Kynurenic Acid and Gpr35 Regulate Adipose Tissue Energy Homeostasis and Inflammation
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(2) Cell Metabolism. Article Kynurenic Acid and Gpr35 Regulate Adipose Tissue Energy Homeostasis and Inflammation Leandro Z. Agudelo,1 Duarte M.S. Ferreira,1 Igor Cervenka,1 Galyna Bryzgalova,2 Shamim Dadvar,1 Paulo R. Jannig,1 Amanda T. Pettersson-Klein,1 Tadepally Lakshmikanth,3,4 Elahu G. Sustarsic,5 Margareta Porsmyr-Palmertz,1 Jorge C. Correia,1 Manizheh Izadi,1 Vicente Martı́nez-Redondo,1 Per M. Ueland,6,7 Øivind Midttun,8 Zachary Gerhart-Hines,5 Petter Brodin,3,4 Teresa Pereira,2 Per-Olof Berggren,2 and Jorge L. Ruas1,9,* 1Department. of Physiology and Pharmacology, Molecular and Cellular Exercise Physiology, Karolinska Institutet, 17177 Stockholm, Sweden Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden 3Science for Life Laboratory, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden 4Department of Newborn Medicine, Karolinska University Hospital, Stockholm, Sweden 5Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark 6Department of Clinical Science, University of Bergen, Bergen, Norway 7Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway 8Bevital A/S, Laboratoriebygget, 5021 Bergen, Norway 9Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.cmet.2018.01.004 2Rolf. SUMMARY. The role of tryptophan-kynurenine metabolism in psychiatric disease is well established, but remains less explored in peripheral tissues. Exercise training activates kynurenine biotransformation in skeletal muscle, which protects from neuroinflammation and leads to peripheral kynurenic acid accumulation. Here we show that kynurenic acid increases energy utilization by activating G protein-coupled receptor Gpr35, which stimulates lipid metabolism, thermogenic, and anti-inflammatory gene expression in adipose tissue. This suppresses weight gain in animals fed a high-fat diet and improves glucose tolerance. Kynurenic acid and Gpr35 enhance Pgc-1a1 expression and cellular respiration, and increase the levels of Rgs14 in adipocytes, which leads to enhanced beta-adrenergic receptor signaling. Conversely, genetic deletion of Gpr35 causes progressive weight gain and glucose intolerance, and sensitizes to the effects of high-fat diets. Finally, exercise-induced adipose tissue browning is compromised in Gpr35 knockout animals. This work uncovers kynurenine metabolism as a pathway with therapeutic potential to control energy homeostasis.. INTRODUCTION The kynurenine (KYN) pathway of tryptophan (TRP) degradation is the major catabolic pathway for this essential amino acid. It is responsible for metabolizing up to 95% of free TRP and generates a group of metabolites collectively referred to as ‘‘kynurenines’’ with NAD+ as the end product (Cervenka et al., 2017). KYN and its metabolites participate in several physiological and pathophysiological processes, most notably in the CNS,. where they are involved in the etiology of several mental health disorders such as depression and schizophrenia (Schwarcz et al., 2012). The actions of KYNs in peripheral tissues are less well established, but some have been shown to be strong modulators of immune cell function (Mándi and Vécsei, 2012; Stone et al., 2013). We have previously shown that exercised skeletal muscle can increase the conversion of KYN to kynurenic acid (KYNA) (Agudelo et al., 2014), a metabolite that cannot cross the blood-brain barrier (Fukui et al., 1991). Activation of this pathway in skeletal muscle is dependent on the transcriptional coactivator Pgc-1a1 (Correia et al., 2015) and protects the brain from stress-induced KYN accumulation and associated deleterious effects (i.e., neuroinflammation and changes in synaptic plasticity associated with depression) (Agudelo et al., 2014). Since KYNA is not transported across the blood-brain barrier it €ller and Schwarz, is locally produced in the CNS from KYN (Mu 2007) by KYN aminotransferases (KATs). Actions of KYNA in the CNS are related to its activity as an antagonist for N-methyl-D-aspartate (NMDA) and cholinergic a7 nicotinic receptors (Vécsei et al., 2013), and agonist for the recently deorphanized G protein-coupled receptor (GPCR) 35 (Gpr35) (Divorty et al., 2015). Interestingly, we and others have shown that even in the absence of a stress challenge, endurance exercise training is sufficient to activate skeletal muscle KYN to KYNA conversion, resulting in considerably elevated circulating KYNA levels (Agudelo et al., 2014; Lewis et al., 2010; Schlittler et al., 2016). In the periphery, high levels of KYNA have been observed in patients with inflammatory bowel disease and other pathologies of the gastrointestinal tract (GIT) (Forrest et al., 2002). Finally, KYNA has been reported to have anti-inflammatory properties and to modulate cytokine release from invariant natural killer T (iNKT) cells through Gpr35 activation (Fallarini et al., 2010). Since KYNA concentrations are increased by aerobic exercise training, we have investigated if it plays a role in peripheral tissue metabolism. Here we show that KYNA regulates adipose tissue energy homeostasis through activation of Gpr35. Activation of this network stimulates the expression of lipid metabolism,. 378 Cell Metabolism 27, 378–392, February 6, 2018 ª 2018 Elsevier Inc..
(3) B. Vehicle KYNA. *. 4000. 2000. Carbon Dioxide Production (ml/kg/h). Oxygen Consumption (ml/kg/h). 6000. 4000. 2000. 0. C. Ni gh t4 -5. Da y. 45. 0. 4. 6000. 2000. 0. 5. Days. Locomotion. PBS. *. 4000. D. KYNA. 4000. 2000. 0. 4. 5. Days. Feeding. PBS. 0.15. 3000. KYNA. PBS 6000. Da y. Oxygen Consumption (ml/kg/h). 6000. VCO2. KYNA. PBS. Carbon Dioxide Production (ml/kg/h). VO2. 45 Ni gh t4 -5. A. KYNA. 25. 2000. 0.00. 5. * 1.5. *. 50. 0.0. 0.0. 0. 32 30. 3 W ee. k. 2 W ee. k. 1 k W ee. D. ay. 0. 28. 0. iWAT 2.5. eWAT. 1. 4. J. 8. TG. 1.5. 1.5. *. 2.0. Relative levels. *. *. 34. 100. 0.5. 1.0. 1.5. * 1.0. 0.5. 1.0. * 0.5. 0.5 0.0. 0.0. PB S KY NA. KYNA. * 1.0. 0.5. Relative Fat Mass (normalized by body weight). PBS. 150. nM. * 1.0. I 36. KYNA Kinetics 200. 1.5. Day 5. H. G. 2.0. PB S KY NA. 24. eWAT. 5. Days. PB S KY NA. 26. iWAT. 4. PB S KY NA. Relative Fat mass (normalized by body weight). PBS KYNA. 0. Da y. Days. F. Day 0. 10. Ni gh t4 -5. 4. 45. Ni gh t4 -5. Da y. 45. 0. 28. 15. 5. E. Body weight (g). 0.05. 1000. 0. Body weight (g). 0.10. 20. 0.0. PB S KY NA. 1000. 3000. Food intake (g). 2000. Feeding (g). XY_counts. Total_XY_counts. 4000. (legend on next page). Cell Metabolism 27, 378–392, February 6, 2018 379.
(4) thermogenic, and anti-inflammatory genes in the adipose tissue. This suppresses weight gain in animals fed a high-fat diet (HFD) and improves glucose tolerance and adipose tissue inflammation. KYNA/Gpr35 signals through a 2-fold mechanism: (1) activation of Ca2+, ERK, and CREB signaling; stabilization of Pgc-1a1; and induction of downstream genes, and (2) increase of Rgs14 gene expression, a protein G regulatory subunit that mediates a negative feedback loop by interacting with Gai subunits, thus leading to enhanced b-adrenergic receptor (b-AR) signaling. Importantly, these effects are lost upon genetic deletion of Gpr35, which causes progressive weight gain and glucose intolerance. In addition, exercise-induced adipose tissue browning is compromised in Gpr35 knockout animals. Together, our data uncover a novel muscle to adipose tissue and immune system crosstalk mediated by KYNA and Gpr35, with impact on systemic energy expenditure and inflammation. RESULTS KYNA Regulates Systemic Energy Expenditure To evaluate the effects of KYNA on systemic energy metabolism, we administered a single daily intraperitoneal (i.p.) dose of 5 mg/kg body weight to C57BL/6J mice during 7 consecutive days. During treatment, animals were placed in a Comprehensive Laboratory Animal Monitoring System (CLAMS) and evaluated for indirect calorimetry as well as locomotion and food and water intake. After 3 days of KYNA administration, we could observe an increase in O2 consumption, and in CO2 and heat production, indicative of higher energy expenditure (Figures 1A, 1B, S1A, and S1B). Importantly, we did not observe changes in locomotion or food or water intake at any time point (Figures 1C, 1D, S1C, and S1D). Finally, although at this point we did not observe changes in body weight (Figure 1E), analysis of the subcutaneous/inguinal white (iWAT), visceral/epididymal white (eWAT), and brown (BAT) adipose tissues revealed a reduction in subcutaneous and visceral adiposity (Figure 1F). No changes were seen in BAT weight (Figure S1E). One-week KYNA administration did not affect triglyceride (TG) levels when compared to vehicle-treated mice (Figure S1F). To evaluate the kinetics of daily i.p. KYNA treatment, we quantified plasma KYNA up to 4 hr post-injection. We verified that, similar to what has been seen in aerobic exercise interventions (Lewis et al., 2010; Schlittler et al., 2016), circulating KYNA levels tripled within an hour after injection (Figure 1G). We next evaluated if chronic daily KYNA administration affected the weight of mice fed a chow. diet. To this end, C57BL/6J mice were given the same daily KYNA dose and followed for 3 weeks. As before (Figure 1E), 1 week of KYNA administration had no effect on body weight (Figure 1H). However, after 2 weeks we could observe a significant reduction in body weight when compared to PBS-injected controls (Figure 1H). The reduction in body weight was reflected by a reduction in iWAT and eWAT and in circulating TGs (Figures 1I and 1J). To evaluate the effect of chronic KYNA administration on circulating metabolites of the KYN pathway, we analyzed plasma of KYNA- and PBS-treated animals. We found that 3 weeks of KYNA treatment reduced the plasma levels of quinolinic acid (QUIN), picolinic acid (PA), and 3-hydroxykynurenine (3HK; Figure S6A) while increasing nicotinic acid (NA; Figure S1G). KYNA Induces a Beige Adipocyte Transcriptional Signature We next analyzed the expression of key metabolic genes in the different adipose depots. In BAT, we detected an increase in the levels of beige genes (Wu et al., 2012) and smaller changes in Ppara and fatty acid oxidation genes. We did not observe significant changes in genes involved in thermogenesis, lipolysis, oxidative phosphorylation (OXPHOS), or lipogenesis (Figures 2A and 2B). However, the adipose depots that showed reduction in weight also showed dramatically increased expression of genes involved in all biochemical pathways we analyzed (Figures 2C–2F and S2A). Most notably, we could observe a robust increase in Ucp1 expression in iWAT and eWAT, as well as other important thermogenic genes such as Pgc-1a, Prdm16, and Cidea (among others) (Figures 2C–2E). Both iWAT and eWAT showed significantly increased transcription of genes involved in fatty acid oxidation and OXPHOS (Figures 2D–2F). Additionally, although all three depots showed positive changes in the expression of genes involved in antioxidant response and glucose uptake, iWAT displayed the most robust increases (Figure S2B). Analysis of liver and skeletal muscle from the same animals revealed only minor changes in Nrf1 and Pdk4 expression, respectively (Figures S2C–S2F), indicating that, under these conditions, KYNA targets mainly the adipose compartments. The Adipose Depots of KYNA-Treated Mice Reveal Gene Expression Signatures of Anti-inflammatory Immune Cells KYNA has been shown to work as an immune modulator, albeit at much higher concentrations than those used in this study (i.e.,. Figure 1. KYNA Regulates Systemic Energy Expenditure C57BL/6J mice were injected daily with KYNA intraperitoneally and monitored using a Comprehensive Lab Animal Monitoring System (CLAMS) (n = 7). (A) VO2, oxygen consumption. (B) VCO2, carbon dioxide production. (C) Locomotion (x and y axes). (D) Accumulated food intake. (E) Body weight at day 0 (before treatment) and at day 5 (n = 7). (F) Boxplot showing fat mass of the different fat depots relative to body weight (n = 7). (G) Plasma levels of KYNA, measured at different time points after a single dose of 5 mg/kg KYNA (n = 4–5). (H) Determination of body weight at day 0 (before treatment) and weekly from mice treated daily with 5 mg/kg KYNA or PBS (n = 5–6). (I) Boxplot of fat depot mass after 3 weeks of KYNA administration, relative to body weight (n = 5–6). (J) Relative plasma TG levels in mice treated during 3 weeks with 5 mg/kg KYNA or PBS (n = 5–6). Bars depict mean values and error bars indicate SEM. Student’s t test was used for statistical analysis, *p < 0.05. See also Figure S1.. 380 Cell Metabolism 27, 378–392, February 6, 2018.
(5) Thermogenesis. Lipolysis. Oxphos. Beige. Fatty acid oxidation. Lipogenesis. A. B 5. PBS KYNA. 4. BAT. * *. 3. *. 2. *. 1. BAT. * Relative transcript levels. Relative transcript levels. 5. PBS KYNA. 4. 3. *. 2. * * * * * 1. * 0. C. D 35. *. iWAT. 25. *. 20 15. *. 10. *. *. 5. *. *. * *. * *. *. * *. * *. *. 20. *. * *. 10. *. *. *. *. *. * *. 2. * * * *. *. 1. *. 0. At. U c Pg p 1 c Pg 1a Pr c1 dm b 1 C 6 y C tc id C ea pt 1b N rf1 A A d p2 ip o Es q Pp rra a Ac ra ox D 1 C io2 d Tm 1 3 em 7 2 Tb 6 x Kl 1 hl Ea 3 C r2 Sl d 4 c2 0 7a 1. g Hl sl M C g gi l -5 Pl 8 Ac in1 a A t1 Ac cat2 ad Ac m Ac ad ad l O vl x P ct N dk du 4 N fa du 9 C fs8 ox At 5a p Pp f5l C arg Sr e b eb p a p1 c Fa Ac s c D 1 g D at1 Aggat pa 2 t1. 0. E. F 10. 10. eWAT. 6. * 4. * *. 2. *. * *. * *. *. *. *. *. *. U c Pg p1 c Pg 1a Pr c1 dm b 1 C 6 y C tc id C ea pt 1b N rf1 A A d p2 ip o Es q Pp rra a Ac r a ox D 1 C io2 d Tm 13 em 7 2 Tb 6 x Kl 1 hl Ea 3 C r2 Sl d4 c2 0 7a 1. 0. *. 8. 6. 4. * *. * *. * *. * * *. *. *. *. *. 2. * 0. g Hl sl C Mg gi l -5 Pl 8 Ac in1 a A t1 Ac cat2 ad Ac m Ac ad ad l O vl x P ct N dk du 4 N fa du 9 C fs8 ox At 5a p Pp f5l C arg Sr e b eb p a p1 c Fa Ac s c D 1 g D at1 Ag gat pa 2 t1. * 8. Relative transcript levels. eWAT. At. Relative transcript levels. iWAT. 30. 30. Relative transcript levels. Relative transcript levels. At. U c Pg p 1 c Pg 1a Pr c1 dm b 1 C 6 yt C c id C ea pt 1b N rf1 A A d p2 ip o Es q r Pp ra a Ac ra ox D 1 C io2 Tm d 1 3 em 7 2 Tb 6 x Kl 1 hl Ea 3 C r2 Sl d 4 c2 0 7a 1. gl C Hs gi l -5 8 M P l gl Ac in1 a A t1 Ac cat2 ad Ac m Ac ad ad l O vl x P ct N dk du 4 N fa du 9 C fs8 ox At 5a p Pp f5l C arg Sr e b eb p a p1 c Fa Ac s c D 1 g D at1 Aggat pa 2 t1. 0. Figure 2. KYNA Induces a Beige Adipocyte Transcriptional Signature Analysis of gene expression by quantitative real-time PCR in brown (A and B), inguinal (C and D), and visceral (E and F) adipose tissue (BAT, iWAT, and eWAT, respectively) (n = 6–10). Bars depict mean values and error bars indicate SD. Student’s t test was used for statistical analysis, *p < 0.05. See also Figure S2.. 50–500 mg/kg) (Moroni et al., 2012). To assess whether chronic KYNA administration had a general effect on the immune compartment, we first analyzed the spleens of KYNA- and PBS-treated animals by mass cytometry (Bandura et al., 2009). This revealed no changes in the size of the different immune. populations (Figure S3A). We next analyzed the different adipose depots of KYNA- or PBS-treated mice for the expression of immune cell markers and cytokines associated with inflammatory and anti-inflammatory phenotypes (Brestoff and Artis, 2015; Lackey and Olefsky, 2016). Both iWAT and eWAT from Cell Metabolism 27, 378–392, February 6, 2018 381.
(6) A Itgam. Ptgdr2. Itgax. Ifng. Retnla Il33 II10 Ccr3. Il13. Cd274. Ccl6. Il18. Cd19. Retnla. Cxcl2. Cxcl10. Cd36. 10-3. Ccr1. Ccr1 Il33. Il5 Ccl6. 0.01. 0.1. 1. 10. 10-4 0.25. 100. 0.5. 1. C. D. BAT. 10-1. Il11 Ccl2 Ccr2. Cxcr2. Tnf. 10-2. Cd4. Ccl6. Il10. Cxcl10. Il16 Cd27. Ccr9. Cd68. 4. WAT-Spearman correlation. 10. eWAT Fold-change KYNA/PBS. 100. 2. Log Fold-change KYNA/PBS. Log Fold-change KYNA/PBS. p-value. Il4. Ccl25 Arg1. Il4. Il13 Ccr3 Mrc1. Foxp3. 10-2. Arg1. Clec10a. 10-5 10-6. Clec10a. Cxcl2. Cd36. Tnf. 10-4. Cd163. 10-1. Cd27. 10-3. Il10 Il2. Foxp3. p-value. p-value. Csf2 Cd25. Ccl5. Cd62l. eWAT 100. Cd86. Cxcl12. 10-1 10-2. B. iWAT. 100. Ccl25 Cd86 10-3. Cxcl10. r = 0.39 p = 0.0015. Il13 Il10. Il33 Ccr3. Ccl6. Il4 Ccr1 1. Tnf. Cxcl12 10-4 0.31622776601684. 0.1 1. 0.01. 0.1. Log Fold-change KYNA/PBS. ILC2 and VAT Tregs enriched. 100. *. 4. 8 3. *. * 6. 2. 1. C. tla 4 Ar eg G itr Irf 4 Ir G s2 at a kl 3 rg 1. 0. 4. 2. *. * *. iWAT. 14. 10. *. 12. *. 4. PBS KYNA. eWAT *. 3. 10 8. *. 2. *. 6 4. *. 1. 2. 0. 0. 0. Il4 r Il5 ra Il1 0r a Il1 rl1. -log p-value. eWAT KYNA. r Il5 r Il1 a 0r a Il1 rl1. Granulocyte Adhesion Innate and Adaptive Immune Cells T Helper Cell Differentiation IL-10 Signaling PPAR Signaling CCR3 Signaling in Eosinophils. PBS. Il4. 10 20. iWAT *. tla 4 Ar eg G itr Irf 4 Ir G s2 at a Kl 3 rg 1. 0. Relative transcript levels. 5. Canonical pathway. 10. Type II immune signaling. Relative transcript levels. F. C. E. 1. iWAT Fold-change KYNA/PBS. Figure 3. Adipose Depots of KYNA-Treated Mice Reveal Gene Expression Signatures of Anti-inflammatory Immune Cells (A–C) Volcano plots for analysis of gene expression by quantitative real-time PCR in iWAT (A), eWAT (B), and BAT (C) from mice treated with PBS or KYNA for 6 days (n = 4–6). (D) Fold change correlation between KYNA-regulated genes in eWAT and iWAT. (E) Ingenuity pathway analysis (IPA) of KYNA-regulated genes shown in (A) and (C). (F) Analysis of gene expression by quantitative real-time PCR in iWAT and eWAT from the same animals as in (A). Bars depict mean values and error bars indicate SD. Student’s t test was used for statistical analysis, *p < 0.05. See also Figure S3.. KYNA-treated mice showed a large enrichment in antiinflammatory gene expression (Figures 3A–3D). In particular, there was an increase in the expression of cytokines involved in type 2 immune responses such as Il4, Il13, Il33, and Il10 (Figures 3A–3D) (Lackey and Olefsky, 2016). We also observed that KYNA reduced the expression of cellular markers 382 Cell Metabolism 27, 378–392, February 6, 2018. and cytokines related to inflammation or type 1 immune response (Figures 3A–3D). Analysis of the gene profiles induced by KYNA in both white adipose depots revealed that those genes tend to group within pathways associated with specific immune populations, such as Il10, PPAR, and CCR3 signaling (Figure 3E)..
(7) A. B. Thermogenesis. Beige/Brite 5. 6. *. 4. *. *. *. 0. *. *. *. 2. *. *. 3. * *. *. 2. *. *. 1. Oxphos. d Tm 1 3 7 em 26 Tb x1 Kl hl 3 Ea r2 C d4 Sl 0 c2 7a 1. 2 io. C. D. Lipogenesis. 400. 8. PBS. Oligomycin. KYNA. FCCP. Antimycin. *. 7 *. 5. OCR (pmoles/min). *. 6. * *. 4. *. 3. *. 2. *. *. *. *. *. *. *. * *. 300. 200. 100. PBS KYNA. 1 0. 0. At. gl H s Ml C gl gi -5 Pl 8 i Ac n1 a Ac t1 Ac at2 a Ac dm ad v O l x Pd ct N k4 du N fa9 du C fs8 ox A 5a Sr tpf eb 5l p1 c Fa Ac s M c og 1 a D t1 ga D t1 g Ag at2 pa t1. 0. 20. 60. 80. G - log p-value. 16. TISSUE DEVELOPMENT 14. CELL DIFFERENTIATION. 3. CELL PROLIFERATION 12. CELLULAR COMPONENT BIOGENESIS. 2. Dr 1. Er r1. Nr Pp f1 ar _D r1. TF enrichment. Relative transcript levels. 6. POSITIVE GENE EXPRESSION. 4. 1. 40. Time (minutes). F. Affymetrix Inguinal Primary Adipocytes. -log p-value. *. 4. D. ar. Ac. Pp. Ad. C. Fatty acid oxidation. ox 1. a. oq. 2. ip. Ap. 1b pt. ea id C. Pr. dm. C yt c. 16. b. a. c1. c1. 1 cp. Pg. U. Lipolysis. E. PBS KYNA. 0. C. Relative transcript levels. Relative transcript levels. PBS KYNA. * 8. Pg. Relative transcript levels. 10. PBS KYNA. 4. * *. 2. *. *. *. 0. 0 -1.5. -1.0. 0. 1.0. 1.5. 2.0. Fold-change KYNA/PBS. 20. Pp a C rg eb p C a eb pb N rf1 Es rra. -2.0. 10. - log p-value. I. ILK SIGNALING LXR/RXR ACTIVATION THYROID HORMONE METABOLISM. NOR- & ADRENALINE DEGRADATION PHENYLALANINE DEGRADATION RETINOATE BIOSYNTHESIS. M em br an e re M gi em on br an e Ce or ll s ga ur ni za fa ce tio Re n ce pt or in te ra ct io n. NICOTINE DEGRADATION. GENE ONTOLOGY. Pl as m co a m me po m ne br nt an e. CANONICAL PATHWAYS. Up Do _ge wn ne _g s en es. H. TRYPTOPHAN DEGRADATION. - log p-value 1.5. 2.0. 2.5. 20. 15. 10. - log p-value (legend on next page). Cell Metabolism 27, 378–392, February 6, 2018 383.
(8) eWAT Treg- and ILC2-Specific Gene Signatures Include KYNA-Induced Genes Interestingly, comparing KYNA-induced genes with gene expression profiles for different immune cell populations (Heng et al., 2008) produced a clear association with alternatively activated macrophages (AAMs), regulatory T cells (Tregs), and type 2 innate lymphoid cells (ILC2s) (Figure S3B), all known to act primarily in type 2 immune responses (Brestoff and Artis, 2015). Both Tregs and ILC2s have been shown to be the initial regulators of this type of immune response in WAT (Cipolletta, 2014; Lackey and Olefsky, 2016). Indeed, we could determine that KYNA-induced genes in WAT partially overlap with eWAT Treg and ILC2 gene signatures (Figure S3D). These include Foxp3, Ccr1, Ccr2, Ccr3, and Cxcl2 (Figure S3D). This prompted us to assess if KYNA affects the expression of genes that are specific and related to the function of both immune cell populations (Figure S3D) (Cipolletta et al., 2012; Monticelli et al., 2011). Both iWAT and eWAT from KYNA-treated mice showed elevated expression of markers such as Ctla4, Areg, Gitr, and Klrg1 (Figure 3F), as well as increased expression of direct modulators of type 2 immune responses such as Il10ra and Il1rl1 (Figure 3F). To further analyze if KYNA affected any other immune populations of the white adipose compartment, we extracted the stromal vascular fraction (SVF) of iWAT and analyzed it by mass cytometry. We observed that KYNA increased the number of monocytes in iWAT but had no effect on T, B, NK, and dendritic cell population size (Figure S3E). In line with this, white fat depots of KYNA-treated mice displayed an increase in AAM markers such as Arg1, Retnla, Mrc1, and Clec10a (Figure S3F). Collectively, these results indicate that KYNA acts as an immune modulator of adipose-resident immune cells. Cell-Autonomous Actions of KYNA in Primary Adipocytes To evaluate if the effects of KYNA in the adipose compartment were adipocyte cell autonomous, we prepared inguinal primary adipocyte cultures from C57BL/6J mice. Upon complete differentiation, adipocytes were treated with KYNA for 12 hr and processed for analysis of gene expression by quantitative real-time PCR. KYNA treatment of cultured primary adipocytes resulted in a gene expression pattern similar to that we observed in vivo (Figures 4A–4C). These changes in gene expression were reflected by an increase in maximal respiratory capacity, as determined by extracellular flux analysis of KYNA-treated adipocytes (Figures 4D and S4A). To better understand the general effects of KYNA in adipocytes, we performed global analysis of gene expression (Figure 4E). Gene ontology analysis. of KYNA-regulated genes scored highly under transcriptional regulatory processes (Figure 4F). In addition, the promoter regions of KYNA-regulated genes showed enrichment in conserved regulatory motifs (Xie et al., 2005) for transcription factors such as ESRRA, NRF1, and PPAR (Figure 4F), important regulators of adipocyte biology (Scarpulla, 2008). In turn, the expression of several of these transcription factors was also enhanced by KYNA treatment (Figure 4G). Additional pathway analysis was performed to determine some of the top canonical pathways affected by KYNA treatment (Figures 4H and 4I). For example, genes induced by KYNA clustered in pathways related to liver X receptor (LXR) and thyroid hormone receptor (TR) (known metabolic regulators). To understand if the KYNA-mediated gene expression profile is fundamentally different from other well-known inducers of adipocyte differentiation and browning, we compared our gene sets with data available for rosiglitazone (Step et al., 2014; Uldry et al., 2006). Interestingly, of 2,258 genes differentially regulated by KYNA, 527 were shared with rosiglitazone and clustered under PPARg-mediated adipogenesis (Liberzon et al., 2015) (Figure S4B). In line with the gene expression data, treating adipocytes with KYNA potentiates the effects of rosiglitazone on gene expression and mitochondrial respiration (Figures S4C and S4D). Given the changes in basal respiration after KYNA and rosiglitazone treatment, we determined if chronic KYNA treatment influences the cellular content in mitochondria. Interestingly, we found that 3 weeks of daily KYNA i.p. administration increases mitochondrial mass and mitochondrial/nuclear DNA ratio (Figures S4E and S4F). Activation of Adipocyte Gpr35 by KYNA Leads to Increased Pgc-1a1 Expression KYNA has been shown to be a Gpr35 agonist (Wang et al., 2006). By determining Gpr35 expression in an array of mouse tissues, we verified that it has high expression levels in the GIT (Wang et al., 2006) and in iWAT (less in eWAT and even less in BAT) (Figure 5A). These relative expression levels match the magnitude of effects we observed in the different adipose tissue depots when treating mice with KYNA (i.e., iWAT > eWAT > BAT). This prompted us to analyze whether KYNA could elicit signaling downstream of Gpr35. Given the impact of Gpr35 activation on Ca2+ transients (Taniguchi et al., 2006), we next quantified if KYNA could elicit Ca2+ release using primary adipocytes under control conditions or upon Gpr35 gene silencing. We found that treating primary adipocytes with KYNA resulted in elevated Ca2+ levels, which were comparable to that obtained by adrenaline treatment (Figure 5B). This effect was blunted upon silencing Gpr35 gene. Figure 4. Cell-Autonomous Action of KYNA in Primary Adipocytes (A–C) Analysis of gene expression by quantitative real-time PCR in inguinal primary adipocytes treated with PBS or KYNA for 12 hr (n = 3). Graphs show relative expression data for genes involved in thermogenesis (A), beige fat (B), and energy metabolism (C). (D) Oxygen consumption rate (OCR) from inguinal primary adipocytes treated as in (A). OCR at basal and in the presence of 1 mM oligomycin, 1 mM FCCP, or 2 mM antimycin. (E) Volcano plot of microarray analysis of gene expression in inguinal primary adipocytes treated as in (A). (F) Gene ontology cluster for differentiation. Transcription factor binding sites in the promoter of genes regulated by KYNA. (G) Analysis of gene expression by quantitative real-time PCR in inguinal primary adipocytes treated with PBS or KYNA for 12 hr (n = 3). (H and I) Ingenuity pathway analysis (H) and gene ontology (I) of KYNA-regulated genes shown in (E). Bars depict mean values and error bars indicate SEM. Student’s t test was used for statistical analysis, *p < 0.05. See also Figure S4.. 384 Cell Metabolism 27, 378–392, February 6, 2018.
(9) A. B. D. E. C. F. G. I. H. J. K. Figure 5. Crosstalk between Gpr35 and b-AR Is Mediated by Rgs14 (A) Gpr35 transcript levels in different mouse tissues analyzed by quantitative real-time PCR and normalized by its expression in the liver (n = 3–4). (B) Relative Ca2+ levels from inguinal primary adipocytes treated for 5 min with PBS, adrenaline (1 mM), or KYNA (50 mM). Right panel shows inguinal primary adipocytes transfected with small interfering RNA (siRNA) for Gpr35 (si_Gpr35) or a scrambled (Scr) control 96 hr before Ca2+ determination. (C) Immunoblots from inguinal primary adipocytes treated with 50 mM KYNA and harvested at different time points (0, 5, 15, and 30 min). (D) cAMP levels (5 min after isoproterenol treatment) in primary adipocytes pre-treated during 2 days with different concentrations of KYNA. Inguinal primary adipocytes were transfected with siRNA for Gpr35 (siGpr35) or a scrambled control (Scr). (legend continued on next page). Cell Metabolism 27, 378–392, February 6, 2018 385.
(10) expression. Surprisingly, Gpr35 silencing enhanced adrenalineinduced Ca2+ release (Figure 5B), suggesting the existence of a crosstalk between the two GPCRs. Primary adipocyte treatment with KYNA also induced signaling pathways associated with thermogenic gene expression, which included an increase in ERK and CREB phosphorylation levels (Figure 5C) (Collins, 2012). We could also observe KYNA- and Gpr35dependent Pgc-1a1 stabilization (Figure S5A). Activation of Pgc-1a1 has been previously shown to result in thermogenic gene expression in adipocytes (Puigserver et al., 1998; Wu et al., 1999). A Crosstalk between Gpr35 and b-AR Mediated by Rgs14 Considering our gene expression data analysis (Figures 4H and 4I), and since crosstalk between GPCRs has been described (Wettschureck and Offermanns, 2005), we decided to investigate if Gpr35 could interfere with b-AR function in adipocytes. To this end, we determined cAMP levels in adipocytes treated with KYNA in the presence or absence of a b-AR agonist (isoproterenol) or of an antagonist (propranolol). Interestingly, we found that acute KYNA treatment dampens the effect of isoproterenol on cAMP levels (Figure S5B). Functional respiratory assays confirmed that acute KYNA treatment decreased the effects of isoproterenol on respiration (Figure S5C). On the other hand, we observed that the reduction in cAMP levels induced by propranolol was further reduced by KYNA (Figure S5D) in a Gpr35-dependent manner (Figure S5E). Since our observations show that KYNA and Gpr35 exert an acute dampening effect on b-AR activity, we next aimed to determine whether chronic KYNA treatment could have an effect on the response elicited by a b-AR agonist. Interestingly, we found that pre-treating adipocytes with KYNA at different concentrations elevates the levels of cAMP in response to isoproterenol treatment (Figure 5D). This effect was blunted upon Gpr35 silencing (Figure 5D). In agreement, KYNA pre-treatment increased the effects of isoproterenol on adipocyte respiration (Figure 5E), which was abolished when Gpr35 was silenced (Figure 5F). Gene expression profiles displayed similar potentiating effects of KYNA on isoproterenol response, which was dependent on Gpr35 (Figure 5G). Accordingly, treating primary adipocytes with KYNA for 6 days followed by a single low dose of isoproterenol (50 nM) increased oxygen consumption under basal conditions and in the presence of oligomycin (Figure S5F). These observations suggest that KYNA and Gpr35 have a sensi-. tizing effect on b-AR signaling. By re-examining gene expression in KYNA-treated adipocytes for GPCR signaling components, we found increased levels of Rgs14 (regulator of G protein signaling 14) (Figure 5H). This was confirmed by quantitative real-time PCR (Figure S5G) and proved to be dependent on Gpr35 expression (Figure S5H). Rgs14 attenuates G protein signaling as it binds to GTP-bound Gai subunits and promotes the formation of inactive Gai:Gb/g heterodimers (Berman et al., 1996; Cho et al., 2000). Although Rgs14 has only been reported to be expressed in the brain (Snow et al., 1997), our analysis of gene expression in mouse tissues revealed high expression also in spleen and iWAT (Figure 5H). Importantly, Rgs14 silencing in adipocytes reduced or eliminated the sensitizing effect of KYNA on the isoproterenol response (Figures 5I, 5J, S5H, and S5I). The synergistic effect of KYNA pre-treatment and lowdose isoproterenol could also be observed in mice treated with KYNA for 6 days, followed by a single dose of isoproterenol (Figure 5K). Gpr35-Deficient Mice Are Resistant to KYNA- and Exercise-Mediated Beiging of Subcutaneous Fat To investigate if Gpr35 is necessary for KYNA-mediated regulation of metabolism in vivo, we used mice with genetic Gpr35 deletion (Gpr35KO) (Ryder et al., 2013; White et al., 2013). When fed a chow diet, Gpr35KO mice start by showing impaired glucose tolerance around 15 weeks of age (Figure 6A), and eventually increased body weight (after 16 weeks of age; Figure 6B), when compared to littermate controls. Importantly, KYNA administration arrests body weight gain in wild-type mice, but not in Gpr35KO (Figure 6C). In the same way, we could observe a reduction in inguinal and visceral fat mass in wild-type mice treated with KYNA, but not in Gpr35KO mice (Figures 6D and S6A). Analysis of gene expression in the white adipose depots showed that deletion of Gpr35 abolishes the effect of KYNA on thermogenic and lipid metabolism genes, Rgs14, and anti-inflammatory genes (Figures 6E, 6F, and S6B–S6E). Interestingly, Gpr35KO mice show elevated TNFa and Mcp1 levels in the subcutaneous adipose tissue (Figure 6F). To verify if Gpr35 participates in the molecular adaptations in subcutaneous fat elicited by endurance exercise training, we analyzed Gpr35KO mice and littermate controls after 4 weeks of free wheel running. This intervention showed that, compared to control littermates, Gpr35KO mice are less responsive to the exercise-mediated effects on white adipose depots (Figures 6G and 6H).. (E) Oxygen consumption rate (OCR) from inguinal primary adipocytes pre-treated with 50 mM KYNA for 2 days. (F) OCR from inguinal primary adipocytes transfected with siRNA for Gpr35 (siGpr35) or a scrambled control (Scr) and pre-treated with 50 mM KYNA for 2 days. (G) Analysis of gene expression by quantitative real-time PCR in inguinal primary adipocytes transfected with siRNA for Gpr35 (siGpr35) or a scrambled control (Scr). Adipocyte cultures were pre-treated with PBS or 50 mM KYNA for 2 days followed by isoproterenol (1 mM) for 12 hr (n = 3). (H) Volcano plot of the microarray data shown in Figure 3D. In yellow, Rgs14 levels. Rgs14 expression levels in an array of mouse tissues determined by quantitative real-time PCR and normalized to its expression in the liver (n = 3–4). (I) Analysis of gene expression by quantitative real-time PCR in inguinal primary adipocytes transfected with siRNA for Rgs14 (si_Rgs14) or a scrambled control (Scr). Adipocytes were treated daily with 50 mM KYNA for 6 days followed by a single dose of isoproterenol (12 hr, 50 nM) 24 hr after the last KYNA treatment (n = 3). (J) OCR from inguinal primary adipocytes transfected with siRNA for Rgs14 (siRgs14) or a scrambled control (Scr). (K) Analysis of gene expression by quantitative real-time PCR in iWAT. Bars depict mean values and error bars indicate SEM. Unpaired Student’s t test was used when two groups were compared, and one-way ANOVA followed by Fisher’s least significance difference (LSD) test for post hoc comparisons was used to compare multiple groups, *p < 0.05. See also Figure S5.. 386 Cell Metabolism 27, 378–392, February 6, 2018.
(11) C. *. 30. 30. 10.0. 10.0 *. 7.5. 7.5 *. 5.0. *. 5.0. *. *. *. 2.5. Uc p1 Pg c1 a Pr 1 dm 16 Cy tc Ci de a Cp t1 b Nr f1 Ap 2 Ad ip oq Es rra Pp ar a Ac ox 1 Di o2. iWAT. 2.5. *. *. *. *. iWAT. Relative transcript levels. Gpr35 KO_Sed Gpr35 KO_FWR. Uc p Pg 1 c1 Pr a1 dm 16 Cy tc Ci de a Cp t1 b Nr f1 Ap Ad 2 ip oq Es rra Pp ar a Ac ox 1 Di o2. Ctrl_Sed Ctrl_FWR. 5.0. H. Rg s1 4. G. * 7.5. 0.0. 0.0. Rg s1 4. 2.5. 0.0. Relative transcript levels. 12.5. Il1 0. Gpr35 KO + KYNA. 0.0. 10.0. Il1 0. Gpr35 KO + PBS. 0.5. iWAT. F *. Il4. Ctrl + KYNA. 1.0. Ctrl + KYNA Gpr35 KO + KYNA. Il4. *. 1.5. Tn fa. iWAT 12.5. 25. Il3 3. 24. 7. 20. Age (weeks). E Relative transcript levels. 16. y. Time (minutes). 12. 0. 8. da. 120. y. 60. da. 45. Il1 3. 30. Il1 3. 15. Relative transcript levels. 0. G pr 35. 0. KO. 20. *. Tn fa. 10. 40. 35. 2.0. M cp 1. *. *. Relative Fat Mass. 20. KYNA Body weight (g). *. Body weight (g). Glucose (mM). *. iWAT. Gpr35 KO + KYNA. 50 *. D. Ctrl + KYNA. Gpr35 KO. M cp 1. Ctrl. Gpr35 KO. (normalized by body weight). B Ctrl. Il3 3. A. Figure 6. Gpr35-Deficient Mice Are Resistant to Exogenous KYNA- and Exercise-Mediated Beiging of Subcutaneous Fat (A) Glucose homeostasis was evaluated by i.p. glucose tolerance tests (n = 6). (B) Body weight (g) evolution (n = 6). (C) Body weight before treatment (BT) and after daily treatment with either KYNA or PBS (n = 6). (D) Boxplot of relative fat mass from the different fat depots, normalized by body weight (n = 6). (E and F) Analysis of gene expression by quantitative real-time PCR in iWAT after KYNA i.p. injections (n = 4). Graphs show relative expression data for genes involved in adipocyte browning and energy metabolism (E) and inflammation (F). (G and H) Analysis of gene expression by quantitative real-time PCR iWAT from sedentary (Sed) and exercise-trained mice (FWR) (n = 5). Graphs show relative expression data for genes involved in adipocyte browning and energy metabolism (G) and inflammation (H). Bars depict mean values and error bars indicate SEM. Unpaired Student’s t test was used when two groups were compared, and one-way ANOVA followed by Fisher’s least significance difference (LSD) test for post hoc comparisons was used to compare multiple groups, *p < 0.05. See also Figure S6.. KYNA Induces Loss of Adiposity and Weight in Mice Fed Chow or High-Fat Diets We next sought to investigate whether KYNA could be used to stop weight gain in animals fed an HFD. To this end, C57BL/6J. mice were fed an HFD for 8 weeks, until animals showed clear weight differences from chow controls and showed signs of impaired glucose tolerance. At that point, we started daily KYNA i.p. administration and monitored weight and glucose Cell Metabolism 27, 378–392, February 6, 2018 387.
(12) A. B. E. C. D. F. G. H. I. (legend on next page). 388 Cell Metabolism 27, 378–392, February 6, 2018.
(13) tolerance weekly. After 2 weeks of KYNA administration, we could observe an arrest in weight gain and a significant difference in weight when compared to animals on HFD injected with vehicle (Figure 7A). Accordingly, chronic KYNA administration improved glucose tolerance (Figures 7B and S7B) and resulted in reduced subcutaneous adipose tissue mass (Figure 7C) and a remarkable reduction in circulating TG levels, which became comparable to chow-fed controls (Figure 7D). KYNAtreated animals on HFD also showed lower fasting insulin levels (data not shown). Analysis of gene expression in the different adipose tissue depots revealed that KYNA administration prevents the HFD-induced reduction in the expression of key thermogenic genes such as Ucp1, Pgc-1a, and Cidea, among others (Figures 7E, S7C, and S7D). This was accompanied by KYNA-mediated regulation of genes involved in lipid metabolism (Figure S7F). At the same time, KYNA increased expression of anti-inflammatory genes and reduced the levels of Mcp1 and Tnfa, well-known pro-inflammatory markers (Figures 7E and S7C–S7E). Conversely, Gpr35KO mice on an HFD became more glucose intolerant and insulin resistant (Figure 7F), as shown by i.p. glucose tolerance test (GTT), insulin tolerance test (ITT), and plasma insulin levels (Figure 7G). Taken together, these results show that Gpr35 is necessary to mediate the actions on KYNA on adipose tissue energy homeostasis and inflammation. DISCUSSION Although KYNA has been reported to accumulate in peripheral tissues in the nM to mM range (Badawy and Bano, 2016; Forrest et al., 2002; Olenchock et al., 2016), its biological activities in those tissues are less well known. To mimic a post-exercise situation, we used a single daily dose of KYNA, which elevates its plasma levels to what we and others have previously reported in exercised mice and humans (Agudelo et al., 2014; Lewis et al., 2010; Schlittler et al., 2016). This resulted in changes in the adipose tissue that increase systemic energy expenditure. Importantly, these changes proved to be strictly dependent on Gpr35 as genetic deletion of this GPCR renders KO mice refractory to the metabolic effects of KYNA. These effects seem to be selective to the subcutaneous and visceral compartments, and to have only minimal effects on classical BAT. This selectivity has been observed before for other mediators of skeletal muscle to adipose tissue communication induced by exercise training such as Irisin (Boström et al., 2012), Meteorin-like (Rao et al., 2014), b-aminoisobutyric acid (Roberts et al., 2014), Il6 (Knudsen. et al., 2014), and lactate (Carrière et al., 2014). The immediate KYNA/Gpr35 actions are mediated by intracellular Ca2+ release, ERK1/2 phosphorylation, and Pgc-1a1 activation, a well-established pathway in the regulation of adipocyte energy expenditure (Lindquist et al., 2000; Puigserver et al., 1998; Wang et al., 2013; Zemel et al., 2000). Indeed, the adipocyte gene expression signature we observe downstream of KYNA signaling includes several known players in adipose tissue beiging. This suggests that the adipocyte-autonomous effects of KYNA are dependent on molecules such as Pgc-1a and PRDM16, which have been shown to have essential roles in adipose tissue beiging (Cohen et al., 2014; Kleiner et al., 2012). Interestingly, we observed that KYNA enhances the antagonist effect of propranolol on adipocyte b-ARs, but only in the presence of Gpr35. This is in agreement with the fact that Gpr35 has been shown to signal through Gaq/11/Ca2+ and Gai/0, which stimulates ERK1/2 but inhibits cAMP signaling (Mackenzie et al., 2011). However, chronic treatment of adipocytes with KYNA elevates Rgs14 levels, which alleviates the dampening effect of Gpr35 on b-ARs. Rgs14 is a structurally and functionally unique member of the RGS family of proteins as it contains RGS, Ras/Rap-binding, and GPR/GoLoco domains (Berman et al., 1996; Cho et al., 2000). The RGS and GPR domains mediate inhibitory interactions with activated and inactive Ga proteins, thus temporally limiting G protein signaling. This crosstalk might have important therapeutic implications as it offers an alternative way to regulate adipocyte b-AR activity and energy expenditure by combining Gpr35 and b-AR agonists at lower doses (thus reducing unwanted systemic effects). We could demonstrate this in vitro and in vivo by combining KYNA with a low dose of isoproterenol. Of the three adipose depots we analyzed, iWAT expresses the highest Rgs14 levels (47-fold versus liver), followed by eWAT and BAT. This correlates with the levels of Gpr35 expression and with the magnitude of KYNA effects we observe. Notably, a Drosophila melanogaster mutant with reduced expression of Loco (the fly homolog for Rgs14) shows increased fat content and reduced cAMP signaling (Lin et al., 2011). In addition, it has been previously shown that browning of the adipose tissue can be induced even in the absence of b-ARs (de Jong et al., 2017; Razzoli et al., 2015; Ye et al., 2013), which opens interesting windows of opportunity for the use of Gpr35 agonists as browning agents. KYNA has been shown to regulate iNKT cytokine release (Fallarini et al., 2010) and at high concentrations to reduce. Figure 7. KYNA Prevents HFD-Induced Weight Gain and Improves Glucose Homeostasis (A) Body weight before treatment (BT) and weekly from animals described in Figure S7A, treated daily with either KYNA or PBS (n = 6). (B) Glucose homeostasis was evaluated by weekly i.p. GTTs. Graph shows the results at week 4 (n = 6). (C) Boxplot of relative fat mass from the different fat depots, normalized by body weight (n = 6). (D) Plasma TG levels in mice fed an HFD treated during 7 days with 5 mg/kg KYNA or PBS (Veh) relative to chow-fed controls (Chow) injected with vehicle (n = 4–6). (E) Analysis of gene expression by quantitative real-time PCR in iWAT (n = 4). (F) i.p. GTTs (left panel). Graph shows the results after 5 weeks of HFD (n = 6). ITT (right panel) (n = 6). (G) Plasma insulin levels (n = 6). (H) GPCRs relevant for beige adipose tissue were selected based on a published study (Klepac et al., 2016). For analysis we used RNA sequencing data from the Genotype-Tissue Expression project (archived at http://www.genenetwork.org/) (Lonsdale et al., 2013). Data are presented as a heatmap. (I) Pearson’s r correlations for Gpr35 with PRDM16 and NRF1 in human WAT. Bars depict mean values and error bars indicate SEM. Unpaired Student’s t test was used when two groups were compared, and one-way ANOVA followed by Fisher’s least significance difference (LSD) test for post hoc comparisons was used to compare multiple groups, *p < 0.05. See also Figure S7.. Cell Metabolism 27, 378–392, February 6, 2018 389.
(14) LPS-induced TNFa release from cultured peripheral blood mononuclear cells (Wang et al., 2006). Our results show that elevating KYNA to ‘‘exercised’’ levels is sufficient to promote an anti-inflammatory phenotype in adipose tissue with increased expression of anti-inflammatory cytokines involved in type 2 immune responses. In addition, we observed a decrease in the expression of inflammatory markers such as TNFa. Altogether, our data show a KYNA-induced increase in markers associated with Treg and ILC2 populations, both of which have been shown to regulate adipose tissue inflammation and insulin resistance (Brestoff and Artis, 2015; Lackey and Olefsky, 2016). The contribution of adipose tissue-resident immune cells to the browning phenotype, and to the regulation of energy homeostasis, has become the subject of active research (Kohlgruber et al., 2016). In this context, it is interesting that iNKT cells have been shown to be present in significant numbers in the lean adipose tissue and to be reduced in the obese state, and thus suggested to contribute to maintaining a state of low inflammation and insulin sensitivity (van Eijkeren et al., 2018). Among the proposed mediators of these effects are Il4, Il10, and Il13, which we observed to be induced by KYNA and with known anti-inflammatory properties. However, the contribution of Il4 for WAT browning and thermogenesis has been recently questioned (Fischer et al., 2017). Of note, KYNA also induced Il33 and ILC2 cellular markers, which have anti-inflammatory properties and promote adipose tissue beiging even in the absence of Il4 signaling (Brestoff et al., 2015). Importantly, mice fed an HFD and treated daily with KYNA show reduced weight gain, improved glucose tolerance, and remarkably reduced circulating TG levels. This phenotype (also partially observed in chow-fed mice) was concomitant with an HFD-induced reduction in the expression of adipose tissue thermogenic genes, which was rescued by KYNA administration. Importantly, the effects of KYNA are lost in a Gpr35KO mouse model, which develops glucose intolerance and weight gain, and is more susceptible to the effects of HFD feeding. In addition, Gpr35KO mice show reduced browning of the subcutaneous adipose tissue induced by aerobic exercise. Interestingly, genome-wide association studies have previously linked Gpr35 with type 2 diabetes (Horikawa et al., 2000), although through unknown mechanisms. In human adipose tissue, GPR35 expression correlates with genes involved in transcriptional regulation of adipocyte browning, such as PRDM16 (Figures 7H and 7I). In sum, this work identifies a novel role for KYNA and Gpr35 in the regulation of energy metabolism that can potentially be explored therapeutically. Limitations of Study The identification of KYNA and Gpr35 as regulators of energy expenditure opens exciting opportunities to explore from both a physiological and therapeutic perspective. As the debate continues about the identity of the endogenous ligands for Gpr35, it will be important to study the interplay between KYNA and other Gpr35 activators. In addition, and as discussed above, Gpr35 is expressed in adipocytes and in other cells such as some immune cell populations. Further work delineating the contribution of the different cell types for systemic energy expenditure will be necessary before exploring the therapeutic potential of this mechanism. 390 Cell Metabolism 27, 378–392, February 6, 2018. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d. d. d d. KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animal Experiments B Inguinal primary adipocytes B HEK293 cells METHOD DETAILS B Metabolic Parameters B Exercise Training B Intraperitoneal glucose tolerance test (IPGTT) B Insulin tolerance test (ITT) B Triglyceride quantification B Determination of Insulin levels B High-fat Diet B Chemical Treatments B cAMP quantification B Extracellular Flux Analysis (Seahorse) Assays B Analysis of gene expression B Immunoblotting B Mass Cytometry B Mass Spectrometry B Mitochondrial mass B Mitochondria/Genomic DNA Ratio B Calcium measurements B Bioinformatic analysis QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and two tables and can be found with this article online at https://doi.org/10.1016/j.cmet.2018. 01.004.. ACKNOWLEDGMENTS We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line (Gpr35tm1b(EUCOMM)hmgu), and INFRAFRONTIER/EMMA (https://www. infrafrontier.eu/). Funding information may be found at http://www.sanger. ac.uk/science/collaboration/mouse-resource-portal and associated primary phenotypic information at http://www.mousephenotype.org/. This work was supported by grants from Karolinska Institutet (J.L.R., P.-O.B., and P.B.), the Swedish Research Council (J.L.R., P.-O.B., and P.B.), the Novo Nordisk Foundation (J.L.R. and P.-O.B.), the Strategic Research Programs in Diabetes (J.L.R. and P.-O.B.) and in Regenerative Medicine (J.L.R.) at Karolinska Institutet, the Swedish Diabetes Association (J.L.R. and P.-O.B.), the European Research Council (P.-O.B., ERC-2013-AdG 338936-BetaImage; P.B. and Z.G.-H., aCROBAT ERC-St 639382), the Swedish Society for Medical Research (P.B.), the Family Knut and Alice Wallenberg Foundation (P.-O.B.), Skandia Insurance Company (P.-O.B.), Diabetes and Wellness Foundation (P.-O.B.), the Bert von Kantzow Foundation (P.-O.B.), the Stichting af Jochnick Foundation (P.-O.B.), and the Family Erling-Persson Foundation (P.-O.B.). J.L.R. was recipient of a Marie Curie Career Integration Grant, D.M.S.F. was supported in part by a postdoctoral fellowship from the Wenner-Gren Foundations (Sweden, CIG-294232), and J.C.C. is recipient of a postdoctoral fellowship from the Swedish Society for Medical Research (SSMF)..
(15) AUTHOR CONTRIBUTIONS L.Z.A. and J.L.R. conceived, coordinated, and designed the study. J.L.R. supervised the study. L.Z.A. performed and analyzed animal experiments, tissue culture, in vitro experiments, and gene expression with contributions from D.M.S.F., I.C., P.R.J., A.T.P.-K., S.D., E.G.S., M.P.-P., J.C.C., M.I., and V.M.-R. L.Z.A. performed bioinformatics analysis. D.M.S.F. performed and analyzed calcium measurements. D.M.S.F. and L.Z.A. performed and analyzed seahorse respirometry experiments. P.R.J. performed and analyzed immunoblots from inguinal primary adipocytes. I.C. performed Pgc-1a stabilization experiments. T.L. and P.B. performed and analyzed mass cytometry experiments. P.M.U. and Ø.M. performed and analyzed determination of metabolites levels in plasma. G.B. and T.P. performed HFD and IGTT experiments. P.-O.B., Z.G.-H., and T.P. edited the manuscript. L.Z.A. and J.L.R. wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. DECLARATION OF INTERESTS J.L.R. is a member of the scientific advisory board and a paid consultant for Metabrain Research. Received: October 24, 2017 Revised: November 30, 2017 Accepted: January 10, 2018 Published: February 6, 2018 REFERENCES Agudelo, L.Z., Femenı́a, T., Orhan, F., Porsmyr-Palmertz, M., Goiny, M., Martinez-Redondo, V., Correia, J.C., Izadi, M., Bhat, M., Schuppe-Koistinen, I., et al. (2014). Skeletal muscle PGC-1a1 modulates kynurenine metabolism and mediates resilience to stress-induced depression. Cell 159, 33–45. Badawy, A.A., and Bano, S. (2016). Tryptophan metabolism in rat liver after administration of tryptophan, kynurenine metabolites, and kynureninase inhibitors. Int. J. Tryptophan Res. 9, 51–65. Bandura, D.R., Baranov, V.I., Ornatsky, O.I., Antonov, A., Kinach, R., Lou, X., Pavlov, S., Vorobiev, S., Dick, J.E., and Tanner, S.D. (2009). 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(17) STAR+METHODS KEY RESOURCES TABLE. REAGENT or RESOURCE. SOURCE. IDENTIFIER. Mouse monoclonal anti-HA. BioLegend. Cat # 90150. Polyclonal anti-ERK1/2. Cell Signaling Technology. Cat # 9102. Polyclonal anti-phospho-ERK1/2 (Thr202/Tyr204). Cell Signaling Technology. Cat # 4377. Phospho-CREB (Ser133) Rabbit mAb. Cell Signaling Technology. Cat # 9198. Monoclonal anti-beta-actin. Sigma Aldrich. Cat # A5441. Antibodies. Monoclonal anti-tubulin. Sigma Aldrich. Cat # T6199. Anti-PGC1a mouse mAb. Calbiochem. Cat # ST1202. Mass cytometry antibodies. This paper. See Table S1. Chemicals, Peptides, and Recombinant Proteins Kynurenic acid. Tocris Bioscience. Cat # 0223. Rosiglitazone. Sigma Aldrich. Cat # R2408. Isoproterenol. Sigma Aldrich. Cat # I5627. Fluo-2, AM, Calcium indicator. Thermo Fisher. Cat # F1242. Adrenalin. Sigma Aldrich. Cat # Y0000882. Propranolol. Sigma Aldrich. Cat # P3500000. Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Sigma Aldrich. Cat # C2920. Antimycin. Sigma Aldrich. Cat # A8674. Oligomycin. Sigma Aldrich. Cat # 75351. Lipofectamine RNAMax. Thermo Fisher. Cat # 12778030. Lipofectamine 2000. Thermo Fisher. Cat # 11668027. cAMP-Glo assay. Promega. Cat # V1501. Triglyceride Quantification. Abcam. Cat # ab65336. Ultra Sensitive Mouse Insulin ELISA kit. Crystal Chem. Cat # 90080. Isol-RNA Lysis Reagent. 5 PRIME. Cat # 2302700. Amplification grade DNase I. Thermo Fisher. Cat # 18068015. Critical Commercial Assays. cDNA Reverse Transcription Kit. Thermo Fisher. Cat # 4368814. SYBR Green PCR Master Mix. Thermo Fisher. Cat # 4309155. Deposited Data Raw and analyzed data. This paper. GEO: GSE108158. Adipocytes treated with rosiglitazone. Step et al., 2014. GEO: GSE56747. Visceral adipose regulatory T cells. Cipolletta et al., 2012. GEO: GSE37535. Innate lymphoid cells. Monticelli et al., 2011. GEO: GSE46468. Experimental Models: Cell Lines HEK293T. ATCC. CRL-3216. Inguinal primary adipocytes. Kajimura et al., 2009. N/A. Wellcome Trust Sanger Institute. Colony prefix: PMCR Allele: Gpr35tm1b(EUCOMM)hmgu. Experimental Models: Organisms/Strains Gpr35_KO mouse: Gpr35tm1b(EUCOMM)hmgu JAX C57BL/6J. Charles River. N/A. C57BL/6JRj. Janvier Labs. N/A. si_RNA targeting Gpr35 gene. Applied Biosystems. ID # s82125. si_RNA targeting Rgs14 gene. Applied Biosystems. ID # 184492. Primer sequences for qPCR gene-expression analysis. This paper. See Table S2. Oligonucleotides. (Continued on next page). Cell Metabolism 27, 378–392.e1–e5, February 6, 2018 e1.
(18) Continued REAGENT or RESOURCE. SOURCE. IDENTIFIER. Mouse Pgc-1a1-Flag plasmid. Ruas et al., 2012. N/A. N-term 3xHA-tagged human Gpr35 plasmid. Addgene. Cat # GPR035TN00. Prism Graphpad. https://www.graphpad.com/ scientific-software/prism/. N/A. ImageJ. https://imagej.nih.gov/ij. N/A. GeneNetworks. https://genenetwork.org/. N/A. Ingenuity pathway analysis. QIAGEN. N/A. DAVID. https://david.ncifcrf.gov/. N/A. Molecular signatures database. http://software.broadinstitute.org/ gsea/msigdb/index.jsp. N/A. Immunological genome project. https://www.immgen.org/. N/A. Recombinant DNA. Software and Algorithms. CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jorge L. Ruas ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Animal Experiments All experiments and protocols were approved by the regional animal ethics committee of Northern Stockholm. Mice were housed in plastic cages (3 - 5 per cage) at 24 ± 1 o C, 12/12 h controlled light conditions with ad-libitum access to water and food, unless indicated. All experiments were approved by the regional animal ethics Committee of Northern Stockholm, Sweden. Wild-type mice (C57BL/6J) were procured from Charles River (Germany) and Janvier Labs (France). The Gpr35 knockout (Gpr35tm1b(EUCOMM)hmgu) animals were procured from the Wellcome Trust Sanger Institute (Ryder et al., 2013; White et al., 2013). All animals were male and of 2-months of age or older (as indicated in figure legends), except for Figure S2 where results were verified in 2-month-old female mice. Inguinal primary adipocytes For primary adipocytes, SVF from inguinal fat depots of 6-12-week-old male mice were isolated and prepared as previously described (Kajimura et al., 2009). The differentiation cocktail was used during the first 2 days of culture, followed by rosiglitazone and Insulin for 6 to 8 days. HEK293 cells HEK293T cells (female gender, ENCODE) were grown at 37 C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and 1% antibiotics (Penicilin/Streptomycin). HEK293T cells were seeded at density of 105 cells/ml into 12-well plates and transfected as indicated 24 hours after seeding with Pgc-1a1-Flag and Gpr35-HA using Lipofectamine 2000 (Life Technologies) in a stoichiometry of 2:1. After 24 hours, cells were treated with 10 mM proteasome inhibitor MG-132 to stabilize Pgc-1a1 or 150 mM KYNA for 8 hours, lysed in 1x sample buffer and analyzed by western blotting. METHOD DETAILS Metabolic Parameters Mice (12 - 16 weeks of age) were acclimated during 24 h in single cages. After this period, kynurenic acid- or PBS-treated mice were monitored for 7 days using CLAMS (Comprehensive Laboratory Animal Monitoring System; Columbus Instruments, Columbus, OH) with access to food ad libitum except for a 12 h fasting period during the last night. The following parameters were monitored continuously: Food intake, drinking volume, O2 consumption (VO2), CO2 production (VCO2), heat and locomotion. Exercise Training 10-14 weeks Gpr35 knockout and control littermates were single-housed and divided into 2 groups: control and exercise. After an acclimation period of 5 days the exercise group was given access to a running wheel with a counter that monitored revolutions for 4 weeks. e2 Cell Metabolism 27, 378–392.e1–e5, February 6, 2018.
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