Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle

Abstract

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

Involvement of the protein kinase Akt2 in insulin-stimulated Rac1 activation leading to glucose uptake in mouse skeletal muscle

“…Plasmid DNAs and siRNAs were introduced into mouse gastrocnemius muscle by electroporation as previously described [16]. Adult male C57BL/6 mice were anesthetized by intraperitoneal injection of a solution of medetomidine (0.3 mg/kg of body weight), midazolam (4.0 mg/kg of body weight), and butorphanol (5.0 mg/kg of body weight).…”

“…A constitutively activated mutant of FLJ00068 indeed stimulated GLUT4 translocation in skeletal muscle of wild-type, but not m-rac1-KO, mice [15]. Moreover, Rac1 activation and GLUT4 translocation caused by ectopic expression of a constitutively activated mutant of PI3K or Akt2 were completely abrogated by small interfering RNA (siRNA)-mediated knockdown of FLJ00068 in mouse skeletal muscle [16].…”

“…Actually, we have not yet tested Rac1 activation and plasma membrane translocation of GLUT4 in Akt2-deficient mouse skeletal muscle due to unavailability of Akt2 knockout mice in our laboratory. However, we recently established siRNA-mediated knockdown and in situ detection of Rac1 activation in mouse skeletal muscle [14, 16, 19], which enabled us to directly examine the involvement of Akt2 in insulin-stimulated activation of Rac1. In this study, we aim to provide additional in vivo evidence for the involvement of Akt2 in Rac1 activation in skeletal muscle insulin signaling by using a mouse model.…”

Promoting Glucose Transporter-4 Vesicle Trafficking along Cytoskeletal Tracks: PAK-Ing Them Out

“…A recent paper shows in an inducible skeletal muscle-specific Rac1 KO mouse the requirement for Rac1 in exercise-induced GLUT4 translocation ( 76 ). Although most evidence shows independent activation of Rac1 and Akt downstream of PI3K, there are reports of AKT2 regulating Rac1 activity to impact GLUT4 translocation in skeletal muscle cells ( 77 , 78 ), involving the guanine nucleotide exchange factor FLJ00068 ( 79 , 80 ). Rac1 superactivation has also been shown to activate AKT to trigger induce GLUT4 translocation in a manner independent of insulin ( 81 ).…”

Simvastatin inhibits glucose uptake activity and GLUT4 translocation through suppression of the IR/IRS-1/Akt signaling in C2C12 myotubes

“…Without prenylation, the small GTPases are unable to anchor to the cell membrane [33]. Recent studies revealed that Rac1, belonging to the family of Rho GTPases, governs the glucose uptake through regulation of GLUT4 translocation both in response to insulin or to exercise in vivo [3434,35]. Our studies indicate that the impairment of glucose uptake in response to insulin after treatment with simvastatin may be associated with the lack of Rac1active form.…”