Combined Administration of Metformin and Amprolium to Rats Affects Metabolism of Free Amino Acids in the Brain, Altering Behavior, and Heart Rate

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Abstract

The risk of developing diabetes and cardiometabolic disorders is associated with increased levels of alpha-aminoadipic acid and disturbances in the metabolism of branched-chain amino acids. The side effects of the widely used antidiabetic drug metformin include impaired degradation of branched-chain amino acids and inhibition of intracellular thiamin transport. These effects may be interconnected, as thiamine deficiency impairs the functioning of thiamine diphosphate (ThDP)-dependent dehydrogenases of 2-oxo acids involved in amino acids degradation, while diabetes is often associated with perturbed thiamine status. In this work, we investigate the action of metformin in rats with impaired thiamine availability. The reduction in the thiamine influx is induced by simultaneous administration of the thiamine transporters inhibitors metformin and amprolium. After 24 days of combined metformin/amprolium administration, no significant changes in the total brain levels of ThDP or activities of ThDP-dependent enzymes of central metabolism are observed, but the affinities of transketolase and 2-oxoglutarate dehydrogenase to ThDP increase. The treatment also significantly elevates the brain levels of free amino acids and ammonia, reduces the antioxidant defense, and alters the sympathetic/parasympathetic regulation, which is evident from changes in the ECG and behavioral parameters. Strong positive correlations between brain ThDP levels and contents of ammonia, glutathione disulfide, alpha-aminoadipate, glycine, citrulline, and ethanolamine are observed in the metformin/amprolium-treated rats, but not in the control animals. Analysis of the obtained data points to a switch in the metabolic impact of ThDP from the antioxidant and nitrogen-sparing in the control rats to the pro-oxidant and hyperammonemic in the metformin/amprolium-treated rats. As a result, metformin administration along with the amprolium-reduced thiamine supply significantly perturb the metabolism of amino acids in the rat brain, altering behavioral and ECG parameters.

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About the authors

A. V. Graf

Lomonosov Moscow State University

Email: bunik@belozersky.msu.ru
Russian Federation, 119234, Moscow

A. V. Artiukhov

Lomonosov Moscow State University; Sechenov Medical University

Email: bunik@belozersky.msu.ru
Russian Federation, 119234, Moscow; 105043, Moscow

O. N. Solovyeva

Lomonosov Moscow State University

Email: bunik@belozersky.msu.ru
Russian Federation, 119234, Moscow

A. L. Ksenofontov

Lomonosov Moscow State University

Email: bunik@belozersky.msu.ru
Russian Federation, 119234, Moscow

V. I. Bunik

Lomonosov Moscow State University; Sechenov Medical University

Author for correspondence.
Email: bunik@belozersky.msu.ru
Russian Federation, 119234, Moscow; 105043, Moscow

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3. Fig. 1. Schematic of animal experiments. Metformin (M) and amprolium (A) injections were administered on the indicated days for 24 days. The first three injections of metformin at a dose of 200 mg/kg body weight were followed by 15 injections of metformin at a dose of 70 mg/kg body weight. Each of the metformin injections was followed by a second injection of amprolium at a dose of 40 mg/kg body weight. Physiological and biochemical tests were performed on the indicated days as described in the Materials and Methods section

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4. Fig. 2. Levels of TDP, NAD+, antioxidant peptides carnosine and glutathione (GSH), oxidised glutathione (GSSG), and the ratio of reduced and oxidised glutathione in the cerebral cortex of rats treated with metformin/amprolium (M+A) compared with control animals

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5. Fig. 3. Activation of TKT (a), MAC (b) and OGDC (c) by addition of TDP to the reaction medium measuring enzymatic activities in the cerebral cortex of control and metformin/amprolium-treated rats (M+A). In the upper panel, hollow and shaded dots indicate activities measured without and in the presence of TDF, respectively (statistical analysis using ANOVA with repeated measures). The lower panel shows the comparison of the fractions of endogenous TCT apoenzymes, MAC and OGDC in treated and control samples calculated as [1 - (Activity without TDP)/(Activity in the presence of TDP)] × 100%. The outlier excluded from the analysis is denoted by the symbol ‘x’

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6. Fig. 4. Changes (in %) in the content of amino acids and related compounds in the cerebral cortex of rats treated with metformin/amprolium, compared with control animals (n = 12 in each group). Outliers excluded from statistical analyses are not shown because they are outside the range of the y-axis. * Significant difference from control values taken as 100%

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7. Fig. 5. Changes in rat behaviour in the open-field test (a, b) and ECG parameters (c) as a function of duration of chronic administration of metformin/amprolium (M+A) compared to control rats. Significant differences between groups are shown in the graphs; statistically significant factors such as ‘treatment’ with metformin/amprolium and ‘duration of treatment’ and their interaction are shown below the graphs (according to ANOVA with repeated measures and Shidak's posteriori test)

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8. Fig. 6. Correlation matrices of physiological and biochemical parameters in control rats. The highlighted horizontal lines divide the parameters into five groups: (1) ECG parameters, (2) anxiety parameters, (3) exploratory and locomotor activity parameters, (4) activities of TDP-dependent enzymes together with TDP level, and (5) levels of NAD+, amino acids and related compounds

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9. Fig. 7. Correlation matrices of physiological and biochemical parameters after chronic administration of metformin/amprolium. The highlighted horizontal lines divide the parameters into five groups: (1) ECG parameters, (2) anxiety parameters, (3) exploratory and locomotor activity parameters, (4) TDP-dependent enzyme activities together with TDP levels, and (5) levels of NAD+, amino acids and related compounds

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