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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.

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(A) Oil-Red O staining. 3T3-L1-GLUT4myc fibroblasts were stained with Oil-Red O on day 0, day 3, and day 14 after differentiation induction. Bars, 100 μm. Note that adipose droplets in cells are dyed red and that a similar effect was found in four independent experiments. (B) Cells collected on day 0, day 3, and day 14 after differentiation induction were lysed followed by western blotting using antibodies against PPARγ and β-actin. In the blotting membrane shown, an anti-PPARγ antibody produces two signal bands at 53 and 57 kDa, each corresponding to PPARγ1 and PPARγ2. Signal intensities for PPARγ1 and PPARγ2 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PPARγ1 and PPARγ2 (n=4 independent experiments). P values as compared with the expression on day 0, Dunnett's test.
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fig1: (A) Oil-Red O staining. 3T3-L1-GLUT4myc fibroblasts were stained with Oil-Red O on day 0, day 3, and day 14 after differentiation induction. Bars, 100 μm. Note that adipose droplets in cells are dyed red and that a similar effect was found in four independent experiments. (B) Cells collected on day 0, day 3, and day 14 after differentiation induction were lysed followed by western blotting using antibodies against PPARγ and β-actin. In the blotting membrane shown, an anti-PPARγ antibody produces two signal bands at 53 and 57 kDa, each corresponding to PPARγ1 and PPARγ2. Signal intensities for PPARγ1 and PPARγ2 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PPARγ1 and PPARγ2 (n=4 independent experiments). P values as compared with the expression on day 0, Dunnett's test.

Mentions: We initially examined whether 3T3-L1-GLUT4myc fibroblasts are differentiated into adipocytes using Oil-Red O. Adipose tissue is stained with Oil-Red O, to detect adipose conversion (Ramírez-Zacarías et al. 1992). 3T3-L1-GLUT4myc fibroblasts before differentiation induction (day 0), with spindle-like shapes, had no reaction to Oil-Red O (Fig. 1A). On day 3 after differentiation induction a few cells, which had assumed a round shape, were positive to Oil-Red O and on day 14 almost all the cells, which had assumed a round shape, were dyed red (Fig. 1A). This indicates that 3T3-L1-GLUT4myc fibroblasts are readily differentiated into 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)

(A) Oil-Red O staining. 3T3-L1-GLUT4myc fibroblasts were stained with Oil-Red O on day 0, day 3, and day 14 after differentiation induction. Bars, 100 μm. Note that adipose droplets in cells are dyed red and that a similar effect was found in four independent experiments. (B) Cells collected on day 0, day 3, and day 14 after differentiation induction were lysed followed by western blotting using antibodies against PPARγ and β-actin. In the blotting membrane shown, an anti-PPARγ antibody produces two signal bands at 53 and 57 kDa, each corresponding to PPARγ1 and PPARγ2. Signal intensities for PPARγ1 and PPARγ2 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PPARγ1 and PPARγ2 (n=4 independent experiments). P values as compared with the expression on day 0, Dunnett's test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: (A) Oil-Red O staining. 3T3-L1-GLUT4myc fibroblasts were stained with Oil-Red O on day 0, day 3, and day 14 after differentiation induction. Bars, 100 μm. Note that adipose droplets in cells are dyed red and that a similar effect was found in four independent experiments. (B) Cells collected on day 0, day 3, and day 14 after differentiation induction were lysed followed by western blotting using antibodies against PPARγ and β-actin. In the blotting membrane shown, an anti-PPARγ antibody produces two signal bands at 53 and 57 kDa, each corresponding to PPARγ1 and PPARγ2. Signal intensities for PPARγ1 and PPARγ2 were normalized to those for β-actin. In the graph, each column represents the mean (±s.e.m.) normalized expression of PPARγ1 and PPARγ2 (n=4 independent experiments). P values as compared with the expression on day 0, Dunnett's test.
Mentions: We initially examined whether 3T3-L1-GLUT4myc fibroblasts are differentiated into adipocytes using Oil-Red O. Adipose tissue is stained with Oil-Red O, to detect adipose conversion (Ramírez-Zacarías et al. 1992). 3T3-L1-GLUT4myc fibroblasts before differentiation induction (day 0), with spindle-like shapes, had no reaction to Oil-Red O (Fig. 1A). On day 3 after differentiation induction a few cells, which had assumed a round shape, were positive to Oil-Red O and on day 14 almost all the cells, which had assumed a round shape, were dyed red (Fig. 1A). This indicates that 3T3-L1-GLUT4myc fibroblasts are readily differentiated into 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