Biochemical characteristics of the anti-ischemic action of the new structural analog of gamma-butyrobetaine 3-(2,2,2,-trimethylhydrazine)propionate

A structural analogue of gamma-butyrobetaine 3-(2,2,2-trimethylhydrazine)propionate (THP) administered orally in doses of 50 and 150 mg/kg for 10 days prevented isoproterenol-induced increase of the activity of the hepatic isoform of lactate dehydrogenase in the rat blood serum and in a dose of 150 mg/kg prevented an increase of creatine phosphokinase activity. Against a background of the course administration of THP isoproterenol failed to cause the accumulation of acyl-insoluble acylcarnitine in the myocardium. In this case a dose-dependent decrease of free carnitine concentration and accumulation of fatty acids in the myocardium were noted. The cardioprotective effect of THP manifested itself in prevention of a decrease of ATP and ADP concentrations, accumulation of AMP and a reduction of energy charge under the influence of isoproterenol. The ability of THP to decrease the intracellular concentration of free carnitine and to depress as a result carnitine-dependent oxidation of free fatty acids may underlie the anti-ischemic effect of THP.     

The cardioprotective action of carnitine and its structural analog 3-(2,2,2-trimethylhydrazine)propionate on cardiac energy metabolism in experimental occlusion of the coronary artery in rats

The effects of carnitine and its structural analogue 3-(2,2,2-trimethylhydrazine) propionate (THP) were studied in rats with experimental myocardial infarction caused by occlusion of the left descending branch of the coronary artery. After one day in the group of untreated animals the relative lethality was 40.3 +/- 10.5%, the size of the infarction zone was 29.8 +/- 2.0%. Carnitine and THP decreased on the average twice the parameters as well as lactate level in the myocardium. THP prevented a reduction of ATP and AMP levels by 35 and 37%, respectively, and a decrease of adenine nucleotide pool by 30%. In this case carnitine was ineffective. It is suggested that inhibition of beta-oxidation of fatty acids by THP is energetically more beneficial for the myocardium during regional ischemia than substitution therapy with carnitine.     

Alkylglycidic acids: potential new hypoglycemic agents

A series of alkylglycidic acid analogues and derivatives were synthesized and tested for their ability to inhibit long-chain fatty acid oxidation in vitro and to lower blood sugar in rats. The extent of inhibition of carnitine acyl transferase, the enzyme at the mitochondrial membrane necessary to transport long-chain fatty acids into the mitochondria for subsequent beta-oxidation, was determined for the series. Structure-activity relationships using in vitro inhibition of [1-14C]palmitic acid oxidation in rat hemidiaphragm muscle indicate that potent activity resides mainly in 2-alkyl (C12-C16) glycidates. Replacement of the oxirane ring with cyclopropyl, thiirane, or other rings diminishes activity, as does substitution of the glycidate ring at the 3-position. In vivo potency in the rat glucose tolerance test roughly parallels the hemidiaphragm results. The lead compound, methyl 2-tetradecylglycidate (8), is a potent hypoglycemic agent following oral administration to several animal species. The hypoglycemic analogues interfere with fatty acid oxidation by specific and irreversible inhibition of mitochondrial carnitine palmitoyl transferase-A.     

Reactivation of peroxisome proliferator-activated receptor alpha in spontaneously hypertensive rat: age-associated paradoxical effect on the heart

Prevention of left ventricular hypertrophy remains a challenge in the prevention of hypertension-induced adverse cardiac remodeling. Cardiac hypertrophy is associated with a shift in energy metabolism from predominantly fatty acid to glucose with a corresponding reduction in the expression of fatty acid oxidation enzyme genes. Although initially adaptive, the metabolic switch seems to be detrimental in the long run. This study was taken up with the objective of examining whether the stimulation of fatty acid oxidation by the activation of peroxisome proliferator-activated receptor alpha (PPARα), a key regulator of fatty acid metabolism, can prevent cardiac hypertrophy. Fenofibrate was used as the PPARα agonist. Spontaneously hypertensive rats (SHRs) in the initial stages of hypertrophy (2 months) and those with established hypertrophy (6 months) were treated with fenofibrate (100 mg·kg·d for 60 days). Cluster of differentiation 36 (CD36)-responsible for myocardial fatty acid uptake, carnitine palmitoyl transferase 1β-a mitochondrial transporter protein and medium chain acyl-Co-A dehydrogenase-a key enzyme in beta oxidation of fatty acids were selected as indicators of fatty acid metabolism. Hypertrophy was apparent at 2 months and metabolic shift at 4 months of age in SHRs. The treatment prevented cardiac remodeling in young animals but aggravated hypertrophy in older animals. Hypertrophy showed a positive association with malondialdehyde levels and cardiac NF-κB gene expression, signifying the role of oxidative stress in the mediation of hypertrophy. Expression of carnitine palmitoyl transferase 1β and medium chain acyl-Co-A dehydrogenase was upregulated on treatment. However, CD36 showed an age-dependent variation on treatment, with no change in expression in young rats and downregulation in older animals. It is inferred that the stimulation of PPARα before the initiation of metabolic remodeling may prevent cardiac hypertrophy, but reactivation after the metabolic adaptation aggravates hypertrophy. Whether the downregulation of CD36 is mediated by decreased substrate availability remains to be explored. Age-dependent paradoxical effect on the heart in response to fenofibrate, used as a lipid-lowering drug, can have therapeutic implications.     

Carnitine transport into muscular cells. Inhibition of transport and cell growth by mildronate

Carnitine is involved in the transfer of fatty acids across mitochondrial membranes. Carnitine is found in dairy and meat products, but is also biosynthesized from lysine and methionine via a process that, in rat, takes place essentially in the liver. After intestinal absorption or hepatic biosynthesis, carnitine is transferred to organs whose metabolism is dependent on fatty acid oxidation, such as heart and skeletal muscle. In skeletal muscle, carnitine concentration was found to be 50 times higher than in the plasma, implicating an active transport system for carnitine. In this study, we characterized this transport in isolated rat myotubes, established mouse C2C12 myoblastic cells, and rat myotube plasma membranes and found that it was Na(+)-dependent and partly inhibited by a Na(+)/K(+) ATPase inhibitor. L-carnitine analogues such as D-carnitine and gamma-butyrobetaine interfere with this system as does acyl carnitine. Among these inhibitors, the most potent was mildronate (3-(2,2,2-trimethylhydrazinium)propionate), known as a gamma-butyrobetaine hydroxylase inhibitor. It also induced a marked decrease in carnitine transport into muscle cells. Removal of carnitine or treatment with mildronate induced growth inhibition of cultured C2C12 myoblastic cells. These data suggest that myoblast growth and/or differentiation is dependent upon the presence of carnitine.     

Carnitine Palmitoyltransferase-I,a New Target for the Treatment of Heart Failure

Although the heart is capable of extracting energy from different types of substrates such as fatty acids and carbohydrates, fatty acids are the preferred fuel under physiological conditions. In view of the presence of diverse defects in myocardial metabolism in the failing heart, changes in metabolism of glucose and fatty acids are considered as viable targets for therapeutic modification in the treatment of heart failure. One of these changes involves the carnitine palmitoyltransferase (CPT) enzymes, which are required for the transfer of long chain fatty acids into the mitochondrial matrix for oxidation. Since CPT inhibitors have been shown to prevent the undesirable effects induced by mechanical overload, e.g. cardiac hypertrophy and heart failure, it was considered of interest to examine whether the inhibition of CPT enzymes represents a novel approach for the treatment of heart disease. A shift from fatty acid metabolism to glucose metabolism due to CPT-I inhibition has been reported to exert beneficial effects in both cardiac hypertrophy and heart failure. Since the inhibition of fatty acid oxidation is effective in controlling abnormalities in diabetes mellitus, CPT-I inhibitors may also prove useful in the treatment of diabetic cardiomyopathy. Accordingly, it is suggested that CPT-I may be a potential target for drug development for the therapy of heart disease in general and heart failure in particular.     

Value of carnitine therapy in kidney dialysis patients and effects on cardiac function from human and animal studies

Cardiovascular complications are the leading cause of mortality, accounting for 50% of all deaths among patients with end-stage renal disease (ESRD). The majority of these deaths are from cardiac causes. The mechanisms underlying the enhanced susceptibility to myocardial ischaemia and subsequent morbidity in ESRD remain ill-defined. Numerous metabolic derangements accompany myocardial ischaemia and reperfusion and play a pivotal role in the development of concurrent myocardial dysfunction. Carnitine plays a critical role in myocardial energy metabolism, as the transporter of long chain fatty acyl intermediates across the inner mitochondrial membrane for β oxidation and as a central regulator of carbohydrate metabolism. Myocardial carnitine is significantly depleted during ischaemia and more particularly in uraemic patients and those on dialysis therapy. Carnitine treatment has cardiovascular benefits including modulation of myocardial metabolism, reduction in necrotic cell death and infarct size, decrease in the incidence of arrhythmias and preservation of mechanical function. This review details the profile of substrate metabolism in the uraemic heart and the impact of carnitine supplementation on metabolism and function of the reperfused heart and finally the experimental and clinical evidence for carnitine replacement therapy, in particular its impact on the uraemic heart via modulation of function and energetics.     

Influence of theophylline on urinary excretion of total, free and acyl carnitine in rats.

BACKGROUND: Carnitine plays a critical role in lipid metabolism. Carnitine deficiency may adversely affect the oxidation of fatty acids and further aggravate abnormal lipid metabolism. OBJECTIVE: To investigate the effect of oral theophylline administration on urinary excretion of total (TC), free (FC), acyl (AC) carnitine as well as the ratio of AC to FC in rats. METHOD: The study was a randomized, controlled animal study. Theophylline was given at 100 mg/kg b.w./day and effects were monitored after a treatment period that lasted between one week and five weeks. RESULTS: Theophylline treatment caused significantly increased food intake and urinary excretion as compared to either control or placebo, P < 0.01. The results indicated that a significant increase in urinary TC, FC and AC excretion as compared to those of control and placebo groups (P < 0.01). Furthermore, the ratio of AC to FC was significantly increased (P < 0.01) as compared to either control or placebo group. CONCLUSION: Theophylline administration to rats leads to significant changes in the urinary excretion of carnitine. These changes may result from theophylline-affected alteration renal tubular re-absorption of carnitine.     

Hypocarnitinaemia induced by sodium pivalate in the rat is associated with left ventricular dysfunction and impaired energy metabolism

Carnitine is a naturally occurring compound that is essential in energy metabolism of the mammalian heart. In addition to its essential role in facilitating beta-oxidation, carnitine eliminates excess toxic acyl residues and regulates the mitochondrial acetyl coenzyme A (CoA)/CoA ratio. Thus, it is not surprising that patients with carnitine deficiency syndromes exhibit defects in energy metabolism and in some cases demonstrate left ventricular dysfunction. Pivalic acid is commonly used to create prodrugs, such as pivampicillin and pivmecillinam, to facilitate enteral absorption and increase oral bioavailability. Pivalic acid released from the drug following absorption readily forms an ester with carnitine, which is then excreted as pivaloylcarnitine. Sustained loss of carnitine in the form of this ester induces a state of carnitine deficiency, exemplified by low plasma and tissue carnitine content. This review examines the effects in the rat of short- and long-term sodium pivalate treatment on: (1) cardiac carnitine content; (2) in vitro mechanical function; (3) markers of glycolytic and fatty acid metabolism; and (4) energy substrate metabolism. Treatment with sodium pivalate induces a gradual loss of cardiac carnitine content for up to 12 weeks. Doubling the duration of treatment is not associated with any further decrease in cardiac carnitine content. While heart function following short-term treatment (2 weeks) is normal under aerobic conditions, impaired recovery of function following ischaemia is seen. In contrast, long-term treatment (11-28 weeks) is associated with impaired heart function, which is dependent on workload and substrate availability. Impaired heart function is also associated with reductions in activity of 3-hydroxyacyl CoA dehydrogenase and rates of fatty acid oxidation. However, to maintain adenosine triphosphate production, glucose metabolism, expressed as hexokinase activity and glucose oxidation, is increased in carnitine-deficient hearts. Hearts from sodium pivalate-treated animals demonstrate a cardiomyopathy that is dependent on duration of treatment, workload and substrate supply. This model of hypocarnitinaemia may thus be useful to study the metabolic and cardiac consequences of carnitine-deficiency syndromes.     

Effect of the calcium antagonists ryodipine and verapamil on the carnitine-dependent metabolism of fatty acids in the rat heart

Riodipine (10 days, 10 mg/kg) was shown to prevent in rats an isoproterenol-induced increase of free fatty acid concentration in the blood serum and heart and to promote normalization of long-chain acylcarnitine content in the heart. Under the same conditions verapamil caused an increase in free acid concentration in the blood serum and prevented their accumulation in the heart. Its ability to limit accumulation of long-chain acylcarnitine manifested itself in a lesser degree as compared to riodipine. Riodipine exerted no effect on oxidation of 1-14-C-palmitic acid by the rat heart homogenate, verapamil suppressed oxidation of this fatty acid.     

Effects of 5-iodotubercidin on hepatic fatty acid metabolism mediated by the inhibition of acetyl-CoA carboxylase

Diverse mechanisms of action have been proposed for 5-iodotubercidin, although it is widely used as an adenosine kinase inhibitor that consequently interferes with the metabolism of adenosine and adenine nucleotides. Incubation of rat hepatocytes with iodotubercidin produced important effects on lipid metabolism. (i) Both acetyl-CoA carboxylase and fatty acid synthesis de novo were inhibited in parallel by iodotubercidin, with no change in the activity of fatty acid synthase. The inhibition of both activities showed a comparable dependence on iodotubercidin concentration and was accompanied by a similar decrease (about 60%) in the intracellular malonyl-CoA concentration. (ii) Iodotubercidin stimulated palmitate oxidation, although octanoate oxidation was unaffected. However, this effect can be attributed to the decrease of malonyl-CoA concentration and the concomitant relief of the inhibition of carnitine palmitoyltransferase I, because the activity of this enzyme was found unaltered when determined in cells permeabilized with digitonin. (iii) Iodotubercidin also inhibited cholesterol synthesis de novo. Results, thus, indicate that iodotubercidin increases fatty acid oxidation activity of the liver at the expense of lipogenesis, and we suggest that these effects on fatty acid metabolism are mediated by the inhibition of acetyl-CoA carboxylase, probably due to a more than twice increase in the AMP/ATP ratio and the concomitant stimulation of the AMP-activated protein kinase.     

延胡索乙素对大鼠实验性心肌缺血的保护作用

目的 研究延胡乙素（dl-THP)对大鼠实验性心肌缺血的保护作用。方法 观察dl-THP对垂体后叶素（pit)所致大鼠心电图急性缺血性改变的预防作用以及对异丙肾上腺素（Iso)引起心肌损伤大鼠的保护作用。结果 dl-THP对垂体后叶素所致心电图改变有明显预防作用。能对抗Iso所致ECG的ST段升高。显著抑制心肌组织中磷酸肌激酶（CPK）、乳酸脱氢酶（LDH）的释放,降低血清CPK和LDH水平,保护心肌组织超氧化物歧化酶（SOD）活性,减少丙二醛（MDA）生成。结论 dl-THP对实验性心肌缺血有明显的保护作用。     

Effect of 4-(4′-chlorobenzyloxy)benzyl nicotinate (KCD-232) on triglyceride and fatty acid metabolism in rats

The effects of KCD-232, a new hypolipidemic agent with a structure of 4-(4'-chlorobenzyloxy) benzyl nicotinate, on triglyceride (TG) and fatty acid (FA) metabolism were studied in rats. KCD-232 dose-dependently reduced both liver and serum TG levels. From in vivo and in vitro studies, the hypotriglyceridemic action of KCD-232 was shown to be based on the inhibition of hepatic TG synthesis due to both decreased FA synthesis and increased FA oxidation in the liver. Of two metabolites of KCD-232, i.e. 4-(4'-chlorobenzyloxy)benzoic acid (MII) and nicotinic acid, MII was found to be responsible for the decreased synthesis and increased oxidation of FA in the liver, the latter apparently being due to increased mitochondrial oxidation activated by MII. MII was demonstrated to form a xenobiotic TG in which one fatty acid moiety was substituted by MII and to form a thioester with CoA by rat liver microsomes. This thioester, MII-CoA, inhibited fatty acid syntheses from [¹⁴C]acetate, [¹⁴C] acetyl-CoA and [¹⁴C]malonyl-CoA in cell-free enzyme systems from rat liver both with and without an NADPH-generating system, whereas MII as such showed no effect. MII-CoA was therefore considered to be a chemical entity for the inhibition of hepatic fatty acid synthesis by KCD-232 and was suggested to inhibit fatty acid synthetase directly.     

Potent Peroxisome Proliferators Inhibit β-Oxidation in the Isolated Perfused Rat Liver

It is unknown whether peroxisome proliferators decrease hepatic fatty acid oxidation via uncoupling of respiration or if they inhibit extramitochondrial fatty acyl CoA synthesis. Therefore, the purpose of this study was to examine both processes simultaneously using the isolated perfused liver, a whole cell preparation where enzymes and biochemical processes can be monitored continuously under nearly physiological conditions. Accordingly, ketone body formation (β-hydroxybutyrate + acetoacetate) from lipid metabolism and oxygen uptake, which is increased by uncoupling agents, were monitored at the same time. 2-Bromooctanoate, a known inhibitor of acyl CoA synthetase, decreased ketone body formation in a dose-dependent manner without altering cellular respiration (half-maximal inhibition, 25 μ ) and concomitantly increased protein kinase C nearly fourfold also in a dose-dependent fashion. Ketogenesis was also blocked maximally 50–66% with mono(ethylhexyl)phthalate, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643), and nafenopin, potent peroxisome proliferators and tumor promoters. These compounds also increased protein kinase C three- to fourfold without altering oxygen uptake significantly. Thus, lipid metabolism appears to be the prime target of potent peroxisome proliferators most likely on actions via acyl CoA synthetase rather than oxidative phosphorylation. In contrast, weak peroxisome proliferators and tumor promoters, di(ethylhexyl)phthalate and 2-ethylhexanol, did not affect ketogenesis, oxygen consumption, or protein kinase C at similar concentrations. Additionally, octanoate increased ketone body formation in the presence of nafenopin. Because octanoate is metabolized by mitochondrial acyl CoA synthetase independent of carnitine acyltransferase, these results indicate that nafenopin does not inhibit mitochondrial β-oxidation. Taken together, it is concluded that potent peroxisome proliferators preferentially block ketogenesis without altering cellular respiration in the liver. This phenomenona, which occurs due to inhibition of acyl CoA synthetase, leads to an elevation of free fatty acids that stimulates protein kinase C and promotes cell proliferation.     

The effect of mildronate on disorders of the cardiac contractile function in rats caused by an excess of free fatty acids and ischemia

Oral administration of mildronate, 3-(2,2,2-trimethylhydrazine)propionate, an inhibitor of carnitine-dependent metabolism, in a dose of 50-100 mg/kg for 10 days promoted a rapid restoration of contractility of Langendorf perfused rat heart preparations during postischemic perfusion and protected the rat hearts against inhibition of contractile function resulting from continuous perfusion with palmitic acid. Mildronate inhibits gamma-butyrobetaine hydroxylase, depresses carnitine biosynthesis and reduces carnitine-dependent fatty acid metabolism. The cardioprotective effect of mildronate is particularly manifest upon continuous administration.     

Reduced carnitine and ketogenesis in the pivampicillin treated rat.

Pivampicillin (630 mg/kg body wt) given daily by stomach tube induced carnitine deficiency in the rat. The carnitine concentrations after 24 days were significantly reduced to (mean +/- SD) 34 +/- 2, 27 +/- 7, 70 +/- 18, 75 +/- 16 and 49 +/- 4% of controls in plasma, liver, muscle, heart and kidney, respectively, without any further reduction after 36 days. Pivampicillin treatment reduced the carnitine concentrations in the liver of the 48 hr fasted rat to about 1/2 of the controls after 6 days. The concentration of beta-hydroxybutyrate was significantly reduced up to 14 days of treatment, and again increased. There was no significant difference in the free fatty acid concentrations between treated and control rats. Thus, pivampicillin treatment induced carnitine deficiency in the rat, but not as pronounced as seen in humans. This is possibly caused by adjustment of bacterial flora in the gut or altered renal mechanisms. The pivampicillin-treated rat, therefore, is not a useful model for pronounced carnitine deficiency in humans.     

Biochemical study on the protective potential of Nardostachys jatamansi extract on lipid profile and lipid metabolizing enzymes in doxorubicin intoxicated rats

Nardostachys jatamansi is a medicinally important herb of Indian origin used for centuries in Ayurvedic and Unani systems of medicine for the treatment of various ailments. The aim of the present work is to evaluate the effect of ethanolic extract of Nardostachys jatamansi rhizomes on doxorubicin induced myocardial injury with respect to lipid metabolism in serum and heart of Wistar albino rats. Altered lipid metabolism alters the cardiac function which is mainly due to changes in the property of the cardiac cell membrane. Doxorubicin exhibits cardiotoxicity by inhibition of fatty acid oxidation in the heart. The rats treated with a single dose of doxorubicin (15 mg/kg) intraperitoneally showed an increase in serum and cardiac lipids (cholesterol, triglycerides, free fatty acids and phospholipids), along with a significant rise in serum low density lipoproteins (LDL), very low density lipoproteins (VLDL) and drop in high density lipoproteins (HDL) levels, resulting in alteration of serum and cardiac lipid metabolizing enzymes. Pretreatment with a extract of Nardostachys jatamansi (500 mg/kg) orally for seven days to doxorubicin induced rats showed a significant prevention in the lipid status with the activities of the lipid metabolizing enzymes. Histopathological observations were also in correlation with the biochemical parameters. These findings suggest that the protective and hypolipidemic effect of Nardostachys jatamansi against doxorubicin induced myocardial injury in rats could possibly be mediated through its anti lipid peroxidative properties.     

Targets for modulation of fatty acid oxidation in the heart

Fatty acids are a major source of fuel used by the heart to provide large amounts of energy necessary to sustain contractile function. In the healthy heart, a balance between fatty acid and carbohydrate use ensures that energy supply to the heart matches demand. However, myocardial ischemia causes profound changes in metabolism, including alterations in glucose and fatty acid metabolism that can lead to excessive myocardial fatty acid oxidation, which occurs at the expense of glucose oxidation. This contributes to cellular acidosis, a decrease in cardiac efficiency and contractile dysfunction in the ischemic heart. Inhibition of fatty acid oxidation has recently emerged as a promising approach to the prevention of these adverse effects of fatty acids. As a result, a number of key enzymes involved in the metabolism of fatty acids are potential targets for therapeutic intervention in myocardial ischemia. This includes inhibition of fatty acid uptake into the myocyte, inhibition of mitochondrial fatty acid uptake and direct inhibition of fatty acid beta-oxidation. This review describes these potential targets for modulation of energy metabolism in the heart.     

Inhibition of long-chain acyl-CoA synthetase by the peroxisome proliferator perfluorodecanoic acid in rat hepatocytes

Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator and is known to affect hepatic lipid metabolism in rats. The effects of PFDA on fatty acid utilization were examined in isolated rat hepatocyte suspensions and in rat liver mitochondria and microsomes. PFDA inhibited the oxidation of palmitic acid but not octanoic or pyruvic acids when hepatocytes were incubated with 1 mM PFDA. At this PFDA concentration the esterification of palmitic acid into triacylglycerols was also reduced. The activity of long-chain acyl-CoA synthetase (ACS), an enzyme essential for both oxidation and esterification of fatty acids, was reduced in hepatocytes incubated with 1 mM PFDA. Carnitine palmitoyltransferase (CPT), an important enzyme for the oxidation of long-chain fatty acids, was not altered in hepatocytes incubated with this PFDA concentration. In rat liver mitochondria, palmitate oxidation and ACS activity were reduced significantly (P less than 0.01) at a PFDA concentration that had no effect on CPT activity. The inhibition of ACS by PFDA was similar in liver mitochondria and microsome preparations. In mitochondria incubated with PFDA, the inhibition of ACS appears to be noncompetitive for the substrates palmitic acid and CoA. However, the ACS inhibition by PFDA appeared to be competitive for the ATP binding site of the enzyme. Several chain length perfluorinated fatty acids were examined for their ability to inhibit mitochondrial ACS. Short-chain perfluorinated fatty acids (perfluoroproprionic and -butyric acid) did not inhibit ACS activity. However, medium-chain perfluorinated acids (perfluorooctanoic, -ananoic and -decanoic acid) were found to be potent inhibitors of ACS in isolated mitochondria. Whether ACS inhibition is causally related to PFDA-induced peroxisome proliferation and altered lipid metabolism seen in vivo is yet to be determined.