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Objectives: Zinc excitotoxicity and thiamine pyrophosphate deficiency (TD) are known pathogenic signals contributing to mechanism of different encephalopathies through inhibition of enzymes responsible for energy metabolism such as pyruvate dehydrogenase, aconitase or ketoglutarate dehydrogenase. The aim of this work was to investigate whether subclinical Zn excess and TD, frequent in aging brain, may combine yielding overt neuronal impairment. Results: Clonal SN56 cholinergic neuronal cells of septal origin were used as the model of brain cholinergic neurons, which are particularly susceptible to neurodegeneration in the course of Alzheimer's disease, hypoxia and other dementia-linked brain pathologies. Neither subtoxic concentration of Zn (0.10 mM) nor mild 20-25% TD deficits alone caused significant negative changes in cultured cholinergic neurons viability and their acetyl-CoA/acetylcholine metabolism. However, cells with mild TD accumulated Zn in excess, which impaired their energy metabolism causing a loss of neurons viability and their function as neurotransmitters. These negative effects of Zn were aggravated by amprolium which is an inhibitor of thiamine intracellular transport. Conclusion: Our data indicate that TD may amplify otherwise non-harmful border-line Zn excitotoxic signals yielding progress of neurodegeneration.

Background: Hyperactivation of blood platelets is an essential factor in the pathomechanism of diabetes-evoked angiopathies. The aim of this work was to investigate whether blood platelets hyperactivation resulting from type 2 diabetic hyperglycaemia-increased pyruvate dehydrogenase complex activity and excessive acetyl-CoA accumulation may be brought to the normal range by the enzyme inhibitors., Methods: Platelets were isolated from the blood of 9 type 2 diabetic patients and 10 healthy donors. Effects of 3-bromopyruvate and 3-nitropropionate on pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase activities, as well as levels of acetyl-CoA, ATP, thiobarbituric acid reactive species and aggregation were assessed in non-activated and thrombin-activated platelets., Results: In type 2 diabetic patients fasting plasma glucose and fructosamine levels were 61 and 64% higher, respectively, than in the healthy group (p < 0.001). In non-activated diabetic platelets PDHC activity, PDHC-E2, acetyl-CoA and ATP levels were 66, 70, 68 and 60%, higher, respectively, than in platelets from healthy controls (p < 0.01). 3-bromopyruvate (0.1 mM) decreased pyruvate dehydrogenase activity in healthy and diabetic platelets by 42% and 59%, respectively. Similar inhibitory effects were observed for acetyl-CoA and ATP levels, aggregation and TBARS accumulation rates. Succinate dehydrogenase activity was inhibited by 3-nitropropionate (10 mM) to 38 and 41% of control values in healthy and diabetic platelets, respectively, but affected neither function nor acetyl-CoA metabolism in platelets of both groups., Conclusions: These data indicate that inhibition of pyruvate dehydrogenase excessive activity in diabetic platelets by 3-bromopyruvate may normalise their functional parameters through adjustment of acetyl-CoA/ATP levels to control values. Platelets from blood of diabetic patients display higher activities of pyruvate dehydrogenase complex (PDHC), higher levels of dihydrolipoate transacetylase (DLAT, E2 subunit of PDHC) as well as higher levels of acetyl-CoA yielding greater ATP/ADP accumulation than in platelets of normoglycemic subjects. Therefore, in diabetic platelets, thrombin caused higher release of ATP/ADP triggering excessive production of reactive oxygen species (ROS) and stronger aggregation compared to control platelets. In diabetic platelets, relative excess of DLAT in PDHC made them highly susceptible to 3-bromopyruvate (3BrP) inhibition. Resulting limitation of acetyl-CoA provision by 3-BrP normalised activity of diabetic platelets.

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well., Competing Interests: The authors have declared that no competing interests exist.

Brain neurons, to support their neurotransmitter functions, require a several times higher supply of glucose than non-excitable cells. Pyruvate, the end product of glycolysis, through pyruvate dehydrogenase complex reaction, is a principal source of acetyl-CoA, which is a direct energy substrate in all brain cells. Several neurodegenerative conditions result in the inhibition of pyruvate dehydrogenase and decrease of acetyl-CoA synthesis in mitochondria. This attenuates metabolic flux through TCA in the mitochondria, yielding energy deficits and inhibition of diverse synthetic acetylation reactions in all neuronal sub-compartments. The acetyl-CoA concentrations in neuronal mitochondrial and cytoplasmic compartments are in the range of 10 and 7 μmol/L, respectively. They appear to be from 2 to 20 times lower than acetyl-CoA Km values for carnitine acetyltransferase, acetyl-CoA carboxylase, aspartate acetyltransferase, choline acetyltransferase, sphingosine kinase 1 acetyltransferase, acetyl-CoA hydrolase, and acetyl-CoA acetyltransferase, respectively. Therefore, alterations in acetyl-CoA levels alone may significantly change the rates of metabolic fluxes through multiple acetylation reactions in brain cells in different physiologic and pathologic conditions. Such substrate-dependent alterations in cytoplasmic, endoplasmic reticulum or nuclear acetylations may directly affect ACh synthesis, protein acetylations, and gene expression. Thereby, acetyl-CoA may regulate the functional and adaptative properties of neuronal and non-neuronal brain cells. The excitotoxicity-evoked intracellular zinc excess hits several intracellular targets, yielding the collapse of energy balance and impairment of the functional and structural integrity of postsynaptic cholinergic neurons. Acute disruption of brain energy homeostasis activates slow accumulation of amyloid-β 1-42 (Aβ). Extra and intracellular oligomeric deposits of Aβ affect diverse transporting and signaling pathways in neuronal cells. It may combine with multiple neurotoxic signals, aggravating their detrimental effects on neuronal cells. This review presents evidences that changes of intraneuronal levels and compartmentation of acetyl-CoA may contribute significantly to neurotoxic pathomechanisms of different neurodegenerative brain disorders.

There are several systemic and intracerebral pathologic conditions, which limit provision and utilization of energy precursor metabolites in neuronal cells. Energy deficits cause excessive depolarization of neuronal cells triggering glutamate-zinc evoked excitotoxic cascade. The intracellular zinc excess hits several intraneuronal targets yielding collapse of energy balance and impairment functional and structural impairments cholinergic neurons. Disturbances in metabolism of acetyl-CoA, which is a direct precursor for energy, acetylcholine, N-acetyl-L-aspartate and acetylated proteins synthesis, play an important role in these pathomechanisms. Disruption of brain homeostasis activates slow accumulation of amyloid-β 1-42 , which extra and intracellular oligomeric deposits disrupt diverse transporting and signaling processes in all membrane structures of the cell. Both neurotoxic signals may combine aggravating detrimental effects on neuronal cell. Different neuroglial and neuronal cell types may display differential susceptibility to similar pathogenic insults depending on specific features of their energy and functional parameters. This review, basing on findings gained from cellular and animal models of Alzheimer's disease, discusses putative energy/acetyl-CoA dependent mechanism in early and late stages of neurodegeneration.

Adjuvant chemotherapy with 5-fluorouracil remains the basic treatment for patients with advanced colorectal carcinoma. The major obstacle in successful treatment is the ability of CRC cells to acquire chemoresistance. Here we examined the impact of ID1 silencing on the sensitivity of CRC cells to 5-FU. To suppress ID1 expression in HT-29 and HCT-116 cells the cells were transduced with a lentiviral vector carrying the ID1 silencing sequence. Cells with silenced ID1 showed altered expression of epithelial and mesenchymal markers and exhibited increased proliferation rate compared to the parental cells. HCT-116 cells with suppressed ID1 became sensitized to 5-FU and this was not observed in HT-29 cells. Silencing ID1 resulted in altered expression of genes encoding enzymes metabolizing 5-FU. HT-29 cells with suppressed ID1 had significantly reduced mRNA level for thymidine phosphorylase, uridine-cytydine kinase 2 and dihydropyrimidine dehydrogenase. ID1 suppression in HCT-116 cells resulted in an increase of mRNA level for thymidine phosphorylase, thymidine kinase and uridine-cytydine kinase 2 with concurrent drop of dihydropyrimidine dehydrogenase and thymidylate synthetase mRNA levels. In conclusion, ID1 expression impacts the sensitivity of colon cancer cells to 5-FU and may be considered as a potential predictive marker in CRC treatment.

Pyruvate dehydrogenase reaction utilizing glucose-derived pyruvate is an almost exclusive source of acetyl-CoA in different cell mitochondrial compartments of the brain. In neuronal mitochondria, the largest fraction of acetyl-CoA is utilized for energy production and the much smaller one for N-acetyl-L-aspartate (NAA) synthesis. Cholinergic neurons, unlike others, require additional amounts of acetyl-CoA for acetylcholine synthesis. Therefore, several neurotoxic signals, which inhibit pyruvate dehydrogenase, generate deeper shortages of acetyl-CoA and greater mortality of cholinergic neurons than noncholinergic ones. NAA is considered to be a marker of neuronal energy status in neuropathic brains. However, there is no data on putative differential fractional distribution of the acetyl-CoA pool between energy producing and NAA or acetylcholine synthesizing pathways in noncholinergic and cholinergic neurons, respectively. Therefore, the aim of this study was to investigate whether zinc-excess, a common excitotoxic signal, may evoke differential effects on the NAA metabolism in neuronal cells with low and high expression of the cholinergic phenotype. Differentiated SN56 neuronal cells, displaying a high activity of choline acetyltransferase and rates of acetylcholine synthesis, contained lower levels of acetyl-CoA and NAA, being more susceptible to ZnCl2 exposition that the nondifferentiated SN56 or differentiated dopaminergic SHSY5Y neuronal and astroglial C6 cells. Differentiated SN56 accumulated greater amounts of Zn2 + from extracellular space than the other ones, and displayed a stronger suppression of pyruvate dehydrogenase complex activity and acetyl-CoA, NAA, ATP, acetylcholine levels, and loss of viability. These data indicate that the acetyl-CoA synthesizing system in neurons constitutes functional unity with energy generating and NAA or acetylcholine pathways of its utilization, which are uniformly affected by neurotoxic conditions.

The 5-fluorouracil (5FU)-based adjuvant chemotherapy improves the survival of patients with colorectal cancer, however the main obstacle affecting its effectiveness is a drug resistance. Our study aimed to investigate the impact of TWIST1 silencing on the sensitivity of cancer cells to 5FU. The suppression of TWIST1 expression in human colon cancer HT29 and HCT116 cell lines was achieved by transduction with lentiviral vector carrying the TWIST1 silencing sequence (pLL3.7-shTWIST1). The suppression of TWIST1 expression induced changes in the expression pattern of epithelial to mesenchymal transition markers, reduced the cells proliferation rate, increased their sensitivity to serum withdrawn, and increased the cytotoxic effect of 5FU. However, significantly higher 5FU cytotoxicity was observed in HT29 cell cultures. Cells with silenced TWIST1 displayed altered expression of enzymes metabolizing 5FU. The expression level of dihydropyrimidine dehydrogenase, and thymidylate synthase decreased significantly in HT29 shTWIST1 cells, but not in HCT116 shTWIST1 cells. On the other hand, significant increases in the expression levels of thymidine phosphorylase, and uridine phosphorylase 1 were seen in both cell lines with suppressed expression of TWIST1. The changes in enzymes expression were mirrored by enzymatic activities. In conclusion, our observations point to TWIST1 as a target protein to enhance the sensitivity of colorectal cancer cells to 5FU., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that endogenously inhibited pyruvate dehydrogenase complex (PDHC), aconitase, and α-ketoglutarate dehydrogenase complex. However, it caused none or small suppressions of acetyl-CoA and microglial viability, respectively. Microglia-derived NO inhibited same enzymes in cholinergic neuronal cells causing marked viability loss because of acetyl-CoA deficits evoked by its competitive consumption by energy producing and acetylcholine/N-acetyl-l-aspartate (NAA) synthesizing pathways., (© 2014 International Society for Neurochemistry.)

Retinoic acid is a potent cell differentiating factor, which through its nuclear receptors affects a vast range of promoter sites in brain neuronal and glial cells in every step of embryonic and postnatal life. Its capacities, facilitating maturation of neurotransmitter phenotype in different groups of neurons, pave the way for its application as a potential therapeutic agent in neurodegenerative diseases including Alzheimer's disease. Retinoic acid was found to exert particularly strong enhancing effects on acetylcholine transmitter functions in brain cholinergic neurons, loss of which is tightly linked to the development of cognitive and memory deficits in course of different cholinergic encephalopathies. Here, we review cholinotrophic properties of retinoic acid and its derivatives, which may justify their application in the management of Alzheimer's disease and the related neurodegenerative conditions.

The pyruvate-derived acetyl-CoA is a principal direct precursor substrate for bulk energy synthesis in the brain. Deficits of pyruvate dehydrogenase in the neocortex are common features of Alzheimer's disease and other age-related encephalopathies in humans. Therefore, amyloid-β overload in brains of diverse transgenic Alzheimer's disease model animals was investigated as one of neurotoxic compounds responsible for pyruvate dehydrogenase inhibition yielding deficits of cholinergic neurotransmission and cognitive functions. Brains of aged, 14-16-month-old Tg2576 mice contained 0.6 μmol/kg levels of amyloid-β1 - 42. Activities of pyruvate dehydrogenase complex, choline acetyltransferase, and several enzymes of acetyl-CoA and energy metabolism were found to be unchanged in both forebrain mitochondria and synaptosomes of Tg2576 mice, indicating preservation of structural integrity at least in cholinergic neuronal cells. However, in transgenic brain synaptosomes, pyruvate utilization, mitochondrial levels, and cytoplasmic acetyl-CoA levels, as well as acetylcholine content and its quantal release, were all found to be decreased by 25-40% . On the contrary, activation of pyruvate utilization was detected and no alterations in acetyl-CoA content and citrate or α-ketoglutarate accumulation were observed in transgenic whole brain mitochondria. These data indicate that amyloid-β evoked deficits in acetyl-CoA are confined to mitochondrial and cytoplasmic compartments of Tg2576 nerve terminals, becoming early primary signals paving the path for further stages of neurodegeneration. On the other hand, acetyl-CoA synthesis in mitochondrial compartments of glial cells seems to be activated despite amyloid-β accumulated in transgenic brains.

The biological properties of vanadium complexes have become an object of interest due to their therapeutic potential in several diseases. However, the mechanisms of action of vanadium salts are still poorly understood. Vanadium complexes are cofactors for several enzymes and also exhibit insulin-mimetic properties. Thus, they are involved in the regulation of glucose metabolism, including in patients with diabetes. In addition, vanadium salts may also normalize blood pressure and play a key role in the metabolism of the thyroid and of iron as well as in the regulation of total cholesterol, cholesterol HDL and triglyceride (TG) levels in blood. Moreover, in cases of hypoxia, vanadium compounds may improve cardiomyocytes function. They may also exhibit both carcinogenic and anti-cancer properties. These include dose- and exposure-time-dependent induction and inhibition of the proliferation and survival of cancer cells. On the other hand, the balance between vanadium's therapeutic properties and its side effects has not yet been determined. Therefore, any studies on the potential use of vanadium compounds as supplements to support the treatment of a number of diseases must be strictly monitored for adverse effects.

Intramitochondrial decarboxylation of glucose-derived pyruvate by PDHC (pyruvate dehydrogenase complex) is a principal source of acetyl-CoA, for mitochondrial energy production and cytoplasmic synthetic pathways in all types of brain cells. The inhibition of PDHC, ACO (aconitase) and KDHC (ketoglutarate dehydrogenase complex) activities by neurodegenerative signals such as aluminium, zinc, amyloid β-peptide, excess nitric oxide (NO) or thiamine pyrophosphate deficits resulted in much deeper losses of viability, acetyl-CoA and ATP in differentiated cholinergic neuronal cells than in non-differentiated cholinergic, and cultured microglial or astroglial cell lines. In addition, in cholinergic cells, such conditions caused inhibition of ACh (acetylcholine) synthesis and its quantal release. Furthermore, cholinergic neuronal cells appeared to be resistant to high concentrations of LPS (lipopolysaccharide). In contrast, in microglial cells, low levels of LPS caused severalfold activation of NO, IL-6 (interleukin 6) and TNFα (tumour necrosis factor α) synthesis/release, accompanied by inhibition of PDHC, KDHC and ACO activities, and suppression of acetyl-CoA, but relatively small losses in their ATP contents and viability parameters. Compounds that protected these enzymes against inhibitory effects of neurotoxins alleviated acetyl-CoA and ATP deficits, thereby maintaining neuronal cell viability. These data indicate that preferential susceptibility of cholinergic neurons to neurodegenerative insults may result from competition for acetyl-CoA between mitochondrial energy-producing and cytoplasmic ACh-synthesizing pathways. Such a hypothesis is supported by the existence of highly significant correlations between mitochondrial/cytoplasmic acetyl-CoA levels and cell viability/transmitter functions respectively.

Glucose-derived pyruvate is a principal source of acetyl-CoA in all brain cells, through pyruvate dehydogenase complex (PDHC) reaction. Cholinergic neurons like neurons of other transmitter systems and glial cells, utilize acetyl-CoA for energy production in mitochondria and diverse synthetic pathways in their extramitochondrial compartments. However, cholinergic neurons require additional amounts of acetyl-CoA for acetylcholine synthesis in their cytoplasmic compartment to maintain their transmitter functions. Characteristic feature of several neurodegenerating diseases including Alzheimer's disease and thiamine diphosphate deficiency encephalopathy is the decrease of PDHC activity correlating with cholinergic deficits and losses of cognitive functions. Such conditions generate acetyl-CoA deficits that are deeper in cholinergic neurons than in noncholinergic neuronal and glial cells, due to its additional consumption in the transmitter synthesis. Therefore, any neuropathologic conditions are likely to be more harmful for the cholinergic neurons than for noncholinergic ones. For this reason attempts preserving proper supply of acetyl-CoA in the diseased brain, should attenuate high susceptibility of cholinergic neurons to diverse neurodegenerative conditions. This review describes how common neurodegenerative signals could induce deficts in cholinergic neurotransmission through suppression of acetyl-CoA metabolism in the cholinergic neurons.

Several pathologic conditions are known to cause thiamine deficiency, which induce energy shortages in all tissues, due to impairment of pyruvate decarboxylation. Brain is particularly susceptible to these conditions due to its high rate of glucose to pyruvate-driven energy metabolism. However, cellular compartmentalization of a key energy metabolite, acetyl-CoA, in this pathology remains unknown. Pyrithiamine-evoked thiamine deficiency caused no significant alteration in pyruvate dehydrogenase and 30% inhibition of α-ketoglutarate dehydrogenase activities in rat whole forebrain mitochondria. It also caused 50% reduction of the metabolic flux of pyruvate through pyruvate dehydrogenase, 78% inhibition of its flux through α-ketoglutarate dehydrogenase steps, and nearly 60% decrease of intramitochondrial acetyl-CoA content, irrespective of the metabolic state. State 3 caused a decrease in citrate and an increase in α-ketoglutarate accumulation. These alterations were more evident in thiamine-deficient mitochondria. Simultaneously thiamine deficiency caused no alteration of relative, state 3-induced increases in metabolic fluxes through pyruvate and α-ketoglutarate dehydrogenase steps. These data indicate that a shortage of acetyl-CoA in the mitochondrial compartment may be a primary signal inducing impairment of neuronal and glial cell functions and viability in the thiamine-deficient brain., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The decrease of pyruvate and ketoglutarate dehydrogenase complex activities is the main cause of energy and acetyl-CoA deficits in thiamine deficiency-evoked cholinergic encephalopathies. However, disturbances in pathways of acetyl-CoA metabolism leading to appearance of cholinergic deficits remain unknown. Therefore, the aim of this work was to investigate alterations in concentration and distribution of acetyl-CoA and in acetylcholine metabolism in brain nerve terminals, caused by thiamine deficits. They were induced by the pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase. The thiamine deficit reduced metabolic fluxes through pyruvate and ketoglutarate dehydrogenase steps, yielding deficits of acetyl-CoA in mitochondrial and cytoplasmic compartments of K-depolarized nerve terminals. It also inhibited indirect transport of acetyl-CoA though ATP-citrate lyase pathway being without effect on its direct Ca-dependent transport to synaptoplasm. Resulting suppression of synaptoplasmic acetyl-CoA correlated with inhibition of quantal acetylcholine release (r = 0.91, p = 0.012). On the other hand, thiamine deficiency activated non-quantal acetylcholine release that was independent of shifts in intraterminal distribution of acetyl-CoA. Choline acetyltransferase activity was not changed by these conditions. These data indicate that divergent alterations in the release of non-quantal and quantal acetylcholine pools from thiamine deficient nerve terminals could be caused by the inhibition of acetyl-CoA and citrate synthesis in their mitochondria. They in turn, caused inhibition of acetyl-CoA transport to the synaptoplasmic compartment through ATP-citrate lyase pathway yielding deficits of cholinergic functions., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

Excessive accumulation of zinc in the brain is one of putative factors involved in pathomechanism of cholinergic encephalopathies. The aim of this work was to investigate whether short-term increase of zinc concentration in the extracellular space may affect energy and acetylcholine metabolism in SN56 cholinergic cells of septal origin. Short 30 min exposition of SN56 cells to increasing zinc levels caused greater loss of viability of differentiated (DC, [EC(0.4)] 0.09 mM) than nondifferentiated cells (NC, [EC(0.4)] 0.14 mM). Concentration-dependent accumulation of zinc displayed exponential non-saturable kinetics. Zinc accumulation caused the decrease of calcium accumulation in mitochondria and its increase in cytoplasmic compartment of SN56 cells. Significant inverse and direct correlations were found between zinc accumulation and calcium levels in mitochondrial (r=-0.96, p=0.028) and cytoplasmic (r=0.97, p=0.028) compartments of DC, respectively. Zinc exerted similar inhibition of pyruvate dehydrogenase, aconitase and isocitrate dehydrogenase both in NC and DC homogenates, at Ki values equal to about 0.07, 0.08 and 0.005 mM, respectively. On the other hand, ketoglutarate dehydrogenase activity in DC was inhibited by zinc (Ki 0.0005 mM) 8 times stronger that in NC (Ki 0.004 mM). Also zinc-evoked decreases in acetylcholine content and its release were significantly greater in DC than in NC. Same conditions caused suppression of cytoplasmic and mitochondrial content of acetyl-CoA, that positively correlated with inhibition of transmitter functions (r=0.995, p=005) and loss of cell viability (r=0.990, p=0.0006), respectively. Significant correlations were also found in zinc-challenged cells between pyruvate dehydrogenase activity and both mitochondrial acetyl-CoA content and cell viability. These data indicate that pyruvate dehydrogenase-dependent acetyl-CoA synthesis in neuronal mitochondria may be a primary target for short-term neurotoxic effects of zinc. In consequence, shortages of acetyl-CoA in the mitochondrial compartment would cause fast loss of functional and structural integrity of cholinergic neurons., (Copyright 2009. Published by Elsevier Ltd.)

Age-dependent accumulation of lead in brain has been implicated in the pathomechanisms of Alzheimer's disease. The aim of this work was to investigate whether cholinotoxic effects of lead may result from alterations in acetyl-CoA metabolism. One day exposure of differentiated SN56 cholinergic neuroblastoma cells to 0.5 micromol/L lead or 0.01 mmol/L amyloid-beta1-42, increased fraction of nonviable cells to about 2%. Suppression of choline acetyltransferase activity occurred only in the presence of fresh amyloid-beta1-42, whereas lead was ineffective. All agents in combination caused suppression of acetyl-CoA in cytoplasm and mitochondria down to 19% and 34% of controls, respectively. Inverse correlation was observed between whole cell acetyl-CoA level and fraction of nonviable cells at different combinations of lead and other neurotoxic compounds. It indicates that lead had no primary suppressive effect on cholinergic phenotype but, at least in part, exerted cytotoxic influence on cholinergic neurons through the decrease of their acetyl-CoA.

Zinc is a trace element necessary for proper development and function of brain cells. However, excessive accumulation of zinc exerts several cytotoxic effects in the brain. The aim of this work was to see whether cytotoxic effects of zinc are quantitatively correlated with changes in acetyl-CoA metabolism. The zinc levels up to 0.20 mmol/L caused concentration-dependent inhibition of pyruvate dehydrogenase (PDH) activity that correlated with the increase in trypan blue-positive fraction and the decrease in cultured cell number (r = 0.96, p = 0.0001). Chronic exposure of cells to 0.15 mmol/L zinc decreased choline acetyltransferase and aconitase activities, cytoplasmic acetyl-CoA and whole cell ATP level by 38%, 57%, 35%, and 62%, respectively but caused no change in mitochondrial acetyl-CoA level and activities of other enzymes of glycolytic and tricarboxylic acid cycle. dl-alpha-lipoamide when added simultaneously with zinc to cultured cells or their homogenates attenuated its chronic or acute suppressive effects. In homogenates of chronically Zn-treated cells, lipoamide overcame PDH but not aconitase inhibition. Presented data indicate that acute-transient elevation of zinc caused reversible inhibition of PDH, aconitase activities and acetyl-CoA metabolism, which when prolonged could lead to irreversible enzyme inactivation yielding decrease in cell viability and secondary suppression of their cholinergic phenotype.

Increased activity of blood platelets contributes to vascular complications in patients with diabetes. The aim of this work was to investigate whether persisting hyperglycemia in diabetic patients generates excessive accumulation of ATP/ADP, which may underlie platelet hyperactivity. Platelet ATP and ADP levels, thiobarbituric acid-reactive species synthesis, and aggregation of platelets from patients with diabetes were 18-82% higher than in platelets from healthy participants. In patients with diabetes, platelet stimulation with thrombin caused about two times greater release of ATP and ADP than in the healthy group while decreasing intraplatelet nucleotide content to similar levels in both groups. This indicates that the increased content of adenylate nucleotides in the releasable pool in the platelets of diabetic patients does not affect their level in metabolic cytoplasmic/mitochondrial compartments. Significant correlations between platelet ATP levels and plasma fructosamine, as well as between platelet ATP/ADP and platelet activities, have been found in diabetic patients. In conclusion, chronic hyperglycemia-evoked elevations of ATP/ADP levels and release from blood platelets of patients with diabetes may be important factors underlying platelet hyperactivity in the course of the disease.