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Insulin induced translocation of Na+/K+ -ATPase is decreased in the heart of streptozotocin diabetic rats.

Rosta K, Tulassay E, Enzsoly A, Ronai K, Szantho A, Pandics T, Fekete A, Mandl P, Ver A - Acta Pharmacol. Sin. (2009)

Bottom Line: The insulin-induced translocation was wortmannin sensitive.This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat.Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary. roskla@hotmail.com

ABSTRACT

Aim: To investigate the effect of acute insulin administration on the subcellular localization of Na(+)/K(+)-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.

Methods: Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na(+)/K(+)-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.

Results: In control rat heart muscle alpha1 isoform of Na(+)/K(+) ATPase resides mainly in the plasma membrane fraction, while alpha2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of alpha1 isoform (25 %, P<0.05) in plasma membrane and alpha2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha2- but not alpha1-subunits was detected. Alpha1-subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of alpha2 increased by 3.3-fold, alpha1 by 1.37-fold and beta1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.

Conclusion: This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

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Related in: MedlinePlus

The relative amount of Na+/K+-ATPase α1 (A), α2 (B), β1 (C) subunits and GLUT4 (D) in control and STZ diabetic rat hearts with or without insulin treatment. Crude membranes (CM) were prepared as described in material and methods. Values are mean±SEM of 5 experiments. Relative density was calculated from optical density film negative of Western blot corrected for the amount of total protein blotted onto the nitrocellulose for each gel lane. Results are expressed in arbitrary units relative to the value in crude membrane. bP≤0.05 vs control (C) and streptozotocin-diabetic rats (D).
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fig2: The relative amount of Na+/K+-ATPase α1 (A), α2 (B), β1 (C) subunits and GLUT4 (D) in control and STZ diabetic rat hearts with or without insulin treatment. Crude membranes (CM) were prepared as described in material and methods. Values are mean±SEM of 5 experiments. Relative density was calculated from optical density film negative of Western blot corrected for the amount of total protein blotted onto the nitrocellulose for each gel lane. Results are expressed in arbitrary units relative to the value in crude membrane. bP≤0.05 vs control (C) and streptozotocin-diabetic rats (D).

Mentions: In crude membrane (CM) preparations diabetes decreased, while acute insulin treatment did not affect total Na+/K+-ATPase subunit isoform and GLUT4 content (Figure 1A, Figure 2). The effect of insulin on the distribution of subunit isoforms of Na+/K+-ATPase among plasma and internal membranes was also studied by Western blot analysis (Figure 1B). The α1 isoform was found mostly in the plasma membrane and was reduced by 17.3% in D rats. No detectable effect of insulin could be observed. The α2 isoform was found in the intracellular membrane fractions both in C and D hearts, and the amount of the α2 isoform decreased by 40.2% (P<0.01) in diabetes.


Insulin induced translocation of Na+/K+ -ATPase is decreased in the heart of streptozotocin diabetic rats.

Rosta K, Tulassay E, Enzsoly A, Ronai K, Szantho A, Pandics T, Fekete A, Mandl P, Ver A - Acta Pharmacol. Sin. (2009)

The relative amount of Na+/K+-ATPase α1 (A), α2 (B), β1 (C) subunits and GLUT4 (D) in control and STZ diabetic rat hearts with or without insulin treatment. Crude membranes (CM) were prepared as described in material and methods. Values are mean±SEM of 5 experiments. Relative density was calculated from optical density film negative of Western blot corrected for the amount of total protein blotted onto the nitrocellulose for each gel lane. Results are expressed in arbitrary units relative to the value in crude membrane. bP≤0.05 vs control (C) and streptozotocin-diabetic rats (D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007495&req=5

fig2: The relative amount of Na+/K+-ATPase α1 (A), α2 (B), β1 (C) subunits and GLUT4 (D) in control and STZ diabetic rat hearts with or without insulin treatment. Crude membranes (CM) were prepared as described in material and methods. Values are mean±SEM of 5 experiments. Relative density was calculated from optical density film negative of Western blot corrected for the amount of total protein blotted onto the nitrocellulose for each gel lane. Results are expressed in arbitrary units relative to the value in crude membrane. bP≤0.05 vs control (C) and streptozotocin-diabetic rats (D).
Mentions: In crude membrane (CM) preparations diabetes decreased, while acute insulin treatment did not affect total Na+/K+-ATPase subunit isoform and GLUT4 content (Figure 1A, Figure 2). The effect of insulin on the distribution of subunit isoforms of Na+/K+-ATPase among plasma and internal membranes was also studied by Western blot analysis (Figure 1B). The α1 isoform was found mostly in the plasma membrane and was reduced by 17.3% in D rats. No detectable effect of insulin could be observed. The α2 isoform was found in the intracellular membrane fractions both in C and D hearts, and the amount of the α2 isoform decreased by 40.2% (P<0.01) in diabetes.

Bottom Line: The insulin-induced translocation was wortmannin sensitive.This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat.Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary. roskla@hotmail.com

ABSTRACT

Aim: To investigate the effect of acute insulin administration on the subcellular localization of Na(+)/K(+)-ATPase isoforms in cardiac muscle of healthy and streptozotocin-induced diabetic rats.

Methods: Membrane fractions were isolated with subcellular fractionation and with cell surface biotinylation technique. Na(+)/K(+)-ATPase subunit isoforms were analysed with ouabain binding assay and Western blotting. Enzyme activity was measured using 3-O-methylfluorescein-phosphatase activity.

Results: In control rat heart muscle alpha1 isoform of Na(+)/K(+) ATPase resides mainly in the plasma membrane fraction, while alpha2 isoform in the intracellular membrane pool. Diabetes decreased the abundance of alpha1 isoform (25 %, P<0.05) in plasma membrane and alpha2 isoform (50%, P<0.01) in the intracellular membrane fraction. When plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha2- but not alpha1-subunits was detected. Alpha1-subunit translocation was only detectable by cell surface biotinylation technique. After insulin administration protein level of alpha2 increased by 3.3-fold, alpha1 by 1.37-fold and beta1 by 1.51-fold (P<0.02) in the plasma membrane of control, and less than 1.92-fold (P<0.02), 1.19-fold (not significant) and 1.34-fold (P<0.02) in diabetes. The insulin-induced translocation was wortmannin sensitive.

Conclusion: This study demonstrates that insulin influences the plasma membrane localization of Na(+)/K(+)-ATPase isoforms in the heart. alpha2 isoform translocation is the most vulnerable to the reduced insulin response in diabetes. alpha1 isoform also translocates in response to insulin treatment in healthy rat. Insulin mediates Na(+)/K(+)-ATPase alpha1- and alpha2-subunit translocation to the cardiac muscle plasma membrane via a PI3-kinase-dependent mechanism.

Show MeSH
Related in: MedlinePlus