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

We would like to thank Dr. Emilie Martinez and Jill Murray for their excellent technical assistance and animal care.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.Yeshttp://www.plosone.org/static/editorial#pee

Validation of the insulin-deficient (type 1) diabetic animal model.

<p><b>A) Mean ± SE venous blood glucose concentration</b> obtained at baseline and up to 8 weeks in type 1 diabetic (T1Dx) and age-matched control (Con) mice (n = 9-11/group). <b>B) Mean ± SE body weight</b> obtained at baseline and up to 8 weeks after induction of type 1 diabetes (n = 9-11/group). <b>C) Mean ± SE serum insulin concentration</b> obtained at 8 weeks after induction of type 1 diabetes (n = 6-8/group). T1Dx: type 1 diabetic; Con: control; *P<0.05 vs. control; # P<0.05 vs. baseline.</p

Intact insulin signaling pathway in the atria of insulin-deficient diabetic animals.

<p><b>Type 1 diabetes (T1 Dx) did not alter A) Akt or B) AS160 phosphorylation in the atria</b>. Top panels: representative Western blot from total lysate of mouse atria; calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of protein expression (values expressed relative to control; n = 4-5/group). <b>Insulin stimulates C) GLUT4 and D) GLUT8 trafficking to the atrial cell surface in type 1 diabetic (T1 Dx) subjects</b>. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to control basal labeled; n = 4-6/group). Methods: Intact mouse hearts were perfused with and without insulin, and photolabeled with bio-LC-ATB-BGPA to determine the amount of cell surface (L: labeled fraction) and intracellular (UL: unlabeled fraction) content. T1Dx: type 1 diabetic; *P<0.05 vs. control; # P<0.05 vs. basal.</p

Analysis of the downstream insulin signaling pathways in the healthy atria.

<p><b>A) Insulin stimulates phosphorylation of Akt at s473 and Th308 site</b> 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>B) Significant linear positive linear correlation between Akt phosphorylation (at s473 and Th308 site</b>) <b>and GLUT4 expression</b> in the healthy atria. <b>C) Insulin stimulates phosphorylation of AS160</b> 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. <b>D) Significant linear correlation between AS160 phosphorylation and GLUT-4 and -8 expression</b> in the healthy atria.</p