GPR17 Gene Disruption Does Not Alter Food Intake Or Glucose Homeostasis in Mice

GPR17 gene disruption does not alter food intake or glucose homeostasis in mice

Jason Mastaitisa,1, Soo Mina, Ralf Elvertb, Aimo Kanntb, Yurong Xina, Francisca Ochoaa, Nicholas W. Galea, David M. Valenzuelaa, Andrew J. Murphya, George D. Yancopoulosa,1, and Jesper Gromadaa

aRegeneron Pharmaceuticals, Tarrytown, NY 10591; and bSanofi Diabetes Research and Translational Medicine, D-65926 Frankfurt am Main, Germany

Contributed by George D. Yancopoulos, December 31, 2014 (sent for review December 18, 2014; reviewed by Tung Fong and Jacob Jelsing) G protein-coupled receptor 17 (GPR17) was recently reported to be single exon contained entirely within the coding region of the Lims2 a Foxo1 target in agouti-related peptide (AGRP) neurons. Intra- gene (NCBI Reference Sequence: NM_144862.3), albeit on the cerebroventricular injection of GPR17 agonists induced food in- opposite strand. We deleted a 1,032-bp segment with the lacZ fused take, whereas administration of an antagonist to the receptor in frame just after Gpr17’s ATG without deleting any exons from reduced feeding. These data lead to the conclusion that pharma- Lims2 (Fig. 1A). No Gpr17 mRNA was detected in the hypothal- cological modulation of GPR17 has therapeutic potential to treat amus of knockout (KO) animals (Fig. 1B). Lims2 expression was − − − − − − obesity. Here we report that mice deficient in Gpr17 (Gpr17 / ) reduced by 29% in Gpr17 / mice (Fig. 1B). Gpr17 / mice were have similar food intake and body weight compared with their born in the expected Mendelian ratios. Reporter expression was −/− wild-type littermates. Gpr17 mice have normal hypothalamic found in multiple areas of the brain and the hypothalamus (Fig. Agrp mRNA expression, AGRP plasma levels, and metabolic rate. 1C), consistent with a previous report (16). Gpr17 is expressed in GPR17 deficiency in mice did not affect glucose homeostasis or few peripheral tissues at lower levels than in the hypothalamus (Fig. − − prevent fat-induced insulin resistance. These data do not support 1D). Gpr17 / and wild-type mice had similar levels of hypothalamic a role for GPR17 in the control of food intake, body weight, or Agrp mRNA and circulating AGRP in the fasted state (Fig. 1 B and − − glycemic control. E). Refeeding of Gpr17 / and control mice produced an equivalent decrement in the AGRP plasma levels (Fig. 1E). GPR17 | AGRP | diabetes | body weight | food intake − − Gpr17 / Mice Have Normal Food Intake, Body Weight, and Metabolic ypothalamic neurons expressing agouti-related peptide Rate. Mice lacking GPR17 had normal body weight compared H(AGRP) play an important role in feeding (1–3). AGRP with their wild-type littermates (Fig. 2A). Consistent with this acts as an inverse agonist on melanocortin 4 receptors (4, 5). data, there were no differences in the light and dark phases of Leptin and insulin are anorectic hormones, which partly regulate the light cycle in food intake (Fig. 2B) or energy expenditure food intake and energy homeostasis by inhibiting AGRP neurons (Fig. 2C). Data for oxygen consumption (VO2), carbon dioxide – (6 8). The transcription factor Foxo1 is expressed in AGRP production (VCO2), respiratory quotient (RER), energy expen- neurons and represents a shared mediator of the pathways ac- diture, and food intake in the light and dark phases of the light tivated by insulin and leptin to suppress food intake (9, 10). cycle are shown in Fig. S1 A–E. Locomotor activity was similar − − Accordingly, Foxo1 expression is reduced by insulin and leptin between both groups of mice (Fig. 2D and Fig. S1F). Gpr17 / and these hormones’ effect on feeding is inhibited following and wild-type mice fed a HFD for 10 wk had a 70% increase in hypothalamic Foxo1 overexpression (9). Moreover, activation of body weight, but showed no difference in food intake or energy Foxo1 in the hypothalamus increases food intake and body expenditure (Fig. 2 E–G and Fig. S1 G–K). There were no sig- weight, whereas inhibition of Foxo1 decreases both (9). Finally, nificant differences in body weight between the KO mice and ablation of Foxo1 in AGRP neurons results in reduced food controls at any time point over the first 9 wk of high-fat feeding. intake, leanness, and improved glycemic control (11). Locomotor activity was slightly reduced in HFD mice (Fig. 2H The G protein-coupled receptor, GPR17, is a prominent and Fig. S1L). No genotypic differences in body composition Foxo1 target and highly expressed in AGRP neurons (11). GPR17 is phylogenetically related to nucleotide P2Y and cys- Significance teinyl-leukotriene receptors (12). It has been reported that uridyl-diphosphate glucose (UDP glucose) and leukotriene D4 (LTD4) activate GPR17, whereas the P2Y12 antagonist, Cangrelor, Hypothalamic agouti-related peptide (AGRP) neurons control suppresses receptor activity (13). Consistent with these findings, food intake and body weight. G protein-coupled receptor 17 (GPR17) was recently shown to be expressed in these neurons intracerebroventricular injectionofUDPglucoseandLTD4inmice MEDICAL SCIENCES stimulated food intake, whereas administration of Cangrelor sup- and controls their activity, thereby reducing body weight and pressed it (11). However, other studies have failed to demonstrate food intake in mice. In the current study, we demonstrate that activation of GPR17 with UDP glucose and LTD4 (14, 15). In this Gpr17-deficient mice have normal hypothalamic and circulating − − study, we generated and fed Gpr17-deficient (Gpr17 / )micea AGRP levels. Body weight, food intake, and glucose homeo- normal and a high-fat diet (HFD). Under these experimental stasis appear normal in the GPR17-deficient mice. The current conditions, we did not observe an effect of GPR17 on food intake, data do not validate GPR17 as a therapeutic target for obesity body weight, or glycemic control. These data call into question the or type 2 diabetes. therapeutic potential of inhibiting GPR17 to treat obesity and Author contributions: J.M., A.K., Y.X., F.O., N.W.G., D.M.V., and J.G. designed research; related metabolic disorders. J.M., S.M., R.E., A.K., and F.O. performed research; R.E., A.K., and Y.X. analyzed data; and J.M., N.W.G., D.M.V., A.J.M., G.D.Y., and J.G. wrote the paper. Results Reviewers: T.F., Biopharma Consulting; and J.J., Gubra. Gpr17 Reporter Gene Expression from the Locus and AGRP Plasma Levels The authors declare no conflict of interest. Gpr17−/− −/− in and Wild-Type Mice. Gpr17 mice were developed by 1To whom correspondence may be addressed. Email: [email protected] or homologous recombination using Regeneron’s VelociGene tech- [email protected]. nology. The murine Gpr17 gene [National Center for Biotechnology This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Information (NCBI) Reference Sequence: NM_001025381.2] is a 1073/pnas.1424968112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1424968112 PNAS | February 10, 2015 | vol. 112 | no. 6 | 1845–1849 Downloaded by guest on October 2, 2021 − − Fig. 1. Locus organization, brain lacZ reporter gene expression, and AGRP plasma levels of Gpr17 / mice. (A) Gpr17 is nested inside Lims2 in the reverse strand such that its single coding exon is fully contained in the long intron 4 (9,879 bp) of Lims2. Coding exons are shown as black boxes, UTR as white boxes, introns as straight line. Deletion of the VG12229 allele is well contained inside the Lims2 intron, far away from splicing signals (distances shown). LacZ-floxed- Neo cassette inserted in frame after Gpr17’s ATG. Drug resistance coding sequence was driven by human ubiquitin promoter. (B) Gpr17, Lims2, and Agrp − − mRNA levels in hypothalamus from Gpr17 / (KO) and wild-type (WT) mice. Data expressed in reads per kilobase per million (RPKM). (C) LacZ reporter staining in the endogenous Gpr17 loci in whole brain section. Inset shows region of the hypothalamus at 5× magnification. (D) Gpr17 expression using RNAseq in 16 − − mouse tissues of C57BL/6 mice on chow diet (n = 6 per group). (E) AGRP plasma levels in fasted (4 h) and refed Gpr17 / (KO) and wild-type (WT) mice. All groups had seven animals if not otherwise indicated. Values are mean ± SEM. ***P < 0.001; ****P < 0.0001.

were observed in mice on standard chow or HFD, except the glucose homeostasis or protect from glucose intolerance in response − − Gpr17 / mice on the chow diet tended to have higher body fat to a HFD. content (Table 1). Hypothalamic Gpr17 and Agrp mRNA expression did not change in response to high-fat feeding (Fig. Discussion − − S2A). AGRP plasma levels were similar between Gpr17 / and The main findings of the present study are that (i)ablationof wild-type mice fed a HFD (Fig. S2B). These data suggest that GPR17 does not regulate circulating AGRP levels, food in- ablation of GPR17 does not affect food intake or body weight take, metabolic rate, and body weight and (ii)GPR17doesnot in mice. control glucose homeostasis or prevent high-fat–induced insulin resistance. Gpr17−/− Mice Have Normal Glucose Homeostasis and Insulin Sensitivity. GPR17 is abundantly expressed in AGRP neurons and was − − Gpr17 / mice had normal glycemic control as revealed by an oral recently suggested to control feeding using pharmacological glucose tolerance test (oGTT) (Fig. 3A) or insulin tolerance test modulators of the receptor and intracerebroventricular admin- − − (ITT) (Fig. 3B) compared with wild-type mice. Feeding Gpr17 / istration in mice (11). However, these compounds have sub- and wild-type mice a HFD for 10 wk impaired glucose tolerance sequently been questioned as pharmacological modulators of (Fig. 3A) and insulin sensitivity (Fig. 3B). No genotypic differences GPR17 (14, 15). In this study, we confirm that Gpr17 is highly in glucose or insulin tolerance were observed in mice on normal expressed in the hypothalamus of mice. However, wild-type and − − chow or HFD. These data show that GPR17 does not control Gpr17 / mice show no differences in food intake or metabolic

1846 | www.pnas.org/cgi/doi/10.1073/pnas.1424968112 Mastaitis et al. Downloaded by guest on October 2, 2021 − − Fig. 2. Body weight, food intake, energy expenditure, and locomotor activity in Gpr17 / and wild-type mice. (A and E) Body weight was measured in male wild- − − type (WT) and Gpr17 / (KO) mice on chow diet (13 wk of age) and high-fat feeding (HFD; 24 wk of age). Mice were on HFD from weeks 14–24. (B and F)Food intake during dark and light phases of the light cycle in wild-type (WT) and Gpr17−/− (KO) mice on chow diet or HFD. (C and G) Energy expenditure during dark and light phases of the light cycle in wild-type (WT) and Gpr17−/− (KO) mice on chow diet or HFD. (D and H) Locomotor activity during dark and light phases of the − − light cycle in wild-type (WT) and Gpr17 / (KO) mice on chow diet or HFD. All groups had eight animals. Values are mean ± SEM. **P < 0.01; ****P < 0.0001.

rate and can mount a significant and similar increase in body could also negatively impact the adult nervous system following − − weight in response to high-fat feeding. Gpr17 / mice have traumatic injury. Consistent with this data, knockdown of Gpr17 normal Agrp hypothalamic mRNA expression and circulating mRNA levels in a focal ischemia rat model attenuated short- AGRP levels. term neuron loss, brain atrophy, and microglial activation after The Gpr17 gene is contained entirely within the coding region reperfusion (21). Thus, GPR17 seems to control oligodendrocyte of the Lims2 gene, but on the opposite strand. LIMS2 belongs to function rather than AGRP neuronal activity. − − the family of focal adhesion proteins and functions as a compo- The Agrp-Foxo1 / mice (which have low Gpr17 expression) nent of the integrin signaling pathway (17). The small but sig- exhibit reduced hepatic glucose production and improved glucose nificant reduction in Lims2 mRNA expression is unlikely to homeostasis (11). Here we demonstrate a lack of involvement of −/− − − affect the metabolism of Gpr17 mice because Lims2-deficient GPR17 in the regulation of glucose control. Moreover, Gpr17 / mice do not have an apparent phenotype (18) and Agrp expres- mice are not protected from HFD-induced insulin resistance. The −/− sion was similar in Gpr17 and wild-type mice. nature of the Foxo1 target gene(s) mediating the beneficial effects − − β-Galactosidase (lacZ) staining of GPR17 in the brain showed on food intake and glycemic control in the Agrp-Foxo1 / mice widespread expression, suggesting that the receptor has a more remains unknown. general function rather than a specific role in AGRP neurons. In conclusion, the present data do not support a role for Previous studies have shown that GPR17 is expressed in oligo- GPR17 in the regulation of food intake or glucose homeostasis. dendrocyte precursors (19) and negatively regulated by the oli- Thus, GPR17 does not appear to be a viable therapeutic target godendrocyte maturation transcription factor, Olig1 (20). for the treatment of obesity and type 2 diabetes. Whereas ablation of GPR17 in mice caused only a slight advance in central nervous system (CNS) myelination, overexpression of Materials and Methods − − GPR17 inhibited myelinogenesis within the CNS of mice (16), Animals. Gpr17 / mice (100% C57BL/6NTac background) were generated by

suggesting it plays a role not just in development of the CNS, but homologous recombination using Regeneron’s VelociGene technology (22). MEDICAL SCIENCES

− − Table 1. Body composition measurements in Gpr17 / and wild-type mice Chow High-fat diet

WT KO WT KO

Body weight, g 25.9 ± 0.5 26.4 ± 0.4 44.1 ± 0.9 45.9 ± 0.6 Lean volume, cm3 16.9 ± 0.4 16.7 ± 0.3 18.1 ± 0.5 18.6 ± 0.5 Fat volume, cm3 3.02 ± 0.15 3.89 ± 0.24* 20.0 ± 0.78 21.6 ± 0.36 Bone volume, cm3 1.48 ± 0.03 1.44 ± 0.02 1.48 ± 0.03 1.47 ± 0.02 Bone density, mgHA/cm3 304 ± 3.1 300 ± 3.7 306 ± 1.6 305 ± 1.6 Bone mineral content, mgHA 449 ± 13.2 433 ± 8.6 454 ± 10.7 448 ± 7.7 Body fat, % 11.0 ± 0.6 14.0 ± 1.0* 41.7 ± 1.4 43.2 ± 0.7

Body composition was measured using μCT in Gpr17−/− (KO) and wild-type (WT) mice on chow diet (13 wk of age) and following 10 wk of high-fat feeding. All groups had eight animals. Values are mean ± SEM. *P < 0.05.

Mastaitis et al. PNAS | February 10, 2015 | vol. 112 | no. 6 | 1847 Downloaded by guest on October 2, 2021 Metabolic Rate and Food Intake Measurements. Metabolic cage data were generated using the Oxymax Lab Animal Monitoring System: CLAMS (Columbus Instruments). Mice (male; 9 wk of age) were individually monitored in cages with center feeds for 96 h. Food intake was measured continuously and

divided into calories consumed per light and dark phase of the light cycle. VO2

and VCO2 were measured in 17-min intervals over a 4-d span and plotted over time in hours. Energy expenditure was calculated as a function of the respiratory quotient and the oxygen consumption, normalized to body weight.

RNA Preparation and RNA Sequencing Read Mapping. Total RNA was purified using MagMAX-96 for Microarrays Total RNA Isolation kit (Ambion), according to manufacturer specifications. Genomic DNA was removed using Mag- MAXTurboDNase buffer and TURBO DNase from the MagMAX kit listed above. mRNA was purified from total RNA using Dynabeads mRNA Purifi- cation kit (Invitrogen). Strand-specific RNA sequencing (RNA-seq) libraries were prepared using ScriptSeq mRNA-Seq Library Preparation kit (Epicentre). Twelve-cycle PCR was performed to amplify libraries. Sequencing was performed on Illumina HiSeq2000 by a multiplexed single-read run with 33 cycles. Raw sequence data (BCL files) were converted to FASTQ format via Illumina Casava 1.8.2. Reads were decoded based on their barcodes and read quality was evaluated with FastQC (www.bioinformatics.babraham.ac.uk/ projects/fastqc/). Reads were mapped to the mouse transcriptome (NCBI Build37.2) using Bowtie (bowtie-bio.sourceforge.net/index.shtml)allowingtwo mismatches. Reads mapped to the sense-strand exons of a gene were summed − − Fig. 3. Oral glucose and insulin tolerance tests in Gpr17 / and wild-type at the gene level. − − mice. (A) Glucose tolerance test in male wild-type (WT) and Gpr17 / (KO) mice on chow diet (11 wk of age) and high-fat feeding (HFD; 23 wk of age). Serum Chemistry. Serum chemistry analysis was performed using the ADVIA Mice were on HFD from weeks 14 to 24. (B) Insulin tolerance test in wild-type 1800 Clinical Chemistry System (Siemens Healthcare Diagnostics). Whole −/− (WT) and Gpr17 (KO) mice on chow diet and HFD for 11 wk. All groups mouse blood was collected by retroperitoneal bleed under anesthesia into had eight animals. Values are mean ± SEM. a BD Microtainer serum separator tube and stored at −20 °C.

Hypothalamic Gene Expression. Hypothalamus was dissected from whole brain VelociGene allele identification number is VG12229. Male mice were used using an acrylic mouse brain block (Harvard Apparatus), placed in RNAlater throughout this study and housed four to five per cage in a controlled envi- (Qiagen), and homogenized in a Precellys mill using the Precellys Ceramic kit ronment (12-h light/dark cycle, 23 ± 1°C,60–70% humidity) and fed ad libitum 1.4/2.8 mm (Peqlab). Total RNA was isolated with the RNeasy Fibrous Tissue with standard chow (Purina Laboratory Rodent Diet 5001, LabDiet). Some mice were fed a high-fat diet (Research Diets, D12492; 60% fat by calories). In the Mini kit (Qiagen), including DNase I digestion and quantified with the Quant-iT food intake, body weight, metabolic rate, and glucose studies, comparisons RiboGreen RNA assay (Life Technologies). Reverse transcription to cDNA was were made between wild-type and knockout littermates. C57BL/6 mice (males; done using the BioRad iScript cDNA synthesis kit. Gpr17, Agrp, and Lims2 Taconic) were used for Gpr17 expression studies. All animal procedures were cDNA were quantified with the digital droplet PCR method (23) using the conducted in compliance with protocols approved by the Regeneron Phar- ddPCR Supermix for probes (no UDP) kit on a QX-200 system (BioRad). maceuticals Institutional Animal Care and Use Committee. TaqMan primer-probe sets are available upon request.

Glucose Homeostasis Measurements. For the insulin tolerance test, mice (male; LacZ Staining. LacZ histochemistry was performed as described earlier (24). 10 wk of age) were fasted for 4 h before testing. The fast started at 9:00 AM, Slides were scanned on Aperio ScanScope AT Turbo (Leica) and annotated 2 h after initiation of the light cycle. Fasting glucose was measured via tail bleed with Aperio 5ImageScope. using the Accu-Chek Compact Plus (Roche Diagnostics). Insulin stock was prepared from Humulin R (Lilly). Mice on normal chow were injected i.p. with 0.75 Plasma AGRP Measurements. The concentration of circulating AGRP was units insulin/kg body weight. Mice on the HFD (male; 23 wk of age; subjected to measured in mouse EDTA plasma using the mouse AGRP fluorescent im- high-fat feeding for 10 wk) were injected i.p. with 2.0 units insulin/kg body munoassay kit from Phoenix Pharmaceuticals, according to the manu- weight. Blood glucose was measured with the glucometer via tail bleed at 0, 15, facturer’s instructions. Signal calibration was done based on a standard curve 30, 60, and 120 min postinjection. For the oral glucose tolerance test, mice were fasted overnight (16 h) followed by oral gavage of glucose at 2 g/kg body obtained with known AGRP concentrations between 10 and 1,000 pg/mL. weight. The fast started at 5:00 PM, 2 h before initiation of the dark cycle. Blood glucose level was evaluated at 0, 15, 30, 60, and 120 min postinjection. Data Analysis. All data are expressed as mean ± SEM. Two-way ANOVA with Bonferroni’s post hoc analysis was performed on data collected for hypo- Body Composition Assessments. MicroCT (μCT) measurements were per- thalamic gene expression, ITT, oGTT, and metabolic cage studies. Unpaired formed using the Quantum FX micro CT preclinical in vivo imaging system Student’s t test was used to analyze data collected from the μCT and serum (Caliper Life Sciences). Mice (male) fed the chow diet were 13 wk of age at chemistry studies. All analysis was performed using GraphPad PRISM 6.0e time of μCT; mice (male) fed the HFD were 24 wk of age at time of μCT and (GraphPad Software). on the HFD for the last 11 wk. Mice were anesthetized using Forane (Baxter). = = Scans were performed using field of view setting 60 and scan technique ACKNOWLEDGMENTS. We thank Esther Latres, Claire Kammermeier, Martin Dyn17Sec2. Data conversion was performed using the Caliper analysis soft- Stephan, Pierre Wenski, Jörn Wandschneider, Uwe Butty, and Alejo Mujica ware (Caliper Life Sciences). for advice and excellent technical assistance.

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