Overexpression of vesicle-associated membrane protein (VAMP) 3, but not VAMP2, protects glucose transporter (GLUT) 4 protein translocation in an in vitro model of cardiac insulin resistance

Abstract

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.

FIGURE 1.

Palmitate-induced insulin resistance in HL-1 cardiomyocytes. Cells were incubated with 20 μm palmitate and 50 nm insulin (palm/ins) overnight to induce insulin resistance. A, lipids were extracted from cells treated with control (white bars) or palm/ins medium (black bars). Intracellular levels of mono-, di-, and triacylglycerol were determined using HP-TLC. B, upon treatment with palm/ins medium, HL-1 cells were acutely stimulated with insulin for 30 min. Lysates of these cells were subsequently blotted against pAkt. C, cells treated with control (white bars) or palm/ins medium (black bars) were acutely stimulated with insulin for 30 min and subsequently stained for sarcolemmal CD36 and GLUT4myc, respectively. Data are mean values ± S.E. (error bars) of at least three independent experiments (n = 3), with duplicate measurements for each condition. *, statistically different from corresponding basal value (p < 0.05); **, statistically different from corresponding basal value (p < 0.01); #, statistically different from corresponding value in control group (p < 0.05).

FIGURE 2.

Effect of VAMP overexpression on GLUT4myc translocation. Cells were either transfected with GLUT4myc and with GFP as a control, or GFP-VAMP2 and GFP-VAMP3, respectively. Afterward, cells were either cultured with depletion medium (control) (A) or medium containing 20 μm palmitate and 50 nm insulin (palm/ins) (B) for 16 h. To induce GLUT4myc translocation, cells were acutely stimulated with insulin for 30 min. After fixation of the cells, sarcolemmal GLUT4myc was stained and quantified. Sarcolemmal GLUT4myc under basal conditions (white bars) is compared with sarcolemmal GLUT4myc after acute insulin stimulation (black bars). Data are mean values ± S.E. (error bars) of at least three independent experiments (n = 3), with triplicate measurements for each condition. *, statistically different from corresponding basal value with p < 0.05; **, statistically different from corresponding basal value with p < 0.01.

FIGURE 3.

Effect of VAMP overexpression on insulin signaling and GLUT4myc expression. Cells were either transfected with GFP as a control, or GFP-VAMP2 and GFP-VAMP3, respectively. Afterward, cells were either cultured with depletion medium (control) or medium containing 20 μm palmitate and 50 nm insulin (palm/ins) for 16 h. A and B, afterward, HL-1 cells were acutely stimulated with insulin for 30 min. Lysates of these cells were subsequently blotted against caveolin-3 (loading control), pAkt, GLUT4myc, and VAMP2 and VAMP3, respectively. Representative Western blots from at least three different experiments (n = 3) are shown. C, after acute stimulation with insulin, control cells were fixed and stained for confocal microscopy. DAPI was used to stain the nuclei, myc represents GLUT4myc, GFP represents GFP-VAMP2 and GFP-VAMP3, respectively. Representative confocal pictures from at least three different experiments (n = 3) are shown.

FIGURE 4.

Effect of VAMP overexpression on CD36 translocation and lipid accumulation. Cells were either transfected with GFP as a control, or GFP-VAMP2 and GFP-VAMP3, respectively. Afterward, cells were either cultured with depletion medium (control) or medium containing 20 μm palmitate and 50 nm insulin (palm/ins) for 16 h. A, to induce CD36 translocation, cells were acutely stimulated with insulin for 30 min. After fixation of the cells, sarcolemmal CD36 was stained and quantified. Sarcolemmal CD36 under basal conditions (white bars) is compared with sarcolemmal CD36 after acute insulin stimulation (black bars). B–D, lipids were extracted from cells treated with normal depletion medium (control) or palm/ins medium, and quantified using HP-TLC. Levels of mono-, di-, and triacylglycerols were compared between cells overexpressing GFP (white bars), GFP-VAMP2 (gray bars), and GFP-VAMP3 (black bars). Data are mean values ± S.E. (error bars) of at least three independent experiments (n = 3), with triplicate measurements for each condition. *, statistically different from corresponding basal value with p < 0.05; **, statistically different from corresponding basal value with p < 0.01; $, statistically different from corresponding value of the GFP transfection with p < 0.05; #, statistically different from corresponding value of control group with p < 0.05.

FIGURE 5.

Immunoprecipitation of VAMP2 and VAMP3. Freshly isolated rat cardiomyocytes were either treated with insulin or oligomycin for 15 min. A, upon lysis, VAMP2 was immunoprecipitated from insulin-treated cardiomyocytes. Subsequently, the immunoprecipitate was blotted against VAMP2, Akt, pAkt, and PKCζ. B, upon lysis, VAMP3 was immunoprecipitated from oligomycin-treated cardiomyocytes. Subsequently, the immunoprecipitate was blotted against VAMP3, AMPK, pAMPK, PKD, and pPKD. Representative Western blots from at least three different experiments (n = 3) are shown. n.a., no antibody during immunoprecipitation (negative control).

FIGURE 6.

Effect of contraction and VAMP3 overexpression on GLUT4myc translocation under lipotoxic conditions. Cells were either transfected with GLUT4myc plus GFP as a control, or with GLUT4myc plus GFP-VAMP3, respectively. After addition of depletion medium (control) or medium containing 20 μm palmitate and 50 nm insulin (palm/ins), the cells were either left quiescent (A) or paced with 0.3 Hz (B) for 16 h. To induce GLUT4myc translocation, cells were acutely stimulated with insulin for 30 min. After fixation of the cells, sarcolemmal GLUT4myc was stained and quantified. Sarcolemmal GLUT4myc under basal conditions (white bars) is compared with sarcolemmal GLUT4myc after acute insulin stimulation (black bars). Data are mean values ± S.E. (error bars) of at least three independent experiments (n = 3), with triplicate measurements for each condition. *, statistically different from corresponding basal value with p < 0.05; **, statistically different from corresponding basal value with p < 0.01; ***, statistically different from corresponding basal value with p < 0.001; #, statistically different from corresponding value of control group with p < 0.05.