Organic cation transporter 3 (Oct3) is a distinct catecholamines clearance route in adipocytes mediating the beiging of white adipose tissue

Beiging of white adipose tissue (WAT) is a particularly appealing target for therapeutics in the treatment of metabolic diseases through norepinephrine (NE)-mediated signaling pathways. Although previous studies report NE clearance mechanisms via SLC6A2 on sympathetic neurons or proinflammatory macrophages in adipose tissues (ATs), the low catecholamine clearance capacity of SLC6A2 may limit the cleaning efficiency. Here, we report that mouse organic cation transporter 3 (Oct3; Slc22a3) is highly expressed in WAT and displays the greatest uptake rate of NE as a selective non-neural route of NE clearance in white adipocytes, which differs from other known routes such as adjacent neurons or macrophages. We further show that adipocytes express high levels of NE degradation enzymes Maoa, Maob, and Comt, providing the molecular basis on NE clearance by adipocytes together with its reuptake transporter Oct3. Under NE administration, ablation of Oct3 induces higher body temperature, thermogenesis, and lipolysis compared with littermate controls. After prolonged cold challenge, inguinal WAT (ingWAT) in adipose-specific Oct3-deficient mice shows much stronger browning characteristics and significantly elevated expression of thermogenic and mitochondrial biogenesis genes than in littermate controls, and this response involves enhanced β-adrenergic receptor (β-AR)/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP)-responsive element binding protein (Creb) pathway activation. Glycolytic genes are reprogrammed to significantly higher levels to compensate for the loss of ATP production in adipose-specific Oct3 knockout (KO) mice, indicating the fundamental role of glucose metabolism during beiging. Inhibition of β-AR largely abolishes the higher lipolytic and thermogenic activities in Oct3-deficient ingWAT, indicating the NE overload in the vicinity of adipocytes in Oct3 KO adipocytes. Of note, reduced functional alleles in human OCT3 are also identified to be associated with increased basal metabolic rate (BMR). Collectively, our results demonstrate that Oct3 governs β-AR activity as a NE recycling transporter in white adipocytes, offering potential therapeutic applications for metabolic disorders.


Introduction
Obesity, a disease characterized by excess body fat, is a major risk factor for many human diseases, including type 2 diabetes, cardiovascular disease, and hepatic steatosis [1]. In mammals, fat is stored primarily in adipose tissue (AT), and three distinct types of ATs have been characterized: white AT (WAT), brown AT (BAT), and beige AT [2]. The morphology, function, cellular origins, and molecular features of the three types of ATs are quite distinct [3]. WAT stores nutrients as triglycerides in unilocular adipocytes, which can be used to generate free fatty acids (FFAs) by lipolysis [4]. BAT is the main tissue responsible for thermogenesis, especially when stimulated by cold [5]. After prolonged thermogenic induction, brown-like adipocytes can also be found in WAT, thus named beige or brite adipocytes [3]. The heat produced by BAT or beige AT is indispensable for survival during cold acclimatization. Also, the adaptive thermogenic beige AT may have the potential to counteract obesity and its related disorders [6,7].
Beiging of WAT can be induced by various stimuli, including cold challenge, bile acids [8,9], pharmacological agents, and hormones such as norepinephrine (NE) [3,10]. NE activates βadrenergic receptor (β-AR), which is coupled to G-proteins, resulting in increases in cyclic adenosine monophosphate (cAMP) concentration in adipocytes that enhance the activity of cAMP-dependent protein kinase A (PKA) [11]. This signal not only leads to lipolysis by phosphorylating hormone sensitive lipase (Hsl) but also by phosphorylating cAMP-responsive element binding protein (CREB), subsequently increasing the expression levels of thermogenic genes (UCP1, PGC1α, and DIO2) to promote nonshivering thermogenesis [11,12]. The tightly regulated dichotomy of NE release and clearance maintains the balance between NE-induced thermogenesis and excessive NE in the vicinity of adipocytes. Thus, it is critical to elucidate the mechanism of NE clearance in AT. Although previous studies reported NE clearance mechanisms via SLC6A2 on sympathetic neurons [13] or proinflammatory macrophages in AT [14,15], the low catecholamine clearance capacity of SLC6A2 may limit its cleaning efficiency. Catecholamines, including NE, epinephrine, serotonin, and dopamine, act as neuromodulators in the central nervous system and as hormones in ATs and blood circulation [16]. Catecholamines are actively cleared in extracellular environment and are translocated into cells by specialized transporters that belong to two distinct transport mechanisms: neuronal transport (Uptake 1 , which is mediated by SLC6A2, SLC6A3, and SLC6A4) and extraneuronal transport (Uptake 2 , which is mediated by organic cation transporters [OCTs]) [17]. Studies have suggested a role of AT in the clearance and metabolism of catecholamines. Indeed, it has been shown that plasma concentrations of epinephrine and NE decrease after passing through AT [18,19]. In addition, human adipocytes exhibit high expression levels and enzymatic activity of the catecholamine-degrading enzyme monoamine oxidase (MAO) [20]. Transport of catecholamines by adipocytes displays similar characteristics as the Uptake 2 system and can be inhibited by the OCT3 inhibitor disprocynium 24 [20]. Ayala-Lopez et al. identified that perivascular ATs in male Sprague-Dawley rats had a NE uptake mechanism and could be reduced by norepinephrine transporter (NET) or/and OCT3 inhibitors ex vivo [21]. We reason that extraneuronal monoamine transporter OCT3 is an important route in NE clearance and adaptive thermogenesis in adipocytes complementary to sympathetic neurons or macrophages. In addition, the SLC22A3 (OCT3)-LPAL2-LPA gene cluster has been identified as a risk locus for coronary artery disease, implying its potential relationship to lipolysis [22].
In this study, we identified Oct3 as a novel non-neural NE clearance route in adipocytes and demonstrated its role in governing β-AR activity to mediate the beiging of WAT. Adipose-specific Oct3 knockout (KO) mice under cold acclimatization or NE administration exhibited significantly higher expression levels of genes related to thermogenesis, mitochondria biogenesis, and glycolysis in inguinal WAT (ingWAT) compared with Oct3 fl/fl (Ctrl) mice. Of note, through mining of the Genome-Wide Association Studies (GWAS) Catalog, the database of Genotypes and Phenotypes (dbGAP), and UK BioBank databases, we also identified genetic variants of human OCT3 associated with higher basal metabolic rate (BMR). These findings suggest that Oct3 regulates catecholamines levels in the vicinity of the β-AR in adipocytes and therefore plays essential roles in the browning of WAT during adaptive thermogenesis. Inhibition of OCT3 may provide distinct therapeutic application by activating energy expenditure pathways.

Oct3 was predominantly expressed in AT and had a preferred uptake of NE
Oct3 mRNA levels were examined in several C57BL/6J mouse tissues by real-time PCR. The highest expression levels were found in ingWAT, followed by gonadal WAT (gonWAT) ( Fig  1A). In human tissues, OCT3 had the highest transcript levels in skeletal muscle and liver, followed by ATs (S1A Fig). Compared with transcript levels of other catecholamine transporters Slc6a2, Slc6a3, and Slc6a4 in murine WAT, mRNA levels of Oct3 were much higher (Fig 1B), suggesting that Oct3 is the predominant transporter for catecholamines in WAT. We further showed that adipocytes expressed high levels of NE degradation enzymes Maoa, Maob, and Comt, which was as high as the adipocytes abundant gene Adiponectin in ingWAT (Fig 1C), providing the molecular basis on NE clearance by adipocytes itself together with its transporter adipocytes, as its expression was limited to mature adipocytes and was not found in SVC (S1B Fig). Similar expression patterns of Oct3 in mature adipocytes and SVC were also observed in gonWAT (S1C and S1D Fig). Immunofluorescence of WAT localized Oct3 to cell membranes of adipocytes ( Fig 1E).
To compare the Oct3 uptake activity against its main substrates (NE, serotonin, 1-methyl-4-phenylpyridinium (MPP + ), epinephrine, dopamine, and tyramine), substrate uptake assays were performed in HEK-293 Flp-In cells stably expressing Oct3. NE had the highest uptake activity when cells were incubated with equivalent concentrations of catecholamines ( Fig 1F). Human OCT3 and mouse Oct3-overexpressing HEK-293 cells both showed strong NE transport activities compared with empty vector (EV) in vitro, with K m and V max of 0.182 ± 0.0275 mM, 3.57 ± 0.174 nmol/mg protein/min (human) and 0.336 ± 0.0726 mM, 5.77 ± 0.448 nmol/ mg protein/min (mouse), respectively ( Fig 1G). Both K m and V max of OCT3 were more than 600 times greater than previously reported values of the Uptake 1 transporter, SLC6A2 (0.28 ± 0.03 μM; 5.83 ± 0.49 pmol/mg protein/min) [23], consistent with OCT3 being a highcapacity, low-affinity Uptake 2 transporter of NE. These data suggested an important role of OCT3 in regulating NE concentrations in peripheral tissues including AT, especially when a high concentration of NE existed, such as under NE stimulation or cold challenge.
To further examine the interactions between catecholamines and Oct3, we built a threedimensional Oct3 homology model based on human glucose transporter 3 (GLUT3) template. Docking of structurally divergent catecholamines (NE, epinephrine, histamine, dopamine, serotonin, and tyramine) indicated that epinephrine (E = −45.7) and NE (E = −43.6) showed the best docking energy scores, resulting from favorable interactions formed between these two bioamines and polar residues in the primary substrate binding site of Oct3 including K215, Q242, E385, and E446 (S1E Fig).
To address the connection between Oct3, catecholamines, and adipocyte metabolism in vivo, we crossed Oct3 fl/fl mice with Adiponectin-Cre mice to conditionally delete Oct3 in adipocytes ( To determine NE uptake activity in different adipocytes, we performed [ 3 H]-NE uptake assays in primary white and brown adipocytes. When treated with NE at a physiologically relevant concentration (0.20 μM), NE accumulation was significantly reduced (80% reduction) in Oct3 KO white adipocytes compared with controls, but not in brown adipocytes, suggesting that Oct3 is required for NE uptake in white adipocytes ( Fig 1I). Moreover, in vivo NE uptake assay was performed after chemical sympathectomy, which eliminated neural NE uptake in ingWAT (S3A and S3B Fig). Oct3-null WAT showed significantly reduced total radioactivity, which reflected the accumulation of NE and its metabolites in adipocytes (73% and 65% reduction in ingWAT and gonWAT, respectively) ( Fig 1J). Both the in vitro and in vivo data supported the role of Oct3 as the NE transporter in WAT.

NE-induced thermogenesis was augmented in adipose-specific Oct3 KO mice
Given that fact that NE was a preferential Oct3 substrate, we sought to investigate whether genetic deletion of Oct3 would influence NE clearance and NE-induced thermogenesis in vivo. After subcutaneous injection of low-dose NE (0.3 mg/kg), adipose-specific Oct3 KO mice showed significantly higher body temperature (Fig 2A and 2D) and higher maximum body temperature (Fig 2B and 2C) than that of Ctrl mice, as well as prolonged retention of increased body temperature (Fig 2A).
Adipose-specific Oct3 KO and Ctrl mice that received NE injections both demonstrated marked elevations in both whole-body O 2 consumption (Fig 2E and 2F) and heat production  (Fig 2E  and 2F) and heat production (Fig 2G and 2H) in adipose-specific Oct3 KO mice was significantly higher than in the Ctrl littermates. Among the thermogenic markers tested, adipose-specific Oct3 KO mice treated with NE displayed a nearly 82-fold increase in Ucp1, a 5-fold increase in Ppargc1α (Pgc1α), and a 3-fold increase in Dio2, as well as an increasing trend in Hsl and Atgl mRNA expression in ingWAT ( Fig 2I). Ucp1, Pgc1α, and Dio2 were also increased in gonWAT of adipose-specific Oct3 KO mice ( Fig 2J). However, the relative expression of Ucp1 in gonWAT was far lower than that in ingWAT, suggesting that ingWAT might be more responsible for wholebody thermogenesis. No significant alterations in thermogenic genes were detected in BAT of both genotypes after NE injection (S4A Fig). Collectively, these results suggested that Oct3 deficiency improved thermogenic capacity in WAT in the presence of adrenergic stimuli that resulted in an increase in core body temperature, O 2 consumption, and whole-body energy expenditure.

Ablation of Oct3 enhanced catecholamine-stimulated lipolysis and βadrenergic signaling in WAT
Activation of β-AR signaling has been found to promote lipolysis via the PKA-mediated phosphorylation of Hsl. To investigate the role of Oct3 in catecholamine signaling in lipolysis, we tested in vivo lipolysis by measuring serum concentration of FFAs. There was no difference in basal lipolysis, while NE injection enhanced lipolysis more significantly in adipose-specific Oct3 KO mice than in Ctrl ( Fig 3A). Epinephrine had a similar effect on lipolysis in vivo (S4B Fig). Increased levels of Hsl phosphorylation at two PKA-responsive serine residues (pHsl-S563 and pHsl-S660) were observed in ingWAT ( Fig 3B) and gonWAT (Fig 3C) of adiposespecific Oct3 KO mice 4 hours after NE stimulation, which is necessary for Hsl translocation into lipid droplets and NE-induced lipolysis in rodents [24]. No noticeable difference was observed in Hsl phosphorylation 4 hours after injection in BAT between the two genotypes (S4C Fig). One possible explanation for these results was that deletion of Oct3 led to impaired NE uptake into adipocytes and extracellular NE accumulation. Therefore, excess NE would activate β 3 -AR, increase cAMP concentration, and phosphorylate PKA, which would promote lipolytic activity via phosphorylating Hsl at S563 and S660 [25].
Next, we compared basal and NE-stimulated glycerol release from WAT explants derived from adipose-specific Oct3 KO mice and Ctrl mice. The rates of glycerol release were similar under basal conditions in both genotypes, but were enhanced more in Oct3 KO AT when treated with NE ( Fig 3D). Ablation of Oct3 in primary adipocytes led to an elevated response to adrenergic stimulation, as demonstrated by an increased lipolytic response to NE ( Fig 3E) and intracellular signaling molecule activation (pHsl-S563 and pHsl-S660) ( Fig 3F).
To compare NE uptake capacity in Ctrl and Oct3 KO adipocytes, we assayed intracellular NE levels using immunofluorescence. Primary adipocytes were treated with catecholaminedegradation enzyme inhibitors decynium-22 in the presence or absence of NE and were then incubated with anti-NE antibodies. Staining patterns were similar between unstimulated adipocytes; however, Oct3 KO adipocytes had lower signals compared with Ctrl under NE stimulation, indicating that ablation of Oct3 in adipocytes impaired NE uptake ( Fig 3G).
We next investigated the role of Oct3 on lipolysis in vitro by using a gain-of-function approach. Preadipocytes were transfected to produce Oct3-overexpressing 3T3-L1 cells (Oct3-OE). Although Oct3 overexpression did not significantly affect adipocyte differentiation based on Oil Red O staining (S4D Fig) or measurement of basal lipolysis (Fig 3H), Oct3-OE cells displayed attenuated NE-stimulated lipolysis compared with EV cells (Fig 3H), likely a result of enhanced NE transport to adipocytes accompanied by reduction of extracellular NE. In addition, Oct3-OE and EV cells were treated with the adenylyl cyclase agonist forskolin (10 μM), and similar lipolytic capacities were observed (S4E Fig), indicating that cAMP signaling (blocked by forskolin) and downstream lipolytic capacity were not primary causes of the difference in NE-stimulated lipolysis in our model.
In light of the enhanced lipolysis in Oct3 KO AT after NE stimulation, we examined the effect of adipose-specific KO of Oct3 on the liver. Fatty liver was not observed (S4F Fig), and there was no significant difference in liver triglyceride levels between genotypes 4 hours after NE injection (S4G Fig). One possible reason was that single NE injection represented only a short-term effect on lipolysis and FFA flux in blood, and another was that the 4-hour duration time might be insufficient to generate significant differences in fat accumulation in the liver.

Oct3 deficiency induced white to brown fat transition and enhanced thermogenesis
To study the role of Oct3 in physiologically stimulated thermogenesis, adipose-specific Oct3 KO and Ctrl mice were exposed to a 4˚C ambient environment for 1 month. Both groups had similar rectal temperatures under RT ( Fig 4A). However, adipose-specific Oct3 KO mice showed significantly higher rectal temperatures (by 1.7˚C) than those of Ctrl littermates after prolonged cold acclimation (Fig 4A and 4B). As seen in S5A Fig, body weight of adipose-specific Oct3 KO mice had a slight but significant decrease compared with that of Ctrl mice after sustained cold exposure. We also observed a more reddish appearance of ingWAT in adiposespecific Oct3 KO mice compared with Ctrl ( Fig 4C). Notably, adipose-specific Oct3 KO ingWAT contained more clusters of uncoupling protein 1 (Ucp1)-positive multilocular adipocytes than Ctrl after cold exposure ( Fig 4D). Whole-body energy expenditure of mice after cold challenge was measured by Comprehensive Lab Animal Monitoring System (CLAMS). Adipose-specific Oct3 KO mice showed markedly higher O 2 consumption rates (OCRs) ( Fig  4E), CO 2 production rates (Fig 4F), and heat production ( Fig 4G) than Ctrl, indicating a higher BMR. In addition, higher NE levels were observed in Oct3 KO ingWAT (S5B Fig), probably due to accumulation of extracellular NE derived from sympathetic nerve endings during cold challenge. Meanwhile, NE concentration in serum was also slightly increased, probably due to a leakage of extracellular NE in WAT into blood circulation (S5C Fig). These results showed the role of Oct3 in altering NE distribution between adipocytes and blood circulation.
In order to examine global gene expression changes in ingWAT, RNA from ingWAT of both genotypes were subjected to RNA sequencing (RNA-seq) analysis. Ablation of Oct3 led to significantly differential expression of 2,521 genes in ingWAT (adjusted p < 0.05) (S5D Fig and S1 Table). Widespread up-regulation of BAT-selective and beige genes were identified in Oct3 KO ingWAT (Fig 4H), while WAT-selective genes showed a down-regulation tendency ( Fig  4I). Furthermore, expression of genes involved in fatty acid oxidation (FAO) also showed substantial up-regulation ( Fig 4J). Based on the RNA-seq data analysis, we conducted real-time PCR analysis on genes involved in thermogenesis and adipose physiology. Oct3 KO ingWAT from cold-exposed mice had significantly higher mRNA expression levels of thermogenic genes Ucp1, Dio2, Pgc1α, Cidea, Pparα, Pgc1β, Tmem26, and Slc27a2-as well as genes that showed an up-regulation trend, including Tbx1 ( Fig 4H)-whereas WAT-selective genes Gsn, Sncg, and Rarres2 were significantly decreased ( Fig 4I). Several key genes of FAO were significantly upregulated in Oct3 KO ingWAT, including Cpt1a, Hadha, Hadhb, Acdvl1, and Acsl5 ( Fig 4J). Moreover, a negative regulator of FAO, Twist1, was significantly reduced in Oct3 KO ingWAT, by 80% ( Fig 4J). No significant changes in morphology of BAT and gonWAT from both genotypes were observed (S5E Fig Given the close relationships between Oct3, NE, and thermogenesis, we next focused on the NE/β-AR/cAMP pathway. Western blot analysis showed that phospho-PKA (pPKA) substrate ( Fig 4K), phospho-Creb (pCreb), and downstream Ucp1 and Pgc1α levels ( Fig 4L) were significantly elevated in ingWAT from adipose-specific Oct3 KO mice compared with Ctrl. Protein levels of pCreb, Ucp1, and Pgc1α remained unchanged in BAT (S5H Fig Based on the data above, we hypothesized that the enhanced browning effect in adipose-specific Oct3 KO mice during cold challenge may be due to higher metabolic rate and elevated thermogenic gene expression.

Ablation of Oct3 promoted mitochondria biogenesis and a remodeling of energetic metabolism in ingWAT during cold challenge
We next characterized the effect of Oct3 deletion on mitochondria in ingWAT after cold exposure. Electron microscopy showed more accumulated mitochondria and enlarged mitochondrial area (Fig 5A), a larger number of mitochondria per cell area (Fig 5B), and increased mitochondrial DNA content ( Fig 5C) in Oct3 KO adipocytes from ingWAT compared with Ctrl. Larger surface area of mitochondria was also observed in Oct3 KO ingWAT stained with Mitotracker (Fig 5D and 5E). Consistently, the gene ontology (GO) analysis of RNA-seq data revealed that differential genes associated with mitochondrial component were enriched (Fig  5F and S1 Table). Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted specific changes in genes associated with oxidative phosphorylation, glycolysis and fatty acid metabolism, and citric acid cycle (Fig 5G and S1 Table).
In ingWAT of adipose-specific Oct3 KO mice, the increase in cold-induced thermogenesis was coupled with broad up-regulation of electron transport chain (ETC) genes ( Fig 5H). Realtime PCR analysis showed that expression of mitochondrial respiratory chain complexes genes (Cox7b, Uqcrb, Uqcrh, and Cytochrome c) were remarkably increased in Oct3 KO ingWAT compared with Ctrl under cold stimulation (Fig 5I). In line with these changes, western blot analysis showed that protein levels of cytochrome c oxidase subunit 7b (Cox7b), Uqcrb, Uqcrh, and Cytochrome c were significantly elevated in Oct3 KO ingWAT from mice under cold exposure (Fig 5J). We found no difference in the expression of ETC genes in BAT (S5K The profound differences of glycolytic genes in Oct3 KO ingWAT suggested that Oct3 deletion resulted in a metabolic rewiring during cold exposure (Fig 5K). Real-time PCR analysis of ingWAT showed up-regulation of glycolytic genes (Hk2, Pdk4, Pfkl, Eno1, Gapdh, Pfkm, and Pkm1) in adipose-specific Oct3 KO versus Ctrl mice (Fig 5L). The protein levels of a key enzyme of glycolysis, Hk2, and the amount of lactate (the product of glycolysis) were up-regulated in ingWAT from adipose-specific Oct3 KO mice (Fig 5M and 5N). These alterations The numerical data underlying this figure are included in S1 Data. BAT, brown adipose tissue; cAMP, cyclic adenosine monophosphate; cKO, conditional knockout; Creb, cAMP-responsive element binding protein; Ctrl, control; HE, hematoxylin-eosin; ingWAT, inguinal white adipose tissue; Oct3, organic cation transporter 3; pCreb, phosphorylated cAMP-responsive element binding protein; pPKA, phosphorylated protein kinase A; RT, room temperature; Ucp1, uncoupling protein 1; VO 2 , oxygen consumption; WAT, white adipose tissue. Collectively, our results suggested that the elevated browning effect in adipose-specific Oct3 KO mice may be owing to increased energy expenditure, mitochondrial biogenesis, and enhanced glycolysis in ingWAT.

Enhanced thermogenesis and lipolysis resulted from overactivating the NE/ β-AR/PKA signaling pathway in adipose-specific Oct3 KO mice
To further investigate the relationship between Oct3-depletion-induced NE-stimulated thermogenesis and β-AR signaling, we firstly cotreated mice with NE and propranolol, a pharmacological β-AR inhibitor. An elevated thermogenic response in adipose-specific Oct3 KO mice was observed, including a more significant increase in core body temperature (Fig 6A), O 2 consumption (Fig 6B), and heat production ( Fig 6C) following single NE treatment, as described before (Fig 2A-2H). However, propranolol pretreatment abolished the elevation of thermogenic response (Fig 6A-6C), indicating that the increased energy expenditure in adipose-specific Oct3 KO mice was mediated by the β-AR signaling pathway.
To estimate the functional relevance of Oct3 for thermogenic capacity of adipocytes, we performed plate-based respirometry on primary adipocytes from preadipocytes isolated from SVC of ingWAT, which maintained expression of beige-related genes. Although the OCR curves were nearly identical in primary adipocytes of both genotypes in the absence of NE (S6A Fig), Oct3 KO beige adipocytes displayed a higher OCR than that of Ctrl cells after NE stimulation ( Fig 6D). Additionally, pharmacological inhibition of β-AR by propranolol significantly blunted the NE-stimulated OCR increase to a similar extent in both groups (Fig 6D).
Primary adipocytes derived from ingWAT were treated with NE and triiodothyronine (T 3 ) to induce Ucp1 expression [26]. An approximate 1.9-fold increase in Ucp1 mRNA expression in Oct3 KO beige adipocytes was found even in the absence of NE compared with Ctrl, and another 3.7-fold increase occurred in response to NE treatment ( Fig 6E). Meanwhile, acute propranolol-induced inhibition of β-AR similarly blunted the NE-induced increase in Ucp1 transcripts in the beige both groups of adipocytes ( Fig 6E). As a control, no noticeable difference was observed in mRNA expression of the NE-insensitive gene Ap2 between groups (S6B Fig). Additionally, Ucp1 transcripts and Creb phosphorylation showed no response to treatment of other Oct3 substrates (epinephrine, dopamine, and serotonin) (S6C and S6D Fig).
As NE was known to activate β-AR and trigger PKA-Creb signaling, we examined whether the enhanced thermogenesis in Oct3 KO beige adipocytes was regulated by the PKA/Creb pathway. NE-stimulated Oct3 KO adipocytes showed robust phosphorylation of PKA substrates and Creb, without changes in total Creb (Fig 6F). Meanwhile, these effects were largely eliminated by blockade of β-AR signaling (Fig 6F), indicating that β-AR signaling was required for enhanced thermogenic activity under Oct3 deficiency.
We next aimed to determine the requirement of NE/β-AR pathway in lipolysis induced by Oct3 ablation. Propranolol at 10 μM partially but significantly inhibited the enhancing effect of NE on lipolysis and Hsl phosphorylation, while 100 μM propranolol showed complete inhibition in Oct3 KO adipocytes (Fig 6G and 6H), indicating that this effect relied on β-AR function.
Based on these collective results, we propose the following model ( Fig 6I). Oct3 locates to cell membranes of adipocytes and uptakes NE, which thereby decreases NE concentration in AT microenvironments. Oct3 deficiency reduces clearance of extracellular NE, subsequently increasing extracellular NE concentration and leading to enhanced lipolysis, glycolysis, thermogenesis, and WAT browning via β-AR/cAMP/PKA pathway activation. These data showed that thermogenesis and lipolysis under cold stimuli were still enhanced in adipose-specific Oct3 KO mice treated with HFD, and insulin sensitivity was also improved.

Human OCT3 polymorphisms were associated with BMR
To determine the relevance of the studies in mice to human, BMR with polymorphisms within the human OCT3 locus was explored through mining of GWAS Catalog, dbGAP, and UK Bio-Bank databases. Among the 11 single-nucleotide polymorphisms (SNPs) in the OCT3 locus, , and maximal heat production change (ΔHeat) (panel C) of Ctrl and cKO mice (n = 6 in body temperature measurement; n = 4 for oxygen consumption and heat production measurement). NE was injected into mice with propranolol ("NE + prop") or without propranolol (5 mg/kg, s.c.) pretreatment ("NE + vehicle"). (D) The OCR in Oct3 KO beige adipocytes and Ctrl. Cells were acutely stimulated with NE in the absence or presence of propranolol at the indicated time point (arrow). Ctrl or Oct3 KO adipocytes with NE (n = 7 for both); Ctrl or Oct3 KO adipocytes with NE and propranolol (n = 8 for both). (E) Ucp1 mRNA expression in differentiated primary inguinal adipocytes from Ctrl and cKO mice after stimulation with NE and T 3 in the presence or absence of propranolol (n = 3). (F) Western blotting of pPKA substrate, pCreb, and total Creb in primary inguinal adipocytes from Ctrl and cKO mice stimulated with NE in the presence or absence of propranolol. (G) In vitro glycerol release from differentiated SVF-derived adipocytes in the presence or absence of NE and propranolol (n = 3). (H) Western blotting of pHsl-S563, pHsl-S660, and total Hsl in differentiated SVF-derived adipocytes from Ctrl and cKO mice in the presence or absence of NE and propranolol. (I) Schematic diagram of Oct3-regulating thermogenesis and lipolysis in ingWAT. Data in A-E and G were analyzed by one-way ANOVA followed by Tukey's test. Data in I and K were analyzed by Student t test. The numerical data underlying this figure are included in S1 Data. β-AR, β-adrenergic receptor; AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; Creb, cAMP-responsive element binding protein; Ctrl, control; FFA, free fatty acid; G s , G s alpha subunit; Hsl, hormone-sensitive lipase; ingWAT, inguinal white adipose tissue; KO, knockout; NE, norepinephrine; OCR, O 2 consumption rate; Oct3, organic cation transporter 3; pCreb, phosphorylated cAMP-responsive element binding protein; pHsl, phosphorylated hormone-sensitive lipase; PKA, protein kinase A; pPKA, phosph-PKA; prop, propranolol; s.c., subcutaneous; SVF, stromal vascular fraction; T 3 , triiodothyronine; TF, transcription factor; TG, triglyceride; VO 2 , oxygen consumption. four independent SNPs were associated with BMR with p < 5 × 10 −8 (S8A Fig and S3 Table). From the GTEx Portal, we noted that 10 of the 11 SNPs are significantly associated with human OCT3 expression levels in ATs (subcutaneous and/or visceral omentum) (S8B and S8C  Fig). Each of the alleles, which was associated with lower expression levels of OCT3 in AT, was correlated with higher BMR in the UK Biobank participants.
To further confirm the relevance to humans of the studies in mice, we performed experiments on human AT and primary human AT-derived mesenchymal stem cells (ATMSCs) that were induced to differentiate into adipocytes. Both human AT and differentiated ATMSCs showed high expression levels of OCT3 and NE-degrading enzymes (S8D-S8F Fig). These results were consistent with studies on human adipocytes showing that NE uptake existed and was inhibited by OCT3 inhibitors [20]. Furthermore, inhibition of OCT3 led to an increased lipolytic response to NE in differentiated ATMSCs (S8G Fig), similar to the observations in mouse stromal vascular fraction (SVF)-derived adipocyte model (Fig 4E).
Although human functional genomic data and cell studies provided cues for metabolic roles of OCT3, further investigations are needed to define the subtle roles of human OCT3 in AT.

Discussion
Brown and beige AT are two key drivers for the dissipation of chemical energy to stimulate thermogenesis [27]. The current study demonstrates that genetic deletion of Oct3 enhances ingWAT beiging and up-regulates NE-induced thermogenesis, lipolysis, and mitochondria biogenesis by attenuating NE clearance. These findings support the non-neural role of Oct3 as a catecholamine scavenger for beige AT microenvironments in adaptive thermogenesis.
Recent studies have proposed a neural mechanism for NE uptake by Slc6a2 in special macrophages in AT [14,15]. However, a proinflammatory state seems necessary to enhance their NE uptake in macrophages [14]. In addition, considering the high-affinity, low-capacity nature of Slc6a2 (K m : 0.28 ± 0.03 μM; V max : 5.83 ± 0.49 pmol/mg protein/min) [23], a NE transport system with greater transport capacity may be needed to meet increased metabolic demand during long-term cold challenge. With high expression in adipocytes and high capacity for catecholamines, Oct3 (K m : 0.183 ± 0.0275 mM; V max : 3.57 ± 0.174 nmol/mg protein/min) may function in part to clear catecholamines in white AT microenvironments. However, the quantitative contribution and exact conditions of extraneuronal NE uptake through Oct3 versus neuronal NE clearance remains to be investigated.
Cold challenge induces stronger browning and NE/β-AR/PKA signaling in ingWAT of adipose-specific Oct3 KO mice. Consistent with sustained action of catecholamines, protein levels of pPKA substrate, pCreb, and downstream Ucp1 and Pgc1α are all up-regulated. Pgc1α is the crucial transcriptional co-activator that is highly induced after cold exposure [28]. In white adipocytes, ectopic expression of Pgc1α induces Ucp1 expression and essential enzymes in mitochondria ETC and also increases mitochondrial DNA content [28,29]. Mitochondria biogenesis and expression of ETC is significantly increased in ingWAT of adipose-specific Oct3 KO mice after cold challenge, which may result from elevated Pgc1α protein expression. Moreover, genes encoding fatty acid β-oxidation were exclusively up-regulated in ingWAT of Oct3deficient mice after cold exposure. Consequently, more FFAs may be utilized as the primary fuel source for thermogenesis through Ucp1. These chain reactions are likely driven by more NE available due to Oct3 deletion.
During browning of ingWAT in adipose-specific Oct3 KO mice, metabolic rewiring of glucose metabolism to glycolysis is another significant phenomenon observed. Upon cold exposure, key enzymes in glycolysis and pyruvate dehydrogenase kinase 4 (Pdk4, the key regulatory enzyme linking glycolysis to the citric acid cycle) are significantly up-regulated in ingWAT of adipose-specific Oct3 KO mice. Higher expression of Pdk4 restricts the conversion of glucose to acetyl-CoA and reprograms it towards a higher glycolytic metabolism [30]. Along with other key glycolytic enzymes, the enhanced glycolysis in adipose-specific Oct3 KO mice may compensate for the loss of mitochondrial ATP production due to heat-generating mitochondrial uncoupling [31]. Recent reports have demonstrated that expression of glycolytic genes and Pdk4 are induced by β-AR agonists in BAT [32]. The enhanced glycolytic activity in ingWAT of cold-exposed adipose-specific Oct3 KO mice most likely results from stronger adrenergic stimulation through blunted NE catabolism by Oct3 deficiency. To date, there is little information available regarding the role of glucose metabolism in WAT. RNA-seq data also showed that these glycolytic genes are more highly expressed in ingWAT in adipose-specific Oct3 KO mice after cold exposure (S1 Table). These findings revealed that Oct3-mediated glucose metabolism is important to WAT energy homeostasis.
The differential responsiveness of WAT and BAT in adipose-specific Oct3 KO mice may result from several factors. First, Oct3 has a much lower expression in BAT compared with WAT. Second, current studies suggest that BAT is more densely innervated compared with WAT [33], and neuronal Slc6a2 may be largely responsible for the clearance of catecholamines released in BAT after NE injection or cold exposure [34]. Although browning of WAT can lead to metabolic improvements, the marked increase in energy expenditure (Fig 4E-4G) may also come from other sources such as skeletal muscle, cardiac and respiratory work, and liver, stimulated by enhanced circulating NE level [30][31][32]. Under HFD treatment at RT or thermoneutrality, similar body weights between two genotypes are observed. It is probably due to drastically reduced density of sympathetic nerves in ingWAT under HFD [35]. On the other hand, the Uptake 1 system might be sufficient to compensate for the KO effect of Oct3. The overall proposed mechanism for Oct3 effects on WAT browning is summarized in Fig 6I. Exploration of the Gene Atlas database for genetic associations of human BMR with variants in OCT3 shows consistency with some of the results in the adipose-specific Oct3 KO mouse model. In particular, several reduced expression variants of OCT3 are positively correlated with BMR in human. Though a greater baseline BMR is not observed in the adipose-specific Oct3 KO mice, metabolic rate is greater after either NE administration or cold challenge. Our study suggests an important clinical implication as β 3 -AR plays a central role in regulating nonshivering thermogenesis, which enhances energy expenditure and has potential to treat obesity [36]. Inhibition of OCT3 might block NE transport into adipocytes and mimic β 3 -AR agonists, resulting in browning of WAT and enhanced lipolysis. In summary, our results demonstrate that Oct3 is essential in catecholamine clearance in adipocytes of WAT and blunts intracellular responses upon sustained catecholamine stimulation by regulating the activity of β-AR. Development of OCT3-specific inhibitors may shed light on improving metabolic disorders.

Animals
Adiponectin-Cre mice were obtained from the Jackson Laboratory (stock NO.010803). Oct3 fl/fl mice were generated in the Ying Xu lab (Soochow University), backcrossed with C57BL/6J mice for at least 8 generations, and subsequently intercrossed with Adiponectin-Cre mice. Mice were housed under a 12-hour light/dark cycle (light period 7:00-19:00) at constant temperature (22˚C RT). Food and water were available ad libitum. Male mice were 8 to 10 weeks of age when used for experiments.

Uptake assays
HEK-293 Flp-In cells (Invitrogen, Carlsbad, CA) stably overexpressing human or mouse OCT3 were generated, maintained, and used for uptake assays as previously described [37]. For comparison between human OCT3 and mouse Oct3 NE uptake kinetics (Fig 2E), uptake time was 2 minutes at 37˚C. K m and V max were calculated by fitting the data to Michaelis-Menten equations using GraphPad Prism software (La Jolla, CA). For [ 3 H]-NE uptake in primary adipocytes, the same procedures were performed as described above.

Cold challenge and indirect calorimetry
For cold challenge experiments, male mice were placed at 16˚C for 1 week, and then 3 weeks at 4˚C. Metabolic measurements were measured using CLAMS (Columbus Instruments, Columbus, OH). Mice were individually housed in cages. Food and water were provided ad libitum.

Mouse Oct3 three-dimensional-structure model and catecholamine docking
The amino acid sequence of the mouse Oct3 was retrieved from the UNIPROT database (Q9WTW5) [38]. Homology models of Oct3 was generated via four steps. First, the primary sequence of Oct3 was submitted to the RCSB Protein Data Bank [39] to search for suitable template structures using PSI-BLAST [40]. Human glucose transporter 3 (hGLUT3) having its crystal structure determined in complex with D-glucose at 1.5 Å resolution in an outwardoccluded conformation (PDB ID: 4ZW9) [41] is the closest structurally characterized protein to Oct3, sharing 24% sequence identity. Second, a sequence alignment between Oct3 and hGLUT3 was computed by Multiple Sequence Comparison by Log-Expectation (MUSCLE) [42]. Third, a total of 500 homology models were generated for Oct3 with the standard "automodel" class in MODELLER [43] on the basis of hGLUT3 template. Finally, these 500 models were evaluated by the discrete optimized protein energy (DOPE) score [44], and the best ranking model was selected.
Molecular docking screens against the selected Oct3 homology model was performed with a semi-automatic docking procedure. All docking calculations were performed with DOCK 3.6 [45]. The docking database contains seven compounds, dextroamphetamine, NE, epinephrine, dopamine, serotonin, histamine, and tyramine. The docked compounds were ranked by the docking energy that is the sum of van der Waals, Poisson-Boltzmann electrostatic, and ligand desolvation penalty terms.

Histology and immunofluorescence analysis
Fresh tissue samples were fixed in 4% paraformaldehyde, dehydrated in serial alcohol, embedded in paraffin, cut into 5-μm-thick sections, and stained with hematoxylin-eosin (HE). Ucp1 immunohistochemical staining was performed as previously described [46]. Specimens were prepared and observed by transmission electron microscope (TEM) (H-7650; Hitachi, Japan) as previously described [47]. Mitochondria numbers were counted in more than 10 images for each specimen.
Mouse ingWAT frozen sections were then blocked for 30 minutes in PBS containing 10% normal goat serum at RT. For Oct3 staining, sections were incubated with a rabbit anti-mouse Oct antibody (1:200; OriGene Technologies, Rockville, MD) followed by staining with a Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:500; Invitrogen, Carlsbad, CA). Then, sections were stained with DAPI for 10 minutes. Images were obtained with a Nikon A1 confocal microscope (Nikon Corp., Japan).
For mitochondria staining, deparaffinized slices of ingWAT from Ctrl and cKO mice were incubated with 250 nM MitoTracker (Molecular Probes, OR, USA) for 1 hour at RT. Slides were washed with PBS and had coverslips mounted. Images were obtained with a Nikon A1 confocal microscope (Nikon Corp., Japan). Mitochondria area fraction percentage was calculated as previously described [48] using NIS-Elements software (Nikon Corp., Japan). More than 20 fields were scored per group.
For detection of intracellular NE by immunofluorescence, primary adipocytes were incubated with medium containing NE (100 ng/ml) for 20 minutes at 37˚C. After blocking, the adipocytes were next incubated with anti-noradrenaline (ab8887, 1:50, Abcam, Cambridge, MA) overnight at 4˚C. The following procedures of immunofluorescence were described earlier.

Determination of NE-induced thermogenesis
Ctrl and cKO mice (males, 8 weeks, chow diet) were intraperitoneally injected with pentobarbital (75 mg/kg), which does not inhibit nonshivering thermogenesis [51,52]. For NE treatment, mice were subcutaneously injected with 0.3 mg/kg NE under anesthesia. For β-AR inhibition assay, propranolol (5 mg/kg) was given 20 minutes before NE. The core body temperature was recorded with a rectal probe connected to a digital thermometer (Yellow Spring Instruments). Infrared thermal images were obtained using a FLIR E60 compact infrared thermal imaging camera and were analyzed using FLIR Tools software (FLIR, North Billerica, MA).

Determination of NE-induced lipolysis
For in vivo lipolysis, 0.3 mg/kg NE or saline was injected intraperitoneally into Ctrl and cKO mice. Tail blood was taken from mice 20 minutes after injection of NE or saline. Serum was separated for the determination of FFA using NEFA C kits (Wako, Oxoid SA, France).
For ex vivo lipolysis, about 50 mg WAT samples from overnight fasted mice were incubated in KRB buffer (12 mM HEPES, 121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO 4 , and 0.33 mM CaCl 2 ) containing 2% FA-free BSA and 2.5 mM glucose, and then stimulated with 1 μM NE. Glycerol release was measured by using free glycerol reagent (Sigma-Aldrich, USA).

In vivo uptake
Chemical sympathectomy was performed by unilateral denervation in ingWAT on one side and sham-operation on the collateral side to eliminate SNS NE release and uptake, as described previously [54]. The effectiveness of sympathectomy was confirmed by immunofluorescence of tyrosine hydroxylse (Th).
After 3 days of recovery from surgery, mice were given intraperitoneal injection of 0.5 mg/kg of NE spiked with [ 3 H]-NE (PerkinElmer, CT, USA). After 30 minutes, mice were transcardially perfused with ice-cold PBS and washed for 4 to 5 minutes. IngWAT was immediately isolated with the major blood vessels removed and homogenized in Solvable (PerkinElmer CT, USA) overnight. Total radioactivity in ingWAT homogenates was determined by liquid scintillation counting. Protein content of homogenates was measured by BCA assay (Thermo Scientific, USA).

RNA-seq analysis
For RNA-seq analysis of ingWAT, total RNA was extracted using the RNeasy Lipid Mini Kit (Qiagen, Hilden, Germany) based on the manufacturer's instructions. Quality of RNA was analyzed using the Agilent 2100 (Agilent Technologies, Palo Alto, CA). The poly-A-containing mRNA was purified by using poly-T oligo-attached magnetic beads. Purified mRNA was fragmented into small pieces using divalent cations under elevated temperature. RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. A single "A" base was added to cDNA fragments, and the adapter was subsequently ligated. The products were then purified and enriched with PCR amplification. PCR yield was quantified by Qubit, and samples were pooled together to make a single-strand DNA circle (ssDNA circle), which gave the final library.
DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and had 50 bp of single end read through the BGISEQ-500 platform. The DNB-based nanoarrays and stepwise sequencing were combined using Combinational Probe-Anchor Synthesis Sequencing Method and were analyzed.
Heatmap for differential genes was produced using pheatmap package of R. GO enrichment analysis and KEGG analysis for differential genes were performed in clusterProfiler package [55] version 2.2.4 of R using default settings.

Real-time PCR analysis
Total RNA was extracted by RNeasy Mini Lipid Tissue Kit (QIAGEN, Hilden, Germany). Reverse transcription (Tiangen, Beijing, China) and SYBR green quantitative PCR (Transgen, Beijing, China) were performed according to the manufacturer's instructions. Primers for target sequences were as shown in S2 Table. Relative gene expression level was normalized by TATA box-binding protein (Tbp) expression levels, unless otherwise indicated, since TBP expression was stable across ATs [56].
For mitochondrial DNA analysis, mouse tissues were homogenized, and genomic DNA was extracted by TIANamp genomic DNA kit (Tiangen, Beijing, China). SYBR green quantitative PCR (Transgen, Beijing, China) was performed in duplicate using mitochondrial DNA specific primers for mitochondrial Cox2 and normalized by amplification of the nuclear gene ribosomal protein s18 (Rps18).

Lactate concentration
Lactate concentration in the ATs was measured by using a Lactate Assay Kit (Biovision, USA) following the instructions of the manufacturer.

Seahorse experiments
Cellular OCRs were determined using a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Biosciences, Chicopee, MA). Primary adipocytes from ingWAT were seeded at 20,000 cells/ well. Differentiation was induced as described above, and the cells were analyzed on day 6. Before the measurements, the cells were washed twice with assay medium (XF DMEM + 25 mM glucose + 2 mM pyruvate + 4 mM glutamine) and incubated in 175 μL of assay medium for 1 hour in an incubator without CO 2
In Gene Atlas, which housed the results of all the genetic association studies analyses of the UK Biobank cohort (N = 408,455), BMR was available for over 7,000 participants. HaploReg version 4.1 [57] and GTEx Portal [58] were used to determine the location, consequence of the variants, and the effect of the variants on transcript levels of the gene. Information about the methods used for determining BMR of the participants were available online (http://biobank. ctsu.ox.ac.uk/crystal/docs/Anthropometry.pdf).

HFD treatment
Six-week-old adipose-specific Oct3 KO and Ctrl mice were treated with HFD for 12 weeks under RT or thermoneutrality (30˚C). For the groups under thermoneutrality, mice were then cold-stimulated at 4˚C for 7 days. Body temperatures of mice were monitored at the first 8 hours under cold exposure. Body weights were recorded before and after cold exposure. Food intake was recorded daily during cold exposure. Blood was collected into EDTA tubes, and plasma was separated by centrifugation. Plasma triglyceride and total cholesterol levels were determined by respective assay kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China). Commercially available ELISA kits were used to measure plasma adiponectin (Abcam), insulin (ALPCO, Salem, NH), serum and tissue NE (Rocky Mountain Diagnostics, Colorado Springs, CO) levels following instructions from the manufacturers. Plasma FFAs was measured NEFA C kits (Wako, Oxoid SA, France). Liver triglyceride content was measured using Triglyceride Quantification Assay Kit (Abcam).

Oral glucose tolerance test
Oral glucose tolerance test was performed on mice fed with 12-week HFD under thermoneutrality and then acclimated cold exposure for 7 days by oral gavage of glucose (2 g/kg) after 12-hour overnight fasting.

ATMSCs
Human MSCs derived from human AT, a kind gift from Dr. Yanan Du (Tsinghua University), were cultured in human MSC growth medium (Wuhan Viraltherapy Technologies Co. Ltd, Beijing) containing 10% calf serum and penicillin G. Adipogenic differentiation of human MSCs were performed as previously described [59].

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0 (http://www.graphpad.com/ scientific-software/prism). ANCOVA was conducted for in vivo metabolic data. Other analysis was conducted by Student t test (for comparison of two experimental conditions) or one-way ANOVA followed by Tukey's test (for comparison of three or more experimental conditions). Data are represented as mean ± standard deviation. Statistical significance was calculated and indicated ( � p < 0.05, �� p < 0.01, ��� p < 0.001). . (E) Three-dimensional-structure modeling of Oct3 and molecular docking of different monoamines. The three-dimensional Oct3 homology model was based on human GLUT3 template in the outward-facing-occluded ("occluded") conformation in complex with D-glucose. The predicted homology models contained the whole target sequence including the 12 transmembrane helices and the primary substrate binding site. After overall structural quality evaluation with DOPE scores, the best-scored model was further assessed based on its ability to discriminate between catecholamines (NE, epinephrine, histamine, dopamine, serotonin, and tyramine) and d-AMPH, which was not a substrate for Oct3 and only exerted weak inhibitory effects on Oct3-mediated uptake. The numerical data underlying this figure are included in S1 Data. AT, adipose tissue; d-AMPH, dextroamphetamine; DOPE, discrete optimized protein energy; GLUT3, glucose transporter 3; gonWAT, gonadal white adipose tissue; ingWAT, inguinal white adipose tissue; MA, mature adipocyte; NE, norepinephrine; Oct3, organic cation transporter 3; SVC, stromal vascular cell. (A) A regional plot of SLC22A3 locus. SNPs were plotted by chromosome 6 against association with BMR in UK Biobank participants (N~7,000). An SNP, rs555754 (purple circle), and its proxies are the top signals in SLC22A3 locus. Estimated recombination rates (cM/Mb) were plotted in blue to reflect the local LD structure. The SNPs surrounding the significant SNP, rs555754, were color coded to reflect their LD with this SNP. This LD was taken from pairwise r 2 values from the 1000 Genomes Nov 2014 EUR (hg19). Genes, the position of exons, and the direction of the transcription from the UCSC Genome Browser were noted. This plot was created using LocusZoom (http://locuszoom.org/genform.php?type=yourdata). The associations for each variant in this plot (effect size of the reference allele and p-value) were shown in S3 Table. (B, C) SLC22A3 transcript levels in adipose subcutaneous (panel B) and adipose visceral omentum (panel C) were significantly associated with the top two SNPs in the locus zoom plot in panel A. The figure and data were available in GTEx portal (gtexportal.org). The x-axis showed the genotype for the SNP and the y-axis showed the expression levels of SLC22A3 as quantified by RNA-seq method. A full list of all eQTL for each SNPs were available in S3 Table. (D) mRNA expression level of catecholamine transporters and catecholamine degradation enzymes in human AT (n = 3). (E) Oil Red O staining of undifferentiated and adipogenically differentiated ATMSC. (F) mRNA expression level of catecholamine transporters and catecholamine degradation enzymes in differentiated ATMSC (n = 3). (G) In vitro glycerol release from differentiated ATMSC untreated or NE-treated to stimulate lipolysis, incubated with vehicle or OCT3 inhibitor decynium-22 (10 μM) (n = 3). Data in panel G were analyzed by Student t test. The numerical data underlying this figure are included in S1 Data. AT, adipose tissue; ATMSC, AT-derived mesenchymal stem cell; BMR, basal metabolic rate; eQTL, expression quantitative trait locus; LD, linkage disequilibrium; NE, norepinephrine; Oct3, organic cation transporter 3; SNP, single-nucleotide polymorphism. (TIF) S1