Product inhibition of mammalian thiamine pyrophosphokinase is an important mechanism for maintaining thiamine diphosphate homeostasis

peer reviewedBackground: Thiamine diphosphate (ThDP), an indispensable cofactor for oxidative energy metabolism, is syn- thesized through the reaction thiamine + ATP ⇆ ThDP + AMP, catalyzed by thiamine pyrophosphokinase 1 (TPK1), a cytosolic dimeric enzyme. It was claimed that the equilibrium of the reaction is in favor of the for- mation of thiamine and ATP, at odds with thermodynamic calculations. Here we show that this discrepancy is due to feedback inhibition by the product ThDP. Methods: We used a purified recombinant mouse TPK1 to study reaction kinetics in the forward (physiological) and for the first time also in the reverse direction. Results: Keq values reported previously are strongly underestimated, due to the fact the reaction in the forward direction rapidly slows down and reaches a pseudo-equilibrium as ThDP accumulates. We found that ThDP is a potent non-competitive inhibitor (Ki ≈ 0.4 μM) of the forward reaction. In the reverse direction, a true equi- librium is reached with a Keq of about 2 × 10− 5, strongly in favor of ThDP formation. In the reverse direction, we found a very low Km for ThDP (0.05 μM), in agreement with a tight binding of ThDP to the enzyme. General significance: Inhibition of TPK1 by ThDP explains why intracellular ThDP levels remain low after administration of even very high doses of thiamine. Understanding the consequences of this feedback inhibition is essential for developing reliable methods for measuring TPK activity in tissue extracts and for optimizing the therapeutic use of thiamine and its prodrugs with higher bioavailability under pathological conditions

Thiamine and lipophilic thiamine precursors protect against oxidative damage in cultured neuroblastoma cells

Recent evidence suggests that thiamine (vitamin B1) and some of its derivatives can exert prominent neuroprotective effects in the mammalian brain, particularly in mouse models of Alzheimer’s disease and tauopathies. As orally administered thiamine crosses intestinal and blood-brain barriers only slowly, precursors with higher bioavailability e.g. sulbutiamine, benfotiamine and dibenzoylthiamine, have been developed. We investigated the protective effects of thiamine and those precursors in neuroblastoma cells cultured in a medium containing minimal amounts of thiamine (10 nM). We induced oxidative stress by incubating the cells (24h) in the presence of the neurotoxic agent paraquat (0.25 mM). This treatment reduced cell viability by 40%. When thiamine or the precursors were present simultaneously, we observed protective effects by the precursors while free thiamine was ineffective.Dibenzoylthiamine was most efficient, affording complete protection of cells at 10-20 μM. It also caused the highest increase in intracellular thiamine, suggesting that the protection from oxidative damage is linked to increased levels of free thiamine (rather than thiamine diphosphate) in the neuroblastoma cells. These results and others from our laboratory raise the possibility that dibenzoylthiamine might useful as a neuroprotective agent in neurodegenerative disease

Neuroprotective Effects of Thiamine and Precursors with Higher Bioavailability: Focus on Benfotiamine and Dibenzoylthiamine

Thiamine (vitamin B1) is essential for brain function because of the coenzyme role of thiamine diphosphate (ThDP) in glucose and energy metabolism. In order to compensate thiamine deficiency, several thiamine precursors with higher bioavailability were developed since the 1950s. Among these, the thioester benfotiamine (BFT) has been extensively studied and has beneficial effects both in rodent models of neurodegeneration and in human clinical studies. BFT has antioxi- dant and anti-inflammatory properties that seem to be mediated by a mechanism independent of the coenzyme function of ThDP. BFT has no adverse effects and improves cognitive outcome in patients with mild Alzheimer’s disease (AD). Recent in vitro studies show that another thiamine thioester, dibenzoylthiamine (DBT) is even more efficient that BFT, especially with respect to its anti-inflammatory potency. Thiamine thioesters have pleiotropic properties linked to an increase in circulating thiamine concentrations and possibly in hitherto unidentified metabolites in particular open thiazole ring derivatives. The identification of the active neuroprotective derivatives and the clarification of their mechanism of action open extremely promising perspectives in the field of neurodegenerative, neurodevelopmental and psychiatric conditions

Dibenzoylthiamine, a lipophilic thiamine precursor, protects against oxidative damage in neuroblastoma cells

Recent evidence suggests that thiamine (vitamin B1) and some of its derivatives can exert prominent neuroprotective effects in the mammalian brain, particularly in mouse models of Alzheimer’s disease and tauopathies. As orally administered thiamine crosses intestinal and blood-brain barriers only slowly, precursors with higher bioavailability e.g. sulbutiamine, benfotiamine and dibenzoylthiamine, have been developed. We investigated the protective effects of thiamine and those precursors in neuroblastoma cells cultured in a medium containing minimal amounts of thiamine (10 nM), but sufficient to sustain normal growth. We induced oxidative stress by incubating the cells (24 h) in the presence of the neurotoxic agent paraquat (0.25 mM). This treatment reduced cell viability by 40%. When thiamine or the precursors were present simultaneously, we observed protective effects by the precursors while free thiamine was ineffective. Dibenzoylthiamine was most efficient, affording complete protection of cells at 10-20 µM. It also caused the highest increase in intracellular thiamine, suggesting that the protection from oxidative damage is linked to increased levels of free thiamine (rather than thiamine disphophate) in the neuroblastoma cells. The mechanism of this protective effect is presently under investigation. These results and others from our laboratory raise the possibility that dibenzoylthiamine might useful as a neuroprotective agent in neurodegenerative disease

Thiamine deficiency in rats affects thiamine metabolism possibly through the formation of oxidized thiamine pyrophosphate

Background: Thiamine deficiency (TD) has a number of features in common with the neurodegenerative diseases development and close relationship between TD and oxidative stress (OS) has been repeatedly reported in the literature. The aim of this study is to understand how alimentary TD, accompanied by OS, affects the expression and level of two thiamine metabolism proteins in rat brain, namely, thiamine transporter 1 (THTR1) and thiamine pyrophosphokinase (TPK1), and what factors are responsible for the observed changes. Methods: The effects of OS caused by TD on the THTR1and TPK1 expression in rat cortex, cerebellum and hippocampus were examined. The levels of active and oxidized forms of ThDP (enzymatically measured) in the blood and brain, ROS and SH-groups in the brain were also analyzed. Results: TD increased the expression of THTR1 and protein level in all studied regions. In contrast, expression of TPK1 was depressed. TD-induced OS led to the accumulation of ThDP oxidized inactive form (ThDPox) in the blood and brain. In vitro reduction of ThDPox by dithiothreitol regenerates active ThDP suggesting that ThDPox is in disulfide form. A single high-dose thiamine administration to TD animals had no effect on THTR1 expression, partly raised TPK1 mRNA and protein levels, but is unable to normalize TPK1 enzyme activity. Brain and blood ThDP levels were increased in these conditions, but ThDPox was not decreased. General significance: It is likely, that the accumulation of ThDPox in tissue could be seen as a potential marker of neurocellular dysfunction and thiamine metabolic state

Thiamine and benfotiamine protect neuroblastoma cells against paraquat and ß- amyloid toxicity by a coenzyme-independent mechanism

Background : Benfotiamine (BFT) is a synthetic thiamine precursor with high bioavailability. It is efficient in treating complications of type 2 diabetes and has beneficial effects in mouse models of neurodegenerative diseases. The mechanism of action of BFT remains unknown, though it is sometimes suggested that it may be linked to increased thiamine diphosphate (ThDP) coenzyme function. Methods : We used a mouse neuroblastoma cell line (Neuro2a) grown in thiamine-restricted medium. The cells were stressed by exposure to paraquat (PQ) or amyloid 1-42 peptide in the presence or absence of BFT and the cell survival was measured using the MTT method. In each case, BFT was compared with sulbutiamine (SuBT), an unrelated thiamine precursor, and thiamine. Metabolites of BFT were determined by HPLC and mass spectrometry. Results : At 50 μM, BFT protects the cells against PQ and amyloid 1-42 peptide-induced toxicity with the same efficacy. Protective effects were also observed with SuBT and with higher concentrations of thiamine. The main metabolites of BFT were thiamine and S-benzoylthiamine (S-BT). Treatment with both precursors induces a strong increase in intracellular content of thiamine. Protective effects of BFT and SuBT are directly related to thiamine (but not ThDP) levels in Neuro2a cells. Conclusions : BFT, SuBT and thiamine all protect the cells against oxidative stress, suggesting an antioxidant effect of thiamine. Our results are not in favor of a direct ROS scavenging effect of thiamine but rather an indirect effect possibly mediated by some antioxidant signaling pathway. It is however not clear whether this effect is due to thiamine itself, its thiol form or an unknown metabolite. General significance : Our results suggest a role of thiamine in protection against oxidative stress, independent of the coenzyme function of thiamine diphosphate

Dibenzoylthiamine has powerful antioxidant and anti-inflammatory properties in cultured cells and in mouse models of stress and neurodegeneration

Thiamine precursors, the most studied being benfotiamine (BFT), have protective effects in mouse models of neurodegenerative diseases. BFT decreased oxidative stress and inflammation, two major characteristics of neurodegenerative diseases, in a neuroblastoma cell line (Neuro2a) and an immortalized brain microglial cell line (BV2). Here, we tested the potential antioxidant and anti-inflammatory effects of the hitherto unexplored derivative O,S-dibenzoylthiamine (DBT) in these two cell lines. We show that DBT protects Neuro2a cells against paraquat (PQ) toxicity by counteracting oxidative stress at low concentrations and increases the synthesis of reduced glutathione and NADPH in a Nrf2-independent manner. In BV2 cells activated by lipopolysaccharides (LPS), DBT significantly decreased inflammation by suppressing translocation of NF-κB to the nucleus. Our results also demonstrate the superiority of DBT over thiamine and other thiamine precursors, including BFT, in all of the in vitro models. Finally, we show that the chronic administration of DBT arrested motor dysfunction in FUS transgenic mice, a model of amyotrophic lateral sclerosis, and it reduced depressive-like behavior in a mouse model of ultrasound-induced stress in which it normalized oxidative stress marker levels in the brain. Together, our data suggest that DBT may have therapeutic potential for brain pathology associated with oxidative stress and inflammation by novel, coenzyme-independent mechanisms.“5–100” Russian Research Excellence Progra

Thiamine and benfotiamine counteract stress-induced aggression, normalize AMPA receptor expression and plasticity markers, and reduce oxidative stress in mice

The negative societal impacts associated with the increasing prevalence of violence and aggression is increasing, and, with this rise, is the need to understand the molecular and cellular changes that underpin ultrasound-induced aggressive behavior. In mice, stress-induced aggression is known to alter AMPA receptor subunit expression, plasticity markers, and oxidative stress within the brain. Here, we induced aggression in BALB/c mice using chronic ultrasound exposure and examined the impact of the psychoactive anti-oxidant compounds thiamine (vitamin B1), and its derivative benfotiamine, on AMPA receptor subunit expression, established plasticity markers, and oxidative stress. The administration of thiamine or benfotiamine (200 mg/kg/day) in drinking water decreased aggressive behavior following 3-weeks of ultrasound exposure and benfotiamine, reduced floating behavior in the swim test. The vehicle-treated ultrasound-exposed mice exhibited increases in protein carbonyl and total glutathione, altered AMPA receptor subunits expression, and decreased expression of plasticity markers. These ultrasound-induced effects were ameliorated by thiamine and benfotiamine treatment; in particular both antioxidants were able to reverse ultrasound-induced changes in GluA1 and GluA2 subunit expression, and, within the prefrontal cortex, significantly reversed the changes in protein carbonyl and polysialylated form of neural cell adhesion molecule (PSA-NCAM) expression levels. Benfotiamine was usually more efficacious than thiamine. Thus, the thiamine compounds were able to counteract ultrasound-induced aggression, which was accompanied by the normalization of markers that have been showed to be associated with ultrasound-induced aggression. These commonly used, orally-active compounds may have considerable potential for use in the control of aggression within the community

Thiamine and benfotiamine prevent stress-induced suppression of hippocampal neurogenesis in mice exposed to predation without affecting brain thiamine diphosphate levels

Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200 mg/kg/day in drinking water for 20 days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3β (GSK-3β) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent stress-induced inhibition of hippocampal neurogenesis and accompanying physiological changes. The present data suggest that thiamine precursors with high bioavailability might be useful as a complementary therapy in several neuropsychiatric disorders.“5-100” Russian Research Excellence progra

Loss of Notch signaling in skeletal stem cells enhances bone formation with aging

Abstract Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton