Variabilité glycémique : exploration in vitro des fonctions cellulaires et mitochondriales sur la lignée de cardiomyocyte HL-1

Diabetes mellitus is associated with higher risk of cardiovascular disease and metabolism dysregulation. Glycemic variability (GV) has been suggested as a risk factor in diabetic complication. In order to characterize dysfunctions induced by GV, we developed an in vitro model that transpose GV on the cardiac cell line HL-1. We exposed our cells to a treatment of 12 hours miming hypoglycemia, normoglycemia, hyperglycemia and GV. The exploration of signaling pathways didn’t allow us to show a deleterious effect of glucose fluctuation. However we were able to point mitochondrial alteration under glucose fluctuation. HL-1 cells mitochondria exhibit a higher membrane potential and an increase of superoxide anion production. Although we didn’t show any alteration in mitochondrial respiration after 12 hours of exposition, we showed that after 72 hours of glucose fluctuation, HL-1 cells showed a decrease in mitochondrial respiration. We finally studied the impact of glucose fluctuation on the susceptibility to develop hypoxic injuries. We showed that after 36 hours of hypoxia, injuries were higher for cells exposed to glucose fluctuation. Our results indicate a deleterious effect of GV, but additional experiments are needed to better characterize the mechanisms.Le diabète est associé à une augmentation de risque de maladie cardiovasculaire et une dérégulation du métabolisme. Il a été suggéré que la variabilité glycémique (VG) pouvait avoir un rôle dans le développement des complications du diabète. Afin d’étudier et de caractériser les dysfonctions induites par la VG, nous avons mis au point un modèle in vitro mimant la VG sur la lignée de cardiomyocytes HL-1. Nous avons ainsi développé un traitement de 12 heures, mimant hypoglycémie, normoglycémie, hyperglycémie et VG. L’étude de la signalisation cellulaire ne nous a pas permis de montrer un rôle délétère de la VG. Nous avons toutefois mis en évidence que la VG participait à des dysfonctions mitochondriales. En effet en situation de fluctuations en glucose, les mitochondries des cellules HL-1 présentent une augmentation de leur potentiel de membrane, ainsi qu’une augmentation de la production d’anions superoxydes. Bien que nous n’ayons pas réussi à montrer de perturbation de la chaîne respiratoire après 12 heures d’exposition, nous avons pu montrer que 72 heures d’exposition provoquaient une baisse de la respiration mitochondriale. Nous avons enfin étudié l’impact des fluctuations en glucose sur la susceptibilité au développement de lésions d’hypoxie, et avons montré que les lésions sont majorées après 36 heures d’hypoxie en cas d’exposition à des fluctuations en glucose. Nos résultats montrent un rôle délétère de la VG, néanmoins des expériences complémentaires sont nécessaires afin de caractériser de manière plus précise les mécanismes impliqués

Glycemic variability : in vitro exploration of mitochondrial and cellular functions on HL-1 cardiomyocyte cell line

Le diabète est associé à une augmentation de risque de maladie cardiovasculaire et une dérégulation du métabolisme. Il a été suggéré que la variabilité glycémique (VG) pouvait avoir un rôle dans le développement des complications du diabète. Afin d’étudier et de caractériser les dysfonctions induites par la VG, nous avons mis au point un modèle in vitro mimant la VG sur la lignée de cardiomyocytes HL-1. Nous avons ainsi développé un traitement de 12 heures, mimant hypoglycémie, normoglycémie, hyperglycémie et VG. L’étude de la signalisation cellulaire ne nous a pas permis de montrer un rôle délétère de la VG. Nous avons toutefois mis en évidence que la VG participait à des dysfonctions mitochondriales. En effet en situation de fluctuations en glucose, les mitochondries des cellules HL-1 présentent une augmentation de leur potentiel de membrane, ainsi qu’une augmentation de la production d’anions superoxydes. Bien que nous n’ayons pas réussi à montrer de perturbation de la chaîne respiratoire après 12 heures d’exposition, nous avons pu montrer que 72 heures d’exposition provoquaient une baisse de la respiration mitochondriale. Nous avons enfin étudié l’impact des fluctuations en glucose sur la susceptibilité au développement de lésions d’hypoxie, et avons montré que les lésions sont majorées après 36 heures d’hypoxie en cas d’exposition à des fluctuations en glucose. Nos résultats montrent un rôle délétère de la VG, néanmoins des expériences complémentaires sont nécessaires afin de caractériser de manière plus précise les mécanismes impliqués.Diabetes mellitus is associated with higher risk of cardiovascular disease and metabolism dysregulation. Glycemic variability (GV) has been suggested as a risk factor in diabetic complication. In order to characterize dysfunctions induced by GV, we developed an in vitro model that transpose GV on the cardiac cell line HL-1. We exposed our cells to a treatment of 12 hours miming hypoglycemia, normoglycemia, hyperglycemia and GV. The exploration of signaling pathways didn’t allow us to show a deleterious effect of glucose fluctuation. However we were able to point mitochondrial alteration under glucose fluctuation. HL-1 cells mitochondria exhibit a higher membrane potential and an increase of superoxide anion production. Although we didn’t show any alteration in mitochondrial respiration after 12 hours of exposition, we showed that after 72 hours of glucose fluctuation, HL-1 cells showed a decrease in mitochondrial respiration. We finally studied the impact of glucose fluctuation on the susceptibility to develop hypoxic injuries. We showed that after 36 hours of hypoxia, injuries were higher for cells exposed to glucose fluctuation. Our results indicate a deleterious effect of GV, but additional experiments are needed to better characterize the mechanisms

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AimsGlycemic variability has been suggested as a risk factor for diabetes complications but the precise deleterious mechanisms remain poorly understood. Since mitochondria are the main source of energy in heart and cardiovascular diseases remain the first cause of death in patients with diabetes, the aim of the study was to evaluate the impact of glucose swings on mitochondrial functions in the cardiomyocyte cell line HL-1.MethodsHL-1 cells were exposed to low (LG, 2.8 mmol/l), normal (NG, 5.5 mmol/l), high (HG, 25 mmol/l) or intermittent high glucose (IHG, swing between low and high) every 2h during 12h (short-time treatment) or every 12h during 72h (long-time treatment). Anaerobic catabolism of glucose was evaluated by measuring glucose consumption and lactate production, oxidative phosphorylation was evaluated by polarography and ATP measurement, mitochondrial superoxide anions and the mitochondrial membrane potential (MMP) were analysed using fluorescent probes, and the protein oxidation was measured by oxyblot.ResultsIHG and HG increased glucose consumption and lactate production compared to LG and NG but without any difference between short- and long-time treatments. After 72h and unlike to LG, NG and HG, we didn’t observe any increase of the mitochondrial respiration in the presence of succinate upon IHG treatment. IHG, and to a lesser extent HG, promoted a time-dependent decrease of the mitochondrial membrane potential compared to LG and NG treatments. HG and IHG also increased superoxide anion production compared to LG and NG both at 12 and 72h but with a higher increase for IHG at 72h. At last, both HG and IHG stimulated protein oxidation at 72h compared to LG and NG treatments.ConclusionsOur results demonstrated that exposure of HL-1 cells to glucose swings promoted time-dependent mitochondrial dysfunctions suggesting a deleterious effect of such condition in patients with diabetes that could contribute to diabetic cardiomyopathy.</div

Evaluation of medium lactate production in HL-1 cells culture with high and normal glucose.

HL-1 cells were cultured at least during 3 weeks either with normal (5.5 mmol/l) or high (25 mmol/l) glucose then submitted to 4 different regimens for 12h: LG, NG, HG or IHG. Lactate concentration was measured in the culture medium at 0, 2, 6, 10 and 12h to calculate lactate production. Data are the means ± S.E.M of 4 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of time treatment in HL-1 cells culture in normal or high glucose. For LG treatment, Ftreatment (1, 24) = 1.09, P = 0.3064; Ftime (3, 24) = 0.18, P = 0.9057; FtreatmentXtime (3, 24) = 0.43, P = 0.7270. For NG treatment, Ftreatment (1, 24) = 1.17, P = 0.2883; Ftime (3, 24) = 1.09, P = 0.3688; FtreatmentXtime (3, 24) = 0.03, P = 0.9914. For HG treatment, Ftreatment "F (1, 24) = 1.89, P = 0.1814; Ftime (3, 24) = 2.13, P = 0.1222; FtreatmentXtime (3, 24) = 0.09, P = 0.9605. For IHG treatment, Ftreatment (1, 24) = 0.05, P = 0.8237; Ftime (3, 24) = 1.47, P = 0.2467; FtreatmentXtime (3, 24) = 0.20, P = 0.8929. (PPTX)</p

Comparison of glucose consumption and lactate production in culture medium of HL-1 cells exposed to LG, NG, HG and IHG between short- (12h) and long-time (72h) glucose treatment.

HL-1 cells were cultured at least during 3 weeks with normal glucose (5.5 mmol/l) then submitted to LG, NG, HG or IHG for 12h with medium change every 2h or 72h with medium change every 12h (see Fig 1). Glucose (A, B, C and D) and lactate (E, F, G and H) concentrations were measured in the culture medium at different times (see Fig 1). Data are the means ± S.E.M of 4–6 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of glucose and time treatment. *, P 2 vs 12h (1, 32) = 1.362, P = 0.252; Fglucose treatment (3, 32) = 48.340, P2 vs 12hXglucose treatment (3, 32) = 0.480, P = 0.698; F 6 vs 36h (1, 32) = 0.017, P = 0.895; Fglucose treatment (3, 32) = 18.82, P6 vs 36hXglucose treatment (3, 32) = 0.584, P = 0.629; F 10 vs 60h (1, 32) = 0.190, P = 0.665; Fglucose treatment (3, 32) = 37.83, P10 vs 60hXglucose treatment (3, 32) = 1.069, P = 0.376; F 12 vs 72h (1, 31) = 7.478, P = 0.010; Fglucose treatment (3, 31) = 30.51, P12 vs 72hXglucose treatment (3, 31) = 0.223, P = 0.8792. For lactate production, F 2 vs 12h (1, 32) = 34.96, Pglucose treatment (3, 32) = 22.41, P2 vs 12hXglucose treatment (3, 32) = 0.500, P = 0.684; F 6 vs 36h (1, 32) = 1.186, P = 0.284; Fglucose treatment (3, 32) = 9.598, P = 0.0001; F 6 vs 36hXglucose treatment (3, 32) = 0.918, P = 0.442; F 10 vs 60h F (1, 32) = 0.160, P = 0.691; Fglucose treatment (3, 32) = 13.93, P10 vs 60hXglucose treatment F (3, 32) = 1.247, P = 0.309; F 12 vs 72h (1, 31) = 2.582, P = 0.118; Fglucose treatment (3, 31) = 11.01, P12 vs 72hXglucose treatment (3, 31) = 1.254, P = 0.307.</p

Comparison of oxidized proteins extracted from HL-1 cells exposed to LG, NG, HG and IHG between short- (12h) and long-time (72h) glucose treatment.

HL-1 cells were cultured at least during 3 weeks with normal glucose (5.5 mmol/l) then submitted to LG, NG, HG or IHG for 12h with medium change every 2h or 72h with medium change every 12h (see Fig 1). Proteins were derivatized (+) or not (-) with DNPH then separated by SDS-PAGE. Total proteins were revealed in free-stain gel and oxidized proteins were revealed by anti-DNP antibody (A). Immunoreactive proteins were quantified by densitometric analysis using Fiji 1.0 and normalized to total stained protein. Data are the means ± S.E.M of 5 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of glucose and time treatment **, P $$, P $$ $, P §§, P §§§, P 12 vs 72h (1, 32) = 18.15, P = 0.0002; Fglucose treatment (3, 32) = 7.220, P = 0.0008; F 12 vs 72hXglucose treatment (3, 32) = 10.39, P<0.0001.</p

Schematic representation of short- and long-time treatment of HL-1 cells with different glucose concentrations.

Cells were exposed to low (2.8 mmol/l, LG), normal (5.5 mmol/l, NG), high (25 mmol/l, NG), and intermittent high glucose (25 followed by 2.8 mmol/l, IHG) either during 12 or 72h. The culture medium was changed either every 2h or every 12h for short- and long-time treatment, respectively. Arrows indicate glucose and lactate measurements.</p

Detection of oxidized proteins by oxyblot in HL-1 cells exposed for 12 or 72h to LG, NG, HG or IHG.

HL-1 cells were cultured at least during 3 weeks with normal (5.5 mmol/l) glucose then submitted to 4 different regimens (LG, NG, HG or IHG) for either 12 (A) or 72h (B). The oxidized proteins were detected with the oxyblot assay. Total proteins were detected in stain free gels and oxidized proteins after derivatization by DNPH. Results were expressed as the ratio of oxidized proteins/total proteins. Data are the means ± S.E.M of 5 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons test. *, P $, P (PPTX)</p

Effects of insulin on glucose consumption and lactate production.

HL-1 cells were seeded at similar density in T75 flasks, cultured in 25 mmol/l glucose and then exposed or not to 3 IU insulin for 24h. Glucose and lactate concentrations were determined at 0 and 24h in the culture medium to calculate glucose consumption (A) and lactate production (B). Data are the means ± S.E.M of 7 independent experiments. Statistical analysis was conducted using the Wilcoxon test. *, P < 0.05.</p