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

Takenaka N, Nihata Y, Satoh T - PLoS ONE (2016)

Bottom Line: This Rac1 activation was also abrogated by FLJ00068 knockdown.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

FLJ00068-dependent activation of Rac1 in mouse gastrocnemius muscle fibers.(A) The expression vector for FLJ68ΔN was introduced into gastrocnemius muscle fibers of wild-type mice. Endogenous Rac1 was detected by immunofluorescent staining with an anti-Rac1 antibody. FLJ68ΔN was detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3 or GST-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (B) Expression vectors for Myr-p110α and Myr-Akt2, together with either one of two siRNA duplexes against mouse FLJ00068 (#1 and #2) and a mixture of NC siRNA duplexes, were introduced into gastrocnemius muscle fibers of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous Rac1 and FLJ00068 were detected by immunofluorescent staining with anti-Rac1 and anti-FLJ00068 antibodies, respectively. Myr-p110α and Myr-Akt2 were detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (C) Activation of Rac1 shown in (B) was quantified. Gray, orange, and blue bars represent the treatment with NC, #1, and #2 siRNA duplexes, respectively. Data are shown as means ± S.E. (n = 6). *P < 0.001.
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pone.0155292.g003: FLJ00068-dependent activation of Rac1 in mouse gastrocnemius muscle fibers.(A) The expression vector for FLJ68ΔN was introduced into gastrocnemius muscle fibers of wild-type mice. Endogenous Rac1 was detected by immunofluorescent staining with an anti-Rac1 antibody. FLJ68ΔN was detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3 or GST-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (B) Expression vectors for Myr-p110α and Myr-Akt2, together with either one of two siRNA duplexes against mouse FLJ00068 (#1 and #2) and a mixture of NC siRNA duplexes, were introduced into gastrocnemius muscle fibers of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous Rac1 and FLJ00068 were detected by immunofluorescent staining with anti-Rac1 and anti-FLJ00068 antibodies, respectively. Myr-p110α and Myr-Akt2 were detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (C) Activation of Rac1 shown in (B) was quantified. Gray, orange, and blue bars represent the treatment with NC, #1, and #2 siRNA duplexes, respectively. Data are shown as means ± S.E. (n = 6). *P < 0.001.

Mentions: To further examine whether FLJ00068 is implicated in insulin signaling as an activator for Rac1, the activation state of Rac1 in gastrocnemius muscle was examined. Recently, we were successful in detecting the activated form of Rac1 in isolated muscle fibers by immunofluorescent microscopy by the use of a polypeptide probe that specifically recognizes activated Rac1 [25]. Specific interaction of the activation-specific Rac1 probe GST-POSH(251–489)-V5×3, but not the control polypeptide GST-V5×3, with GTP-bound activated Rac1, which were generated by ectopic expression of the constitutively activated FLJ00068 mutant FLJ68ΔN [21], was confirmed in gastrocnemius muscle fibers (Fig 3A). Rac1 activation following intravenous injection of insulin was remarkably inhibited by siRNA-mediated knockdown of FLJ00068 as revealed by the above immunofluorescent microscopy-based overlay assay (Fig 3B and 3C). Furthermore, Rac1 activation in response to ectopic expression of constitutively activated PI3K or Akt2 was highly sensitive to the inhibitory effect of FLJ00068 knockdown (Fig 3B and 3C). Taken together, these results strongly support the notion that FLJ00068 in fact acts as a specific Rac1 activator downstream of Akt2 in insulin signaling.


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

Takenaka N, Nihata Y, Satoh T - PLoS ONE (2016)

FLJ00068-dependent activation of Rac1 in mouse gastrocnemius muscle fibers.(A) The expression vector for FLJ68ΔN was introduced into gastrocnemius muscle fibers of wild-type mice. Endogenous Rac1 was detected by immunofluorescent staining with an anti-Rac1 antibody. FLJ68ΔN was detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3 or GST-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (B) Expression vectors for Myr-p110α and Myr-Akt2, together with either one of two siRNA duplexes against mouse FLJ00068 (#1 and #2) and a mixture of NC siRNA duplexes, were introduced into gastrocnemius muscle fibers of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous Rac1 and FLJ00068 were detected by immunofluorescent staining with anti-Rac1 and anti-FLJ00068 antibodies, respectively. Myr-p110α and Myr-Akt2 were detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (C) Activation of Rac1 shown in (B) was quantified. Gray, orange, and blue bars represent the treatment with NC, #1, and #2 siRNA duplexes, respectively. Data are shown as means ± S.E. (n = 6). *P < 0.001.
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pone.0155292.g003: FLJ00068-dependent activation of Rac1 in mouse gastrocnemius muscle fibers.(A) The expression vector for FLJ68ΔN was introduced into gastrocnemius muscle fibers of wild-type mice. Endogenous Rac1 was detected by immunofluorescent staining with an anti-Rac1 antibody. FLJ68ΔN was detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3 or GST-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (B) Expression vectors for Myr-p110α and Myr-Akt2, together with either one of two siRNA duplexes against mouse FLJ00068 (#1 and #2) and a mixture of NC siRNA duplexes, were introduced into gastrocnemius muscle fibers of wild-type mice. Insulin (175.5 μg/kg body weight) was administered intravenously. Endogenous Rac1 and FLJ00068 were detected by immunofluorescent staining with anti-Rac1 and anti-FLJ00068 antibodies, respectively. Myr-p110α and Myr-Akt2 were detected by immunofluorescent staining with an anti-HA antibody. Activated Rac1 (Rac1·GTP) was visualized by immunofluorescent staining with an anti-V5 antibody after incubation with GST-POSH(251–489)-V5×3. Scale bar, 20 μm. The position of the focal plane from which the image was acquired is shown in Fig 2B. (C) Activation of Rac1 shown in (B) was quantified. Gray, orange, and blue bars represent the treatment with NC, #1, and #2 siRNA duplexes, respectively. Data are shown as means ± S.E. (n = 6). *P < 0.001.
Mentions: To further examine whether FLJ00068 is implicated in insulin signaling as an activator for Rac1, the activation state of Rac1 in gastrocnemius muscle was examined. Recently, we were successful in detecting the activated form of Rac1 in isolated muscle fibers by immunofluorescent microscopy by the use of a polypeptide probe that specifically recognizes activated Rac1 [25]. Specific interaction of the activation-specific Rac1 probe GST-POSH(251–489)-V5×3, but not the control polypeptide GST-V5×3, with GTP-bound activated Rac1, which were generated by ectopic expression of the constitutively activated FLJ00068 mutant FLJ68ΔN [21], was confirmed in gastrocnemius muscle fibers (Fig 3A). Rac1 activation following intravenous injection of insulin was remarkably inhibited by siRNA-mediated knockdown of FLJ00068 as revealed by the above immunofluorescent microscopy-based overlay assay (Fig 3B and 3C). Furthermore, Rac1 activation in response to ectopic expression of constitutively activated PI3K or Akt2 was highly sensitive to the inhibitory effect of FLJ00068 knockdown (Fig 3B and 3C). Taken together, these results strongly support the notion that FLJ00068 in fact acts as a specific Rac1 activator downstream of Akt2 in insulin signaling.

Bottom Line: This Rac1 activation was also abrogated by FLJ00068 knockdown.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka, Japan.

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.

No MeSH data available.


Related in: MedlinePlus