Deficiency of the GPR39 receptor is associated ... - The FASEB Journal

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Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism Pia Steen Petersen,*,1 Chunyu Jin,*,1 Andreas Nygaard Madsen,* Maria Rasmussen,* Rune Kuhre,* Kristoffer L. Egerod,* Lars Bo Nielsen,† Thue W. Schwartz,*,‡ and Birgitte Holst*,‡,2 *Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology; † Department of Clinical Biochemistry; and ‡Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark GPR39, a constitutively active 7TM receptor important for glucose-induced insulin secretion and maturation of pancreatic ␤-cell function, is upregulated in adipose tissue on abstinence from food and chemically induced diabetes. In the present study, we investigated the effect of GPR39 deficiency on body weight and adipocyte metabolism. GPR39-deficient mice were subjected to a high-fat diet and body composition, glucose tolerance, insulin secretion, food intake, and energy expenditure were evaluated. The cell biology of adipocyte metabolism was studied on both mRNA and protein levels. A significant increase in body weight corresponding to a 2-fold selective increase in fat mass was observed in GPR39-deficient mice fed a high-fat diet as compared with wild-type littermate controls fed the same diet. The GPR39-deficient animals had similar food intake but displayed almost eliminated diet-induced thermogenesis, measured by the oxygen consumption rate (VO2) on change from normal to high-fat diet. Analysis of the adipose tissue for lipolytic enzymes demonstrated decreased level of phosphorylated hormone-sensitive lipase (HSL) and a decreased level of adipose triglyceride lipase (ATGL) by 35 and 60%, respectively, after food withdrawal in the GPR39-deficient mice. Extracellular signal-regulated kinases (ERK1/2), a signaling pathway known to be important for lipolysis, was decreased by 56% in the GPR39-deficient mice. GPR39 deficiency is associated with increased fat accumulation on a high-fat diet, conceivably due to decreased energy expenditure and adipocyte lipolytic activity.—Petersen, P. S., Jin, C., Madsen, A. N., Rasmussen, M., Kuhre, R., L. Egerod, K. L., Nielsen, L. B., Schwartz. T. W., Holst, B. Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism. FASEB J. 25, 3803–3814 (2011). www.fasebj.org

ABSTRACT

Key Words: 7TM receptor signaling 䡠 lipolysis 䡠 high-fat diet

Obesity constitutes a major global health problem, as it is increasing in prevalence and is associated with increased risk of metabolic disorders, including type 2 diabetes and cardiovascular diseases (1). Increased 0892-6638/11/0025-3803 © FASEB

accumulation of adipose tissue may arise from increased deposit or decreased removal and utilization of triglycerides or a combination of these mechanisms. Tight regulation by a cascade of enzymes and transcription factors is important to maintain the body weight regulation both in the central brain centers and in the peripheral metabolic tissues (2– 4). In rodent and human studies, central modulation of appetite and energy expenditure, especially by the hypothalamus, has been demonstrated to be crucial for the development of obesity (5). Interestingly, modulation of fat metabolism by hypothalamic factors such as ghrelin and melanocortin seems to be mediated primarily through the adrenergic sympathetic system (6, 7). Severe obesity has been observed in mice with a combined deficiency in all 3 types of ␤-adrenergic receptors. These mice developed obesity within 6 d of high-fat diet (HFD) treatment and were resistant to the diet-induced adaptive thermogenesis (8). Modulation of the expression of the intracellular adipocyte lipases also affects the development of obesity, although the redundancy in lipases makes the system complicated (9 –12). Furthermore, it has recently been reported that the process of lipolysis is under local paracrine control both by metabolites such as lactate and other ketone bodies as well as by locally produced prostaglandins (13–15). GPR39 is an orphan 7TM receptor that is structurally and functionally related to the ghrelin receptor (16). However, GPR39 has a broader signaling repertoire than that of the ghrelin receptor, as it couples both to the G␣q pathway with activation of phospholipase C, to the G␣s with activation of adenylate cyclase, and apparently also to G␣12/13 (16, 17). Notably, like the ghrelin receptor, GPR39 is constitutively active; accordingly, an increase in receptor expression will induce increased 1

These authors contributed equally to this work. Correspondence: Laboratory for Molecular Pharmacology, Department of Pharmacology, University of Copenhagen, Blegdamsvej 3b, DK-2200, Copenhagen, Denmark. E-mail: [email protected] doi: 10.1096/fj.11-184531 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

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signaling, even without any ligand present (16). However, GPR39 signaling is stimulated by zinc ions, which may constitute a modulator of an as yet unidentified, selective endogenous agonist (17). In contrast to the ghrelin receptor, GPR39 is mainly expressed in peripheral tissues with important metabolic activity, such as the liver, the gastrointestinal tract, the endocrine pancreas, and adipose tissue (18). GPR39 has previously been shown to modulate the function of the endocrine pancreas, as GPR39-deficient mice displayed impaired glucose-stimulated insulin secretion both in vivo and in vitro (for example, in isolated perifused islets), and their islets expressed decreased levels of the crucial transcription factors HNF-1a and PDX-1 (19, 20). In addition, it has been demonstrated that GPR39 is important for gastric emptying, gastric fluid secretion, and gastrointestinal transit time (21). The exact function of GPR39 in adipose tissue has not previously been characterized, but the expression of GPR39 in white adipose tissue is up-regulated both during abstinence from food and in streptozotocin-induced diabetic rats (18). In addition, it is known that mouse embryo fibroblasts capable of differentiating into both white and brown adipocytes (22) express high levels of GPR39, and this expression is decreased by differentiation into both white and brown fat cells (18). In the present study, we have challenged GPR39deficient mice with HFD and observed increased body weight and increased fat mass compared with wild-type (WT) littermate control mice. The mice were exposed to calorimetric analysis to understand the contribution from food intake, physical activity, and energy expenditure to the increased fat accumulation. Furthermore, the gene expression, protein levels, and phosphorylation levels of various enzymes involved in the metabolism of the adipocytes were studied. Finally, the important signaling pathways downstream of GPR39 were also evaluated. MATERIALS AND METHODS Animals GPR39-deficient mice were generated by Deltagen (San Mateo, CA, USA; ref. 20) and backcrossed into C57BL/6 background for 5 generations. Male GPR39-deficient mice and WT mice obtained from heterozygous mating were used in this study. Genotypes were verified as described previously (20). The mice were housed in an animal facility with a 12-h light-dark cycle (6 AM to 6 PM) and had free access to water and diet, either a standard chow diet or an HFD with 60% calories derived from the fat (5.24 kcal/g; D12492; Research Diets, New Brunswick, NJ, USA). For mice fed HFD, the food was switched from chow to HFD when they were 7 wk old. Body weight was recorded once a week. All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Experiments Inspectorate in Denmark. Body composition After being maintained on an HFD for 12 wk, fat and lean mass of live, unanesthetized GPR39-deficient and WT mice 3804

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were assessed by quantitative magnetic resonance imaging (MRI) using EchoMRI (Echo Medical Systems, Houston, TX, USA). Oral glucose tolerance test (OGTT) OGTT was carried out in mice fed an HFD for 12 wk. The animals were deprived of food overnight for 16 –18 h with free access to water. Glucose (1.5 g/kg body wt) was administered by an oral gavage tube. Blood glucose was monitored in samples obtained from tail punctures using a handheld glucometer (Ascensia Contour Glucometer; Bayer, Kiel, Germany) before and after glucose administration. At time points of 0 and 15 min, blood was collected from the orbital sinus, and plasma was isolated by centrifugation for insulin measurement. Insulin was measured using sensitive insulin RIA kit (Linco Research, St. Charles, MO, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated with glucose and insulin concentrations obtained using the following formula: [fasting glucose (mM) ⫻ fasting insulin (␮U/ml)]/22.5. In vivo lipolysis measurement The 15- to 18-wk-old female GPR39-deficient and WT mice fed a standard chow diet were deprived of food for 3 h at the start of the light phase, and a blood sample was drawn from the orbital sinus for measurement of basal free fatty acid levels. A dose of isoproterenol (5 mg/kg i.p.) was injected 1 h later. At 15 min postinjection, another blood sample was taken. Plasma was isolated by centrifugation and used for free fatty acid measurement (NEFA-2HR kit; Wako Chemicals, Neuss, Germany). Ex vivo lipolytic activity Epididymal adipose tissue was dissected from 9- to 13-wk-old GPR39-deficient and WT mice (age-matched with no more than 1 wk difference in age within each experiment). Fat explants of ⬃20 mg were incubated in 1 ml buffer containing 120 nM NaCl, 4 mM KH2PO4, 1 mM MgSO4, 0.75 mM CaCl2, 10 mM NaHCO3, and 30 mM HEPES (pH 7.4). Isoproterenol (10 nM) or insulin (20 ng/ml) was added to stimulate or inhibit lipolysis. Samples were incubated at 37°C for 4 h, and free fatty acid and glycerol concentrations in the buffer were measured using a NEFA-2HR kit (Wako Chemicals) and serum triglyceride determination kit (Sigma-Aldrich; St. Louis, MO, USA). Protein content of the adipose explants was measured using the Pierce BCA protein assay kit (Thermo Scientific, Slangerup, Denmark) after the tissue was homogenized with lysis buffer containing 1% Triton X-100, 150 nM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1% Nonidet P-40. Tissue collection At the end of the study, the animals fed an HFD for 17–18 wk were euthanized after being deprived of food overnight for 16 –18 h. Blood was sampled from the orbital sinus, and plasma was separated. Plasma triglycerides, cholesterol, and glycerol were measured by HPLC. Free fatty acids were measured using NEFA-2HR kit from Wako Chemicals. Adiponectin was measured using an immunoassay kit from Invitrogen. White adipose tissues (inguinal subcutaneous, retroperitoneal, mesenteric, and epididymal) were dissected and weighed. Adipose tissues for Western blot were snapfrozen in liquid nitrogen and stored at ⫺80°C until use, and

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PETERSEN ET AL.

tissues for mRNA analysis were incubated overnight in RNAlater RNA stabilization reagent (Qiagen, Germantown, MD, USA) at 4°C and then stored at ⫺80°C until quantitative PCR analysis. Long-term food intake measurements A separate cohort of mice was used for long-term food intake studies. Mice were individually housed. Diet was switched from chow to HFD at 7 wk of age, and the animals were fed HFD for 11 wk. Body weight was recorded weekly. Food intake was measured 3⫻/wk, and average daily intake was calculated. Feed efficiency [weight gain (mg)/food consumption (kcal)] was calculated. Indirect calorimetry Indirect calorimetry was performed in a 16-chamber indirect calorimetry system (PhenoMaster; TSE Systems, Bad Homburg, Germany) to examine the response of GPR39-deficient mice to an HFD. A separate cohort of mice initially fed chow diet was used in this study at 7– 8 wk of age. Mice were individually housed and placed in the chambers for 10 d; the first 5 d was considered the acclimation phase, and data were analyzed only for the last 5 d. Diet was switched from chow to HFD after 3 d of measurements. Oxygen consumption rate (Vo2; ml/h/kg), respiratory exchange ratio (RER), total activity (beam breaks), and food intake were simultaneously measured for each mouse. The oxygen consumption rate was also measured in another cohort of mice fed HFD for 16 wk. Western blot Epididymal fat pads were dissected from overnight fooddeprived mice and snap-frozen in liquid nitrogen. The frozen fat pads were homogenized in 500 ␮l ice-cold lysis buffer (1% Triton X-100; 150 mM NaCl; 10 mM Tris-HCl, pH 7.5; 1 mM EDTA; 1% Nonidet P-40; and minicomplete protease inhibitor cocktail; Roche, Mannheim, Germany) with a mechanical homogenizer, and debris was cleared by centrifugation at 10,000 g at 4°C for 10 min. The supernatant was collected, and the amount of protein was determined with a BCA kit (Thermo Scientific). Equal amounts of protein per well were diluted in NuPage LDS 4* sample buffer (Invitrogen, Carlsbad, CA, USA) containing DTT and boiled for 5 min. Protein extracts (20 ␮g/lane) were separated on 10% SDS-PAGE and electroblotted to PDVF membranes. Membranes were blocked for 30 min in TBS ⫹ 0.1%Tween (TBST), with 2% BSA (Calbiochem, Damstadt, Germany) and incubated with primary antibody [hormone-sensitive lipase (HSL), extracellular signal-regulated kinases (ERK1/2), adipose triglyceride lipase (ATGL), perilipin A (1:1000), or ␤-actin (1:5000) in TBST⫹2% BSA], overnight at 4°C. Membranes were washed 3 ⫻ 10 min in TBST before developing with a horseradish peroxidase-conjugated-goat anti-rabbit IgG or goat antimouse IgG (1:10,000 dilution in TBST⫹2% nonfat milk powder; Bio-Rad, Copenhagen, Denmark) for 2 h at room temperature. Membranes were washed 3 ⫻ 10 min in TBST again, and bands were visualized by enhanced chemiluminescence reagent [SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) and FluorChem HD2 (Cell Biosciences, Santa Clara, CA, USA)]. Bands were quantified by densitometry (Alpha Easy Innotech software; Cell Biosciences). Equal loading was ensured with use of ␤-actin antibody. All antibodies used were from Cell Signaling (Danvers, MA, USA) or Sigma-Aldrich. GPR39 REGULATES LIPOLYSIS AND ENERGY EXPENDITURE

Quantitative real-time PCR Relative mRNA levels were measured by the quantitative PCR method (QPCR) using the Mx3000P from Stratagene (La Jolla, Ca, USA) and SYBRPremix Ex Taq (Takara, Otsu, Japan). The relative levels of genes from different samples were compared by the ⌬⌬Ct method using the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ␨ polypeptide (YWHAZ) as reference gene. Before the ⌬⌬Ct value was calculated, primer efficiency was validated by standard curve measurements, and primers with ⬎95% efficiency were used (18). A calibrator sample was included in each assay for normalization between runs. Primer sequences are listed in Supplemental Fig. S1. RNA was extracted with RNeasy lipid tissue mini kit (Qiagen), and cDNA was synthesized by reverse transcription using the ImProm-IITM reverse transcriptase (Promega, Madison, WI). Statistical analyses Statistical analyses were performed using GraphPad Prism 4.0 statistical software (GraphPad, San Diego, CA, USA). Data were analyzed using 2-tailed Student’s t test, 1-way or 2-way ANOVA, followed by Bonferroni post hoc tests. All data are presented as means ⫾ se. Values of P ⱕ 0.05 were considered significant.

RESULTS To investigate the cellular and molecular importance of GPR39 for the control of body weight and adipose tissue metabolism, the previously described GPR39deficient mice (20) were challenged with HFD for 17 wk from the age of 7 wk. The body weight of the mice was measured every week, and after 12 wk of HFD, more detailed measurements were started, including OGTT, measurement of body composition by MRI scanning, and calorimetric measurement in TSE systems. The study was terminated after ⬃17 wk of HFD, when the mice were deprived of food for 16 h and plasma metabolic parameters, adipose tissue gene, and protein expression were determined. Body weight and body composition At study entry, no difference in body weight was observed between the WT and GPR39-deficient mice, but after 7 wk of HFD, the GPR39-deficient mice started to display a higher body weight. After 11 wk of HFD, this difference reached a significant level that continued for the following 3 wk, when the GPR39-deficient mice had a body weight that was 5.0 g higher than that of the WT mice (P⫽0.008; Fig. 1A). These data were reproduced in 2 independent cohorts of GPR39-deficient mice studied over a period of 2 yr (data from the second cohort are shown in Supplemental Fig. S2; data from a third cohort of singly housed mice are shown in Fig. 3A). After 12 wk of HFD, MRI scanning demonstrated a significantly higher fat content in the GPR39deficient mice, where the fat mass was doubled compared with the WT littermates (12.5⫾1.1 vs. 6.4⫾1.2 g; Fig. 1B). In contrast, the lean body weight did not differ 3805

Figure 1. Diet-induced weight gain. A) Body weight gain in GPR39-deficient (solid circles) and WT (open circles) mice fed an HFD. Mean body weight of the GPR39-deficient and WT mice when started on the diet treatment was 23.1 ⫾ 0.6 and 23.8 ⫾ 0.7 g, respectively. After 14 wk of HFD, body weight of the mice was 31.6 ⫾ 0.7 and 36.6 ⫾ 1.5 g, respectively; n ⫽ 9 –13. B) Fat mass measured with MRI 4-in-1 body composition scanner in GPR39deficient (⫺/⫺; 12.5⫾1.1 g) and WT (⫹/⫹; 6.4⫾1.2 g) mice fed an HFD for 12 wk; n ⫽ 14 –15. C) Lean mass in GPR39-deficient (27.9⫾2.1 g) and WT (28.0⫾2.1 g) mice. D) Fat compartment mass in GPR39-deficient (solid bars; n⫽21) and WT (open bars; n⫽14) mice after HFD feeding for 17–18 wk. Unilateral inguinal subcutaneous fat, GPR39-deficient (0.5⫾0.08 g) vs. WT (0.2⫾0.04 g), P ⫽ 0.04. Unilateral retroperitoneal fat pad, GPR39-deficient (0.7⫾0.05 g) vs. WT (0.4⫾0.07 g), P ⫽ 0.002. Mesenteric fat pad, GPR39-deficient (1.30⫾0.1 g) vs. WT (0.6⫾0.1 g), P ⫽ 0.003. Bilateral epididymal fat pad, GPR39-deficient (2.2⫾0.1 g) vs. WT (1.6⫾0.2 g), P ⫽ 0.02. Data are shown as means ⫾ se. *P ⱕ 0.05; **P ⱕ 0.01; ***P ⱕ 0.001.

between the groups (Fig. 1C), indicating that HFD caused a selective increase in body fat content without any effect on the general growth pattern in GPR39deficient mice. By the end of the study, fat pads from each compartment were dissected from the mice. Increased fat accumulation was observed in all compartments, both in the subcutaneous and in the visceral fat compartments. The subcutaneous inguinal fat in the GPR39-deficient mice was 0.46 ⫾ 0.08 g in comparison to 0.24 ⫾ 0.04 g in the WT mice (P⫽0.039; Fig. 1D). In the well-defined visceral fat compartments, the difference between WT and GPR39-deficient mice reached a higher degree of significance, for example, in the retroperitoneal fat compartment, where the weight for WT vs. GPR39-deficient mice was 0.71 ⫾ 0.05 and 0.43 ⫾ 0.07 g (P⫽0.0019), respectively (Fig. 1D). In summary, on HFD, the GPR39-deficient mice gained more body weight than WT control mice, mainly due to a selective accumulation of fat. Glucose homeostasis In previous studies (19, 20), it has been demonstrated that GPR39 is important for the function of the endocrine pancreas, as GPR39-deficient mice suffered from impaired insulin secretion in response to glucose challenges. After 12 wk of HFD, the animals were deprived of 3806

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food overnight and challenged with an oral glucose load (Fig. 2A). The GPR39-deficient mice had significantly higher basal glucose levels after 16 h of food deprivation and were unable to clear the glucose load as efficiently as observed for the WT littermates, where blood glucose did not reach basal levels even 2 h after the glucose load (Fig. 2A). The plasma insulin level was significantly elevated in GPR39-deficient mice after 16 h of food deprivation (Fig. 2B, C). However, the increment in plasma insulin level observed 15 min after glucose administration was slightly lower but not significantly different from that observed in the WT littermate control mice. The glucose-induced insulin secretion has previously been described to be decreased in GPR39-deficient mice fed chow diet (20), and in the present study the insulin secretion was not sufficient in the HFD-treated GPR39-deficient mice to overcome the potential insulin resistance. To assess the insulin sensitivity, we calculated the HOMA-IR. This empirical method, based on fasting insulin and glucose levels (Fig. 2C, D), was originally developed for humans and verified by hyperinsulinemic-euglycemic clamp data. More recently, it has been shown to be a valuable assessment for insulin sensitivity in rodents as well (23, 24). HOMA-IR based on 16 h food-deprivation data was higher in the GPR39-deficient mice, indicating lower insulin sensitivity in these mice compared with the control mice (Fig. 2E).


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Figure 2. Glucose regulation in mice fed an HFD. Solid circles and bars indicate GPR39-deficient mice; open circles and bars indicate WT mice. Data are shown as means ⫾ se. A) OGTT performed in GPR39-deficient mice and WT after 12 wk of HFD. *P ⱕ 0.05; **P ⱕ 0.01. B) Plasma insulin levels before and 15 min after an oral glucose load of 1.5 g/kg body wt. **P ⫽ 0.0041; ## P ⱕ 0.01, ###P ⱕ 0.001 vs. corresponding baseline control. C) Plasma insulin after overnight food deprivation in WT (⫹/⫹; 0.20⫾0.07 ng/ml) and GPR39-deficient mice (⫺/⫺; 0.56⫾0.07 ng/ml). **P ⫽ 0.0041. D) Blood glucose after overnight food deprivation in WT (4.5⫾0.3 mM) and GPR39-deficient mice (7.4⫾1.0 mM). *P ⫽ 0.024. E) HOMA-IR calculated as [fasting glucose (mM) ⫻ fasting insulin (␮U/ml)]/22.5. *P ⫽ 0.0103.

These data suggest that the GPR39-deficient mice treated with HFD have a primary phenotype of increased fat accumulation with secondary impaired glucose tolerance caused by obesity-induced insulin resistance and decreased insulin secretion. Chronic food intake and feed efficiency In principle, the increased fat accumulation may be caused either by increased energy intake or by decreased energy expenditure. Over a period of 11 wk, a separate cohort of GPR39-deficient mice and WT

littermate controls were treated with HFD but singly housed to measure food intake. In accordance with the previous observation for group-housed mice, the GPR39-deficient mice demonstrated increased body weight gain compared with the control mice (Fig. 3A). In contrast, no difference was observed in the daily food intake (Fig. 3B). The feed efficiency, defined as body weight gain per kilocalorie of food consumption (25), was significantly higher in the GPR39-deficient mice (Fig. 3C). It is therefore concluded that the GPR39-deficient mice have normal food intake but increased energy storage for the

Figure 3. Long-term study of feeding behavior and body weight. GPR39-deficient mice display same food intake as WT mice but gain more weight (n⫽8 –10). A) Cumulative weight gain after 11 wk of HFD. B) Daily food intake. C) Feed efficiency ⫽ weight gain (mg)/food consumption (kcal); 15.6 ⫾ 0.84 and 12 ⫾ 1 mg/kcal for GPR39-deficient and WT mice, respectively. Data are shown as means ⫾ se. *P ⱕ 0.05. GPR39 REGULATES LIPOLYSIS AND ENERGY EXPENDITURE

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same energy intake, which may contribute to the increased fat accumulation. Acute food intake, locomotor activity, and energy expenditure The 7- to 8-wk-old lean mice were placed in habituation cages and 5 d later transferred to calorimetry cages. During the first 3 d in the calorimetry cages, the mice were fed standard chow diet; subsequently, the diet was shifted to HFD for 2 d. It is well recognized that exposure to HFD increases the energy expenditure, i.e., diet-induced thermogenesis (8, 26), a process that to a large degree is dependent on the sympathetic nervous system and the presence of ␤-adrenergic receptors (8). The normal diurnal rhythm, with high activity during the dark phase and low activity during the light phase, was observed for all parameters measured in the calorimetry cages (Fig. 4). Food intake did not differ significantly between the two groups of mice, either in the dark or the light phase; however, a trend toward increased food intake was observed in the light phase under both feeding conditions for mice deficient in GPR39 (Fig. 4A). Similarly, activity did not differ significantly between the groups, although, during the darkactive phase, a trend toward decreased activity was observed for GPR39-deficient mice under both feeding conditions (Fig. 4B). There was no difference in RER between the two groups (Fig. 4C).

The expected increase in Vo2 consumption in response to HFD exposure was observed in the WT group of animals, particularly during the light phase, as the Vo2 consumption during light phase increased from 4200 ⫾ 120 ml/h/kg on regular chow diet to 4989 ⫾ 182 ml/h/kg after a shift to HFD (P⬍0.05). In contrast, the GPR39-deficient mice only responded with a small insignificant increase in energy expenditure (Fig. 4D, E). Thus, the diet-induced thermogenesis is almost eliminated in the GPR39-deficient mice. Energy expenditure after 16 wk of HFD feeding After 16 wk exposure to HFD, the mice were placed in the calorimetry cages under nonstressed conditions for 5 d, as described for the young mice. At this time point, significantly lower Vo2 consumption was observed in the GPR39-deficient mice (3060⫾110 ml/h/kg) as compared with WT mice (3610⫾170 ml/h/kg; P⫽ 0.001). Furthermore, the diurnal fluctuations observed between light and dark phase were less pronounced for GPR39-deficient mice compared with WT mice (Fig. 4F). Expression of genes involved in adipose tissue differentiation and regeneration After the 17–18 wk of HFD treatment, the mice were deprived of food for 16 h, the study was terminated,

Figure 4. Indirect calorimetry. The 7- to 8-wk-old lean mice were placed in habituation cages and 5 d later transferred to calorimetry cages. During the first 3 d in the calorimetry cages, mice were fed standard chow diet; subsequently, the diet was shifted to HFD for 2 d. A–C) Average 12 h food intake (A), ambulatory activity (B), and RER (C) in the dark and light period for WT (⫹/⫹; open bar) and GPR39-deficient (⫺/⫺; solid bar) mice before and after switching from chow diet to HFD. D) Vo2 (ml/h/kg) in the dark and light period for WT (⫹/⫹; gray trace) and GPR39-deficient (⫺/⫺; solid black trace) mice before and after switching from chow diet to HFD. Light gray areas indicate dark periods. E) Average 12 h Vo2 (ml/h/kg) in the light period for WT (⫹/⫹; open bar) and GPR39-deficient (⫺/⫺; solid bar) mice before and after switching the diet from chow to HFD. *P ⱕ 0.05, 1-way ANOVA with Bonferroni post hoc test. F) After 16 wk of exposure to HFD, mice were placed in the calorimetry cages after habituation for 5 d; Vo2 (ml/h/kg) in the dark and light period for WT (⫹/⫹; gray trace) and GPR39-deficient (⫺/⫺; solid black trace) mice. Data are shown as means ⫾ se. 3808

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and visceral adipose tissue depots were dissected. Gene expression was evaluated by quantitative PCR to understand the cellular background of the increased accumulation in adipose tissue. GPR39-associated changes in adipocyte differentiation were suggested by previous studies where we observed the highest GPR39 expression in the undifferentiated state of the mouse embryonic fibroblast cell line (Rb⫹/⫺) that is capable of differentiating into both brown and white fat-like cells (22). The decreased GPR39 expression in both the brown and white adipose tissue-like cell line suggested a role for GPR39 in the differentiation process (18). To test for the importance of GPR39 in the differentiation of preadipocytes into mature, fully functional adipocytes, we measured the expression level of some of the main factors driving the transcription cascade of adipocyte differentiation, specifically CCAAT/enhancer-binding protein ␣ (C/ EBP-␣) and peroxisome proliferator-activated receptor ␥ (PPAR-␥), sterol regulatory element binding protein (SREBP), and C/EBP-␤ (27, 28). However, we observed no significant difference between GPR39-deficient and littermate WT mice for any of these genes (Fig. 5A). Preadipocyte factor 1 (Pref-1) is highly expressed in preadipocytes but completely absent in mature adipocytes (29). However, Pref-1 was not changed in the HFD-treated GPR39-deficient mice (Fig. 5A). PPAR␥ coactivator 1␣ (PGC-1␣) is one of the master regulators of mitochondrial biogenesis that activate and induce expression of genes involved in coordinated regulation of nuclear- and mitochondrial-encoded genes (30). The mRNA expression levels of PGC-1␣ and the mitochondrial expressed citrate synthase (CS) were similar in GPR39-deficient and WT mice (Fig. 5A).

Expression of genes involved in adipocyte metabolism The two adrenergic receptors expressed in the dissected adipocytes were both decreased in the GPR39deficient mice. For the ␤2 adrenergic receptor (␤2adrR), this decrease reached a significant level, whereas the ␤3 adrenergic receptor (␤3-adrR), which is more abundantly expressed in adipocytes, showed only a trend toward decrease in the GPR39-deficient mice (Fig. 5B). The expression of 3 major genes involved in the fatty acid synthesis, acetyl-coenzyme A carboxylase (ACC), phosphoenolpyruvate carboxykinase (PEPCK), and fatty acid synthase (FASN), were found not to be altered significantly in the GPR39-deficient mice (Fig. 5B). The same was the case for fatty acid-binding protein (FABP4), which is involved in fatty acid transport and storage and for carnitine palmitoyltransferase I (CPT-1b), which is involved in fat oxidation (31). Expression of genes and proteins involved in lipolysis HSL, an important enzyme controlling the adipocyte lipolytic activity, known to be highly regulated by, for example, insulin and the sympathetic nervous system acting through adrenergic receptors, was found to be significantly altered in the GPR39-deficient mice as compared with WT mice, as shown in Fig. 5B. Since the enzyme activity and the translocation of HSL to lipid droplets are dependent on phosphorylation, we studied this by Western blot analysis. In accordance with the observation at the mRNA level, the total amount of HSL protein expressed in epididymal adipose tissue was found to be decreased in the GPR39-deficient mice as compared with WT (Fig. 6A). Notably, we observed a

Figure 5. Expression of genes involved in adipocyte differentiation and lipid metabolism in GPR39-deficient mice (solid bars) vs. WT littermate control mice (open bars). A) C/EBP-␣, PPAR-␥, SREBP, C/EBP-␤, Pref-1, PGC-1␣, and CS. B) ␤2-adrR, ␤3-adrR, ACC, PEPCK, FASN, FABP4, CPT-1b, perilipin (Plin1), desnutrin/ATGL, HSL, and adiponectin (Adipo). Data are shown as means ⫾ se; n ⫽ 5– 8. Level of mRNA is relative to the reference gene; level of WT is set to 1. *P ⫽ 0.049; **P ⫽ 0.0046. GPR39 REGULATES LIPOLYSIS AND ENERGY EXPENDITURE

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Figure 6. Expression of proteins involved in lipid metabolism. Phosphorylation and total protein expression with representative Western blots. Lanes 1, 3, and 5 are 3 representative samples from WT mice; lanes 2, 4, and 6 are 3 representative samples from GPR39-deficient mice. Graphs represent the average of 7 and 9 independent Western blots from epididymal fat in WT mice (open bars) and GPR39-deficient mice (solid bars), respectively. A) HSL. ***P ⫽ 0.0003. B) Perilipin A. *P ⫽ 0.048. C) ATGL. **P ⫽ 0.0044. D) ERK. ***P ⫽ 0.0004. Similar data have been obtained from adipose tissue from the retroperitoneal compartment.

significant 35% decrease also in the degree of HSL phosphorylation level (P⫽0.0003; Fig. 6A). Perilipin A is a protein covering the lipid droplets and serving as an essential interaction partner for HSL. The mRNA level of this protein showed a trend toward a decrease (Fig. 5B), and a significantly decreased protein level was seen in the GPR39-deficient mice, i.e., down to 66% of that observed in the WT mice (P⫽0.048; Fig. 6B). The other main lipase involved in the control of adipocyte lipolysis is desnutrin/ATGL, which, like HSL, is regulated by adrenergic receptor activity. Also, in the case of ATGL, we found that GPR39deficient mice fed an HFD for 17–18 wk had significantly lower levels of ATGL protein in their adipocytes, decreased to 40% (P⫽0.0044; Fig. 6C), a difference that was only observed as a trend at mRNA level (Fig. 5B). The total protein level of ACC, which is important for the synthesis of fatty acids, was strongly decreased (Supplemental Fig. S3; P⫽0.01). This down-regulation of one of the key enzymes involved in fatty acid synthesis may be compensatory to the decreased lipolysis, a mechanism also observed in HSL-deficient mice (32). ERK signaling in adipocytes

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Fat explants from the visceral fat compartments were dissected from WT and GPR39-deficient mice, and ex vivo lipolysis was studied under basal and isoproterenolstimulated conditions (Fig. 7A, B). Under basal conditions, the lipolysis measured as release of free fatty acids was decreased 35% in the GPR39-deficient mice (Fig. 7A). The lipolytic ability was measured in vivo after 3 h of food deprivation. We observed a small significant difference between the two groups in free fatty acid release after 3 h of food deprivation (P⫽0.04). Administration of a full agonist for all 3 different ␤-adrenergic receptors induced a lipolytic response that was not significantly altered in the GPR39-deficient mice (Fig. 7C). The notion that GPR39-deficient animals display decreased lipolytic activity was supported by measurements of plasma levels of free fatty acids and glycerol after 16 h of food deprivation, which were not increased in GPR39-deficient mice compared with WT mice, whereas the triglycerides and cholesterol levels were significantly higher in HFD-fed GPR39-deficient mice (Table 1).

DISCUSSION

Adrenergic regulation of phosphorylation of HSL is dependent on protein kinase A and on ERK1/2 (33), which previously has also been shown in recombinant in vitro systems to be a major signaling pathway for GPR39 (16). As shown in Fig. 6D, we found that ERK1/2 phosphorylation was decreased by 56% in the GPR39-deficient mice (P⫽0.0004). 3810

Adipocyte lipolytic activity

In the present study, we found that the receptor GPR39 is involved in the development of obesity. Significant increases in body weight were observed in GPR39deficient mice fed an HFD, where we observed an ⬃2-fold increase in fat mass compared with littermate control mice. The fat accumulation was located both in the subcutaneous and in the visceral fat compartments,


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Figure 7. Lipolytic activity. Rate of lipolysis was measured on fat explants from GPR39-deficient (solid bars) and WT (open bars) mice. Free fatty acids (FFA; A) and glycerol (B) were measured in the incubation buffer. Lipolysis was stimulated by isoproterenol (Iso; 10⫺8), and stimulation was inhibited by simultaneous addition of insulin (20 ng/ml). C) Lipolysis measured in vivo where FFAs were measured in the collected plasma samples. *P ⱕ 0.05.

and the expected deregulation of the glucose homeostasis was observed, including increased fasting glucose and insulin levels, as well as insulin resistance. Analysis of adipose tissue from mice fed an HFD showed decreased expression and phosphorylation of HSL and decreased expression was also observed for the other hormonally regulated lipase, ATGL. In addition, the protein expression level of the accessory protein important for lipolysis, perilipin A, was significantly lower in the GPR39-deficient mice compared with the WT mice. Accordingly, 3 h of food deprivation in vivo and basal level lipolysis on fat explants in vitro caused lower release of free fatty acids in GPR39-deficient mice compared with control mice. Since one of the signaling pathways downstream of the ␤-adrenergic receptors involved in lipolysis, ERK1/2, showed significantly decreased phosphorylation levels in the GPR39-deficient mice, it is suggested that GPR39 exerts its modulation of lipolysis through the same intracellular pathway as described previously for ␤-adrenergic receptors (Fig. 8). The original reason for exposing GPR39-deficient mice to HFD was the notion that GPR39 could be important in protecting against lipid-induced ␤-cell damage. In our initial study on the GPR39-deficient mice, we found decreased expression of transcription factors important for ␤-cell development, suggesting that the mice had immature and possibly more vulnerable ␤-cells. Furthermore, Dittmer et al. (34) demonstrated that GPR39 in neuronal cell lines was critical for resistance against oxidative and endoplasmic reticulum stress. Moreover, Tremblay et al. (19) reported that a diet supplemented with both high fat and high sucrose (HFHS diet) impaired the secretion of insulin from GPR39-deficient mice in vivo, presumably due to a lack of adaptive ␤-cell proliferation. In the present study,

where we supplemented the diet with high fat but normal levels of sucrose, the observed phenotype was, however, primarily related to the increased accumulation of fat. In addition, we observed a relatively insufficient insulin secretion in GPR39-deficient mice exposed to HFD, i.e., compared with the obesity-induced insulin resistance and glucose intolerance observed during OGTT. In view of the report that a diet supplemented with high levels of both fat and sucrose did not induce increased body weight in GPR39-deficient mice as compared with littermate control mice (19), we wanted to confirm that the obese phenotype we observed with a pure HFD was reproducible; therefore, we recruited a separate cohort including 39 WT mice and 51 GPR39deficient mice. This larger cohort confirmed that the GPR39-deficient mice were more susceptible to dietinduced obesity, as shown in Supplemental Fig. S2. Moreover, the single-housed GPR39-deficient mice also became more obese when fed HFD than littermate controls. One explanation for the difference observed in mice exposed to HFD vs. the HFHS diet is that the treatment with HFD in our studies was initiated when mice were only 7 wk old, whereas the HFHS treatment was initiated when the mice were 16 wk old (19). It has previously been reported that the age of initial exposure to the diet can be essential to reveal such differences. For example, mice deficient for the structurally related ghrelin receptor or deficient for ghrelin itself display decreased body weight and fat accumulation in comparison with controls, primarily when they were exposed to HFD at an early age (35, 36). Another explanation could be that the high sucrose concentration used in the study by Tremblay et al. (19) could have induced an overall higher energy intake in both the

TABLE 1. Plasma metabolic parameters Group

WT KO

Adiponectin (␮g/ml)

Triglycerides (mM)

Cholesterol (mM)

Glycerol (mM)

Free fatty acids (mg/dl)

49.56 ⫾ 5.34 40.26 ⫾ 2.18

0.37 ⫾ 0.05* 0.54 ⫾ 0.05*

3.49 ⫾ 0.42* 4.67 ⫾ 0.38*

0.74 ⫾ 0.06 0.82 ⫾ 0.03

47.99 ⫾ 2.32 49.47 ⫾ 2.74

Values are means ⫾ se; n ⱖ 7. Plasma adiponectin, triglycerides, cholesterol, glycerol, and free fatty acids were measured in mice deprived of food overnight after being fed an HFD for 17–18 wk. KO, knockout. *P ⱕ 0.05; Student’s t test.

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Figure 8. Regulation of lipolysis in adipocytes. Desnutrin/ ATGL initiates lipolysis by hydrolyzing triacylglycerol (TAG) to diacylglycerol (DAG). HSL hydrolyzes DAG to monoacylglycerol (MAG), which is subsequently hydrolyzed by MAG lipase (MGL) to generate glycerol. Three fatty acids (FA) generated during lipolysis can be released into the circulation for use by other organs or oxidized within adipocytes. During abstinence from food, catecholamines, by binding to G␣scoupled ␤-adrenergic receptors (␤AR), activate adenylate cyclase to increase cAMP and activate protein kinase A (PKA). In addition, they also potently stimulate phosphorylation of the MAPK pathway ERK1/2. It is suggested that GPR39, through intracellular crosstalk with the signaling pathways employed by the ␤ARs, could modulate lipolysis via ERK phosphorylation. In addition, GPR39 couples to the G␣sprotein and may also activate PKA.

GPR39-deficient and the WT animal groups due to palatability of the diet, which may have masked the effect of the GPR39 deficiency observed with the pure HFD. Based on the observation that important markers of pancreatic differentiation are significantly decreased in GPR39-deficient mice, we initially aimed our adipose tissue gene expression studies toward a series of similar markers of adipocyte differentiation (20, 27). However, surprisingly, none of these genes important for differentiation of preadipocytes were observed to be different in the GPR39-deficient mice exposed to HFD. This indicates that GPR39 probably does not play an important role in the differentiation of adipocytes during HFD feeding, despite the fact that the expression of GPR39 was higher in preadipocytes compared with mature white or brown adipocytes (18). The most prominent dysregulation observed in GPR39-deficient mice exposed to HFD was an increase in body weight corresponding to a highly selective accumulation of fat. We did not observe any change in a number of genes involved in fatty acid synthesis, storage, etc. In contrast, the main enzymes controlling lipolysis, HSL and ATGL, were both strongly reduced. This indicates that the increased fat accumulation observed in GPR39-deficient mice is due to impaired lipolysis, as we observed a decrease in both expression 3812

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level and phosphorylation of HSL and decreased protein expression of ATGL. The lipolysis is initiated by ATGL, which catalyzes hydrolysis of triglycerides, (37, 38), whereas HSL catalyzes the hydrolysis of diacylglycerol (39, 40) (Fig. 8). Accordingly, mice deficient in ATGL develop severe obesity with accumulation of triglycerides in various tissues, whereas deficiency of HSL results in a phenotype characterized by resistance to diet-induced obesity and accumulation of diacylglycerol (41– 43). The activity of HSL is dependent on interaction with auxiliary proteins, of which perilipin, which was also decreased in GPR39-deficient mice, is important for the recruitment of HSL to the lipid droplets (40). Stimulation of the ␤-adrenergic receptors in white adipose tissue is regarded as the major activator of lipolysis, whereas insulin is the best-characterized blocker of this process (40). The hormonal regulation of HSL is well known; however, the activity of ATGL is also modulated by the same hormones, although the molecular mechanism and functional importance of phosphorylation are not yet fully understood (40). Activation of ERK 1/2, which constitutes an important mediator of the hormonal regulation of the lipolytic process, was also strongly decreased in the GPR39-deficient mice, suggesting a crosstalk between intracellular signaling pathways activated via GPR39 and ␤-adrenergic receptors (Fig. 8). However, GPR39 was not required for the effect of a full agonist for all three ␤-adrenergic receptors, isoproterenol, and the decreased mRNA level of ␤2-adrenergic receptor observed in GPR39-deficient mice did not affect the isoproterenol induced lipolysis (Figs. 5B and 7). In contrast, lipolysis induced by 3 h of food deprivation was reduced in GPR39-deficient mice, which is interesting, considering the fact that ␤-adrenergic receptors are not required for food-deprivation-induced lipolysis (44). Thus, it is suggested that GPR39 represents a novel receptor that stimulates lipolysis and may influence the lipolytic response, for example, induced by food deprivation (Fig. 8). The other highly noticeable phenotypic trait associated with GPR39 deficiency in the present study was the near elimination of diet-induced thermogenesis. ␤-Adrenergic stimulation has been shown to be crucial for this adaptive process characterized by increased energy expenditure observed when the diet is switched from normal diet to HFD (26, 45). However, compensatory mechanisms take over when only a single type of ␤-adrenergic receptor is deleted, and the diet-induced thermogenesis is eliminated only when all 3 types of ␤-adrenergic receptor are knocked out (26). Interestingly, GPR39 is apparently also required for this shift in metabolism. Thus, it could be argued that GPR39 modulates the diet-induced thermogenesis in a similar manner to that described for ␤-adrenergic receptors. The results place GPR39 in a group of 7TM receptors modulating adipose tissue metabolism in particular lipolysis. The complexity of the lipolytic process has been illustrated recently by the revelation of novel antilipolytic mediators, such as prostaglandin PGE2


PETERSEN ET AL.

[produced by adipocyte phospholipase A (AdPLA)] and ketone bodies, acting in an autocrine or paracrine manner in the adipose tissue (15, 46, 47). For example, both AdPLA- and GPR81-deficient mice display a phenotype characterized by resistance to diet-induced obesity (15, 46, 47). Thus, the opposite phenotype of GPR39 deficiency was observed by elimination of the antilipolytic PGE2 or the receptor for lactate, which signals through G␣i-coupled receptors and thereby negatively affects the level of phosphorylated HSL and lipolysis (15). The present data indicate that GPR39 is involved in the regulation of body weight and fat accumulation, apparently through the modulation of intracellular signaling pathways and enzymes controlling lipolysis and that the receptor is required for diet-induced thermogenesis. It is therefore tempting to suggest that a GPR39 agonist would increase lipolysis and energy expenditure, especially under exposure to HFD, and consequently would be an interesting novel antiobesity agent. However, the increased lipolysis may induce a chronically high level of circulating fatty acids that correlates with adverse metabolic effects, such as insulin resistance. This would occur if fatty acids are generated at a rate that exceeds the oxidative capacity, with consequent potential ectopic storage of triglycerides and increased insulin resistance. Hence, partial stimulation of lipolysis, which is expected by stimulation of GPR39, in contrast to the ␤-adrenergic receptor, may provide an interesting novel target. Combined with the previous observation that GPR39 stimulation improves the glucose-induced insulin secretion and maturation of pancreatic ␤-cells (19, 20), this study indicates that GPR39 is an important novel drug target for the treatment of metabolic syndrome and obesity. The authors are grateful to Bente Friis and Line Olsen for expert technical assistance and to Dr. Helen Cox for critically reading the manuscript. The study was supported by research grant from the Danish Medical Research Council, the Novo Nordisk Foundation, the Lundbeck Foundation, and the Alfred Benzon Foundation and by a grant from the Novo Nordisk Foundation to the Novo Nordisk Foundation Center for Basic Metabolic Research. This work is carried out as a part of the research program of the UNIK: Food, Fitness & Pharma for Health and Disease. The UNIK project is supported by the Danish Ministry of Science, Technology, and Innovation.

4. 5. 6.

7.

8.

9.

10.

11.

12. 13. 14.

15.

16.

17.

18.

REFERENCES 1.

Grundy, S. M. (2008) Metabolic syndrome pandemic. Arterioscler. Thromb. Vasc. Biol. 28, 629 – 636 2. Gaidhu, M. P., Anthony, N. M., Patel, P., Hawke, T. J., and Ceddia, R. B. (2010) Dysregulation of lipolysis and lipid metabolism in visceral and subcutaneous adipocytes by high-fat diet: role of ATGL, HSL, and AMPK. Am. J. Physiol. Cell. Physiol. 298, C961–C971 3. Martinez-Botas, J., Anderson, J. B., Tessier, D., Lapillonne, A., Chang, B. H., Quast, M. J., Gorenstein, D., Chen, K. H., and Chan, L. (2000) Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice. Nat. Genet. 26, 474 – 479

GPR39 REGULATES LIPOLYSIS AND ENERGY EXPENDITURE

19.

20.

Woods, S. C. (2009) The control of food intake: behavioral versus molecular perspectives. Cell Metab. 9, 489 – 498 Woods, S. C., and D’Alessio, D. A. (2008) Central control of body weight and appetite. J. Clin. Endocrinol. Metab. 93, S37–S50 Nogueiras, R., Wiedmer, P., Perez-Tilve, D., Veyrat-Durebex, C., Keogh, J. M., Sutton, G. M., Pfluger, P. T., Castaneda, T. R., Neschen, S., Hofmann, S. M., Howles, P. N., Morgan, D. A., Benoit, S. C., Szanto, I., Schrott, B., Schurmann, A., Joost, H. G., Hammond, C., Hui, D. Y., Woods, S. C., Rahmouni, K., Butler, A. A., Farooqi, I. S., O’Rahilly, S., Rohner-Jeanrenaud, F., and Tschop, M. H. (2007) The central melanocortin system directly controls peripheral lipid metabolism. J. Clin. Invest. 117, 3475– 3488 Theander-Carrillo, C., Wiedmer, P., Cettour-Rose, P., Nogueiras, R., Perez-Tilve, D., Pfluger, P., Castaneda, T. R., Muzzin, P., Schurmann, A., Szanto, I., Tschop, M. H., and Rohner-Jeanrenaud, F. (2006) Ghrelin action in the brain controls adipocyte metabolism. J. Clin. Invest. 116, 1983–1993 Bachman, E. S., Dhillon, H., Zhang, C. Y., Cinti, S., Bianco, A. C., Kobilka, B. K., and Lowell, B. B. (2002) betaAR signaling required for diet-induced thermogenesis and obesity resistance. Science 297, 843– 845 Huijsman, E., van de Par, C., Economou, C., van der Poel, C., Lynch, G. S., Schoiswohl, G., Haemmerle, G., Zechner, R., and Watt, M. J. (2009) Adipose triacylglycerol lipase deletion alters whole body energy metabolism and impairs exercise performance in mice. Am. J. Physiol. Endocrinol. Metab. 297, E505–E513 Ahmadian, M., Duncan, R. E., Varady, K. A., Frasson, D., Hellerstein, M. K., Birkenfeld, A. L., Samuel, V. T., Shulman, G. I., Wang, Y., Kang, C., and Sul, H. S. (2009) Adipose overexpression of desnutrin promotes fatty acid use and attenuates diet-induced obesity. Diabetes 58, 855– 866 Haemmerle, G., Zimmermann, R., Hayn, M., Theussl, C., Waeg, G., Wagner, E., Sattler, W., Magin, T. M., Wagner, E. F., and Zechner, R. (2002) Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J. Biol. Chem. 277, 4806 – 4815 Zechner, R., and Madeo, F. (2009) Cell biology: another way to get rid of fat. Nature 458, 1118 –1119 Ahmed, K., Tunaru, S., and Offermanns, S. (2009) GPR109A, GPR109B and GPR81, a family of hydroxy-carboxylic acid receptors. Trends Pharmacol. Sci. 30, 557–562 Ahmed, K., Tunaru, S., Tang, C., Muller, M., Gille, A., Sassmann, A., Hanson, J., and Offermanns, S. (2010) An autocrine lactate loop mediates insulin-dependent inhibition of lipolysis through GPR81. Cell Metab. 11, 311–319 Jaworski, K., Ahmadian, M., Duncan, R. E., Sarkadi-Nagy, E., Varady, K. A., Hellerstein, M. K., Lee, H. Y., Samuel, V. T., Shulman, G. I., Kim, K. H., de Val, S., Kang, C., and Sul, H. S. (2009) AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency. Nat. Med. 15, 159 –168 Holst, B., Holliday, N. D., Bach, A., Elling, C. E., Cox, H. M., and Schwartz, T. W. (2004) Common structural basis for constitutive activity of the ghrelin receptor family. J. Biol. Chem. 279, 53806 –53817 Holst, B., Egerod, K. L., Schild, E., Vickers, S. P., Cheetham, S., Gerlach, L. O., Storjohann, L., Stidsen, C. E., Jones, R., BeckSickinger, A. G., and Schwartz, T. W. (2007) GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 148, 13–20 Egerod, K. L., Holst, B., Petersen, P. S., Hansen, J. B., Mulder, J., Hokfelt, T., and Schwartz, T. W. (2007) GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue. Mol. Endocrinol. 21, 1685–1698 Tremblay, F., Richard, A. M., Will, S., Syed, J., Stedman, N., Perreault, M., and Gimeno, R. E. (2009) Disruption of G protein-coupled receptor 39 impairs insulin secretion in vivo. Endocrinology 150, 2586 –2595 Holst, B., Egerod, K. L., Jin, C., Petersen, P. S., Ostergaard, M. V., Hald, J., Sprinkel, A. M., Storling, J., Mandrup-Poulsen, T., Holst, J. J., Thams, P., Orskov, C., Wierup, N., Sundler, F., Madsen, O. D., and Schwartz, T. W. (2009) G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction. Endocrinology 150, 2577–2585

3813

21.

22.

23.

24.

25.

26. 27.

28. 29. 30. 31.

32.

33.

34.

3814

Moechars, D., Depoortere, I., Moreaux, B., de Smet, B., Goris, I., Hoskens, L., Daneels, G., Kass, S., Ver Donck, L., Peeters, T., and Coulie, B. (2006) Altered gastrointestinal and metabolic function in the GPR39-obestatin receptor-knockout mouse. Gastroenterology 131, 1131–1141 Hansen, J. B., Jorgensen, C., Petersen, R. K., Hallenborg, P., De Matteis, R., Boye, H. A., Petrovic, N., Enerback, S., Nedergaard, J., Cinti, S., te Riele, H., and Kristiansen, K. (2004) Retinoblastoma protein functions as a molecular switch determining white versus brown adipocyte differentiation. Proc. Natl. Acad. Sci. U. S. A. 101, 4112– 4117 Muniyappa, R., Chen, H., Muzumdar, R., Einstein, F. H., Yan, X., Yue, L. Q., Barzilai, N., and Quon, M. J. (2009) Comparison between surrogate indexes of insulin sensitivity/resistance and hyperinsulinemic euglycemic clamp estimates in rats. Am. J. Physiol. Endocrinol. Metab. 297, E1023–E1029 Lee, S., Muniyappa, R., Yan, X., Chen, H., Yue, L. Q., Hong, E. G., Kim, J. K., and Quon, M. J. (2008) Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice. Am. J. Physiol. Endocrinol. Metab. 294, E261–E270 Shklyaev, S., Aslanidi, G., Tennant, M., Prima, V., Kohlbrenner, E., Kroutov, V., Campbell-Thompson, M., Crawford, J., Shek, E. W., Scarpace, P. J., and Zolotukhin, S. (2003) Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats. Proc. Natl. Acad. Sci. U. S. A. 100, 14217–14222 Lowell, B. B., and Bachman, E. S. (2003) Beta-Adrenergic receptors, diet-induced thermogenesis, and obesity. J. Biol. Chem. 278, 29385–29388 Armani, A., Mammi, C., Marzolla, V., Calanchini, M., Antelmi, A., Rosano, G. M., Fabbri, A., and Caprio, M. (2010) Cellular models for understanding adipogenesis, adipose dysfunction, and obesity. J. Cell. Biochem. 110, 564 –572 White, U. A., and Stephens, J. M. (2010) Transcriptional factors that promote formation of white adipose tissue. Mol. Cell. Endocrinol. 318, 10 –14 Wang, Y., and Sul, H. S. (2009) Pref-1 regulates mesenchymal cell commitment and differentiation through Sox9. Cell Metab. 9, 287–302 Scarpulla, R. C. (2008) Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol. Rev. 88, 611– 638 Lopez, M., Lage, R., Saha, A. K., Perez-Tilve, D., Vazquez, M. J., Varela, L., Sangiao-Alvarellos, S., Tovar, S., Raghay, K., Rodriguez-Cuenca, S., Deoliveira, R. M., Castaneda, T., Datta, R., Dong, J. Z., Culler, M., Sleeman, M. W., Alvarez, C. V., Gallego, R., Lelliott, C. J., Carling, D., Tschop, M. H., Dieguez, C., and Vidal-Puig, A. (2008) Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin. Cell Metab. 7, 389 –399 Zimmermann, R., Haemmerle, G., Wagner, E. M., Strauss, J. G., Kratky, D., and Zechner, R. (2003) Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue. J. Lipid Res. 44, 2089 –2099 Collins, S., Cao, W., and Robidoux, J. (2004) Learning new tricks from old dogs: beta-adrenergic receptors teach new lessons on firing up adipose tissue metabolism. Mol. Endocrinol. 18, 2123–2131 Dittmer, S., Sahin, M., Pantlen, A., Saxena, A., Toutzaris, D., Pina, A. L., Geerts, A., Golz, S., and Methner, A. (2008) The constitutively active orphan G-protein-coupled receptor GPR39 protects from cell death by increasing secretion of pigment epithelium-derived growth factor. J. Biol. Chem. 283, 7074 –7081

Vol. 25

November 2011

35.

36.

37.

38.

39. 40.

41.

42.

43.

44.

45.

46.

47.

Wortley, K. E., del Rincon, J. P., Murray, J. D., Garcia, K., Iida, K., Thorner, M. O., and Sleeman, M. W. (2005) Absence of ghrelin protects against early-onset obesity. J. Clin. Invest. 115, 3573–3578 Zigman, J. M., Nakano, Y., Coppari, R., Balthasar, N., Marcus, J. N., Lee, C. E., Jones, J. E., Deysher, A. E., Waxman, A. R., White, R. D., Williams, T. D., Lachey, J. L., Seeley, R. J., Lowell, B. B., and Elmquist, J. K. (2005) Mice lacking ghrelin receptors resist the development of diet-induced obesity. J. Clin. Invest. 115, 3564 –3572 Villena, J. A., Roy, S., Sarkadi-Nagy, E., Kim, K. H., and Sul, H. S. (2004) Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis. J. Biol. Chem. 279, 47066 – 47075 Zimmermann, R., Strauss, J. G., Haemmerle, G., Schoiswohl, G., Birner-Gruenberger, R., Riederer, M., Lass, A., Neuberger, G., Eisenhaber, F., Hermetter, A., and Zechner, R. (2004) Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306, 1383–1386 Vaughan, M., Berger, J. E., and Steinberg, D. (1964) Hormonesensitive lipase and monoglyceride lipase activities in adipose tissue. J. Biol. Chem. 239, 401– 409 Zechner, R., Kienesberger, P. C., Haemmerle, G., Zimmermann, R., and Lass, A. (2009) Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. J. Lipid Res. 50, 3–21 Harada, K., Shen, W. J., Patel, S., Natu, V., Wang, J., Osuga, J., Ishibashi, S., and Kraemer, F. B. (2003) Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice. Am. J. Physiol. Endocrinol. Metab. 285, E1182–E1195 Haemmerle, G., Lass, A., Zimmermann, R., Gorkiewicz, G., Meyer, C., Rozman, J., Heldmaier, G., Maier, R., Theussl, C., Eder, S., Kratky, D., Wagner, E. F., Klingenspor, M., Hoefler, G., and Zechner, R. (2006) Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science 312, 734 –737 Osuga, J., Ishibashi, S., Oka, T., Yagyu, H., Tozawa, R., Fujimoto, A., Shionoiri, F., Yahagi, N., Kraemer, F. B., Tsutsumi, O., and Yamada, N. (2000) Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc. Natl. Acad. Sci. U. S. A. 97, 787–792 Jimenez, M., Leger, B., Canola, K., Lehr, L., Arboit, P., Seydoux, J., Russell, A. P., Giacobino, J. P., Muzzin, P., and Preitner, F. (2002) Beta(1)/beta(2)/beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting. FEBS Lett. 530, 37– 40 Wijers, S. L., Saris, W. H., and van Marken Lichtenbelt, W. D. (2009) Recent advances in adaptive thermogenesis: potential implications for the treatment of obesity. Obes. Rev. 10, 218 –226 Cai, T. Q., Ren, N., Jin, L., Cheng, K., Kash, S., Chen, R., Wright, S. D., Taggart, A. K., and Waters, M. G. (2008) Role of GPR81 in lactate-mediated reduction of adipose lipolysis. Biochem. Biophys. Res. Commun. 377, 987–991 Liu, C., Wu, J., Zhu, J., Kuei, C., Yu, J., Shelton, J., Sutton, S. W., Li, X., Yun, S. J., Mirzadegan, T., Mazur, C., Kamme, F., and Lovenberg, T. W. (2009) Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81. J. Biol. Chem. 284, 2811–2822


Received for publication April 3, 2011. Accepted for publication July 11, 2011.

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