Enhancement of brain choline levels by nicotinamide: mechanism of action

Neuroscience Letters 249 (1998) 111–114

Enhancement of brain choline levels by nicotinamide: mechanism of action Christina Erb, Jochen Klein* Pharmakologisches Institut der Universitat Mainz, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany Received 20 January 1998; received in revised form 24 April 1998; accepted 8 May 1998

Abstract Following the subcutaneous (s.c.) administration of nicotinamide (10 mmol/kg), the brain and CSF levels of nicotinamide were increased to millimolar concentrations, but the concentrations of N-methylnicotinamide (NMN) in the CSF, and of NMN and NAD+ in brain tissue were not significantly altered. Concomitantly, nicotinamide caused increases of the choline levels in the venous brain blood. In hippocampal slices, nicotinamide (1–10 mM) induced choline release in a calcium- and mepacrine-sensitive manner and, in [3H]choline-labelled slices, increased the levels of [3H]lyso-phosphatidylcholine and [3H]glycerophosphocholine. We conclude that nicotinamide enhances brain choline concentrations by mobilising choline from choline-containing phospholipids, presumably via activation of phospholipase A2, while the formation of NMN does not contribute to this effect.  1998 Published by Elsevier Science Ireland Ltd. All rights reserved

Keywords: Choline; Nicotinamide; N-methylnicotinamide; Phospholipase A2

Nicotinamide is a vitamin of the B complex (‘vitamin B3’) due to its role as a precursor of NAD(H) and NADP(H), two cofactors of oxidoreductases [4]. NAD+ is also a cosubstrate in mono- and poly-ADP-ribosylation reactions which occur, e.g. during DNA replication and repair, and nicotinamide (1–10 mM) has been routinely used as an inhibitor of poly-ADP-ribose polymerase (PARP) in a variety of in-vitro studies. PARP inhibition may also underlie the present therapeutic use of nicotinamide in humans as a radiosensitiser in tumour therapy [14] and as a possible prophylactic for juvenile diabetes [12]; in these studies, up to 6 g of nicotinamide are tolerated without major signs of toxicity, and plasma nicotinamide levels in the low millimolar range are attained. Nicotinamide has also raised interest as a drug which is capable of elevating brain choline levels. Jenden and coworkers [5,15] had first reported increases of the choline concentration in total brain tissue and in CSF after nicotinamide administration. Using the microdialysis technique, we had subsequently demonstrated increases of the brain * Corresponding author. Tel.: +49 6131 174393; fax +49 6131 176611; e-mail: [email protected]

extracellular choline concentration [9] and a facilitation of evoked acetylcholine release in the hippocampus [10] following nicotinamide administration. Jenden and co-workers had speculated that nicotinamide may act via transformation to N-methylnicotinamide (NMN) in the brain [5,15]. NMN is a major metabolite of the hepatic metabolism of nicotinamide in rats and humans [4,7]. In the brain, NMN has been described as a competitive inhibitor of the choline transporter which is involved in the clearance of choline from the extracellular space of the brain. As a quaternary cation, NMN inhibits the uptake of choline by the choroid plexus with a Ki value of 720 mM [1], and millimolar concentrations of NMN were found to decrease the clearance of choline during ventriculo–cisternal perfusion in rabbits [11]. While NMN does not penetrate the blood–brain barrier, Vargas and Jenden recently reported that rat brain homogenate contains an enzymatic activity which converts nicotinamide to NMN [15]. However, it is not known which concentrations of NMN in the brain are formed after nicotinamide administration, and whether these concentrations are sufficient to interfere with choline clearance. In order to measure the extent of NMN formation after nicotinamide administration, we treated six male Wistar rats

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(Charles River, Sulzfeld, Germany) with 10 mmol/kg nicotinamide (dissolved in water) by subcutaneous (s.c.) injection; this dose was previously shown to maximally enhance brain choline concentrations [9]. Two hours after nicotinamide injection, i.e. when the effects on brain choline were maximal [9], the animals were anaesthetised with pentobarbital (40–80 mg/kg), arterial blood was withdrawn from the femoral artery, and CSF samples (40–80 ml) were obtained by puncture of the cisterna magna. Finally, the animals were decapitated, and total brain and aliquots of liver tissue were removed. Blood, CSF and tissue levels of nicotinamide and NMN were determined using a fluorimetric procedure [2].

Fig. 2. Arterial plasma choline levels (Chart) and arteriovenous differences (AVD) of choline across the brain 2 h after s.c. administration of 10 mmol/kg nicotinamide. AVD values for choline were calculated from the differences between arterial (A. femoralis) and venous (sinus transversus) blood choline levels analysed by HPLC. The ‘matched group’ is a group of animals which were treated with 6 mg/kg choline chloride to obtain similar Chart levels as in the nicotinamide-treated group. Data are means ± SEM from 6–10 experiments. **Significant difference (P , 0.01) from control group of animals.

Fig. 1. Concentrations of nicotinamide, NMN, and NAD+ 2 h after s.c. administration of 10 mmol/kg nicotinamide. (A) Concentrations of nicotinamide and NMN in plasma and CSF. (B) Concentrations of nicotinamide, NMN and NAD+ in brain and liver homogenate. Data are means ± SEM from six experiments. *P , 0.05, **P , 0.01 versus saline (t-test).

For the measurement of the tissue levels of NAD+, aliquots of liver and brain tissue were extracted in acidic medium, and NAD+ was determined by HPLC on a Hypersil ODS column (eluent: 0.2 M ammonium phosphate (pH 5.25), 3% methanol; tR = 8.3 min) [6]. The assay was linear from 10 to 100 pmol NAD. The results of the metabolite determinations are summarised in Fig. 1. The application of nicotinamide increased the concentrations of free nicotinamide by a factor of 1000 in blood plasma and cerebrospinal fluid (CSF), to levels of 5–10 mM (Fig. 1A). Concomitantly, the tissue levels of nicotinamide were increased more than 10-fold in liver and more than 30-fold in brain (Fig. 1B), indicating a rapid equilibration between intracellular and extracellular compartments. In contrast, increases of NMN concentrations were only significant in the periphery. NMN levels were significantly enhanced in arterial plasma (by almost 20-fold) and in liver homogenate (+163%). Total brain NMN was increased by 28% (P . 0.1), and NMN levels in the CSF (1.3 mM) were unchanged. A similar behaviour was noted for NAD+. The tissue levels of NAD+ were doubled in liver (P , 0.05) but only slightly increased in brain (+34%; P . 0.1). Two findings deserve special attention. First, the application of nicotinamide led to millimolar concentrations of nicotinamide not only in the plasma, but also in the CSF and in the brain intracellular space (Fig. 1). Previous work had shown that, at physiological (micromolar) plasma concentrations, the penetration of nicotinamide through the blood–brain barrier occurs via a high-affinity transport at

C. Erb, J. Klein / Neuroscience Letters 249 (1998) 111–114

the choroid plexus; however, a non-saturable component of nicotinamide transport into the brain had been observed as well [13]. Our data clearly show that nicotinamide, when administered in high doses, is capable to penetrate freely through the blood–brain barrier. Second, NMN formation is only a minor metabolic route of nicotinamide in the brain and, therefore, cannot explain the central actions of nicotinamide. In fact, brain NMN concentrations were slightly enhanced in brain homogenate; thus, in agreement with a previous study [15], brain cells may have a limited capacity to N-methylate nicotinamide. However, NMN, as a quaternary cation, seems to leave the cytoplasmic space slowly and, therefore, does not significantly increase the NMN concentration in the extracellular fluid and its adjacent compartment, the CSF. In agreement with earlier studies [4,7], NMN is a major metabolite in the periphery (as indicated by an almost 20-fold increase of NMN plasma levels), but plasma NMN does not contribute to the NMN levels in the brain because NMN cannot penetrate the blood–brain barrier. Since NMN formation apparently could not explain nicotinamide’s effect on brain choline, we turned our attention to a possible mobilisation of choline from bound stores by nicotinamide itself. For this purpose, we measured the arterio–venous difference (AVD) of choline across the brain. Rats were treated as described above, and 2 h after nicotinamide application (10 mmol/kg s.c.), venous blood of the brain was withdrawn from the transverse sinus, and arterial blood was collected from the femoral artery. The levels of free choline were measured by HPLC, and brain choline AVD values which reflect choline uptake and/or release from the brain were calculated from the difference of arterial and venous blood levels (for methods, see [8]). Untreated animals had plasma choline levels of approximately 10 mM and negative AVD values indicating a net release of choline from the brain into the venous blood (Fig. 2). Nicotinamide administration increased blood choline levels by approximately 2-fold. Concomitantly, the AVD of choline across the brain was markedly enhanced to −4.8 mM (Fig. 2; P , 0.01). As increasing plasma choline concentrations lead to more positive AVD values [8], we analysed a third group of rats which had received 6 mg/kg choline chloride 10 min before blood withdrawal. These rats (‘matched group’) displayed a plasma choline levels of a similar size as the nicotinamide-treated rats, but exhibited AVD values of +1.4 mM on average. The comparison of matched and nicotinamide-treated groups demonstrates that nicotinamide leads to a prominent release of choline from the brain in vivo, rather than to an inhibition of release as previously hypothesised [15]. To further characterise the action of nicotinamide on choline release, we used hippocampal slices as an in-vitro model. Rats (200–250 g) were killed by decapitation, hippocampal slices (400 mm) were prepared and superfused with Tyrode solution, and choline release was determined by a luminometric method as described previously [9]. As shown in Fig. 3A, nicotinamide increased choline release

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from brain slices in a delayed fashion which is reminiscent of its delayed effect in vivo [9]. The nicotinamide concentrations required for these effects (1 and 10 mM) were similar to those which were obtained in brain and CSF in vivo after exogenous nicotinamide administration (Fig. 1). Previous experiments with up to 0.1 mM of nicotinamide had not revealed any effects on choline release [9]. The release

Fig. 3. In-vitro experiments with hippocampal slices. (A) Increase of choline efflux in the presence of nicotinamide (nico; 1 and 10 mM) and 3-aminobenzamide (3-ABA; 1 mM). (B) Attenuation of the effect of 10 mM nicotinamide in the presence of low (0.2 mM) calcium (low Ca) and 0.1 mM mepacrine (mep). Hippocampal slices were prepared according to standard procedures, and choline efflux was followed by luminometry. The data are means ± SEM from 5–7 experiments. (C) Increased formation of [3H]lyso-phosphatidylcholine (lyso-PC) and [3H]glycerophosphocholine (GPCh) in slices which were prelabelled with [3H]choline (20 Ci, 2 h). The slices were superfused with Tyrode solution (Ctr) or nicotinamide (Nico; 10 mM in Tyrode solution) for 60 min, and workup and TLC separation of metabolites was as described in [8]. Data are means ± SEM from four experiments and are expressed as % label associated with lyso-PC and GPCh divided by the label associated with [3H]phosphatidylcholine in individual experiments.

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of choline by nicotinamide was dependent on extracellular calcium (Fig. 3B) and was strongly inhibited by mepacrine, an inhibitor of phospholipase A2 (PLA2). More direct evidence for PLA2 activation was obtained in slices which were prelabelled with [3H]choline (Fig. 3C). In these slices, nicotinamide (10 mM, 60 min) significantly increased the label associated with lyso-phosphatidylcholine (lyso-PC) (+23%; P , 0.05) and glycerophosphocholine (GPCh) (+51%; P , 0.01), compared to controls (for methods, see [8]). As lyso-PC is the direct product of PLA2, and GPCh is a metabolite which is only formed after breakdown of PC by A-type phospholipases, these data strongly suggest an activation of a calcium-dependent subform of phospholipase A2, presumably a type II- or type IV-subtype [3]. In separate experiments, we excluded the possibility that nicotinamide may affect choline release by activating phospholipase D in the brain. In hippocampal slices prelabelled with [3H]glycerol and superfused in the presence of 2% propanol, the level of [3H]phosphatidylpropanol (0.49 ± 0.07% of lipid phase; n = 5), a PLD-specific reaction product, was not significantly changed in the presence of 1 mM (0.41 ± 0.11%) or 10 mM (0.62 ± 0.07%) of nicotinamide (not illustrated). It must be noted that an increased release of choline from hippocampal slices was also observed with 3-aminobenzamide (Fig. 3B), a drug which, like nicotinamide, is also an inhibitor of PARP (see above). Importantly, 3-aminobenzamide is not a precursor of NAD+; therefore, PARP inhibition, but not the limited increase of brain NAD+ described above, is likely involved in nicotinamide’s effect on brain PLA2. Presently, the possible signalling between PARP and PLA2 is speculative; it may be of interest that both enzymes are substrates of caspases, the mediators of apoptotic cell death [16]. In conclusion, nicotinamide, under conditions known to increase brain extracellular choline and acetylcholine [9,10], affects brain choline levels by activating a phospholipase A2 which releases choline from bound stores. The formation of NMN in the brain, although present to a minor degree (Fig. 1), does not appreciably contribute to this effect. The mechanism of nicotinamide’s effects on PLA2 will remain elusive until the signalling cascades leading to PLA2 activation are more completely understood. [1] Aquilonius, S. and Winbladh, B., Cerebrospinal fluid clearance of choline and some other amines, Acta Physiol. Scand., 85 (1972) 78–90.

[2] Clark, B.R., Halpern, R.M. and Smith, R.A., A fluorimetric method for quantitation in the picomole range of N1-methylnicotinamide and nicotinamide in serum, Anal. Biochem., 68 (1975) 54–61. [3] Dennis, E.A., Diversity of group types, regulation, and function of phospholipase A2, J. Biol. Chem., 269 (1994) 13057–13060. [4] Henderson, L.M., Niacin, Ann. Rev. Nutr., 3 (1983) 289–307. [5] Jenden, D.J., Rice, K., Roch, M., Booth, R.A. and Lauretz, D., Effects of nicotinamide on choline and acetylcholine levels in rats, Adv. Neurol., 51 (1990) 131–138. [6] Kalhorn, T.F., Thummel, K.E., Nelson, S.D. and Slattery, J.T., Analysis of oxidized and reduced pyridine dinucleotides in rat liver by high-performance liquid chromatography, Anal. Biochem., 151 (1985) 343–347. [7] Kang-Lee, Y.A.E., McKee, R.W., Wright, S.M., Swendseid, M.E., Jenden, D.J. and Jope, R.S., Metabolic effects of nicotinamide administration in rats, J. Nutr., 113 (1983) 215–221. [8] Klein, J., Ko¨ppen, A., Lo¨ffelholz, K. and Schmitthenner, J., Uptake and metabolism of choline by rat brain after acute choline administration, J. Neurochem., 58 (1992) 870–876. [9] Ko¨ppen, A., Klein, J., Holler, T. and Lo¨ffelholz, K., Synergistic effect of nicotinamide and choline administration on extracellular choline levels in the brain, J. Pharmacol. Exp. Ther., 266 (1993) 720–725. [10] Ko¨ppen, A., Klein, J., Erb, C. and Lo¨ffelholz, K., Acetylcholine release and choline availability in rat hippocampus: effects of exogenous choline and nicotinamide, J. Pharmacol. Exp. Ther., 282 (1997) 1139–1145. [11] Lanman, R.C. and Schanker, L.S., Transport of choline out of the cranial cerebrospinal fluid spaces of the rabbit, J. Pharmacol. Exp. Ther., 215 (1980) 563–568. [12] Mandrup-Poulsen, T., Reimers, J.I., Andersen, H.U., Pociot, F., Karlsen, A.E., Bjerre, U. and Nerup, J., Nicotinamide treatment in the prevention of insulin-dependent diabetes mellitus, Diabetes/Metab. Rev., 9 (1993) 295–309. [13] Spector, R. and Kelley, P., Niacin and niacinamide accumulation by rabbit brain slices and choroid plexus in vitro, J. Neurochem., 33 (1979) 291–298. [14] Stratford, M.R.L., Rojas, A., Hall, D.W., Dennis, M.F., Dische, S., Joiner, M.C. and Hodgkiss, R.J., Pharmacokinetics of nicotinamide and its effect on blood pressure, pulse and body temperature in normal human volunteers, Radiother. Oncol., 25 (1992) 37–42. [15] Vargas, H.M. and Jenden, D.J., Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N-methylnicotinamide in brain tissue, Life Sci., 58 (1996) 1995–2002. [16] Wissing, D., Mouritzen, H., Egeblad, M., Poirier, G.G. and Jaattela, M., Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis, Proc. Natl. Acad. Sci. (USA), 94 (1997) 5073–5077.