β3-adrenergic receptors stimulate glucose uptake in brown adipocytes by two mechanisms independently of GLUT4 translocation

• To identify the mechanisms whereby norepinephrine induces glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. Proliferating brown adipocytes had high levels of glucose transporter (GLUT) 1 mRNA and low levels of GLUT4 mRNA. The ratio of GLUT4/GLUT1 mRNA expression increased during differentiation, and mature brown adipocytes had high levels of GLUT4 mRNA. The endogenous adrenergic neurotransmitter norepinephrine induced a potent increase in GLUT1 mRNA and a decrease of GLUT4 mRNA in mature brown adipocytes. The norepinephrine effect was mimicked by isoprenaline and CL 316243 and was thus mediated by beta(3)-adrenergic receptors. The cAMP analog 8-bromoadenosine-cAMP partly mimicked the response on GLUT1 mRNA increase and fully mimicked the GLUT4 mRNA decrease. We found no involvement of alpha(1) or alpha(2)-adrenergic receptors on GLUT1 or GLUT4 mRNA transcription. Norepinephrine treatment led to a large increase of GLUT1 protein amount in brown adipocytes as visualized with immunocytochemical staining and subcellular fractionation. A large part of the newly synthesized GLUT1 was found in the plasma membrane (PM). The potent transcriptional inhibitor actinomycin D fully abolished this increase of GLUT1 protein at all time points examined. Norepinephrine treatment shifted GLUT4 from the PM to an intracellular vesicular compartment. Norepinephrine increased 2-deoxy-D-glucose uptake 2-fold at an early time point ( 1 h) and 4-fold at later time point ( 5 h). Addition of actinomycin D did not block the early phase but blocked a large part of the later phase of 2-deoxy-D-glucose uptake. These results imply that adrenergic stimulation through beta(3)-adrenergic receptors induces glucose uptake in brown adipocytes via two mechanisms: 1) a mechanism not dependent on GLUT1 and GLUT4 translocation, 2) a mechanism that is dependent on de novo synthesis of GLUT1 protein and increase of GLUT1 protein at the PM.

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