High l-carnitine concentrations do not prevent late diabetic complications in type 1 and 2 diabetic patients

Increased intake of l-carnitine, a cofactor in cellular energy metabolism, is recommended for diabetic patients with late complications. However, its clinical benefits remain controversial. We hypothesized that patients with low l-carnitine levels would have an increased rate of diabetic complications. To test this hypothesis, we evaluated the relationship of l-carnitine concentrations in blood with the prevalence and severity of late diabetic complications in type 1 and 2 diabetic patients. Human blood samples were collected from 93 and 87 patients diagnosed as having type 1 or type 2 diabetes, respectively, and 122 nondiabetic individuals. The determination of free l-carnitine concentrations in whole blood lysates was performed using ultra-performance liquid chromatography with tandem mass spectrometry. In diabetic patients, diabetic complications such as neuropathy, retinopathy, nephropathy, or hypertension were recorded. The average l-carnitine concentration in the blood of control subjects was 33 ± 8 nmol/mL, which was not significantly different from subgroups of patients with type 1 (32 ± 10 nmol/mL) or type 2 diabetes (36 ± 11 nmol/mL). Patients with low (<20 nmol/mL) l-carnitine levels did not have increased occurrences of late diabetic complications. In addition, patient subgroups with higher l-carnitine concentrations did not have decreased prevalence of late diabetic complications. Our results provide evidence that higher l-carnitine concentrations do not prevent late diabetic complications in type 1 and 2 diabetic patients.     

Effects of Long‐Term Mildronate Treatment on Cardiac and Liver Functions in Rats

Abstract: 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 γ‐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.     

Investigations on the pharmacology of the cardioprotective guanidine ME10092

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine), which possesses a strong cardioprotective effect to ischemia-reperfusion, was assessed for different pharmacological actions that may underlie its cardioprotective effect. In the living rat ME10092 decreased the blood pressure and heart rate in a dose-dependent manner. We found ME10092 to bind to alpha 1- and alpha 2-adrenoreceptors with moderate affinity (Ki values 1-4 microM), and to block adrenaline-elicited contractile responses in isolated guinea pig aortas. Our results indicate that ME10092 possesses a certain anti-oxidant profile. Thus, in a competitive manner and with low affinity it inhibited the bovine milk xanthine oxidase enzyme, as well as NAD(P)H oxidase driven oxyradical formation in membrane fractions isolated from the rat brain. By using electron paramagnetic resonance we here show that, after its systemic administration, ME10092 modulates the nitric oxide (NO) content in several tissues of the rat in a time-dependent manner. However, in vitro ME10092 inhibited the activities of nitric oxide synthases nNOS and eNOS, but not that of iNOS. Our data give evidence that the cardioprotective effect of ME10092 could be mediated through pharmacological mechanisms that include some modulation of NO production, as well as possible inhibition of radical formation during ischemia-reperfusion.     

The novel guanidine ME10092 protects the heart during ischemia-reperfusion

The novel guanidine N-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine [ME10092; a metabolite to the strongly cardioprotective hydroxyguanidine N-(3,4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine (PR5)] was administered intravenously to rats subjected to left coronary artery clamping followed by reperfusion. Administration of 1-10 mg/kg of ME10092 1 or 5 min before 10 min of coronary artery occlusion followed by 20 min reperfusion significantly and dose-dependently inhibited the reperfusion-induced burst of arrhythmia, and markedly improved the survival of the animals. This dose schedule also dose-dependently and significantly inhibited the ST-segment elevation seen on the ECG during the artery occlusion, and attenuated the secondary rise in ST-segment during the reperfusion. Even when ME10092 was administered 5 min after the start of the reperfusion, the ST-segment elevation became significantly attenuated. Administration of ME10092 (3 plus 1.5 mg/kg) to animals subjected to 1 h left coronary occlusion followed by 2 h reperfusion reduced the heart infarction size by about 40%. ME10092 also dose-dependently reduced the heart rate, both during normal conditions and during ischemia and reperfusion. Moreover, the highest dose of ME10092 used (10 mg/kg) strongly attenuated the reduction in blood pressure seen during 10 min left coronary occlusion, as well as it attenuated the rebound rise in blood pressure seen during the 20 min reperfusion phase; that is, resulting in a normalisation of the blood pressure disturbances caused by the ischemia-reperfusion. We also showed that after its p.o. administration, the PR5 hydroxyguanidine became completely metabolised to its guanidine ME10092, with no detectable traces of PR5 being present 30 and 60 min after the administration. Moreover, after the p.o. administration of ME10092, no signs of the formation of PR5 were seen on analysis of the rats' plasma. In view of the practically indistinguishable pharmacological effects of ME10092 and PR5, we suggest the strong cardioprotective effects of these compounds to be mediated by a direct effect by ME10092 per se.     

Application of hydrophilic interaction chromatography for simultaneous separation of six impurities of mildronate substance

The possibility of separating the impurities of mildronate, an antiischemic drug, by hydrophilic interaction chromatography (HILIC) was investigated on different polar stationary phases (silica, amino, cyano and zwitterionic sulfobetaine). The investigations have shown that HILIC is a useful alternative to reversed phase and ion-pair chromatography. The impact of HILIC separation conditions (acetonitrile content, buffer pH in mobile phase) on retention and selectivity has been systematically studied. Importance of these factors was found to be dependent on the structural properties of solutes. A HILIC method using a zwitterionic sulfobetaine stationary phase was developed and validated to determine six impurities in the drug substance. The method was validated in terms of specificity, limit of quantitation, limit of detection, linearity, accuracy and precision.     

Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction

The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.     

Inhibition of L-carnitine biosynthesis and transport by methyl-γ-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction

Background and Purpose

The important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia–reperfusion injury.

Key Results

In this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits γ-butyrobetaine dioxygenase (IC₅₀ 3 μM) and organic cation transporter 2 (OCTN2, IC₅₀ 3 μM), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5 or 20 mg·kg⁻¹) decreased the infarct size by 45–48%. In vivo pretreatment with Methyl-GBB (20 mg·kg⁻¹) attenuated the infarct size by 45% and improved 24 h survival of rats by 20–30%.

Conclusions and Implications

Reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia–reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism.     

The heart is better protected against myocardial infarction in the fed state compared to the fasted state

Objective

A variety of calorie restriction diets and fasting regimens are popular among overweight people. However, starvation could result in unexpected cardiovascular effects. Therefore, it is necessary to evaluate the short-term effects of diets on cardiovascular function, energy metabolism and potential risk of heart damage in case of myocardial infarction. The objective of the present study was to investigate whether the increased level of glucose oxidation or reduction of fatty acid (FA) load in the fed state provides the basis for protection against myocardial infarction in an experimental rat model of ischemia–reperfusion.

Materials/Methods

We tested the effects of the availability of energy substrates and their metabolites on the heart functionality and energy metabolism under normoxic and ischemia–reperfusion conditions.

Results

In a fasted state, the heart draws energy exclusively from FAs, whereas in a fed state, higher concentration of circulating insulin ensures a partial switch to glucose oxidation, while the load of FA on heart and mitochondria is reduced. Herein, we demonstrate that ischemic damage in hearts isolated from Wistar rats and diabetic Goto–Kakizaki rats is significantly lower in the fed state compared to the fasted state.

Conclusions

Present findings indicate that postprandial or fed-state physiology, which is characterised by insulin-activated glucose and lactate utilisation, is protective against myocardial infarction. Energy metabolism pattern in the heart is determined by insulin signalling and the availability of FAs. Overall, our study suggests that even overnight fasting could provoke and aggravate cardiovascular events and high-risk cardiovascular patients should avoid prolonged fasting periods.     

A short-term high-dose administration of sodium pivalate impairs pyruvate metabolism without affecting cardiac function

The pivalate moiety of some oral antibiotics enhances their intestinal absorption, but liberated pivalic acid decreases tissue carnitine concentration and could lead to impaired energy metabolism. The present study investigated the effects of short-term sodium pivalate administration on cardiac functionality and mitochondrial energy metabolism. Wistar rats received sodium pivalate (40?mM) in their drinking water for 14?days, and the carnitine content was measured in heart tissues. The activities of carnitine-dependent enzymes, including carnitine acetyltransferase (CrAT) and carnitine palmitoyltransferase I (CPT I), and the mitochondrial respiration rate were also measured. The isolated rat heart ischemia?Creperfusion injury assay was performed based on the Langendorff technique through the reversible occlusion of the left anterior descending coronary artery. The administration of sodium pivalate decreased carnitine concentration in the myocardium by 37?%. Sodium pivalate significantly decreased mitochondrial respiration on pyruvate/malate by 28?%. The activities of CrAT and CPT I in sodium pivalate-treated animals were decreased by 34 and 30?%, respectively. No differences were observed in the infarct size or in the heart functional parameters between the groups. Together, these results indicate that the short-term administration of a high dose of sodium pivalate impairs cardiac mitochondrial energy metabolism without depressing cardiac function during ischemia?Creperfusion injury.     

Activated peroxisomal fatty acid metabolism improves cardiac recovery in ischemia-reperfusion

Depressed oxidation of long chain fatty acids (LCFA) in heart ischemia leads to acute accumulation of LCFA metabolites that impair the functioning of the mitochondria. We hypothesized that reduced activity of carnitine palmitoyltransferase-I (CPT-I) might activate peroxisomal LCFA oxidation and protect mitochondrial function in ischemia and reperfusion. In the present study, despite the long-term threefold reduction in L-carnitine content by 3-(2,2,2-trimethylhydrazinium)-propionate, the uptake and oxidation rates of LCFA in the heart in normoxia were not significantly influenced. The significant increase in PPARα and PGC1α nuclear content, observed in this study, were followed by increased expression of genes involved in peroxisomal fatty acid oxidation (FAO) which compensated for the limited CPT-I-dependent FA transport into the mitochondria. In ischemia followed by reperfusion, the redirection of LCFA oxidation from mitochondria to peroxisomes protected the mitochondria from the accumulation of LCFA. In turn, the recovery of FAO resulted in significant reduction of myocardial infarct size. In conclusion, the decreased L-carnitine content in the heart preserves its peroxisomal and mitochondrial function after ischemia and improves cardiac recovery during reperfusion. The functional interplay between the decrease in L-carnitine and the PPARα/PGC1α pathway-induced redirection of FA metabolism protects the mitochondria against LCFA overload and provides a foundation for novel cardioprotective mechanisms.