Abstract
Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.
Highlights
Vation of cAMP-dependent protein kinase (PKA)2, whose key targets of phosphorylation are hormone-sensitive lipase (HSL) and perilipin A
When we reported that the 3AR in adipocytes is coupled to the stimulation of both PKA and extracellular signal-regulated kinases and 2 (ERK) [12] we proposed that one consequence of this event could be a convergence of these path
In earlier work on the activation of ERK by the 3AR we Catecholamines are the major regulators of triacylglycerol described an essential role for a direct interaction between pro- hydrolysis in adipocytes, and this lipolytic process is mediated by line-rich intracellular portions of the receptor and the cytosolic the adrenergic receptor (AR) [27]. AR signaling is the principal physiological pathtyrosine kinase Src [13]
Summary
Materials—Chicken anti-rat HSL antisera were described previously [15]. 3T3-L1 cells were obtained from Dr Howard Green [16]. Lipolysis—Differentiated 3T3-L1 adipocytes were washed with PBS and incubated in Kreb’s Ringer buffer containing 2% fatty acid-free BSA at 37 °C for 3 h (for experiments with E2, cells were incubated overnight). This medium was replaced with fresh medium containing various concentrations (0, 10 –50 M) of the PKA inhibitors H89 or Rp-cAMPS (0.5 mM) and/or the MEK1/2 inhibitor PD098059 (0, 10 –50 M) for 1 h. Protein Kinase Activity—PKA activity was measured in vitro from whole cell extracts prepared from differentiated 3T3-L1 adipocytes that had been pretreated as described above for the lipolysis experiments After this preincubation, CL or NE (0, 0.1–1000 nM) was added to the cells for 5 min. The slides were covered with glass coverslips, mounted using Vectashield mounting medium from Vector Laboratories (Burlingame, CA), and visualized by laser scanning confocal microscope (Zeiss, Thornwood, NY) at 585 nm
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