Characterizing Adrenergic Regulation of Glucose Transporter 4-Mediated Glucose Uptake and Metabolism in the Heart

Abstract

Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac β₂ adrenoreceptor (β₂AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac β₂AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of β₂AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.

Keywords: Akt substrate of 160 kDa; G-protein receptor kinase 2; adrenergic receptor; glucose oxidation; glucose transporter 4; glucose uptake.

Graphical abstract

Figure 1

Effects of β₂AR Deletion on Glucose and Oleate Oxidation in Heart Under Adrenergic Stimulation (A, B) Time course and peak response in glucose oxidation before and after stimulation with isoproterenol (ISO) (100 nmol/L). (C, D) Time course and peak response in oleate oxidation before and after stimulation with ISO (100 nmol/L). (A to D) Data represent mean ± SEM, n = 8; P values were analyzed using 2-way analysis of variance with Tukey’s multiple comparison test. (E) Western blot showing glucose transporter 4 (GLUT4) localization after intraperitoneal ISO injection (60 μg/kg) for 30 minutes. (F, G) The Western blots were quantified using ImageJ, and arbitrary units were defined as the ratio of intensity of GLUT4 over the intensity of insulin receptor β (IRβ) for plasma membrane (PM) fractions (F) and the ratio of intensity of GLUT4 over the intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for total protein level (G). Data represent mean ± SEM, n = 3; ∗P < 0.05 and P < 0.0001 were analyzed using 2-way analysis of variance with Tukey’s multiple comparison test. β₂AR = β₂ adrenoreceptor; cKO = cardiac specific b2AR knockout; F/F = b2AR flox mice.

Figure 2

Inhibition of β₂AR Decreases Adrenergic Stimulation of GLUT4 Translocation and Glucose Uptake in AVMs (A) Western blots showing phosphorylation of protein kinase B (Akt) at Ser473 and Ser474 and phosphorylation (p) of Akt substrate of 160 kDa (AS160) at Thr172 in rat adult ventricular cardiomyocytes (AVMs) after stimulation with ISO (100 nmol/L, 10 minutes) or after pretreatment with β₂AR antagonist ICI118551 (ICI) (100 nmol/L, 5 minutes) or β₁AR antagonist CGP20712a (CGP) (300 nmol/L, 5 minutes) followed by stimulation with ISO. The Western blots were quantified using ImageJ, and arbitrary units were defined as the ratio of intensity of pAkt over the intensity of total (t) Akt (B, C) or as the ratio of intensity of pAS160 over the intensity GAPDH (D). (B to D) Data represent mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 were analyzed using 1-way analysis of variance with Tukey’s multiple comparison test. (E-H) Representative confocal images of GLUT4 staining and quantification of GLUT4 translocation (I) after stimulation with ISO (100 nmol/L, 30 minutes) or after pretreatment with β₂AR antagonist ICI (100 nmol/L, 5 minutes) or β₁AR antagonist CGP (300 nmol/L, 5 minutes) followed by stimulation with ISO. (J) Representative confocal images of 2-(N-(7-nitrobenz-2-oxa—1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) uptake and 2-NBDG uptake quantification (K) after stimulation with ISO (100 nmol/L, 30 minutes) or after pretreatment with β₂AR antagonist ICI (100 nmol/L, 5 minutes) or β₁AR antagonist CGP (300 nmol/L, 5 minutes) followed by stimulation with ISO. (I, K) Data represent mean ± SEM of myocytes from 3 mice; P < 0.0001 were obtained using 1-way analysis of variance with Tukey’s multiple comparison test. Cont = control; other abbreviations as in Figure 1.

Figure 3

Inhibition of Akt Blocks Adrenergic Stimulation of GLUT4 Translocation and Glucose Uptake in AVMs (A) Western blots showing pAkt at Ser473 and Ser474 and pAS160 at Thr172 in rat AVMs after stimulation with ISO (100 nmol/L, 10 minutes) or after pretreatment with Akt inhibitor MK2206 (10 μmol/L, 30 minutes) or adenosine monophosphate–activated protein kinase inhibitor CompoundC (CompC) (10 μmol/L, 30 minutes) followed by stimulation with ISO. (B to D) The Western blots were quantified using ImageJ, and arbitrary units were defined as the ratio of intensity of pAkt over the intensity of tAkt (B, C) or as the ratio of intensity of pAS160 over the intensity GAPDH (D). Data represent mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and P < 0.0001 were analyzed using one-way analysis of variance with Tukey’s multiple comparison test. (E to H) Representative confocal images of GLUT4 staining and quantification of GLUT4 translocation (I) after stimulation with ISO (100 nmol/L, 30 minutes) or after pretreatment with MK2206 (10 μmol/L, 30 minutes) or CompC (10 μmol/L, 30 minutes) followed by stimulation with ISO. (J) Representative confocal images of 2-NBDG uptake and 2-NBDG uptake quantification (K) after stimulation with ISO (100 nmol/L, 30 minutes) or after pretreatment with MK2206 (10 μmol/L, 30 minutes) or CompC (10 μmol/L, 30 minutes) followed by stimulation with ISO. (I, K) Data represent mean ± SEM of myocytes from 3 mice; P < 0.0001 were analyzed using 1-way analysis of variance with Tukey’s multiple comparison test. WT = wild type; other abbreviations as in Figures 1 and 2.

Figure 4

Activation of β₂AR Induces the Gi Pathway for Glucose Uptake (A) Western blots showing pAkt at Ser473 and Ser474 and pAS160 at Thr172 in rat AVMs after stimulation with ISO (100 nmol/L, 10 minutes) or after pretreatment with G inhibitory (Gi) inhibitor, pertussis toxin (PTX) (500 ng/mL, 3 hours) followed by stimulation with ISO. The Western blots were quantified using ImageJ, and arbitrary units were defined as the ratio of intensity of pAkt over the intensity of tAkt (B, C) or as the ratio of intensity of pAS160 over the intensity of GAPDH (D). (B to D) Data represent mean  ± SEM of myocytes from 3 mice; ∗P < 0.05 and ∗∗∗P < 0.001 were analyzed using 1-way analysis of variance with Tukey's multiple comparison test. (E to G) Representative confocal images of GLUT4 staining and (H) quantification of GLUT4 translocation in rat AVMs after stimulation with ISO (100 nmol/L, 10 minutes) or after pretreatment with PTX (500 ng/mL, 3 hours) followed by stimulation with ISO. (I) Representative confocal images of 2-NBDG uptake and (J) 2-NBDG uptake quantification in rat AVMs after stimulation with ISO (100 nmol/L, 10 minutes) or after pretreatment with PTX (500 ng/mL, 3 hours) followed by stimulation with ISO. (H, J) Data represent mean ± SEM, n = 3; ∗∗∗∗P < 0.0001 were analyzed using 1-way analysis of variance with Tukey's multiple comparison test. NS = not significant; other abbreviations as in Figures 1, 2, and 3.

Figure 5

β₂AR Deletion Decreases Adrenergic Stimulation of GLUT4 Translocation and Glucose Uptake in AVMs (A to D) Representative confocal images of GLUT4 staining and quantification of GLUT4 translocation (E) in AVMs isolated from β₂AR flox or β₂AR CKO mice after stimulation with ISO (100 nmol/L, 30 minutes). (F) Representative confocal images of 2-NBDG uptake and 2-NBDG uptake quantification (G) in AVMs isolated from β₂AR flox or β₂AR CKO mice after stimulation with ISO (100 nmol/L, 30 minutes). (E, G) Data represent mean ± SEM of myocytes from 3 mice; P < 0.0001 were analyzed using two-way analysis of variance with Tukey’s multiple comparison test. (H, L) Western blots showing pAkt at Ser473 and Ser474 and pAS160 at Thr172 after intraperitoneal ISO injection (60 μg/kg) 10 minutes prior to organ harvest in β₂AR flox, β₂AR-CKO, WT, and β₂AR whole body knockout (KO), respectively. (I to K, M to O) Data represent mean ± SEM, n = 3-4; ∗P < 0.05 and ∗∗P < 0.01 were obtained using two-way analysis of variance with Tukey’s multiple comparison test. Abbreviations as in Figures 1, 2, 3, and 4.

Figure 6

GRK2 Phosphorylation of β₂AR Is Necessary for Adrenergic Stimulation of GLUT4 Translocation and Glucose Uptake in AVMs (A to D) Representative confocal images of GLUT4 staining and (F) representative confocal images of 2-NBDG uptake in rat AVMs following stimulation with ISO (100 nmol/L, 30 minutes) or after pretreatment with GRK2 inhibitor paroxetine (PARO) (50 μmol/L, 5 minutes) or fluoxetine (FLUO) (10 μmol/L, 30 minutes) followed by stimulation with ISO. (E, G) Data represent mean ± SEM of myocytes from 3 mice; P < 0.0001 were analyzed using 1-way analysis of variance with Tukey’s multiple comparison test. (H to K) Representative confocal images of GLUT4 staining and (M) representative confocal images of 2-NBDG uptake in AVMs isolated from β₂AR-CKO mice and expressing WT or mutant β₂AR lacking the GRK phosphorylation sites at Ser355/356 (GRKmut-β₂AR) after infection with recombinant adenovirus and after stimulation with ISO (100 nmol/L, 30 minutes). (L, N) Data represent mean ± SEM of myocytes from 5 mice; P < 0.0001 were analyzed using Student’s t-test. Abbreviations as in Figures 1, 2, and 3.

Figure 7

Deletion of GRK2 Phosphorylation Sites of β₂AR Blocks Adrenergic Stimulation of Glucose Uptake in AVMs and in Animal Hearts (A to D) Representative confocal images of GLUT4 staining and (F) representative confocal images of 2-NBDG uptake in AVMs isolated from WT or GRKD mice and after stimulation with ISO (100 nmol/L, 30 minutes). (E, G) Data represent mean ± SEM of myocytes from 3 mice; P < 0.0001 were analyzed using two-way analysis of variance with Tukey’s multiple comparison test. (H to K) Time course and peak response in glucose oxidation (H,I) and oleate oxidation (J, K) before and after stimulation with ISO (100 nmol/L) in WT and GRKD mice. Data represent mean ± SEM, n = 8; ∗P < 0.05 were obtained using two-way analysis of variance with Tukey’s multiple comparison test.