Limits...
PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway.

Tsuchiya A, Kanno T, Nishizaki T - J. Endocrinol. (2013)

Bottom Line: Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2.Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1.In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309.

View Article: PubMed Central - PubMed

Affiliation: Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan.

ABSTRACT
Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1  nM-1  μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation.

Show MeSH

Related in: MedlinePlus

Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PI3K (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PDK1 (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842210&req=5

fig5: Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PI3K (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PDK1 (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Mentions: To knock-down PI3K and PDK1, we constructed siRNAs for PI3K and PDK1. In the western blot analysis, expression of PI3K and PDK1 proteins was significantly reduced in 3T3-L1-GLUT4myc adipocytes transfected with the PI3K siRNA and the PDK1 siRNA, respectively, as compared with that for cells transfected with each NC siRNA (Fig. 5A and B), confirming knocking-down of PI3K and PDK1. Insulin (100 nM)-induced phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 was apparently suppressed by knocking-down PI3K (Fig. 5A), but otherwise no inhibition was obtained by knocking-down PDK1 (Fig. 5B). This further supports the notion that insulin stimulates Akt1/2 activation in a PI3K-dependent manner, without PDK1, in adipocytes.


PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway.

Tsuchiya A, Kanno T, Nishizaki T - J. Endocrinol. (2013)

Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PI3K (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PDK1 (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842210&req=5

fig5: Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PI3K (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PDK1 (n=4 independent experiments). P value, unpaired t-test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (±s.e.m.) normalized intensity for pAkt1/2 at each site (n=4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.
Mentions: To knock-down PI3K and PDK1, we constructed siRNAs for PI3K and PDK1. In the western blot analysis, expression of PI3K and PDK1 proteins was significantly reduced in 3T3-L1-GLUT4myc adipocytes transfected with the PI3K siRNA and the PDK1 siRNA, respectively, as compared with that for cells transfected with each NC siRNA (Fig. 5A and B), confirming knocking-down of PI3K and PDK1. Insulin (100 nM)-induced phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 was apparently suppressed by knocking-down PI3K (Fig. 5A), but otherwise no inhibition was obtained by knocking-down PDK1 (Fig. 5B). This further supports the notion that insulin stimulates Akt1/2 activation in a PI3K-dependent manner, without PDK1, in adipocytes.

Bottom Line: Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2.Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1.In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309.

View Article: PubMed Central - PubMed

Affiliation: Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan.

ABSTRACT
Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1  nM-1  μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation.

Show MeSH
Related in: MedlinePlus