metabolite of this path-way, has an alternative occupation as a signaling moleculefor Sucnr1 coupling.

Information regarding the function of Sucnr1 is limited.Previous studies have shown that Sucnr1 is distributed inorgans with high metabolic demand, including the kidneyand liver (19). Signaling of Sucnr1 appears to be an earlyresponse to hyperglycemic conditions in the kidney andleads to activation of the intrarenal renin-angiotensin sys-tem, resulting in hypertension and potential tissue injury(37). Here we show that Sucnr1 is expressed in high levelsin the adipocyte and that loss of Sucnr1 leads to dichoto-mous effects on body weight and metabolism. Althoughnot conspicuously different during early growth, after16–20 weeks on SD (at 21–25 weeks of age) Sucnr1 KO

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mice exhibited a modestly lean phenotype compared withWT mice, with significantly reduced levels of plasma lep-tin and reduced WAT mass containing smaller adipocytesand decreased triglyceride content. This decrease in

adiposity was not associated with ectopic triglyceride stor-age in liver or plasma (Fig. 5) or skeletal muscle (notshown) but correlated with a significant increase in glu-cose tolerance and elevated metabolic activity, including

Figure 6—Sucnr1 KO mice show diet-dependent alterations in glucose homeostasis. A: Plasma levels of fasting (16 h) and fed glucose inSucnr1-deficient mice (KO) and WT littermates under SD (left) and HFD (right) (n = 8–12 mice per group). B: Plasma insulin levels after 16-hfast (n = 8–12 mice per group). Plasma glucose during IPGTT (left) and IPITT (right) in SD (C) and HFD (D) (n = 8–12 mice per group).E: Phosphorylation of Akt (Ser473) in liver, WAT, and skeletal muscle of HFD mice. After an overnight fast, mice were injected with PBS (–)or insulin (+) (1.5 units/kg) for 10 min. F: Serum insulin and GLP-1 concentrations measured from fasted WT and Sucnr1-deficient (KO) miceon HFD, measured after intraperitoneal glucose (1.5 g/kg) injection (n = 8 mice per group). G: Representative hematoxylin and eosin–stainedsections of paraffin-embedded pancreas taken from WT and KO mice after 18 weeks on diets (1003magnification), used to determine isletarea and b-cell number. H: Quantification of islet size measured as a percentage of total pancreas area (n = 6 mice per group).I: Quantification of b-cell density was assessed by counting the number of nuclei in a 1,740-mm2 islet center area (n = 6 mice per group).J: Insulin content from acid-extracted pancreata of HFD-fed mice (n = 12 mice per group). K: Representative insulin immunostaining (403magnification) of pancreas from Sucnr1-deleted mice and WT littermates on HFD (left panels). Immunofluorescence detection (1003magnification) of insulin (green) and glucagon (red) in islets from Sucnr1-deleted mice and WT littermates on HFD (right panels).L: Representative Ki67 and caspase-3 staining of pancreas from Sucnr1-deleted mice and WT littermates on HFD. Islets are denotedby a dotted line, immunopositive cells are marked with an arrowhead. Data represent mean 6 SEM. IPGTT/IPITT analysis was assessed byrepeated-measures ANOVA. *P < 0.05; ***P < 0.001. pAkt, phospho-Akt; prot, protein; S. muscle, skeletal muscle.

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increased energy expenditure, which might partly explainwhy Sucnr1 KO mice were lean despite hyperphagia. How-ever, this phenotype was seen in the setting of normallevels of BAT content and tissue expression of UCPs,which are often elevated in models of increased energyexpenditure (38). Decreased weight gain in Sucnr1 KOmice was also observed in the HFD study, particularlyduring the initial 10 weeks. This finding supports ourobservations on SD and strongly suggests that leannessis a primary outcome of Sucnr1 deletion. Since adipogen-esis was not affected by deletion of Sucnr1 and an anti-lipolytic effect of succinate had been previously described,this metabolically favorable phenotype might be explained,in part, by the release from succinate-inhibited lipolysis.Notably, experiments both in vitro and in vivo verifiedthat succinate inhibited the breakdown of triglycerides,although basal and isoproterenol-induced lipolysis wasunchanged in Sucnr1 KO animals. This phenotype, inpart, is consistent with other mouse models of “unre-strained” lipolysis and shows that release from Gi-coupledinhibition of fat mobilization results in leanness (39–41)and increased energy expenditure (41). After prolongedhigh-fat feeding, Sucnr1 KO mice displayed a rather morecomplex phenotype, presumably a result of nutritionalstress induced by the HFD. Under these conditions,end-of-study body weights of Sucnr1 KO mice were, inthe main, comparable with WT controls, but WAT wassignificantly heavier. Furthermore, after 16 weeks onHFD, Sucnr1 KO mice had significantly greater levels ofblood glucose and failed to secrete insulin in glucose tol-erance tests; however, pancreas architecture was not dif-ferent than in WT mice. Ostensibly, this hyperglycemicstate may have resulted from the failure to buffer serumglucose, although this inference is tempered by the resultthat insulin sensitivity was unchanged in Sucnr1 KO mice.Still, compensatory mechanisms such as neuronal orendocrine modulation may account for this discrepancybetween insulin secretion and resistance.

Although our original hypothesis presupposed thata lean phenotype would result from augmented WATlipolysis, our findings of increased metabolic activity,under basal conditions, in Sucnr1 KO mice points toa more systemic effect of Sucnr1 deletion. Whether thesephenotypes are triggered directly by differences in adiposemass in Sucnr1 KO mice or through a more circuitousroute awaits further study. Nevertheless, adiponectin lev-els, which are typically inversely correlated with obesity,were decreased in Sucnr1 KO mice and might reflect spe-cific changes in adipocytokine signaling (42), leading us tohazard that a hyperglycemic phenotype might occur whenfat content reaches a critical mass.

Many mechanisms can account for leanness, but theyall generally result from deregulation of energy balance, asnoted here. We can speculate on several potentialmechanisms that may promote this phenotype in Sucnr1KO mice (43): hypothalamic signal transduction (44), re-duced intestinal absorption of foods leading to decreased

energy availability (45), enhanced energy metabolism inperipheral tissues, or a defect in adipose tissue storagecapability. It remains to be determined whether one ormore of these mechanisms are operating in our model.

Finally, it should be noted that an underlying conceptin Sucnr1 signaling is hypoxia-induced modulation ofreceptor engagement through local increases in succinatelevels. This is particularly manifest in ischemic retinopa-thy, where microvascular degeneration results in anabnormal hypoxia-driven succinate-induced neovasculari-zation through Sucnr1-dependent upregulation of proan-giogenic factors (13,46). Interestingly, hyperglycemia canalso result in regional increases in succinate (16,46), mostlikely through overactivation of the Krebs cycle (47). In-deed, adipose tissue succinate has been shown to increase(twofold) in mice under HFD (48), and hypoxia has beendetected during expansion of adipose tissue in obesity(49,50), doubtless occurring when vascularization is in-sufficient to maintain oxygen levels. Thus increases insuccinate, hypoxia, and/or hyperglycemia might triggerSucnr1-induced signaling at the level of the adipocyte.Clearly, because Sucnr1 is expressed in organs with highmetabolic demands, tissue-selective KOs will be requiredto identify the specific cell type(s) in which Sucnr1 playsa direct role; nevertheless, our results indicate that acti-vation of Sucnr1 may have therapeutic implications in thetreatment of obesity-related disorders.

Acknowledgments. The authors thank Guadalupe Sabio and TamasRözer for helpful discussions and Roisin Brid Doohan, Ana Belén Ricote, andJesús María Ruiz-Cabello for technical assistance (all from CNIC).Funding. This work was supported in part by a research grant from theMinisterio de Ciencia e Innovación (SAF2009-07965 to K.J.M. and SAF2010-15239 to B.G.G.). The CNIC is supported by Ministerio de Economía y Compet-itividad and the Pro-CNIC Foundation.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. K.J.M. and A.M.C. conceived the project, performedexperiments, interpreted data, and wrote the paper. S.E., B.G.G., M.B., A.d.M., andP.S. performed experiments and interpreted data. A.M.C. is the guarantor of thiswork and, as such, had full access to all the data in the study and takes responsibilityfor the integrity of the data and the accuracy of the data analysis.

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