Metabolite signals from browning adipocytes increase expression of brown adipocyte-associated genes in primary adipocytes
Adipocyte browning was induced in primary adipocytes differentiated from the stromal vascular fraction of subcutaneous (inguinal) WAT of mice using two distinct canonical signaling mechanisms, an adenylate cyclase activator (forskolin), and Peroxisome Proliferator-Activated Receptor δ (PPARδ) agonist (GW0742)16. Cells were washed and fresh serum-free media was conditioned on the cells for 24hr. Conditioned media was transferred to naïve primary adipocytes (Fig. 1a) and induced expression of brown adipocyte-associated genes in naive primary adipocytes (Fig. 1b & 1c). Expression of brown-adipocyte associated genes, including Ucp1, peroxisome proliferator-activated receptor γ co-activator1α (Ppargc1a; Pgc1α), cell death-inducing DFFA-like effector a (Cidea), carnitine palmitoyltransferase 1b (Cpt1b), acyl-CoA dehydrogenase very long chain (Acadvl) and cytochrome C (Cycs) was further enhanced following media protein denaturation by boiling, implicating a non-protein small molecule mediator(s) (Fig. 1b & c). These data may also indicate the presence of a secreted protein inhibitor of browning. To define the physicochemical nature of the small molecule mediators, aqueous-soluble metabolites were extracted from media conditioned on activated beige adipocytes using solvent partition. The aqueous soluble metabolites were reconstituted in fresh media and transferred to naïve primary adipocytes (Fig 1d). Expression of brown-adipocyte associated genes was induced by aqueous-soluble metabolites released from browning adipocytes.
To identify candidate metabolites that may induce browning, we applied both Gas Chromatography – Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS) metabolic profiling to media conditioned on browning adipocytes. GW0742 and forskolin were not detected in conditioned media. We focused on identifying metabolites commonly enriched in both PPARδ agonism and forskolin-treated adipocyte media to determine common species. Multivariate statistical models of the metabolic profiling data identified common metabolite species enriched in the media by both cAMP and PPARδ-induced browning (Fig. 1e). The concentration of sugar species and the branched-chain amino acids (BCAAs) valine and isoleucine was decreased in the media of browning adipocytes (Fig 1f). Concomitantly the concentration of 5-oxoproline (5OP) and the BCAA catabolites α-hydroxyisocaproic acid (HIC), α-ketoisovaleric acid (AKV), α-hydroxyisovaleric acid (AHI), 3-methyl-2-oxovaleric acid (MOVA), β-hydroxyisobutyric acid (BHIBA) and β-hydroxyisovaleric acid (BHIVA) was increased in the media. Glycerol, a marker of lipolysis, was also increased.
Next, we examined whether physiological plasma concentrations of the BCAA metabolites and 5OP increased the expression of brown adipocyte-associated genes in primary adipocytes (Fig. 1g) 17-20. Physiological plasma concentrations of the metabolites are given in supplementary table 1. MOVA, 5OP, BHIBA and BHIVA significantly and robustly induced expression of brown adipocyte-associated genes including Ucp1, Cidea and Cpt1b. The media of primary mouse canonical brown adipocytes treated with either forskolin (1 μM) or the PPARδ agonist (100 nM) was also analyzed using our metabolomics approach (Fig. 1h). MOVA, 5OP, BHIBA and BHIVA were also enriched in the media of primary brown adipocytes following cAMP or PPARδ-mediated induction of brown adipocyte-associated gene expression (Fig. 1h & i).
Therefore, the metabolites MOVA, 5OP, BHIBA and BHIVA are released from primary white and brown adipocytes in response to thermogenic stimuli and stimulate the expression of brown-adipocyte associated genes in naïve adipocytes.
Metabolite signals are secreted from browning human adipocytes and induce a brown adipocyte-like functional phenotype
We determined whether the secretion of the candidate metabokines from browning adipocytes was conserved in human cells. Human primary adipocytes were treated with either forskolin or a PPARδ agonist to induce a brown-adipocyte like phenotype (Supplementary Fig. 1a – h). MOVA, 5OP, BHIBA and BHIVA were enriched in the media of forskolin (1 μM) and PPARδ agonist (100 nM GW0742) treated human adipocytes (Fig. 2a). Treatment of primary human adipocytes with physiological plasma concentrations (Supplementary Table 1) of MOVA (20 μM), 5OP (20 μM), BHIBA (20 μM) and BHIVA (10 μM) induced expression of a panel of brown adipocyte-associated genes including UCP1, CIDEA and PGC1α (Fig. 2b). Induction of UCP1 expression in primary human adipocytes treated with metabolites in the physiological micromolar range occurred in a dose-dependent manner (Supplementary Fig 2 a – d). To determine whether changes in the expression of brown adipocyte-associated genes were translated to the level of protein, the concentrations of UCP1 protein in metabokine-treated human primary adipocytes were determined by ELISA (Fig 2c). We further investigated whether the metabolites induced functional effects consistent with browning on energy expenditure in human primary adipocytes. Both the basal and succinate-stimulated (complex II) oxygen consumption rates of adipocytes treated with MOVA (20 μM), 5OP (20 μM), BHIBA (20 μM) and BHIVA (10 μM) were increased (Fig. 2d). Primary human adipocytes were treated with MOVA (20 μM), 5OP (20 μM), BHIBA (20 μM) and BHIVA (10 μM) and incubated in serum-free media containing U-13C-palmitate to monitor adipocyte fatty acid β-oxidation. The labeled palmitate is catabolized via β-oxidation, releasing labeled acetyl-CoA, which enters the TCA cycle (Supplementary Fig 2e). GC-MS analysis identified increased relative enrichment of downstream TCA cycle metabolites citrate, fumarate, and malate in MOVA, 5OP and BHIBA-treated adipocytes (Fig. 2e – g), confirming fatty acid β-oxidation is increased in these cells. Browning is accompanied by an increase in glucose and fatty acid uptake into adipocytes. The uptake of glucose and fatty acid into human primary adipocytes treated with the metabolites was measured with the fluorescent glucose analog 6-NDBG or the fluorescent fatty acid analog BODIPY-FA (Fig. 2h & i) (Supplementary Fig 2f – m). The metabolites increased adipocyte glucose and fatty acid uptake. We sought to further characterize the transcriptional programme induced in adipocytes by the candidate metabolite signals, and to confirm that the effects on brown adipocyte-associated gene expression are conserved in an independent in vitro model of human adipose tissue. A gene expression array of key adipocyte and brown adipocyte-associated genes was used to probe immortalized human white preadipocytes isolated from neck fat and differentiated to mature adipocytes in the presence of MOVA (20 μM) (Supplementary Table 2), 5OP (20 μM) (Supplementary Table 3), BHIBA (20 μM) (Supplementary Table 4) or BHIVA (10 μM) (Supplementary Table 5). Confocal imaging of immortalized human adipocytes treated with the candidate metabokines identified MOVA, 5OP and BHIBA significantly increased cellular UCP1 protein content (Fig. 2j & k). Functionally, leak respiration and electron transport chain uncoupling were increased in immortalized human adipocytes treated with the metabolites (Fig 2l-o).
These data indicate that MOVA, 5OP, BHIBA and, to a lesser extent, BHIVA induce gene and protein expression and a functional phenotype consistent with browning in two human adipocyte models.
Transcriptional analysis and 13C-isotope substrate tracing reveal mechanisms of metabokine biosynthesis and secretion by browning adipocytes
BCAAs were depleted in the media of browning adipocytes (Fig 1f). MOVA, BHIBA and BHIVA are generated through the degradation of the BCAAs isoleucine, valine and leucine, respectively. These pathways share multiple enzymes. 5OP is synthesized from glutamate. We examined mechanisms by which adipocyte browning may increase the concentrations of MOVA, BHIBA, BHIVA and 5OP. U-13C-labeled isoleucine, valine, leucine, and glutamate were used to monitor stable isotope enrichment through the biosynthetic pathways and into extracellular accumulation of the candidate metabokines produced by human primary adipocytes treated with forskolin. Concomitantly, we performed RNA-Seq on human primary adipocytes treated with forskolin. Induction of the browning response increased both the intracellular and extracellular (culture media) 13C-enrichment of MOVA (Supplementary Fig. 3a – d), BHIBA (Supplementary Fig. 3e – j), BHIVA (Supplementary Fig. 3k – n) and 5OP (Supplementary Fig. 3o – u). The expression of the BCAA catabolic enzymes, branched chain amino acid transaminase 2 (BCAT2), branched chain keto acid dehydrogenase E1 subunit beta (BCKDHB), acyl-CoA dehydrogenase short chain (ACADS), acyl-CoA dehydrogenase medium chain (ACADM), Enoyl-Coenzyme A, Hydratase/3-Hydroxyacyl Coenzyme A Dehydrogenase (EHHADH), hydroxyacyl-CoA dehydrogenase (HADHA) and Enoyl-CoA Hydratase, Short Chain 1 (ECHS1) was increased in forskolin-treated adipocytes (Supplementary Fig. 3a – n). The expression of the 5OP biosynthetic enzymes glutathione synthetase (GSS), γ-glutamyltransferase 7 (GGT7) and γ-glutamylcyclotransferase (GGCT) were also increased in browning adipocytes (Supplementary Fig. 3o – u).
These data identify that browning induces a transcriptional program upregulating expression of the metabokine biosynthetic enzymes and driving adipocyte synthesis and release of MOVA, 5OP, BHIBA and BHIVA.
Metabokine signals are exported from browning adipocytes via monocarboxylate transporters
Next, we investigated the mechanisms through which the browning adipocytes export the metabolite signals. MOVA, 5OP, BHIBA and BHIVA, structurally, share a common single carboxyl group. Our RNA-seq analysis identified that monocarboxylate transporter1 (MCT1) expression was increased in human primary adipocytes treated with forskolin (SLC16A1, Log Fold-change = 0.37, P < 0.05, n = 3). MCT1 functions to both export and import monocarboxylates through the plasma membrane. We used a pharmacological MCT inhibitor (α-cyano-4-hydroxycinnamate) to determine the involvement of MCTs in browning-mediated secretion of the metabokines. Inhibition of MCTs abrogated forskolin-induced secretion of the metabokine signals, decreasing MOVA, 5OP, BHIBA and BHIVA extracellular concentration whilst increasing their intracellular concentration (Figure 3a – d). Therefore MCTs are required for metabokine export from browning adipocytes.
MOVA, 5OP and BHIBA regulate metabolism in skeletal myocytes
In murine models of both adipose tissue browning and BAT activity, fatty acid oxidation in skeletal muscle is increased8-10, representing an adipose-muscle metabolic signaling axis. We hypothesized that the metabolites secreted from browning adipocytes may contribute to the functional link between browning adipose tissue and muscle. First, we reconstituted this adipose tissue-muscle functional relationship in vitro. As previously described, conditioned serum-free media was collected from primary mouse adipocytes treated with forskolin (1 μM). Conditioned media was transferred to mouse C2C12 myotubes and induced expression of transcriptional regulators of metabolism (Pparα, Pgc1α), fatty acid β-oxidation genes including Cpt1b, and Acadvl, and mitochondrial genes Cycs and respiratory chain complex 1 component NADH:Ubiquinone Oxidoreductase Core Subunit S1 (Ndufs1) (Fig. 4a). The metabolites MOVA and 5OP robustly induced expression of the metabolic gene panel in mouse myotubes (Fig. 4b).
The adipose tissue-muscle in vitro signaling model was translated to human primary cells. Conditioned media from browning human adipocytes induced expression of key fatty acid metabolism genes in human myocytes (Fig. 4c). The effect of MOVA and 5OP on metabolic, mitochondrial, and fatty acid oxidation gene expression was conserved in human primary skeletal myocytes and was dose responsive in the physiological low micromolar range (Fig. 4d) (Supplementary Fig 4a-d). BHIBA was also observed to increase expression of PPARα and CPT1b in human primary skeletal myocytes. To confirm that transcriptional changes in human myocytes are accompanied by a dose-dependent change in functional phenotype, the oxygen consumption rates of primary myocytes treated with MOVA (5 μM and 20 μM), 5OP (5 μM and 20 μM), BHIBA (5 μM and 20 μM) and BHIVA (2.5 μM and 10 μM) were measured. Basal respiration rates of the myocytes were increased by MOVA (Supplementary Fig. 4e), 5OP (Supplementary Fig. 4f) and BHIBA (Supplementary Fig. 4g), but not BHIVA (Supplementary Fig. 4h). MOVA and 5OP induced the greatest increase in oxidative gene expression in both mouse and human primary myocytes. These metabolites were selected for characterization in primary myocytes using a substrate-inhibitor high-resolution respirometry protocol. MOVA and 5OP increased respiratory capacity in permeabilized human myocytes supported by substrates for mitochondrial complex I (glutamate and malate), fatty acid β-oxidation (octanoylcarnitine) and mitochondrial complex II (succinate) respiration (Fig 4e & f). 5OP also increased maximal electron transport chain capacity (FCCP-uncoupled) in myocytes (Fig 4f). Murine models of adipose browning and thermogenesis activate fatty acid and glucose catabolism in skeletal muscle8,9,11. Therefore we investigated the effect of the metabokines on uptake of both glucose and fatty acid into human primary myocytes using the fluorescent glucose analog 6-NDBG and the fluorescent fatty acid analog BODIPY-FA, respectively (Fig. 4h & i) (Supplementary Fig. 4i – n).
These analyses identify that MOVA, 5OP, and to a lesser extent BHIBA, regulate both murine and human skeletal myocyte metabolism consistent with an adipose-muscle metabolic signaling axis.
Metabolite signals are enriched by cold conditioning and depleted by obesity in vivo
To determine if MOVA, 5OP, BHIBA and BHIVA function as brown and beige adipocyte metabokines in vivo we examined their concentrations in the BAT, subcutaneous inguinal WAT and blood plasma of physiological models of increased and decreased adipose thermogenic function. We examined mice housed at thermoneutrality, room temperature and under thermogenic conditions with cold exposure at 8˚C for a period of one week and one month. As expected, cold exposure robustly induced a thermogenic phenotype in mouse BAT and subcutaneous WAT gene expression, UCP1 protein expression and adipocyte morphology (Supplementary Fig. 5a – f). The concentrations of MOVA, 5OP, BHIBA and BHIVA were increased in the BAT and subcutaneous WAT of cold challenged mice (Fig. 5a & b). Consistent with the increase in the metabolite concentrations in the tissues, the expression of the MOVA, BHIBA and BHIVA biosynthetic genes (Bcat2, Bckdhb, Acads, Acadm, Ehhadh, Hadha and Echs1) and the 5OP biosynthetic genes (Gss, Ggct) were increased in the BAT (Fig. 5c) and subcutaneous WAT (Fig. 5d) of cold challenged mice. The expression of the monocarboxylate transporter, Mct1, was also induced by cold challenge in the BAT (Fig. 5c) and subcutaneous WAT (Fig. 5d) of mice.
In line with their potential as secreted brown and beige adipocyte paracrine and endocrine metabokines, plasma concentrations of the metabolites were also increased in mice housed in a cold environment (Fig. 5e).
Brown and beige adipose tissue are lost during the so called “whitening” effect associated with obesity, leading BAT to morphologically and metabolically resemble WAT (22). We determined if the metabolites MOVA, 5OP, BHIBA and BHIVA are decreased by diet-induced obesity. Diet-induced obese mice, fed a 60% fat diet for 17 weeks, had greater body weight and impaired glucose tolerance compared to matched chow-fed controls (Supplementary Fig. 5g & 5h). Obese mice exhibited markers of whitening within their intrascapular BAT, with decreased expression of thermogenic genes (Supplementary Fig. 5i) and a white-adipocyte like morphology (Supplementary Fig. 5j). In agreement with these observations, the BAT concentrations of MOVA, 5OP, BHIBA and BHIVA were decreased by diet-induced obesity (Supplementary Fig. 5k).
Therefore the metabokine signals are modulated in adipose depots and systemically in in vivo physiological models of altered thermogenic function.
The metabokines MOVA, 5OP and BHIBA increase systemic energy expenditure and regulate the adipose tissue and skeletal muscle metabolic phenotype in vivo
Browning of WAT and activation of BAT thermogenesis may regulate metabolism in WAT and skeletal muscle through secreted small molecule signals which alter systemic energy balance. MOVA, 5OP, BHIBA and BHIVA are secreted from browning adipocytes, increase in adipose tissue and plasma in vivo in response to canonical thermogenesis activated by cold, decrease in response to thermoneutrality and BAT “whitening”, and regulate browning and fatty acid metabolism in adipocytes and myocytes respectively. We investigated the effect of MOVA, 5OP, BHIBA and BHIVA on the in vivo metabolic phenotype of mice. Six-week-old mice fed standard chow were either treated with MOVA (100 mg/kg/day), 5OP (100 mg/kg/day), BHIBA (150 mg/kg/day) or BHIVA (125 mg/kg/day) in drinking water for 17 weeks (based on preliminary dose escalation studies) or remained untreated (control mice). Treatment increased the plasma concentrations of the metabolites in the mice within the low micromolar physiological range (Supplementary Fig 6a – d). Water intake was not different between groups (Supplementary Fig 6e). Weight gain of 5OP and MOVA treated mice was decreased compared with controls (Supplementary Fig 6f). Analysis with metabolic cages indicated BHIBA, MOVA and 5OP increased energy expenditure (Supplementary Fig 6g – j) and oxygen consumption (Supplementary Fig 6 k – n) independent of body mass. Metabolite treatment did not affect the activity of the mice (Supplementary Fig 6o). Food intake was increased in the 5OP and BHIBA treated groups which likely underpin the lack of difference in weight between BHIBA treated mice and control (Supplementary Fig 6p). BHIVA had no effect on the metabolic parameters independent of body mass.
MOVA, 5OP and BHIBA increased systemic energy expenditure in mice. We examined the expression of thermogenic and mitochondrial metabolism genes in BAT, subcutaneous inguinal WAT and skeletal muscle of the metabokine treated mice (Supplementary Fig 6q – t). Metabolite treatment also increased citrate synthase activity, a marker of mitochondrial density and TCA cycle flux, in the BAT, inguinal WAT and muscle of metabokine treated mice (Supplementary Fig 6u – x).
MOVA, 5OP and BHIBA decrease weight gain, increase systemic energy expenditure and regulate glucose homeostasis in a mouse model of obesity and diabetes
The candidate metabokines 5OP, MOVA and BHIBA increased energy expenditure and markers of oxidative metabolism in muscle and thermogenesis in adipose tissue in mice. Therefore, we investigated the effect of MOVA, 5OP, and BHIBA on the metabolic phenotype in a high fat feeding mouse model of obesity and T2DM. Six-week-old mice were treated with the metabokines (MOVA 100 mg/kg/day; 5OP 100 mg/kg/day and BHIBA 150 mg/kg/day) in drinking water for 17 weeks while fed a 60% fat diet. Plasma concentrations of the metabolites were significantly increased in treated mice (Supplementary Fig. 7a – c). MOVA, 5OP and BHIBA significantly reduced weight gain in fat fed mice (Fig. 6a – c). Adiposity of the MOVA and 5OP treated mice was observed to be reduced by 17.1% and 19.4%, respectively, using Computed Tomography (CT) (Fig. 6d). Consistent with the effect on adiposity and body weight, analysis with metabolic cages indicated that whole-body energy expenditure (Fig. 6e – g) and oxygen consumption (Supplementary Fig. 7d – f) were increased in the MOVA, 5OP and BHIBA treated high fat-fed mice, independent of body mass (as determined by ANCOVA). There was no significant difference in activity, food intake or water intake (Supplementary Fig. 7g – i).
Next, the mice were challenged with an insulin tolerance test (ITT) (Fig. 6 h – j) (Supplementary Fig. 7 j) and intraperitoneal glucose tolerance test (IPGTT) (Supplementary Fig. 7 k - n). 5OP and BHIBA significantly improved both the insulin sensitivity and the glucose tolerance in the mice. MOVA treatment demonstrated a mild but significant improvement in insulin sensitivity.
MOVA, 5OP and BHIBA increased systemic energy expenditure and improved glucose homeostasis in mice. The metabolites also altered the metabolic phenotypes of adipocytes and skeletal myocytes in vitro and in chow fed mice in vivo. Therefore, we examined markers of thermogenesis and mitochondrial metabolism in BAT and subcutaneous WAT of the MOVA, 5OP and BHIBA treated mice. The activity of citrate synthase was significantly increased in the BAT of mice following MOVA, 5OP and BHIBA treatment, suggesting increased mitochondrial biogenesis (Fig 6k). Consistent with these data, IHC analysis of the BAT of MOVA, 5OP and BHIBA treated mice indicated increased concentrations of UCP1 (Supplementary Fig. 7o), which were confirmed by ELISA (Fig. 6l). PGC1α protein concentration was also increased in the BAT of BHIBA treated mice (Fig 6l). Citrate synthase activity was increased in subcutaneous WAT of mice following MOVA, 5OP and BHIBA treatment (Fig 6m). IHC analysis of inguinal subcutaneous WAT from these mice also indicated increased UCP1 concentrations following 5OP and BHIBA treatment (Supplementary Fig. 7o), which were again confirmed by ELISA (Fig. 6n). 5OP and BHIBA also increased the concentration of PGC1α protein in inguinal WAT (Fig. 6n), with 5OP, BHIBA and MOVA all increasing CPT1 concentrations (Fig. 6n). MOVA and 5OP decreased adipocyte hypertrophy, significantly reducing adipocyte size within the inguinal WAT depot, consistent with effects of the metabolites on weight gain (Supplementary Figure. 7p).
MOVA, 5OP, and BHIBA, increased expression of mitochondrial and metabolic genes in skeletal myocytes in vitro and in vivo. Consequently we investigated markers of mitochondrial metabolism in the soleus muscle of the MOVA, 5OP and BHIBA treated murine model of obesity. Mitochondrial density was increased in skeletal muscle by all three metabolite signals (Fig. 6o). Protein concentrations of PGC1α and NDUFS1 were significantly increased in the muscle of metabolite-treated mice (Fig. 6p).
PET/CT imaging identifies increased glucose uptake into skeletal muscle and brown adipose tissue of metabolite treated mice
Positron Emission Tomography / Computed Tomography (PET/CT) was used to determine the tissue-specific metabolic effects of MOVA, 5OP and BHIBA treatment in vivo in the mouse model of obesity and T2DM. PET/CT is commonly used to determine BAT location and activity21. Mice either treated with MOVA (100 mg/kg/day), 5OP (100 mg/kg/day) or BHIBA (150 mg/kg/day) were imaged using PET/CT and the glucose analogue 18F-FDG (Fig. 6q). The metabolic activity of BAT was significantly increased in BHIBA and MOVA treated mice (Fig. 6r). Hind limb skeletal muscle metabolic activity was increased in BHIBA, MOVA and 5OP treated mice (Fig. 6s), with forelimb muscle metabolic activity significantly increased in MOVA and 5OP treated mice (Fig. 6t). Together, these data show the metabokines increase energy expenditure, reduce weight gain, improve glucose homeostasis and increase glucose and fatty acid catabolism in BAT, WAT and skeletal muscle.
MOVA and 5OP have additive effects on body weight and glucose metabolism in mice
Physiologically the candidate metabokines are generated in browning adipocytes and concomitantly increased in the plasma by stimulation of thermogenesis in mice. MOVA and 5OP produced the most robust and significant reduction in weight gain and adiposity in high fat fed mice and induced the greatest changes in metabolic phenotype in human adipocytes and myocytes. Therefore, we examined whether these metabolites would have combinatorial anti-obesity and anti-diabetic effects on systemic metabolism. Six-week-old mice were treated with a combination of MOVA (100 mg/kg/day) and 5OP (100 mg/kg/day) in drinking water for 17 weeks and fed a 60% fat diet. The combination of metabolites additively reduced weight gain when compared to either 5OP or MOVA treatments alone (Supplementary Fig. 8a). CT analysis identified that a combination of MOVA and 5OP reduced body fat by 24.6 % in treated mice compared with controls (Supplementary Fig. 8b). Glucose tolerance was further improved by a combination of 5OP and MOVA treatment (Supplementary Fig. 8c). PET/CT analysis using 18F-FDG indicated that mice treated with both MOVA and 5OP had enhanced glucose uptake into the hind limb skeletal muscle when compared to the singly administered treatments (Supplementary Fig. 8d & e). These data suggest MOVA and 5OP function through disparate mechanisms and that the small molecule adipokine-like signals function in concert to mediate systemic metabolism and anti-obesity effects on release from brown/beige adipose tissue.
MOVA and 5OP signal through cAMP-PKA-p38 MAPK and BHIBA via mTOR to regulate adipocyte and myocyte metabolic gene expression
We then determined whether the metabokines function extracellularly or intracellularly at the human adipocyte to induce expression of UCP1. MCTs can function to both import and export monocarboxylate species22. Concomitant treatment of primary adipocytes with an MCT inhibitor (MCTi; α-cyano-4-hydroxycinnamate) and 5OP resulted in an abrogation of 5OP-induced UCP1 expression, suggesting that 5OP requires import into the adipocyte to induce molecular signals leading to increased UCP1 expression (Fig 7a). Conversely, inhibition of MCT activity did not impair MOVA or BHIBA-mediated UCP1 expression, with dual metabokine and MCT inhibitor treatment trending towards increased UCP1 expression compared to metabokine treatment alone (Fig 7b & c). These data suggest MOVA and BHIBA function through extracellular signal transduction and may require a receptor in the adipocyte membrane.
Canonical activation of adipocyte thermogenesis through β3-adrenergic signaling requires intracellular signal transduction by cyclic AMP (cAMP) and downstream activation of Protein Kinase A (PKA)1. Using LC-MS we measured the intracellular cAMP content in human adipocytes treated with 5OP, MOVA and BHIBA (Fig 7d). The concentration of cAMP was unchanged in BHIBA treated adipocytes but increased in 5OP and MOVA-treated cells. We then analyzed the cAMP content of BAT (Supplementary Fig 9a), subcutaneous WAT (Supplementary Fig 9b) and soleus skeletal muscle (Supplementary Fig 9c) of 5OP, MOVA and BHIBA-treated mice. The cAMP content was increased in BAT, subcutaneous WAT and skeletal muscle of 5OP and MOVA-treated mice. We then co-treated primary adipocytes with either MOVA or 5OP and the selective PKA inhibitor H89. Inhibition of PKA impaired MOVA and 5OP-induced expression of brown adipocyte-associated genes in the adipocytes (Fig 7e & f) (Two-way ANOVA, P < 0.0001 Control vs MOVA, P < 0.0001 MOVA vs MOVA + PKAi; P < 0.0001 Control vs 5OP, P = 0.007 5OP vs 5OP + PKAi).
The downstream signaling pathways induced by MOVA, 5OP and BHIBA in skeletal muscle and adipose tissue were investigated using phosphokinase profiling of BAT, subcutaneous WAT and soleus muscle from metabolite treated mice. Treatment with MOVA increased phosphorylation of members of the p38 mitogen-activated protein kinase family (p38 MAPK) in BAT (Supplementary Fig. 9d), subcutaneous WAT (Supplementary Fig. 9e) and soleus (Supplementary Fig. 9f). In soleus, phosphorylation of the p38 MAPK substrate glycogen synthase kinase 3 beta (GSK-3β) was also increased by MOVA. In BAT, MOVA treatment increased phosphorylation of mitogen-activated protein kinase 3 (MKK3) located upstream of the p38 MAPKs in cellular signaling cascades. 5OP was also observed to increase phosphorylation of p38 MAPKs in BAT (Supplementary Fig. 9g), subcutaneous WAT (Supplementary Fig. 9h) and soleus (Supplementary Fig. 9i). These findings are consistent with the requirement for p38MAPK in cAMP-mediated expression of UCP123. BHIBA increased phosphorylation of both the mammalian target of rapamycin (mTOR) and mTOR’s downstream substrate p70S6 kinase (p70S6K) in BAT, subcutaneous WAT and soleus (Supplementary Fig. 9j – l) and increased the phospho mTOR / total mTOR ratio (Supplementary Fig 9m & n). Inhibition of p38 MAPK signaling by a pan p38 MAPK inhibitor (500 nM BIRB 796) abrogated MOVA (Fig. 7g) and 5OP (Fig. 7h) induced metabolic gene expression (PPARα, CPT1b, ACADvl) in human primary skeletal myocytes (Two-way ANOVA, Control vs MOVA P < 0.01, MOVA vs MOVA + p38 MAPKi P < 0.0001; Control vs 5OP P < 0.0001, 5OP vs 5OP + p38 MAPKi P < 0.0001). Inhibition of mTOR (temsirolimus 500 nM) in skeletal myocytes impaired BHIBA-induced metabolic gene expression (Fig. 7i) (Two-way ANOVA, Control vs BHIBA P < 0.01, MOVA vs MOVA + mTORi P < 0.01). In human primary adipocytes, the inhibition of p38 MAPK signaling reduced MOVA and 5OP-induced brown adipocyte associated gene expression (Fig. 7j & 7k) (Two-way ANOVA, Control vs MOVA P < 0.0001, MOVA vs MOVA + p38MAPKi P < 0.01, Control vs 5OP P < 0.0001, 5OP vs 5OP + p38MAPKi P < 0.0001). Inhibition of mTOR signaling with temsirolimus reduced BHIBA-induced expression of brown adipocyte-associated genes in human primary adipocytes (Fig. 7l) (Two-way ANOVA, Control vs BHIBA P < 0.0001, BHIBA vs BHIBA + mTORi P < 0.0001).
These data suggest that MOVA and 5OP function differentially via extracellular and intracellular mechanisms, respectively, to induce metabolic reprogramming in skeletal myocytes and adipocytes via a cAMP-PKA-p38 MAPK-mediated signaling pathway. BHIBA induces the thermogenic genes in adipocytes and mitochondrial gene expression in myocytes through extracellular activation of downstream mTOR signaling.
Concentrations of the adipokine-like metabolites in adipose tissue and plasma are inversely correlated with body mass index in humans
We investigated the association between genetic variants in the genes encoding the metabokine biosynthetic enzymes and body mass index (BMI) in a large-scale Genome Wide Association Study (GWAS) database in Genetic Investigation of ANthropometric Traits (GIANT) and UK Biobank Meta-analysis 24, included in the 795,640 subjects in the Type 2 Diabetes Knowledge Portal (http://www.type2diabetesgenetics.org/). We found that common noncoding variants in the MOVA, BHIBA and BHIVA biosynthetic genes (BCAT2, BCKDHB, ACADS and HADHA), the 5OP biosynthetic genes (GSS GGCT1) and the gene for MCT1 were significantly associated with BMI (Supplementary Table 6). The most significant variants in each gene for BMI were: BCKDHB rs13220420, P = 0.00000750; BCAT2 rs73587808, P = 0.000488; ACADS rs12369156, P = 0.000131; HADHA rs559393527, P = 0.0000341; GSS rs2236270, P = 3.60e-8; GGCT rs549124813, P = 0.0000875 and MCT1/SLC16A1 rs186286251, P = 0.000471).
We then examined the association of subcutaneous WAT MOVA, 5OP, BHIVA and BHIBA concentrations with BMI in human volunteers (Supplementary Table 7). The WAT concentration of MOVA, 5OP and BHIVA were significantly inversely correlated with BMI (Fig. 8 a – d). Plasma concentrations of the metabolite adipokine-like signals were also inversely correlated with BMI (Supplementary Fig. 10 a – d).
The association of metabokine concentrations with beige adipose tissue in humans was also interrogated. RNA was isolated from the adipose tissue of 30 volunteers and the expression of UCP1 and CPT1b measured using RT-qPCR. Associations between the adipose tissue metabolite concentrations and the expression of UCP1 (Fig 8 e – h) and CPT1b (Fig. 8 i – l) were analyzed. Concentrations of MOVA, 5OP and BHIVA were significantly correlated with tissue expression of UCP1 and CPT1b.
These data suggest that the metabokines are functionally associated with human physiology and may influence body mass phenotypes.