Michael Joubert,1,2,3 Benoît Jagu,1 David Montaigne,4 Xavier Marechal,4

Angela Tesse,1 Audrey Ayer,1 Lucile Dollet,1 Cédric Le May,1 Gilles Toumaniantz,1

Alain Manrique,3 Flavien Charpentier,1 Bart Staels,4 Jocelyne Magré,1

Bertrand Cariou,5 and Xavier Prieur1

The Sodium–Glucose Cotransporter2 Inhibitor Dapagliflozin PreventsCardiomyopathy in a DiabeticLipodystrophic Mouse ModelDiabetes 2017;66:1030–1040 | DOI: 10.2337/db16-0733

Type 2 diabetes mellitus (T2DM) is a well-recognizedindependent risk factor for heart failure. T2DM is asso-ciated with altered cardiac energy metabolism, leadingto ectopic lipid accumulation and glucose overload, theexact contribution of these two parameters remainingunclear. To provide new insight into the mechanismdriving the development of diabetic cardiomyopathy, westudied a unique model of T2DM: lipodystrophic Bscl22/2

(seipin knockout [SKO]) mice. Echocardiography and car-diac magnetic resonance imaging revealed hypertrophiccardiomyopathy with left ventricular dysfunction in SKOmice, and these two abnormalities were strongly corre-lated with hyperglycemia. Surprisingly, neither intramyo-cardial lipid accumulation nor lipotoxic hallmarks weredetected in SKO mice. [18F]Fludeoxyglucose positronemission tomography showed increased myocardial glu-cose uptake. Consistently, the O-GlcNAcylated proteinlevels were markedly increased in an SKO heart, suggest-ing a glucose overload. To test this hypothesis, we treatedSKO mice with the hypoglycemic sodium–glucose co-transporter 2 (SGLT2) inhibitor dapagliflozin and the insu-lin sensitizer pioglitazone. Both treatments reduced theO-GlcNAcylated protein levels in SKO mice, and dapagli-flozin successfully prevented the development of hy-pertrophic cardiomyopathy. Our data demonstrate thatglucotoxicity by itself can trigger cardiac dysfunction andthat a glucose-lowering agent can correct it. This resultwill contribute to better understanding of the potential car-diovascular benefits of SGLT2 inhibitors.

Type 2 diabetes mellitus (T2DM) is a well-recognizedindependent risk factor for heart failure (HF) (1,2). Whereasthe prevalence of HF in the general population is 1–4%, itreaches ;12% in patients with T2DM (3). In 1972, Rubleret al. (4) reported a specific diabetes-associated cardiac in-jury called diabetic cardiomyopathy. This cardiomyopathy isdefined by ventricular dysfunction occurring without coro-nary disease or hypertension (1,5). Diabetic cardiomyopathyis also characterized by left ventricular (LV) hypertrophy,diastolic dysfunction, and myocardial fibrosis (1,5).

A large body of work indicates that diabetic cardiomy-opathy is associated with altered cardiac energy metabo-lism (6). Indeed, in obese patients with T2DM, heart lipiduptake is increased (7). Several studies support that freefatty acid (FFA) accumulation leads to the increased pro-duction of diacylglycerol (DAG), ceramides, and reactiveoxygen species (ROS), affecting cardiac insulin sensitivity(8,9) and cardiac contractility (6,10,11).

On the other hand, hyperglycemia and glucose overloadhave been involved in cardiac hypertrophy and dysfunctionin the context of T2DM and obesity (12). The diabeticheart is simultaneously characterized by impaired insulin-stimulated glucose uptake (13) and obvious signs of glucoseoverload, such as ROS and advanced glycation end product(AGE) production as well as hexosamine pathway chronicactivation (12,14). Interestingly, when comparing obesepatients with and without diabetes, we previously demon-strated that hyperglycemia per se plays a central role in the

1L’Institut du Thorax, INSERM, CNRS, Université de Nantes, Nantes, France2Endocrinologie, CHU Caen, Caen, France3EA 4650, UNICAEN, GIP Cyceron, Caen, France4Universite Lille, INSERM, CHU Lille, Institut Pasteur de Lille, U1011–EuropeanGenomic Institute for Diabetes, Lille, France5L’Institut du Thorax, INSERM, CNRS, Université de Nantes, CHU Nantes, Nantes,France

Corresponding author: Xavier Prieur, xavier.prieur@univ-nantes.fr.

Received 14 June 2016 and accepted 16 December 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0733/-/DC1.

© 2017 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. More information is available at http://www.diabetesjournals.org/content/license.

1030 Diabetes Volume 66, April 2017

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impaired cardiac mitochondrial activity associated withmyocardial contractile dysfunction (15).

The exact contribution of lipotoxicity and glucotoxicityremains unclear. Most rodent studies have been per-formed in obese animals, and it remains difficult, in thissetting, to distinguish the respective contribution of obe-sity, insulin resistance, and hyperglycemia. To provide newinsight into the mechanism driving the development of dia-betic cardiomyopathy, we aimed to use a unique nonobesemodel of diabetes: the lipodystrophic Bscl22/2 (seipin knock-out [SKO]) mouse model.

Berardinelli-Seip congenital lipodystrophy (BSCL) ischaracterized by an almost complete lack of adipose tissuefrom birth or early infancy. In ;50% of the cases, BSCL isthe result of mutations in BSCL2 encoding the proteinSeipin (16). BSCL is associated with major metabolic com-plications, such as insulin resistance progressing to overtdiabetes, hypertriglyceridemia, and liver steatosis (17). Inaddition, cardiomyopathy frequently occurs in patientswith BSCL (18). Most patients have concentric LV hyper-trophy, equally split into mild, moderate, and severe, of-ten associated with diastolic dysfunction but generallywith preserved systolic function. About half of the pa-tients (54%) display abnormal electrocardiogram (ECG)with prolonged QT intervals and nonspecific T-wave ab-normalities (18). It is not clear yet whether this cardiomyop-athy is associated with ectopic myocardial lipid accumulation(19–21). Thus, the phenotype of lipodystrophic cardiomy-opathy is poorly characterized, and the underlying molec-ular mechanism remains unknown.

We and others (22–24) have previously shown that SKOmice are severely lipodystrophic and display insulin resis-tance, diabetes, and massive liver steatosis. In this study,we performed cardiac phenotyping of the SKO mice, andwe used dapagliflozin treatment, a sodium–glucose cotrans-porter 2 (SGLT2), and the insulin sensitizer pioglitazone toreverse the hyperglycemia that appears to be central to thedevelopment of cardiomyopathy in these animals.

RESEARCH DESIGN AND METHODS

Animals, Physical Activity, and Dapagliflozin TreatmentSKO mice were generated as previously described (24) andhoused at 21°C with a 12:12-h light/dark cycle with freeaccess to food and water. The experimental procedureswere approved by the regional ethics committee (Comitésd’éthique en expérimentation animale, Pays de la Loire,France) according to Directive 2010/63/EU of the Euro-pean Union. For physical activity assessment, the micewere submitted to graduated aerobic exercise training,performed on a motor treadmill (slop 0°) over 4 weeks.Dapagliflozin, an SGLT-2 inhibitor, was obtained fromInterchim (Montluçon, France) and added to the drinkingwater at a concentration of 1 mg/kg for 8 weeks. Piogli-tazone (Interchim) was added to the diet at a concen-tration of 300 mg/kg (45 mg/kg/day) for 8 weeks. Micestarted the treatment at 6 weeks of age. All animals werekilled in a random-fed state.

ECGECGs were performed as previously described (25). Briefly,under isoflurane anesthesia, a six-lead ECG was recordedwith 25-gauge subcutaneous electrodes on a computerthrough an analog-digital converter (IOX 1.585; EMKATechnologies, Paris, France) for monitoring and offlineanalysis (ECG Auto v3.2.0.2; EMKA Technologies).

EchocardiographyMice were anesthetized with isoflurane, and two-dimensionalechocardiography was performed on mice using Vivid 7Dimension ultrasonography (GE Healthcare) with a14-MHz transducer. The Doppler-derived mitral deceler-ation time, the early diastolic (E), late diastolic (A), andE/A ratio were obtained to assess diastolic dysfunction.

Cardiac Magnetic Resonance and Cardiac FunctionPostanalysisCardiac magnetic resonance (CMR) was performed using a7-Tesla Bruker Pharmascan magnetic resonance imager(Bruker Biospin, Ettlingen, Germany) interfaced with adedicated small-animal electrocardiographic and respi-ratory triggering system (SA Instruments). Ventricularfunction and wall deformation were assessed using atriggered cine FLASH sequence and tagged CMR frames,respectively, as previously described (26).

Micro–Positron Emission Tomography ComputedTomographyMice were fasted for 2 h and then anesthetized with isoflurane(1.5–3%) in an oxygen/nitrous oxide mixture (0.6 L/min) viaspontaneous breathing. A 10-min computed tomography scanwas first acquired for subsequent attenuation correction andanatomical identification. Then, a 60-min positron emissiontomography (PET) acquisition was initiated, along with10-s tail vein injection of [18F]fludeoxyglucose (FDG) or[11C]acetate (15–20 MBq in 50–100 mL for both radiolabeledmarkers). For myocardial glucose uptake (MGU; mmol/g/min)and myocardial VO2 (mmol/100 g/min) analysis, [18F]FDGand [11C]acetate PET acquisition data were sorted in 24 and56 dynamic frames, respectively, secondarily analyzed,and quantified with Carimas software (Turku PET Center,Turku, Finland) using Patlak kinetic analysis and a one-tissue compartment model as described elsewhere (27).

In Vivo Lipid Metabolism[3H]VLDL was produced as previously described (24). Wild-type (WT) and SKO mice were anesthetized with a xylazine/ketamine/PBS (1:1:8) solution and received 100,000 cpm[3H]VLDL. The mice were killed, and the hearts were washedin cold PBS 5 min after injection. The tissues were digestedwith Solvable (PerkinElmer, Courtaboeuf, France), and theradioactivity was counted.

Insulin SignalingTwelve-week-old SKO and WT mice were fasted for 2 h, anda single dose of 0.75 IU/kg human recombinant insulin(Umuline rapide; Eli Lilly, Suresnes, France) was administeredby intravenous injection. The mice were killed 5 min afterinjection, and the hearts were collected and washed in PBS.

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Membrane Isolation, Western Blot, ROS Production,and RNA AnalysisMembrane isolation of heart lysates was performed byultracentrifugation (75 min at 110,000 3 g), and the pel-lets were resuspended in solubilization buffer (50 mmol/LTris [pH 7.4], 100 mmol/L NaCl, 50 mmol/L LiCl, 5 mmol/LEDTA, 0.5% Triton X-100, 0.5% deoxycholic acid, and0.05% SDS). For Western blot analyses, we used phospho–Ser473-AKT/AKT, phospho–Thr172–AMP kinase (AMPk)/AMPk, FOXO1, and GLUT1 antibodies from Cell SignalingTechnology; SERCA2, Ser16-P-phospholamban (PLN), andPLN antibodies were from Santa Cruz Biotechnology;GLUT4 and Tubulin antibodies from Sigma-Aldrich; andan O-GlcNAc Western blot kit from Thermo Fisher Scien-tific. For O-GlcNAcylated protein isolation, 750 mg lysateswas incubated with wheat germ agglutinin (Sigma-Aldrich)agarose conjugate and then analyzed by Western blot. TheAGEs were quantified using the oxiSelect AGE competitiveELISA (Cell Biolabs), and ROS production was measuredby incubating heart lysates with dichlorodihydrofluoresceindiacetate (Life Technologies) for 30 min at 37°C as previ-ously described (28). RNA expression was analyzed aspreviously described (24).

Tissue Lipid Extraction and MeasurementTriglyceride (TG) measurement was performed using acolorimetric assay after cyclohexane/isopropanol (3:2, vol-ume for volume) extraction. For ceramide measurement,samples were analyzed using a 1290 UPLC system coupledto a G6460 triple quadripole spectrometer (Agilent Tech-nologies) using MassHunter software for data acquisitionand analysis. For neutral lipids, 1 mL lipid extract wasanalyzed by gas-liquid chromatography on a FOCUSThermo Electron system (Thermo Fisher Scientific) usingZebron-1 Phenomenex fused silica capillary columns (29).

Evaluation of Ex Vivo Myocardial FunctionFreshly excised mouse hearts were immediately mountedonto a Langendorff apparatus, as previously described(30). Briefly, hearts were perfused in 0.7, 1.4, or2.5 mmol/L Ca2+ using Krebs-Henseleit bicarbonate buffersupplemented with two different energetic substrates (i.e.,glucose [11 mmol/L] alone or glucose [11 mmol/L] in thepresence of octanoate [1.5 mmol/L]). The oxygen partialpressure was measured in the coronary perfusate and ef-fluent using specific electrodes to calculate the myocardialO2 uptake (in mL $ min21 $ g21).

Statistical AnalysisAll data are reported as the mean 6 SEM. Data sets wereanalyzed for statistical significance using a nonparametricMann–Whitney U test, Kruskal Wallis, or two-way ANOVAanalysis when specified.

RESULTS

SKO Mice Display Hypertrophic CardiomyopathyWith Diastolic DysfunctionAs previously described (24), 10-week-old SKO mice arehyperglycemic, insulin-resistant, mildly hypotriglyceridemic,

and normocholesterolemic (Supplementary Fig. 1). Therefore,we performed cardiac phenotyping in WT and SKO micefrom 10–24 weeks of age. As differences between both geno-types were quite stable with aging (data not shown), we pre-sent in this study the data from 14-week-old animals. Theratio between heart weight and body weight (HW/BW) (Fig.1A) as well as between heart weight and tibias length (Sup-plementary Fig. 1E) were both increased in SKO comparedwith WT mice. ECG showed significant prolonged QT intervalduration in SKO versus WT animals (Table 1), whereas theheart rate was similar in both groups (Fig. 1B). On Dopplerflow analysis, LV diastolic function parameters were markedlyaffected in SKO mice: the E/A ratio was increased, and boththe deceleration time and the isovolumetric filling time wereincreased (Fig. 1C–F). Consistently, CMR imaging highlightedan increased diastolic wall thickness in SKO mice (Fig. 1G)despite a normal end diastolic volume (EDV) (Fig. 1H). Rightventricular volumes and function were normal (data notshown). In addition, SKO mice exhibited a mild ejection frac-tion (EF) decrease (52 vs. 64% in WT animals) (Fig. 1I) and ablunted longitudinal strain (Fig. 1J). Longitudinal strain cor-related positively with EF (Fig. 1K) and negatively with enddiastolic wall thickness (EDW) (Fig. 1L), highlighting the re-lationship among LV hypertrophy, wall deformation abnor-malities, and altered EF. Finally, correlations were foundamong random-fed mean plasma glucose, EDW, and EF,with the most hyperglycemic animals displaying the thickestLV walls and the lowest EFs (Fig. 1M and N).

Absence of Lipotoxic Profile in the Heart of SKO MiceAs ectopic lipid deposition and lipotoxicity have been exten-sively described in diabetic cardiomyopathy, we studiedcardiac lipid metabolism in SKO mice. Previously, usingradiolabeled TG-rich lipoproteins, we demonstrated elevatedTG uptake and a massive ectopic lipid deposition in SKOliver in vivo (24). Using the same methodology, we showedin this study that cardiac TG uptake was similar in SKO andWT mice (Fig. 2A). Cardiac lipidomic profiles showed similarTG and ceramide levels in both SKO and WT animals (Fig.2B and C), and DAG levels were only modestly reduced inthe SKO heart (Fig. 2D). In addition, we did not observe anychange in the expression levels of the procatabolic nuclearreceptor peroxisome proliferator–activated receptor a or ofits target genes involved in FFA oxidation in SKO mice(Supplementary Fig. 2A). These data suggest that the seipin-deficient heart unexpectedly exhibits neither a lipotoxicprofile nor a major impairment in lipid metabolism.

Insulin Resistance Coexists With Increased GlucoseUptake in the SKO HeartAs insulin resistance and chronic exposure to hyperglycemiaare also involved in the pathophysiology of diabetic cardio-myopathy, we assessed the insulin sensitivity and glucoseuptake of the SKO heart. The insulin-induced phosphory-lation of AKT was blunted by 70% in the hearts of SKOcompared with WT mice (Fig. 2E). Consistently, in the fedstate, the total and membrane GLUT4 protein levels were50 and 40% lower, respectively, in SKO animals (Fig. 2F).

1032 Cardiomyopathy in Lipodystrophic Mice Diabetes Volume 66, April 2017

GLUT4 mRNA tended to be lower in SKO, but this trend wasnot significant (Supplementary Fig. 2B). The GLUT1 protein(Fig. 2F) and mRNA levels (Supplementary Fig. 2B) werecomparable in both genotypes. Using [18F]FDG PET scan, amarked increase in in vivo MGU was found in SKOhearts (Fig. 2G). To evaluate the fate of the glucose, weused [11C]acetate PET scan–monitored oxidation to modeloxygen consumption (31–33). Our data revealed a 30% de-crease in heart oxygen consumption, suggesting a decreaseof all oxidative metabolism, including glucose (Fig. 2H).This latter result was further confirmed using an ex vivo–perfused heart setup, with a significant reduction of oxygen

consumption in SKO hearts whether they were supplied withglucose or glucose plus octanoate (Fig. 2I). AMPk plays acentral role in energetic substrate catabolism and oxygenconsumption regulation. In accordance with a defect in ener-getic substrate utilization, a 60% decrease in phosphorylatedAMPk was observed in SKO heart lysates (Fig. 2J). However,phosphorylation levels of the acetyl-coA carboxylase, oneAMPk target, were not significantly decreased in SKO sam-ples (data not shown). Together, these data demonstrate thatin SKO hearts, despite marked insulin resistance, glucoseuptake is elevated but oxygen consumption is reduced.

Chronic Activation of the Hexosamine Pathway Leadsto Cardiac Molecular Alterations in the SKO HeartBecause glucose uptake was increased in SKO hearts, wetested different pathways that are classically activated byglucose overload in the heart. First, we observed no changein ROS production in AGE or p47-phox plasma membranelevels in SKO mice compared with WT mice (Supplemen-tary Fig. 3). Second, we assessed the hexosamine biosyn-thetic pathway (HBP), which leads to the covalent additionof O-GlcNAc residues on various cellular proteins. The

Figure 1—SKOmice display hypertrophic cardiomyopathy with systolic and diastolic dysfunction. Cardiac phenotyping of 14-week-old WT (whitebars) and SKO (black bars) mice was investigated as described in RESEARCHDESIGNANDMETHODS. Ratio of HW (g) to BW (g) (A) and echocardiographyDoppler flow analysis in WT and SKO mice (n = 8/group): heart rate (B), representative images (C), E/A ratio (D), deceleration time (E), andisovolumetric relaxation (F). CMR analysis in SKO (n = 12) and WT (n = 10) mice: EDW (G), EDV (H), EF (I), and longitudinal strain (J). Correlationsusing CMR parameters: EF to longitudinal strain (K), EDW to longitudinal strain (L), EDW to random-fed glycemia (M), and EF to random-fedglycemia (N). Significant differences between WT and SKO mice are represented as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Table 1—ECG characteristics of WT and SKO mice

n RR (ms) P (ms) PR (ms) QRS (ms) QT (ms)

WT 10 124 6 3 12 6 0 35 6 0 11 6 0 51 6 1

SKO 10 127 6 4 11 6 1 36 6 1 11 6 0 60 6 2**

Data are mean 6 SEM. P, P wave duration; PR, PR intervalduration; QRS, QRS complex duration; QT, QT interval duration;RR, RR interval duration. **P , 0.01 vs. WT.

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o-GlcNAcylated protein levels measured by Westernblot were increased by 2.5-fold in SKO mice compared withWT mice (Fig. 3A). As chronic HBP activation has beenpreviously shown to alter insulin signaling (34), we isolatedO-GlcNAcylated protein using wheat germ agglutinin andanalyzed these protein samples by Western blot. Importantly,the O-GlcNAcylated level of the transcription factor FOXO1,a key player in insulin sensitivity, was twofold increased,whereas its total level remains stable (Fig. 3B). In contrast,O-GlcNAcylated level of AKT was unaltered in SKO heart. Inaddition, the phosphorylation level of PLN, a regulator of theendoplasmic reticulum calcium pump SERCA2a, was markedlyblunted in SKO hearts, whereas SERCA2 levels tended to bemarginally lower in SKO heart samples (Fig. 3C). Finally, inSKO hearts, the myosin heavy chain (MHC) b/a ratio was

threefold increased (Fig. 3D), and the number of positive cellsin TUNEL staining was increased by 50% (Fig. 3E). Notably, nosign of fibrosis was found in SKO mouse hearts (Supplemen-tary Fig. 3D). Together, these data demonstrate that activationof the hexosamine pathway by glucose overload is associatedwith molecular signs of cardiac dysfunction in SKO mice.

Dapagliflozin Improves SKO Mouse Heart Functionby Limiting Cardiac Glucose OverloadAs cardiac parameters were strongly correlated with plasmaglucose levels, we hypothesized that targeting hyperglycemiamight correct the cardiac dysfunction observed in SKO mice.To that purpose, we used the SGLT2 inhibitor dapagliflozin,which reduces hyperglycemia by increasing glycosuria. Six-week-old SKO mice were treated for 8 weeks with 1 mg/kg

Normalized

Figure 2—Energy metabolism in SKO mice. A: 3H-palmitate–labeled TRL uptake was evaluated 5 min after injection by counting total amountof radioactivity in the heart in 14-week-old WT (white bars) and SKO (black bars) mice (n = 6/group). Total intracardiac TG (B), ceramides (C),and DAG (D) levels were measured in heart from 12-week-old WT and SKO mice (n = 6/group). E: Insulin signaling: 12-week-old WT and SKOmice were fasted for 2 h, then received a 0.75 IU/kg dose of insulin intravenously, and killed 5 min after (n = 7 insulin-injected mice and3 saline-injected mice). The activation of AKT was measured by Western blot on heart lysates. Representative Western blots are shown, withquantification expressed as fold stimulation by insulin of the activated/phosphorylated-to-total protein ratio. F: Protein expression of GLUT4and GLUT1 were determined by Western blot in heart lysates from 14-week-old WT and SKOmice. GAPDH and transferrin receptor were usedas housekeeping protein respectively for total extract and membrane fraction (n = 8/group). [18F]FDG or [11C]acetate micro-PET/computedtomography were performed to measure glucose uptake (G) and oxygen consumption (H) (n = 7 or 4/5 WT/KOmice per group, respectively, foroxygen consumption). I: Ex vivo oxygen consumption evaluated with heart mounted in Langendorff setup as described in RESEARCH DESIGN AND

METHODS. J: The AMPk activation levels have been evaluated by Western blot. Tubulin was used as an index of the protein loading. Significantdifferences between WT and SKO mice are represented as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

1034 Cardiomyopathy in Lipodystrophic Mice Diabetes Volume 66, April 2017

dapagliflozin. Dapagliflozin treatment massively increasedglycosuria (Supplementary Fig. 4A), normalized plasmaTG levels (Supplementary Fig. 4B), and limited hyperglyce-mia (Supplementary Fig. 4C), but had no effect on insulinsensitivity, liver steatosis, or body weight (SupplementaryFig. 4D–F). The HW/BW ratio displayed a nonsignificanttrend to be reduced (Supplementary Fig. 4G). Echocardiog-raphy and CMR imaging analyses revealed that dapagliflozinimproved the E/A ratio, decreased the isovolumetric relaxa-tion time, reduced the deceleration time, and normalized theEDW as well as the EF (Fig. 4A–F). Finally, we tested whetherthe normalization of random-fed glycemia by dapagliflozindecreased the cardiac molecular consequences of glucoseoverload. Interestingly, dapagliflozin-treated SKO mice hadsignificantly lower total levels of O-GlcNAcylated proteinsand specifically the O-GlcNAcylated FOXO1 form as com-pared with untreated SKO mice (Fig. 4G and H). Moreover,the activation state of PLN was normalized, and the

phosphorylation level of AMPk was also increased with dapa-gliflozin (Fig. 4I). These results suggest that dapagliflozinpartly corrected cardiac dysfunction by decreasing the glucoseoverload and the subsequent hexosamine pathway activation.

Pioglitazone Lowers Glucose Overload but OnlyPartially Improves Cardiac FunctionTo further investigate the link between glucose overload andcardiac dysfunction, we used the insulin sensitizer pioglita-zone as an alternative hypoglycemic drug. We previouslypublished that pioglitazone treatment increases adiposetissue mass, insulin sensitivity, and random-fed hyperglyce-mia in SKO mice (24) (Supplementary Fig. 5A and B). Re-garding the effect on cardiac function, we first noticed thatpioglitazone increased the HW/BW ratio (SupplementaryFig. 5D). Cardiac echography and magnetic resonance imag-ing exams showed that pioglitazone treatment improvedE/A ratio, deceleration time, and EDW in SKO treated ascompared with SKO control mice (Fig. 5A, C, and D). Incontrast, isovolumetric relaxation time was unchanged,and EF tended to be reduced, even though this decreasedid not reach significance (Fig. 5B and E). Consistent withits hypoglycemic action, pioglitazone reduced the HBP path-way activation and the O-GlcNAcylated FOXO1 level (Fig.5G and H). However, the improvements of AMPk and PLNactivation did not reach statistical significance, despite astrong trend (Fig. 5I). Altogether, these data confirm thatglucose-lowering agents protect SKO mice from glucotoxic-ity, but with pioglitazone, the cardiac dysfunction is onlypartially corrected compared with dapagliflozin.

DISCUSSION

In this study, we performed for the first time a completecardiac phenotyping of SKO mice. We showed that SKO miceexhibit LV hypertrophy, along with diastolic/systolic dysfunc-tion and prolonged QT intervals. Whereas no signs of lipidaccumulation or lipotoxicity were observed, we report analteration in glucose metabolism in the SKO heart, withincreased glucose uptake and chronic hexosamine pathwayactivation. The SGLT2 inhibitor dapagliflozin, and in a lesserextent the insulin sensitizer pioglitazone, alleviates theglucose overload burden, leading to a marked reduction inthe O-GlcNAcylated protein levels and an improvement ofthe cardiac phenotype.

In our rodent model of lipodystrophy, cardiac pheno-typing revealed a decreased E/A ratio and an increasedisovolumetric filling time, which are both signs of de-creased heart compliance, leading to diastolic dysfunction.Consistently, the LV was found to be hypertrophic andstiff, as reflected by increased EDW along with an increasein heart mass and by blunted longitudinal strain.

Regarding the mechanisms involved in the associatedheart disease, we first asked the question of whether acardiomyocyte-autonomous effect of seipin deficiencyexisted. Seipin is expressed at very low levels in the heart ofWT animals, and we also report that the b/a MHC ratio, amarker of pathological cardiac remodeling, was comparable

Figure 3—Accumulation of O-GlcNAcylated protein and cardiac mo-lecular remodeling. O-GlcNAcylated protein levels (A), O-GlcNAcylatedFOXO1 levels (B), SERCA2a levels, and Ser-PLN (Pser. PLN) phosphor-ylation levels (C) have been evaluated by Western blot. D: MHC isoformwere evaluated by quantitative PCR. E: Apoptosis was evaluated byTUNEL staining. Significant differences between WT and SKO miceare represented as follows: **P < 0.01.

diabetes.diabetesjournals.org Joubert and Associates 1035

between 6-week-old SKO mice, which were normoglycemic atthis time, and WT mice (data not shown). Conversely,at 10 weeks of age, when the hyperglycemic phenotypearose, the b/a MHC ratio was elevated, and the firstcardiac imaging abnormalities (decrease in E/A ratio) be-gan to appear in SKO mice. The positive correlation be-tween random-fed glycemia and cardiac abnormalitiesreinforce our hypothesis that systemic energy metabolismhas a deleterious impact on cardiac function. The im-provement of the cardiac phenotype with an antihyper-glycemic drug is also consistent with this insight.

Lipotoxicity has been largely involved in diabetic cardio-myopathy (9,11), and we have previously shown that SKOmice display massive hepatic ectopic lipid deposition andincreased hepatic triacylglycerol-rich lipoprotein (TRL) up-take (24). Surprisingly, we did not see any changes in TRLuptake or intracardiac lipid levels (TG and ceramides) in theSKO heart. This finding is consistent with several autopsy

reports that did not show ectopic lipid deposition in thehearts of patients with BSCL (19,20). Overall, the SKOmouse is not a model for heart steatosis and lipotoxicity-induced cardiac dysfunction.

As glucotoxicity has also been implicated in T2DM-associated cardiac dysfunction (14,15), we studiedcarbohydrate metabolism in SKO hearts. Using [18F]FDGPET imaging, we established that SKO hearts displayedincreased MGU compared with WT hearts. Literatureis quite puzzling and heterogeneous regarding heartglucose uptake in diabetic animals. In one study, Zuckerdiabetic fatty rats displayed lower glucose uptake underhyperinsulinemic-euglycemic clamp conditions comparedwith lean rats (35). In another study, Zucker diabetic fattyrats were fasted for 6 h, and glucose uptake PET moni-tored was found to be unaffected, with a trend toward anincrease (33). Nutritional status obviously affects glucoseuptake, and in our case, the glucose uptake assay was

Figure 4—Dapagliflozin improves SKO heart function by limiting cardiac glucose overload. Six-week-old SKO mice were treated withdapagliflozin (1 mg/kg) for 8 weeks. Echocardiography Doppler flow analysis was performed: E/A ratio (A), isovolumetric relaxation (B), anddeceleration time (C ). White bars represent WT mice (n = 8), black bars represent nontreated SKO mice (n = 13), and gray bars representdapagliflozin-treated SKO mice (KO+DAPA; n = 7). CMR analysis performed in SKO (black bars) and KO+DAPA (gray bars) mice andcompared with previously obtained data from WT mice: EDW (D), EF (E), and EDV (F ). Total O-GlcNAcylated protein levels (G), FOXO1O-GlcNAcylated protein levels (H), and AMPk activation and PLN phosphorylation levels (I) evaluated by Western blot. Pser.PLN, phospho-Ser-PLN. Significant differences between WT and SKO mice are represented as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Significantdifferences between SKO control and KO+DAPA were represented as follows: #P < 0.05; ##P < 0.01; ###P < 0.001.

1036 Cardiomyopathy in Lipodystrophic Mice Diabetes Volume 66, April 2017

performed after a short 2-h fasting period to avoid majorhyperglycemia that would alter PET analysis. We alsoavoided longer fasting, which has been previously shownto alter SKO mouse insulin sensitivity (36). In our condi-tions, compared with WT animals, SKO mice are massivelyhyperinsulinemic (23,24,37). This latter point, along withhyperglycemia, probably explains why MGU is increasedin vivo despite strong insulin resistance. A similar resultwas obtained by Kaczmarczyk et al. (38) in GLUT4 partiallydeficient C57BL/6 mice. Despite a 50% decrease in GLUT4levels compared with WT animals, these mice presented ahigher baseline MGU, related to their hyperinsulinemicstate. Thus, we consider that, in our setting, PET mea-surement of glucose uptake appropriately represents thephysiological balance among cardiac insulin resistance,hyperinsulinemia, and hyperglycemia and realistically re-flects the entry of glucose into cardiomyocytes. As noted,

the discrepancy between the lower GLUT4 protein levelsand the unchanged mRNA levels was unexpected, as, inmost T2DM mice models, both are concomitants (39). In-terestingly, using [11C]acetate measurement, we demon-strated that oxygen consumption was decreased in SKOhearts. When entering the cell, [11C]acetate is quickly me-tabolized into acetyl-CoA and then is usually catabolized inthe Krebs cycle to produce ATP (40). Our data clearly in-dicate that the Krebs cycle activity is low in SKO mice butdo not determine whether FFA or glucose oxidation isprimarily affected. The ex vivo data, in isolated hearts inthe presence of glucose only, confirmed that glucose oxida-tion is deficient in SKO hearts. This would be consistentwith a large body of work demonstrating that glucose oxi-dation is impaired in the diabetic heart (6,33,41,42). Inaddition, the decreased AMPk activity in SKO hearts isconsistent with altered aerobic catabolism, given that

Figure 5—Pioglitazone lowers glucose overload but only partially improves cardiac function. Six-week-old SKO mice were treated withpioglitazone-enriched diet (300 mg/kg) for 8 weeks. Echocardiography Doppler flow analysis was performed: E/A ratio (A), isovolumetricrelaxation (B), and deceleration time (C ). White bars represents WT mice (n = 8), black bars represent nontreated SKO mice (n = 14), andgray bars represent pioglitazone-treated SKO mice (KO PIO; n = 8). CMR analysis performed in SKO (black bars) and KO PIO (gray bars)mice and compared with previously obtained data from WT mice: EDW (D), EF (E), and EDV (F ). Total O-GlcNAcylated protein levels (G),FOXO1 O-GlcNAcylated protein levels (H), and AMPk activation and PLN phosphorylation levels (I) evaluated by Western blot. PSer-PLN,phospho-Ser-PLN. Significant differences between WT and SKO mice are represented as follows: *P < 0.05; **P < 0.01; ***P < 0.001.Significant differences between SKO control and KO PIO were represented as follows: #P < 0.05; ##P < 0.01.

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AMPk is known to promote FFA and glucose cardiac oxida-tion (43). Interestingly, the TG levels and the expressionlevels of the main genes involved in FA oxidation were notaltered in SKO mice, which supports the hypothesis thatthe alteration occurs mainly in glucose oxidation.

We tried to highlight how glucose overload maycontribute to cardiac dysfunction. Several publicationshave demonstrated that glucotoxicity induces oxidativestress (14,41), but using several methods, we found nooxidative stress in SKO hearts. We next investigated theinvolvement of the other well-known glucotoxic pathway,the HBP (44). Indeed, we found a marked increase inO-GlcNAcylated protein levels in the SKO heart, and weinterpreted this finding as a clear sign of glucose overload.The HBP pathway is activated in vitro and in vivo in thecontext of hyperglycemia and diabetes (44). Several studieshave sought to elucidate whether HBP activation has a di-rect causal impact on cardiac dysfunction. Indeed, pharma-cological inhibition of the HBP in db/db cardiomyocytesattenuated hypertrophic signaling (45). Hyperglycemia hasbeen shown to alter the b/a MHC ratio both ex vivo andin vivo. The increase of uridine diphosphate N-acetyl glu-cosamine production, the main metabolite of the HBP path-way, is suspected to be involved in the overexpression of theb form of the MHC (46), a phenomenon that could explainthe increased ratio reported in SKO mice. Chronic activationof the HBP by glucose overload has also been shown to altercalcium signaling and PLN phosphorylation levels (47). Con-sistently, PLN Ser phosphorylation levels were decreased inSKO mice. In addition, dysregulation of O-GlcNAcylationalters mitochondrial function, oxygen consumption, andATP synthesis (48). Thus, increased O-GlcNAcylated proteinlevels would alter mitochondrial function, contributing tothe reduced oxygen consumption in SKO hearts.

We specifically highlighted in this study the increasedO-GlcNAcylated levels of FOXO1. This is particularly inter-esting for two main reasons. Firstly, FOXO1O-GlcNAcylationincreases its activity (49), and this activation has been sug-gested to be a key mediator of glucotoxicity in differentorgans (50). Secondly, FOXO1 has been involved in metabol-ically induced cardiac dysfunction, especially insulin resis-tance, and FOXO1 knockdown appears to be protective ina model of diet-induced cardiomyopathy (51). Our workshows for the first time that FOXO1 O-GlcNAcylation isassociated with heart insulin resistance and cardiac dysfunc-tion. Together, these data lead us to hypothesize that in ourmodel, glucose overload is the main trigger of cardiac dys-function and that chronic HBP activation is a central mech-anism in glucose-adverse effects.

To test this hypothesis, we used two hypoglycemicagents: the SGLT2 inhibitor dapagliflozin and the insulinsensitizer pioglitazone. In SKO mice, pioglitazone reduceglycemia through insulin-sensitivity enhancement (24),whereas dapagliflozin does not improve the insulin resis-tance. Both treatments lowered glycemia and reversedthe HBP chronic activation, including the increase inO-GlcNAcylated FOXO1 levels, suggesting an antiglucotoxic

effect. In term of cardiac function, SKO mice treated withdapagliflozin presented a decrease in ventricular wall hy-pertrophy and an improvement of both diastolic and sys-tolic function. In contrast, pioglitazone corrected onlypartially the cardiac dysfunction in SKO mice, improvingthe E/A ratio and ventricular wall hypertrophy, but withno effect on the EF and the isovolumetric relaxation time.Of note, pioglitazone induced an increase of the HW/BWratio and a trend to increase the EDV. Therefore, despitea similar antiglucose overload action, these treatments actdifferently because of either a deleterious side effect ofpioglitazone or an additional beneficial effect of dapagliflozineand potentially by a combination of both. Thiazolidine-diones have been described to induce cardiac hypertrophythrough volume expansion (52). This is further supportedby a large meta-analysis showing that the use of pioglitazonewas associated with increased risk of nonfatal chronic HFrequiring hospitalization (53). In striking contrast, SGLT2inhibitors exert osmotic diuretic and natriuretic effects con-tributing to plasma volume contraction (54). In addition, arecent study suggests that empagliflozin, another SGLT2inhibitor, increases the production of ketone bodies thatare more easily used by the heart (55).

In absence of steatosis and fibrosis, the origin ofcardiac hypertrophy remains poorly understood in ourmodel. Chronic activation of the HBP in the context ofdiabetes is tightly correlated with cardiac hypertrophywith several transcription factors regulating prohypertro-phic genes being activated (56). However, dapagliflozinonly tends to lower the HW/BW ratio, suggesting thateither the reduction of the O-GlcNAcylation was not suf-ficient or that other mechanisms are involved. Futurework is needed to highlight the contribution of the HBPinduction to the hypertrophy in our SKO heart.

Although the precise mechanism remains yet to be com-pletely elucidated, our results support a beneficial cardiaceffect of dapagliflozin and therefore are consistent withrecent clinical studies. Indeed, the BI 10773 (Empagliflozin)Cardiovascular Outcome Event Trial in Type 2 DiabetesMellitus Patients (EMPA-REG OUTCOME) trial has recentlydemonstrated that empagliflozin strongly decreased bothcardiovascular and total mortality (30–40% relative risk re-duction) (57) in a large population of patients with T2DM athigh cardiovascular risk. This protective effect of the SGLT2inhibitor occurred very quickly after a few months and wassustained over the 4-year study. In line with our findings, itis tempting to speculate that SGLT2 inhibitors improve car-diac abnormalities at least partly by limiting cardiac glucoseoverload, HBP activation, and the functional consequenceson calcium signaling and aerobic metabolism. Previously,Hong et al. (58) showed that the hypoglycemic SGLT1/SGLT2 inhibitor phlorizin improves the ventricular thick-ness in nonobese T2DM Akita mice. However, to our knowl-edge, our study is the first to demonstrate that dapagliflozinmight have beneficial cardiac effects by protecting the heartfrom chronic HBP activation, especially at least throughFOXO1 O-GlcNAcylation.

1038 Cardiomyopathy in Lipodystrophic Mice Diabetes Volume 66, April 2017

In conclusion, using a unique model for metaboliccardiomyopathy with no lipotoxic features, we highlightthat glucotoxicity by itself can trigger cardiac dysfunctionand that agents reducing glucose exposure can improvethis cardiomyopathy, with a superiority of dapagliflozineover pioglitazone in this specific model.

Acknowledgments. The authors thank M. Takahashi, J.-F. Deleuze, andM. Lathrop (Centre de Génotypage–Commissariat à l’Énergie Atomique, Evry,France) for donating the mouse model; C. Dumet, S. Suzanne, S. Lemarchand-Minde (animal facility, L’Institut du Thorax, Nantes, France), and B. Haelewyn(CURB, UNICAEN, Caen, France) for animal care; Dr. Benjamin Lauzier and Dr.Mickael Dergangeon (L’Institut du Thorax, Nantes, France) for fruitful scientificdiscussions; and Pia Tager (EA 4650, UNICAEN, GIP Cyceron) for assistance duringCMR certification exams. The authors also thank the MicroPICell core facility(Structure Fédérative de Recherche François Bonamy, Nantes, France) for micros-copy. Lipidomic analyses were performed at Toulouse INSERM MetaToul-Lipidomique Core Facility (MetaboHub ANR-11-INBS-0010).Funding. This work was supported by grants from INSERM and Frenchassociations Aides aux Jeunes Diabétiques, Société Francophone du Diabète, andFondation de France. X.P. was awarded the European Foundation for the Study ofDiabetes/Lilly Research Fellowship Programme 2015.Duality of Interest. M.J. conducted clinical trials as coinvestigator,provided advisory services, and/or attended conferences as contributor for Lilly,Novo Nordisk, Sanofi, Takeda, Bristol-Myers Squibb, Novartis, AstraZeneca,Boehringer Ingelheim, and Janssen-Cilag. B.C. is taking part in advisory boardsfor AstraZeneca, Lilly, Sanofi, and Takeda. No other potential conflicts of interestrelevant to this article were reported.Author Contributions. M.J. researched data and wrote the manuscript.B.J., D.M., X.M., A.T., A.A., L.D., G.T., and A.M. researched data. D.M. and B.S.contributed to the scientific discussion and edited the manuscript. C.L.M. andF.C. contributed to scientific discussion. J.M. wrote the manuscript. B.C.contributed to scientific design and wrote the manuscript. X.P. researcheddata, designed the experiments, and wrote the manuscript. X.P. 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 76th Scientific Sessions of the American Diabetes Association, New Orleans,LA, 10–14 June 2016.

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