The GPR 120 agonist TUG ‐891 promotes metabolic health by stimulating mitochondrial respiration in brown fat
2018; Springer Nature; Volume: 10; Issue: 3 Linguagem: Inglês
10.15252/emmm.201708047
ISSN1757-4684
AutoresMaaike Schilperoort, Andrea D. van Dam, Geerte Hoeke, Irina G. Shabalina, Anthony Okolo, Aylin C. Hanyaloglu, Lea Dib, Isabel M. Mol, Natarin Caengprasath, Yi‐Wah Chan, Sami Damak, Anne Reifel Miller, Tamer Coşkun, Bharat Shimpukade, Trond Ulven, Sander Kooijman, Patrick C.N. Rensen, Mark Christian,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoResearch Article17 January 2018Open Access Transparent process The GPR120 agonist TUG-891 promotes metabolic health by stimulating mitochondrial respiration in brown fat Maaike Schilperoort Corresponding Author Maaike Schilperoort [email protected] orcid.org/0000-0002-8597-7675 Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Andrea D van Dam Andrea D van Dam Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Geerte Hoeke Geerte Hoeke Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Irina G Shabalina Irina G Shabalina orcid.org/0000-0002-2915-6450 Department of Molecular Biosciences, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden Search for more papers by this author Anthony Okolo Anthony Okolo Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Search for more papers by this author Aylin C Hanyaloglu Aylin C Hanyaloglu Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Search for more papers by this author Lea H Dib Lea H Dib Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Isabel M Mol Isabel M Mol Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Natarin Caengprasath Natarin Caengprasath Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Search for more papers by this author Yi-Wah Chan Yi-Wah Chan Lymphocyte Development Group, MRC London Institute of Medical Sciences, Hammersmith Campus, Imperial College London, London, UK Search for more papers by this author Sami Damak Sami Damak Nestlé Research Center, Lausanne, Switzerland Search for more papers by this author Anne Reifel Miller Anne Reifel Miller Lilly Research Laboratories, Diabetes/Endocrine Department, Lilly Corporate Center, Indianapolis, IN, USA Search for more papers by this author Tamer Coskun Tamer Coskun Lilly Research Laboratories, Diabetes/Endocrine Department, Lilly Corporate Center, Indianapolis, IN, USA Search for more papers by this author Bharat Shimpukade Bharat Shimpukade Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark Search for more papers by this author Trond Ulven Trond Ulven Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark Search for more papers by this author Sander Kooijman Sander Kooijman Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Patrick CN Rensen Patrick CN Rensen Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Mark Christian Corresponding Author Mark Christian [email protected] orcid.org/0000-0002-1616-4179 Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Maaike Schilperoort Corresponding Author Maaike Schilperoort [email protected] orcid.org/0000-0002-8597-7675 Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Andrea D van Dam Andrea D van Dam Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Geerte Hoeke Geerte Hoeke Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Irina G Shabalina Irina G Shabalina orcid.org/0000-0002-2915-6450 Department of Molecular Biosciences, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden Search for more papers by this author Anthony Okolo Anthony Okolo Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Search for more papers by this author Aylin C Hanyaloglu Aylin C Hanyaloglu Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Search for more papers by this author Lea H Dib Lea H Dib Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK Search for more papers by this author Isabel M Mol Isabel M Mol Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Natarin Caengprasath Natarin Caengprasath Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK Search for more papers by this author Yi-Wah Chan Yi-Wah Chan Lymphocyte Development Group, MRC London Institute of Medical Sciences, Hammersmith Campus, Imperial College London, London, UK Search for more papers by this author Sami Damak Sami Damak Nestlé Research Center, Lausanne, Switzerland Search for more papers by this author Anne Reifel Miller Anne Reifel Miller Lilly Research Laboratories, Diabetes/Endocrine Department, Lilly Corporate Center, Indianapolis, IN, USA Search for more papers by this author Tamer Coskun Tamer Coskun Lilly Research Laboratories, Diabetes/Endocrine Department, Lilly Corporate Center, Indianapolis, IN, USA Search for more papers by this author Bharat Shimpukade Bharat Shimpukade Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark Search for more papers by this author Trond Ulven Trond Ulven Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark Search for more papers by this author Sander Kooijman Sander Kooijman Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Patrick CN Rensen Patrick CN Rensen Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands Search for more papers by this author Mark Christian Corresponding Author Mark Christian [email protected] orcid.org/0000-0002-1616-4179 Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK Search for more papers by this author Author Information Maaike Schilperoort *,1,2,3, Andrea D Dam2,3, Geerte Hoeke2,3, Irina G Shabalina4, Anthony Okolo5, Aylin C Hanyaloglu5, Lea H Dib6, Isabel M Mol2,3, Natarin Caengprasath5, Yi-Wah Chan7, Sami Damak8, Anne Reifel Miller9, Tamer Coskun9, Bharat Shimpukade10, Trond Ulven10, Sander Kooijman2,3, Patrick CN Rensen2,3 and Mark Christian *,1 1Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK 2Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands 3Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands 4Department of Molecular Biosciences, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden 5Department of Surgery and Cancer, Institute of Reproductive and Developmental Biology, Imperial College London, London, UK 6Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK 7Lymphocyte Development Group, MRC London Institute of Medical Sciences, Hammersmith Campus, Imperial College London, London, UK 8Nestlé Research Center, Lausanne, Switzerland 9Lilly Research Laboratories, Diabetes/Endocrine Department, Lilly Corporate Center, Indianapolis, IN, USA 10Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark *Corresponding author. Tel: +31-71-5265304. E-mail: [email protected] *Corresponding author. Tel: +44-24-76-968585. E-mail: [email protected] EMBO Mol Med (2018)10:e8047https://doi.org/10.15252/emmm.201708047 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein-coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120-mediated signaling in BAT. We found that activation of GPR120 by the selective agonist TUG-891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content and increased nutrient uptake by BAT, confirming increased BAT activity. Consistent with these observations, GPR120 deficiency reduced expression of genes involved in nutrient handling in BAT. Stimulation of brown adipocytes in vitro with TUG-891 acutely induced O2 consumption, through GPR120-dependent and GPR120-independent mechanisms. TUG-891 not only stimulated GPR120 signaling resulting in intracellular calcium release, mitochondrial depolarization, and mitochondrial fission, but also activated UCP1. Collectively, these data suggest that activation of brown adipocytes with the GPR120 agonist TUG-891 is a promising strategy to increase lipid combustion and reduce obesity. Synopsis This study demonstrates that the GPR120 agonist TUG-891 improves metabolic health by activation of brown fat. Mechanistically, TUG-891 promotes respiration in brown adipocytes by stimulating GPR120-dependent Ca2+ release and mitochondrial fragmentation, thereby activating UCP1. The GPR120 agonist TUG-891 acutely increases fat oxidation and decreases body weight and fat mass in mice. Beneficial metabolic effects of TUG-891 are related to increased brown fat activity, reflected by an increased uptake of fatty acids by brown adipose tissue in vivo. TUG-891 increases mitochondrial respiration in brown adipocytes in vitro, via both GPR120- dependent and -independent mechanisms. Introduction Brown adipose tissue (BAT) is present and active in human adults and contributes to total energy expenditure (EE) (Cypess et al, 2009; van Marken Lichtenbelt et al, 2009; Virtanen et al, 2009). This contrasts with white adipose tissue (WAT), which primarily serves as a site of energy storage. Cold exposure, the natural stimulus of BAT, increases the volume and activity of metabolically active BAT and reduces fat mass in adult human subjects (van der Lans et al, 2013; Yoneshiro et al, 2013; Blondin et al, 2014). Cold exposure stimulates the sympathetic nervous system to release norepinephrine, which in turn activates brown adipocytes through the β3-adrenergic receptor (ADRB3) (Argyropoulos & Harper, 2002). Activation of brown adipocytes initiates intracellular signaling cascades, resulting in the breakdown of triglycerides (TG) stored in intracellular lipid droplets to yield fatty acids (FA) and glycerol (Cannon & Nedergaard, 2004). The FAs are subsequently transported to the mitochondria where they are either oxidized or used to allosterically activate uncoupling protein-1 (UCP1), which is present in the inner membrane of mitochondria (Fedorenko et al, 2012; Nicholls, 2017). UCP1 disrupts the proton gradient that is required for ATP synthesis, resulting in the release of energy as heat instead of ATP: a process called thermogenesis (Trayhurn, 2017). Since activated BAT burns high amounts of FAs, it is considered an attractive target to combat obesity and related disorders. Therefore, novel targets to increase BAT activity are highly warranted. A potential target is G protein-coupled receptor 120 (GPR120), also termed free FA receptor 4 (FFAR4). We have previously shown that GPR120 is highly expressed in BAT and cold exposure further increases its expression in both BAT and subcutaneous WAT of mice (Rosell et al, 2014), suggesting that GPR120 contributes to the thermogenic capacity of BAT. GPR120 is activated by both medium-chain FA (MCFA) and long-chain FA (LCFAs) (Hirasawa et al, 2005; Christiansen et al, 2015) and is coupled to Gαq, which activates several intracellular signaling pathways. Recent studies have revealed that through these signaling mechanisms, GPR120 plays an important role in energy metabolism, hormonal regulation, and the immune system. For example, Oh et al (2010) demonstrate that GPR120 mediates the anti-inflammatory actions of ω-3 FAs. GPR120 deficiency leads to obesity, glucose intolerance, and hepatic steatosis in mice fed a high-fat diet (Ichimura et al, 2012). In humans, GPR120 expression is higher in obese compared to lean subjects, and individuals carrying a mutation associated with decreased GPR120 signaling have an increased risk of obesity (Ichimura et al, 2012). Given the high GPR120 expression in BAT, it is likely that BAT contributes to the metabolic effects of GPR120 observed in these studies. Indeed, a very recent study by Quesada-López et al (2016) confirmed a role for GPR120 in BAT activation. However, therapeutic potential and underlying signaling of GPR120-mediated BAT activation remain to be elucidated. Therefore, the aims of this study were to further investigate the therapeutic potential of GPR120 agonism and to address GPR120-mediated intracellular signaling in BAT. We found that stimulation of GPR120 by the agonist TUG-891 increases fat oxidation and lipid uptake by BAT thereby reducing fat mass, while GPR120 deficiency reduces expression of genes involved in nutrient handling. Mechanistically, we show that TUG-891 acts in a GPR120-dependent manner to induce intracellular Ca2+ release which could result in mitochondrial depolarization and fragmentation. In addition, our data reveal that TUG-891 activates mitochondrial UCP1, which may act synergistically with mitochondrial fragmentation to increase respiration. Taken together, our data indicate that by acutely increasing lipid combustion by BAT, GPR120 agonism may be a promising therapeutic strategy to reduce obesity. Results The GPR120 agonist TUG-891 increases lipid oxidation and reduces fat mass in mice To investigate the effect of GPR120 activation on energy metabolism in vivo, mice were injected with the GPR120 agonist TUG-891 daily for a period of 2.5 weeks. This compound was selected due to higher selectivity for GPR120 over GPR40 compared to other agonists, including GW9508 and NCG21 (Shimpukade et al, 2012; Hudson et al, 2013). TUG-891 reduced total body weight (Fig 1A), which was due to a large reduction in fat mass (−73%; Fig 1B) and a minor reduction in lean mass (−9.9%; Fig 1C) at week 2.5 compared to vehicle. The reduced lean mass could be due to increased muscle turnover, as TUG-891 non-significantly increased expression of markers for both muscle atrophy and regeneration (Appendix Fig S1). During the first week of treatment, food intake was similar in the control and treatment groups (Fig 1D), while fat mass was already reduced by 19% in the TUG-891-treated group at day 5. Longer treatment reduced food intake, which further contributed to body weight and fat mass loss. To investigate whether TUG-891 enhances EE or alters substrate utilization, mice were housed in metabolic cages during the first week of treatment. TUG-891 treatment did not increase total EE (Appendix Fig S2A) nor did it affect physical activity levels (Appendix Fig S2B). However, TUG-891 acutely lowered the respiratory exchange ratio (RER) upon injection, which persisted throughout the dark period (Fig 1E). Accordingly, TUG-891 lowered glucose oxidation (Fig 1F) and largely increased fat oxidation (Fig 1G). This increase in fat oxidation was supported by histological analysis of adipose tissues, revealing that TUG-891 administration reduced lipid content in BAT (−28%; Fig 2A), and adipocyte size in both sWAT (−47%; Fig 2B) and gWAT (−38%; Fig 2C). In addition, total organ weights of iBAT (−31%), gWAT (−44%), and liver (−14%) were reduced in TUG-891-treated mice as compared to controls (Fig 2D). Plasma TG levels were increased at the end of the study, possibly as a result of increased lipolysis (Appendix Fig S3A). Protein (Appendix Fig S3B–E) and gene (Appendix Fig S3F) expressions of markers for lipolysis, adipogenesis, proliferation, and thermogenesis were largely unaffected in BAT. However, Ucp1 gene expression (Appendix Fig S3H) and protein staining (Appendix Fig S4) were increased in gWAT of TUG-891-treated animals, suggesting GPR120-mediated browning. Figure 1. The GPR120 agonist TUG-891 decreases body weight and fat mass, and increases fat oxidation A–D. C57Bl/6J mice on chow diet were treated with the GPR120 agonist TUG-891 (35 mg/kg) or vehicle (n = 8) for 2.5 weeks. Body weight, fat mass, lean mass, and food intake were measured at indicated time points. E–G. Vehicle- and TUG-891-treated mice (n = 8) were housed in fully automated metabolic cages in which respiratory exchange ratio (RER) (E), glucose oxidation (F), and fat oxidation (G) were measured. Injection of the GPR120 agonist TUG-891 (35 mg/kg) or vehicle is indicated by dotted lines, and light and gray areas represent the light and dark phase, respectively. For bar graph analysis, mean results in light and dark phase were calculated. Data information: Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the vehicle group, according to the two-tailed unpaired Student's t-test. The exact P-value for each significant difference can be found in Appendix Table S5. Download figure Download PowerPoint Figure 2. TUG-891 decreases lipid content of BAT and WAT A–C. Representative images of hematoxylin and eosin (H&E)-stained interscapular BAT (iBAT), subcutaneous WAT (sWAT), and gonadal WAT (gWAT) of mice treated with vehicle or the GPR120 agonist TUG-891 (n = 8). Stained slides were digitalized, and lipid droplet content of BAT and adipocyte size in WAT was analyzed using ImageJ software. D. After mice treated with vehicle or the GPR120 agonist TUG-891 (n = 8) were sacrificed, iBAT, gWAT, and liver were collected and weighed (n = 8). Data information: Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the vehicle group, according to the two-tailed unpaired Student's t-test. The exact P-value for each significant difference can be found in Appendix Table S5. Download figure Download PowerPoint As TUG-891 also has affinity for GPR40 (Hudson et al, 2013), we aimed to confirm that the effects of TUG-891 on body composition and substrate utilization were mediated by GPR120. To this end, metabolic effects of TUG-891 were also assessed in GPR120 KO mice and WT littermates. In GPR120 KO mice, TUG-891 non-significantly reduced body weight (Fig 3A) and fat mass (Fig 3B), but not to the same extent as in WT mice. TUG-891 decreased food intake similarly in WT and GPR120 KO mice (Fig 3C). The modest decrease in fat mass in TUG-891 GPR120 KO mice compared to non-treated WT mice may be related to diminished food intake. Lean mass was unchanged in all treatment groups (Fig 3D). In addition, while RER and fat oxidation did not differ between WT and GPR120 KO mice at baseline (Appendix Fig S5), TUG-891 non-significantly (P = 0.136) decreased RER (Fig 3E) and increased fat oxidation (Fig 3F) during the dark period in WT mice but not in GPR120 KO mice. Figure 3. Metabolic effects of TUG-891 are reduced or absent in GPR120-deficient mice A–D. GPR120 KO mice and WT littermates (n = 6–8) were treated with the GPR120 agonist TUG-891 (35 mg/kg) or vehicle for 12 days. At the beginning (day 0) and end (day 12) of this treatment period, body weight, fat mass, and lean mass were measured. Food intake was determined after 5 and 12 days of treatment. E–F. GPR120 KO mice and WT littermates (n = 6–8) were treated with the GPR120 agonist TUG-891 (35 mg/kg) or vehicle. Respiratory exchange ratio (RER) and fat oxidation were determined by housing the mice in metabolic cages. Injection of TUG-891 or vehicle is indicated by dotted lines, and light and gray areas represent the light and dark phase, respectively. For bar graph analysis, mean results in the light and dark phase were calculated. Data information: Data represent means ± SEM. *P < 0.05, **P < 0.01 compared to the vehicle group, according to two-way ANOVA with Tukey's post hoc test. The exact P-value for each significant difference can be found in Appendix Table S5. Download figure Download PowerPoint The GPR120 agonist TUG-891 stimulates fatty acid uptake by BAT Hereafter, we aimed to elucidate which organs were responsible for the increased fat oxidation in TUG-891-treated WT animals. As increased fat oxidation subsequently leads to increased FA uptake, the tissue-specific uptake of FAs derived from intravenously injected lipoprotein-like particles labeled with glycerol tri[3H]oleate was determined. In WT mice, TUG-891 treatment markedly increased the uptake of [3H]oleate in both iBAT and subscapular BAT (sBAT) as compared to vehicle (Fig 4A), suggesting increased BAT activity. TUG-891 also increased the uptake of [14C]deoxyglucose in iBAT and sBAT (Fig 4B). However, when the uptake data were corrected for organ weight (for organs that could be removed quantitatively within an acceptable time frame), the difference in glucose uptake was lost. FA uptake in whole iBAT remained approximately twice as high in treated WT mice versus controls (Appendix Fig S6), showing an independency of organ weight. In line with these data, TUG-891 decreased total organ weights of iBAT and sWAT depots as compared to vehicle in WT mice, but not in GPR120 KO mice (Fig 4C). Figure 4. TUG-891 increases the uptake of nutrients by BAT A, B. WT and GPR120 KO mice (n = 6–8) treated with vehicle or the GPR120 agonist TUG-891 were intravenously injected with [3H]TO-labeled lipoprotein-like emulsion particles and [14C]deoxyglucose ([14C]DG). After 15 min, mice were sacrificed and uptake of [3H]TO- and [14C]DG-derived radioactivity per gram tissue was determined in various organs, including gonadal WAT (gWAT), subcutaneous WAT (sWAT), interscapular BAT (iBAT), and subscapular BAT (sBAT). C. After WT and GPR120 KO mice (n = 6–8) treated with vehicle or the GPR120 agonist TUG-891 were sacrificed, organs were collected and weighed. Data information: Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the vehicle group or indicated control group, according to two-way ANOVA with Tukey's post hoc test (A, B) or the two-tailed unpaired Student's t-test (C). The exact P-value for each significant difference can be found in Appendix Table S5. Download figure Download PowerPoint GPR120 alters expression of genes involved in nutrient handling To evaluate how GPR120 modulates lipid handling by BAT, we investigated the effects of GPR120 deficiency on the global gene expression profile in BAT by performing a microarray on BAT of GPR120 KO and WT littermates. Clustering of genes was observed between GPR120 KO and WT mice (Fig 5A). The top 50 of genes that were either upregulated or downregulated in the absence of GPR120 are listed in Appendix Table S3. Selected genes were validated, and expression of genes associated with inflammation (Fig 5B), adipocyte biology (Fig 5C), glucose metabolism (Fig 5D), and lipid metabolism (Fig 5E) was investigated by qRT–PCR. Expression of inflammatory genes tended to be increased in GPR120 KO BAT. GPR120 deficiency upregulated Sncg, encoding synuclein-γ which is involved in lipid droplet dynamics in white adipocytes and is negatively regulated by PPARγ (Dunn et al, 2015). On the other hand, GPR120 deficiency downregulated Mlxipl, which encodes the carbohydrate response element-binding protein (ChREBP), a transcriptional inducer of glucose metabolism and de novo lipogenesis (Witte et al, 2015). Of the genes associated with glucose metabolism, Glut4, Insr, Adcy4, and Gys2 were downregulated in GPR120 KO BAT. Gys2 encodes glycogen synthase 2 and is PPARγ-regulated in adipocytes (Mandard et al, 2007). Of the genes that determine lipid metabolism, those involved in both lipogenesis (Acc1, Acc2, Fas, Scd2) and intracellular lipolysis (Hsl, Atgl, Pnpla3) were lower in GPR120 KO BAT. Figure 5. GPR120 deficiency alters the expression of genes involved in glucose and lipid metabolism in BAT A. Probe sets for WT and GPR120 KO BAT from microarray analysis are colored according to average expression levels across all samples, with green denoting a higher expression level and red denoting a lower expression level. The probe sets shown in the heat map passed the threshold of absolute value of the logFC > 0.5 and P-adjusted < 0.05. B–E. Expression of genes involved in inflammation, adipocyte biology, glucose metabolism, and lipid metabolism in BAT from GPR120 KO mice (n = 5) and WT littermates (n = 6) was determined through qRT–PCR (N.D. = non-detectable). Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the WT control group, according to the two-tailed unpaired Student's t-test. The exact P-value for each significant difference can be found in Appendix Table S5. Download figure Download PowerPoint Functional annotation clustering using DAVID (https://david.ncifcrf.gov/) (Dennis et al, 2003) revealed that genes downregulated in the absence of GPR120 were associated with mitochondrial function, FA metabolism, nucleotide binding, and mRNA processing (Appendix Table S4). The set of upregulated genes was associated with immune responses, as well as antigen processing and ribosomes. Gpr120 expression promotes brown adipocyte differentiation and is increased in "browned" white adipocytes Using a conditionally immortalized model of brown adipocytes (Rosell et al, 2014), we investigated the expression profile of Gpr120 in preadipocytes differentiated to fully mature adipocytes over 7 days. Like Ucp1 expression, Gpr120 expression was highly induced during differentiation of brown adipocytes, reaching maximum levels on day 6 (Fig 6A). This is consistent with high GPR120 expression in BAT compared to other organs (Appendix Fig S7). Treatment of differentiated adipocytes with the β3-adrenergic agonist CL induced both Gpr120 (ninefold) and Ucp1 (53-fold) expression (Fig 6A). Differentiation also increased expression of adipocyte markers aP2, Cidea, and Adrb3, and decreased expression of the preadipocytes marker Pref1, validating our brown adipocyte cell line (Appendix Fig S8). Figure 6. GPR120 is involved in differentiation of brown adipocytes and browning of white adipocytes Immortalized murine brown adipocytes (n = 3) were differentiated for 0, 2, 4, 6, or 7 days after which expression of Gpr120 and Ucp1 was determined by qRT–PCR. On day 7, a subset of adipocytes (n = 3) was stimulated with CL (10 μM) or vehicle. Expression of Gpr120 and Ucp1 was measured in undifferentiated (Undiff), differentiated (Diff), and CL-treated (Diff + CL) brown adipocytes (BA), subcutaneous white adipocytes (sWA), and sWA treated with the browning agent rosiglitazone (sWA brite) (n = 3). WT and GPR120 KO brown adipocytes (n = 3) were treated with vehicle or TUG-891 (10 μM) throughout differentiation and stained at day 0, 3, 7, and 9 of differentiation with Oil Red O. Absorbance of the staining at 520 nm was quantified. A representative image at day 8 of differentiation was taken with a phase-contrast microscope (Leica) at 20-fold magnification. As in (C), WT and GPR120 KO brown adipocytes (n = 3) were treated with vehicle or TUG-891 throughout differentiation to analyze expression patterns of aP2 and Ucp1. Data information: Data represent means ± SEM. **P < 0.01 compared to the vehicle group, ***P < 0.001 compared to the WT control group or indicated controls, according to the two-taile
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