17 research outputs found
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
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
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
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
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
Measurement of ATP production 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) either for 12 (A) or 72h (B). The mitochondrial ATP production was measured under basal (no substrate) or stimulated conditions (pyruvate + malate or succinate + rotenone). After subtracting ATP level obtained without stimulation, results were normalized to the activity of the citrate synthase activity and expressed in %. Data are the means ± S.E.M of 3–5 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of substrates or glucose treatments. ***, P §§§, P substrate (1, 22) = 21.000, P = 0.001; Ftreatment (3, 22) = 3.776, P = 0.025; FsubstrateXtreatment (3, 22) = 0.984, P = 0.418. For 72h treatment, Fsubstrate (1, 26) = 57.910, Ptreatment (3, 26) = 15.880, PsubstrateXtreatment (3, 26) = 22.200, P (PPTX)</p
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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
Mitochondrial respiration of 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) either for 12 (A) or 72h (B). Oxygen consumption was measured by polarography using 3 different substrates. Data are the means ± S.E.M of 5–7 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of substrates or glucose treatments. *, P $, P substrate (2, 56) = 39.66, Ptreatment (3, 56) = 13.15, PsubstrateXtreatment (6, 56) = 0.453, P = 0.8394. For 72h treatment, Fsubstrate (2, 48) = 48.59, Ptreatment (3, 48) = 2.038, P = 0.1210; FsubstrateXtreatment (6, 48) = 1.443, P = 0.2182. (PPTX)</p
Measurement of the MMP 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) either for 12 (A) or 72h (B). The MMP was measured using TMRE under basal or stimulated conditions (succinate, palmitate or pyruvate). Results were normalized to the NG condition. Data are the means ± S.E.M of 3–7 independent experiments. Two-way ANOVA followed by the Tukey’s multiple comparisons test when evaluating the effect of substrates or glucose treatments. *, P $, P substrate (3, 56) = 1.107, P = 0.3541; Ftreatment (3, 56) = 2.212, P = 0.0967; FsubstrateXtreatment (9, 56) = 1.799, P = 0.0887. For 72h treatment, Fsubstrate (3, 64) = 0.5330, P = 0.6613; Ftreatment (3, 64) = 11.53, PsubstrateXtreatment (9, 64) = 1.619, P = 0.1286. (PPTX)</p