Andrea R. Nawrocki,1 Carlos G. Rodriguez,1 Dawn M. Toolan,2 Olga Price,1 Melanie Henry,1 Gail Forrest,1 Daphne Szeto,1

Carol Ann Keohane,1 Yie Pan,1 Karen M. Smith,3 Izzat T. Raheem,4 Christopher D. Cox,4 Joyce Hwa,1 John J. Renger,2 andSean M. Smith2

Genetic Deletion andPharmacological Inhibition ofPhosphodiesterase 10AProtects Mice From Diet-Induced Obesity and InsulinResistance

Phosphodiesterase 10A (PDE10A) is a noveltherapeutic target for the treatment ofschizophrenia. Here we report a novel role ofPDE10A in the regulation of caloric intake andenergy homeostasis. PDE10A-deficient mice areresistant to diet-induced obesity (DIO) andassociated metabolic disturbances. Inhibition ofweight gain is due to hypophagia after mice are feda highly palatable diet rich in fats and sugar but nota standard diet. PDE10A deficiency producesa decrease in caloric intake without affecting mealfrequency, daytime versus nighttime feedingbehavior, or locomotor activity. We tested THPP-6,a small molecule PDE10A inhibitor, in DIO mice.THPP-6 treatment resulted in decreased food intake,body weight loss, and reduced adiposity at dosesthat produced antipsychotic efficacy in behavioralmodels. We show that PDE10A inhibition increasedwhole-body energy expenditure in DIO mice feda Western-style diet, achieving weight loss andreducing adiposity beyond the extent seen with foodrestriction alone. Therefore, chronic THPP-6treatment conferred improved insulin sensitivity and

reversed hyperinsulinemia. These data demonstratethat PDE10A inhibition represents a novelantipsychotic target that may have additionalmetabolic benefits over current medications forschizophrenia by suppressing food intake,alleviating weight gain, and reducing the risk for thedevelopment of diabetes.Diabetes 2014;63:300–311 | DOI: 10.2337/db13-0247

Atypical antipsychotic medications constitute the front-line treatment for schizophrenia; however, they havea high rate of discontinuation resulting from dissatis-faction with efficacy and a general lack of tolerability (1).Several atypical antipsychotics induce weight gain andproduce adverse metabolic effects that contribute tomorbidity and drive patient noncompliance with pre-scribed medications (2). Increases in weight gain vary andrange from modest changes with aripiprazole to signifi-cant increases with olanzapine (3,4). Metabolic risks as-sociated with these medications include increased insulinresistance, hyperglycemia, dyslipidemia, and type 2 di-abetes (3–5). Therefore, there is a pressing need to

1In Vivo Pharmacology, Merck Research Laboratories, Rahway, NJ2Neuroscience, Merck Research Laboratories, West Point, PA3In Vivo Pharmacology, Merck Research Laboratories, West Point, PA4Medicinal Chemistry, Merck Research Laboratories, West Point, PA

Corresponding author: Sean M. Smith, sean_smith2@merck.com.

Received 12 February 2013 and accepted 29 September 2013.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0247/-/DC1.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

300 Diabetes Volume 63, January 2014

PHARMACOLOGYAND

THERAPEUTIC

S

identify novel therapeutics to treat schizophrenia that donot induce weight gain or have an increased risk forproducing metabolic adverse effects in patients.

Cyclic nucleotide phosphodiesterases (PDEs) area family of enzymes that selectively degrade cyclic AMP(cAMP) and cyclic GMP (cGMP). There are 11 differentfamilies of PDEs that vary in their substrate specificity,kinetic properties, modes of regulation, intracellular lo-calization, and tissue expression patterns (6–8). PDE 10A(PDE10A) is a dual-substrate PDE that degrades bothcAMP and cGMP. PDE10A is encoded by a single genethat is highly expressed in the brain and has limitedexpression in the peripheral tissues (6,9). In the brain,PDE10A is highly concentrated in the striatum and isexpressed at lower levels in other brain regions (10–12).In peripheral tissues, measurable levels of PDE10A ex-pression are limited to the testis and pancreatic islet cells(9,10,13,14).

Substantial evidence has been generated supportingthe hypothesis that PDE10A may be a novel therapeutictarget for the treatment of schizophrenia. PDE10A-deficient mice show reduced exploratory behavior whenplaced in a novel environment and have a blunted re-sponse to the psychomotor activating effects ofN-methyl-D-aspartate receptor (NMDA) receptor antag-onists phencyclidine and MK-801 (15,16). SelectivePDE10A inhibitors decrease psychomotor activity, re-verse deficits in prepulse inhibition, and inhibit condi-tioned avoidance responding in rodents, models thatare predictive of antipsychotic activity in the clinic(17–21). PDE10A inhibitors are also efficacious in pre-clinical assays that test cognitive domains impaired inschizophrenia (17,20,22,23) and models of negativesymptoms (17).

Here we describe a novel role for PDE10A in theregulation of caloric intake and energy homeostasis. Us-ing PDE10A knockout (PDE10A2/2) mice and a selectivesmall molecule PDE10A inhibitor, we investigated therole of PDE10A in regulating body weight, feeding be-havior, locomotor activity, and energy expenditure inmice fed a standard diet or a highly palatable diet rich infats and carbohydrates. We show that pharmacologicalinhibition of PDE10A activity leads to a dose-dependentsuppression of food intake and increased energy expen-diture in obese mice with subsequent reduction of adi-posity and improved insulin sensitivity. Thus, wedescribe a novel role for PDE10A in the homeostaticregulation of energy metabolism, possibly by integratingperipheral satiety and adiposity signals with centralregulation of food intake.

RESEARCH DESIGN AND METHODS

Animals

Experiments were performed in lean and diet-inducedobese (DIO) C57Bl/6NTac mice (Taconic, Germantown,NY). PDE10A2/2 mice were generated by replacinga fragment of 2,756 nucleotides (bp 1,947 to 2,000) with

the Neo cassette between exons 16 and 17 of thePDE10A gene. After backcrossing into C57Bl/6Ntac,99.6% of the C57Bl/6 genome was confirmed. Animalswere individually housed under a 12-h light/dark cycleand fed ad libitum a standard diet (Teklad 7012; HarlanTeklad, Indianapolis, IN), a high-fat diet (HFD; D12492:60% kcal from fat; Research Diets, New Brunswick, NJ),or a Western-style diet containing 32% kcal from milk fatand corn-oil (D12266B) with ad libitum water. All pro-tocols for the use of these animals were approved by theMerck Research Laboratories Institutional Animal Careand Use Committee.

Metabolic Phenotyping

At 8 weeks of age, male PDE10A2/2 and wild-type (WT)mice (n = 10–13 per group) were switched on HFD adlibitum for 10 weeks. Whole-body composition wasquantified by magnetic resonance analysis (Echo MedicalSystems, Houston, TX). Leptin and adiponectin (MesoScale Discovery, Gaithersburg, MD), insulin (PerkinElmer,Waltham, MA), and glucose (OneTouch Ultra, LifeScan,Milpitas, CA), were measured using commercially availableassays. Triglycerides, cholesterol, aspartate aminotrans-ferase (AST), and alanine aminotransferase (ALT) levelswere measured using an automated analyzer (Roche,Indianapolis, IN).

THPP-6 [2-(6-chloropyridin-3-yl)-4-[(2S)-1-methoxypropan-2-yl]oxy-N-(6-methylpyridin-3-yl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxamide]was synthesized in-house (18) and was dosed by oralgavage in 5% Tween80/0.25% methylcellulose. AM251[N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]was purchased from (Tocris, Ellisville, MO). For glucosetolerance tests (GTTs), 6-h fasted mice were given1.5 g/kg glucose intraperitoneally (i.p.), and glucose wasmeasured by glucometer (OneTouch Ultra) at 0, 20, 40,60 and 120 min. Blood (2.5 mL) was collected into 13PBS/EDTA (12.5 mL) for insulin determination.

Data were analyzed using repeated-measures ANOVAwith Bonferroni post hoc analysis or unpaired Studentt tests. Homeostasis model assessment of insulin re-sistance (HOMA-IR) was calculated as [fasting insulin(mU/L) 3 fasting glucose (mmol/L)]/22.5.

Indirect Calorimetry

Male PDE10A2/2 mice and their littermates were ana-lyzed at 10 weeks of age (n = 12 per group). They wereacclimated to individual boxes within the OxyMax sys-tem (Columbus Instruments, Columbus, OH). Total andambulatory locomotor activity, food intake, and Vo2 andVCO2 were measured in 30-min intervals, and the re-spiratory exchange ratio (RER) was calculated as VCO2/VO2. Heat (energy expenditure) was calculated as CV 3VO2 subject 3 body weight0.75, with CV = 3.815 + 1.2323 RER (24). Total locomotor activity was expressed asone count per two consecutive x-axis infrared beam

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breaks, and fine movement was defined a single-beambreak. Food intake and locomotor activity data were ex-cluded 30 min before and after handling of the mice.

For pair-feeding studies, C57Bl/6 mice were fed anHFD (D12492) for 16 weeks and then switched toD12266B for 4 weeks before study. Mice were acclimatedto pair-feeding for 1 week. The average feeding activitypattern of the group treated with 10 mg/kg THPP-6 wasimposed on the pair-fed group in 10-min intervals, andpair-feeding was achieved by automatically controllingaccess to the food hopper.

Pharmacokinetic Studies

Plasma concentration of THPP-6 was sampled from 2 to24 h after oral administration and quantified usinga SCIEX API5000 Q-Trap mass spectrometer (AppliedBiosystems, Concord, ON, Canada). Plasma samples wereseparated on an Acquity UPLC HSS T3 C18 column(50 mm 3 2.1 mm 3 1.8 mm), with a mobile phaseconsisting of solvent A (water with 0.1% formic acid) andsolvent B (acetonitrile with 0.1% formic acid). THPP-6and the internal standard were monitored in the positiveion mode at the transition from m/z 469.10 to 134.90and m/z 309.2 to 205.1, respectively. The concentrationof THPP-6 in the samples was determined using Multi-Quant 2.1 based on standard curve and quality controlsamples.

In Vitro PDE Activity Assays

PDE activity was determined in duplicate at room tem-perature using an IMAP FP kit (Molecular Devices, Sun-nyvale, CA) (20). Human PDE10A2 and rhesus monkeyPDE2A3 enzymes were prepared from cytosolic fractionsof transiently transfected AD293 cells, as described (20).All other PDEs (PDE1A, PDE3A, PDE4A1A, PDE5A1,PDE6C, PDE7A, PDE8A1, PDE9A2, and PDE11A4) wereglutathione S-transferase–tagged human enzymesexpressed in insect cells and were obtained from BPSBioscience (San Diego, CA). The apparent inhibitionconstant (Ki) for THPP-1 against the 11 PDEs was de-termined using the apparent KM values for each enzyme,as described by Smith et al. (20).

Measurement of MK-801–Induced PsychomotorActivity

Male C57Bl/6 mice were used to examine the influence ofTHPP-6 on MK-801–induced locomotor activity. Afterhabituation, animals were given vehicle (10% Tween80/90% water) or THPP-6 (1, 3, 10, or 30 mg/kg orally),followed 120 min later by vehicle (saline) or MK-801(0.25 mg/kg i.p.). Animals were placed in locomotor ac-tivity monitors (21 3 42 cm; Kinder Scientific, Julian,CA) 90 min after injection of THPP-6 and left in theactivity monitors 90 min after being given MK-801. Totaldistance traveled was used to assess the influence ofTHPP-6 administration on activity during habituationand after MK-801 treatment. Locomotor activity wasanalyzed with one-way ANOVA (group as a between-subjects

factor), and group differences were further examinedusing post hoc comparisons (Fisher least significantdifference).

Statistical Analysis

Data are expressed as means 6 SEM. Statistical analysiswas conducted using ANOVA or unpaired Studentt test, as indicated. Statistical significance was definedas P , 0.05.

RESULTS

PDE10A2/2 Mice Are Protected From HFD-InducedWeight Gain and Associated Metabolic Disturbances

To explore a functional role of PDE10A in energy me-tabolism and feeding behavior, we challenged PDE10A2/2

mice and WT controls with an HFD, which was high insaturated fats (60% kcal from lard and soybean oil, 20%kcal carbohydrates). Diet-induced body weight gain wasinhibited [ANOVA, F(1,21) = 41.26, P , 0.01] inPDE10A2/2 mice and associated with a reduction in ca-loric intake (Fig. 1A, Table 1). Cumulative food intakewas significantly reduced in PDE10A2/2 compared withWT mice [ANOVA, F(1,21) = 28.30, P, 0.01; Fig. 1B]. DIOresistance correlated with reduced fat (t test, P , 0.01)and lean mass (t test, P , 0.01; Fig. 1C ) and reducedleptin levels in PDE10A2/2 mice (16.8 6 3.4 ng/mL)versus WT controls (65.04 6 4.1 ng/mL; Fig. 1D).

Feeding of an HFD leads to modest diet-induced in-sulin resistance and dyslipidemia in C57Bl/6 mice. After10 weeks on the HFD, WT control mice developed hy-perglycemia (286 6 13 mg/dL) and mild hyper-insulinemia (6.5 6 1.7 ng/mL; Table 1). Deletion ofPDE10A was associated with a normalization of themetabolic profile evidenced by improved fed plasmaglucose (232 6 7 mg/dL; t test vs. WT, P , 0.01) andinsulin levels (1.1 6 0.3 ng/mL; t test vs. WT, P , 0.05;Table 1). Improved insulin sensitivity was indicated bya reduction in HbA1c (Table 1). PDE10A

2/2 mice showedlower total cholesterol levels in plasma and a trend to-ward reduced triglyceride levels in plasma compared withcontrols, indicative of improved lipid metabolism (Table1). A significant reduction of AST levels (t test, P , 0.01)suggested overall improved liver function in PDE10A2/2

mice.

Diet-Dependent Suppression of Food Intake inPDE10A2/2 Mice

To investigate the involvement of PDE10A in the regu-lation of food intake and whole-body energy metabolism,we placed PDE10A2/2 mice in metabolic rate chambersand determined feeding behavior, locomotor activity,energy expenditure, and RER in mice when fed a regularchow and after switching them to the HFD. No genotypicdifferences were found in cumulative food intake or dailyfood intake when mice were fed a regular chow diet(Fig. 2A–C). As observed previously, introduction ofa highly palatable HFD suppressed food intake inPDE10A2/2 mice [ANOVA, F(1,19) = 4.44; P , 0.05],

302 PDE10A Inhibition Decreases Weight Gain Diabetes Volume 63, January 2014

resulting in resistance to diet-induced body weight gain[ANOVA, F(1,22) = 58.77; P , 0.001; Fig. 2A and B]. Thesuppression of food intake was not associated witha change in circadian behavior or meal patterns; however,30-min food intake was significantly reduced [ANOVA,F(1,22) = 20.11; P, 0.01; Fig. 2D]. Total distance traveledand fine locomotor activity was unaffected by genotypeor diet (Fig. 2E and F). Leptin levels (16.3 6 1.74 vs.4.3 6 0.66 ng/mL, respectively; t test, P , 0.001) andinsulin (6.5 6 1.7 vs. 1.1 6 0.3 ng/mL, respectively;t test, P , 0.05) were also reduced in PDE10A2/2 micecompared with WT mice.

Using indirect calorimetry, we investigated the im-plication of a PDE10A deficiency on whole-body energyexpenditure and nutrient utilization. Net metabolic ratewas unchanged on the chow diet or after switchingPDE10A2/2 mice to an HFD (Supplementary Fig. 1Aand B). We hypothesize that potential effects on energyexpenditure may have been masked by compensatorydecreases in energy expenditure due to reduced caloricintake. The RER ( CO2-to-O2 ratio) was similar mice fedthe chow diet (Supplementary Fig. 1C) but was reduced

in PDE10A2/2 mice within 24 h after switching to theHFD [ANOVA, F(1,22) = 17.79, P , 0.001]. These findingssuggest that a shift in nutrient partitioning toward in-creased fatty acid oxidation relative to glucose oxidationlikely contributed to obesity resistance (SupplementaryFig. 1D).

Inhibition of PDE10A Causes Weight Loss andMetabolic Improvements in Obese Mice

A small molecule PDE10A inhibitor, THPP-6, was used tofurther characterize the effects of PDE10A inhibition onbody weight, glucose, and lipid metabolism in DIO mice.THPP-6 (Fig. 3A) is a potent inhibitor of PDE10A (Ki =0.92 6 0.08 nmol/L) as determined by a fluorescencepolarization assay measuring the ability of test com-pounds to inhibit hydrolysis of cAMP (25). THPP-6 isgreater than 300-fold selective over the other 10 PDEfamilies (Table 2) and did not have any significant ac-tivity when tested against a panel of more than 150receptors, enzymes, and ion channels (Panlabs Screen,MDS Pharma). The antipsychotic potential of THPP-6was evaluated in the psychostimulant-induced locomotoractivity assay, and THPP-6 produced a full attenuation of

Figure 1—PDE10A2/2 mice are resistant to DIO. A: Changes in body weight of male PDE10A2/2 (n = 10) and their littermate controls (WT,n = 13) after being fed an HFD (60% kcal from fat) for 10 weeks. Initial body weights were 24.2 6 0.5 g for WT and 20.4 6 0.7 g forPDE10A2/2 mice. B: Weekly HFD consumption expressed as cumulative food intake. C: Body composition at the end of the study wasassessed as the absolute amount of fat and lean mass as determined by quantitative nuclear magnetic resonance (qNMR) analysis.D: Plasma leptin levels after 10 weeks of HFD feeding. *P < 0.05 vs. WT control.

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MK-801 (NMDA receptor antagonist) activity in mice ata dose (10 mg/kg, orally) that yielded a total plasmaconcentration of 3.46 6 0.91 mmol/L (Fig. 3B). DIOmice were dosed with THPP-6 daily before the feedingperiod began. Steady-state plasma concentrations ofTHPP-6 were 0.28 6 0.03, 1.98 6 0.18, and 13.12 61.51 mmol/L after oral administration of 3, 10, and30 mg/kg THPP-6, respectively.

THPP-6 treatment caused significant body weightloss within 3 days at all doses [ANOVA, F(4,35) = 60.92,P , 0.01; Fig. 4A] comparable to efficacy with a CB1neutral antagonist, AM251, at full target engagement(Fig. 4A). THPP-6 treatment also decreased food intake[ANOVA, F(4,29) = 16.04, P , 0.01; Fig. 4B] and reducedfat mass (Fig. 4C and D). Reduced leptin [ANOVA,F(4,35) = 30.02, P , 0.01] and an increase in total adi-ponectin (Fig. 4E and F) was observed with THPP-6 butnot AM251 treatment [ANOVA, F(4,35) = 12.59, P, 0.001].Body weight loss was associated reduced fasting insulin[ANOVA, F(4,35) = 8.57, P , 0.001; Fig. 5A] and HOMA-IR[ANOVA, F(4,35) = 6.5, P, 0.001; Fig. 5B]. GTT results werenot affected by THPP-6 in this study (Fig. 5C). However,glucose-stimulated insulin release was reduced during theGTT [ANOVA, F(4,35) = 9.48, P , 0.001; Fig. 5D]. Insummary, PDE10A inhibition improved insulin sensitiv-ity by mechanisms that are likely secondary to bodyweight loss.

In a separate experiment, we investigated the effect offeeding of a Western-style diet on PDE10A-mediatedfood intake in obese mice. We confirmed that equivalentdoses of THPP-6 caused comparable suppression of food

intake and body weight loss independently of the sourceor the amount of the fat in the diet, that is, when fedlard- (60% kcal from fat) or milk fat–based (32% kcalfrom fat) diets (Figs. 4 and 6). DIO mice (initial bodyweight, 43.9 6 0.6 g) were treated with vehicle, 10 mg/kgTHPP-6, or were vehicle pair-fed to the average food in-take of the THPP-6–treated group for 13 days (n = 7 pergroup). Caloric intake was indistinguishable betweenTHPP-6–treated and pair-fed groups (average 212%cumulative reductions compared with vehicle group;Fig. 6A). Despite equal food intake, body weight loss wassignificantly greater in the THPP-6–treated group, in-dicating a feeding-independent mechanism (Studentt test; Fig. 6B). Notably, the first dose of THPP-6 causeda marked suppression of food intake (SupplementaryFig. 2) that was sustained for ;8 h postdose, followed bya compensatory increase on days 2 and 3. Food intakethen stabilized for the remainder of the study (85% ofthe control groups). Lean mass was not significantlydifferent between the THPP-6–treated mice and theirpair-fed group (Fig. 6C); however, loss in adipose tissuewas greater in the THPP-6 group than in the pair-fedgroup, suggesting that PDE10A inhibition promotesutilization fat independent of food intake.

Pair-feeding allowed us to uncover a PDE10A-relatedincrease in energy expenditure (Fig. 6D). Energy expen-diture was decreased in the pair-fed group as result ofreduced food intake but was reversed by THPP-6 treat-ment to normal levels. The differential drug effect washighly reproducible after 5 days of dosing and in-dependent of food intake and body weight. THPP-6treatment did not independently affect RER and trackedclosely with food intake (Fig. 6E and SupplementaryFig. 2). Ambulatory activity was comparable to vehicle(Fig. 6F), with the exception of a transient reductionafter the first dose that subsided quickly at steady-stateconcentrations of the drug. Our results indicate thatPDE10A inhibition affects body weight and adiposity dueto a combination of hypophagia and increased restingand active metabolic rate. In addition, the PDE10A in-hibitor THPP-6 causes hypophagia and increases energyexpenditure unrelated to the source of lipids and/orcomposition of the diets (Figs. 4 and 6).

DISCUSSION

Schizophrenia is a debilitating disorder estimated to ef-fect up to 1% of the general population (26). In additionto the well-defined psychiatric symptoms, schizophrenicpatients have an increased prevalence of obesity (27,28).A number of atypical antipsychotic medications have alsobeen reported to induce significant weight gain and in-crease the likelihood of developing type 2 diabetes andcardiovascular disease (3,4,29). Therefore, developingnovel antipsychotic medications that do not induceweight gain or that have the potential to reverse weightgain and metabolic effects produced by marketed anti-psychotic drugs is of utmost importance.

Table 1—PDE10A2/2 mice are protected from HFD-inducedhyperglycemia and hyperinsulinemia

10 weeks’ HFD

WT(n = 9)

PDE10A2/2

(n = 12) P

DBody weight (g) 22.1 6 0.9 10.8 6 1.1 ,0.01**

Cumulative foodintake (g) 187.1 6 3.8 170.9 6 5.2 ,0.01**

Ambient glucose(mg/dL) 286 6 13 232 6 7 ,0.01**

Insulin (ng/mL) 6.457 6 1.7 1.120 6 0.31 ,0.05*

Total cholesterol(mg/dL) 226.8 6 8.7 148.9 6 7.3 ,0.01**

Triglyceride(mg/dL) 77.2 6 4.4 71.1 6 7.2

Adiponectin(mg/mL) 28.01 6 0.97 27.02 6 1.23

HbA1c (%) 4.98 6 0.03 4.87 6 0.03 ,0.05*

ALT (units/L) 80 6 9.3 25 6 1.7 ,0.01**

AST (units/L) 80 6 5.4 69 6 8.6

*P , 0.05 vs. age-matched WT control. **P , 0.01.

304 PDE10A Inhibition Decreases Weight Gain Diabetes Volume 63, January 2014

In this study, we describe a novel role for PDE10A inthe regulation of caloric intake and energy expenditure.We found that deletion of PDE10A in mice leads to a pro-found suppression of food intake after exposure to highlypalatable Western-style diets containing an excess of fatsand sugars. This significant reduction of food consumptionconferred resistance to diet-induced weight gain inPDE10A2/2 mice and protection against the developmentof comorbidities typically associated with obesity such asinsulin resistance and dyslipidemia. PDE10A2/2 micehad normalized lipid profiles and a significant reductionin fasting insulin and glucose-stimulated insulin secre-tion compared with obese DIO controls (Table 1). These

changes correlated with improved HbA1c levels anddemonstrate a protective effect against the developmentof insulin resistance associated with obesity.

Using indirect calorimetry, we show that PDE10Aregulates feeding behavior and also influences overallmetabolic rate. Under conditions of HFD feeding,PDE10A2/2 mice exhibited a small but significant shift inRER toward increased lipid oxidation, whereas total heatwas unchanged. The increase in lipid oxidation at therelative cost of glucose utilization is reflective of a stateof reduced caloric intake when increased mobilization oflipids is necessary to maintain energy balance (24). Suchan increase in lipid oxidation may have contributed to

Figure 2—Deletion of PDE10A suppresses food intake when mice are fed a highly palatable HFD but not a standard low-fat diet (chow).Daily measures of body weight (A) and food intake (B) in PDE10A2/2 and littermate controls (WT) are shown. Food intake on chow (C) orHFD (D) was measured in 30-min intervals after 2 days of acclimation to the newly introduced diet. Locomotor activity was comparable inPDE10A2/2 and WT mice fed chow (E) or the HFD (F ). The shaded areas indicate active period (lights off). *P < 0.05 vs. WT control.

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a preservation of lean mass and a reduction of fat massin PDE10A2/2 mice. From these findings we speculatethat effects on total energy utilization in PDE10A2/2

mice were masked by a compensatory decrease in energyexpenditure associated with reduced caloric intake. Thenet balance therefore contributing to the obesity re-sistance in PDE10A2/2 mice is likely a combination ofreduced intake and increased metabolism.

Findings based on genetically deficient PDE10A micewere mirrored pharmacologically with a PDE10A in-hibitor, THPP-6. Chronic THPP-6 treatment of DIO micecaused significant body weight loss and suppression offood intake at similar plasma exposures that produceantipsychotic-like activity in animal models. Body com-position analysis indicated that body weight loss wasdriven by a reduction in body fat while lean mass wasmaintained. Inhibition of PDE10A activity producedbody weight loss significantly greater than data from thepair-fed group, revealing a previously not appreciatedrole of PDE10A in the regulation of energy expenditure.Importantly, these effects were sustained after multipledoses and chronic THPP-6 treatment caused metabolicimprovements in the absence of effects on locomotivebehavior.

Our data also indicate that the type of substrates usedby the body is not affected by PDE10A inhibition. Unlikethe results from the PDE10A2/2 mice, RER was over-lapping with the pair-fed group, suggesting that the com-pound did not affect substrate preference. However, thelack of an effect may be attributable to food restriction andcannot be dissected in this particular paradigm. Our pair-feeding experiments unequivocally demonstrate a food-independent component of PDE10A inhibition leading to

increased overall energy utilization. The mechanism forthis increase in metabolic rate is yet to be dissected ona molecular level, and greater understanding of the linkbetween PDE10A inhibition and peripheral oxidative ca-pacity is needed to fully interpret the utility of thismechanism. In any case, enhanced nutrient combustion inthe absence of cardiovascular effects (data not shown) andin combination with significant caloric restriction may beof great therapeutic interest.

Pharmacological inhibition of PDE10A was also asso-ciated with an improved metabolic profile in insulin-resistant DIO mice. Similar to PDE10A2/2 mice, fastinginsulin levels were reduced, and there was a trend towardimproved glucose disposal with reduced glucose-stimulatedinsulin release during a GTT. It is noteworthy that fasting

Figure 3—A: Chemical structure of the PDE10A inhibitor THPP-6. Papp, apparent permeability; Pgp, P-glycoprotein substrate; PPB,plasma protein binding. B: THPP-6 dose-dependently attenuated psychomotor activity induced by the NMDA receptor antagonist MK-801. Mean plasma concentrations of THPP-6 for each dosing group are listed above the dataset on the graph. **P < 0.01 vs. vehicle(V) + MK-801.

Table 2—Selectivity of THPP-6 for PDE10A compared withother human PDE families

PDE family Substrate Selectivity vs. PDE10A

PDE1A cGMP .1,0003

PDE2A cAMP .1,0003

PDE3A cAMP .1,0003

PDE4A cAMP .1,0003

PDE5A cGMP .3003

PDE6A cGMP .3003

PDE7A cAMP .1,0003

PDE8A cAMP .1,0003

PDE9A cGMP .1,0003

PDE11A cAMP .3003

306 PDE10A Inhibition Decreases Weight Gain Diabetes Volume 63, January 2014

insulin was reduced to a greater extent upon inhibition ofPDE10A than with similar body weight loss associatedwith AM251 treatment at full-target engagement, sug-gesting superior insulin-sensitizing efficacy. The CB1 re-ceptor antagonist AM251 is thought to drive metabolicimprovements predominantly through central regulationof appetite. Although CB1 receptors are expressed in

peripheral tissue, in our hands, non–brain-penetrantantagonists do not have insulin-sensitizing properties in-dependently of body weight loss (data not shown). To thateffect, reductions in fasting insulin correlated with an in-crease in circulating total adiponectin levels after THPP-6treatment that was absent in mice treated with our an-orexic control compound AM251. Adiponectin levels

Figure 4—Chronic dosing of a PDE10A inhibitor THPP-6 inhibited food intake in obese mice, leading to body weight loss and reducedadiposity. A: Daily change in body weight relative to baseline (average, 44 g). B: Cumulative food consumption over 13 days. Fat mass (C )and lean mass (D) were determined by quantitative magnetic resonance analysis on day 13. Plasma leptin (E) and total adiponectin (F )were measured after DIO mice received 13 days of THPP-6 treatment (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle.

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inversely correlate with adipose tissue inflammation, he-patic glucose output and peripheral glucose uptake, andadiposity (30). Taken together, our data indicate a poten-tial for additional body weight–independent benefits re-lated to PDE10A inhibition on handling of glucose thatshould be further investigated. However, whether PDE10Aactivity directly impacts metabolic parameters independentlyof CNS-mediated effects remains to be examined.

PDE10A mRNA is expressed in pancreatic islet cells,and researchers have reported that small-moleculePDE10A inhibitors function as insulin secretagogues invitro (31,32). Furthermore, Cantin et al. (31) demon-strated that PDE10A inhibitors improved glucose toler-ance and reduced insulin secretion in Wistar rats aftera glucose challenge, suggesting that the effects of THPP-6in DIO and HFD studies may be partly related to alteredinsulin secretion. In this study, we examined the effectsof THPP-6 on glucose-stimulated insulin release andshowed that THPP-6 did not significantly affect glucose-stimulated insulin release in isolated mouse islets (datanot shown). These data support the hypothesis that

THPP-6–mediated effects on glucose metabolism andinsulin levels are conferred predominantly by bodyweight loss and not through direct regulation of insulinsecretion.

Preclinical data pointing to nutrient-dependent effectson feeding behavior suggest that PDE10A plays a role incentral orexigenic pathways and/or with a rewardmechanism related to the palatability of various types ofnutrient and caloric content of food. This is supported bythe abundant expression of PDE10A in the brain andlimited distribution in peripheral tissue. Specifically,PDE10A is expressed at high levels in the mesolimbic andmesocortical dopamine systems, including the caudate,nucleus accumbens, hippocampus, and prefrontal cortex(10,11). Preclinical studies have linked cues from highlypalatable foods to multiple aspects of dopaminergic sig-naling. Mesolimbic dopaminergic neurons that innervatethe nucleus accumbens have been associated with moti-vation for highly palatable food and related rewardbehaviors (33–36). Furthermore, mesocortical dopami-nergic neurons innervate brain regions that regulate the

Figure 5—Pharmacological inhibition of PDE10A inhibition improved glucose metabolism by reducing fasting and postprandial insulin.Fasting insulin (A) and glucose (B) after 2 weeks of treatment with THPP-6 are shown. HOMA-IR was calculated for each treatment group.C: GTT after 13 days of treatment with THPP-6 in obese mice. Glucose (1.5 g/kg) was injected intraperitoneally after a 6-h fast, and bloodwas collected from the tail vein for glucose measurements. The inset shows area under the curve (AUC) values for 0–120 min of glucosechallenge. D: Reduced glucose-dependent insulin response after chronic THPP-6 treatment measured 20 min postglucose challenge(n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle control.

308 PDE10A Inhibition Decreases Weight Gain Diabetes Volume 63, January 2014

emotional response to eating, and dopaminergic neuronsthat innervate the caudate influence sensory-motoraspects of feeding (34,36,37). In addition to the directeffects of palatable foods on central dopaminergic sys-tems, a number of peripheral metabolic signals, includingleptin, insulin, and ghrelin, influence dopaminergic sys-tems (34,38,39). Taken together, these studies suggest

that PDE10A is positioned to play an important role inthe central regulation of feeding behavior.

In summary we have described a novel role forPDE10A in the regulation of feeding behavior and theregulation of metabolic rate. We have demonstrated thatgenetic deletion and pharmacological inhibition ofPDE10A both have significant effects on body weight and

Figure 6—THPP-6 increases energy expenditure and causes body weight loss in mice fed a Western-style diet independent of foodintake. Effect of vehicle, 10 mg/kg THPP-6, or pair-feeding to THPP-6 on daily food intake (A) and body weight (B) is shown. Linesindicate P < 0.05 vs. vehicle, *P < 0.05, **P < 0.01, ***P < 0.001 THPP-6 vs. pair-fed group. C: Changes in body compositionmeasured by quantitative nuclear magnetic resonance NMR at baseline and after 13 days of THPP-6 treatment. *P < 0.05, **P <0.01, ***P < 0.001 vs. vehicle. Energy expenditure (D), respiratory quotient (E ), and X-ambulatory activity (F ) are shown in 1-h bins forbaseline, day 1, and day 2 of dosing and for the last 4 days of steady-state dosing. Baseline and THPP-6 treatment periods areindicated by shaded areas.

diabetes.diabetesjournals.org Nawrocki and Associates 309

food intake in rodents and on improved insulin sensi-tivity. Furthermore, PDE10A inhibition increased energyexpenditure, leading to reduced adiposity and greaterweight loss than seen under conditions of equal caloricintake. These findings suggest that PDE10A inhibitorsmay be novel therapeutics for the treatment of schizo-phrenia with the potential to address weight gain andmetabolic issues associated with this disorder.

Funding. All work in the manuscript was funded by Merck ResearchLaboratories.

Duality of Interest. All authors are employees of Merck ResearchLaboratories. No other potential conflicts of interest relevant to this article werereported.

Author Contributions. A.R.N. and S.M.S. wrote and edited themanuscript. C.G.R., D.M.T., O.P., M.H., G.F., D.S., C.A.K., Y.P., K.M.S., I.T.R.,and C.D.C. generated data for the manuscript and contributed to discussion.J.H. and J.J.R. contributed to discussion and edited the manuscript. S.M.S. isthe guarantor of this work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the data and the accuracy ofthe data analysis.

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