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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

Insulin-dependent subcellular translocation of FLJ00068 to the plasma membrane in L6-GLUT4 cells and mouse gastrocnemius muscle.(A) Serum-starved L6-GLUT4 cells were stimulated with insulin for indicated times. Endogenous FLJ00068 was detected by immunofluorescent staining with an anti-FLJ00068 antibody. Arrow heads indicate the localization of FLJ00068 in the tip of membrane ruffle-like structures. Scale bar, 50 μm. (B) Localization of FLJ00068 in cytosol (C) and crude plasma membrane (P) fractions of insulin-stimulated and unstimulated L6-GLUT4 cells and mouse gastrocnemius muscle was examined by immunoblot analysis with an anti-FLJ00068 antibody. Α-tubulin and Na+/K+-ATPase were visualized as marker proteins for cytosol and crude plasma membrane fractions, respectively, by specific antibodies.
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pone.0155292.g005: Insulin-dependent subcellular translocation of FLJ00068 to the plasma membrane in L6-GLUT4 cells and mouse gastrocnemius muscle.(A) Serum-starved L6-GLUT4 cells were stimulated with insulin for indicated times. Endogenous FLJ00068 was detected by immunofluorescent staining with an anti-FLJ00068 antibody. Arrow heads indicate the localization of FLJ00068 in the tip of membrane ruffle-like structures. Scale bar, 50 μm. (B) Localization of FLJ00068 in cytosol (C) and crude plasma membrane (P) fractions of insulin-stimulated and unstimulated L6-GLUT4 cells and mouse gastrocnemius muscle was examined by immunoblot analysis with an anti-FLJ00068 antibody. Α-tubulin and Na+/K+-ATPase were visualized as marker proteins for cytosol and crude plasma membrane fractions, respectively, by specific antibodies.

Mentions: It is important to elucidate the molecular basis of insulin-dependent activation of FLJ00068, and therefore, as a first step, subcellular localization of FLJ00068 was examined. Translocation of FLJ00068 to the cell periphery, particularly to the tip of membrane ruffle-like structures, was observed within 5 minutes after the addition of insulin in L6-GLUT4 cells (Fig 5A). The peripheral localization of FLJ00068 was also observed after 20 minute stimulation and maintained at least for 60 minutes (Fig 5A and data not shown). The plasma membrane localization of FLJ00068 in insulin-stimulated L6-GLUT4 cells was also confirmed by subcellular fractionation and immunoblotting (Fig 5B). Similar results were obtained from subcellular fractionation of gastrocnemius muscle isolated from insulin-administered mice, supporting the notion that subcellular translocation of FLJ00068 to the plasma membrane in fact occurs following insulin stimulation in mouse skeletal muscle (Fig 5B). This change of subcellular localization of FLJ00068 is not obvious in images obtained by immunofluorescent microscopy due to the very thin cytoplasmic region in skeletal muscle fibers. (Figs 2B, 3B and 4B). Taken together, these findings may provide important clues to understand the regulatory mechanisms of FLJ00068 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)

Insulin-dependent subcellular translocation of FLJ00068 to the plasma membrane in L6-GLUT4 cells and mouse gastrocnemius muscle.(A) Serum-starved L6-GLUT4 cells were stimulated with insulin for indicated times. Endogenous FLJ00068 was detected by immunofluorescent staining with an anti-FLJ00068 antibody. Arrow heads indicate the localization of FLJ00068 in the tip of membrane ruffle-like structures. Scale bar, 50 μm. (B) Localization of FLJ00068 in cytosol (C) and crude plasma membrane (P) fractions of insulin-stimulated and unstimulated L6-GLUT4 cells and mouse gastrocnemius muscle was examined by immunoblot analysis with an anti-FLJ00068 antibody. Α-tubulin and Na+/K+-ATPase were visualized as marker proteins for cytosol and crude plasma membrane fractions, respectively, by specific antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862641&req=5

pone.0155292.g005: Insulin-dependent subcellular translocation of FLJ00068 to the plasma membrane in L6-GLUT4 cells and mouse gastrocnemius muscle.(A) Serum-starved L6-GLUT4 cells were stimulated with insulin for indicated times. Endogenous FLJ00068 was detected by immunofluorescent staining with an anti-FLJ00068 antibody. Arrow heads indicate the localization of FLJ00068 in the tip of membrane ruffle-like structures. Scale bar, 50 μm. (B) Localization of FLJ00068 in cytosol (C) and crude plasma membrane (P) fractions of insulin-stimulated and unstimulated L6-GLUT4 cells and mouse gastrocnemius muscle was examined by immunoblot analysis with an anti-FLJ00068 antibody. Α-tubulin and Na+/K+-ATPase were visualized as marker proteins for cytosol and crude plasma membrane fractions, respectively, by specific antibodies.
Mentions: It is important to elucidate the molecular basis of insulin-dependent activation of FLJ00068, and therefore, as a first step, subcellular localization of FLJ00068 was examined. Translocation of FLJ00068 to the cell periphery, particularly to the tip of membrane ruffle-like structures, was observed within 5 minutes after the addition of insulin in L6-GLUT4 cells (Fig 5A). The peripheral localization of FLJ00068 was also observed after 20 minute stimulation and maintained at least for 60 minutes (Fig 5A and data not shown). The plasma membrane localization of FLJ00068 in insulin-stimulated L6-GLUT4 cells was also confirmed by subcellular fractionation and immunoblotting (Fig 5B). Similar results were obtained from subcellular fractionation of gastrocnemius muscle isolated from insulin-administered mice, supporting the notion that subcellular translocation of FLJ00068 to the plasma membrane in fact occurs following insulin stimulation in mouse skeletal muscle (Fig 5B). This change of subcellular localization of FLJ00068 is not obvious in images obtained by immunofluorescent microscopy due to the very thin cytoplasmic region in skeletal muscle fibers. (Figs 2B, 3B and 4B). Taken together, these findings may provide important clues to understand the regulatory mechanisms of FLJ00068 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