300 Diabetes Volume 63, January 2014 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 and Sean M. Smith2 Genetic Deletion and Pharmacological Inhibition of Phosphodiesterase 10A Protects Mice From Diet- Induced Obesity and Insulin Resistance Phosphodiesterase 10A (PDE10A) is a novel reversed hyperinsulinemia. These data demonstrate therapeutic target for the treatment of that PDE10A inhibition represents a novel schizophrenia. Here we report a novel role of antipsychotic target that may have additional PDE10A in the regulation of caloric intake and metabolic benefits over current medications for energy homeostasis. PDE10A-deficient mice are schizophrenia by suppressing food intake, resistant to diet-induced obesity (DIO) and alleviating weight gain, and reducing the risk for the associated metabolic disturbances. Inhibition of development of diabetes. weight gain is due to hypophagia after mice are fed Diabetes 2014;63:300–311 | DOI: 10.2337/db13-0247 a highly palatable diet rich in fats and sugar but not PHARMACOLOGY AND THERAPEUTICS a standard diet. PDE10A deficiency produces a decrease in caloric intake without affecting meal Atypical antipsychotic medications constitute the front- frequency, daytime versus nighttime feeding line treatment for schizophrenia; however, they have behavior, or locomotor activity. We tested THPP-6, a high rate of discontinuation resulting from dissatis- a small molecule PDE10A inhibitor, in DIO mice. faction with efficacy and a general lack of tolerability (1). THPP-6 treatment resulted in decreased food intake, Several atypical antipsychotics induce weight gain and body weight loss, and reduced adiposity at doses produce adverse metabolic effects that contribute to that produced antipsychotic efficacy in behavioral morbidity and drive patient noncompliance with pre- models. We show that PDE10A inhibition increased scribed medications (2). Increases in weight gain vary and whole-body energy expenditure in DIO mice fed range from modest changes with aripiprazole to signifi- a Western-style diet, achieving weight loss and cant increases with olanzapine (3,4). Metabolic risks as- reducing adiposity beyond the extent seen with food sociated with these medications include increased insulin restriction alone. Therefore, chronic THPP-6 resistance, hyperglycemia, dyslipidemia, and type 2 di- treatment conferred improved insulin sensitivity and abetes (3–5). Therefore, there is a pressing need to 1In Vivo Pharmacology, Merck Research Laboratories, Rahway, NJ Corresponding author: Sean M. Smith, [email protected]. 2 Neuroscience, Merck Research Laboratories, West Point, PA Received 12 February 2013 and accepted 29 September 2013. 3In Vivo Pharmacology, Merck Research Laboratories, West Point, PA This article contains Supplementary Data online at http://diabetes 4Medicinal Chemistry, Merck Research Laboratories, West Point, PA .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. diabetes.diabetesjournals.org Nawrocki and Associates 301 identify novel therapeutics to treat schizophrenia that do the Neo cassette between exons 16 and 17 of the not induce weight gain or have an increased risk for PDE10A gene. After backcrossing into C57Bl/6Ntac, producing metabolic adverse effects in patients. 99.6% of the C57Bl/6 genome was confirmed. Animals Cyclic nucleotide phosphodiesterases (PDEs) are were individually housed under a 12-h light/dark cycle a family of enzymes that selectively degrade cyclic AMP and fed ad libitum a standard diet (Teklad 7012; Harlan (cAMP) and cyclic GMP (cGMP). There are 11 different Teklad, Indianapolis, IN), a high-fat diet (HFD; D12492: families of PDEs that vary in their substrate specificity, 60% kcal from fat; Research Diets, New Brunswick, NJ), kinetic properties, modes of regulation, intracellular lo- or a Western-style diet containing 32% kcal from milk fat calization, and tissue expression patterns (6–8). PDE 10A and corn-oil (D12266B) with ad libitum water. All pro- (PDE10A) is a dual-substrate PDE that degrades both tocols for the use of these animals were approved by the cAMP and cGMP. PDE10A is encoded by a single gene Merck Research Laboratories Institutional Animal Care that is highly expressed in the brain and has limited and Use Committee. expression in the peripheral tissues (6,9). In the brain, PDE10A is highly concentrated in the striatum and is Metabolic Phenotyping 2 2 expressed at lower levels in other brain regions (10–12). At 8 weeks of age, male PDE10A / and wild-type (WT) In peripheral tissues, measurable levels of PDE10A ex- mice (n =10–13 per group) were switched on HFD ad pression are limited to the testis and pancreatic islet cells libitum for 10 weeks. Whole-body composition was (9,10,13,14). quantified by magnetic resonance analysis (Echo Medical Substantial evidence has been generated supporting Systems, Houston, TX). Leptin and adiponectin (Meso the hypothesis that PDE10A may be a novel therapeutic Scale Discovery, Gaithersburg, MD), insulin (PerkinElmer, target for the treatment of schizophrenia. PDE10A- Waltham, MA), and glucose (OneTouch Ultra, LifeScan, deficient mice show reduced exploratory behavior when Milpitas, CA), were measured using commercially available placed in a novel environment and have a blunted re- assays. Triglycerides, cholesterol, aspartate aminotrans- sponse to the psychomotor activating effects of ferase (AST), and alanine aminotransferase (ALT) levels N-methyl-D-aspartate receptor (NMDA) receptor antag- were measured using an automated analyzer (Roche, onists phencyclidine and MK-801 (15,16). Selective Indianapolis, IN). PDE10A inhibitors decrease psychomotor activity, re- THPP-6 [2-(6-chloropyridin-3-yl)-4-[(2S)-1- verse deficits in prepulse inhibition, and inhibit condi- methoxypropan-2-yl]oxy-N-(6-methylpyridin-3-yl)-7,8- tioned avoidance responding in rodents, models that dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxamide] are predictive of antipsychotic activity in the clinic was synthesized in-house (18) and was dosed by oral (17–21). PDE10A inhibitors are also efficacious in pre- gavage in 5% Tween80/0.25% methylcellulose. AM251 clinical assays that test cognitive domains impaired in [N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4- schizophrenia (17,20,22,23) and models of negative dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] symptoms (17). was purchased from (Tocris, Ellisville, MO). For glucose Here we describe a novel role for PDE10A in the tolerance tests (GTTs), 6-h fasted mice were given regulation of caloric intake and energy homeostasis. Us- 1.5 g/kg glucose intraperitoneally (i.p.), and glucose was 2 2 ing PDE10A knockout (PDE10A / ) mice and a selective measured by glucometer (OneTouch Ultra) at 0, 20, 40, small molecule PDE10A inhibitor, we investigated the 60 and 120 min. Blood (2.5 mL) was collected into 13 role of PDE10A in regulating body weight, feeding be- PBS/EDTA (12.5 mL) for insulin determination. havior, locomotor activity, and energy expenditure in Data were analyzed using repeated-measures ANOVA mice fed a standard diet or a highly palatable diet rich in with Bonferroni post hoc analysis or unpaired Student fats and carbohydrates. We show that pharmacological t tests. Homeostasis model assessment of insulin re- inhibition of PDE10A activity leads to a dose-dependent sistance (HOMA-IR) was calculated as [fasting insulin suppression of food intake and increased energy expen- (mU/L) 3 fasting glucose (mmol/L)]/22.5. diture in obese mice with subsequent reduction of adi- posity and improved insulin sensitivity. Thus, we Indirect Calorimetry 2 2 describe a novel role for PDE10A in the homeostatic Male PDE10A / mice and their littermates were ana- regulation of energy metabolism, possibly by integrating lyzed at 10 weeks of age (n = 12 per group). They were peripheral satiety and adiposity signals with central acclimated to individual boxes within the OxyMax sys- regulation of food intake. tem (Columbus Instruments, Columbus, OH). Total and ambulatory locomotor activity, food intake, and Vo and RESEARCH DESIGN AND METHODS 2 VCO2 were measured in 30-min intervals, and the re- Animals spiratory exchange ratio (RER) was calculated as VCO2/ Experiments were performed in lean and diet-induced VO2. Heat (energy expenditure) was calculated as CV 3 0.75 obese (DIO) C57Bl/6NTac mice (Taconic, Germantown, VO2 subject 3 body weight , with CV = 3.815 + 1.232 2 2 NY). PDE10A / mice were generated by replacing 3 RER (24). Total locomotor activity was expressed as a fragment of 2,756 nucleotides (bp 1,947 to 2,000) with one count per two consecutive x-axis infrared beam 302 PDE10A Inhibition Decreases Weight Gain Diabetes Volume 63, January 2014 breaks, and fine movement was defined a single-beam factor), and group differences were further examined break. Food intake and locomotor activity data were ex- using post hoc comparisons (Fisher least significant cluded 30 min before and after handling of the mice. difference). For pair-feeding studies, C57Bl/6 mice were fed an Statistical Analysis HFD (D12492) for 16 weeks and then switched to Data are expressed as means 6 SEM. Statistical analysis D12266B for 4 weeks before study. Mice were acclimated was conducted using ANOVA or unpaired Student to pair-feeding for 1