Edoardo Arrigoni-Martelli

Regulation by carnitine of myocardial fatty acid and carbohydrate metabolism under normal and pathological conditions

Abstract This review focuses on the regulation of myocardial fatty acids and glucose metabolism is physiological and pathological conditions, and the role of L-carnitine and of its derivative, propionyl-L-carnitine.Fatty acids are the major oxidation fuel for the heart, while glucose and lactate provide the remaining need. Fatty acids in cytoplasm are transformed to long-chain acyl-CoA and transferred into the mitochondrial matrix by the action of three carnitine dependent enzymes to produce acetyl-CoA through the β-oxidaton pathway. Another source of mitochondrial acetyl-CoA is from the oxidation of carbohydrates. The pyruvate dehydrogenase (PDH) complex, the key irreversible rate limiting step in carbohydrate oxidation, is modulated by the intra-mitochondrial ratio acetyl-CoA/CoA. An increased ratio results in the inhibition of PDH activity. A decreased ratio can relieve the inhibition of PDH as shown by the transfer of acetyl groups from acetyl-CoA to carnitine, forming acetylcarnitine, a reaction catalyzed by carnitine acetyl-transferase. This activity of L-carnitine in the modulation of the intramitochondrial acety-CoA/CoA ratio affects glucose oxidation.Myocardial substrate metabolism during ischemia is dependent upon the severity of ischemia. A very severe reduction of blood flow causes a decrease of substrate flux through PDH. When perfusion is only partially reduced there is an increase in the rate of glycolysis and a switch from lactate uptake to lactate production. Tissue levels of acyl-CoA and long-chain acylcarnitine increase with important functional consequences on cell membranes. During reperfusion fatty acids oxidation quickly recovers as the prevailing source of energy, while pyruvate oxidation is inhibited.A considerable body of experimental evidence suggests that L-carnitine exert a protective effect in in vitro and in vivo models of heart ischemia and hypertrophy. Clinical trials confirm these beneficial effects although controversial results are observed. The actions of L-carnitine and propionyl-L-carnitine cannot be explained as exclusively dependent on the stimulation of fatty acid oxidation but rather on a marked increase in glucose oxidation, via a relief of PDH inhibition caused by the elevated acetyl-CoA/CoA ratio. Enhanced pyruvate flux through PDH is beneficial for the cardial cells since less pyruvate is converted to lactate, a metabolic step resulting in the acidification of the intracellular compartment. In addition, L-carnitine decreases tissue levels of acyl moieties, a mechanism particularly important in the ischemic phase.

Neural Dysfunction and Metabolic Imbalances in Diabetic Rats: Prevention by Acetyl-L-Carnitine

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4–6 weeks duration were that 24-h urinary carnitine excretion was increased ∼ twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10–15%) and sciatic nerve changes in Na+-K+-ATPase activity (decreased 50%), Mg2+-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.

N″-cyano-N-4-pyridyl-N′-1,2,2-trimethylpropylguanidine, monohydrate (P 1134): A new, potent vasodilator

N″-cyano-N-4-pyridyl-N′-1,2,2-trimethylpropylguanidine, monohydrate (P 1134) is a new agent which induces a marked and sustained hypotensive response in normotensive and renal, neurogenic, and spontaneously hypertensive rats, as well as in normotensive and renal hypertensive dogs. The overall potency of this compound is 2–3 times greater than that of hydralazine. The fall of blood pressure is accompanied by an increase in heart rate and cardiac output and a decrease in total peripheral resistance. The hypotensive effect appears to be due primarily to a direct relaxant effect on vascular smooth muscle.

Sites of action of carnitine and its derivatives on the cardiovascular system: interactions with membranes

Carnitine plays an essential role in the regulation of long-chain fatty acid metabolism in skeletal and cardiac muscle, a process that is mediated by well-characterized enzymatic mechanisms. Here, Irving Fritz and Edoardo Arrigoni-Martelli review the evidence that carnitine and its O-acyl derivatives also influence membrane fluidity, ion channel functions, smooth muscle contractility, membrane stability and cardiac functions. The authors present the view that direct interactions of carnitine derivatives with cell membranes are independent of reactions catalysed by carnitine acyltransferases. They propose that the novel actions discussed are implicated in the mechanisms by which carnitine and its derivatives protect perfused hearts subjected to ischaemia or to oxidative stress, and help people suffering from certain types of myocardial ischaemia or peripheral arterial disease.

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl‐ choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μM 2‐tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μM TDGA CPT activity dropped more rapidly than between 10 and 40 μM. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose‐dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl‐dl‐ aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation‐deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA‐treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.

Acyl-trafficking in membrane phospholipid fatty acid turnover : the transfer of fatty acid from the acyl-L-carnitine pool to membrane phospholipids in intact human erythrocytes

In this work we have investigated the transfer of radioactive palmitic acid between membrane phospholipids and acyl-L-carnitines in intact human erythrocytes. During the incubation period of labeled erythrocyte in non-defatted bovine serum albumin, radioactivity in phosphatidylcholine and phosphatidylethanolamine increased. On the contrary, a decrease of radioactivity in erythrocyte palmitoyl-L-carnitine was observed. 2-Tetradecylglycidic acid, an irreversible erythrocyte carnitine palmitoyltransferase inhibitor, abolished any radioactivity changes in both phospholipids and palmitoyl-L-carnitine. Similar findings were obtained by using erythrocytes labeled with radioactive oleic acid. Our data suggest that in human erythrocytes a carnitine palmitoyltransferase-catalyzed acyl transfer from acyl-L-carnitine to phospholipids, rather than a previously described fatty acid transfer from phosphatidylcholine to phosphatidylethanolamine, is operative.

Effects of l-carnitine and its acetate and propionate esters on the molecular dynamics of human erythrocyte membrane

EPR and fluorescence probes were used in this study to define the effects of L-carnitine and its short-chain esters, acetyl-L-carnitine and propionyl-L-carnitine, on the natural fluidity gradient and molecular packing of phospholipid headgroups of erythrocyte membrane in intact cells. Purified erythrocyte suspensions, labeled with different stearic acid derivatives containing a stable doxyl radical ring at the C-5, C-7, C-12 and C-16, were incubated with 0.5-5 mM L-carnitine and its esters for 60 min at 37 degrees C and washed twice with an isosmotic buffer. A decrease in the order parameter, calculated from the EPR spectra of the 5-doxylstearic acid derivative, was observed at all the concentrations of propionyl-L-carnitine and the extent of the decrease was dose and temperature dependent. An increase of the chain length between the doxyl ring and the carboxylic group of the spin label, resulted in a much lower efficacy of propionyl-L-carnitine in decreasing the order parameter. Acetyl-L-carnitine also showed a significant effect of decreasing the molecular order but only at the lower temperatures of red cells labeled with 5-doxyl and treated with the highest concentration of the drug. L-Carnitine did not modify the molecular dynamics at all the temperatures and concentrations used in this study. L-Carnitine and its short-chain derivatives did not alter significantly membrane fluidity of deeper regions of the erythrocyte membrane, measured by means of the excimer/monomer fluorescence intensity ratio of pyrene incorporated into the membrane of intact erythrocytes. However, these compounds were all capable of loosening the molecular packing of the polar head of erythrocyte membrane phospholipids evaluated by the membrane binding fluorescence properties of merocyanine-540. The binding of the fluorescent probe decreased in the order propionyl-L-carnitine > acetyl-L-carnitine > L-carnitine. Our findings suggest that this category of compounds affect the molecular dynamics of a membrane bilayer region close to the glycerol backbone of phospholipids, which might be relevant for the expression of membrane functions.

Interaction of cartinine with mitochondrial cardiolipin

Abstract The physiological role of l -cartinine is to determine the transport of acyl-CoA through the mitochondrial membrane. However, some observations may also suggest a direct effect of the molecule per se on the physical properties of the membrane, most probably at the level of the binding site. This possibility has been investigated by studying the influence of adriamycin. a drug that binds to cardiolipin, on the effect of cartinine on isolated rat liver mitochondria. It has been found that adriamycin almost abolishes the activating effect of carnitine on state 2 respiration. The effect and its inhibition is seen by using either the l -form of carnitine of the d -form or both. Cardiolipin removes the effect of adriamycin and restores the activation by carnitine. It is proposed that some effects of carnitine on mitochondrial properties may be the result of interaction of carnitine with cardiolipin at the membrane level.

Vascular effects in dogs of pinacidil (P 1134), a novel vasoactive antihypertensive agent

In pentobarbital sodium-anaesthetized dogs, pinacidil was infused for approximately 5 min into the carotid, coronary, femoral or renal artery at a rate of 10 micrograms/kg per min. The infusion, which did not affect systemic blood pressure, rapidly and markedly increased blood flow to any of the regions studied. When given i.v., 0.2 mg/kg pinacidil caused a moderate reduction in mean arterial blood pressure (15-20 mmHg) associated with an increase in coronary and renal blood flow while femoral and carotid blood flow remained unchanged; 0.5 mg/kg led to a marked (40-60 mmHg) reduction in blood pressure associated with an increase in coronary blood flow whereas renal, carotid and femoral blood flow stabilized at control levels. Indomethacin (2.5 mg/kg i.v.) failed to reverse the hypotension induced by pinacidil. The results are in accord with the concept that the vascular effect of pinacidil is due to direct smooth muscle relaxation which does not depend on prostaglandin synthesis.

Effect of propionyl-L-carnitine treatment on membrane phospholipid fatty acid turnover in diabetic rat erythrocytes

In this work we have examined the effect of the oral administration of propionyl-L-carnitine (PLC) on the membrane phospholipid fatty acid turnover of erythrocytes from streptozotocin-induced diabetic rats. A statistically significant reduction in radioactive palmitate, oleate, and linoleate, but not arachidonate, incorporation into membrane phosphatidylcholine (PC) of diabetic rat erythrocytes with respect to control animals was found. Changes in radioactive fatty acid incorporation were also found in diabetic red cell phosphatidylethanolamine (PE), though they were not statistically significant. Oral propionyl-L-carnitine (PLC) treatment of diabetic rats partially restored the ability of intact red cells to reacylate membrane PC with palmitate and oleate, and reacylation with linoleate was fully restored. The analysis of the membrane phospholipid fatty acid composition revealed a consistent increase of linoleate levels in diabetic rat red cells, and a modest decrease of palmitate, oleate and arachidonate. The phospholipid fatty acid composition of diabetic red blood cells was not affected by the PLC treatment. Lysophosphatidylcholine acyl-CoA transferase (LAT) specific activity measured with either palmitoyl-CoA or oleyl-CoA was significantly reduced in diabetic erythrocyte membranes in comparison to controls. In addition LAT kinetic parameters of diabetic erythrocytes were altered. The reduced LAT activity could be partially corrected by PLC treatment of diabetic rats. Our data suggest that the impaired erythrocyte membrane physiological expression induced by the diabetic disease may be attenuated by the beneficial activity of PLC on the red cell membrane phospholipid fatty acid turnover.