In situ detection of the activation of Rac1 and RalA small GTPases in mouse adipocytes by immunofluorescent microscopy following in vivo and ex vivo insulin stimulation.

Rac1 has been implicated in insulin-dependent glucose uptake by mechanisms involving plasma membrane translocation of the glucose transporter GLUT4 in skeletal muscle. Although the uptake of glucose is also stimulated by insulin in adipose tissue, the role for Rac1 in adipocyte insulin signaling remains controversial. As a step to reveal the role for Rac1 in adipocytes, we aimed to establish immunofluorescent microscopy to detect the intracellular distribution of activated Rac1. The epitope-tagged Rac1-binding domain of a Rac1-specific target was utilized as a probe that specifically recognizes the activated form of Rac1. Rac1 activation in response to ex vivo and in vivo insulin stimulations in primary adipocyte culture and mouse white adipose tissue, respectively, was successfully observed by immunofluorescent microscopy. These Rac1 activations were mediated by phosphoinositide 3-kinase. Another small GTPase RalA has also been implicated in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Similarly to Rac1, immunofluorescent microscopy using an activated RalA-specific polypeptide probe allowed us to detect intracellular distribution of insulin-activated RalA in adipocytes. These novel approaches to visualize the activation status of small GTPases in adipocytes will largely contribute to the understanding of signal transduction mechanisms particularly for insulin action.

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

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.

Immunofluorescent detection of the activation of the small GTPase Rac1 in mouse skeletal muscle fibers.

The small GTPase Rac1 acts as a molecular switch of intracellular signaling in mammals. For understanding the regulatory mechanism, it is important to identify subcellular locations in which Rac1 is activated following multiple extracellular stimuli. However, it is difficult to detect Rac1 activation in situ in animal tissues, and thus a novel method is highly desirable. Here, we report a simple method to visualize the activation of endogenous Rac1 in mouse skeletal muscle fibers. In this assay, specific interaction between activated Rac1 and a binding polypeptide is detected by immunofluorescent microscopy. This approach is readily applicable to other small GTPases.

Role of the guanine nucleotide exchange factor in Akt2-mediated plasma membrane translocation of GLUT4 in insulin-stimulated skeletal muscle.

The small GTPase Rac1 plays a key role in insulin-promoted glucose uptake mediated by the GLUT4 glucose transporter in skeletal muscle. Our recent studies have demonstrated that the serine/threonine protein kinase Akt2 is critically involved in insulin-dependent Rac1 activation. The purpose of this study is to clarify the role of the guanine nucleotide exchange factor FLJ00068 in Akt2-mediated Rac1 activation and GLUT4 translocation in mouse skeletal muscle and cultured myocytes. Constitutively activated FLJ00068 induced GLUT4 translocation in a Rac1-dependent and Akt2-independent manner in L6 myocytes. On the other hand, knockdown of FLJ00068 significantly reduced constitutively activated Akt2-triggered GLUT4 translocation. Furthermore, Rac1 activation and GLUT4 translocation induced by constitutively activated phosphoinositide 3-kinase were inhibited by knockdown of FLJ00068. In mouse gastrocnemius muscle, constitutively activated FLJ00068 actually induced GLUT4 translocation to the sarcolemma. GLUT4 translocation by constitutively activated FLJ00068 was totally abolished in rac1 knockout mouse gastrocnemius muscle. Additionally, we were successful in detecting the activation of Rac1 following the expression of constitutively activated FLJ00068 in gastrocnemius muscle by immunofluorescence microscopy using an activation-specific probe. Collectively, these results strongly support the notion that FLJ00068 regulates Rac1 downstream of Akt2, leading to the stimulation of glucose uptake in skeletal muscle.

A critical role of the small GTPase Rac1 in Akt2-mediated GLUT4 translocation in mouse skeletal muscle.

Insulin promotes glucose uptake in skeletal muscle by inducing the translocation of the glucose transporter GLUT4 to the plasma membrane. The serine/threonine kinase Akt2 has been implicated as a key regulator of this insulin action. However, the mechanisms whereby Akt2 regulates multiple steps of GLUT4 translocation remain incompletely understood. Recently, the small GTPase Rac1 has been identified as a skeletal muscle-specific regulator of insulin-stimulated glucose uptake. Here, we show that Rac1 is a critical downstream component of the Akt2 pathway in mouse skeletal muscle as well as cultured myocytes. GLUT4 translocation induced by constitutively activated Akt2 was totally dependent on the expression of Rac1 in L6 myocytes. Moreover, we observed the activation of Rac1 when constitutively activated Akt2 was ectopically expressed. Constitutively activated Akt2-triggered Rac1 activation was diminished by knockdown of FLJ00068, a guanine nucleotide exchange factor for Rac1. Knockdown of Akt2, on the other hand, markedly reduced Rac1 activation by a constitutively activated mutant of phosphoinositide 3-kinase. In mouse skeletal muscle, constitutively activated mutants of Akt2 and phosphoinositide 3-kinase, when ectopically expressed, induced GLUT4 translocation. Muscle-specific rac1 knockout markedly diminished Akt2- or phosphoinositide 3-kinase-induced GLUT4 translocation, highlighting a crucial role of Rac1 downstream of Akt2. Taken together, these results strongly suggest a novel regulatory link between Akt2 and Rac1 in insulin-dependent signal transduction leading to glucose uptake in skeletal muscle.