CV Protection in the EMPA-REGOUTCOME Trial: A “ThriftySubstrate” HypothesisDiabetes Care 2016;39:1108–1114 | DOI: 10.2337/dc16-0330

The striking and unexpected relative risk reductions in cardiovascular (CV) mortality(38%), hospitalization for heart failure (35%), and death from any cause (32%)observed in the EMPA-REG OUTCOME trial using an inhibitor of sodium–glucosecotransporter 2 (SGLT2) in patients with type 2 diabetes and high CV risk have raisedthe possibility that mechanisms other than those observed in the trialdmodestimprovement in glycemic control, small decrease in body weight, and persistentreductions in blood pressure and uric acid leveldmay be at play. We hypothesizethat under conditions of mild, persistent hyperketonemia, such as those that prevailduring treatment with SGLT2 inhibitors, b-hydroxybutyrate is freely taken up by theheart (among other organs) and oxidized in preference to fatty acids. This fuelselection improves the transduction of oxygen consumption into work efficiencyat the mitochondrial level. In addition, the hemoconcentration that typically followsSGLT2 inhibition enhances oxygen release to the tissues, thereby establishing apowerful synergy with the metabolic substrate shift. These mechanisms would co-operatewithother SGLT2 inhibition–induced changes (chiefly, enhanced diuresis andreduced blood pressure) to achieve the degree of cardioprotection revealed in theEMPA-REG OUTCOME trial. This hypothesis opens up new lines of investigation intothe pathogenesis and treatment of diabetic and nondiabetic heart disease.

First among cardiovascular (CV) end point trials of glucose-lowering agents (1), theEMPA-REG OUTCOME trialdusing 10 or 25 mg/day sodium–glucose cotransporter2 (SGLT2) inhibitor empagliflozin against placebo in 7,020 patients with type 2diabetes (T2D) who were at increased CV riskdreported a 14% reduction in majorCV events and marked relative risk reductions in CV mortality (38%), hospitalizationfor heart failure (35%), and death from any cause (32%) over amedian time period of2.6 years (2). Of note, all the pathologic categories of CV death (ischemic, pumpfailure, arrhythmic, embolic) contributed to the overall reduction in CV death in apatient cohort well treated with the use of renin-angiotensin-aldosterone inhibi-tors, statins, and acetylsalicylic acid. Furthermore, separation of the cumulativeincidence functions between pooled-dose groups and placebo was already evidentmonths after randomization. This unusual time course and the discrepancy betweenthe relative risk reduction of the primary end point (nonfatal myocardial infarction,stroke, and CV mortality) and CV mortality itself suggests that active treatmentaffected case fatality rates more than event rates. In other words, empagliflozintreatment appeared mostly to rescue patients from impending cardiac decompen-sation. This interpretation is supported by the recent post hoc analyses of heartfailure, documenting a large benefit in first and recurrent heart failure hospitaliza-tion across virtually every patient subgroup (3). Despite the fact that the diagnosis ofheart failure was based on investigator reporting, this outcome of the EMPA-REG

1CNR Institute of Clinical Physiology, Pisa, Italy2Cardiometabolic Disease Research, BoehringerIngelheim Pharma GmbH & Co. KG, Biberach,Germany

Corresponding author: Ele Ferrannini, ferranni@ifc.cnr.it.

Received 16 February 2016 and accepted 28March 2016.

E.F. and E.M. contributed equally to this study.

© 2016 by the American Diabetes Association.Readersmayuse this article as longas thework isproperly cited, the use is educational and not forprofit, and the work is not altered.

Ele Ferrannini,1 Michael Mark,2 and

Eric Mayoux2

1108 Diabetes Care Volume 39, July 2016

DIABETES

CARESYMPOSIUM

OUTCOME trial stands out against the rec-ognized lack of evidence on the safety orefficacy of glucose-lowering drugs in pa-tients with heart failure (4,5); indeed, arisk signal of incident heart failure aftertreatment with saxagliptin emerged fromthe SAVOR-TIMI trial (1).As expected fromprevious clinical stud-

ies, in the EMPA-REG OUTCOME trial thedifference in glycemic control betweenthe treatment and placebo arms (withHbA1c averaging 0.54–0.60% at 12 weeksand 0.24–0.36% at study end) was accom-panied by a small decrease in bodyweightand persistent reductions in blood pres-sure anduric acid level. Aheadof the studyend, the EMPA-REGOUTCOME trial inves-tigators themselves had outlined addi-tional risk factors that the use of SGLT2inhibitors influences positively: visceraladiposity, hyperinsulinemia, arterialstiffness, albuminuria, and oxidative stress(6). However, isolated reductions in HbA1clevel, body weight, or uricemia of the de-gree seen in the trial have been gener-ally reputed to be insufficient to explainthe outcome (7–11). For example, ameta-analysis of macrovascular outcomes inintensive glucose control trials (12)reported a 9% reduction in major CVevents for an average HbA1c reduction of0.7%, but no significant reduction in CV orall-cause mortality. By their mode ofaction, SGLT2 inhibitors induce natriure-sis and osmotic diuresis (13,14) and adrop in systolic and diastolic blood pres-sure levels (2); indeed, these effects havebeen hypothesized to play a pivotal role inthe CV benefit shown by the EMPA-REGOUTCOME trial (10,11), especially duringthe early weeks of treatment when thesystolic blood pressure gradient betweenthe arms was largest (5 mmHg) (10).Clearly, such hemodynamic changes canbe beneficial, particularly in patients withhigh CV risk, insofar as a reduced bloodvolume lowers the preload and a re-duced blood pressure lowers the postloadburden to the heart. There remain, how-ever, inconsistencies between EMPA-REGOUTCOME trial outcomes and the CV ben-efit profile of antihypertensive anddiuretictherapy. For example, a recent systematicreview of blood pressure lowering in T2Dpatients (15) reported significant reduc-tions inmajor CV events (including stroke),but smaller reductions in heart failure andmortality associated with a 10 mmHg de-crease in systolic blood pressure. Likewise,in systematic meta-analyses (16,17),

diuretic agents appear to be associatedwith a smaller (;20%) reduction in CVmortality than that seen in the EMPA-REG OUTCOME trial and a clear riskreduction in stroke, which was not ob-served in the EMPA-REG OUTCOME trial.In a recent trial (18), the use of themineralocorticoid receptor antagonisteplerenone in patients with systolicheart failure resulted in a reduction inhospitalization for heart failure similarin size and time course to the corre-sponding outcome in the EMPA-REGOUTCOME trial. The eplerenone-treatedpatients, however, did not have diabe-tes, were selected on the basis of highlevels of B-type natriuretic peptide(BNP) and N-terminal pro-BNP, andthe trial outcome showed no protectionagainst nonfatal myocardial infarctionor stroke. The EMPA-REG OUTCOMEtrial cohort (2) was composed of olderpatients with long-standing T2D whoseheart failure, when reported, was notcharacterized either for ejection frac-tion or levels of natriuretic peptides.Also, most patients in the EMPA-REGOUTCOME trial were being treatedwith antihypertensive drugs, includingdiuretic agents, so SGLT2 inhibitionconferred a benefit for heart failureabove and beyond antihypertensivetreatment. Interestingly, in a small,short-term (12-week) mechanistic studyin T2D patients (19), treatment with da-pagliflozin (another selective SGLT2 in-hibitor) resulted in a smaller reductionin blood pressure than did treatmentwith a comparator diuretic (25 mg hy-drochlorothiazide), but also in a 7% de-crease in plasma volume (as directlymeasured with the use of 125I-labeledalbumin) and some increment inN-terminalpro-BNP levels, neither of which wereobserved with the treatment with thediuretic agent. Thiazide diuretic agentsdecrease plasma volume only transiently,whereas in the EMPA-REG OUTCOMEtrial the observed increase in hematocritpersisted for the entire duration of thetrial (20).

Collectively, these considerationssuggest that other mechanisms trig-gered by SGLT2 inhibition may contributeto the CV outcomes of the EMPA-REGOUTCOME trial.

HYPOTHESIS

Raised circulating levels of b-hydroxybuty-rate offered significant cardioprotection

to the high-risk diabetes patients inthe trial.

RATIONALE

In diet-induced obese rats treated withdapagliflozin (21), ipragliflozin (22), ortofogliflozin (23), lipolysis is acceleratedand circulating ketone body levels areincreased, especially in the fasting stateor when animals are fed in pairs. In pa-tients with T2D, empagliflozin-inducedglycosuria lowers plasma glucose and in-sulin levels and raises fasting and postmealglucagon concentrations. The subtractionof large amounts of glucose from the glu-cose pool, coupled with the dual hormonalchanges, results in a 25% restriction of glu-cose utilization, and a concomitant in-crease in lipid mobilization and usage forenergy production (24). Under conditionsof reduced portal insulin-to-glucagon ratio,the increased delivery of free fatty acids(FFAs) to the liver stimulates ketogenesis(25), resulting in a metabolic conditionresembling a prolonged fast (26). In well-controlled Caucasian patients with T2Dwho were receiving stable doses of met-formin or were drug-naive (27), a 4-weekcourse of treatment with 25mg empagli-flozin was associated with raised fastingand postmeal circulating FFA and glyc-erol levels, indicating enhanced lipolysis.Concomitantly, both fasting and post-meal plasma b-hydroxybutyrate concen-trations were increased twofold tothreefold; these changes were similarin time course, though attenuated in ex-tent, in a group of nondiabetic volun-teers receiving the drug. Similar resultshave been reported in Japanese patientswith T2D with the use of empagliflozin,tofogliflozin, luseogliflozin, or canagliflozin(28–32). For example, in a 24-week phaseIII study of drug-naive patients with T2D(32), plasma ketones rose dose depen-dently with the administration of 100 or200 mg canagliflozin versus placebothroughout the study period. Of note,though mean plasma ketone levels areonly modestly elevated, in a sizeable pro-portion of subjects they rise into the mil-limolar range (27–32), particularly in themore insulinopenic patients (27).

Circulating b-hydroxybutyrate is takenup (through the monocarboxylate trans-porter, which also transports pyruvate) inproportion to its plasma concentrationinto most organs, including heart, brain,and kidney, by a saturable transportmech-anism (33). In humans, b-hydroxybutyrate

care.diabetesjournals.org Ferrannini, Mark, and Mayoux 1109

uptake by forearm tissues is not influ-enced by local hyperinsulinemia (34)(Fig. 1), and exogenous b-hydroxybuty-rate infusion does not interfere with in-sulin-mediated glucose utilization (35).Thus, b-hydroxybutyrate transport is in-sulin independent. In the fasting state,b-hydroxybutyrate is taken up by thehuman heart, along with glucose, lac-tate, pyruvate, glycerol, and FFA, withthe highest avidity per unit mass amongbody tissues and with a fractional extrac-tion (;40%) comparable to that of pyru-vate and far higher than that of glucose(;2%) or FFA (15–20%) (36) (Fig. 2). Bycombining mass uptake (;10 mmol/min)with the heat of combustion (Table 1), itcan be calculated that in the overnightfasted state b-hydroxybutyrate contrib-utes 15% of resting cardiac energy expen-diture comparedwith 45%of FFAs and 8%of glucose/lactate/pyruvate. When therate pressure product is increased by in-cremental atrial pacing (Fig. 3), the frac-tional extraction of b-hydroxybutyrateremains highat;40%, thereby continuingto support external cardiac work (37).In perfused working rat hearts, b-

hydroxybutyrate supplementation in-hibits pyruvate oxidation by deactivatingpyruvate dehydrogenase (38), mimics in-sulin action (39), and competes with oxi-dation of FFAs (possibly also by impedingtheir transport into cells [40]). Within thecell, after conversion to acetoacetate(catalyzed by the mitochondrial isoform

of 3-hydroxy-3-methylglutaryl-CoA syn-thase) and breakdown to acetyl-CoA,b-hydroxybutyrate enters the tricarbox-ylic acid cycle (TCA) to be oxidized. Byexpanding the mitochondrial acetyl-CoA pool, b-hydroxybutyrate com-petes for entry into the TCA cycle withthe acetyl-CoA originating from FFAoxidation and glucose-derived pyruvate(Fig. 4) (39,40). Thus, during fasting andstarvation, b-hydroxybutyrate partiallyreplaces glucose as a fuel, which is criti-cal for the brain in circumstances oflow glucose availability (26). Impor-tantly, the energetics of mitochondrialb-hydroxybutyrate oxidation comparesfavorably with the oxidation of pyruvate(Table 1); in fact, when b-hydroxybutyrateis added to the perfusion medium ofworking rat hearts, the heat of combus-tion per unit of carbon has been calcu-lated to be 31% increased and theoxygen cost of this energy output to be27% decreased (41). This advantage isgranted by a more efficient oxidation ofthe mitochondrial coenzyme Q coupleand an increase in the free energy ofcytosolic ATP hydrolysis. As a conse-quence, in the isolated working heartb-hydroxybutyrate increases externalcardiac work at the same time as it re-duces oxygen consumption, therebyimproving cardiac efficiency by 24%.Furthermore, a persistently elevatedrate of FFA oxidation generates reactiveoxygen species in excess of scavenging

capacity (42); the resulting oxidativestress contributes to mitochondrialdamage in a range of pathologies (43).In in vitro systems, b-hydroxybutyratehas been shown to suppress oxidativestress (44) by inhibiting histone deace-tylases. Finally, ketone bodies havebeen shown to upregulate mitochondri-al biogenesis and, by stabilizing cellmembrane potential, to exhibit antiar-rhythmic potential (45).

In the fed state, as insulin restrainslipolysis and stimulates glucose oxida-tion, the circulating concentrations ofb-hydroxybutyrate decline along withFFA flux and oxidation. In the normalhuman heart, systemic insulin adminis-tration at physiologic concentrationsstimulates myocardial glucose uptake,both directly and by suppressing plasmaFFA concentrations, without alteringcoronary blood flow; the uptake ofb-hydroxybutyrate and its contributionto cardiac energy expenditure drop tonil (Fig. 2) (36). However, in patientswith T2D (27,46) as well as in patientswithout diabetes with coronary arterydisease (47) or stress-induced ische-mia (48), whole-body and myocardialinsulin-mediated glucose utilization areimpaired (i.e., insulin resistance), and alarger (.80%) than normal (50–70%)proportion of energy is derived fromthe oxidation of fatty substrates (49).FFAs require ;8% more oxygen thanglucose to produce the same numberof calories, and the external power ofthe left ventricle for a given oxygen con-sumption is higher when rates of fattyacid b-oxidation are low relative toglucose and lactate oxidation (50). Thiscoupling between substrate selectionand mechanical efficiency is especiallyrelevant to heart failure, regardless ofits nature (ischemic or nonischemic)and functional manifestation (reducedor preserved ejection fraction) (51).Whether primarily or secondary to sys-temic changes induced by cardiac failureitself (e.g., adrenergic activation [47]), thefailing heart is an “engine out of fuel”(52), especially when the energy de-mand increases, such as it does duringexercise. Indeed, most kinds of cardiacinjurydischemic, myopathic, and re-perfusion injury (53)dconverge on in-sufficient mitochondrial energy outputand contractile failure as the basic mecha-nism. Enhancing oxygen use and mechan-ical efficiency through the long-term

Figure 1—Fractional exchange of substrates by the human forearm under basal conditions andduring local physiological hyperinsulinemia. Note the insulin-induced increased net release oflactate and the switch from net uptake to net release of pyruvate in the face of a constantextraction of b-hydroxybutyrate. Data were recalculated from the study by Natali et al. (34).

1110 CV Protection in EMPA-REG OUTCOME Trial Diabetes Care Volume 39, July 2016

provision of an energetically thrifty sub-strate should therefore benefit most con-ditions of extensive CV damage in arelatively short time frame, precisely aswas the case in the EMPA-REG OUTCOMEtrial. In this context, b-hydroxybutyratecan be viewed as a constitutive mitochon-drial helper, just as is its closest kin of en-ergetics, pyruvate (54,55) (Table 1). Ofnote is that, in a mouse model of heartfailure, metabolite signatures of fattyacid oxidation are reduced, whereas signa-tures of the oxidation of ketone bodies(e.g., b-hydroxybutyrate dehydrogenase)are increased (56). Furthermore, in pa-tients with heart failure, the use of circu-lating ketones is impaired by .50% inskeletal muscle but is preserved in myo-cardial tissues (57). Finally, in a study ofmyocardial tissue obtained from patientswith advanced heart failure at the timeof cardiac transplantation (58), levels ofacyl-CoAs were reduced and those ofacetyl-CoA (and succinyl-CoA:3-oxoacid

CoA transferase expression) were in-creased, as predicted by the metabolicsequence sketched in Fig. 4. Thus, thefailing heart attempts to cope with theincreased energy expenditure (59) byturning to ketone use at the expenseof fatty acid use (60).

A contributing factor to the CV out-comes of the EMPA-REG OUTCOME trialcould be represented by an increaseddelivery of oxygen to tissues. In fact,SGLT2 inhibitors cause an increase inhematocrit (61), which in the EMPA-REG OUTCOME trial averaged 5% in ab-solute values, and 11% in percentagepoints (2). This change likely reflectsthe hemoconcentration associatedwith the diuretic effect. However, pre-liminary results in patients with T2Dwho were receiving treatment with da-pagliflozin (19) have shown that redblood cell mass (as measured by 51Cr-labeled erythrocytes) was expanded by;6%, a change that was preceded by a

transient increase in erythropoietin con-centrations and reticulocyte count. Thus,the observed increase in hematocritmight result in part from the stimulationof erythropoiesis, an intriguing possibilitythat awaits confirmation. Interestingly,the blood volume contraction was not as-sociated with a significant increase inheart rate (2), suggesting that cardiac out-put was maintained at least at pretreat-ment levels. A higher hematocrit for thesame blood flow is expected to delivermore oxygen to tissues (62). In quantita-tive terms, experiments in hamsterstransfused with packed red blood cells(63) demonstrate that short-term incre-ments in the hematocrit (up to ;10%)induce a 20% rise in oxygen delivery totissues, coupled with a 10% drop in ar-terial blood pressure and an increase incardiac output. In patients (includingpatients with diabetes) with decompen-sated heart failure and renal dysfunc-tion, hemoconcentration induced withvery high doses of a loop diuretic hasbeen associated with a substantially im-proved survival time despite the deteri-oration of renal function (64–67).

CAVEATS

The metabolic mechanism laid outhere is basic, and as such it should ap-ply across organs (foremost, the kidneyand the brain) and pathologies (ische-mic and nonischemic). However, at pre-sent crucial information is missing. Thus,we do not know 1) the dose-responserelationship between raised ketonebodies and cardiac function in humans,2) the time course of hyperketonemiaduring SGLT2 inhibitor treatment, and3) the impact of other medications. Forinstance, the combination of an SGLT2inhibitor with glucose-lowering agentsthat stimulate insulin secretion (sulfo-nylureas, dipeptidyl peptidase-4 in-hibitors, and glucagon-like peptide 1receptor agonists) or with exogenous in-sulin itself may quench or obliterate theglucagon and/or the ketone response.Furthermore, our hypothesis posits thatthe combination of improved oxygenuse/supply with sodium/volume reduc-tion should work best toward heartfailure and organ ischemia (as well asarrhythmias), but in the EMPA-REGOUTCOME trial these end points wereaffected differentially in size and, possi-bly, in time course (2). Thus, the incidenceof stroke was, if anything, numerically

Figure 2—Fractional extraction of substrates by the human heart under basal (overnight fast)and systemic hyperinsulinemia (euglycemic-hyperinsulinemic clamp). Note the increased netextraction of glucose, lactate, and pyruvate in the face of a marked reduction in the net extrac-tion of FFA and b-hydroxybutyrate (due to insulin-induced suppression of whole-body lipolysis).Data were recalculated from the study by Ferrannini et al. (36).

Table 1—Comparative mitochondrial energetics of b-hydroxybutyrate oxidation

Glucose Palmitate Pyruvate b-HB

O2 used (L/mol) 134 515 56 101

Heat of combustion (kcal/mol) 670 2,385 279 487

O2 cost of calorie (mL/kcal) 200 216 201 207

Heat of combustion per C2 (kcal/mol)¶ 224 298 186 244

In terms of oxygen (O2) cost, the energy yield ofb-HB oxidation is comparable to those of glucoseand pyruvate, and lower than that of palmitate. If the energy yield is calculated per C2 unit,b-HB oxidation is superior to glucose and far better than palmitate. b-HB, b-hydroxybutyrate.¶According to Sato et al. (41).

care.diabetesjournals.org Ferrannini, Mark, and Mayoux 1111

higher in the treatment arm than in theplacebo arm (hazard ratio 1.24 [95% CI0.92–1.67], P = 0.16), although deathfrom stroke was recorded in 0.34% oftreated patients versus 0.47% of placebopatients, and transient ischemic attackswere recorded in 0.8% of treated patients

versus 1.0% of placebo participants.Given the small number of these latterevents, a satisfactory explanation is notat hand, but it is theoretically possiblethat in vulnerable patients the increasedblood viscosity associated with hemo-concentration may play a role. More ad-judicated events and/or a longer trialduration and better definition of endpoints (e.g., type of heart failure), aswell as measures of circulating sub-strates (e.g., b-hydroxybutyrate) andbiomarkers (e.g., natriuretic peptides),would be needed to refine the quantita-tive aspects of any hypothesis. We hopethat ongoing CV outcomes trials withother SGLT2 inhibitors (1) will provideadditional information on these impor-tant issues.

Starting from the evolutionary role ofketosis (26), other biology of ketone bod-ies (e.g., their effects on cardiac remodel-ing, oxidative stress, and mitochondrialbiogenesis [42–45]) has generated someenthusiasm for their therapeutic exploita-tion (68–70). Low-carbohydrate ketogenic

diets are still widely used, mainly forweight loss, but their long-term impacton CV function is still uncertain and con-troversial (71). In patients with type 1 di-abetes or insulin-treated patients withT2D who are receiving therapy withSGLT2 inhibitors, inappropriate reduc-tions in exogenous insulin administrationor intercurrent illness may precipitate ep-isodes of nonhyperglycemic ketoacidosis(72). It is conceivable that the overallphysiological impact of ketone bodiesfollows an inverted U-shaped curve,whereby mild elevations are beneficial,but higher levelsmay be harmful. Accord-ing to the present evidence, treatmentwith SGLT2 inhibitors appears to superim-pose intermittent mild hyperketonemiaon an otherwise normal daily pattern offast/feeding, with consistent but limitedrestriction of carbohydrate usage (24).More drastic changes in circulating ke-tone levels, carbohydrate availability,and fat composition may be counterpro-ductive. Above all, it should not be for-gotten that the cardioprotection of theEMPA-REG OUTCOME trial was seen in acohort of patients with T2D who were athigh CV risk.

FUTURE STUDIES

A viable hypothesis should be not justplausible but verifiable. To this end,the presence, amount, and time courseof SGLT2-induced ketonemia could bemeasured and related to cardiac out-comes in adequately powered clinicalstudies in patients with T2D. Short-term, low-rate b-hydroxybutyrate infu-sions could be used in hospital settingsto test their effects on metabolic andfunctional parameters in patients withdecompensated heart failure (59–63).The failing heart is insulin resistant (46)(Fig. 5) because of increased adrenergictone and the release of natriuretic pep-tides and inflammatory molecules (73).Furthermore, there is evidence thatmyocardial insulin resistance is an inde-pendent risk factor for mortality in sta-ble patients with chronic heart failure(74). Also, myocardial insulin resistanceis associated with a mismatch betweeninsulin-mediated glucose uptake andmyocardial blood flow, possibly as a re-sult of remodeling (75). However, we donot know whether reducing myocardialinsulin resistance would restore con-tractile function and improve prognosis.Functional studies (three-dimensional

Figure 3—b-hydroxybutyrate (b-HB) extrac-tion by the normal human heart duringgraded atrial pacing. RPP, rate pressureproduct. Data were recalculated from thestudy by Camici et al. (37).

Figure 4—Raised circulating FFAs are taken up by the liver and metabolized via b-oxidation.Under circumstances of reduced insulin/glucagon ratio, in liver mitochondria the acetyl-CoA iscondensed to form acetoacetate (AcAc, catalyzed by the mitochondrial isoform of 3-hydroxy-3-methylglutaryl synthase [mHMG-CoA]) and b-hydroxybutyrate (b-HB) (catalyzed by b-hydrox-ybutyrate dehydrogenase [HBD]). b-HB is then exported into the bloodstream, from which it isavidly taken up into the heart (through the monocarboxylate transporter [MCT]). In heartmitochondria, b-HB is converted to AcAc (by HBD), AcAc-CoA (catalyzed by succinyl-CoA:3-oxoacid CoA transferase [SCOT], with succinyl-CoA as the CoA donor), and finally acetyl-CoA(by mitochondrial thiolase [Th]), which then enters the TCA for oxidative phosphorylation (OxPhos). The excess acetyl-CoA restrains (dotted lines) further generation of acetyl-CoA frompyruvate (by inhibiting pyruvate dehydrogenase [PDH]) and from b-oxidation of fatty acids.Ketone body oxidation results in a more efficient oxidation of the mitochondrial coenzymeQ couple and an increase in the free energy of cytosolic ATP hydrolysis. The increase in hematocritcontributes to the improvement in cardiac efficiency by releasing more oxygen (O2) to themuscle.

1112 CV Protection in EMPA-REG OUTCOME Trial Diabetes Care Volume 39, July 2016

echocardiography and magnetic reso-nance spectroscopy) and imaging studies(MRI, positron emission tomography) in pa-tientswith diabeteswhohave ahighCV riskload could be performed before and afteran SGLT2 treatment course. Finally, if thethrifty substrate paradigm held up in high-risk patients, studies in patients with lowerCV riskwould generate clinically useful datato inform therapeutic strategies.

SUMMARY

The hypothesis posits that, under con-ditions of mild but persistent hyper-ketonemiadsuch as those that prevailduring treatment with SGLT2 inhibitorsdb-hydroxybutyrate is freely taken up bythe heart and oxidized in preference tofatty acids. This substrate selection im-proves the transduction of oxygen con-sumption into work efficiency in theendangered myocardium (and may alsoimprove metabolic status and function ofother organs, mainly the kidney). Thismechanism should cooperate with otherSGLT2-induced changes (reduced bloodpressure [10] and enhanced diuresis [11])to achieve the degree of cardioprotectionrevealed by the EMPA-REGOUTCOME trial.In addition, enhanced oxygen release tothemyocardium through hemoconcentra-tionwould be in powerful synergywith thesubstrate shift.

Funding. This work was aided in part by the Ital-ian Ministry of Education, University, and Re-search (grant #2010329EKE).Duality of Interest. E.F. has been a speaker andconsultant for Merck Sharp & Dohme, Sanofi, EliLilly and Company, Boehringer Ingelheim, Johnson& Johnson, and AstraZeneca. M.M. and E.M. areemployees of Boehringer Ingelheim PharmaGmbH & Co. KG, Biberach, Germany. No other

potential conflicts of interest relevant to this arti-cle were reported.Prior Presentation. Parts of this article werepresented at the 76th Scientific Sessions of theAmerican Diabetes Association, New Orleans,LA, 10–14 June 2016.

References1. Ferrannini E, DeFronzo RA. Impact of glu-cose-lowering drugs on cardiovasculare diseasein type 2 diabetes. Eur Heart J 2015;36:2288–22962. Zinman B,Wanner C, Lachin JM, et al.; EMPA-REG OUTCOME Investigators. Empagliflozin,cardiovascular outcomes, and mortality intype 2 diabetes. N Engl J Med 2015;373:2117–21283. Fitchett D, Zinman B,Wanner C, et al.; EMPA-REG OUTCOME trial investigators. Heart failureoutcomes with empagliflozin in patients withtype 2 diabetes at high cardiovascular risk: re-sults of the EMPA-REG OUTCOME trial. EurHeart J. 26 January 2016 [Epub ahead of print].DOI: 10.1093/eurheartj/ehv7284. McMurray JJ, Adamopoulos S, Anker SD,et al.; Task Force for the Diagnosis and Treat-ment of Acute and Chronic Heart Failure 2012of the European Society of Cardiology; ESCCommittee for Practice Guidelines. ESC guide-lines for the diagnosis and treatment of acuteand chronic heart failure 2012: The Task Forcefor the Diagnosis and Treatment of Acute andChronic Heart Failure 2012 of the EuropeanSociety of Cardiology. Developed in collabo-ration with the Heart Failure Association(HFA) of the ESC. Eur J Heart Fail 2012;14:803–8695. Ryden L, Grant PJ, Anker SD, et al.; Authors/Task Force Members; ESC Committee for Prac-tice Guidelines (CPG); Document Reviewers.ESC Guidelines on diabetes, pre-diabetes, andcardiovascular diseases developed in collabora-tion with the EASD: the Task Force on diabetes,pre-diabetes, and cardiovascular diseases of theEuropean Society of Cardiology (ESC) and devel-oped in collaboration with the European Asso-ciation for the Study of Diabetes (EASD). EurHeart J 2013;34:3035–30876. Inzucchi SE, Zinman B, Wanner C, et al. SGLT-2inhibitors and cardiovascular risk: proposed path-ways and review of ongoing outcome trials. DiabVasc Dis Res 2015;12:90–1007. Muskiet MHA, van Raalte DH, van Bommel E,Smits MM, Tonneijck L. Understanding EMPA-REG OUTCOME. Lancet Diabetes Endocrinol2015;3:928–9298. Ceriello A, Genovese S, Mannucci E, GrondaE. Understanding EMPA-REG OUTCOME. LancetDiabetes Endocrinol 2015;3:929–9309. Gilbert RE, Connelly KA. UnderstandingEMPA-REGOUTCOME. Lancet Diabetes Endocri-nol 2015;3:930–93110. DeFronzo RA. The EMPA-REG study: whathas it told us? A diabetologist’s perspective.J Diabetes Complications 2016;30:1–211. McMurray J. EMPA-REG - the “diuretichypothesis”. J Diabetes Complications 2016;30:3–412. Turnbull FM, Abraira C, Anderson RJ, et al.;Control Group. Intensive glucose control andmacrovascular outcomes in type 2 diabetes. Di-abetologia 2009;52:2288–2298

13. Komoroski B, Vachharajani N, Feng Y, Li L,Kornhauser D, Pfister M. Dapagliflozin, a novel,selective SGLT2 inhibitor, improved glycemiccontrol over 2 weeks in patients with type 2 di-abetes mellitus. Clin Pharmacol Ther 2009;85:513–51914. Cherney DZ, Perkins BA, Soleymanlou N,et al. Renal hemodynamic effect of sodium-glucose cotransporter 2 inhibition in patientswith type 1 diabetes mellitus. Circulation2014;129:587–59715. Emdin CA, Rahimi K, Neal B, Callender T,Perkovic V, Patel A. Blood pressure lowering intype 2 diabetes: a systematic review and meta-analysis. JAMA 2015;313:603–61516. Psaty BM, Lumley T, Furberg CD, et al.Health outcomes associated with various anti-hypertensive therapies used as first-line agents:a networkmeta-analysis. JAMA2003;289:2534–254417. Olde Engberink RH, Frenkel WJ, van denBogaard B, Brewster LM, Vogt L, van den BornBJ. Effects of thiazide-type and thiazide-like di-uretics on cardiovascular events and mortality:systematic review and meta-analysis. Hyperten-sion 2015;65:1033–104018. Zannad F, McMurray JJ, Krum H, et al.;EMPHASIS-HF Study Group. Eplerenone in pa-tients with systolic heart failure and mild symp-toms. N Engl J Med 2011;364:11–2119. Lambers Heerspink HJ, de Zeeuw D, Wie L,Leslie B, List J. Dapagliflozin a glucose-regulatingdrug with diuretic properties in subjects withtype 2 diabetes. Diabetes Obes Metab 2013;15:853–86220. Roush GC, Kaur R, Ernst ME. Diuretics: areview and update. J Cardiovasc Pharmacol Ther2014;19:5–1321. Devenny JJ, Godonis HE, Harvey SJ, RooneyS, Cullen MJ, Pelleymounter MA. Weight lossinduced by chronic dapagliflozin treatment isattenuated by compensatory hyperphagia indiet-induced obese (DIO) rats. Obesity (SilverSpring) 2012;20:1645–165222. Yokono M, Takasu T, Hayashizaki Y, et al.SGLT2 selective inhibitor ipragliflozin reducesbody fat mass by increasing fatty acid oxidationin high-fat diet-induced obese rats. Eur J Phar-macol 2014;727:66–7423. SuzukiM, TakedaM, Kito A, et al. Tofogliflozin,a sodium/glucose cotransporter 2 inhibitor, atten-uates body weight gain and fat accumulation indiabetic and obese animal models. Nutr Diabetes2014;4:e12524. Ferrannini E, Muscelli E, Frascerra S, et al.Metabolic response to sodium-glucose cotrans-porter 2 inhibition in type 2 diabetic patients.J Clin Invest 2014;124:499–50825. McGarry JD, Foster DW. Regulation of he-patic fatty acid oxidation and ketone body pro-duction. Annu Rev Biochem 1980;49:395–42026. Cahill GF Jr. Fuel metabolism in starvation.Annu Rev Nutr 2006;26:1–2227. Ferrannini E, Baldi S, Frascerra S, et al. Shiftto fatty substrates utilization in response to so-dium-glucose co-transporter-2 inhibition innondiabetic subjects and type 2 diabetic pa-tients. Diabetes. 9 February 2016 [Epub aheadof print]. DOI: 10.2337/db15-135628. Nishimura R, Tanaka Y, Koiwai K, et al. Effectof empagliflozin monotherapy on postprandialglucose and 24-hour glucose variability in

Figure 5—Direct association between insu-lin-induced myocardial glucose uptake andejection fraction in control subjects and T2Dpatients. Redrawn with permission fromIozzo et al. (46).

care.diabetesjournals.org Ferrannini, Mark, and Mayoux 1113

Japanese patients with type 2 diabetes mellitus:a randomized, double-blind, placebo-controlled,4-week study. Cardiovasc Diabetol 2015;14:1129. Nishimura R, Osonoi T, Kanada S, et al. Effectsof luseogliflozin, a sodium-glucose co-transporter2 inhibitor, on 24-h glucose variability assessedby continuous glucose monitoring in Japanesepatients with type 2 diabetes mellitus: a ran-domized, double-blind, placebo-controlled,crossover study. Diabetes Obes Metab 2015;17:800–80430. Nishimura R, Omiya H, Sugio K, Ubukata M,Sakai S, Samukawa Y. The sodium-glucose co-transporter 2 inhibitor luseogliflozin improvesglycaemic control assessed by continuous glu-cose monitoring even on a low-carbohydratediet. Diabetes Obes Metab. 6 December 2015[Epub ahead of print]. DOI: 10.1111/dom.1261131. Ikeda S, Takano Y, Cynshi O, et al. A noveland selective sodium-glucose cotransporter-2inhibitor, tofogliflozin, improves glycaemic con-trol and lowers body weight in patients withtype 2 diabetes mellitus. Diabetes Obes Metab2015;17:984–99332. Inagaki N, Kondo K, Yoshinari T, TakahashiN, Susuta Y, Kuki H. Efficacy and safety of cana-gliflozin monotherapy in Japanese patients withtype 2 diabetes inadequately controlled withdiet and exercise: a 24-week, randomized, dou-ble-blind, placebo-controlled, phase III study.Expert Opin Pharmacother 2014;15:1501–151533. Balasse EO, Fery F. Ketone body productionand disposal: effects of fasting, diabetes, andexercise. Diabetes Metab Rev 1989;5:247–27034. Natali A, Buzzigoli G, Taddei S, et al. Effectsof insulin on hemodynamics and metabolism inhuman forearm. Diabetes 1990;39:490–50035. Bratusch-Marrain PR, DeFronzo RA. Failureof hyperketonemia to alter basal and insulin-mediated glucose metabolism in man. HormMetab Res 1986;18:185–18936. Ferrannini E, Santoro D, Bonadonna R,Natali A, Parodi O, Camici PG. Metabolic andhemodynamic effects of insulin on humanhearts. Am J Physiol 1993;264:E308–E31537. Camici PG, Marraccini P, Lorenzoni R, et al.Coronary hemodynamics and myocardial me-tabolism in patients with syndrome X: responseto pacing stress. J Am Coll Cardiol 1991;17:1461–147038. Garland PB, Newsholme EA, Randle PJ. Reg-ulation of glucose uptake by muscle. 9. Effectsof fatty acids and ketone bodies, and of alloxan-diabetes and starvation, on pyruvate metabo-lism and on lactate-pyruvate and L-glycerol3-phosphate-dihydroxyacetone phosphate con-centration ratios in rat heart and rat diaphragmmuscles. Biochem J 1964;93:665–67839. Kashiwaya Y, King MT, Veech RL. Substratesignaling by insulin: a ketone bodies ratiomimics insulin action in heart. Am J Cardiol1997;80:50A–64A40. Stanley WC, Meadows SR, Kivilo KM, RothBA, Lopaschuk GD. b-Hydroxybutyrate inhibitsmyocardial fatty acid oxidation in vivo indepen-dent of changes in malonyl-CoA content. Am JPhysiol Heart Circ Physiol 2003;285:H1626–H163141. Sato K, Kashiwaya Y, Keon CA, et al. Insulin,ketone bodies, and mitochondrial energy trans-duction. FASEB J 1995;9:651–658

42. Boveris A, Oshino N, Chance B. The cellularproduction of hydrogen peroxide. Biochem J1972;128:617–63043. Murphy MP. How mitochondria producereactive oxygen species. Biochem J 2009;417:1–1344. Shimazu T, Hirschey MD, Newman J, et al.Suppression of oxidative stress by b-hydroxybu-tyrate, an endogenous histone deacetylase in-hibitor. Science 2013;339:211–21445. Cotter DG, Schugar RC, Crawford PA. Ke-tone body metabolism and cardiovascular dis-ease. Am J Physiol Heart Circ Physiol 2013;304:H1060–H107646. Iozzo P, Chareonthaitawee P, Dutka D,Betteridge DJ, Ferrannini E, Camici PG. Indepen-dent association of type 2 diabetes and coro-nary artery disease with myocardial insulinresistance. Diabetes 2002;51:3020–302447. Paternostro G, Camici PG, Lammerstma AA,et al. Cardiac and skeletal muscle insulin resis-tance in patients with coronary heart disease.A study with positron emission tomography.J Clin Invest 1996;98:2094–209948. Camici P, Marraccini P, Lorenzoni R, et al.Metabolic markers of stress-induced myocardi-al ischemia. Circulation 1991;83(Suppl.):III8–III1349. Camici P, Ferrannini E, Opie LH. Myocardialmetabolism in ischemic heart disease: basicprinciples and application to imaging by posi-tron emission tomography. Prog CardiovascDis 1989;32:217–23850. Lopaschuk GD, Ussher JR, Folmes CDL,Jaswal JS, StanleyWC.Myocardial fatty acid me-tabolism in health and disease. Physiol Rev2010;90:207–25851. Stanley WC, Recchia FA, Lopaschuk GD.Myocardial substrate metabolism in the normaland failing heart. Physiol Rev 2005;85:1093–112952. Neubauer S. The failing heartdan engineout of fuel. N Engl J Med 2007;356:1140–115153. Chouchani ET, Pell VR, Gaude E, et al. Is-chaemic accumulation of succinate controls re-perfusion injury through mitochondrial ROS.Nature 2014;515:431–43554. TanakaM, Nishigaki Y, Fuku N, Ibi T, SahashiK, Koga Y. Therapeutic potential of pyruvatetherapy for mitochondrial diseases. Mitochon-drion 2007;7:399–40155. Fujii T, Nozaki F, Saito K, et al. Efficacy ofpyruvate therapy in patients with mitochondrialdisease: a semi-quantitative clinical evaluationstudy. Mol Genet Metab 2014;112:133–13856. Aubert G, Martin OJ, Horton JL, et al. Thefailing heart relies on ketone bodies as a fuel.Circulation 2016;133:698–70557. Janardhan A, Chen J, Crawford PA. Alteredsystemic ketone body metabolism in advancedheart failure. Tex Heart Inst J 2011;38:533–53858. Bedi KC Jr, Snyder NW, Brandimarto J, et al.Evidence for intramyocardial disruption of lipidmetabolism and myocardial ketone utilizationin advanced human heart failure. Circulation2016;133:706–71659. Du Z, Shen A, Huang Y, et al. 1H-NMR-basedmetabolic analysis of human serum revealsnovel markers of myocardial energy expendi-ture in heart failure patients. PLoS One 2014;9:e88102

60. Kolwicz SC Jr, Airhart S, Tian R. Ketones stepto the plate: a game changer for metabolic re-modeling in heart failure? Circulation 2016;133:689–69161. Mudaliar S, Polidori D, Zambrowicz B, HenryRR. Sodium-glucose cotransporter inhibitors:effects on renal and intestinal glucose trans-port: from bench to bedside. Diabetes Care2015;38:2344–235362. Testani JM, Chen J, McCauley BD, KimmelSE, Shannon RP. Potential effects of aggressivedecongestion during the treatment of decom-pensated heart failure on renal function andsurvival. Circulation 2010;122:265–27263. McBride BF, White CM. Anemia manage-ment in heart failure: a thick review of thindata. Pharmacotherapy 2004;24:757–76764. Martini J, Tsai AG, Cabrales P, Johnson PC,Intaglietta M. Increased cardiac output and mi-crovascular blood flow during mild hemocon-centration in hamster window model. Am JPhysiol Heart Circ Physiol 2006;291:H310–H31765. Mentz RJ, O’Connor CM. Pathophysiologyand clinical evaluation of acute heart failure.Nat Rev Cardiol 2016;13:28–3566. Greene SJ, Gheorghiade M, VaduganathanM, et al.; EVEREST Trial investigators. Haemo-concentration, renal function, and post-dischargeoutcomes among patients hospitalized for heartfailure with reduced ejection fraction: insightsfrom the EVEREST trial. Eur J Heart Fail 2013;15:1401–141167. van der Meer P, Postmus D, Ponikowski P,et al. The predictive value of short-term changesin hemoglobin concentration in patients pre-senting with acute decompensated heart fail-ure. J Am Coll Cardiol 2013;61:1973–198168. Cahill GF Jr, Veech RL. Ketoacids? Goodmedicine? Trans Am Clin Climatol Assoc 2003;114:149–16169. Veech RL. The therapeutic implications ofketone bodies: the effects of ketone bodies inpathological conditions: ketosis, ketogenic diet,redox states, insulin resistance, and mitochon-drial metabolism. Prostaglandins Leukot EssentFatty Acids 2004;70:309–31970. Veech RL, Chance B, Kashiwaya Y, Lardy HA,Cahill GF Jr. Ketone bodies, potential therapeu-tic uses. IUBMB Life 2001;51:241–24771. Astrup A,Meinert Larsen T, Harper A. Atkinsand other low-carbohydrate diets: hoax or aneffective tool for weight loss? Lancet 2004;364:897–89972. Rosenstock J, Ferrannini E. Euglycemic di-abetic ketoacidosis: a predictable, detectable,and preventable safety concern with SGLT2 in-hibitors. Diabetes Care 2015;38:1638–164273. Doehner W, Frenneaux M, Anker SD. Met-abolic impairment in heart failure: the myocar-dial and systemic perspective. J Am Coll Cardiol2014;64:1388–140074. Doehner W, Rauchhaus M, Ponikowski P,et al. Impaired insulin sensitivity as an indepen-dent risk factor for mortality in patients withstable chronic heart failure. J Am Coll Cardiol2005;46:1019–102675. Iozzo P, Chareonthaitawee P, Rimoldi O,Betteridge DJ, Camici PG, Ferrannini E. Mismatchbetween insulin-mediated glucose uptake andblood flow in the heart of patients with type IIdiabetes. Diabetologia 2002;45:1404–1409

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