Baiba Svalbe

Protective effects of mildronate in an experimental model of type 2 diabetes in Goto-Kakizaki rats

Background and purpose:  Mildronate [3‐(2,2,2‐trimethylhydrazinium) propionate] is an anti‐ischaemic drug whose mechanism of action is based on its inhibition of L‐carnitine biosynthesis and uptake. As L‐carnitine plays a pivotal role in the balanced metabolism of fatty acids and carbohydrates, this study was carried out to investigate whether long‐term mildronate treatment could influence glucose levels and prevent diabetic complications in an experimental model of type 2 diabetes in Goto‐Kakizaki (GK) rats.

Mildronate decreases carnitine availability and up-regulates glucose uptake and related gene expression in the mouse heart

AIMS l-carnitine has been shown to play a central role in both fat and carbohydrate metabolisms. This study investigated whether acute and long-term treatments with an l-carnitine biosynthesis inhibitor, mildronate (3-(2,2,2-trimethylhydrazinium) propionate), modulate glucose uptake. MAIN METHODS The effects of acute and long-term administration of mildronate at a dose of 200 mg/kg (i.p. daily for 20 days) were tested in mouse blood plasma and heart. KEY FINDINGS Acute administration of mildronate in vivo, or in vitro administration with perfusion buffer in isolated heart experiments, did not induce any effects on glucose blood concentration and uptake in the heart. Mildronate long-term treatment significantly decreased carnitine concentration in plasma and heart tissues, as well as increased the rate of insulin-stimulated glucose uptake by 35% and the expression of glucose transporter 4, hexokinase II, and insulin receptor proteins in mouse hearts. In addition, expression of both carnitine palmitoyltransferases IA and IB were significantly increased. Mildronate long-term treatment statistically significantly decreased fed state blood glucose from 6+/-0.2 to 5+/-0.1 mM, but did not affect plasma insulin and C-peptide levels. SIGNIFICANCE Our experiments demonstrate for the first time that long-term mildronate treatment decreases carnitine content in the mouse heart and leads to increased glucose uptake and glucose metabolism-related gene expression.

Effect of inhibiting carnitine biosynthesis on male rat sexual performance.

l-carnitine has a documented role as a cofactor in cellular energy metabolism and fatty acid beta-oxidation pathways and it has also been considered to function in reproductive biology. We investigated whether decreasing concentrations of L-carnitine using an inhibitor of its biosynthesis, mildronate (3-(2,2,2-trimethylhydrazinium)-propionate), would influence the sexual behavior or sperm quality in male rats. Mildronate treatment induced a significant decrease in carnitine concentration and an increase in gamma-butyrobetaine (GBB) concentration in both plasma and testes extracts. However, the expression of carnitine palmitoyltransferase I in testes and testosterone concentration in plasma was not changed in mildronate treated rat. Behavioral experiments demonstrated that mildronate treatment did not decrease the sexual motivation in both sexually naive and sexually experienced rats. The densities of spermatozoa in the cauda epididymis, as well as motility, were unchanged after mildronate treatment at a dose of 100 mg/kg. In conclusion, our study provides experimental evidence that mildronate induces decrease in the free carnitine concentration in rat testes, but does not decrease the sexual activity or sperm quality of male rats.

Anti-diabetic effects of mildronate alone or in combination with metformin in obese Zucker rats

Mildronate is a cardioprotective drug, the mechanism of action of which is based on the regulation of l-carnitine concentration. We studied the metabolic effects of treatment with mildronate, metformin and a combination of the two in the Zucker rat model of obesity and impaired glucose tolerance. Zucker rats were p.o. treated daily with mildronate (200mg/kg), metformin (300 mg/kg), and a combination of both drugs for 4 weeks. Weight gain and plasma metabolites reflecting glucose metabolism were measured. The expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ and target genes was measured in rat heart and liver tissues. Each treatment decreased the blood glucose concentration during the fed and fasted states by 1 to 2 mmol/l. Treatment with mildronate and metformin decreased the plasma insulin concentration by 31 and 29%, respectively, while the combination of both drugs significantly reduced fed insulin concentration by about 47%. Mildronate treatment increased the expression of PPAR-α in the heart tissue and PPAR-γ in the heart and liver tissues. In addition, treatment increased the expression of PPAR target genes in the heart, but not in the liver tissue. In contrast to monotherapy, treatment with the combination of mildronate and metformin significantly decreased weight gain by 19% and did not affect food intake. In conclusion, our results demonstrate that mildronate, an inhibitor of l-carnitine biosynthesis, improves adaptation to hyperglycemia- and hyperlipidemia-induced metabolic disturbances and increases PPAR-α activity.

Mildronate treatment improves functional recovery following middle cerebral artery occlusion in rats.

Mildronate (3-(2,2,2-trimethylhydrazinium) propionate) is an inhibitor of l-carnitine biosynthesis and an anti-ischemic drug. In the present study, we investigated the effects of mildronate in rats following focal cerebral ischemia. Male Wistar rats were subjected to transient occlusion of the middle cerebral artery (MCAO) for 90min, followed by the intraperitoneal administration of mildronate at doses of 100 and 200mg/kg 2h after reperfusion and then daily for an additional 14days. The beam-walking, rota-rod and cylinder tests were used to assess sensorimotor function, and vibrissae-evoked forelimb-placing and limb-placing tests examined responses to tactile and proprioceptive stimulation. Following behavioural testing, the infarct volume was measured. The cerebellar concentrations of l-carnitine, γ-butyrobetaine (GBB) and mildronate were also measured. The results showed that saline-treated MCAO rats had minor or no spontaneous recovery in sensorimotor and proprioceptive function up to 14days post-stroke. Treatment with mildronate at a dose of 200mg/kg was found to accelerate recovery of motor and proprioceptive deficits in limb-placing, cylinder and beam-walking tests. Analysis of rat cerebellar tissue extracts revealed that l-carnitine and GBB concentrations changed with mildronate treatment; the concentration of l-carnitine was significantly decreased by mildronate treatment, whereas the concentration of GBB was significantly increased. Cerebellar concentrations of mildronate also increased in a dose-dependent manner following systemic administration. Infarct size did not differ among the experimental groups on post-stroke day 14. The present study suggests that mildronate treatment improves the functional outcome in MCAO rats without influencing infarct size.

Effects of long-term mildronate treatment on cardiac and liver functions in rats.

Mildronate is a cardioprotective drug that improves cardiac function during ischaemia and functions by lowering l-carnitine concentration in body tissues and modulating myocardial energy metabolism. The aim of the present study was to characterise cardiovascular function and liver condition after long-term mildronate treatment in rats. In addition, changes in the plasma lipid profile, along with changes in the concentration of mildronate, l-carnitine and gamma-butyrobetaine were monitored in the rat tissues. Wistar rats were perorally treated daily with a mildronate dose of either 100, 200 or 400 mg/kg for 4, 8 or 12 weeks. The l-carnitine-lowering effect of mildronate was dose-dependent. However, the carnitine levels reached a plateau after about four weeks of treatment. During the additional weeks of treatment, the carnitine levels were not considerably changed. The obtained results provide evidence that even a high dose of mildronate does not alter cardiovascular parameters and the function of isolated rat hearts. Furthermore, the histological evaluation of liver tissue cryosections and measurement of biochemical markers of hepatic toxicity showed that all the measured values were within the normal reference range. Our results provide evidence that long-term mildronate administration induces significant changes in carnitine homeostasis, but it is not associated with cardiac impairment or disturbances in liver function.

The sensorimotor and cognitive deficits in rats following 90- and 120-min transient occlusion of the middle cerebral artery.

Middle cerebral artery occlusion (MCAO) is the most commonly used method to study the neurological and histological outcomes and the pathological mechanisms of ischaemic stroke. The current work compares sensorimotor and cognitive deficits and the infarct volume in rats following a transient 90- or 120-min MCAO, which allows the appropriate behavioural tests to be chosen based on the goal and design of the experiment. In the beam-walking test, we found significant differences between the 90- and 120-min MCAO groups in the number of foot faults made with the impaired hindlimb on post-stroke days 3, 7 and 14. In the cylinder test, a difference between the 90- and 120-min groups was observed on post-operation day 14. The responses to tactile and proprioceptive stimulation were impaired to a similar extent after 90- and 120-min MCAO in the vibrissae-evoked forelimb-placing and limb-placing tests. Moreover, we found significant memory impairment in the 120-min MCAO group 6 days after the acquisition trial. The brain tissue damage was significantly higher after 120-min occlusion of the MCA compared with 90-min occlusion; the infarct volumes were 13% and 25% of the contralateral hemispheres, respectively. In conclusion, both the 90- and 120-min occlusion models result in a significant impairment of sensorimotor, tactile and proprioceptive function, but memory impairment is only observed in the 120-min MCAO group. The beam-walking and cylinder tests detected neurological dysfunction after the 120-min MCAO, whereas the limb-placing and vibrissae-evoked forelimb-placing tests were able to evaluate the neurological dysfunction in rats after 90- and 120-min MCAO.

Administration of L-carnitine and mildronate improves endothelial function and decreases mortality in hypertensive Dahl rats.

Hypertension is a well established risk factor for the development of cardiovascular diseases and increased mortality. This study was performed to investigate the effects of the administration of L-carnitine or mildronate, an inhibitor of L-carnitine biosynthesis, or their combination on the development of hypertension-related complications in Dahl salt-sensitive (DS) rats fed with a high salt diet. Male DS rats were fed laboratory chow containing 8% NaCl from 7 weeks of age. Experimental animals were divided into five groups and treated for 8 weeks with vehicle (water; n = 10), L-carnitine (100 mg/kg, n = 10), mildronate (100 mg/kg, n = 10) or a combination of L-carnitine and mildronate at the doses above (n = 10). During the experiment, control group animals continued to consume a diet with normal salt content. Administration of the combination significantly improved the survival rate for 50% of the population. None of the tested compounds or their combination influenced high salt intake-induced hypertension, while treatment with mildronate and the combination for 8 weeks significantly decreased resting heart rate by 12% and 10%, respectively. Feeding with high salt diet had no influence on systolic function of the heart, but it induced thickening of the ventricular walls and development of heart hypertrophy that was not improved by the administration of tested compounds. In addition, administration of the combination attenuated the development of endothelial dysfunction in isolated aortic rings. In conclusion, our results demonstrate that treatment with a combination of L-carnitine and mildronate is protective against hypertension-induced complications in an experimental model of salt-induced hypertension.

Decreased acylcarnitine content improves insulin sensitivity in experimental mice models of insulin resistance

The important pathological consequences of insulin resistance arise from the detrimental effects of accumulated long-chain fatty acids and their respective acylcarnitines. The aim of this study was to test whether exercise combined with decreasing the content of long-chain acylcarnitines represents an effective strategy to improve insulin sensitivity in diabetes. We used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB), treatment and exercise to decrease acylcarnitine contents in the plasma and muscles in the insulin resistance models of high fat diet (HFD) fed C57BL/6 mice and db/db mice. The methyl-GBB treatment induced a substantial decrease in all acylcarnitine concentrations in both fed and fasted states as well as when it was combined with exercise. In the HFD fed mice methyl-GBB treatment improved both glucose and insulin tolerance. Methyl-GBB administration, exercise and the combination of both improved insulin sensitivity and reduced blood glucose levels in db/db mice. Methyl-GBB administration and the combination of the drug and exercise activated the PPARα/PGC1α signaling pathway and stimulated the corresponding target gene expression. Insulin insensitivity in db/db mice was not induced by significantly increased fatty acid metabolism, while increased insulin sensitivity by both treatments was not related to decreased fatty acid metabolism in muscles. The pharmacologically reduced long-chain acylcarnitine content represents an effective strategy to improve insulin sensitivity. The methyl-GBB treatment and lifestyle changes via increased physical activity for one hour a day have additive insulin sensitizing effects in db/db mice.

Investigation into Stereoselective Pharmacological Activity of Phenotropil

Phenotropil [N-carbamoylmethyl-4-aryl-2-pyrrolidone (2-(2-oxo-4-phenyl-pyrrolidin-1-yl) acetamide; carphedon)] is clinically used in its racemic form as a nootropic drug that improves physical condition and cognition. The aim of this study was to compare the stereoselective pharmacological activity of R- and S-enantiomers of phenotropil in different behavioural tests. Racemic phenotropil and its enantiomers were tested for locomotor, antidepressant and memory-improving activity and influence on the central nervous system (CNS) using general pharmacological tests in mice. After a single administration, the amount of compound in brain tissue extracts was determined using an ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method in a positive ion electrospray mode. In the open-field test, a significant increase in locomotor activity was observed after a single administration of R-phenotropil at doses of 10 and 50 mg/kg and S-phenotropil at a dose of 50 mg/kg. In the forced swim test, R-phenotropil induced an antidepressant effect at doses of 100 and 50 mg/kg, and S-phenotropil was active at a dose of 100 mg/kg. R-phenotropil significantly enhanced memory function in a passive avoidance response test at a dose of 1 mg/kg; the S-enantiomer did not show any activity in this test. However, the concentrations of R- and S-phenotropils in brain tissue were similar. In conclusion, the antidepressant and increased locomotor activity relies on both R- and S-phenotropils, but the memory-improving activity is only characteristic of R-phenotropil. These results may be important for the clinical use of optically pure isomers of phenotropil.