Takaya Satoh

A fraction of the insulin-stimulated uptake of long-chain fatty acids (FAs) is mediated by the FA translocase cluster of differentiation 36 (CD36) in white adipocytes. Intracellular vesicle-localized CD36 is redistributed to the plasma membrane following insulin stimulation, enhancing the uptake of long-chain FAs across the plasma membrane. We previously developed an epitope-tagged CD36 reporter, which enabled the visualization and quantification of the plasma membrane translocation of CD36. Herein, we demonstrate that the insulin-stimulated CD36 translocation is regulated by the phosphoinositide 3-kinase (PI3K)/Akt2/Rac1/RalA axis in adipocytes of subcutaneous white adipose tissue (WAT) in living mice. The uptake of long-chain FAs by insulin was completely abrogated in white adipocytes isolated from adipocyte-specific rac1-knockout (adipo-rac1-KO) mice. Correspondingly, the translocation of CD36 to the plasma membrane by insulin was also totally inhibited in Rac1-deficient white adipocytes. PI3K and Akt2 acted upstream of Rac1, and the guanin nucleotide exchange factor FLJ00068 served as a regulator for Rac1. Furthermore, the involvement of another small GTPase RalA was suggested by inhibitory effects of a dominant-negative mutant. Taken together, these results support the notion that insulin regulates the plasma membrane translocation of CD36 by mechanisms similar to those for the translocation of the glucose transporter GLUT4 in white adipocytes.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lep^ob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lep^ob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lep^ob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lep^ob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lep^ob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

White adipocytes act as lipid storage, and play an important role in energy homeostasis. The small GTPase Rac1 has been implicated in the regulation of insulin-stimulated glucose uptake in white adipocytes. Adipocyte-specific rac1-knockout (adipo-rac1-KO) mice exhibit atrophy of subcutaneous and epididymal white adipose tissue (WAT); white adipocytes in these mice are significantly smaller than controls. Here, we aimed to investigate the mechanisms underlying the aberrations in the development of Rac1-deficient white adipocytes by employing in vitro differentiation systems. Cell fractions containing adipose progenitor cells were obtained from WAT and subjected to treatments that induced differentiation into adipocytes. In concordance with observations in vivo, the generation of lipid droplets was significantly attenuated in Rac1-deficient adipocytes. Notably, the induction of various enzymes responsible for de novo synthesis of fatty acids and triacylglycerol in the late stage of adipogenic differentiation was almost completely suppressed in Rac1-deficient adipocytes. Furthermore, the expression and activation of transcription factors, such as the CCAAT/enhancer-binding protein (C/EBP) β, which is required for the induction of lipogenic enzymes, were largely inhibited in Rac1-deficient cells in both early and late stages of differentiation. Altogether, Rac1 is responsible for adipogenic differentiation, including lipogenesis, through the regulation of differentiation-related transcription.

Insulin stimulates glucose uptake in adipose tissue and skeletal muscle by inducing plasma membrane translocation of the glucose transporter GLUT4. Although the small GTPase Rac1 is a key regulator downstream of phosphoinositide 3-kinase (PI3K) and the protein kinase Akt2 in skeletal muscle, it remains unclear whether Rac1 also regulates glucose uptake in white adipocytes. Herein, we investigated the physiological role of Rac1 in white adipocytes by employing adipocyte-specific rac1 knockout (adipo-rac1-KO) mice. Subcutaneous and epididymal white adipose tissues (WATs) in adipo-rac1-KO mice showed significant reductions in size and weight. Actually, white adipocytes lacking Rac1 were smaller than controls. Insulin-stimulated glucose uptake and GLUT4 translocation were abrogated in rac1-KO white adipocytes. On the other hand, GLUT4 translocation was augmented by constitutively activated PI3K or Akt2 in control, but not in rac1-KO, white adipocytes. Similarly, to skeletal muscle, the involvement of another small GTPase RalA downstream of Rac1 was demonstrated. In addition, mRNA levels of various lipogenic enzymes were down-regulated in rac1-KO white adipocytes. Collectively, these results suggest that Rac1 is implicated in insulin-dependent glucose uptake and lipogenesis in white adipocytes, and reduced insulin responsiveness due to the deficiency of Rac1 may be a likely explanation for atrophy of WATs.

Diverse GTPases act as signal transducing enzymes in a variety of organisms and cell types

Insulin stimulates glucose uptake via the translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes. Several lines of evidence suggest that the small GTPase Rac1 plays an important role in insulin‐stimulated glucose uptake in skeletal muscle and adipocytes. The purpose of this study is to investigate the mechanisms whereby Rac1 is regulated in adipocyte insulin signaling. Here, we show that knockdown of the guanine nucleotide exchange factor FLJ00068 inhibits Rac1 activation and GLUT4 translocation by insulin and a constitutively activated form of the protein kinase Akt2. Furthermore, constitutively activated FLJ00068 induced Rac1 activation and Rac1‐dependent GLUT4 translocation. Collectively, these results suggest the involvement of FLJ00068 downstream of Akt2 in insulin‐stimulated glucose uptake signaling in adipocytes.

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.