Metformin overdose causes platelet mitochondrial dysfunction in humans

Alessandro Protti, Anna Lecchi, Francesco Fortunato, Andrea Artoni, Noemi Greppi, Sarah Vecchio, Gigliola Fagiolari, Maurizio Moggio, Giacomo Pietro Comi, Giovanni Mistraletti, Barbara Lanticina, Loredana Faraldi, Luciano Gattinoni, Alessandro Protti, Anna Lecchi, Francesco Fortunato, Andrea Artoni, Noemi Greppi, Sarah Vecchio, Gigliola Fagiolari, Maurizio Moggio, Giacomo Pietro Comi, Giovanni Mistraletti, Barbara Lanticina, Loredana Faraldi, Luciano Gattinoni

Abstract

Introduction: We have recently demonstrated that metformin intoxication causes mitochondrial dysfunction in several porcine tissues, including platelets. The aim of the present work was to clarify whether it also causes mitochondrial dysfunction (and secondary lactate overproduction) in human platelets, in vitro and ex vivo.

Methods: Human platelets were incubated for 72 hours with saline or increasing doses of metformin (in vitro experiments). Lactate production, respiratory chain complex activities (spectrophotometry), mitochondrial membrane potential (flow-cytometry after staining with JC-1) and oxygen consumption (Clark-type electrode) were then measured. Platelets were also obtained from ten patients with lactic acidosis (arterial pH 6.97 ± 0.18 and lactate 16 ± 7 mmol/L) due to accidental metformin intoxication (serum drug level 32 ± 14 mg/L) and ten healthy volunteers of similar sex and age. Respiratory chain complex activities were measured as above (ex vivo experiments).

Results: In vitro, metformin dose-dependently increased lactate production (P < 0.001), decreased respiratory chain complex I activity (P = 0.009), mitochondrial membrane potential (P = 0.003) and oxygen consumption (P < 0.001) of human platelets. Ex vivo, platelets taken from intoxicated patients had significantly lower complex I (P = 0.045) and complex IV (P < 0.001) activity compared to controls.

Conclusions: Depending on dose, metformin can cause mitochondrial dysfunction and lactate overproduction in human platelets in vitro and, possibly, in vivo.

Trial registration: NCT 00942123.

Trial registration: ClinicalTrials.gov NCT00942123.

Figures

Figure 1
Figure 1
Effects of metformin on human platelet mitochondrial function. Platelets from healthy donors were incubated in plasma with saline (white bar) or metformin diluted in saline (concentration: 1.66 mg/L, grey bar; 166 mg/L, dark grey bar; or 16,600 mg/L, black bar). After 72 hours, (a) plasma pH (P < 0.001; one-way ANOVA), (b) lactate (P < 0.001; ANOVA on ranks) and (c) glucose (P < 0.001; ANOVA on ranks) concentrations, (d) platelet complex I (relative to citrate synthase, CI/CS) activity (P = 0.009; one-way ANOVA), (e) the proportion between normally polarized and abnormally depolarized mitochondria (JC-1 fluorescence ratio) (P = 0.003; one-way ANOVA) and (f) oxygen consumption (VO2) (P < 0.001; one-way ANOVA) were measured. Data are mean and SD, from four to eight experiments. * P < 0.05 versus saline (Holm-Sidak method). ANOVA, analysis of variance; SD, standard deviation.
Figure 2
Figure 2
Effects of pH on human platelet oxygen consumption. Platelets from healthy donors were incubated in plasma with saline (white bar) or metformin diluted in saline (16,600 mg/L; grey bar), lactic acid (to mimic metformin-induced lactic acidosis; dark grey bar) or metformin diluted in saline (16600 mg/L) plus sodium bicarbonate (to correct metabolic acidosis; black bar). After 72 hours, (a) plasma pH (P < 0.001; one-way ANOVA), (b) lactate (P < 0.001; one-way ANOVA) and (c) bicarbonate (P < 0.001; one-way ANOVA) concentrations and (d) platelet oxygen consumption (P < 0.001; ANOVA on ranks) (VO2) were measured. Data are mean and SD, from four experiments. * P < 0.05 versus saline (Holm-Sidak method). ANOVA, analysis of variance; SD, standard deviation.
Figure 3
Figure 3
Platelet mitochondrial function of metformin-intoxicated patients. Ten healthy subjects (white bars) and ten metformin-intoxicated patients (black bars) were studied. The activity of platelet respiratory chain (a) complex I (CI) (P = 0.045; rank sum test), (b) complex II and III (CII+III) (P = 0.571; rank sum test) and (c) complex IV (CIV; t test) (P < 0.001) are expressed relative to that of (d) citrate synthase (CS) (P = 0.307; rank sum test). (e) The proportion between normally polarized and abnormally depolarized mitochondria was assessed in terms of JC-1 fluorescence ratio (FL2/FL1) (healthy subjects, n = 6; metformin-intoxicated, n = 4) (P = 0.051; t test). Data are mean and SD. * P < 0.05 versus healthy subjects;° P = 0.05 versus healthy subjects. SD, standard deviation.

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