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Citrate as the precursor of acetyl moiety of acetylcholine
Journal of Neurochemistry
Abstract
— Rat brain cortex slices were incubated with glucose labeled with either 3H or 14C in the 6-position. The 3H/14C ratios and the incorporation of radioactivity into lactate, citrate, malate and acetylcholine were determined. While the 3H/14C ratio of lactate was close to that of glucose, the ratios in the acetyl moiety of acetylcholine and the acetyl (C-4,5) portion of citrate decreased in a similar proportion. This was interpreted as indirect evidence for the participation of citrate as a precursor to the acetyl moiety of acetylcholine. Two inhibitors of the citrate cleavage pathway: n-butylmalonate, an inhibitor of citrate transport and (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase were studied for their effect on acetylcholine synthesis. N-butylmalonate (10 mM) and (-)-hydroxycitrate (7.5 mM) led to a decrease in the per cent of 14C recovered as acetylcholine. In each instance the 3H/14C ratio in acetylcholine was higher in the presence of inhibitor while the corresponding ratios in lactate and citrate (C-4.5) remained unchanged. From the results, it is suggested that citrate is involved in the transport mechanism of acetyl units from its site of synthesis in mitochondria to the site of acetylcholine synthesis in the cytosol.
... Hence, PC is considered to have an impact on a multitude of processes, which are directly or indirectly dependent on the availability of oxaloacetate and other members of the TCA cycle, i.e., gluconeogenesis [4] and glycogen synthesis [5,6], lipogenesis [7,8], biosynthesis of non-essential amino acids with impact on the cellular/tissue nitrogen balance [9,10], the metabolism and the mitochondriacytosol shuttling of acetyl-CoA [4], and the regeneration of NADPH [11]. Due to its anaplerotic role, PC in brain may contribute to maintaining a cytosolic level of acetyl-CoA that is required for the synthesis of the neurotransmitters acetylcholine [12], glutamate, and GABA [13,14]. ...
The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the "distal class" of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.
... The intramitochondrial acetyl-CoA can pass the inner mitochondrial membrane in the form of citrate, delivering acetyl-CoA to cytosol due to the action of citrate lyase (citrate oxaloacetate-lyase, EC 4.1.3.6). It was, however, demonstrated that citrate pathway supplied not more than 30% of acetyl moieties for acetylcholine synthesis in mammalian brain (Gibson and Shimada, 1980 ;Turek et al., 1981 ;Sterling and O'Neill, 1978). The other possible pathway of acetyl-CoA transfer through the inner mitochondrial membrane might be the carnitine shuttle. ...
Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 microM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of beta-oxidation might be correlated with the lower level of acetylcholine synthesis.
Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer’s disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy and favors amyloid-β (Aβ) aggregation. Citrate inhibits Aβ25–35 and Aβ1–40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aβ aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need to study if citrate reverses Aβ deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD’s silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.
As previous reviews on brain mitochondrial properties appeared in the first edition of the Handbook in Neurochemistry (Volumes 2 and 3, 1969–70), we confine ourselves mainly to those reports appearing in the last 10–12 years. Certain aspects of what may legitimately be considered as mitochondrial activity have been left out, as they form subjects in their own right which are being treated elsewhere, e.g., mitochondrial protein synthesis, monoamine oxidase activity. The emphasis of this review is therefore, in the main, concerned with the properties of mitochondria derived from the main cell types, neurons and glia; the metabolic properties of these preparations particularly with respect to energy metabolism; the role of the mitochondria in the development of brain function; and clinical disorders that are considered to be the result of a primary mitochondrial lesion in the brain.
In mammalian brain, acetylcholine is synthesised by the enzyme choline acetyl transferase from the precursors choline and acetyl CoA. Since the brain ‘in situ’ appears to be unable to synthesise choline de novo (see Tucek, 1978), much interest has centred around the supply of acetyl CoA for acetylcholine synthesis. Although early work suggested that choline acetyl transferase might be bound to a particulate fraction in brain, Fonnum (1968) established that this was an artefact of isolation and the bulk of the enzyme is now generally accepted as being cytosolic, With approximately half of its activity located at the nerve ending (see Tucek, 1978). The work of Gibson and Blass (1976) and Jope and Jenden (1977) has demonstrated that conditions which reduce the availability of acetyl CoA in general bring about proportional reductions in acetyl choline synthesis although the flux into the acetyl group of acetyl choline is less than 1% of the total carbon throughput in whole brain. Two immediate questions which arise from this are: a) Is choline supply the principal control point in acetylcholine syntheis (Simon & Kuhar, 1976) b) What is the nature of the carbon pool which supplies the acetyl CoA for acetylcholine synthesis. Observations by Lefresne et al. (1973) and other groups (Browning & Schulman, 1968; Grewaal & Quastel, 1973) that radioactive pyruvate is incorporated into acetyl choline with little or no dilution implies that the endogenous pool is extremely small with a rapid turnover rate.
Oxidative metabolism is essential for normal neuronal function. The brain consumes 20% of the oxygen that is used by the body even though it represents only 2% of the total body mass. Most of this oxygen is utilized for the catabolism of glucose and the production of ATP. However, mild to moderate decreases in the availability or utilization of oxygen or glucose impair brain function without reducing the levels of energy metabolites (i.e., ATP).1–4 The cerebral dysfunction that accompanies impaired oxidative metabolism is associated with changes in neurotransmitter metabolism. Thus, an understanding of the pathophysiological basis of altered neuronal function requires knowledge of the relationship of metabolism to neurotransmission.
The concentration of acetylcholine (ACh) in brain is maintained within narrow limits even though the turnover rate is several fold that estimated for other neurotransmitters (16). The precise mechanisms regulating ACh metabolism are complex and there is considerable disagreement as to which factor(s) is rate-limiting to synthesis. Regulation of ACh levels can be separated into several categories, no one of which by itself is controlling but collectively can regulate ACh synthesis: 1) Choline acetyltransferase (ChAT) activity; 2) Availability of its precursors, choline or AcCoA and; 3) Indirectly, by changes in ACh release.
Acetylcholine (ACh) is one of the most investigated synaptic transmitters. It would be impossible to make a comprehensive review of all data concerning its synthesis within the available space; instead, an attempt has been made to provide summarizing information on the most important facets of the topic and to give references to more specialized reviews.
Garcinia cambogia is an exotic fruit grown in the southern parts and Western ghats of India. Hydroxy citric acid is the active component present in this fruit which imparts the characteristic sour taste. Hydroxy citric acid is found to be physiologically active. It is a significant factor in reducing obesity. Hydroxy citric acid is an alpha, beta dihydroxy tricarboxylic acid, which is less stable and easily converted into its lactone. Both hydroxy citric acid and its lactone are estimated using RP amide C16 HPLC column and is described in this paper. Ethylene diamine salt of hydroxy citric acid is used as the reference material. The percentage of hydroxy citric acid varies from 45-65% in different salts of hydroxy citric acid.
This chapter provides an overview of twenty years of progress of the synthesis of acetylcholine. The synthesis of acetylcholine (ACh) proceeds according to the equation described in the chapter and is catalyzed by the enzyme choline acetyltransferase. All the components of the reaction were identified by 1960s. But the sources of both substrates used for the synthesis of ACh, the chemical nature of choline acetyltransferase (ChAT), the mechanism of its action, its distribution in neurons and among neurons, the factors affecting its expression and the effects of diseases upon the synthesis of ACh and upon the cholinergic neurons are much better understood at present. Four methods have mainly assisted the progress in the field: histochemistry of cholinesterases, particularly that performed at intervals after the administration of a strong cholinesterase inhibitor; retrograde tracing of markers like horse radish peroxidase, fluorescent dyes and lectins; immunohistochemical detection of ChAT; and biochemical measurements performed after specific lesions. The discovery of the basal forebrain cholinergic system is another important finding that was achieved. The factors which induce the expression of cholinergic features, other factors have been found to enhance their expression.
Soon after the discovery that acetylcholine (ACh) mediates synaptic transmission in sympathetic ganglia, Brown and Feldberg (1936) noted that the amount of ACh released from perfused stimulated ganglia during their experiments was several-fold higher than the amount of ACh that had been present in the ganglia at the start of the experiments. It became apparent from this and other observations that ACh is synthesized in the nerve terminals which use it as their transmitter and that the rate of its synthesis varies so as to keep the stores of ACh in the nerve terminals at a constant level under most physiological conditions. Systematic studies of the process of ACh synthesis were started by Quastel and Mann (Quastel et al. 1936, Mann et al. 1938, 1939) and by Stedman and Stedman (1937). In a few years, Nachmansohn and Machado (1943) proved able to demonstrate the synthesis of ACh in cell-free tissue extracts with added ATP, acetate and choline; they ascribed the synthesis to the activity of a new enzyme which they called ‘choline acetylase’. It has been clarified in the following work that at least two enzymes must have been active in the system used by Nachmansohn and Machado (1943), one enzyme producing acetylcoenzyme A (AcCoA) from acetate, coenzyme A (CoA) and ATP (AcCoA synthetase by the present nomenclature), and another enzyme producing ACh from AcCoA and choline. The name of choline acetylase was preserved for the latter enzyme and was modified to choline acetyltransferase (acetyl-CoA: choline-O-acetyltransferase, EC 2.3.1.6) (ChAT) in the EC-IUB enzyme nomenclature.
Purified rat brain mitochondria were incubated in the presence of pyruvate or (l-14 C)pyruvate and the output of the pyruvate-generated acetylcoen zyme A (acetyl-CoA) from the mitochondria into the medium was measured and compared with the rate of (l-"*C)pyruvate decarboxylation. When CaCl 2 (1 mmol-/l) was added to the incubation medium, the output of acetyl-CoA from the mitochondria was increased 3.8—6 times; at the same time, the rate of pyruvate decarboxylation (and of the intramitochondrial acetyl-Co A production) increased only 1.3 times. After repeated freezing and thawing, the output of acetyl-CoA into the medium was higher, but the stimulatory effect of Ca 2+ ions was consider ably diminished. It is concluded that Ca 2+ ions increase the output of acetyl-CoA from the mitochondria by altering the permeability of mitochondrial membranes rather than by increasing the activity of the pyruvate dehydrogenase complex. Possible physiological role of the observed effect of Ca 2+ ions in the control of acetylcholine synthesis in the nerve terminals is discussed.
: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl2, 1.2 mM-NaH2PO4, 25 mM-NaHCO3, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-14C]citrate by 82–86%, but that from [U-14C]glucose by only 33%, from [2-14C]pyruvate by 24% and from [1-14C-acetyl]carnitine by 8%; the production of 14CO2 from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-14C]glucose and from [1-14C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca2+ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.
: Electrolytic lesions made in the medial septum of the rat brain caused an 80% decrease in the activity of choline acetyltransferase and a 33% reduction in ATP-citrate lyase activity in the synaptosomal fraction from the hippocampus. Decreases in the activities of the two enzymes in the cytosol (S3) fraction were 70 and 13%, respectively. The activities of pyruvate dehydrogenase, citrate synthase, acetyl-CoA synthase, and carnitine acetyltransferase in crude hippocampal homogenates and in subcellular fractions were not affected by septal lesions. The data indicate that ATP-citrate lyase is linked to the septal-hippocampal pathway and that the enzyme is preferentially located in cholinergic nerve endings that terminate within the hippocampus.
: The activities of choline acetyltransferase and ATP-citrate lyase were significantly correlated (r = 0.995) in fractions of small and large synaptosomes isolated from rat hippocampus and cerebellum. The activities of these two enzymes did not correlate with those of pyruvate dehydrogenase, carnitine acetyltransferase, citrate synthase, acetyl-CoA synthetase, lactate dehydrogenase, or with the rate of high-affinity glutamate uptake in the synaptosomal fractions. The results provide additional evidence linking ATP-citrate lyase to the cholinergic system in the brain.
— Rat brain cortex slices were incubated with 10 mm-glucose and trace amounts of [6-3H]glucose and [3-14C]β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and 3H:14C ratios calculated. Incorporation of [14C]β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of 14C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [3H-,14C]-glucose. (–)-Hydroxycitrate (5.0 mM) reduced incorporation of [3H]glucose and [14C]β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm) showed a decrease in 14C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm) also produced a decreased 14C incorporation into ACh and its precursors. At 10 mm, acetoacetate reduced 3H and 14C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.
: The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyl-transferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulo-interpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30–409%, whereas the activities of the other enzymes decreased 5–15%. ChAT activity was in all cases less than 159% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.
Data on acetylcholine (ACh) synthesis in nerve cells are summarized and the mechanism of regulation of this process is described. Under conditions of relative rest on moderate synaptic activity the ACh concentration in the compartment of its synthesis in cholinergic nerve endings is probably maintained at a level corresponding to equilibrium of the reaction catalyzed by the enzyme choline-acetyltransferase (CAT). ACh release is followed by its transport from the compartment of synthesis into the compartment of secretion and automatic resynthesis of new ACh, until equilibrium is restored in the compartment of synthesis. At the same time synaptic activity and ACh release promote synthesis of new ACh by the following pathways. First, a fall in the ACh concentration in the nerve endings disinhibits carriers for choline, and facilitates choline transfer from the extracellular fluid into the cell in accordance with the electrochemical gradient. Second, hydrolysis of liberated ACh increases the choline concentration in the extracellular fluid in the neighborhood of the nerve endings. Third, postactivation hyperpolarization of the nerve endings facilitates transport of choline and an increase in its concentration in the nerve endings. Fourth, there are grounds for considering that stimulation of muscarine receptors promotes a further increase in the choline concentration in the region of the nerve endings by intensification of phosphatidylcholine hydrolysis in postsynaptic cells. Fifth, a decrease in the acetyl-CoA content on account of ACh resynthesis increases pyruvate dehydrogenase activity and acetyl-CoA production. Sixth, it is possible that an increase in the Ca++ concentration in nerve endings promotes direct transport of acetyl-CoA from the mitochondria into the cytosol of nerve endings, where ACh is synthesized. It is postulated that under conditions of intensive synaptic activity the rate of supply of acetyl-CoA and choline and also CAT activity in the nerve endings may be factors limiting the velocity of ACh resynthesis.
:Dibutyryl cyclic AMP and butyrate inhibited growth of S-20 (cholinergic) and NIE-115 (adrenergic) neuroblastoma clones. Both these drugs resulted in a parallel increase of choline acetyltransferase and ATP-citrate lyase activities in S-20 neuroblastoma cells. On the other hand, the increase in tyrosine hydroxylase activity in NIE-115 caused by these drugs was not accompanied by a significant change in ATP-citrate lyase activity. Both dibutyryl cyclic AMP and butyrate caused a decrease in fatty acid synthetase activity in both cell lines. The activities of pyruvate dehydrogenase, citrate synthase, choline acetyltransferase, and lactate dehydrogenase in both S-20 and NIE-115 cells were not significantly influenced by the drugs. ATP-citrate lyases from S-20 and NIE-115 had similar kinetic and immunological properties, and their subunits had the same molecular weight as the rat liver enzyme. These data indicate that the differential regulation of ATP-citrate lyase activity in cholinergic and adrenergic cells does not result from the existence of different molecular forms of the enzyme in these cell lines. They also provide further evidence to support the hypothesis that ATP-citrate lyase activity increases during maturation of normal cholinergic neurons and decreases in noncholinergic cells of the brain.
The effects of (-)-hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 mM K+, 0 or 2.5 mM Ca2+, and 0, 1, or 10 mM EGTA. OHC diminished the concentration of acetyl-CoA in the slices under all conditions used: in experiments with 2.5 mM OHC, the concentration of acetyl-CoA was lowered by 25-38%. Citrate, in contrast, had no effect on the level of acetyl-CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 mM K+ and 1 mM EGTA, they had different effects on the content of ACh during incubations in the presence of Ca2+. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca2+ and 31.2 or 6.2 mM K+, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP-citrate lyase; with glucose as the main metabolic substrate, ATP-citrate lyase appears to provide about one-third of the acetyl-CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca+2, is interpreted to be a consequence of the chelation of intracellular Ca2+; this interpretation is supported by the observation of a similar effect caused by 10 mM EGTA.
Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5-14C]citrate, [1-14C-acetyl]carnitine, [1-14C]acetate, [2-14C]pyruvate, or [U-14C]glucose. The synthesis of acetyl-labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2-14C]pyruvate > [U-14C]glucose > [1-14C-acetyl]carnitine > [1,5-14C]citrate > [1-14C]acetate. The utilization of [1,5-14C]citrate and [1-14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of and 14C-labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5-14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcar-nitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brain; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl-CoA to the extramitochondrial space in cerebral cholinergic neurons.
Until the mid-1960s, studies on the metabolism of brain mitochondria were hampered by the lack of suitable methodologies for
brain mitochondrial isolation. In the majority of the studies up to this time, crude mitochondrial preparations were isolated
from mammalian brain homogenates using differential centrifugation techniques that were adapted from those that had originally
been designed for isolating liver mitochondria. During the early 196Os, two groups of workers [see Whittaker (1969 and 1984)
and De Robertis and de Lores Arnaiz (1969) for discussions] independently designed relatively elaborate subcellular fractionation
procedures, whereby the crude mitochondrial fraction derived from brain homogenates was subfractionated, using sucrose density
gradients, into three or more discrete fractions, including pinched-off nerve-ending particles or “synaptosomes” and myelinated
axon fragments (usually referred to as “myelin”), in addition to “free” (i.e., nonsynaptic) mitochondria. Thus, the structural
heterogeneity of the crude mitochondrial fraction derived from brain homogenates was revealed. Consequently, many of the metabolic
properties (e.g., glycolysis) that were erroneously attributed to isolated brain mitochondria in the studies in that era could
be accounted for by the presence of vesicular and other membranous particles (e.g., synaptosomes, myelin, and micro-somes)
that contain cytosolic material to a greater or lesser extent [see Clark and Nicklas (1970) for a discussion].
The relationships between pyruvate and derived citrate metabolism and acetylcholine (ACh) synthesis in synaptosomes were examined. In the presence of 30 mM KCl, 0.1 mM Ca2+ caused 31 and 63% inhibition of pyruvate utilization and citrate accumulation, respectively. Verapamil and EGTA (0.5 mM) brought about no change in pyruvate consumption but increased rate of citrate accumulation, and overcame inhibitory effect of Ca2+. The rates of citrate accumulation in the presence of verapamil or EGTA were three to six times, respectively, higher than those in the presence of Ca2+. (−) Hydroxycitrate increased rate of citrate accumulation under all experimental conditions. The value of this activation appeared to be stable (0.20–0.28 nmol/min/mg of protein) and independent of changes in the basic rate of citrate accumulation. Ca2+ caused no significant changes in [14C]ACh synthesis, but it inhibited 14CO2 production by synaptosomes. These activities were inhibited by verapamil by 33 and 60%, respectively. Ca2+ did not modify these effects of the drug. On the other hand, (−)hydroxycitrate resulted in 22 and 29% inhibition of [14C]ACh synthesis in Ca2+ free and Ca2+ supplemented medium, respectively. These data indicated that rates of acetyl-CoA synthesis in synaptoplasm, via ATP-citrate lyase and probably by another pathways are independent of Ca-evoked changes in pyruvate oxidation and citrate supply from intraterminal mitochondria. This property might play a significant role in maintenance of stable level of ACh in active cholinergic nerve endings.
This review describes recent advances made in the understanding of the regulation of acetylcholine synthesis in brain with regard to the availability of its two precursors, choline and acetylCoA. Choline availability appears to be regulated by the high affinity choline transport system. Investigations of the localization and inhibition of this system are reviewed. Procedures for measuring high affinity choline transport and their shortcomings are described. The kinetics and effects of previous in vivo and in vitro treatments on high affinity choline transport are reviewed. Kinetic and direct coupling of the transport and acetylation of choline are discussed. Recent investigations of the source of acetylCoA used for the synthesis of acetylcholine are reviewed. Three sources of acetylCoA have recently received support: citrate conversion catalyzed by citrate lyase, direct release of acetylCoA from mitochondria following its synthesis from pyruvate catalyzed by pyruvate dehydrogenase, and production of acetylCoA by cytoplasmic pyruvate dehydrogenase. Investigations indicating that acetylCoA availability may limit acetylcholine synthesis are reviewed. A model for the regulation of acetylcholine synthesis which incorporates most of the reviewed material is presented.
Eleven regions of mouse brain and twelve layers of monkey retina were assayed for choline acetyl transferase (ChAT), acetylcholine esterase (AChE), and 4 enzymes that synthesize acetyl CoA. The purpose was to seek evidence concerning the source of acetyl CoA for acetylcholine generation. In brain ATP citrate lyase was strongly correlated with ChAT as well as AChE (r = 0.914 in both cases). Weak, but statistically significant correlation, was observed between ChAT and both cytoplasmic and mitochondrial thiolase, whereas there was a significant negative correlation between ChAT and acetyl thiokinase. In retina ChAT was essentially limited to the inner plexiform and ganglion cell layers, whereas substantial AChE activity extended as well into inner nuclear, outer plexiform and fiber layers, but no further. ATP citrate lyase activity was also highest in the inner four retinal layers, but was not strongly correlated with either ChAT or AChE (r = 0.724 and 0.761, respectively). Correlation between ChAT and acetyl thiokinase was at least as strong (r = 0.757), and in the six inner layers of retina, the correlation between ChAT and acetylthiokinase was very strong (r = 0.932).
The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.
Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high-affinity, sodium- and temperature-dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure-activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC-3, acetylsecohemicholinium, and HC-15 are potent inhibitors of HAChU, HC-3 being the most potent (I50 = 6.1 X 10(-8) M). In the present study, the most potent quinuclidinyl derivative was the N-methyl-3-quinuclidinone (I50 = 5.6 X 10(-7) M). This compound had approximately 100-fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3-hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N-functional group from a methyl to an allyl in the alcohol led to a 10-fold increase in activity. However, removal of the quaternizing N-methyl group yielding the tertiary amine, 3-quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2-benzylidene-3-quinuclidinone derivatives studied, only the m-chloro derivative significantly reduced HAChU.
beta beta'-Methyl-substituted, C14-C18, alpha, omega-dicarboxylic acids (MEDICA 14-18) were found to inhibit fatty acids and cholesterol synthesis in cultured rat hepatocytes. Maximum inhibition was observed with MEDICA 16, amounting to a 50% decrease in 3H2O and acetate incorporation into fatty acids and cholesterol in the presence of 0.08 mM of the drug added to the culture medium. Inhibition of lipogenesis was not accompanied by inhibition of palmitate or glycerol esterification into neutral lipids and phospholipids. The respective capacities of MEDICA homologues of varying acyl chain length as inhibitors of fatty acid and cholesterol synthesis in cultured rat hepatocytes and in vivo (Bar-Tana, J., Rose-Kahn, G., and Srebnik, M. (1985) J. Biol. Chem. 260, 8404-8410) correlated well with their respective inhibitory effect on liver ATP-citrate lyase. Thus, MEDICA 16 inhibited liver ATP-citrate lyase competitively to citrate with a Ki of 16 microM as compared to a Km of 0.8 mM for the citrate substrate.
1.1. The activity of CCE during ontogenesis of the cerebellum decreased from 29.5 to 7.3 μmol/ hr/g tissue, while in the cerebrum it remained on the level of 30 μmol/hr/g tissue.2.2. In the same regions the activity of ChAT did not change and rose 12 times, respectively.3.3. The developmental patterns of remaining enzymes involved in acetyl-CoA metabolism were similar, in these two parts of brain.4.4. The activity of fatty acid synthetase decreased by about 60%, while that of acetyl-CoA synthetase, citrate synthase, and carnitine acetyltransferase increased from 2 to 5 times, respectively.
The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 mu mol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 mu mol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and Bp. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.
The activity of ATP-citrate lyase in homogenates of five selected rat brain regions varied from 2.93 to 6.90 nmol/min/mg of protein in the following order: cerebellum less than hippocampus less than parietal cortex less than striatum less than medulla oblongata and that of the choline acetyltransferase from 0.15 to 2.08 nmol/min/mg of protein in cerebellum less than parietal cortex less than hippocampus = medulla oblongata less than striatum. No substantial differences were found in regional activities of lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase or acetyl-CoA synthase. High values of relative specific activities for both choline acetyltransferase and ATP-citrate lyase were found in synaptosomal and synaptoplasmic fractions from regions with a high content of cholinergic nerve endings. There are significant correlations between these two enzyme activities in general cytocol (S3), synaptosomal (B) and synaptoplasmic (Bs) fractions from the different regions (r = 0.92-0.99). These data indicate that activity of ATP-citrate lyase in cholinergic neurons is several times higher than that present in glial and noncholinergic neuronal cells.
Dibutyryl cyclic AMP and butyrate inhibited growth of S-20 (cholinergic) and NIE-115 (adrenergic) neuroblastoma clones. Both these drugs resulted in a parallel increase of choline acetyltransferase and ATP-citrate lyase activities in S-20 neuroblastoma cells. On the other hand, the increase in tyrosine hydroxylase activity in NIE-115 caused by these drugs was not accompanied by a significant change in ATP-citrate lyase activity. Both dibutyryl cyclic AMP and butyrate caused a decrease in fatty acid synthetase activity in both cell lines. The activities of pyruvate dehydrogenase, citrate synthase, choline acetyltransferase, and lactate dehydrogenase in both S-20 and NIE-115 cells were not significantly influenced by the drugs. ATP-citrate lyases from S-20 and NIE-115 had similar kinetic and immunological properties, and their subunits had the same molecular weight as the rat liver enzyme. These data indicate that the differential regulation of ATP-citrate lyase activity in cholinergic and adrenergic cells does not result from the existence of different molecular forms of the enzyme in these cell lines. They also provide further evidence to support the hypothesis that ATP-citrate lyase activity increases during maturation of normal cholinergic neurons and decreases in noncholinergic cells of the brain.
Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5-14C] citrate, [1-14C-acetyl]carnitine, [1-14C]acetate, [2-14C]pyruvate, or [U-14C]glucose. The synthesis of acetyl-labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2-14C]pyruvate greater than [U-14C]glucose greater than [1-14C-acetyl]carnitine greater than [1,5-14C]citrate greater than [1-14C]acetate. The utilization of [1,5-14C]citrate and [1-14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of 14CO2 and 14C-labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5-14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcarnitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brian; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl-CoA to the extramitochondrial space in cerebral cholinergic neurons.
Rat-liver mitochondria were incubated with [1,5-14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion-exchange chromatography. Incorporation of 14C into acetyl-l-carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol. mg−1. min−1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg−1 . min−1.
We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.
The effect of chronic low-level lead (Pb2+) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb2+-exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs-Ringer bicarbonate buffer with 10 mM glucose and tracer amounts of [6-3H]glucose and either [6-14C]glucose or [3-14C] beta-hydroxybutyrate. Altering the concentration of Pb2+ in the drinking water produced a dose-related increase in blood and brain lead levels. When tissue from Pb2+-exposed rats was incubated with mixed-label glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the 3H/14C rations. Similar effects of Pb2+ were found when 14C-labeled beta-hydroxybutyrate was substituted for the [14C]glucose. It appears from these data that Pb2+ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb2+-induced changes observed in cholinergic function.
1.1. Synaptosomes utilizing glucose or glucose plus malate produced citrate with rates of 2.4 and 7.8 nmol/hr/mg of protein, respectively.2.2. (−)Hydroxycitrate increased citrate net synthesis 4 times and inhibited acetylcholine synthesis by 40%.3.3. Oxygen and glucose consumption as well as lactate and CO2 production were not changed by this inhibitor.4.4. (−)Hydroxycitrate inhibited utilization of exogenous citrate in synaptosomes by 50%.
Labeled acetylcholine derived from labeled pyruvate in a synaptosomal preparation from rat brain, incubated with nicotinamide
adenine dinucleotide as well as coenzyme A, is stimulated by calcium ions in the absence but not in the presence of Triton
X-100. Whereas citrate is taken up by cholinergic synaptosomes because it suppresses the formation of acetylcholine from pyruvate,
it is not itself converted into acetylcholine. The evidence suggests that there is a calcium-dependent transfer of mitochondrial
acetyl coenzyme A into the cholinergic synaptoplasm, which is apparently devoid of the citrate cleavage enzyme, and is there
converted into acetylcholine. The permeability of the inner mitochondrial membrane to coenzyme A and acetyl coenzyme A seems
to be enhanced by calcium ions, and this effect may be mediated by mitochondrial phospholipase A2.
This chapter discusses the stereochemistry and preparation of hydroxycitrate. In nonruminant mammals the acetyl-CoA used for lipogenesis is generated largely from citrate in a reaction catalyzed by ATP: citrate lyase. Hydroxycitrate is a competitive inhibitor of the reaction with respect to citrate. The de novo syntheses of long-chain fatty acids and 3-β-hydroxysterols are inhibited to about the same extent by hydroxycitrate. Both pathways are extramitochondrial and both use acetyl-CoA as a carbon source. In nonruminant mammals extramitochondrial acetyl-CoA is made predominantly via the citrate cleavage reaction. Hydroxycitrate can therefore be expected to inhibit both pathways. The degrees to which both pathways are inhibited will depend on the relative affinity of their acetyl-CoA-utilizing enzymes for acetyl-CoA. Metabolite analyses of perfused livers show that addition of either hydroxycitrate or oleate causes a crossover at the phosphofructokinase reaction. This indicates a slowing down of glycolysis, which is regulated through phosphofructokinase. It is found that ketone production by perfused livers from fed rats, which occurs at a considerably slower rate, is inhibited by hydroxycitrate.
Glucose and pyruvate are the most effective precursors of the acetyl moiety of acetylcholine in mammalian brain; the metabolic intermediates between pyruvate and acetylcholine, however, are unknown. The following data suggest that citrate is not the sole intermediate of the acetyl group for acetylcholine synthesis in rat brain slices or synaptosomes: (1) 2.5 mM (−)-hydroxycitrate decreased acetylcholine synthesis from [U-14C]glucose by only 25 per cent; (2) inhibition of citrate transport out of mitochondria by n-butylmalonate or 1,2,3-benzenetricarboxylate variably affected acetylcholine synthesis; and (3) high concentrations of nonradioactive citrate decreased the synthesis of acetylcholine but did not decrease the specific activity of the acetylcholine synthesized from [U-14C]glucose. even though the uptake of citrate into the synaptosomes under these experimental conditions was approximately five times greater than the uptake of glucose. Other possible acetyl donors altered acetylcholine synthesis. Acetylcarnitine stimulated synthesis in brain slices, and carnitine stimulated synthesis by synaptosomes.The specificactivity of the acetylcholine synthesized from [U-14Cglucose by synaptosomes was decreased by N-acetyl-l-aspartate (10mM), acetyl CoA (1 mM), and acetyl phosphate (10mM) which is consistent with these compounds acting as direct acetyl donors. Acetate (10 mM) did not affect either the amount or specific activity of the acetylcholine synthesized. Further evidence of compartmentation of cytoplasmic acetyl CoA is presented. The cytoplasmic acetyl CoA for acetylcholine synthesis is distinguishable from the cytoplasmic acetyl CoA for lipid synthesis. (−)-Hydroxycitrate inhibited acetylcholine synthesis without inhibiting lipid synthesis from [U-14C]glucose. However, when 3-hydroxy[3-14C]butyrate was used as substrate, (−)-hydroxycitrate inhibited incorporation into lipids twice as much as incorporation into acetylcholine. [U-14C]Glucose metabolism by infant brain slices was more sensitive than adult brain slices to (−)-hydroxycitrate. However, the response to the other compounds which interfere with citrate metabolism was similar in slices from adult and infant brains.
Acetylcholine release by the phrenic nerve was measured in the isolated, perfused rat hemidiaphragm. In controls, the rate of release of acetylcholine increases with time; however, when (--)-hydroxycitrate, an inhibitor of ATP-citrate lyase, is added to the perfusate, the release of acetylcholine stabilizes at a level 40% below the final control value. In this preparation, the capacity of the citrate cleavage pathway to transfer acetyl coenzyme A from the nerve cell mitochondria to the cytosol increases with time; this is not the case for other transport processes.
An influence of carnitine on acetylcholine synthesis from radiolabeled glucose was monitored in neuroblastoma NB-2a cells. Upon addition of carnitine the distribution of its derivatives was found significantly different than the values published for brain, the level of long-chain acyl derivatives being much higher and reaching 60%. Carnitine itself did not change acetylcholine level. Together with choline (20 microM), carnitine was observed to stimulate (by 36%) acetylcholine synthesis in a synergistic way, which indicated that both substrates could be limiting factors of this process in NB-2a cell line of neuroblastoma.
Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) seems to fulfill in the brain a different role than in peripheral tissues. Carnitine is accumulated by neural cells in a sodium-dependent way. The existence of a novel transporter in plasma membrane, specific to compounds with a polar group in the beta-position with respect to carboxyl group, has been postulated. The presence of a carnitine carrier in the inner mitochondrial membrane has been proven and the protein has been purified. It is postulated that its major role in adult brain would be translocation of acetyl moieties from mitochondria into the cytoplasm for acetylcholine synthesis. The latter process is stimulated by carnitine and choline in a synergistic way in cells utilizing glucose as the main energetic substrate. Carnitine metabolism in neural cells leads to accumulation of different acyl derivatives of carnitine. Palmitoylcarnitine can influence directly the activity of protein kinase C. An involvement of carnitine in a decrease of palmitate pool used for palmitoylation of regulatory proteins has been postulated.
(-)-Hydroxycitric acid [(-)-HCA] is the principal acid of fruit rinds of Garcinia cambogia, Garcinia indica, and Garcinia atroviridis. (-)-HCA was shown to be a potent inhibitor of ATP citrate lyase (EC 4.1.3.8), which catalyzes the extramitochondrial cleavage of citrate to oxaloacetate and acetyl-CoA: citrate + ATP + CoA --> acetyl-CoA + ADP + P(i) + oxaloacetate. The inhibition of this reaction limits the availability of acetyl-CoA units required for fatty acid synthesis and lipogenesis during a lipogenic diet, that is, a diet high in carbohydrates. Extensive animal studies indicated that (-)-HCA suppresses the fatty acid synthesis, lipogenesis, food intake, and induced weight loss. In vitro studies revealed the inhibitions of fatty acid synthesis and lipogenesis from various precursors. However, a few clinical studies have shown controversial findings. This review explores the literature on a number of topics: the source of (-)-HCA; the discovery of (-)-HCA; the isolation, stereochemistry, properties, methods of estimation, and derivatives of (-)-HCA; and its biochemistry, which includes inhibition of the citrate cleavage enzyme, effects on fatty acid synthesis and lipogenesis, effects on ketogenesis, other biological effects, possible modes of action on the reduction of food intake, promotion of glycogenesis, gluconeogenesis, and lipid oxidation, (-)-HCA as weight-controlling agent, and some possible concerns about (-)-HCA, which provides a coherent presentation of scattered literature on (-)-HCA and its plausible mechanism of action and is provocative of further research.
Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid), a compound necessary for a transfer of fatty acids for their oxidation within the cell, accumulates in brain although beta-oxidation of fatty acids is very low in neurons. Carnitine accumulates to lower extent in the brain than in peripheral tissues and the mechanism of its transport through the blood-brain barrier is discussed, with the involvement of two transporters, OCTN2 and B(0,+) being presented. A limitation by the blood-brain barrier of carnitine supply for the brain and the mechanism of its transport to neural cells by a protein belonging to neurotransmitters' transporters superfamily is further discussed. Due to the beneficial effects of administration of acetylcarnitine in case of patients with dementia, the role of this acylcarnitine is presented in the context of neuronal cell metabolism and the role of acetylcarnitine in the synthesis of acetylcholine. The roles of long-chain acyl derivatives of carnitine, in particular palmitoylcarnitine, responsible for interaction with the membranes, lipids acylation and specific interactions with proteins have been summarized. Stimulation of protein palmitoylation and a possibility of changing the acylation status of G proteins is described, as well as interaction of palmitoylcarnitine with protein kinase C. Diminished interaction of the isoform delta of this kinase with GAP-43 (B-50, neuromodulin), whose expression increases upon accumulation of either carnitine or palmitoylcarnitine points to a possible regulation of differentiation by these compounds and their role in neuroregeneration.
Some of the kinetic properties of carnitine acetyltransferase in brain homogenates have been reported with the use of a new, sensitive radiometric method for the determination of acetylcarnitine. Those properties and the distribution pattern of carnitine acetyltransferase in various rat tissues, subcellular fractions, and discrete areas of the brain have been compared with those obtained for choline acetyltransferase and found to be markedly different. In subcellular fractions of brain, the carnitine enzyme remained associated with the mitochondrial fragments after “hypoosmotic” shock, whereas the choline enzyme showed enrichment in the “nerve ending particle” fraction. In developing brain cortex, the choline enzyme reaches adult levels of activity earlier than the carnitine enzyme. Thus, we have concluded that the two enzymes appear to be associated with different morphological structures. Further, the uniform distribution of carnitine acetyltransferase within the nervous system, as well as its association with the mitochondrial fraction, argues against the hypothesis that acetylcarnitine functions in brain as a neurohumoral agent. These data support the concept that carnitine acetyltransferase is involved in the transport of acyl groups across mitochondrial membranes.
—Cortex slices of rat brain were incubated with glucose mixed-labelled with 3H and 14C in the 6-position and the 3H/14C ratios of lactate, acetate, citrate and acetylcholine were determined. The values obtained were: lactate 0·95, acetate 0·85, citrate 0·65 and acetylcholine 0·67 when expressed in relation to a glucose 3H/14C ratio of 1·00. When brain slices were incubated with [2-14C, 2-3H]acetate in the presence of unlabelled glucose, labelled acetylcholine was formed with a 3H/14C ratio not significantly different from the labelled substrate. The results indicate that citrate is a precursor to the acetyl moiety of acetylcholine.
The subcellular localization of the AcCoA compartment supplying the cytoplasmic choline acetyltransferase (ChAc, EC 2.3.1.6) was investigated using a purified preparation of rat striatal synaptosomes (B fraction). It was first demonstrated that the SRA of the [14C]ACh synthesized during a 10 min incubation period was equal to the SRA of the [2-14C] and the [3-14C]pyruvate added to the isolated nerve terminal suspension. The experimental results can be summarised as follows: (i) No modification in the amount of [14C]ACh synthesized from [2-14C]pyruvatetion in the amount of [14C]ACh synthesized from [2-13C]pyruvate could be detected after the addition of high concentrations of either carnitine, acetylcarnitine or acetyl phosphate to the synaptosomal suspension. (ii) Under experimental conditions in which the amount of [1,5-14C]citrate taken up by passive diffusion into the cholinergic nerve endings would allow detection of the possible formation of the labelled ester, no [14C]ACh could be recovered. (iii) The SRA's of the individual carbon atoms of the Krebs cycle intermediary compounds when the cycle is fed with [2-14C] and [3-14C]pyruvate were calculated as a function of the STA's of each of these two precursors (a and a' respectively), of the number of 14CO2 dpm produced in the Krebs cycle from each of these two labelled compounds (D2 and D3 respectively), and as the function of the rate y of exchanges of molecules between the tricarboxylic acid cycle and other metabolic compartments. The experimental value obtained from a 10 min incubation, after the nerve endings had reached a steady metabolic activity, indicate that if the acetyl moiety of ACh was derived from some Krebs cycle intermediary compounds, its SRA could never exceed 55 per cent that of the [2-14C]pyruvate from which it is produced, (iv) No correlation could be found between the rate of [14C]ACh formation and changes in the Krebs cycle activity induced by sodium cyanide, 2-4 dinitrophenol and Ca2+ free medium. (v) The lack of significant [14C]ACh synthesis from [1-14C]acetate in striatal synaptosomes is consistent with the failure of fluoroacetate to modify the amounts of 14CO2 as well as of [14C]ACh formed from [2-14C]pyruvate. These results were interpreted as a confirmation of the presence of a low AcCoA synthetase activity in the nerve terminals. To reconcile all these data, it is proposed that pyruvate is transformed into AcCoA outside the mitochondria by the action of some cytoplasmic pyruvate dehydrogenase-like enzyme.
The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.
The effects of two citrate analogues, (—)hydroxycitrate and 1,2,3-tricarboxybenzene, were studied using rat liver enzymes which interact with citrate.
The most pronounced effect of 1,2,3-tricarboxybenzene was inhibition of acetyl-CoA carboxylase (Ki 20 μM). It also inhibited the mitochondrial citrate transporter (50% inhibition at 3 mM), but was not a substrate for this transporter.
ATP-citrate lyase was markedly inhibited by both free (—)hydroxycitrate (Ki 8 μM) and (—)hydroxycitrate lactone (Ki 50 to 100 μM). Acetyl-CoA carboxylase was activated by both the forms of (—)hydroxycitrate (Ka 0.7 mM and 1.6 mM, respectively). (—)Hydroxycitrate is a substrate for the mitochondrial citrate transporter, but its rate of transport is less than 10% of that of citrate.
Other citrate metabolizing enzymes also were inhibited by 1,2,3-tricarboxybenzene and (—)hydroxycitrate but much higher concentrations were required.
An enzymic procedure has been developed for the determination of micro quantities of tissue citrate. The reactions involve the cleavage of citrate by citrate lyase to oxalacetate and its subsequent oxidation of NADH by malic dehydrogenase. The NAD+ thus produced is cycled with lactate, α-ketoglutarate, and, ammonium ion, in the presence of the enzymes lactate dehydrogenase and glutamic dehydrogenase. Conversion of the cycling product (i.e., pyruvate) plus NADH to NAD+ and lactate leads to a strongly fluorescent substance when heated in strong alkali. The fluorescence thus produced from NAD+ can be equated by means of known standards, with the citrate originally present in tissue samples. Data obtained from several tissues are presented to demonstrate the potential applicability of the method to very small tissue samples and even to single cells.
— In any assay for the determination of acetylcholine based on the conversion of choline to a product, the immediate problem is the removal of endogenous choline. Other published enzymatic assays have taken advantage of electrophoresis to accomplish this goal. In the assay to be described, this is accomplished by the enzymatic phosphorylation of endogenous choline by choline kinase. Once this reaction is complete, endogenous acetylcholine is simultaneously hydrolysed and then phosphorylated with [32P]ATP. The labelled product [32P]phosphorylcholine is separated from the labelled substrate by precipitation of the ATP and further separation is accomplished on microcolumns of ion exchange resin. Using this methodology, picomole amounts of acetylcholine, derived from tissue, can be measured.
The origin of the acetyl group in acetyl‐CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate + cis ‐aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1‐ ¹⁴ C]acetate, [2‐ ¹⁴ C]acetate, [4‐ ¹⁴ C]acetoacetate, [1‐ ¹⁴ C]butyrate, [1, 5‐ ¹⁴ C]citrate, [2‐ ¹⁴ C]glucose, [5‐ ¹⁴ C]glutamate, 3‐hydroxy[3‐ ¹⁴ C]butyrate, [2‐ ¹⁴ C]lactate, [U‐ ¹⁴ C]leucine, [2‐ ¹⁴ C]pyruvate and [ ³ H]acetylaspartate.
The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2‐ ¹⁴ C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3‐hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5‐ ¹⁴ C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh.
The incorporation of label from [1, 5‐ ¹⁴ C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2‐ ¹⁴ C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1‐ ¹⁴ C]acetate into brain lipids and lowered its incorporation into ACh.
Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra‐arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1‐ ¹⁴ C]acetate, [2‐ ¹⁴ C]acetate, [1‐ ¹⁴ C]butyrate, [1,5‐ ¹⁴ C]citrate, [ ³ H]acetylaspartate, [U‐ ¹⁴ C]leucine, and also after [2‐ ¹⁴ C]pyruvate and [4‐ ¹⁴ C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine‐synthesizing (‘small’) compartment. Increasing the amount of injected [2‐ ¹⁴ C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio.
The incorporation of ¹⁴ C from [1‐ ¹⁴ C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3‐hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1‐ ¹⁴ C]acetate than after [2‐ ¹⁴ C]pyruvate.
The results indicate that the [1‐ ¹⁴ C]acetyl‐CoA arising from [1‐ ¹⁴ C]acetate does not enter the same pool as the [1‐ ¹⁴ C]acetyl‐CoA arising from [2‐ ¹⁴ C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate‐utilizing and glutamine‐synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1‐ ¹⁴ C]acetate was different from that after [1, 5‐ ¹⁴ C]citrate. This suggests that [1‐ ¹⁴ C]acetate and [1, 5‐ ¹⁴ C]citrate are utilized in different subdivisions of the ‘;small’ compartment.
1. Labelled precursors of choline, namely ethanolamine, dimethylaminoethanol and methionine and also labelled choline itself were injected intraperitoneally into the adult female rat and the incorporation into lipids and water-soluble fractions was traced in liver, blood and brain. 2. No significant free choline was detected and no labelling of the phosphorylcholine of blood. There was, however, considerable labelling of the phosphorylcholine of brain and liver. 3. After intracerebral injection, [1,2-(14)C]dimethylaminoethanol was rapidly phosphorylated and converted into phosphatidyldimethylaminoethanol, presumably by the cytidine pathway. 4. In view of the pattern of labelling and the amount of phosphatidylcholine in the tissues examined, it seems highly likely that choline is transported to the brain by the blood in a lipid-bound form.
Citrate cleavage enzyme shows atypical kinetics at low chloride concentrations, but normal kinetics at high chloride concentrations. Tricarballylate is a substrate for citrate cleavage enzyme. The reaction with tricarballylate can be demonstrated both by the disappearance of CoA and, in the presence of hydroxylamine, by the appearance of a hydroxamate. This indicates that tricarballylyl-CoA is formed in the reaction. When hydroxamate formation is used to assay activity, the apparent Km for tricarballylate is about three times greater than that for citrate, while the maximum reaction velocity with tricarballylate is about 90% of that observed with citrate. One of the stereoisomers of hydroxycitrate is a powerful inhibitor of citrate cleavage enzyme.
1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.
The role of ATP citrate lyase (ATP: citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephosphorylating), EC 4.1.3.8) in the transfer of acetyl groups out of the mitochondria was investigated in rat liver its anti-enzyme.With a reconstructed system from normal rat liver (fed state), containing both the supernatant and the mitochondria, treatment of the supernatant with the antiserum reduced the acetylation of either sulfanilamide or p-toluidine by pyruvate to about 15% of the obtained by treatment of the supernatant with control serum. These drops in the rate of acetylation were completely reversed by addition of excess purified ATP citrate lyase. With mitochondria alone, it was found that the acetylation of these compounds was parallel to the amount of ATP citrate lyase added. Similar degrees of inhibition of the acetylation of p-toluidine by the antibody were obtained with preparations from the livers of fasted or diabetic rats. In addition, neither (−)- nor (+)-carnitine affected the acetylation rate under any conditions. From these findings, it was concluded that more than 80% of the extramitochondrical acetyl-CoA, derived from pyruvate via the mitochondria, is supplied through the ATP citrate lyase pathway in rat liver, even in the fasted or diabetic state.
A technique has been described for the resolution of multiple overlapping chromatography peaks, eluted from Dowex-formate columns. The method is based upon systematic removal of interfering components by prior addition of appropriate enzymes. Details are presented for applying the method to specific activity measurements of four key glycolytic intermediates in brain.
An accurate and convenient method for determination of tritium water in
body fluids by liquid scintillation spectrometry is described. The method is
based on distillation of body fluids with benzene to provide an almost pure
sample of H/sup 3/-water, and use of an effective solvent (dioxanenaphthalene) to
transfer the excitation energy of tritium to the phosphors, 2,5-diphenyloxazole
and 1,4-bis-2 (5-phenyloxazolyl)-benzene. When applied to determination of
tritium water in urine and plasma, the method gave recoveries ranging from 98 to
104%. By the new method, metabolism of intravenously administered H/sup 3/- beta
-sitosterol to H/sup 3/-water was demonstrated in the guinea pig. (auth)
The role of central cholinergic synapses in excitable tissue is suggested from the results described. Six cholinolytic drugs, including Ditran (JB-329), are shown to block stimulated respiration of rat and guinea pig cerebral cortex (at concentrations 5 × 10−4 to 10−5 M) without demonstrable effect on unstimulated tissue. One of these drugs (JB-840) was studied for effects on electrically stimulated (10 to 12V, 120 pulses/sec) and potassium ion (105 mM) stimulated glycolysis and respiration which are selectively depressed at similar concentrations. In addition a simple device for sectioning brain tissue is described.
The Metabolic Roles of Citrate
- J. M. Lowenstein