Recent findings in adipocytes suggest that mitogen-activated protein kinase (MAPK)/extracellular-regulated signaling kinase (ERK) kinase 1/2 (MEK1/2) signaling regulates regulated in development and DNA damage 1 (REDD1) protein expression. Similarly, our previous work show that a lack of REDDI protein expression, and associated hyperactive basal mechanistic target of rapamycin (mTOR) signaling, limits skeletal muscle's response to insulin. Therefore, we sought to determine: 1) if MEKI/2 inhibition is sufficient to reduce REDD1 protein expression and subsequently insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation via negative feedback of hyperactive mTOR in REDDI wild-type (WT) mice and 2) if rapamycin-mediated mTOR inhibition is sufficient to improve IRS-i tyrosine phosphorylation in REDDI knockout (KO) mice. REDD1 WT mice were injected with 10 mg/kg BW of the MEKI/2 noncompetitive inhibitor, PD184352, 3 h prior to acute insulin treatment. In separate studies, REDD1 KO mice were injected with 5 mg/kg BW of the mTOR inhibitor, rapamycin, 3 h prior to acute insulin treatment. Following the inhibitor treatment period, markers of insulin signaling activation (IRS-1 Y1222, MEKI/2 S217/221, ERKI/2 T202/Y204), REDDI, and mTOR signaling activation (S6K1 T389, rpS6 S240/244) were examined in skeletal muscle collected before and after a 10 min insulin treatment. PD184352 treatment reduced MEK/ERK phosphorylation and REDDI protein expression, independent of insulin. This reduction in REDDI protein expression was associated with elevated basal S6K1 and rpS6 phosphorylation and reduced insulin stimulated IRS-1 phosphorylation. Conversely, rapamycin inhibited S6K1 and rpS6 activation, and significantly improved insulin stimulated activation of IRS-I and MEKI/2 in KO mice. These data support that REDDI is required for normal insulin-stimulated signaling, and that a subtle balance exists between MEKI/2, REDDI, and mTOR for the proper regulation of insulin signaling. (C) 2016 Elsevier Inc. All rights reserved.