Abstract
Rat perivascular adipose tissue (PVAT) and rat adipocytes contain the organic cation transporter 3 (OCT3), a transporter of norepinephrine (NE). This is physiologically relevant because PVAT reduces vasoconstriction through the uptake of NE. We hypothesized that an accepted model of adipocytes, 3T3‐L1 cells, take up NE through OCT3 or a similar catecholamine transporter. Mouse 3T3‐L1 fibroblasts were induced into adipocytes by adding 0.5 mM IBMX and 0.25 mM dexamethasone for 48 hours. AdipoRedTM assay and Oil Red O staining confirm that induction was successful (15 times more fluorescent than preadipocytes and 95% of cells exhibit lipid droplet staining, respectively) on the 10th day after the induction. NE was added to 3T3‐L1 adipocytes at 0, 1, 10, and 100 mM concentrations in physiological saline solution (PSS) with NE metabolism inhibitors [monoamine oxidase, pargyline (10−5 M), catecholamine‐O‐methyltransferase, Ro 41‐0960 (10−6 M), semicarbazide‐sensitive amine oxidase, semicarbazide hydrochloride (10−3 M)] for 30 minutes at 37 °C. Cells were washed with PSS, collected in tissue buffer, and analyzed for NE content using HPLC. There was an increase in NE, normalized for protein content, from vehicle to 10 mM and 100 mM (0.09 ± 0.01 pg/mg, 5.97 ± 1.48 pg/mg, 18.89 ± 3.37 pg/mg, respectively, p < 0.05, N=6). PCR was performed on cDNA isolated from 3T3‐L1 adipocytes to test for known NE transporters: norepinephrine transporter (NET), OCT3, serotonin transporter (SERT), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and plasma membrane monoamine transporter (PMAT), using beta‐actin as the reference gene. OCT3, SERT, DAT, and VMAT2 did not reach the detectable threshold after 40 amplification cycles (CT value > 40, N=4). NET and PMAT had 1 sample out of 4 with a detected CT value (37.45 ± 0.15 and 37.41 ± 0.05, respectively). The positive controls had detectable amplification (heart: 26.94 ± 0.02 for OCT3, brain: 27.53 ± 0.10 for SERT, 27.90 ± 0.03 for DAT, and 26.97 ± 0.03 for PMAT, adrenal: 25.80 ± 0.15 for VMAT2, pons: 24.59 for NET). These data suggest that NE is entering 3T3‐L1 adipocytes via mechanisms other than the canonical transporters of NE. Learning how NE interacts with adipocytes, and therefore PVAT, is essential for studying the relation between vasoconstriction and obesity.Support or Funding InformationNIH NHLBI P01HL70687IPSTP 5 T32 GM 92715‐4NIH NRSA F31 HL128035‐01
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