Norepinephrine (NE) bitartrate monohydrate is generally considered to be a β1-subtype selective adrenergic agonist. Norepinephrine(NE) also has direct activity at the β2-adrenoceptor in higher concentrations[1]. Adipocytes from the inguinal fat pad (iWA) or the interscapular fat pad (BA) are isolated from neonatal wild-type C57BL/6J mice and cultured. To examine the effect of activating AT2 upon β-adrenergic signaling, cAMP production is first assessed in response to Norepinephrine (NE, 10 µM) with or without CGP (10 nM) co-treatment. Norepinephrine (NE) increases cAMP as expected in iWA, and CGP does not alter this effect. Norepinephrine (NE) is also known to induce lipolysis, and liberated fatty acids are required to functionally activate UCP1 protein and to stimulate heat production. CREB phosphorylation at Ser133 is increased after Norepinephrine (NE) treatment and significantly attenuated with CGP co-treatment in mouse iWA[2].
Kinase Assay
Cell Assay
Subcutaneous preadipocytes derived from a 38-year old non-diabetic female donor are immortalized with TERT and HPV E6/E7. For the current studies, a stable diploid clone (referred to as clone B) with consistent differentiation capacity is isolated by ring cloning. Cells are grown in preadipocyte PGM2 media. Once cells are confluent, differentiation is induced by incubation in differentiation media consisting of dexamethasone, IBMX, indomethacin, and additional insulin. Cells are differentiated for 10 days. Prior to treatment, media is replaced with PGM2 media for one day and then switched to serum-free media overnight for treatments. Adipocytes are treated for 6 hours with vehicle, Norepinephrine (NE, 10 μM), CGP (10 nM), or Norepinephrine (NE) and CGP[2].
Animal Administration
References
[1]. MacGregor DA, et al. Relative efficacy and potency of beta-adrenoceptor agonists for generating cAMP in human lymphocytes. Chest. 1996 Jan;109(1):194-200.
[2]. Littlejohn NK, et al. Suppression of Resting Metabolism by the Angiotensin AT2 Receptor. Cell Rep. 2016 Aug 9;16(6):1548-60.
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