Rita Ricciolini

Effect of long-term feeding with acetyl-L-carnitine on the age-related changes in rat brain lipid composition: a study by 31P NMR spectroscopy.

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.

Acetyl-l-carnitine modulates glucose metabolism and stimulates glycogen synthesis in rat brain

The effects of acetyl-L-carnitine on cerebral glucose metabolism were investigated in rats injected with differently 14C- and 13C-labelled glucose and sacrificed after 15, 30, 45, and 60 min. Acetyl-L-carnitine was found to reduce total 14CO2 release from [U-14C]glucose along with the decrease in [1-13C]glucose incorporation into cerebral amino acids and tricarboxylic acid cycle intermediates. However the 13C labelling pattern within different carbon positions of glutamate, glutamine, GABA, and aspartate was unaffected by acetyl-L-carnitine administration. Furthermore, the cerebral levels of newly-synthesized proglycogen were higher in rats treated with acetyl-L-carnitine than in untreated ones. These results suggest that acetyl-L-carnitine was able to modulate cerebral glucose utilization and provide new insights on the mechanisms of action of this molecule in the central nervous system.

Role of acetyl-L-carnitine in rat brain lipogenesis: implications for polyunsaturated fatty acid biosynthesis.

Abstract: This study was undertaken to explore the metabolic fate of acetyl‐l‐carnitine in rat brain. To measure the flux of carbon atoms into anabolic processes occurring at regional levels, we have injected [1‐14C]acetyl‐l‐carnitine into the lateral brain ventricle of conscious rats. After injection of [1‐14C]acetyl‐l‐carnitine, the majority of radioactivity was recovered as 14CO2 expired (60% of that injected). The percentage of radioactivity recovered in brain was 1.95, 1.60, 1.30, and 0.93% at 1, 3, 6, and 22 h, respectively. Radioactivity distribution in various lipid components indicated that the fatty acid moiety of phospholipid contained the majority of radioactivity. The radioactive profile of these fatty acids showed that the acetyl moiety of acetyl‐l‐carnitine was incorporated into saturated (60%), monounsaturated (15%), and polyunsaturated (25%) fatty acids [mainly present in 20:4 (5.2%) and 22:6 (7.8%)]. Injection in the brain ventricle of radioactive glucose, the major source of acetyl‐CoA in the CNS, revealed that glucose was a precursor of saturated (85%) and monounsaturated (15%) but not of polyunsaturated fatty acids. Thus, this study demonstrated distinct fates of glucose and acetyl‐l‐carnitine following intracerebroventricular injection. In summary, these data implicate acetyl‐l‐carnitine as an important member of a complex acetate trafficking system in brain lipid metabolism.

The effect of pivalate treatment of pregnant rats on body mass and insulin levels in the adult offspring

Pivalic acid is used as a prodrug to increase gut absorption of a variety of different antibiotics. Pivalic acid is also known to induce a number of metabolic aberrations which may be in part explained by concurrent mild carnitine depletion. Rat pups (5 days old) born to mothers treated throughout their pregnancy and lactation period with sodium pivalate, showed an increase in liver and muscle triglycerides and elevated plasma ketone bodies, compared to controls. A reduction of free carnitine content in liver, muscle and plasma was also observed in the pivalate treated group. In a second study, pups were treated with either pivalate for 24 days (females), or pivalate for 120 days (males). Both groups were fed standard diets. In both groups (male and female), the pivalate treatment showed a statistically significant hyperinsulinaemia and an increase of body mass compared with that of age- and sex-matched control groups. In addition, after a glucose loading, significantly higher levels of insulin in the pivalate-treated group (male) with respect to controls were observed. In conclusion, our data suggest that maternal pivalate treatment may predispose adult offspring to developing insulin-resistance and obesity.

Modulation of the free sphingosin levels in Epstein Barr virus transformed human B lymphocytes by phorbol dibutyrate

The amounts of free sphingosine in Epstein Barr virus transformed B lymphocytes (EBV-B) treated with sphingosine and phorbol-12,13-dibutyrate (PD) has been quantified by high performance liquid chromatography (HPLC). PD treatment did not affect intracellular sphingosine level, while it seems to lessen the removal of this long chain base in sphingosine-treated EBV-B cells. The previous results relative to sphingosine-dependent changes in choline-metabolite levels have to be interpreted on the basis of these results.

Aminocarnitine ureidic derivatives as inhibitors of carnitine palmitoyltransferase I.

Type II diabetes is a complex metabolic disorder characterized by insulin resistance and impaired b-cell function. It arises as a consequence of obesity, sedentary lifestyle and aging, with resulting hyperglycemia, blood pressure elevation and dyslipidemia. Moreover, in type II diabetes, highly increased hepatic fatty acid oxidation generates high levels of acetyl-coenzyme A (acetyl-CoA), ATP and NADH, which in turn upregulate gluconeogenesis and thus hepatic glucose production. The transport of fatty acids into mitochondria is regulated by membrane-bound carnitine palmitoyltransferases (CPT) I and II. CPT I, the outer mitochondrial membrane enzyme that is present as two isoforms known as liver (L-CPT I) and muscle (MCPT I), catalyzes the formation of long-chain acylcarnitines. CPT II, the inner mitochondrial membrane enzyme present as a single isoform, converts long-chain acylcarnitines back into long-chain acetyl-CoA thioesters. CPT inhibitors, by lowering the level of acetyl-CoA, indirectly reduce liver gluconeogenesis. Oxirane carboxylates, such as etomoxir and methyl 2-tetradecylglycidate, previously identified as irreversible inhibitors of CPT, were found to induce cardiac hypertrophy due to a lack of liver and muscle isoform selectivity. In previously published studies, Novartis (formerly Sandoz) described an alkylphosphate derivative of carnitine as a CPT I inhibitor, and we described the identification of highly selective L-CPT I inhibitors. Teglicar (1, ST1326) was chosen from these inhibitors for preclinical and clinical development as an antiketotic and antidiabetic agent. The ureidic functional group present in teglicar was advantageous in terms of efficacy and selectivity towards the liver isoform of CPT, in comparison with other investigated moieties. Taking these findings into consideration, we decided to continue our studies on L-CPT I inhibitors with a series of new aminocarnitine ureidic derivatives, exploring the effects of aromatic functionalities in the straight-chain alkyl group of 1. The aim was to obtain new inhibitors with improved efficacy, while maintaining the high selectivity for the liver over the muscle isoform of CPT I, and/or with limited tensioactivity (an unwanted characteristic of this class of molecules arising from the long alkyl chain and ionic head). Oxygenated substitutions were introduced for their water coordinating properties, which could favorably limit packing of the molecules in micelle formation. Accordingly, the compounds were synthesized starting from aminocarnitine and isocyanates or the corresponding carboxylic acids. For the most interesting molecule identified, the phosphonium analogue was also prepared in order to investigate the effects of ammonium group substitution with the bioisoster phosphonium on the activity profile. In order to explore the effects of aryloxy substituents in the alkyl chain of 1, ortho and meta oxygen functionalities on an aromatic ring were inserted in derivatives 2, 3 and 5, in an attempt to obtain a lower packing of the molecules. These hexyloxy-phenoxyalkyl derivatives were prepared from carboxylic acids synthesized according to the standard procedures described in Scheme 1, subsequently transformed into isocyanates using diphenyl phosphoryl azide, or alternatively following classical activation as acyl chlorides, substitution with sodium azide and Curtius transposition. The phosphonium analogue 4 was also prepared following the same procedure, using (R)-4-trimethylphosphonio-3-aminobutyrate, prepared from d-aspartic acid as described in the literature, instead of aminocarnitine. Moreover, the effect of the aryl group adjacent to the ureidic functionality was explored in derivatives 6, 10 and 11, having an alkyloxy or a small alkyl chain as the substituent, and with a methylene group spacer between the ureido and aryl group, as in derivatives 7 and 8, having an alkyloxy and/or a benzyloxy substituent. Conversely, a derivative with an aryloxy group at the end of a long chain was also prepared (compound 9). All of these compounds were prepared starting from the isocyanate or the corresponding carboxylic acid, according to the procedure summarized in Scheme 2 (see Supporting Information for more details). A three-dimensional homology model for human liver CPT I (hL-CPT I) was built using the crystallographic structure of murine carnitine acetyltransferase (CAT) co-crystallized with CoA and hexanoylcarnitine (PDB code: 2H3W). The sequence identity between the two enzymes is 33 %. Moreover, the acyl pocket in human liver CPT I is characterized by an insertion of 14 amino acids (between 690 and 707), compared with other [a] Dr. E. Tassoni, Dr. G. Gallo, Dr. S. Vincenti, Dr. N. Dell’Uomo, Dr. L. Mastrofrancesco, Dr. W. Cabri Chemistry & Analytical Department, Sigma-Tau S.p.A. Via Pontina Km 30.400, 00040 Pomezia (Italy) Fax: (+ 39) 069-139-3638 E-mail : emanuela.tassoni@sigma-tau.it [b] Dr. R. Conti, Dr. R. Ricciolini, Dr. F. Giannessi Endocrinology & Metabolism Department, Sigma-Tau S.p.A. Via Pontina Km 30.400, 00040 Pomezia (Italy) Fax: (+ 39) 069-139-3988 E-mail : fabio.giannessi@sigma-tau.it [c] Dr. P. Carminati Director of Research & Development Department, Sigma-Tau S.p.A. (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.200900535.

The Carnitine System and Acyl Trafficking in CNS

The carnitine system may be defined as a family of different short and long-chain acyltransferases, translocases, and their related substrates, whose common denominator is L-carnitine. The carnitine system is best known for the role played in mitochondrial metabolism, though the presence of carnitine-dependent short and long-chain acyltransferases in extra-mitochondrial compartments raises a number of intringuing question about their role. We have recently proposed that carnitine palmitoyltransferase (CPT) may be important in the pathway of phospholipid and triglyceride fatty acid turnover in neurons. The CPT action is accomplished by modulating the size and composition of the acyl-CoA pool between the activation step of the fatty acid and its transfer into complex lipids. In addition, studies on the metabolic fate of the acetate moiety of acetyl-L-carnitine revealed that the lipogenic acetyl-CoA pools present in different cellular compartments of rat brain are not necessary homogeneous. Taken together, these data suggest that the carnitine system may influence key regulatory points of lipid biosynthetic pathways.

Cerebral Metabolic Compartmentation as Revealed by 13C NMR Analysis of [1-13C

Nuclear magnetic resonance spectroscopy (MRS) has been shown to be a powerful non invasive technique that can be used to study cerebral metabolism in vivo (1). 31P and 1H NMR spectra have yielded information on the concentration of cerebral metabolites and on their response to various pathological states (1,2).