Abstract

Cyclic nucleotides cAMP and cGMP are important second messengers for the regulation of adaptive thermogenesis. Their levels are controlled not only by their synthesis, but also their degradation. Since pharmacological inhibitors of cGMP-specific phosphodiesterase 9 (PDE9) can increase cGMP-dependent protein kinase signaling and uncoupling protein 1 expression in adipocytes, we sought to elucidate the role of PDE9 on energy balance and glucose homeostasis in vivo. Mice with targeted disruption of the PDE9 gene, Pde9a, were fed nutrient-matched high-fat (HFD) or low-fat diets. Pde9a−/− mice were resistant to HFD-induced obesity, exhibiting a global increase in energy expenditure, while brown adipose tissue (AT) had increased respiratory capacity and elevated expression of Ucp1 and other thermogenic genes. Reduced adiposity of HFD-fed Pde9a−/− mice was associated with improvements in glucose handling and hepatic steatosis. Cold exposure or treatment with β-adrenergic receptor agonists markedly decreased Pde9a expression in brown AT and cultured brown adipocytes, while Pde9a−/− mice exhibited a greater increase in AT browning, together suggesting that the PDE9-cGMP pathway augments classical cold-induced β-adrenergic/cAMP AT browning and energy expenditure. These findings suggest PDE9 is a previously unrecognized regulator of energy metabolism and that its inhibition may be a valuable avenue to explore for combating metabolic disease.

Introduction

The lack of effective therapies for the obesity epidemic is a serious challenge, as recent studies report that ∼40% of Americans are clinically obese, with the prevalence of obesity rising worldwide (1). Obesity is comorbid with insulin resistance and type 2 diabetes along with other chronic illnesses, such as cardiovascular diseases, nonalcoholic fatty liver disease, asthma, and certain cancers (2). Thus, finding ways to treat or reduce obesity are important for decreasing diabetes and related diseases. At its simplest, obesity can be managed by either reducing the calories consumed or by increasing energy expenditure. Changes in diet and exercise are the most commonly recommended methods to treat obesity, but rarely succeed over the long-term (3). Much effort has been directed toward reducing caloric intake, including pharmacological approaches such as appetite suppressants and blockade of fat absorption (4). These are frequently accompanied by unacceptable side effects or eventual lack of efficacy. Invasive procedures such as bariatric surgery have also been prescribed (4), and while to date, this approach has proven effective at ameliorating insulin resistance and providing sustained weight loss, the risks associated with such radical surgeries should not be underestimated (5). Alternatively, one potential means to increase energy expenditure aside from physical exercise is energy-consuming futile metabolic cycles and uncoupled respiration (6).

Brown adipose tissue (BAT) in mammals is a mechanism for maintaining body temperature. Brown adipocytes can regulate uncoupled respiration via oxidative metabolism that generates heat without ATP synthesis. These adipocytes contain a unique mitochondrial protein called uncoupling protein 1 (UCP1). When activated, UCP1 is a gated pore that allows H+ to pass through the inner mitochondrial membrane, thereby uncoupling oxidative phosphorylation from ATP production (7). While UCP1 is the signature protein of BAT, it can also be expressed in cells of white adipose tissue (WAT) in response to stimuli that increase cyclic nucleotides (i.e., cAMP and cGMP). These WAT adipocytes that have a brownlike phenotype are sometimes called “beige” adipocytes (8,9). During the last decade, there has been growing appreciation that adult humans possess significant amounts of brown and beige adipocytes that are rich in mitochondria and UCP1 (10–13). Thus, activation of brown/beige adipocyte thermogenesis to increase net energy expenditure might be an attractive therapeutic target for obesity and metabolic disease.

Increasing the concentration of cyclic nucleotides cAMP and cGMP in adipocytes is associated with an increase in UCP1 expression and thermogenic activity (14–17). For cAMP, this is classically observed in response to cold temperature, leading to increased secretion of norepinephrine from sympathetic nerves that activate the adipocyte’s β-adrenergic receptors (AR) (18). In addition to the important physiological role of the sympathetic nervous system and catecholamines, we and other have shown that the cardiac natriuretic peptides (NP), atrial NP (ANP) and B-type NP (BNP), are also capable of increasing thermogenic activity of adipocytes via increasing intracellular cGMP and thereby exert antiobesity effects (19–25). Levels of cyclic nucleotides are controlled not only by their synthesis, but also their degradation. The phosphodiesterase (PDE) enzymes break the phosphodiester bond in cyclic nucleotides, rendering them inactive (26). Based on our new findings that cold exposure decreases the expression of cGMP-specific PDE9, one of the most selective PDEs to degrade cGMP over cAMP (27), we asked whether decreasing the degradation of cGMP would have similar prothermogenic and antiobesity effects. The safe clinical utility for pharmacological inhibition of several PDEs has been demonstrated for a number of diseases, and the inhibition of multiple PDEs has been associated with increased adipocyte browning (reviewed in Ref. 17). Several small-molecule PDE9 inhibitors have entered clinical trials for conditions such as Alzheimer disease, schizophrenia, and sickle cell disease in which their safety has been established (28–34).

We hypothesized that the absence of PDE9 would counteract diet-induced weight gain and improve glucose handling via increasing uncoupled energy expenditure. We used a global Pde9a knockout mouse and show that these Pde9a−/− mice resist diet-induced weight gain and have concomitant improvements associated with reduced adiposity. Changes in energy expenditure associated with Pde9a knockout at any given time were small; however, over the extended course of the study, these modest changes became significant, leading to a reduced body weight and adiposity. As most people become obese slowly, gaining ∼0.5–1.0 kg/year (35,36), we posit that inhibition of PDE9 may be useful for counteracting this slow weight gain over time.

Research Design and MethodsAnimals

Animal studies were conducted at Sanford Burnham Prebys Medical Discovery Institute (SBP), Vanderbilt University Medical Center (VUMC), and Johns Hopkins University and School of Medicine (JHSOM). Pde9a−/− mice on a C57BL/6J background were previously described (37). Mice were maintained on a 12-h light/dark cycle with two to five animals per cage and ad libitum food and water. At 6 weeks of age, SBP and VUMC mice received nutrient-matched Surwit diets: a control low-fat diet (LFD) (10.5% calories from fat: D12328; Research Diets) or high-fat diet (HFD) (58.0% calories from fat: D12330; Research Diets). JHSOM mice were fed HFD (60.0% calories from fat: D12492; Research Diets). Mice were weighed twice weekly, and body composition was measured using a Minispec Body Composition Analyzer (Bruker) at SBP and Vanderbilt Mouse Metabolic Phenotyping Center (VMMPC) or EchoMRI-100 (Echo Medical Systems) at JHSOM. Mice were fasted for 5-h prior to euthanasia by CO2 asphyxiation and exsanguination via cardiac puncture. All procedures were approved by the Institutional Animal Care and Use Committees at SBP, VUMC, and JHSOM.

Cold Exposure and β3-AR Agonist

Chow-fed 13–16-week-old male mice were housed at 30°C for 2–4 days prior to habitation at 6°C for the indicated times. Additional mice received intraperitoneal CL-316,243 (1 mg/kg body weight/day) for 1 week as in Liu et al. (38).

Cell Culture

IngJ6 and Bat8 immortalized mouse cell lines (8) and human multipotent adipose-derived stem cells (hMADS) (39) were cultured and differentiated as described.

Quantitative Real-time RT-PCR

RNA was extracted using TRIzol and the aqueous phase purified on Zymo-Spin IIICG Columns. Instruments used were the Roche LightCycler 480 II or Applied Biosystems QuantStudio 6 Flex System. Three replicates of each sample were averaged, normalized to mRplp0 (36B4), and analyzed using the Pfaffl method. Primer sequences are in Supplementary Table 2.

Western Blotting

Western blotting was as described (24). Image acquisition was on Bio-Rad Digital ChemiDoc MP with IR or Typhoon FLA9000 Variable Mode Imager.

Histology

Tissues were fixed in 10% formaldehyde (4°C overnight) and stored in 70% ethanol (4°C). Histology was performed by the Vanderbilt Translational Pathology Shared Resource. Slides were imaged at ×20 original magnification with a Leica SCN400 Slide Scanner in the Digital Histology Shared Resource.

Triglyceride Composition

Lipids were extracted from frozen liver and quantified by the VMMPC Lipid Core.

Glucose and Insulin Sensitivity

For both the glucose tolerance test (GTT) and insulin tolerance test (ITT), mice were fasted for 5-h during the light cycle. Intraperitoneal injections of 1.0 g/kg dextrose in 0.9% saline for GTT or 0.5 units/kg insulin in 0.9% saline for ITT. Glucose was measured from tail-vein blood with a Bayer CONTOUR glucometer and glucose test strips. Clamp studies were done in chronically catheterized conscious mice by the VMMPC.

Fasting Blood Glucose, Plasma Insulin, and Plasma Chemistry

Prior to euthanasia, mice were fasted for 5 h, and glucose was measured from tail-vein blood. Mice were euthanized, and plasma was obtained from blood via cardiac puncture into tubes containing 8 µL of 0.5 mol/L EDTA. Insulin was measured from plasma stored at −80°C by ELISA. ALT, triglyceride, and cholesterol quantification was performed on plasma stored at −20°C by the Vanderbilt Translational Pathology Shared Resource.

Energy Balance

Mice used for energy balance studies were maintained at VUMC and JHSOM and fed HFD for 15 and 26 weeks, respectively. Indirect calorimetry and measurements of food intake and physical activity were performed on mice in the Promethion System (Sable Systems International) by the VMMPC or the Comprehensive Lab Animal Monitoring System (CLAMS) by the Rodent Metabolism Core at JHSOM. Light and dark cycles were analyzed independently.

BAT Respirometry

High-resolution respirometry was performed using Oxygraph-2k (Oroboros Instruments) in the VMMPC. Interscapular BAT (iBAT) from age-matched chow-fed mice was dissected into 1.6–2.5-mg pieces and permeabilized by sequential incubation in biopsy preservation solution (BIOPS), BIOPS plus saponin, and MiR05, each for 20 min at 4°C. Assays were performed in MiR05 at 37°C, and substrates were added in the order indicated. Data were normalized to wet tissue weight.

Statistics

Data are mean ± SEM using Prism version 9.1.0 for Windows 64-bit (GraphPad Software). Unless otherwise stated, analyses were performed using two-way ANOVA. Post hoc analyses of ANOVA used Sidak multiple comparisons test for Pde9a genotype only and indicated on figures with the symbols: *Pde9a+/+ versus Pde9a−/−, †Pde9a+/+ versus Pde9a+/−, and ‡Pde9a+/− versus Pde9a−/−; and one symbol, P < 0.05; two symbols, P < 0.01; or three symbols, P < 0.001. For analyses of body mass and blood glucose over time, two-way ANOVA with repeated measures was performed comparing the factors Pde9a genotype and time, analyzing diet groups independently. Average energy expenditure, VO2, and respiratory quotient for light and dark cycles were analyzed using the least squares method of multiple linear regression with model including the continuous effect of body mass and the categorical effects of diet, genotype, and instrument (i.e., Promethion at VUMC or CLAMS at JHSOM).

Data and Resource Availability

Data/resources are available upon request from the corresponding author.

ResultsLoss of PDE9 Facilitates Cold-Mediated AT Browning

We and others previously established that the cGMP–cGMP-dependent protein kinase (PKG) signaling axis in adipocytes can increase brown and beige adipocyte activity, including mitochondrial biogenesis, UCP1 expression, uncoupled respiration, and energy expenditure (19–25,40–42). In addition, the dynamic control of the ratio between the guanylyl cyclase-containing NP receptor, NPRA, to the clearance receptor, NPRC, is an important regulator of NP signaling (reviewed in Ref. 17), and we previously showed that cold temperature exposure reduces expression of NPRC (20). Given that PDE5 and PDE9 are selective for degrading cGMP, we sought to determine if they also played a role in the brown fat thermogenic program. Pde5a and Pde9a expression were measured in iBAT following both short-term and long-term cold exposure. We found that Pde9a, but not Pde5a, gene expression was significantly suppressed by cold exposure at both time points (Fig. 1A–D). A similar suppression of Pde9a, but not Pde5a, expression in iBAT was found in mice treated with the β3-AR agonist CL-316,243 (Fig. 1E and F). In an immortalized brown adipocyte cell line (Bat8), Pde9a expression was reduced in response to the β-AR agonist isoproterenol in a comparable manner (Fig. 1G and H). These findings indicate that cold-induced β-AR signaling suppresses Pde9a, but not Pde5a, gene expression.

Figure 1

Cold exposure and β3-adrenergic signaling suppresses Pde9a gene expression, while loss of PDE9 amplifies the cold-induced thermogenic program. Chow-fed male mice were exposed to cold for 8 h, and expression of Pde5a (A) and Pde9a (B) was measured in iBAT. N = 3 30°C and N = 3–4 6°C mice. Chow-fed male mice were exposed to cold for 5 days, and iBAT expression of Pde5a (C) and Pde9a (D) was measured. N = 4 30°C and N = 6 6°C mice. Chow-fed male mice were treated with vehicle or β3-AR agonist CL-316,243 (CL) for 1 week, and expression of Pde5a (E) and Pde9a (F) in iBAT was measured. N = 5 vehicle and N = 5 CL mice. Pde5a (G) and Pde9a (H) in Bat8 adipocytes. Cells were serum starved for 2 h prior to treatment with vehicle or 10 μmol/L isoproterenol (ISO) for an additional 4 h. Three independent experiments are shown. All data are mean ± SEM, and analyses were performed using unpaired t test. Histology of iBAT (I), iWAT (J), and eWAT (K) from chow-fed male mice housed at 6°C for 3 days. Representative H-E– and UCP1-stained sections from N = 3 Pde9a+/+ and N = 3 Pde9a−/− mice. **P < 0.01, ***P < 0.001.

As these findings suggest that a reduction in Pde9a may be important for cold-mediated induction of the brown fat thermogenic program, we asked whether global loss of PDE9 would affect AT browning. Male Pde9a+/+ and Pde9a−/− mice were exposed to cold temperature for 3 days to cause a mild induction of the thermogenic program. Pde9a−/− mice demonstrated a significant increase in the amount of UCP1+ adipocytes in their inguinal WAT (iWAT), while both iBAT and epididymal WAT (eWAT) appeared to have a reduction in adipocyte size (Fig. 1I–K). Together, these findings suggest that PDE9 is a negative regulator of the cold-mediated BAT thermogenic program.

PDE9 Inhibition Increases ANP-Evoked PKG Signaling and UCP1 Expression

We asked whether blocking PDE9 activity in adipocytes in vitro using small-molecule inhibitors would increase PKG signaling and the adipocyte thermogenic program. We used mouse cell lines representative of white (IngJ6) and brown (Bat8) adipocytes (8), hMADS (43), and two different PDE9 inhibitors: BAY73–6691 (BAY) and PF-04447943 (PF). As shown in Fig. 2A and B, phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 239, a marker of PKG signaling (44), was increased in both IngJ6 and Bat8 cells treated with BAY above the level seen with ANP alone. BAY also slightly increased VASP phosphorylation in IngJ6 independent of ANP (Fig. 2A). The PF compound was also tested in Bat8 cells, where it increased ANP-evoked VASP(S239) phosphorylation in a dose-dependent manner (Fig. 2C). The hMADS cell line can express thermogenic markers and increase mitochondrial biogenesis after differentiation in response to catecholamines as well as cardiac NPs (20,39,43). We stimulated cGMP-PKG signaling by including the NPs, ANP and BNP, and/or PF during the last 7 days of differentiation. As shown in Fig. 2D, both the NPs and PF were able to increase UCP1 expression. Also shown is that the cell-permeable cGMP analog pCPT-cGMP raised UCP1 expression (Fig. 2D).

Figure 2

PDE9 inhibitors increase PKG signaling and UCP1 expression in adipocytes. IngJ6 (A) and Bat8 (B) adipocytes were serum starved overnight and treated with 5 μmol/L BAY for 90 min and 100 nmol/L ANP, 500 μmol/L pCPT-cGMP, or 1 μmol/L isoproterenol (ISO) for 60 min. Phosphorylation of VASP at Ser239 is an indicator of PKG activity, as this is the major PKG phosphorylation site. The shift from 46 kDa to 50 kDa is due to phosphorylation at Ser157, which can be phosphorylated by both cAMP-dependent protein kinase and PKG (44). Blots are representative of three similar experiments. C: Bat8 adipocytes were serum starved overnight and then treated with increasing concentrations of PF for 60 min and 100 nmol/L ANP or 500 μmol/L pCPT-cGMP for 30 min. Blots are representative of two similar experiments. D: hMADS were differentiated for 9 days, after which 200 nmol/L ANP, 200 nmol/L BNP, 300 nmol/L PF, and 200 μmol/L pCPT-cGMP were added for the remaining 7 days of differentiation. Blots are representative of three similar experiments.

Protection From HFD-Induced Obesity by Pde9a Knockout

To test our hypothesis that PDE9 ablation would improve diet-induced obesity and glucose handling, male Pde9a+/+, Pde9a+/−, and Pde9a−/− mice were fed nutrient-matched control LFD or HFD for 16 weeks beginning at 6 weeks of age. Overall, Pde9a genotype affected body weight and body weight gain throughout the feeding study (Fig. 3A–D and Supplementary Fig. 1). The reduced body weight of HFD-fed Pde9a−/− was attributed to a reduction in fat mass (Fig. 3E and G). Of note, Pde9a+/− mice exhibited an intermediate obesity phenotype when challenged with HFD. These data demonstrate that even haploinsufficiency of Pde9a confers resistance to HFD. Female Pde9a−/− mice also gained less weight than Pde9a+/+ throughout the 16 weeks of HFD feeding (Supplementary Fig. 2). Compared with males, the differences in body weight were not as pronounced, but were also a result of reduced fat mass.

Figure 3

Male Pde9a−/− mice are resistant to diet-induced weight gain and adiposity. A: Male Pde9a+/+, Pde9a+/−, and Pde9a−/− mice were fed an LFD or HFD for 16 weeks beginning at 6 weeks of age. LFD results are in Supplementary Fig. 1. (P < 0.0001, genotype × time interaction for HFD; P = 0.0117, genotype × time interaction for LFD.) B: Terminal body weight (P = 0.0047, effect of genotype). C: Body weight gain. LFD results are in Supplementary Fig. 1. (P < 0.0001, genotype × time interaction for HFD; P = 0.0118, genotype × time interaction for LFD.) D: Cumulative body weight gain (P = 0.0387, effect of genotype). E: Representative images of Pde9a+/+, Pde9a+/−, and Pde9a−/− littermates fed HFD. Body composition at 12 (F) and 16 weeks (G) of HFD feeding. Data are mean ± SEM. Analyses were performed using two-way ANOVA. For A and C, two-way ANOVAs were performed with repeated measures. Post hoc analyses were performed using Sidak multiple comparisons test for Pde9a genotype only and are indicated on figures with * comparing Pde9a+/+ vs. Pde9a−/− and ‡ comparing Pde9a+/− vs. Pde9a−/−. * or ‡P < 0.05; ** or ‡‡P < 0.01. For A–D, N = 10 Pde9a+/+ LFD, 17 Pde9a+/− LFD, 11 Pde9a−/− LFD, 13 Pde9a+/+ HFD, 21 Pde9a+/− HFD, and 16 Pde9a−/−−/− HFD. For F and G, N = 9–10 Pde9a+/+ LFD, 11–12 Pde9a+/− LFD, 10–11 Pde9a−/− LFD, 12–13 Pde9a+/+ HFD, 13–16 Pde9a+/− HFD, and 11–14 Pde9a−/− HFD.

Improvements in Glucose Handling Are Proportional to Reduced Body Weight of Pde9a−/− Mice

Intraperitoneal GTT (IP-GTT) and IP-ITT were used to assess glucose handling and insulin sensitivity in male mice. In mice fed LFD, Pde9a genotype did not affect glucose handling. For HFD-fed mice, glucose tolerance was essentially identical between Pde9a+/+ and Pde9a+/− mice, while Pde9a−/− mice exhibited a modest improvement (Fig. 4A and B and Supplementary Fig. 3A). ITT revealed very little difference between the Pde9a genotypes (Fig. 4C and Supplementary Fig. 3B and C). Fasting glucose and insulin levels were not significantly different (Fig. 4D and E). These findings are consistent with other observations in this study that modest increases in “adipose browning” and energy expenditure are sufficient to mitigate the more overt features of insulin resistance. In female mice on HFD, absence of Pde9a was also associated with a modest improvement in IP-GTT and IP-ITT (Supplementary Fig. 4A–F). Fasting glucose and insulin levels were not different (Supplementary Fig. 4G and H). Hyperinsulinemic-euglycemic clamps were performed in chronically catheterized conscious mice from another cohort of Pde9a+/+ and Pde9a−/− male mice that were fed HFD for 16 weeks. The body weights of these mice were closely matched, thus controlling for the previous weight difference. In these mice, all measurements were largely the same between the Pde9a+/+ and Pde9a−/− mice (Supplementary Fig. 3D–F). Pde9a−/− mice showed an increase in fasting glucose turnover rate; however, the rates of glucose uptake and endogenous glucose production during the hyperinsulinemic clamp were similar (Supplementary Fig. 3G).

Figure 4

Glucose homeostasis is modestly improved in male Pde9a−/− mice. A: IP-GTT in male Pde9a+/+, Pde9a+/−, and Pde9a−/− mice fed HFD for 15 weeks. B: Area under the curve (AUC) of data in A and Supplementary Fig. 3. C: IP-ITT in male Pde9a+/+, Pde9a+/−, and Pde9a−/− mice fed HFD for 14 weeks. Five-hour fasting glucose (D) and insulin (E). F: p-AKT at Ser473 Western blot. Data are mean ± SEM. Analyses were performed using two-way ANOVA. For A and C, two-way ANOVAs were performed with repeated measures. Post hoc analyses were performed using Sidak multiple comparisons test for Pde9a genotype only and are indicated on figures, with ‡P < 0.05 comparing Pde9a+/− vs. Pde9a−/−. For A–E, N = 10 Pde9a+/+ LFD, 17 Pde9a+/− LFD, 10–11 Pde9a−/− LFD, 12–13 Pde9a+/+ HFD, 21 Pde9a+/− HFD, and 15–16 Pde9a−/− HFD.

To assess insulin signaling in AT, phosphorylation of AKT at Ser473 was measured postmortem in eWAT, iWAT, and iBAT (Fig. 4F). Three representative mice were chosen from each genotype × diet treatment group based upon their closeness to the mean terminal body weight of their group. Loss of Pde9a was associated with increased AKT phosphorylation and therefore insulin signaling in AT regardless of dietary treatment (Fig. 4F).

Protection From HFD-Induced AT Expansion and Liver Damage in Pde9a−/− Mice

The reduction in body weight and adiposity in Pde9a−/− male mice was associated with reduced liver mass. Consistent with the observation that overall fat mass was lower in Pde9a−/− mice on HFD, the weights of individual adipose depots that were collected exhibited a similar trend (Fig. 5A–C). This also corresponded to a reduction in adipocyte size (Fig. 5D–F). On HFD, the iBAT of Pde9a+/+ mice had large unilocular adipocytes, whereas Pde9a−/− had smaller multilocular adipocytes (Fig. 5F). In female mice on HFD, tissue weights exhibited a similar trend toward reduced mass in the Pde9a−/−, which was particularly evident for iWAT and iBAT (Supplementary Fig. 5). In the liver, absence of Pde9a led to a greater protection during HFD feeding. Hematoxylin and eosin (H-E) staining of the livers revealed prominent steatosis in the Pde9a+/+ HFD-fed mice, which was significantly reduced in the HFD fed Pde9a−/− livers (Fig. 5G). This corresponded to a significant reduction in liver weight in Pde9a−/− mice (Fig. 5H). Once again, Pde9a+/− mice showed an intermediate reduction in liver weight. As expected, we observed a strong trend toward reduced hepatic triglyceride levels in the HFD-fed Pde9a−/− mice (Fig. 5I), with minor differences in fatty acid composition between genotypes (Supplementary Table 3). Hepatic steatosis frequently leads to liver damage. Circulating levels of plasma ALT, a biomarker of hepatocyte membrane damage, were measured. In the Pde9a−/− mice, ALT levels were significantly reduced (Fig. 5J). Plasma triglyceride levels were unchanged and cholesterol showed a trend toward being reduced in HFD-fed Pde9a−/− mice (Supplementary Fig. 6). Absence of Pde9a was also associated with a significant reduction in heart and spleen weight and a trend toward reduced kidney weight with HFD feeding (Fig. 5K–L).

Figure 5

Male Pde9a−/− mice are protected from HFD-induced AT expansion and liver damage. A: eWAT weights (P = 0.049, effect of genotype). B: iWAT weights. C: iBAT weights. Histology of eWAT (D) and iWAT (E). F: Representative H-E– and UCP1-stained sections of iBAT from HFD-fed mice. G: H-E staining of livers. H: Liver weights (P = 0.039, effect of genotype). I: Ratio of hepatic triglyceride to tissue weight. J: Plasma ALT (P = 0.027, effect of genotype). K: Heart weight (P = 0.027, effect of genotype × diet interaction). L: Spleen weight (P = 0.0003, effect of genotype). M: Kidney weight. Data are mean ± SEM. Analyses were performed using two-way ANOVA. Post hoc analyses were performed using Sidak multiple comparisons test for Pde9a genotype only and are indicated on figures, with * comparing Pde9a+/+ vs. Pde9a−/−, † comparing Pde9a+/+ vs. Pde9a+/−, and ‡ comparing Pde9a+/− vs. Pde9a−/−. * or †P < 0.05; **P < 0.01; ***P < 0.001. N = 10 Pde9a+/+ LFD, 17 Pde9a+/− LFD, 11 Pde9a−/− LFD, 13 Pde9a+/+ HFD, 21 Pde9a+/− HFD, and 16 Pde9a−/− HFD. Images are a representative sample from three mice from each group.

Increased Energy Expenditure in Pde9a−/− Mice

To determine whether there were differences in energy expenditure that would account for the differences in body weights and fat mass in HFD-fed Pde9a−/− mice, indirect calorimetry was measured on male mice at the end of the diet period. As shown in Supplementary Fig. 7, food intake and physical activity cannot account for the decreased weight and fat mass in the Pde9a−/− mice. Importantly, we found significantly increased energy expenditure (P = 0.047) and a trend toward increased oxygen consumption (P = 0.057) in Pde9a−/− mice during the dark cycle (Fig. 6A and B). Respiratory quotient was not changed (Fig. 6C).

Figure 6

Pde9a−/− mice have increased energy expenditure. A: Energy expenditure increase in Pde9a−/− mice in dark cycle (gray; P = 0.047, effect of genotype) and light cycle (white; P = 0.444, effect of genotype). B: Oxygen consumption in Pde9a−/− in dark cycle (P = 0.057, effect of genotype) and light cycle (P = 0.440, effect of genotype). C: Respiratory quotient in Pde9a−/− mice in dark cycle (P = 0.220, effect of genotype) and light cycle (P = 0.836, effect of genotype). Values in the figure are the mean from the Promethion System plotted against time (day:hour:minute). Statistics are calculated using the combined data from Promethion and CLAMS using multiple linear regression. See description in research design and methods. N = 6 Pde9a+/+ LFD, 7 Pde9a−/− LFD, 20 Pde9a+/+ HFD, and 20 Pde9a−/− HFD.

Increased Thermogenesis-Related Gene Expression in the AT of Pde9a−/− Mice

We hypothesized that the increased energy expenditure in Pde9a−/− mice was due to increased PKG signaling and thermogenesis in the AT. Significant changes in resting VASP(S239) phosphorylation were not observed in iBAT or iWAT (Supplementary Fig. 8). It is likely that differences in unstimulated VASP(S239) phosphorylation are difficult to observe in vivo due to the continuous interplay of basal stimuli and dephosphorylation events. However, as shown in Fig. 7, a signa