Abstract
Incubating rat diaphragm muscles with insulin increased the glycogen synthase activity ratio (minus glucose 6-phosphate/plus glucose 6-phosphate) by approximately 2-fold. Insulin increased the activities of mitogen-activated protein (MAP) kinase and the Mr = 90,000 isoform of ribosomal protein S6 kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth factor (EGF) was more effective than insulin in increasing MAP kinase and Rsk activity, but in contrast to insulin, EGF did not affect glycogen synthase activity. The activation of both MAP kinase and Rsk by insulin was abolished by incubating muscles with the MAP kinase kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not significantly reduce the effect of insulin on activating glycogen synthase. Incubating muscles with concentrations of rapamycin that inhibited activation of p70S6K abolished the activation of glycogen synthase. Insulin also increased the phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein) and promoted the dissociation of the PHAS-I*eIF-4E complex. Increasing MAP kinase activity with EGF did not mimic the effect of insulin on PHAS-I phosphorylation, and the effect of insulin on increasing MAP kinase could be abolished with the MEK inhibitor without decreasing the effect of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway appears to be neither necessary nor sufficient for insulin action on glycogen synthase and PHAS-I in rat skeletal muscle. The results indicate that the effects of insulin on increasing the synthesis of glycogen and protein in skeletal muscle, two of the most important actions of the hormone, involve a rapamycin-sensitive mechanism that may include elements of the p70S6K signaling pathway.
Highlights
Glycogen synthesis in skeletal muscle has a key role in the control of blood glucose levels by insulin
Neither insulin nor Epidermal growth factor (EGF) changed the amount of synthase detected, consistent with the observation that total glycogen synthase activity was changed by neither agent (Table I)
Activation of mitogen-activated protein (MAP) kinase by insulin in rat skeletal muscle is neither necessary nor sufficient for the activation of glycogen synthase or the phosphorylation of PHAS-I. Supporting this conclusion are observations that MAP kinase activation by insulin could be abolished by incubating diaphragms with an inhibitor of MAP kinase kinase (MEK), which did not significantly attenuate the effects of insulin on activating glycogen synthase (Table I) or on decreasing PHAS-I binding to eIF-4E (Fig. 5)
Summary
Incubation of Muscle in Vitro—Male rats (60 – 80 g, Sprague-Dawley, Sasco) were fed ad libitum before diaphragms with the surrounding ribs were removed as described by Goldberg et al [39]. Antibodies—ERK-1 antisera were generated by immunizing rabbits with a peptide (CQETARFQPGAPEAP) based on a sequence in the COOH-terminal region of ERK-1, and antibodies were affinity-purified before use as described previously [44]. Activation of ERK-1 and ERK-2 was assessed by using the “in-gel” assay described by Wang and Erikson [49] In this assay, extract samples (25 g) were subjected to SDS-PAGE in gels containing MBP fixed within the matrix of the gel. Rsk activity was measured by mixing beads with 10 l of solution containing 50 mM sodium -glycerophosphate (pH 7.4), 14 mM sodium fluoride, 10 mM MgCl2, 1 mM dithiothreitol, 9 M cAMP-dependent protein kinase inhibitory peptide [51], 20 M calmidazolium, 200 M [␥-32P]ATP (300 –500 cpm/pmol), and either 30 M S6 peptide or 0.1 mg/ml [His6]⌬RGL (described below). Microcystin and okadaic acid were from LC Labs, and rapamycin was purchased from Calbiochem
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