Fig 1.
Regional heterogeneity of the insulin-sensitive GLUT4 and GLUT8 in the healthy myocardium.
Higher total protein expression of A) GLUT4 and lower B) GLUT8 content in the healthy atrial compared to ventricular myocytes. Top panels: representative Western blot from total lysate of isolated rat myocytes; calsequestrin (CLSQ) was used as a loading control; representative bands were obtained from the same membrane. Bottom panels: Mean ± SE of total GLUT protein content (values expressed relative to atria; n = 6/group); * P<0.05 vs. atria. GLUT: glucose transporters. Insulin stimulation increases C) GLUT4 and D) GLUT8 protein content in the healthy atrial myocytes. Top panels: representative Western blot from total lysate of isolated rat myocytes, calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of total GLUT protein content (values expressed relative to basal atria; n = 8-11/group); # P<0.05 vs. basal. Insulin stimulates E) GLUT4 and F) GLUT8 trafficking to the atrial and ventricular cell surface. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to labeled basal atria; n = 3-4/group); # P<0.05 vs. basal; *P<0.05 vs. atria. Methods: Cell surface GLUT measured using biotinylated photolabeling technique in the intact perfused mouse heart. L: Labeled (cell surface fraction); UL: Unlabeled (intracellular fraction).
Fig 2.
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.
Fig 3.
Validation of the insulin-deficient (type 1) diabetic animal model.
A) Mean ± SE venous blood glucose concentration obtained at baseline and up to 8 weeks in type 1 diabetic (T1Dx) and age-matched control (Con) mice (n = 9-11/group). B) Mean ± SE body weight obtained at baseline and up to 8 weeks after induction of type 1 diabetes (n = 9-11/group). C) Mean ± SE serum insulin concentration 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.
Fig 4.
Alteration of the trafficking of the insulin-sensitive GLUTs in the diabetic atria.
Decreased atrial A) GLUT4 and B) GLUT8 content during type 1 diabetes (T1Dx). Top panels: representative Western blot from crude membrane extract of perfused mouse atria, calsequestrin (CLSQ) was used as a loading control. Bottom panels: Mean ± SE of GLUT protein content (values expressed relative to control; n = 7-8/group). Type 1 diabetes decreased C) GLUT4 and D) GLUT8 trafficking to the atrial cell surface. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values expressed relative to control; n = 4-5/group). Majority of E) GLUT4 and F) GLUT8 is intracellular under basal conditions (mean ± SE, values expressed relative to control labeled, n = 5/group). Methods: Intact perfused mouse hearts were 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; Con: control; * P<0.05 vs. control.
Fig 5.
Intact insulin signaling pathway in the atria of insulin-deficient diabetic animals.
Type 1 diabetes (T1 Dx) did not alter A) Akt or B) AS160 phosphorylation in the atria. 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). Insulin stimulates C) GLUT4 and D) GLUT8 trafficking to the atrial cell surface in type 1 diabetic (T1 Dx) subjects. 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.