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

Abstract

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.

Fig 1. Oct3 was highly expressed in AT and had the greatest uptake rate of NE.

(A) Tissue distribution of mouse Oct3 mRNA (8-week-old male C57BL/6 mice, n = 3). (B) mRNA expression of Oct3, Slc6a2, Slc6a3, and Slc6a4 in different mouse WATs (n = 3). (C) mRNA expression of Maoa, Maob, Comt, and adipocytes abundant gene Adiponectin in different mouse WATs (n = 3). (D) mRNA expression of Oct3 in mouse MAs and SVCs of ingWAT (n = 3). (E) Oct3 immunofluorescence staining (green) of mouse primary inguinal adipocytes. DAPI (blue), nucleus. Scale bar, 50 μm. (F) Uptake of different monoamines and MPP⁺ in HEK-293 cells stably overexpressing mouse Oct3. MPP⁺ was used as a model substrate for functional study of Oct3. n = 3 experiments. (G) Saturation kinetics of [³H]-NE transport in HEK-293 Flp-In cells stably expressing human OCT3 or mouse Oct3 (n = 3–4). (H) Immunohistochemical staining of Oct3 in ingWAT of Ctrl and adipose-specific Oct3 KO (cKO) mice. Scale bar, 50 μm. (I) [³H]-NE uptake assay in primary white and brown adipocytes from Ctrl and cKO mice. (J) Total radioactivity in ingWAT and gonWAT from Ctrl and cKO mice after [³H]-NE injection (n = 6). Data in panel B were analyzed by one-way ANOVA followed by Tukey’s test. Data in panels D, I, and J were analyzed by Student t test. The numerical data underlying this figure are included in S1 Data. AT, adipose tissue; BAT, brown adipose tissue; cKO, conditional knockout; Ctrl, control; EV, empty vector; gonWAT, gonadal white adipose tissue; HEK, human embryonic kidney; ingWAT, inguinal white adipose tissue; KO, knockout; MA, mature adipocyte; MPP, methyl-4-phenylpyridinium; NE, norepinephrine; Oct3, organic cation transporter 3; SVC, stromal vascular cell; WAT, white adipose tissue.

Fig 2. Adipose-specific Oct3 KO mice exhibited enhanced NE-induced thermogenesis.

(A–C) Body temperature parameters of Ctrl and cKO mice after NE injection (s.c., 0.3 mg/kg) at the indicated time point (n = 8). (A) Rectal core body temperature; (B) maximal body temperature; (C) maximal body temperature change. (D) Representative thermal images of Ctrl and cKO mice after NE injection. (E–H) Metabolic parameters of Ctrl and cKO mice after NE injection (n = 8). (E, F) Relative O₂ consumption; (G, H) Heat production. (I, J) mRNA expression of thermogenic and lipolytic genes in ingWAT (panel I) and gonWAT (panel J) of Ctrl and cKO mice after NE injection (n = 8). Data in A–C and I–J were analyzed by Student t test. Data in E–H were analyzed by ANCOVA. The numerical data underlying this figure are included in S1 Data. AU, arbitrary unit; cKO, conditional knockout; Ctrl, control; gonWAT, gonadal white adipose tissue; Hsl, hormone-sensitive lipase; ingWAT, inguinal white adipose tissue; KO, knockout; NE, norepinephrine; Oct3, organic cation transporter 3; s.c., subcutaneous; Ucp1, uncoupling protein 1; VO₂, oxygen consumption.

Fig 3. Genetic ablation of Oct3 augmented NE-stimulated lipolysis in WAT.

(A) Basal and NE-stimulated lipolysis by measuring serum FFAs in Ctrl and cKO mice (n = 6). (B, C) Protein levels of Hsl phosphorylated at Ser563 and Ser660 (pHsl-S563 and pHsl-S660, respectively) and total Hsl in ingWAT (panel B) and gonWAT (panel C) of Ctrl and cKO mice with NE (“NE”) or without NE injection (“Ctrl”). (D) Ex vivo glycerol release from AT explants in the absence (“basal”) or presence of NE (“NE”) (n = 4). (E) In vitro glycerol release from differentiated SVF-derived adipocytes from Ctrl and cKO mice in the absence or presence of NE (n = 4). (F) Protein levels of pHsl-S563, pHsl-S660, and total Hsl in differentiated SVF-derived adipocytes from Ctrl and cKO mice in the absence or presence of NE. (G) Immunofluorescence analysis of intracellular NE (green) in differentiated SVF-derived adipocytes from Ctrl and cKO mice. Adipocytes were either untreated or treated with NE (100 nM) for 10 minutes. DAPI (blue), nucleus. Scale bar, 50 μm. (H) Basal and NE-stimulated lipolysis, as measured by glycerol release from differentiated 3T3-L1 adipocytes stably transfected with EVs or Oct3 (Oct3-OE) (n = 3). Data in panels A, D, E, and H were analyzed by Student t test. The numerical data underlying this figure are included in S1 Data. cKO, conditional knockout; Ctrl, control; EV, empty vector; FFA, free fatty acid; gonWAT, gonadal white adipose tissue; Hsl, hormone-sensitive lipase; ingWAT, inguinal white adipose tissue; KO, knockout; NE, norepinephrine; Oct3, organic cation transporter 3; OE, over-expression; pHsl, phosphorylated hormone-sensitive lipase; SVF, stromal vascular fraction; WAT, white adipose tissue.

Fig 4. Oct3 ablation promoted beiging and enhanced thermogenesis in ingWAT.

Ctrl and cKO mice were housed at 4°C for 1 month. (A) Core body temperature of Ctrl and cKO mice at RT and after cold acclimation (n = 6). (B) Representative thermal images of Ctrl and cKO mice after cold acclimation. (C) Gross images of ingWAT of Ctrl and cKO mice. (D) Representative HE and Ucp1 immunohistochemical staining in ingWAT. Scale bar, 50 μm. Shown are representative images of three experiments. (E–G) Metabolic parameters of Ctrl and cKO mice after cold acclimation (n = 4). (E) O₂ consumption; (F) CO₂ production; (G) Heat production. (H–J) Heat maps (left) and real-time PCR analysis (right) of relative gene expression levels in ingWAT of Ctrl and cKO mice after cold acclimation (n = 6). Expression values in heat maps were z-transformed and scaled. (H) BAT selective genes and beige genes; (I) WAT selective genes; (J) Fatty acid β-oxidation genes. (K, L) Protein levels of pPKA substrate (panel K), pCreb, Creb, Ucp1, and Pgc1α (panel L) in ingWAT of Ctrl and cKO mice. Data in A and E–J were analyzed by Student t test. 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; VO₂, oxygen consumption; WAT, white adipose tissue.

Fig 5. Deletion of Oct3 induced mitochondria biogenesis and enhanced mitochondria function and glycolysis in ingWAT.

Ctrl and cKO mice were housed at 4°C for 1 month. (A) TEM images of ingWAT sections. Red arrows indicate mitochondria. (B) Mitochondrial number per nucleus determined from electron micrographs. (C) Mitochondrial DNA content determined in ingWAT, gonWAT, and BAT (n = 4). (D) Representative figures of MitoTracker staining in ingWAT. (E) Positive signals of MitoTracker quantified as percentage total cells. (F, G) ClusterProfiler package in R was used to test for enrichment of cellular component (panel F) and KEGG pathway (panel G). Dot color indicates statistical significance (−log₁₀p.adjust value); dot size represents the gene numbers annotated to each term. (H) Heat maps of relative expression levels of ETC genes in ingWAT of Ctrl and cKO mice. (I) mRNA expression of genes associated with mitochondrial respiratory chain complex genes and other mitochondrial genes in ingWAT (n = 6). (J) Western blots of mitochondrial respiratory chain complex proteins in ingWAT. (K) Heat maps of relative expression levels of glycolytic genes in ingWAT of Ctrl and cKO mice. (L) mRNA expression of genes associated with glycolysis in ingWAT (n = 6). (M) Western blots of proteins associated with glycolysis in ingWAT. (N) The amount of lactate in ingWAT. Data in B–C, E, I, L, and N were analyzed by Student t test. All images shown are representative of three experiments. The numerical data underlying this figure are included in S1 Data. AU, arbitrary unit; BAT, brown adipose tissue; cKO, conditional knockout; Ctrl, control; ETC, electron transport chain; gonWAT, gonadal white adipose tissue; ingWAT, inguinal white adipose tissue; KEGG, Kyoto Encyclopedia of Genes and Genomes; Oct3, organic cation transporter 3; TEM, transmission electron microscope.

Fig 6. Oct3 deficiency induced a thermogenesis and lipolysis program through the NE/β-AR/PKA signaling pathway.

(A–C) Maximal body temperature change (panel A), maximal O₂ consumption change (ΔO₂) (panel B), 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 T₃ 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, O₂ 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; T₃, triiodothyronine; TF, transcription factor; TG, triglyceride; VO₂, oxygen consumption.