The Effects of a Meldonium Pre-Treatment on the Course of the LPS-Induced Sepsis in Rats.

A dysregulated and overwhelming response to an infection accompanied by the exaggerated pro-inflammatory state and metabolism disturbance leads to the fatal outcome in sepsis. Previously we showed that meldonium, an anti-ischemic drug clinically used to treat myocardial and cerebral ischemia, strongly increases mortality in faecal-induced peritonitis (FIP) in rats. We postulated that the same mechanism that is responsible for the otherwise strong anti-inflammatory effects of meldonium could be the culprit of the increased mortality. In the present study, we applied the LPS-induced model of sepsis to explore the presence of any differences from and/or similarities to the FIP model. When it comes to energy production, despite some shared similarities, it is evident that LPS and FIP models of sepsis differ greatly. A different profile of sympathoadrenal activation may account for this observation, as it was lacking in the FIP model, whereas in the LPS model it was strong enough to overcome the effects of meldonium. Therefore, choosing the appropriate model of sepsis induction is of great importance, especially if energy homeostasis is the main focus of the study. Even when differences in the experimental design of the two models are acknowledged, the role of different patterns of energy production cannot be excluded. On that account, our results draw attention to the importance of uninterrupted energy production in sepsis but also call for much-needed revisions of the current recommendations for its treatment.

Effect of l-carnitine and mildronate on the mitochondrial metabolism of heart and bacterial composition of the gut microbiome in ageing mice.

Ageing is the most significant risk factor for cardiovascular diseases. l-Carnitine has a potent cardioprotective effect and its synthesis decreases during ageing. At the same time, there are pharmaceuticals, such as mildronate which, on the contrary, are aimed at reducing the concentration of l-carnitine in the heart and lead to slows down the oxidation of fatty acids in mitochondria. Despite this, both l-carnitine and mildronate are positioned as cardio protectors. We showed that l-carnitine supplementation to the diet of 15-month-old mice increased expression of the PGC-1α gene, which is responsible for the regulation of fatty acid oxidation, and the Nrf2 gene, which is responsible for protecting mitochondria by regulating the expression of antioxidants and mitophagy, in the heart. Mildronate activated the expression of genes that regulate glucose metabolism. Probably, this metabolic shift may protect the mitochondria of the heart from the accumulation of acyl-carnitine, which occurs during the oxidation of fatty acids under oxygen deficiency. Both pharmaceuticals impacted the gut microbiome bacterial composition. l-Carnitine increased the level of Lachnoanaerobaculum and [Eubacterium] hallii group, mildronate increased the level of Bifidobacterium, Rikinella, Christensenellaceae. Considered, that these bacteria for protection the organism from various pathogens and chronic inflammation. Thus, we suggested that the positive effects of both drugs on the mitochondria metabolism and gut microbiome bacterial composition may contribute to the protection of the heart during ageing.

The Effects of a Meldonium Pre-Treatment on the Course of the Faecal-Induced Sepsis in Rats.

Sepsis is a life-threatening condition caused by the dysregulated and overwhelming response to infection, accompanied by an exaggerated pro-inflammatory state and lipid metabolism disturbance leading to sequential organ failure. Meldonium is an anti-ischemic and anti-inflammatory agent which negatively interferes with lipid metabolism by shifting energy production from fatty acid oxidation to glycolysis, as a less oxygen-demanding pathway. Thus, we investigated the effects of a four-week meldonium pre-treatment on faecal-induced sepsis in Sprague-Dawley male rats. Surprisingly, under septic conditions, meldonium increased animal mortality rate compared with the meldonium non-treated group. However, analysis of the tissue oxidative status did not provide support for the detrimental effects of meldonium, nor did the analysis of the tissue inflammatory status showing anti-inflammatory, anti-apoptotic, and anti-necrotic effects of meldonium. After performing tissue lipidomic analysis, we concluded that the potential cause of the meldonium harmful effect is to be found in the overall decreased lipid metabolism. The present study underlines the importance of uninterrupted energy production in sepsis, closely drawing attention to the possible harmful effects of lipid-mobilization impairment caused by certain therapeutics. This could lead to the much-needed revision of the existing guidelines in the clinical treatment of sepsis while paving the way for discovering new therapeutic approaches.

Mildronate protects heart mtDNA from oxidative stress toxicity induced by exhaustive physical exercise.

Exhaustive physical exercises are potentially dangerous for human's physical health and may lead to chronic heart disease. Therefore, individuals involved in such activity require effective and safe cardioprotectors. The goal of this research was to study Mildronate (a cardioprotective drug) effect on the level of oxidative stress markers in hearts of mice under conditions of exhausting physical exercise, such as forced swimming for 1 h per day for 7 days. Forced swimming lead to mtDNA damage accumulation, increase in diene conjugates level and loss of reduced glutathione despite an increase in antioxidant genes expression and activation of mitochondrial biogenesis. Mildronate treatment reduced oxidative stress, probably due to the inhibition of fatty acids transport to mitochondria and an increase in the intensity of glucose oxidation, which in part confirms by increase in glucose transporter expression. Thus, we can assume that Mildronate is an effective cardioprotector in exhaustive physical exercises.

Testing Meldonium: Assessing Soviet pragmatic alternatives to the randomized controlled trial.

BACKGROUND/AIMS: Current research largely tends to ignore the drug-testing model that was developed in the "Second World" as an explicit alternative to the randomized controlled trial. This system can be described as "socialist pharmapolitics," accounting for the specific features of state socialism that influenced the development and testing of experimental drugs. The clinical trials model employed in the "Second World" was heavily influenced by the Soviet Union, which was by far the most influential player in the socialist bloc during the Cold War. Based on extensive archival research, this article presents an empirical case of a late Soviet clinical trial as a pragmatic alternative to the randomized controlled trial model. It accounts for the divergences between the official model prescribed by the Soviet authorities and the messy realities of healthcare practice. It further outlines different factors that ultimately shaped how clinical trials were organized in Soviet institutions "on the ground." Accordingly, this article presents a "real-life" history of "socialist pharmapolitics" and outlines the problems that this system faced in practice. METHODS: Archival research was conducted at the Russian State Archive of Scientific and Technical Documentation in Moscow. Archival files include scientific, technical, and registration documentation such as biochemical, pharmacological, and clinical descriptions of the experimental drug Meldonium, letters between various hospitals, research institutes and the Soviet regulatory body, as well as 26 reports of completed clinical trials. Manual content analysis was used for the interpretation of results. RESULTS: This article presents an empirical case of a late Soviet clinical trial as a pragmatic alternative to the randomized controlled trial model. It demonstrates some key differences from the randomized controlled trial model. This article also highlights some of the discrepancies between the model that was officially prescribed by the Soviet authorities and the realities of experimental drug testing in the Soviet Union in the late 1980s and early 1990s. In particular, it notes some elements of randomization, double-blinding, and the use of placebo that were present in Meldonium trials despite being formally denounced by Soviet bioethics. CONCLUSION: The Soviet model for testing experimental drugs differed from the Western one substantially in a number of respects. This difference was not only proclaimed officially by the Soviet authorities, but was for the most part enforced in clinical trials in practice. At the same time, our research demonstrates that there were important differences between the official model and the clinical realities on the ground.

Hyperpolarized magnetic resonance shows that the anti-ischemic drug meldonium leads to increased flux through pyruvate dehydrogenase in vivo resulting in improved post-ischemic function in the diabetic heart.

The diabetic heart has a decreased ability to metabolize glucose. The anti-ischemic drug meldonium may provide a route to counteract this by reducing l-carnitine levels, resulting in improved cardiac glucose utilization. Therefore, the aim of this study was to use the novel technique of hyperpolarized magnetic resonance to investigate the in vivo effects of treatment with meldonium on cardiac metabolism and function in control and diabetic rats. Thirty-six male Wistar rats were injected either with vehicle, or with streptozotocin (55 mg/kg) to induce a model of type 1 diabetes. Daily treatment with either saline or meldonium (100 mg/kg/day) was undertaken for three weeks. in vivo cardiac function and metabolism were assessed with CINE MRI and hyperpolarized magnetic resonance respectively. Isolated perfused hearts were challenged with low-flow ischemia/reperfusion to assess the impact of meldonium on post-ischemic recovery. Meldonium had no significant effect on blood glucose concentrations or on baseline cardiac function. However, hyperpolarized magnetic resonance revealed that meldonium treatment elevated pyruvate dehydrogenase flux by 3.1-fold and 1.2-fold in diabetic and control animals, respectively, suggesting an increase in cardiac glucose oxidation. Hyperpolarized magnetic resonance further demonstrated that meldonium reduced the normalized acetylcarnitine signal by 2.1-fold in both diabetic and control animals. The increase in pyruvate dehydrogenase flux in vivo was accompanied by an improvement in post-ischemic function ex vivo, as meldonium elevated the rate pressure product by 1.3-fold and 1.5-fold in the control and diabetic animals, respectively. In conclusion, meldonium improves in vivo pyruvate dehydrogenase flux in the diabetic heart, contributing to improved cardiac recovery after ischemia.

The effects of meldonium on the acute ischemia/reperfusion liver injury in rats.

Acute ischemia/reperfusion (I/R) liver injury is a clinical condition challenging to treat. Meldonium is an anti-ischemic agent that shifts energy production from fatty acid oxidation to less oxygen-consuming glycolysis. Thus, we investigated the effects of a 4-week meldonium pre-treatment (300 mg/kg b.m./day) on the acute I/R liver injury in Wistar strain male rats. Our results showed that meldonium ameliorates I/R-induced liver inflammation and injury, as confirmed by liver histology, and by attenuation of serum alanine- and aspartate aminotransferase activity, serum and liver high mobility group box 1 protein expression, and liver expression of Bax/Bcl2, haptoglobin, and the phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells. Through the increased hepatic activation of the nuclear factor erythroid 2-related factor 2, meldonium improves the antioxidative defence in the liver of animals subjected to I/R, as proved by an increase in serum and liver ascorbic/dehydroascorbic acid ratio, hepatic haem oxygenase 1 expression, glutathione and free thiol groups content, and hepatic copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Based on our results, it can be concluded that meldonium represent a protective agent against I/R-induced liver injury, with a clinical significance in surgical procedures.

Protective Effects of Meldonium in Experimental Models of Cardiovascular Complications with a Potential Application in COVID-19.

Right ventricular (RV) and left ventricular (LV) dysfunction is common in a significant number of hospitalized coronavirus disease 2019 (COVID-19) patients. This study was conducted to assess whether the improved mitochondrial bioenergetics by cardiometabolic drug meldonium can attenuate the development of ventricular dysfunction in experimental RV and LV dysfunction models, which resemble ventricular dysfunction in COVID-19 patients. Effects of meldonium were assessed in rats with pulmonary hypertension-induced RV failure and in mice with inflammation-induced LV dysfunction. Rats with RV failure showed decreased RV fractional area change (RVFAC) and hypertrophy. Treatment with meldonium attenuated the development of RV hypertrophy and increased RVFAC by 50%. Mice with inflammation-induced LV dysfunction had decreased LV ejection fraction (LVEF) by 30%. Treatment with meldonium prevented the decrease in LVEF. A decrease in the mitochondrial fatty acid oxidation with a concomitant increase in pyruvate metabolism was noted in the cardiac fibers of the rats and mice with RV and LV failure, respectively. Meldonium treatment in both models restored mitochondrial bioenergetics. The results show that meldonium treatment prevents the development of RV and LV systolic dysfunction by enhancing mitochondrial function in experimental models of ventricular dysfunction that resembles cardiovascular complications in COVID-19 patients.

The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity.

A mismatch between ß-oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to glucose as an energy substrate can be restored by reducing the reliance of the cell on fatty acid oxidation. The inhibition of the carnitine system, limiting the carnitine shuttle to the oxidation of lipids in the mitochondria, allows cells to develop a high plasticity to metabolic rewiring with a decrease in fatty acid oxidation and a parallel increase in glucose oxidation. We found that 3-(2,2,2-trimethylhydrazine)propionate (THP), which is able to reduce cellular carnitine levels by blocking both carnitine biosynthesis and the cell membrane carnitine/organic cation transporter (OCTN2), was reported to improve mitochondrial dysfunction in several diseases, such as Huntington's disease (HD). Here, new THP-derived carnitine-lowering agents (TCL), characterized by a high affinity for the OCTN2 with a minimal effect on carnitine synthesis, were developed, and their biological activities were evaluated in both in vitro and in vivo HD models. Certain compounds showed promising biological activities: reducing protein aggregates in HD cells, ameliorating motility defects, and increasing the lifespan of HD Drosophila melanogaster.

Mitochondrial Fatty Acid ß-Oxidation Inhibition Promotes Glucose Utilization and Protein Deposition through Energy Homeostasis Remodeling in Fish.

BACKGROUND: Fish cannot use carbohydrate efficiently and instead utilize protein for energy supply, thus limiting dietary protein storage. Protein deposition is dependent on protein turnover balance, which correlates tightly with cellular energy homeostasis. Mitochondrial fatty acid ß-oxidation (FAO) plays a crucial role in energy metabolism. However, the effect of remodeled energy homeostasis caused by inhibited mitochondrial FAO on protein deposition in fish has not been intensively studied. OBJECTIVES: This study aimed to identify the regulatory role of mitochondrial FAO in energy homeostasis maintenance and protein deposition by studying lipid, glucose, and protein metabolism in fish. METHODS: Carnitine-depleted male Nile tilapia (initial weight: 4.29 ± 0.12 g; 3 mo old) were established by feeding them with mildronate diets (1000 mg/kg/d) for 6 wk. Zebrafish deficient in the carnitine palmitoyltransferase 1b gene (cpt1b) were produced by using CRISPR/Cas9 gene-editing technology, and their males (154 ± 3.52 mg; 3 mo old) were used for experiments. Normal Nile tilapia and wildtype zebrafish were used as controls. We assessed nutrient metabolism and energy homeostasis-related biochemical and molecular parameters, and performed 14C-labeled nutrient tracking and transcriptomic analyses. RESULTS: The mitochondrial FAO decreased by 33.1-88.9% (liver) and 55.6-68.8% (muscle) in carnitine-depleted Nile tilapia and cpt1b-deficient zebrafish compared with their controls (P < 0.05). Notably, glucose oxidation and muscle protein deposition increased by 20.5-24.4% and 6.40-8.54%, respectively, in the 2 fish models compared with their corresponding controls (P < 0.05). Accordingly, the adenosine 5'-monophosphate-activated protein kinase/protein kinase B-mechanistic target of rapamycin (AMPK/AKT-mTOR) signaling was significantly activated in the 2 fish models with inhibited mitochondrial FAO (P < 0.05). CONCLUSIONS: These data show that inhibited mitochondrial FAO in fish induces energy homeostasis remodeling and enhances glucose utilization and protein deposition. Therefore, fish with inhibited mitochondrial FAO could have high potential to utilize carbohydrate. Our results demonstrate a potentially new approach for increasing protein deposition through energy homeostasis regulation in cultured animals.

The Vasorelaxant and Neuroprotective Effects of Mildronate in A Rabbit Subarachnoid Hemorrhage Model.

AIM: To investigate the effects of an anti-ischemic agent, mildronate, on subarachnoid hemorrhage-induced vasospasm. MATERIAL AND METHODS: Rabbits were randomly divided into four groups: control, subarachnoid hemorrhage (SAH), vehicle, and mildronate (n=8 animals per group). In the treatment group, 200 mg/kg of mildronate were intraperitoneally administered 5 minutes after the procedure and continued for 3 days as daily administrations of the same dose. At the end of the third day, the cerebrum, cerebellum, and brain stem were perfused, fixated, and removed for histopathological examination. Tissues were examined for arterial wall thickness, luminal area, and hippocampal neuronal degeneration. RESULTS: Mildronate group showed significantly increased luminal area and reduced wall thickness of the basilar artery compared with the subarachnoid hemorrhage group. In addition, the hippocampal cell degeneration score was significantly lower in the mildronate group than in the SAH and vehicle groups. CONCLUSION: These results show that mildronate exerts protective effects against SAH-induced vasospasm and secondary neural injury.

Increased fatty acid oxidation and mitochondrial proliferation in liver are associated with increased plasma kynurenine metabolites and nicotinamide levels in normolipidemic and carnitine-depleted rats.

Dysregulation of the tryptophan (Trp)-NAD+ pathway has been related to several pathological conditions, and the metabolites in this pathway are known to influence mitochondrial respiration and redox status. The aim of this project was to investigate if stimulation of beta-oxidation and mitochondrial proliferation by the mitochondrial-targeted compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) would influence metabolites of the Trp-Kyn-NAD+ pathway. We wished to investigate how carnitine depletion by meldonium-treatment influenced these metabolites. After dietary treatment of male Wistar rats with 1-triple TTA for three weeks, increased hepatic mitochondrial- and peroxisomal fatty acid oxidation resulted. The plasma content of total carnitines decreased compared to control animals, whereas hepatic genes involved in CoA biosynthesis were upregulated by 1-triple TTA treatment. The plasma Trp level and individual metabolites in the kynurenine pathway were increased by 1-triple TTA, associated with decreased hepatic gene expression of indoleamine2,3-dioxygenase. 1-triple TTA treatment increased conversion of Trp to nicotinamide (Nam) as the plasma content of quinolinic acid, Nam and N1-methylnicotinamide (mNam) increased, accompanied with suppression of hepatic gene expression of α-amino-α-carboxymuconate-ε-semialdehyde decarboxylase. A positive correlation between mitochondrial fatty acid oxidation and Trp-derivatives was found. Almost identical results were obtained by 1-triple TTA in the presence of meldonium, which alone exerted minor effects. Moreover, the plasma Kyn:Trp ratio (KTR) correlated negatively to mitochondrial function. Whether increased flux through the Trp-NAD+ pathway increased redox status and lowered inflammation locally and systemically should be considered.

Normalization of heart rate variability with taurine and meldonium complex in post-infarction patients with type 2 diabetes mellitus.

The purpose of this study is to scrutiny the Dynamics of heart rate variability (HRV) in patients with PICS with 2nd type DM against the background of Taurine (TN) and meldonium (ME). The results of the investigations prove the decrease of the oxidative stress, which is basis of DACN, under the influence of sulfur-containing amino acid taurine (TN), and meldonium (ME) - a competitive inhibitor of gamma-butyrobetaine hydroxylase. Biochemical mechanisms of synergistic action of ME and TN are also described. The results of the studies of 98 patients with PICS and concomitant 2nd type diabetes mellitus were analyzed. They were distributed by simple randomization method into two groups, comparable according to age and sex: the main group (MG) (n = 68): and group of comparison (GoC) (n = 30). HRV was evaluated twice daily at the Cardiosense HMEGG system: at baseline and after 12 weeks of treatment. For the assessment of HRV the frequency and spectral parameters were used. While evaluating the different methods of treatment, their influence on the range of spectral and time indices of HRV was determined (p = 0.001 by the criterion of Kruskall-Wallis). It was learned that the combined application of ME and TN gives a statistically significant (p <0.01) increase of SDNN, HF at night, pNN - on 50% by day (p <0.01, p <0.001 and p <0.01 respectively), and statistically significant decrease in LF at night, compared to GHG.

Neuroprotective effects of mildronate in a rat model of traumatic brain injury.

OBJECTIVE: Traumatic brain injury (TBI) is one of the most common preventable causes of mortality and morbidity. Inflammation, apoptosis, oxidative stress, and ischemia are some of the important pathophysiological mechanisms underlying neuronal loss after TBI. Mildronate is demonstrated to be beneficial in various experimental models of ischemic diseases via anti-inflammatory, antioxidant, and neuroprotective mechanisms. This study aimed to investigate possible antioxidant, anti-inflammatory, antiapoptotic, and neuroprotective effects of mildronate in a rat model of TBI. METHODS: A total of 46 male rats were divided into three groups of control, saline-treated TBI, and mildronate-treated TBI. Both TBI groups were subjected to closed-head contusive weight-drop injuries followed by treatment with saline or mildronate (100 mg/kg) administered intraperitoneally. The forebrain was removed 24 h after trauma induction, the activities of myeloperoxidase (MPO) and caspase-3, levels of superoxide dismutase (SOD), luminol- and lucigenin-enhanced chemiluminescence were measured, and histomorphological evaluation of cerebral tissues was performed. RESULTS: Increased MPO and caspase-3 activities in the vehicle-treated TBI group (p < 0.001) were suppressed in the mildronate-treated TBI group (p < 0.001). Similarly, increase in luminol and lucigenin levels (p < 0.001 and p < 0.01, respectively) in the vehicle-treated TBI group were decreased in the mildronate-treated TBI group (p < 0.001). Concomitantly, in the vehicle-treated TBI group, TBI-induced decrease in SOD activity (p < 0.01) was reversed with mildronate treatment (p < 0.05). On histopathological examination, TBI-induced damage in the cerebral cortex was lesser in the mildronate-treated TBI group than that in other groups. CONCLUSION: This study revealed for the first time that mildronate, exhibits neuroprotective effects against TBI because of its anti-inflammatory, antiapoptotic, and antioxidant activities.

Meldonium improves Huntington's disease mitochondrial dysfunction by restoring peroxisome proliferator-activated receptor γ coactivator 1α expression.

Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.

A fatty acid analogue targeting mitochondria exerts a plasma triacylglycerol lowering effect in rats with impaired carnitine biosynthesis.

L-carnitine is important for the catabolism of long-chain fatty acids in the mitochondria. We investigated how the triacylglycerol (TAG)-lowering drug 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) influenced lipid metabolism in carnitine-depleted, 3-(2,2,2-trimethylhydrazinium)propionate dehydrate (Mildronate; meldonium)-treated male Wistar rats. As indicated, carnitine biosynthesis was impaired by Mildronate. However, TAG levels of both plasma and liver were decreased by 1-triple TTA in Mildronate-treated animals. This was accompanied by increased gene expression of proteins involved in mitochondrial activity and proliferation and reduced mRNA levels of Dgat2, ApoB and ApoCIII in liver. The hepatic energy state was reduced in the group of Mildronate and 1-triple TTA as reflected by increased AMP/ATP ratio, reduced energy charge and induced gene expression of uncoupling proteins 2 and 3. The increase in mitochondrial fatty acid oxidation was observed despite low plasma carnitine levels, and was linked to strongly induced gene expression of carnitine acetyltransferase, translocase and carnitine transporter, suggesting an efficient carnitine turnover. The present data suggest that the plasma TAG-lowering effect of 1-triple TTA in Mildronate-treated rats is not only due to increased mitochondrial fatty acid oxidation reflected by increased mitochondrial biogenesis, but also to changes in plasma clearance and reduced TAG biosynthesis.

Inhibited fatty acid ß-oxidation impairs stress resistance ability in Nile tilapia (Oreochromis niloticus).

Energy metabolism plays important roles in stress resistance and immunity in mammals, however, such functions have not been established in fish. In the present study, Nile tilapia (Oreochromis niloticus) was fed with mildronate, an inhibitor of mitochondrial fatty acid (FA) ß-oxidation, for six weeks subsequently challenged with Aeromonas hydrophila and ammonia nitrogen exposure. Mildronate treatment reduced significantly l-carnitine concentration and mitochondrial FA ß-oxidation efficiency, while it increased lipid accumulation in liver. The fish with inhibited hepatic FA catabolism had lower survival rate when exposed to Aeromonas hydrophila and ammonia nitrogen. Moreover, fish fed mildronate supplemented diet had lower immune enzymes activities and anti-inflammatory cytokine genes expressions, but had higher pro-inflammatory cytokine genes expressions. However, the oxidative stress-related biochemical indexes were not significantly affected by mildronate treatment. Taken together, inhibited mitochondrial FA ß-oxidation impaired stress resistance ability in Nile tilapia mainly through inhibiting immune functions and triggering inflammation. This is the first study showing the regulatory effects of lipid catabolism on stress resistance and immune functions in fish.

Muscle carnitine availability plays a central role in regulating fuel metabolism in the rodent.

KEY POINTS: Meldonium inhibits endogenous carnitine synthesis and tissue uptake, and accelerates urinary carnitine excretion, although the impact of meldonium-mediated muscle carnitine depletion on whole-body fuel selection, and muscle fuel metabolism and its molecular regulation is under-investigated. Ten days of oral meldonium administration did not impact on food or fluid intake, physical activity levels or body weight gain in the rat, whereas it depleted muscle carnitine content (all moieties), increased whole-body carbohydrate oxidation and muscle and liver glycogen utilization, and reduced whole-body fat oxidation. Meldonium reduced carnitine transporter protein expression across muscles of different contractile and metabolic phenotypes. A TaqMan PCR low-density array card approach revealed the abundance of 189 mRNAs regulating fuel selection was altered in soleus muscle by meldonium, highlighting the modulation of discrete cellular functions and metabolic pathways. These novel findings strongly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo. ABSTRACT: The body carnitine pool is primarily confined to skeletal muscle, where it regulates carbohydrate (CHO) and fat usage. Meldonium (3-(2,2,2-trimethylhydrazinium)-propionate) inhibits carnitine synthesis and tissue uptake, although the impact of carnitine depletion on whole-body fuel selection, muscle fuel metabolism and its molecular regulation is under-investigated. Male lean Zucker rats received water (control, n = 8) or meldonium-supplemented water (meldonium, n = 8) for 10 days [1.6 g kg-1 body mass (BM) day-1 days 1-2, 0.8 g kg-1  BM day-1 thereafter]. From days 7-10, animals were housed in indirect calorimetry chambers after which soleus muscle and liver were harvested. Food and fluid intake, weight gain and physical activity levels were similar between groups from days 7 to 10. Compared to control, meldonium depleted muscle total carnitine (P < 0.001) and all carnitine esters. Furthermore, whole-body fat oxidation was less (P < 0.001) and CHO oxidation was greater (P < 0.05) compared to the control, whereas soleus and liver glycogen contents were less (P < 0.01 and P < 0.01, respectively). In a second study, male Wistar rats received water (n = 8) or meldonium-supplemented water (n = 8) as above, and kidney, heart and extensor digitorum longus muscle (EDL) and soleus muscles were collected. Compared to control, meldonium depleted total carnitine content (all P < 0.001), reduced carnitine transporter protein and glycogen content, and increased pyruvate dehydrogenase kinase 4 mRNA abundance in the heart, EDL and soleus. In total, 189 mRNAs regulating fuel selection were differentially expressed in soleus in meldonium vs. control, and a number of cellular functions and pathways strongly associated with carnitine depletion were identified. Collectively, these data firmly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo.

Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2).

The purpose of this study was to characterize the uptake of carnitine, the physiological substrate, and the uptake of 3-(2,2,2-trimethylhydrazinium)propionate, a consensus substrate by rat Octn2 and human OCTN2 transporters as well as to characterize drug-mediated inhibition of l-carnitine uptake by the rat and human orthologs overexpressed in CHO-K1 cells. l-carnitine and 3-(2,2,2-trimethylhydrazinium)propionate were found to be a lower affinity substrate for rat Octn2 (KM = 32.66 ± 5.11 µM and 23.62 ± 4.99 µM respectively) than for human OCTN2 (KM = 3.08 ± 0.74 µM and 7.98 ± 0.63 µM). The intrinsic clearance (CLint) value for carnitine was higher for the human than for the rat transporter (22.82 ± 5.57 ml/min*mg vs 4.008 ± 0.675 ml/min*mg). For 3-(2,2,2-trimethylhydrazinium)propionate, in contrast, the CLint value for rat Octn2 was higher than for human OCTN2 (323.9 ± 72.8 ml/min*mg vs 65.11 ± 5.33 ml/min*mg). Furthermore, many pharmacologically important drugs were shown to affect l-carnitine transport by Octn2/OCTN2. The correlation between the IC50 datasets for the rat and human transporter resulted in an r value of 0.47 (p > 0.05). However, the greatest difference was less than seven-fold and 13 of 15 compounds yielded a difference less than 3-fold. Thus, the transporters from these two species showed an overlapping but somewhat different substrate and inhibitor specificity.

Carnitine and γ-Butyrobetaine Stimulate Elimination of Meldonium due to Competition for OCTN2-mediated Transport.

Meldonium (3-(2,2,2-trimethylhydrazinium)propionate) is the most potent clinically used inhibitor of organic cation transporter 2 (OCTN2). Inhibition of OCTN2 leads to a decrease in carnitine and acylcarnitine contents in tissues and energy metabolism optimization-related cardioprotective effects. The recent inclusion of meldonium in the World Anti-Doping Agency List of Prohibited Substances and Methods has raised questions about the pharmacokinetics of meldonium and its unusually long elimination time. Therefore, in this study, the rate of meldonium washout after the end of the treatment was tested with and without administration of carnitine, γ-butyrobetaine (GBB) and furosemide to evaluate the importance of competition for OCTN2 transport in mice. Here, we show that carnitine and GBB administration during the washout period effectively stimulated the elimination of meldonium. GBB induced a more pronounced effect on meldonium elimination than carnitine due to the higher affinity of GBB for OCTN2. The diuretic effect of furosemide did not significantly affect the elimination of meldonium, carnitine and GBB. In conclusion, the competition of meldonium, carnitine and GBB for OCTN2-mediated transport determines the pharmacokinetic properties of meldonium. Thus, due to their affinity for OCTN2, GBB and carnitine but not furosemide stimulated meldonium elimination. During long-term treatment, OCTN2-mediated transport ensures a high muscle content of meldonium, while tissue clearance depends on relatively slow diffusion, thus resulting in the unusually long complete elimination period of meldonium.