Tissue lipid accumulation by L-aminocarnitine, an inhibitor of carnitine-palmitoyltransferase-2. Studies in intact rats and isolated mitochondria.

Tissues of fasted animals treated with L-aminocarnitine (L-3-amino-4-trimethylaminobutyrate) showed an accumulation of long-chain acylcarnitines and triacylglycerols. Blood levels of free fatty acids, long-chain acylcarnitines and triacylglycerol-rich lipoproteins were found to be increased, whereas glucose was reduced. The liver mitochondria isolated from rats treated with L-aminocarnitine utilized both pyruvate and succinate normally, but were not able to oxidize palmitoylcarnitine. In vitro oxidation of palmitoylcarnitine by liver mitochondria was inhibited by L-aminocarnitine in a concentration-dependent fashion in contrast to succinate and pyruvate oxidation which was not modified. L-aminocarnitine proved to be a potent and selective inhibitor (IC50 = 805 nM) of the carnitine palmitoyltransferase isoenzyme, located on the inner side of the mitochondrial inner membrane (CPT2). The activity of the carnitine palmitoyltransferase isoenzyme located on the mitochondrial outer membrane inhibitable by malonyl-CoA (IC50 = 19 microM), was not inhibited by 0.8 microM L-aminocarnitine. Both in vitro and in vivo effects of L-aminocarnitine suggest that the substance has a specific and potent inhibitory action on CPT2. Its in vivo inhibition results in a dramatic increase of long-chain acylcarnitines in various organs, that it is why this increase can be considered a very good marker of CPT2 inhibition. A markedly altered lipid metabolism was observed.

Enalapril prevents cardiac fibrosis and arrhythmias in hypertensive rats.

To evaluate the effects of hypertension on cardiac hypertrophy, on myocardial structure, and on ventricular arrhythmias, 27 3-month-old spontaneously hypertensive rats were treated with enalapril (10 mg/kg) daily for 11 months and compared with 26 untreated control rats. Systolic arterial pressure was significantly decreased in treated rats, and at the end of the experiment, it was 199 +/- 3 mm Hg (treated) versus 237 +/- 3 mm Hg (controls) (p less than 0.001). At this time, spontaneous arrhythmias and induced arrhythmias either by programmed electrical stimulation (train of stimuli +1 or 2 extrastimuli) or by trains of eight stimuli at decreasing coupling intervals were observed in isolated heart preparations. Comparing enalapril-treated and control rats, spontaneous arrhythmias (9 of 27 versus 20 of 26, respectively; p less than 0.01), programmed stimulation-induced arrhythmias (3 of 26 versus 12 of 23, respectively; p less than 0.01), and trains of stimuli-induced arrhythmias (4 of 26 versus 14 of 19, respectively, p less than 0.001) were less frequent in the enalapril group. Left ventricular weight was decreased in treated rats by 18% (p less than 0.001). Enalapril administration diminished the fraction of myocardium occupied by foci of replacement fibrosis normally occurring in control rats by 59% (p less than 0.001). Finally, a significant correlation was found between left ventricular weight, the extent of myocardial fibrosis, and the occurrence of ventricular fibrillation. It was concluded that chronic treatment with enalapril, which resulted in attenuation of systemic arterial pressure by limiting cardiac hypertrophy and myocardial fibrosis, decreases the propensity of the heart of hypertensive rats to arrhythmogenesis.

The morphology of lipopigment in rat Purkinje neurons after chronic acetyl-L-carnitine administration: a reduction in aging-related changes.

The aging-related accumulation of neuronal lipopigment is considered to be cellular debris from processes of renewal of cellular constituents, but it can also reflect cell damage and certain diseases. Acetyl-L-carnitine (AC) has been reported to reduce some morphological and behavioral associations of brain aging and the present study investigated the effects of 37 weeks of AC administration on lipopigment in rat Purkinje neurons. Lipopigment was identified by fluorescence microscopy and the area enclosed by an outline of each discrete region of lipopigment was measured. Acetyl-L-carnitine was associated with a significant (p = 0.05) reduction in the number of discrete lipopigment regions and there was a significant (p = 0.001) association of AC administration with numbers of lipopigment regions in various size categories. As AC administration was associated with a reduction in some of the aging-related morphological changes in lipopigment, this compound is a candidate for evaluation as a long-term prophylactic agent for the adverse effects of cerebral aging.

Spatial learning and memory in the radial maze: a longitudinal study in rats from 4 to 25 months of age.

This longitudinal study was designed to investigate whether previous experience may influence performances and strategies of rats tested in the radial maze without external cues when aged 4, 13, and 25 months. Their performances and strategies were compared with those of another group of rats tested only when aged 25 months. Expert old animals showed a good retention of previous experiences, whereas age-matched nonexpert animals exhibited some acquisition deficits. On the contrary, in the course of aging, the animals kept modifying their strategies independently of experience. In summary, we can conclude that previous experience is likely to influence performances of the aged rat but not the strategies adopted which are strictly age-dependent and independent of acquired experience.

Enzyme histochemistry of monoamine oxidase in the heart of aged rats.

The distribution of monoamine oxidase types A and B in the heart of young (3-month-old) and aged (26-month) Wistar rats was studied by histochemical methods. In young rats, MAO activity was higher in the left ventricle than in the right. Enzyme reactivity was present within both muscle cells and the blood vessel wall. Histochemical staining was abolished by clorgyline administration. In the heart of aged rats, we observed a very remarkable increase of clorgyline sensitive MAO activity primarily at the level of myocardial cells. The right ventricle showed the highest increase of MAO reactivity.

Age- and trauma-dependent modifications of neuromuscular junction and skeletal muscle structure in the rat. Effects of long-term treatment with Acetyl-L-Carnitine.

The influence of ageing and crushing of the sciatic nerve on the morphology of the neuromuscular junction (NMJ) and on the muscle fiber composition were studied in the rat soleus muscle using histochemical techniques associated with image analysis. The influence of a 6-month treatment with Acetyl-L-Carnitine (ALCAR, 150 mg/kg/day) on the age- and crushing-dependent changes of the NMJ and on age-related modifications of the muscle fiber composition was assessed as well. In control old and injured young rats a loss of complexity of the NMJ was observed. Treatment with ALCAR resulted in an increased endplate complexity both in old rats and in young rats injured by crushing, in comparison with respective controls. The structure of the rat soleus muscle changes with increasing age. Modification mainly consists in a type II fiber atrophy, and in the alteration of the peculiar mosaic organization of the soleus muscle fibers. In ALCAR-treated old rats, the morphology of the soleus muscle fibers was similar to that observed in adult animals. These findings suggest that treatment with ALCAR has a beneficial effect on NMJ and on muscle fiber structure in ageing or after nerve crushing. The possible mechanism of action of this 'trophic' effect of ALCAR-treatment is discussed.

Propionyl-L-carnitine prevents the progression of atherosclerotic lesions in aged hyperlipemic rabbits.

We have characterized the extent and the phenotype of total and proliferating cell population of aortic plaques in aged rabbits receiving a long-term low-dose cholesterol hyperlipemic diet, which represents an experimental model of atherosclerosis. For nine months, rabbits received the hypercholesterolemic diet alone or in addition to a treatment with propionyl-L-carnitine (PLC), a derivative of carnitine, an intramitochondrial carrier of fatty acids present in most cell types. We observed that, in both PLC-treated and control hyperlipemic rabbits, the ratio between proliferating macrophage-derived and smooth muscle cells was 2:1. PLC in addition to the hypercholesterolemic diet induced a marked lowering of plasma triglycerides, very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) triglycerides, while plasma cholesterol was slightly and transiently reduced. Moreover, PLC-treated hyperlipemic rabbits exhibited a reduction of plaque thickness and extent, a slight but significant reduction of the percentage of macrophage-derived cells as compared to control hyperlipemic animals and a reduction of the number of both proliferating macrophage- and smooth muscle cell-derived foam cells. Finally, both proliferating and non-proliferating plaque cells expressed large amounts of macrophage colony-stimulating factor protein, in particular macrophage-derived foam cells. These results indicate that a modification of plasma lipemic pattern obtained by a long-term oral administration of PLC was associated with a decrease of plaque cell proliferation and severity of aortic atherosclerotic lesions.

Aging and atherosclerosis in the rabbit. 1. Distribution, prevalence and morphology of atherosclerotic lesions.

Aging is considered a risk factor in the pathogenesis of atherosclerosis. It is not clear, however, whether the relationship between aging and atherosclerosis is the result of increased susceptibility of the arterial wall related to intrinsic alterations or the expression of the increase in intensity or duration of exposure to risk factors. In this study, we used aged (median age 46 months) and young (4 months old) New Zealand white rabbits. Nine aged and 11 young rabbits received a hyperlipemic diet enriched with a low dose of cholesterol for 18 months. Eleven aged and 8 young rabbits, fed standard chow for the same period, were used as controls. Using morphologic and morphometric methods, we detected in aged hyperlipemic rabbits (a) a marked prevalence of fibroatheromatous plaques (as opposed to fatty streaks in young hyperlipemic rabbits); (b) aortic lesions more extensive and of greater dimensions than in young hyperlipemic rabbits; (c) fibroatheromatous plaques in carotids and raised fatty streaks in the large subepicardial coronary branches. Our results show an increased susceptibility of the aged arterial wall to hypercholesterolemia.

Transient nigral ubiquinone depletion after single MPTP administration in mice.

The administration of a single injection of 30 mg/kg MPTP to mice produces at 1 hr, a transient significant decrease of the reduced ubiquinol Q10 in the substantia nigra that is normalized afterwards. This suggests a transient stress imposed by MPTP (or more likely MPP+) in the mitochondrial respiratory and/or oxidoreducing system, located in close proximity to the NADH system.

Regional distribution of ubiquinones and tocopherols in the mouse brain: lowest content of ubiquinols in the substantia nigra.

C57 black mice of 3 months of age were sacrificed, and their brain regionally dissected according to a protocol that strickly control for the death-freezing interval of each region. HPLC measurements of tocopherols and oxidized and reduced ubiquinones demonstrated significant regional variations. The substantia nigra had the lowest content of Q10 and a skewed ratio in favor of its oxidized form. Forebrain cholinergic nuclei had also more oxydized than reduced Q10 and in addition the lowest content of tocopherols. These findings suggest that nuclei that show neuronal depletion with age are the ones prone to oxidative stress.

Synthesis and pharmacological activity and some derivatives of 1-phenyl-1,2,3,4-tetrahydro-5H-1,4-benzodiazepin-5-one.

4-N-Alkylamino derivatives and corresponding ammonium quaternary salts of tetrahydro-1,4-benzodiazepin-5-one were synthesized and evaluated for psychotropic activity in mice by ip via. This study was also extended to some nitro and amino derivatives of tetrahydro-1,4-benzodiazepin-5-one. Compounds were devoid of tranquilizing activity and in comparison with two classical benzodiazepines, chlordiazepoxide and diazepam, they showed high toxicity and little or no effect on motor coordination, motor activity, and maximal electroshock. On some "in vitro" tests the compounds exhibited pharmacological properties when they were used at high concentrations.

Effect of long-term acetyl-L-carnitine on stress-induced analgesia in the aging rat.

Cold water swim (CWS) analgesia in the rat is mediated by the hypothalamo-pituitary-adrenocortical (HPA) axis. An age-dependent increase of CWS-induced analgesia was observed in male Sprague-Dawley young (4 months), adult (15 months) and old (26 months) rats. Acetyl-L-Carnitine (ALCAR) chronically administered (75 mg/kg/daily in drinking water for 8 months) to old rats was able to maintain the stress-dependent response at the same levels as in adult rats. This effect may be explained by ALCAR capability of retarding the age-dependent loss of glucocorticoid receptors in the hippocampus, thus maintaining the glucocorticoid competence of this structure which exerts a negative feedback control over the HPA axis activity.

The effect of age on the activity of enzymes of peroxide metabolism in rat brain.

The activity of some enzymes associated with peroxide metabolism and cytochrome oxidase activity was measured in cortex, striatum, hypothalamus, and hippocampus from brains of rats aged either 4, 15, or 27 months. Cytochrome oxidase activity was greatest in the cortex, but no significant age-related changes in the activity of cytochrome oxidase, superoxide dismutase, or glutathione peroxidase were found in any of the brain areas. In contrast, glutathione reductase activity increased as a function of age in all regions. In general, the activity of catalase fell on maturation of the animal to adulthood and then showed a trend to increase with age.

Antioxidant enzyme activities in heart and skeletal muscle of rats of different ages.

The activity of antioxidant enzymes was measured in cardiac and skeletal muscle in rats aged either 4, 15, or 27 months. Generally, regardless of age, heart contains a greater content of these enzymes than skeletal muscle. Whereas skeletal muscle showed age-dependent increases in glutathione peroxidase, glutathione reductase, and catalase activities, heart tissue showed increases in only the glutathione peroxidase activity. Neither tissue showed any significant age-dependent change in cytosolic or mitochondrial superoxide dismutase content or in cytochrome oxidase.

Aging and immune response: a multivariate approach.

This study on the immune response was chosen as the experimental milieu for the aging process. Parameters associated with the immune response were measured in rats of different ages, thus providing multivariate conditions within which multidimensional data analysis procedures could be applied. These methods, specifically designed for analyzing complex situations, allowed for the delineation of some structural characteristics of maturity and senescence.

Levels of carnitines in brain and other tissues of rats of different ages: effect of acetyl-L-carnitine administration.

Male Sprague-Dawley rats, aged 2, 5, 16, 20 and 30 months and normally fed, were used for determination of carnitines in the brain, serum, heart, tibial muscle, liver and urine. With respect to 5-month-old animals, those aged 30 months exhibited a statistically significant decrement of total carnitine levels in the brain, serum, heart and tibial muscle, accompanied by a dramatic increment in the liver. This suggests impaired net transport of carnitines from the liver to the blood in old age. Urinary excretion was similar in the two age groups. One group received from 5 months on daily 75 mg/kg acetyl-L-carnitine in drinking water. At 20 months, the treated animals showed levels of brain, heart and serum carnitines similar to those of 5-month-old animals. The recovery of brain, heart and serum carnitines in the old animals treated with acetyl-L-carnitine indicates that intestinal absorption and tissue uptake remain sufficiently efficient in the course of aging. The lower level of brain lipofuscins due to acetyl-L-carnitine treatment may be related to the effect of the compound on acetylcholine metabolism.

A cluster analysis study of acetyl-L-carnitine effect on NMDA receptors in aging.

Aging is associated with a reduction in the maximum density of n-methyl-d-aspartate (NMDA)-sensitive glutamate binding sites in the hippocampus of Fischer 344 rats. This study was designed to investigate the effect of acetyl-l-carnitine (ALCAR) on NMDA receptors in the old rat (24 months) after chronic or single-dose treatments. The number of NMDA receptors was significantly decreased in the old rat hippocampus by 19.5% compared with the young rat. A six-month treatment with ALCAR in the old rat attenuated the loss of NMDA binding sites in the hippocampus. A single-dose treatment with ALCAR in the old rat increased the Bmax value by 35%, while no change was observed in the young group. We conclude that ALCAR can exert two actions: a trophic/neuro-preserving one when chronically administered during aging, and a stimulatory one when given at a single dose in the aged rat.

Acetyl-L-carnitine treatment increases nerve growth factor levels and choline acetyltransferase activity in the central nervous system of aged rats.

The hypothesis that some neurodegenerative events associated with ageing of the central nervous system (CNS) may be due to a lack of neurotrophic support to neurons is suggestive of a possible reparative pharmacological strategy intended to enhance the activity of endogenous neurotrophic agents. Here we report that treatment with acetyl-l-carnitine (ALCAR), a substance which has been shown to prevent some impairments of the aged CNS in experimental animals as well as in patients, is able to increase the levels and utilization of nerve growth factor (NGF) in the CNS of old rats. The stimulation of NGF levels in the CNS can be attained when ALCAR is given either for long or short periods to senescent animals of various ages, thus indicating a direct effect of the substance on the NGF system which is independent of the actual degenerative stage of the neurons. Furthermore, long-term treatment with ALCAR completely prevents the loss of choline acetyltransferase (ChAT) activity in the CNS of aged rats, suggesting that ALCAR may rescue cholinergic pathways from age-associated degeneration due to lack of retrogradely transported NGF.

Stimulation of nerve growth factor receptors in PC12 by acetyl-L-carnitine.

Acetyl-L-carnitine (ALCAR) prevents some deficits associated with aging in the central nervous system (CNS), such as the aged-related reduction of nerve growth factor (NGF) binding. The aim of this study was to ascertain whether ALCAR could affect the expression of an NGF receptor (p75NGFR). Treatment of PC12 cells with ALCAR increased equilibrium binding of 125I-NGF. ALCAR treatment also increased the amount of immunoprecipitable p75NGFR from PC12 cells. Lastly, the level of p75NGFR messenger RNA (mRNA) in PC12 was increased following ALCAR treatment. These results are in agreement with the hypothesis that there is a direct action of ALCAR on p75NGFR expression in aged rodent CNS.

Valproic acid and bicuculline affect the formation of glycerolipid in rat brain.

To ascertain the effects of bicuculline and of sodium valproate on the incorporation of glycerol into rat brain lipid, rats were divided into 5 groups: (a) controls; (b) treated with sodium valproate (400 mg/kg body wt); (c) treated with bicuculline (12.5 ?mol/kg body wt); (d) treated with sodium valproate as in (b) + bicuculline as in (c); and (e) treated with bicuculline (25 ?mol/kg body wt). Only rats of group (c) had seizures, which lasted until the end of the experiment. Each animal received 20 ?Ci of [2-(3)H]glycerol by intraventricular route and was sacrificed 12 min afterwards. Hippocampi and cerebella were taken and lipid extracted and separated by chromatography. The type of treatment influenced very much the fate of injected, labeled glycerol. Indeed, total recovered radioactivity increased following either convulsions or the administration of valproate, whereas both treatments decreased the amount of radioactivity incorporated into lipid. These effects were more evident in cerebella than in hippocampi. The distribution of radioactivity among lipid classes (diglyceride, triglyceride and total phospholipid) was also affected by seizures, which decreased the labeling ratio phospholipid/neutral lipid. The distribution of radioactivity among phospholipid classes was influenced by bicuculline (both at convulsant and non-convulsant doses) and these effects were sometimes antagonized by valproate. We conclude that some effects of bicuculline are exerted through the systemic modifications due to seizures and that other effects are probably connected to neuronal hyperfiring. The data reported in this paper are consistent with both mechanisms of action proposed for valproate, i.e. increased membrane permeability and modifications of GABAergic systems.