Deletion of prolyl carboxypeptidase attenuates the metabolic effects of diet-induced obesity

Abstract

α-Melanocyte-stimulating hormone (α-MSH) is a critical regulator of energy metabolism. Prolyl carboxypeptidase (PRCP) is an enzyme responsible for its degradation and inactivation. PRCP-null mice (PRCP^gt/gt) showed elevated levels of brain α-MSH, reduced food intake, and a leaner phenotype compared with wild-type controls. In addition, they were protected against diet-induced obesity. Here, we show that PRCP^gt/gt animals have improved metabolic parameters compared with wild-type controls under a standard chow diet (SD) as well as on a high-fat diet (HFD). Similarly to when they are exposed to SD, PRCP^gt/gt mice exposed to HFD for 13 wk showed a leaner phenotype due to decreased fat mass, increased energy expenditure, and locomotor activity. They also showed improved insulin sensitivity and glucose tolerance compared with WT controls and a significant reduction in fasting glucose levels. These improvements occured before changes in body weight and composition were evident, suggesting that the beneficial effect of PRCP ablation is independent of the adiposity levels. In support of a reduced gluconeogenesis, liver PEPCK and G-6-Pase mRNA levels were reduced significantly in PRCP^gt/gt compared with WT mice. A significant decrease in liver weight and hepatic triglycerides were also observed in PRCP^gt/gt compared with WT mice. Altogether, our data suggest that PRCP is an important regulator of energy and glucose homeostasis since its deletion significantly improves metabolic parameters in mice exposed to both SD and HFD.

a precise control of caloric intake and energy expenditure is fundamental for the balance of energy homeostasis. In the mammalian brain, a product of the proopiomelanocortin (POMC) gene, α-melanocyte-stimulating hormone (α-MSH) plays a central role in detecting, integrating, and responding to a range of central and peripheral inputs relating to energy metabolism (1, 4, 13, 14, 20). The use of genetically altered mouse models has shown the importance of α-MSH in the regulation of a broad range of metabolic parameters (9, 17). Mice with ablation of the POMC gene are obese, whereas mice overexpressing α-MSH in the brain showed protection against diet-induced obesity with improved glucose homeostasis.

We have reported recently a molecular mechanism that is responsible for the inactivation and degradation of α-MSH (21). Prolylcarboxypeptidase (PRCP) is a serine protease that cleaves the last amino acid at the COOH terminus of small peptides if proline is the penultimate amino acid. We have shown previously that PRCP is expressed in the lateral hypothalamus, where PRCP-expressing neurons target with their axons the paraventricular nucleus of the hypothalamus, a site where α-MSH is released from POMC terminals to stimulate melanocortin 4 receptor-expressing neurons. PRCP is also expressed in the dorsomedial nucleus of the hypothalamus where melanocortin 4 receptor-expressing neurons are located (21). We have found that animals with PRCP ablation (PRCP^gt/gt) have elevated levels of α-MSH in the hypothalamus, decreased food intake, body weight, and fat mass, and increased energy expenditure compared with their wild-type (WT) controls (8, 21). Moreover, they are protected from diet-induced obesity (21).

To further characterize the metabolic responses to standard and high-fat diets, we analyzed several metabolic parameters in PRCP-null mice and compared them with the WT controls.

MATERIALS AND METHODS

Animals.

All animal work in these studies was approved by the Yale University Institutional Animal Care and Use Committee. Male mice were studied to ≤16 wk of age. All mice studied were on a C57Bl6 background, that being the transgenic mice back-crossed for ≥10 generations. Animals were housed under a 12:12-h light-dark cycle at 25°C and had a free access to either a standard chow diet (SD; Harlan Teklad no. 2018, 18% calories from fat) or a 45% high-fat diet (HFD; Harlan Teklad no. 93075) for ≤13 wk from weaning.

Metabolic chamber recording and body composition.

Animals for each group (n = 8 for both PRCP^gt/gt and WT on HFD for 13 wk and n = 5 for both PRCP^gt/gt and WT on HFD for 7–8 wk) were acclimated in metabolic chambers (TSE System-Core Metabolic Phenotyping Center, Yale University) for 4 days before the start of the recordings. Mice were continuously recorded for 3 days, with the following measurements taken every 30 min: water intake, food intake, ambularoty activity (in the x- and z-axes), and gas exchange (O₂ and CO₂; The TSE LabMaster System). CO₂ production (V̇co₂) and energy expenditure were calculated according to the manufacturer's guidelines (PhenoMaster Software, TSE System). The respiratory exchange rate was estimated by calculating the ratio of V̇co₂ to oxygen consumption (V̇o₂). Values were adjusted by body weight to the power of 0.75 (kg^−0.75) where mentioned. Body composition was measured in vivo by MRI (EchoMRI; Echo Medical Systems, Houston, TX).

Real-time PCR.

Total RNA from brown adipose tissue and liver was extracted from PRCP^gt/gt and WT animals (n = 5 for each group in SD and 13 wk on HFD and n = 4 for 8-wk HFD mice) using Trizol solution (Invitrogen). Uncoupling protein 1 (UCP1) mRNA levels in the brown adipose tissue and glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels in the liver were measured by real-time PCR. A High Capacity cDNA Reverse transcription Kit (Applied Biosystems) was used for the reverse transcription. Real-time PCR (LightCycler 480; Roche) was performed with diluted cDNAs in a 20-μl reaction volume in triplicates. Primers used for this study are as follows: cat. no. Mm 00494069_m1 for UCP1, cat. no. Mm 00839363_m1 for G-6-Pase, cat. no. Mm 01247059_g1 for PEPCK, and cat. no. 4319413E-0810041 for 18S rRNA (Applied Biosystems). The calculations of average Cp values, SDs, and resulting expression ratios for each target gene were based on the Roche LightCycler 480 software.

Measurement of circulating hormones.

Serum from blood samples was obtained by centrifugation at 3,000 rpm for 15 min, and each circulating hormone was determined using a commercially available ELISA kit as follows: leptin (Mouse Leptin ELISA Kit, cat. no. EZML-82K; Millipore), active ghrelin (Rat/Mouse Active Ghrelin ELISA kit, cat. no. EZRGRA-90K; Millipore), insulin (Rat/Mouse Insulin ELISA kit, cat. no. EZRMI-13K; Millipore), triglyceride (cat. no. TR0100; Sigma), adiponectin (Mouse Adiponectin ELISA kit, cat. no. EZMADP-60K; Millipore), and NEFA-HR2 (Wako Diagnostics). All procedures were performed by following the manufacturer's protocol.

Glucose and insulin tolerance tests.

Glucose tolerance test was performed in 16- to 17-h-fasted animals. After the level of blood glucose was determined, fasted animals were injected intraperitoneally with 20% glucose (10 ml/kg; DeltaSelect) in saline. Blood glucose levels were then monitored at 15, 30, 60, and 120 min from the injection.

Insulin tolerance test was performed with mice fed ad libitum. After determination of basal blood glucose levels, each animal received an intraperitoneal injection of insulin, 0.75 U/kg (Actrapid; Novo Nordisk). Blood glucose levels were then measured at 15, 30, 60, and 120 min after insulin injection.

Statistical analysis.

Two-way ANOVA was used to determine the effect of the genotype and diet with the Prism 4.0 software (GraphPad Software, San Diego, CA). For repeated-measures analysis, ANOVA was used when values over different times were analyzed. Significant effects were evaluated (followed) with Fisher's protected least significant difference post hoc test with Bonferroni's correction. When only two groups were analyzed, statistical significance was determined by an unpaired Student t-test. A value of P < 0.05 was considered statistically significant. All data are shown as means ± SE unless stated otherwise.

RESULTS

Body weight, composition, and energy expenditure.

We have shown previously that PRCP^gt/gt mice on SD as well as on HFD have a lower body weight compared with WT mice exposed to the same diet (8, 21). In addition, most recently, we have shown that PRCP^gt/gt mice have lower percentages of body fat and increased percentages of lean mass and overall higher energy expenditure when exposed to SD (8). To assess whether these differences were maintained when exposed to HFD, we measured body weight, body composition, and energy metabolism in PRCP^gt/gt and WT mice exposed to HFD. From the beginning of the HFD at weanning, a significant difference in body weight was found at 9 wk of HFD exposure (25.78 ± 0.64 g in PRCP^gt/gt mice vs. 28.96 ± 1.10 g in WT, P = 0.028), and this difference persisted to the end of our experiment at 13 wk of HFD (27.89 ± 0.85 vs. 31.65 ± 0.99 g, P = 0.0143; Fig. 1A). At 13 wk of HFD, PRCP^gt/gt mice showed lower percentages of fat mass (13.4 ± 1.4 vs. 26.6 ± 1.8%, P < 0.01; Fig. 1B) and increased percentages of lean mass (73.4 ± 1.3 vs. 61.7 ± 1.6%, P < 0.01; Fig. 1C) compared with WT mice. Furthermore, PRCP^gt/gt mice had increased energy expenditure compared with WT mice on HFD (1,480 ± 37.8 kcal·kg⁻¹·h⁻¹ in WT and 1,601 ± 31.9 kcal·kg⁻¹·h⁻¹ in PRCP^gt/gt; n = 4, P = 0.021; Fig. 2, A and B). This phenomenon was observed during both the light (662.3 ± 15.4 kcal·kg⁻¹·h⁻¹ in WT and 712.2 ± 14.0 kcal·kg⁻¹·h⁻¹ in PRCP^gt/gt; n = 4, P = 0.025) and dark phases (817.9 ± 23.4 kcal·kg⁻¹·h⁻¹ in WT and 888.9 ± 19.3 kcal·kg⁻¹·h⁻¹ in PRCP^gt/gt; n = 4, P = 0.029; Fig. 2B). Elevated levels of V̇o₂ (2,426 ± 87.7 ml/h in WT and 2,876 ± 46.1 ml/h in PRCP^gt/gt; n = 4 each, P = 0.0039; Fig. 2C) and V̇co₂ (2,385 ± 108.5 ml/h in WT and 2,822 ± 45.8 ml/h in PRCP^gt/gt; n = 4 each, P = 0.0099; Fig. 2D) were observed in PRCP^gt/gt mice compared with WT mice. No difference in the respiratory exchange rate (0.9785 ± 0.0098 in WT and 0.9772 ± 0.0039 in PRCP^gt/gt; n = 4 each, P = 0.9; Fig. 2E) was observed. The total locomotor activity of PRCP^gt/gt mice on HFD was significantly higher than that of WT (56,690 ± 3,073 beam breaks/48 h in WT and 87,980 ± 10,720 beam breaks/48 h in PRCP^gt/gt; n = 4 each, P = 0.03; Fig. 2F). This difference was due to an increased locomotor activity during the dark phase (37,920 ± 3,556 beam breaks/48 h in WT and 68,930 ± 6,395 beam breaks/48 h in PRCP^gt/gt; n = 4 each, P = 0.005; Fig. 2F) since no differences were observed during the light phase (18,770 ± 3,630 beam breaks/48 h in WT and 19,050 ± 3,955 beam breaks/48 h in PRCP^gt/gt; n = 4 each, P = 0.96; Fig. 2F).

Fig. 1.

Effect of high-fat diet (HFD) on wild-type (WT) and PRCP^gt/gt mice. A: graphs showing body weight changes in WT and PRCP^gt/gt mice during HFD. B and C: graphs showing the %fat (B) and %lean mass (C) in 13-wk HFD mice. *P < 0.05 compared with WT mice; **P < 0.01. PRCP, prolylcarboxypeptide.

Fig. 2.

Effect of PRCP deletion of energy metabolism during HFD. A: results of the energy expenditure in WT and PRCP^gt/gt mice exposed to HFD for 13 wk. Gray area represents dark phases. B: results of the energy expenditure as total in the 24-h cycle and in the dark and light phases of the cycle. C–E: results of the O₂ consumed (V̇o₂), CO₂ produced (V̇co₂), and their ratio. F: results of the locomotor activity of the WT and PRCP^gt/gt mice in 48 h and in the dark and light phases of 2 cycles (48 h). *P < 0.05; **P < 0.01. RQ, respiratory quotient.

Function of PRCP^gt/gt on adipose tissue.

To assess the contribution of the brown adipose tissue in the metabolic phenotype of PRCP^gt/gt mice, we next analyzed UCP1 mRNA expression levels in animals on HFD for 13 wk (Fig. 3). UCP1 mRNA levels were significantly higher in PRCP^gt/gt compared with WT when animals were exposed to the standard chow diet (0.9 ± 0.04 in WT and 1.9 ± 0.34 in PRCP^gt/gt; n = 4 each, P = 0.03). However, when exposed to HFD, no differences in UCP1 mRNA levels were found (1.9 ± 0.2 in WT and 1.6 ± 0.2 in PRCP^gt/gt; n = 5 each, P = 0.4; Fig. 3).

Fig. 3.

Effect of PRCP ablation on brown adipose tissue (BAT). Graph showing the results of uncoupling protein-1 (UCP1) mRNA levels measured by real-time PCR in the BAT of WT and PRCP^gt/gt mice on standard chow diet (SD) or HFD for 13 wk. *P < 0.05.

Circulating hormone levels.

Blood leptin levels were significantly lower in PRCP^gt/gt mice compared with those of WT controls on SD as well as 13-wk HFD. On SD, leptin levels in WT and PRCP^gt/gt were 15.1 ± 3.2 and 4.7 ± 0.9 ng/ml, respectively (n = 13 and 11, respectively, P = 0.008; Fig. 4A). Leptin levels were also statistically different between the two groups under HFD, with PRCP^gt/gt mice having 18.04 ± 4.15 ng/ml and WTs having 40.48 ± 1.12 ng/ml (n = 8 for both groups, P = 0.0001; Fig. 4A).

Fig. 4.

Circulating leptin (A), adiponectin (B), active ghrelin (C), and total ghrelin (D) levels in WT and PRCP^gt/gt mice on either SD or HFD for 13 wk. A: corresponding to the results on the reduced fat mass, PRCP^gt/gt mice showed lower serum leptin levels than the WT controls when exposed to either SD or HFD. B: adiponectin levels were found reduced in PRCP^gt/gt mice compared with their WT controls when exposed to SD; no difference was observed in adiponectin levels in mice exposed to HFD. C: HFD reduced active ghrelin levels in WT and PRCP^gt/gt mice significantly. However, whereas HFD reduced total ghrelin levels in WT mice significantly, no decrease in total ghrelin levels was observed in PRCP^gt/gt mice on HFD compared with PRCP^gt/gt mice on SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Adiponectin levels were significantly lower in PRCP^gt/gt mice on SD (8.2 ± 0.5 μg/ml; n = 5) compared with those of WT mice (10.6 ± 0.6 μg/ml; n = 6, P = 0.03; Fig. 4B). However, no difference in adiponectin levels were observed when the mice were exposed to HFD (8.8 ± 1.3 μg/ml in WT and 9.7 ± 1.5 μg/ml in PRCP^gt/gt mice; n = 6; Fig. 4B).

No differences in active ghrelin levels were found between the two groups under either diet condition (0.20 ± 0.06 pg/ml in WT on SD vs. 0.31 ± 0.05 in PRCP^gt/gt on SD, n = 5 and 4, respectively, P = 0.2; 0.07 ± 0.01 pg/ml in WT on HFD vs. 0.05 ± 0.009 pg/ml in PRCP^gt/gt on HFD, n = 5 and 4 respectively, P = 0.3; Fig. 4C). Anyway, HFD significantly reduced active ghrelin in both groups of animals compared with that observed under the SD diet. Also, no difference in total ghrelin levels was observed under the SD diet (0.17 ± 0.03 ng/ml in WT mice and 0.18 ± 0.04 ng/ml in PRCP^gt/gt mice, n = 5 and 4, respectively, P = 0.8; Fig. 4D).

However, on HFD, PRCP^gt/gt mice had statistically higher total ghrelin levels (0.17 ± 0.01 ng/ml; n = 4) compared with those of WT animals (0.11 ± 0.005 ng/ml; n = 5, P = 0.002; Fig. 4D).

Glucose and fatty acid homeostasis.

Intraperitoneal glucose and insulin tolerance tests were performed to compare glucose metabolism in PRCP^gt/gt mice and WT controls (Fig. 5, A–D). Fasting glucose levels were significantly lower in PRCP^gt/gt mice compared with WT controls when they were exposed to both SD and a 13-wk HFD (time 0; Fig. 5, A and C). On both diets, glucose tolerance was improved significantly in PRCP^gt/gt mice compared with that of WT controls (Fig. 5, A and C). The area under the glucose response curve was reduced in PRCP^gt/gt mice on SD (15%) and 13-wk HFD (25%) compared with WT controls. We then tested insulin tolerance by systemic injection of insulin in fed animals and found that insulin sensitivity was also improved in PRCP^gt/gt mice on SD as well as on 13-wk HFD (Fig. 5, B and D).

Fig. 5.

Effect of PRCP deletion on glucose homeostasis. Glucose (left) and insulin tolerance tests (right) in WT and PRCP^gt/gt mice exposed to either SD (A and B) or 13-wk HFD (C and D). A and C: both PRCP^gt/gt experimental groups showed significantly lower fasting glucose levels compared with their WT control groups. B and D: increased insulin sensitivity was found in PRCP^gt/gt mice when exposed to SD and 13-wk HFD. E–G: no difference in body weight (E), %fat (F), or lean mass (G) between WT and PRCP^gt/gt mice exposed to HFD for 7–8 wk. When glucose and insulin tolerance tests were performed in these mice, a significant difference in glucose tolerance (H) and insulin sensitivity (I) was noticed between WT and PRCP^gt/gt mice, suggesting that the differences in glucose levels observed in SD and 13-wk HFD were not due to changes in adiposity levels.

To test whether the improvement of glucose and insulin tolerance was due to the difference in adiposity between PRCP^gt/gt and WT mice, we analyzed fat mass, glucose tolerance test, and insulin tolerance test in mice exposed to HFD for 8 wk, a time point in which body weight between PRCP^gt/gt and WT mice was not significantly different (27.72 ± 0.82 g in WT and 26.04 ± 0.76 g in PRCP^gt/gt, P = 0.15; Figs. 1A and 5E). MRI analysis showed no difference in fat (17.75 ± 3.75% in WT and 15.17 ± 1.02% in PRCP^gt/gt mice, P = 0.54; Fig. 5F) and lean mass (69.40 ± 2.92% in WT and 71.60 ± 0.84% in PRCP^gt/gt mice, P = 0.51; Fig. 5G) between WT and PRCP^gt/gt mice. Similarly to mice exposed to SD and HFD for 13 wk, PRCP^gt/gt mice on HFD for 7–8 wk showed a significant decrease in fasting glucose levels and improvement in glucose tolerance test (area under the curve was decreased by 28%) and insulin tolerance test compared with WT mice exposed to HFD for 7–8 wk (Fig. 5, H and I).

Since PRCP^gt/gt mice on SD showed low glucose levels, we measured fasting insulin levels. PRCP^gt/gt mice showed significantly lower levels of insulin (0.25 ± 0.03 ng/ml, n = 6) compared with those of WT controls (0.49 ± 0.03 ng/ml, n = 3, P = 0.003; Fig. 6A). However, in the fed state, no differences in insulin levels were found between WT and PRCP^gt/gt mice on SD. WT exposed to 13 wk of HFD showed a significant increase in circulating insulin levels (2.76 ± 0.51 ng/ml) compared with WT and PRCP^gt/gt exposed to SD (0.77 ± 0.13 and 0.67 ± 0.09 ng/ml, respectively, P < 0.01; Fig. 6B) and PRCP^gt/gt on HFD for 13 wk (1.55 ± 0.14 ng/ml, P < 0.05; Fig. 6B).

Fig. 6.

A: results of circulating insulin levels measured in fasted WT and PRCP^gt/gt mice on SD. Insulin levels were significantly lower in PRCP^gt/gt mice compared with WT controls. B: results of insulin levels in fed WT and PRCP^gt/gt mice on SD or 13-wk HFD. Although increased, insulin levels in PRCP^gt/gt mice on 13-wk HFD were not statistically significant compared with WT and PRCP^gt/gt mice on SD. Circulating nonesterified fatty acid (NEFA; C) and triglyceride levels (D) were measured in mice on 13-wk HFD. Note that NEFA levels were reduced significantly in PRCP^gt/gt mice on HFD compared with all other experimental groups. D: HFD induced a significant increase in triglyceride levels in both WT and PRCP^gt/gt mice compared with mice on SD. **P < 0.01; ***P < 0.001.

Nonesterified fatty acid (NEFA) levels were not different between WT and PRCP^gt/gt mice exposed to SD (0.62 ± 0.03 in WT and 0.75 ± 0.08 in PRCP^gt/gt, n = 7 each, P = 0.08; Fig. 6C). However, 13-wk HFD PRCP^gt/gt mice showed lower levels of circulating NEFA (0.32 ± 0.05 meq/l, n = 7) compared with WT controls (0.71 ± 0.08 meq/l, n = 7, P = 0.001; Fig. 6C).

HFD for 13 wk induced an increase in triglyceride levels in both WT and PRCP^gt/gt mice (Fig. 6D). However, no differences in triglyceride levels were found between WT and PRCP^gt/gt mice when exposed to either SD or 13-wk HFD (68.44 ± 4.73 mg/dl in WT on SD and 79.45 ± 12.75 mg/dl in PRCP^gt/gt on SD, n = 8 in both groups, P = 0.43; 109.7 ± 5.33 mg/dl in WT on HFD and 107.0 ± 3.20 mg/dl in PRCP^gt/gt on HFD, n = 11 and 20, respectively, P = 0.64; Fig. 6D).

Liver weight, hepatic lipid accumulation, and gluconeogenesis.

No difference in liver weight was found in mice under SD (1.35 ± 0.11 g in WT, n = 7; 1.27 ± 0.06 g in PRCP^gt/gt mice, n = 10, P >0.05; Fig. 7A). Although liver weight in 7-wk HFD PRCP^gt/gt mice was lower (1.08 ± 0.07 g, n = 4) than that of 7-wk HFD WT mice (1.47 ± 0.15 g in WT, n = 4), it was not statistically significant. However, at 13 wk of HFD, a significantly lower liver weight was found in PRCP^gt/gt mice (1.31 ± 0.09 g, n = 10) compared with WT controls (1.97 ± 0.15 g, n = 10, P < 0.01; Fig. 7A). Hepatic trygliceride levels were significantly lower in PRCP^gt/gt mice on SD as well as on HFD for 13 wk. On SD, WT mice had trygliceride levels of 77.3 ± 9.1 mg/dl, whereas PRCP^gt/gt mice had 49.9 ± 1.9 mg/dl (n = 9 in both groups, P = 0.01). On HFD for 13 wk, trygliceride levels in WT mice were 294.9 ± 45.2 mg/dl, whereas in PRCP^gt/gt mice they were 163.0 ± 16.6 mg/dl (n = 8 and 9, respectively, P = 0.011). Histological analysis of hepatic tissue showed an increase in lipid accumulation specifically in the liver of WT mice exposed to 13 wk of HFD (Fig. 7F) compared with PRCP^gt/gt mice on HFD for 13 wk (Fig. 7E). No lipid accumulation was observed in the liver of PRCP^gt/gt mice on HFD for either 7 (Fig. 7C) or 13 wk (Fig. 7E) or in WT mice on HFD for 7 wk (Fig. 7D).

Fig. 7.

Effect of PRCP deletion on liver. A: liver weight in WT and PRCP^gt/gt mice on SD and HFD for either 7–8 or 13 wk. Note that whereas HFD induced increase in liver weight in WT mice, no difference was observed in PRCP^gt/gt mice. B: hepatic triglyceride (TGL) levels. PRCP^gt/gt mice showed statistically lower TGL levels on both diets (SD and 13 wk-HFD) compared with their WT controls. Thirteen weeks of HFD induced a significant increase in hepatic TGL in both WT and PRCP^gt/gt mice. However, the levels of TGL in 13-wk HFD PRCP^gt/gt mice were significantly lower than those of WT mice exposed to the same diet. C–F: representative pictures of liver histology from PRCP^gt/gt mice (C and E) and WT mice (D and F) on HFD for 7–8 (C and D) or 13 wk (E and F). Note the increase in lipid accumulation in the 13-wk HFD WT mouse (F) compared with the 13-wk HFD PRCP^gt/gt mouse (E). G and H: results from the real-time PCR for liver phosphoenolpyruvate carboxykinase (PEPCK; G) and glucose-6-phosphatase (G-6-Pase; H) in WT and PRCP^gt/gt mice exposed to SD and HFD for 7 and 13 wk. *P < 0.05; **P < 0.01; ***P < 0.001. Bar scale in F (for C–F) represents 50 μm.

As fasting glucose levels were decreased and the insulin sensitivity was increased in PRCP^gt/gt animals, we then compared the mRNA expression levels by real-time PCR of Pepck and G6pase, genes that are involved in liver gluconeogenesis. No differences in Pepck mRNA levels were observed between all PRCP^gt/gt or all WT experimental groups. However, each PRCP^gt/gt experimental group showed reduced Pepck mRNA levels compared with those of their corresponding WT control groups (1.2 ± 0.12 in WT and 0.82 ± 0.02 in PRCP^gt/gt on SD, n = 9 and 4, respectively, P <0.05; 1.11 ± 0.22 in WT and 0.84 ± 0.002 in PRCP^gt/gt on HFD for 7 wk, n = 4 and 3, respectively, P > 0.05; 1.3 ± 0.09 in WT and 0.5 ± 0.09 in PRCP^gt/gt on HFD for 13 wk, n = 5 and 4, respectively, P < 0.05; Fig. 7G), although the difference was not statistically significant in 7-wk HFD mice. G6pase mRNA levels in WT mice were greater when mice were exposed to SD showing decreased levels during the exposure of HFD, reaching a significantly lower level only at 13 wk of HFD (1.0 ± 0.14 in WT on SD, 0.79 ± 0.11 in WT on 7-wk HFD, and 0.34 ± 0.08 in WT on 13-wk HFD; n = 5, 4, and 4, respectively, P < 0.01). However, no changes in G6pase mRNA levels were observed between the experimental groups of PRCP^gt/gt animals (0.47 ± 0.09 on SD, 0.47 ± 0.09 in 7-wk HFD, and 0.26 ± 0.09 in 13-wk HFD; n = 5, 4, and 3, respectively, P > 0.5; Fig. 7G).

DISCUSSION

We have shown that mice lacking PRCP are resistant to the development of obesity and adiposity induced by HFD due to reduced food intake (21) and increased energy expenditure (8). We have also found that deletion of PRCP is associated with improved insulin sensitivity and hyperglycemia induced by the HFD. Furthermore, PRCP-deficient mice are protected against HFD-induced liver steatosis. Liver weight was lower in PRCP^gt/gt mice compared with WT controls, with a concomitant reduction in hepatic lipid accumulation and triglyceride content. In addition, the results on the decreased levels of liver Pepck and G6Pase, important genes involved in liver gluconeogenesis, are also consistent with the reduced circulating glucose levels found in PRCP^gt/gt mice compared with WT controls when exposed to HFD. More importantly, the improvements in glucose tolerance, insulin sensitivity, and liver gluconeogenesis observed between WT and PRCP^gt/gt mice were found already at 7–8 wk-HFD, the time point at which WT and PRCP^gt/gt mice showed no difference in body weight and fat mass deposition, an indication that PRCP deletion has beneficial effects on glucose metabolism independent of adiposity.

PRCP is one of the serine proteases that cleave the last amino acid at the COOH-terminal end of short peptides that contain proline as the penultimate amino acid. We have shown previously that this enzyme, by degrading it, is an important regulator of α-MSH produced by hypothalamic arcuate neurons (21). Thus, the phenotype observed in PRCP^gt/gt mice may be, at least in part, attributed to the increased levels of α-MSH. In agreement with this, previous reports studying α-MSH overexpression (9, 10, 11, 17) showed similar phenotype and resistance against the development of obesity and diabetes that we observed in PRCP^gt/gt mice. Specifically, the studies by Lee et al. (9) and Savontaus et al. (17) have reported that mice overexpressing α-MSH are protected from diet-induced obesity, showing increased energy expenditure and improved glucose metabolism. Furthermore, these mice, similarly to our PRCP^gt/gt mice, present reduced liver weight and triglyceride content and are protected against liver steatosis. The mechanism by which PRCP deletion prevents liver steatosis is unknown. Several reports have suggested that PRCP, which is expressed in a variety of peripheral organs, plays roles in hypertension, immune response, and angiogenesis (2, 7, 16, 18, 22). Thus, we cannot exclude the possibility that the phenotype observed in the liver of these mice is due to its direct effect on other substrates of PRCP in the liver and not via the α-MSH signaling in the central nervous system. On the other hand, data have shown that hypothalamic α-MSH, via its effect on the sympathetic nervous system, plays an important role controlling hepatic gluconeogenesis as well as lipid accumulation in the liver (3, 5, 12). In support of that, POMC-deficient mice show liver steatosis (6, 19, 23).

Our studies also show that deletion of PRCP has an impact on glucose metabolism and insulin sensitivity, which was independent of fat mass. PRCP^gt/gt mice showed reduced fasting glucose levels when exposed to both SD and HFD for 7–8 or 13 wk. Analysis of the area under the glucose response curve showed the improvement in glucose metabolism in PRCP^gt/gt vs. WT mice fed on SD and both high-fat diets. This is consistent with previous reports studying α-MSH-overexpressed animals (9) as well as chronically intracerebroventricularly α-MSH-infused mice (15). In addition, insulin sensitivity was also increased in PRCP^gt/gt mice on all diets compared with their WT controls. Insulin levels were significantly lower in fasted PRCP^gt/gt compared with those of WT mice on SD. No differences in insulin levels were observed in the fed state on a regular chow diet; however, 13-wk HFD fed PRCP^gt/gt mice had lower insulin levels. Furthermore, we also found that hepatic gluconeogenesis was decreased in PRCP^gt/gt mice compared with WT controls under all diets. Whether the improvement in glucose metabolism, like the prevention of liver steatosis in PRCP^gt/gt mice, is centrally mediated or not remains to be elucidated.

Finally, the increased energy expenditure observed in PRCP^gt/gt mice cannot be explained only by the increased locomotor activity. Energy expenditure in PRCP^gt/gt mice was found to be higher during both the light and dark cycles. When locomotor activity was analyzed, we found that it was elevated only in the dark phase but not during the light phase, an indication that the metabolic phenotype is not dependent on the changes in locomotor activity. In support of this, no changes in locomotor activity during either the light or dark phases were observed in PRCP^gt/gt mice exposed to a standard chow diet (8).

In conclusion, our data show that deletion of PRCP induced a reduction of body weight gain, fat mass accumulation, liver steatosis, and improvement in glucose metabolism when mice were exposed to high-fat feeding that was independent of adiposity. Future studies will address whether the deletion of PRCP induces these metabolic improvements via central mechanisms, i.e., higher levels of hypothalamic α-MSH, or by acting on other substrates directly in peripheral tissues.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-084065 (to S. Diano).

DISCLOSURES

There are no conflicts of interest, financial or otherwise, declared by the authors.

AUTHOR CONTRIBUTIONS

J.K.J., G.S., and G.M.R. performed the experiments; J.K.J., G.S., G.M.R., R.M., and S.D. analyzed the data; J.K.J., G.M.R., R.M., and S.D. interpreted the results of the experiments; J.K.J. and S.D. prepared the figures; J.K.J. and S.D. drafted the manuscript; J.K.J., G.S., G.M.R., R.M., and S.D. approved the final version of the manuscript; R.M. and S.D. did the conception and design of the research; S.D. edited and revised the manuscript.