DIABETES, VOL. 47, OCTOBER 1998 1549 Epinephrine and Insulin Stimulate Different Mitogen-Activated Protein Kinase Signaling Pathways in Rat Skeletal Muscle Raffaele Napoli, Lindsay Gibson, Michael F. Hirshman, Marni D. Boppart, Scott D. Dufresne, Edward S. Horton, and Laurie J. Goodyear Little is known about the regulation of the mitogen-acti- vated protein (MAP) kinase signaling cascades by hor- monal stimulation in vivo. The extracellular signal–reg- ulated kinase (ERK) and the c-jun kinase (JNK) are two MAP kinase signaling pathways that could play a role in the cellular response to hormones such as insulin and epinephrine. We studied the effects of insulin (20 U/rat) and epinephrine (25 μg/100 g body wt) injected in vivo on ERK and JNK signaling in skeletal muscle from Sprague-Dawley rats. Insulin significantly increased ERK phosphorylation and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2 (RSK2), by 1.4-fold, but it had no effect on JNK activity. In contrast, epinephrine had no effect on ERK phospho- rylation or RSK2 activity, but it increased JNK activity by twofold, an effect that was inhibited by the presence of combined and blockade. Furthermore, the phos- phorylation of both p46 and p55 isoforms of JNK, meas- ured by phosphospecific antibody, was increased sever- alfold. The activity and phosphorylation of MAP kinase kinase (MKK)-4, an upstream regulator of JNK, was unchanged by epinephrine. Incubation of isolated soleus muscles in vitro with epinephrine (10 –5 mol/l) also increased JNK activity by twofold. These data are the first to demonstrate that epinephrine can increase JNK activity. Insulin and epinephrine have different effects on MAP kinase signaling pathways in skeletal muscle, which may be one of the underlying molecular mecha- nisms through which these hormones regulate opposing metabolic functions. Diabetes 47:1549–1554, 1998 T he extracellular signal–regulated kinase (ERK) and c-jun NH 2 -terminal kinase (JNK) are compo- nents of two parallel mitogen-activated protein (MAP) kinase signaling cascades that have been implicated in the regulation of numerous cellular functions (1–4). Studies in cultured cells have shown that the ERK cas- cade can be activated by several growth-factor receptors through the Shc-Grb2-Sos complex. This complex mediates the activation of Ras and Raf, which in turn causes the sequential phosphorylation and activation of the MAP kinase kinase (MEK-1 and -2) and ERK1 and ERK2 (1,2,5–7). The p90 ribosomal S6 kinase (RSK2) is downstream of the ERKs in this signaling cascade, and both the ERKs and RSK2 can phosphorylate transcription factors (8). The stimulation of JNK is thought to occur through the activation of Rac1/2, Cdc42, MEK kinase-1 and MAP kinase kinase (MKK)-4, although it is still unclear which mechanisms or receptors are triggered by the various cell stressors that activate this pathway (9–15). Activated JNK phosphorylates c-jun at its NH 2 -terminus (11,16), which is thought to play a critical role in the activation of the AP-1 genes (9). Epinephrine regulates growth and hypertrophy in several tissues, including smooth and cardiac muscle (17–22). Epi- nephrine also functions to oppose many of the metabolic effects of insulin on skeletal muscle, and it has been recently shown that insulin and epinephrine can divergently regulate the transcription of important genes that are responsible for the regulation of opposing cell functions in skeletal muscle (23). Because a major cellular response to signaling through the ERK and JNK cascades is believed to be the regulation of gene transcription, we hypothesized that epinephrine and insulin may have different effects on these signaling pathways. To date, few studies have investigated ERK and JNK sig- naling in animals or humans in response to in vivo stimula- tion (4,24,25). In skeletal muscle, insulin can increase ERK and RSK2 activities (4,24), and one report has shown that insulin can transiently activate JNK (4). Although the effects of epi- nephrine on ERK and JNK signaling are not known, studies in vitro have shown that an increase in intracellular cAMP, a second messenger for catecholamine action, increases ERK activity in some cell types (26,27) but inhibits growth-factor stimulation of ERK signaling in other cell types (28–32). In endothelial cells, JNK activity can be increased by stimulation of -adrenoreceptors via a Ras-dependent pathway (33). In the current study, we investigated the effects of epinephrine stimulation in vivo on the ERK and JNK pathways in skele- tal muscle and determined if there are interactions between insulin and epinephrine on these signaling cascades. RESEARCH DESIGN AND METHODS Animals and experimental design In vivo studies. Sprague-Dawley rats weighing 150–200 g were purchased from Taconic Farms (Germantown, NY), housed at 23°C with a 12-h light/dark cycle, and fed ad libitum for several days before the start of the study. All animals were studied in the nonfasted state under anesthesia (Pentobarbital: 60 mg/kg body wt From the Research Division, Joslin Diabetes Center; the Department of Med- icine, Brigham and Women’s Hospital; and the Harvard Medical School, Boston, Massachusetts. Address correspondence and reprint requests to Laurie J. Goodyear, PhD, Research Division, Joslin Diabetes Center, One Joslin Pl., Boston, MA 02215. E-mail: goodyeal@joslab.harvard.edu. Received for publication 30 April 1998 and accepted in revised form 2 July 1998. DTT, dithiothreitol; ECL, enhanced chemiluminescence; ERK, extracel- lular signal–regulated kinase; HRP, horseradish peroxidase; JNK, c-jun NH 2 -terminal kinase; MAP, mitogen-activated protein; MEK, mito- gen-activated protein kinase kinase; MKK, MAP kinase kinase; RSK2, p90 ribosomal S6 kinase.