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Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria.

Maria Z, Campolo AR, Lacombe VA - PLoS ONE (2015)

Bottom Line: In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05).Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria.Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, United States of America.

ABSTRACT
Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.

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Analysis of the downstream insulin signaling pathways in the healthy atria.A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes incubated with (0.01μM) and without (basal) insulin; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 5/group); # P<0.05 vs. basal. B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site) and GLUT4 expression in the healthy atria. C) Insulin stimulates phosphorylation of AS160 in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 6-8/group); # P<0.05 vs. basal. D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression in the healthy atria.
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pone.0146033.g002: Analysis of the downstream insulin signaling pathways in the healthy atria.A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes incubated with (0.01μM) and without (basal) insulin; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 5/group); # P<0.05 vs. basal. B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site) and GLUT4 expression in the healthy atria. C) Insulin stimulates phosphorylation of AS160 in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 6-8/group); # P<0.05 vs. basal. D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression in the healthy atria.

Mentions: In order to investigate the mechanisms regulating the translocation of GLUT-4 and -8 to the atrial cell surface, we then explored the downstream insulin signaling pathway, by incubating rat atrial myocytes with and without (basal) insulin. We reported a significant increase in the phosphorylation of Akt (at s473 and Th308 sites) and AS160 upon insulin stimulation (Fig 2A and 2C). No significant change was observed when phospho AS160 (Fig 2C) was compared to total AS160. In addition, we reported a significant positive linear correlation between total GLUT4 protein content and phosphorylation of Akt (Fig 2B), as well as between total GLUT protein content and AS160 activation (Fig 2D).


Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria.

Maria Z, Campolo AR, Lacombe VA - PLoS ONE (2015)

Analysis of the downstream insulin signaling pathways in the healthy atria.A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes incubated with (0.01μM) and without (basal) insulin; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 5/group); # P<0.05 vs. basal. B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site) and GLUT4 expression in the healthy atria. C) Insulin stimulates phosphorylation of AS160 in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 6-8/group); # P<0.05 vs. basal. D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression in the healthy atria.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697822&req=5

pone.0146033.g002: Analysis of the downstream insulin signaling pathways in the healthy atria.A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes incubated with (0.01μM) and without (basal) insulin; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 5/group); # P<0.05 vs. basal. B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site) and GLUT4 expression in the healthy atria. C) Insulin stimulates phosphorylation of AS160 in atrial myocytes. Top panel: representative Western blot from total lysate of isolated rat atrial myocytes; calsequestrin (CLSQ) was used as a loading control. Bottom panel: Mean ± SE of protein expression (values expressed relative to basal; n = 6-8/group); # P<0.05 vs. basal. D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression in the healthy atria.
Mentions: In order to investigate the mechanisms regulating the translocation of GLUT-4 and -8 to the atrial cell surface, we then explored the downstream insulin signaling pathway, by incubating rat atrial myocytes with and without (basal) insulin. We reported a significant increase in the phosphorylation of Akt (at s473 and Th308 sites) and AS160 upon insulin stimulation (Fig 2A and 2C). No significant change was observed when phospho AS160 (Fig 2C) was compared to total AS160. In addition, we reported a significant positive linear correlation between total GLUT4 protein content and phosphorylation of Akt (Fig 2B), as well as between total GLUT protein content and AS160 activation (Fig 2D).

Bottom Line: In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05).Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria.Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, United States of America.

ABSTRACT
Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.

Show MeSH
Related in: MedlinePlus