Insulin protects apoptotic cardiomyocytes from hypoxia/reoxygenation injury through the sphingosine kinase/sphingosine 1-phosphate axis

Abstract

Objective: Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated.

Methods and results: Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes.

Conclusion: The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.

Figure 1. Effects of insulin on sphingosine kinase activity and subcellular localization.

Serum-starved cardiomyocytes were incubated with 10 mU/L insulin for the indicated periods of time. A: Aliquots of cell extracts (40 µg) were used to assess sphingosine kinase (SphK) activity. Data represent the mean ± SEM of at least three independent experiments, each performed at least in duplicate. The difference between the effects of insulin in challenged and unchallenged cells was found to be statistically significant using Student's t tests (*P<0.05). B: Western blot analyses of SphK1 were performed in membrane and cytosolic fractions. Blots representative of at least three independent experiments are shown. The histograms represent densitometric analysis of three independent experiments. Data reported are expressed as fold increase of the membrane∶cytosol ratio. The insulin-induced increase in the SphK1 content of the membrane was found to be statistically significant by Student's t-test (*P<0.05). C: Cardiomyocytes were transfected with GFP-SphK1 for 24 h, stimulated with insulin, then observed SphK1 under confocal fluorescence microscopy. C1: cardiomyocytes transfected with GFP-SphK1 for 24 h and with 0 mU/L insulin for 10 min; C2: cardiomyocytes transfected with GFP-SphK1 for 24 h and with 5 mU/L insulin for 10 min; C3: cardiomyocytes transfected with GFP-SphK1 for 24 h and with 10 mU/L insulin for 10 min. Arrows point to membrane ruffling.

Figure 2. Dose dependence of the effects of insulin on S1P generation.

Cardiomyocytes were incubated for 30[γ-32P] ATP. After lipid extraction, the radioactive S1P that had been generated was separated using thin-layer chromatography and quantitated. Data are means ± S.E. of three independent experiments carried out in triplicate.

Figure 3. Insulin-induced S1P receptor activation as demonstrated by FRET analysis.

A: Cardiomyocytes transfected with expression plasmids encoding S1P1-CFP and YFP-β-arrestin were treated either with stimuli (buffer vehicle or 500 nM S1P) or with 10 mU/L insulin and were analyzed for FRET in living cells. A representative emission ratio of the two fluorophores (excited at 458 nm) from five independent experiments is shown. B: Cardiomyocytes cotransfected with expression plasmids encoding S1P1-CFP and YFP-β-arrestin plasmids were treated with or without 50 µM HACPT for 30 min and then treated with either empty buffer, 500 nM S1P, or 10 mU/L insulin. They were then analyzed for FRET in living cells. Emissions detected from an increase in donor fluorescence after acceptor photobleaching of puncta of membrane were measured and expressed as FRET efficiency. Insulin and S1P treatment both caused a significant increase in FRET efficiencies (n = 50; a representative experiment of four independent experiments is shown; P<0.05, Student's paired t test). C: In some experiments, cardiomyocytes were transfected with control or rSK1-siRNAs along with plasmids encoding the fluorophore-conjugated proteins. The areas of membrane were photobleached. Then the cardiomyocytes were stimulated with either empty (buffer), 500 nM S1P, or 10 mU/L insulin, fixed, and measured for FRET efficiency. Data are expressed as means and standard errors of the means of three independent experiments carried out in triplicate.

Figure 4. TUNEL-positive cardiomyocytes in all groups shown using confocal fluorescence microscopy.

Cardiomyocytes were evaluated using hypoxia-reoxygenation. During reoxygenation, they were cultured with different concentrations of insulin. 1: 0 mU/L insulin; 2: 5 mU/L insulin; 3: 10 mU/L insulin; 4: 20 mU/L insulin. TUNEL-positive cardiomyocytes/total cardiomyocytes×100% (AI). Data are means ± S.E. of three independent experiments carried out in triplicate.

Figure 5. SphK activity and insulin-induced inhibition of hypoxia-reoxygenation-induced cell death in cardiomyocytes.

A: Effects of HACPT on insulin in cardiomyocytes, cardiomyocytes subjected to hypoxia-reoxygenation, and cardiomyocytes during reoxygenation, all cultured with empty vehicle (vehicle, 0.05% dimethylsulfoxide and 0.05% methanol) with 50 µM HACPT, as indicated by white and gray bars, respectively. Cells were treated with control or SphK1-siRNA and cultured for 72 h. Then cells were subjected to hypoxia and treated either with nothing, S1P, or insulin. B: Cardiomyocytes were transiently transfected and reoxygenated. Cells were stimulated with nothing, S1P, or insulin during reoxygenation. TUNEL-positive cardiomyocytes/total cardiomyocytes×100% (AI). Data are means ± S.E. of five independent experiments carried out in triplicate.

Figure 6. S1P receptor-mediated insulin-induced inhibition of hypoxia-reoxygenation-induced cell death in cardiomyocytes.

Effects of S1P receptor antagonist on insulin in cardiomyocytes, cardiomyocytes subjected to hypoxia-reoxygenation, and cardiomyocytes undergoing reoxygenation, all either cultured with empty vehicle (vehicle, 0.05% dimethylsulfoxide) pretreated with 1 µM VPC23019, an inhibitor of S1P₁ and S1P₃ receptors for 30 minutes. They are shown with white and gray bars, respectively. Then cells were treated either with nothing, S1P, or insulin. Data are means ± S.E. of five independent experiments carried out in triplicate.

Figure 7. Cardioprotective pathway of insulin on acute hypoxia/reoxygenation injury-apoptosis.