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Thiamine (vitamin B1) is a precursor of the well-known coenzyme of central metabolic pathways thiamine diphosphate (ThDP). Highly intense glucose oxidation in the brain requires ThDP-dependent enzymes, which determines the critical significance of thiamine for neuronal functions. However, thiamine can also act through the non-coenzyme mechanisms. The well-known facilitation of acetylcholinergic neurotransmission upon the thiamine and acetylcholine co-release into the synaptic cleft has been supported by the discovery of thiamine triphosphate (ThTP)-dependent phosphorylation of the acetylcholine receptor-associated protein rapsyn, and thiamine interaction with the TAS2R1 receptor, resulting in the activation of synaptic ion currents. The non-coenzyme regulatory binding of thiamine compounds has been demonstrated for the transcriptional regulator p53, poly(ADP-ribose) polymerase, prion protein PRNP, and a number of key metabolic enzymes that do not use ThDP as a coenzyme. The accumulated data indicate that the molecular mechanisms of the neurotropic action of thiamine are far broader than it has been originally believed, and closely linked to the metabolism of thiamine and its derivatives in animals. The significance of this topic has been illustrated by the recently established competition between thiamine and the antidiabetic drug metformin for common transporters, which can be the reason for the thiamine deficiency underlying metformin side effects. Here, we also discuss the medical implications of the research on thiamine, including the role of thiaminases in thiamine reutilization and biosynthesis of thiamine antagonists; molecular mechanisms of action of natural and synthetic thiamine antagonists, and biotransformation of pharmacological forms of thiamine. Given the wide medical application of thiamine and its synthetic forms, these aspects are of high importance for medicine and pharmacology, including the therapy of neurodegenerative diseases.

Thiamine is a crucial cofactor involved in the maintenance of carbohydrate metabolism and participates in multiple cellular metabolic processes within the cytosol, mitochondria, and peroxisomes. Currently, four genetic defects have been described causing impairment of thiamine transport and metabolism: SLC19A2 dysfunction leads to diabetes mellitus, megaloblastic anemia and sensory-neural hearing loss, whereas SLC19A3, SLC25A19, and TPK1-related disorders result in recurrent encephalopathy, basal ganglia necrosis, generalized dystonia, severe disability, and early death. In order to achieve early diagnosis and treatment, biomarkers play an important role. SLC19A3 patients present a profound decrease of free-thiamine in cerebrospinal fluid (CSF) and fibroblasts. TPK1 patients show decreased concentrations of thiamine pyrophosphate in blood and muscle. Thiamine supplementation has been shown to improve diabetes and anemia control in Rogers' syndrome patients due to SLC19A2 deficiency. In a significant number of patients with SLC19A3, thiamine improves clinical outcome and survival, and prevents further metabolic crisis. In SLC25A19 and TPK1 defects, thiamine has also led to clinical stabilization in single cases. Moreover, thiamine supplementation leads to normal concentrations of free-thiamine in the CSF of SLC19A3 patients. Herein, we present a literature review of the current knowledge of the disease including related clinical phenotypes, treatment approaches, update of pathogenic variants, as well as in vitro and in vivo functional models that provide pathogenic evidence and propose mechanisms for thiamine deficiency in humans.

Thiamine helps transketolase in removing toxic metabolites, counteracting high glucose-induced damage in microvascular cells, and progression of diabetic retinopathy/nephropathy in diabetic animals. Diabetic subjects show reduced thiamine levels. Hyperglycemia and reduced thiamine availability concur in impairing thiamine transport inside the blood-retinal barrier, with thiamine transporter-2 (THTR2) primarily involved. Here, we examined the behavior of thiamine transporter-1 (THTR1), THTR2, and their transcription factor Sp1 in response to high glucose and altered thiamine availability in renal cells involved in diabetic nephropathy. Human proximal tubule epithelial cells, podocytes, glomerular endothelial, and mesangial cells were exposed to high glucose and/or thiamine deficiency/oversupplementation. Localization and modulation of THTR1, THTR2, and Sp1, intracellular thiamine, transketolase activity, and permeability to thiamine were examined. Reduced thiamine availability and hyperglycemia impaired thiamine transport and THTR2/Sp1 expression. Intracellular thiamine, transketolase activity, and permeability were strongly dependent on thiamine concentrations and, partly, excess glucose. Glomerular endothelial cells were the most affected by the microenvironmental conditions. Our results confirmed the primary role of THTR2 in altered thiamine transport in cells involved in diabetic microvascular complications. Lack of thiamine concurs with hyperglycemia in impairing thiamine transport. Thiamine supplementation could represent a therapeutic option to prevent or slow the progression of these complications.

A rare cause of megaloblastic anemia (MA) is thiamine-responsive megaloblastic anemia (TRMA), a genetic disorder caused by mutations in SLC19A2 (encoding THTR1), a thiamine transporter. The study objectives were to (1) functionally characterize selected TRMA-associated SLC19A2 variants and (2) determine whether current prescription drugs associated with drug-induced MA (DIMA) may act via inhibition of SLC19A2. Functional characterization of selected SLC19A2 variants was performed by confocal microscopy and isotopic uptake studies of [3H]-thiamine in HEK293 cells. Sixty-three drugs associated with DIMA were screened for SLC19A2 inhibition in isotopic uptake studies. Three previously uncharacterized SLC19A2 variants identified in TRMA patients exhibited disrupted localization to the plasma membrane along with near-complete loss-of-function. Ten of 63 drugs inhibited SLC19A2-mediated thiamine transport ≥ 50% at screening concentrations; however, with the exception of erythromycin, none was predicted to inhibit SLC19A2 at clinically relevant unbound plasma concentrations. Data from electronic health records revealed reduced levels of thiamine pyrophosphate (TPP) in patients prescribed erythromycin, consistent with inhibition of SLC19A2-mediated thiamine transport. Here, we confirmed the role of three SLC19A2 variants in TRMA pathology. Additionally, we report that inhibition of SLC19A2 is a potential, but uncommon mechanism for DIMA. Supplementary Information The online version contains supplementary material available at 10.1208/s12248-021-00562-4.

International audience; Introduction: Janus kinase inhibitors (JAKinibs) constitute an emerging and promising pharmacological class of anti-inflammatory or anti-cancer drugs, used notably for the treatment of rheumatoid arthritis and some myeloproliferative neoplasms. Areas covered: This review provides an overview of the interactions between marketed JAKinibs and major uptake and efflux drug transporters. Consequences regarding pharmacokinetics, drug-drug interactions and toxicity are summarized. Expert opinion: JAKinibs interact in vitro with transporters in various ways, as inhibitors or as substrates of transporters or as regulators of transporter expression. This may theoretically result in drug-drug interactions (DDIs), with JAKinibs acting as perpetrators or as victims, or in toxicity, via impairment of thiamine transport. Clinical significance in terms of DDIs for JAKinib-transporter interactions remains however poorly documented. In this context, the in vivo unbound concentration of JAKinibs is likely a key parameter to consider for evaluating the clinical relevance of JAKinibs-mediated transporter inhibition. Additionally, the interplay with drug metabolism as well as possible interactions with transporters of emerging importance and time-dependent inhibition have to be taken into account. The role drug transporters may play in controlling cellular JAKinib concentrations and efficacy in target cells is also an issue of interest.

Supplemental levels of vitamin B1 (thiamine) have been implicated in tumor progression. Tumor cells adaptively up-regulate thiamine transport during hypoxic stress. Upon uptake, thiamine pyrophosphokinase-1 (TPK1) facilitates the rapid phosphorylation of thiamine into thiamine pyrophosphate (TPP). However, the regulation of TPK1 during hypoxic stress is undefined. Understanding how thiamine homeostasis changes during hypoxia will provide critical insight into the malignant advantage supplemental thiamine may provide cancer cells. Using Western blot analysis and RT-PCR, we have demonstrated the post-transcriptional up-regulation of TPK1 in cancer cells following hypoxic exposure. TPK1 expression was also adaptively up-regulated following alterations of redox status by chemotherapeutic and antioxidant treatments. Although TPK1 was functionally up-regulated by hypoxia, HPLC analysis revealed a reduction in intracellular TPP levels. This loss was reversed by treatment with cell-permeable antioxidants and corresponded with reduced ROS production and enhanced cellular proliferation during supplemental thiamine conditions. siRNA-mediated knockdown of TPK1 directly enhanced basal ROS levels and reduced tumor cell proliferation. These findings suggest that the adaptive regulation of TPK1 may be an essential component in the cellular response to oxidative stress, and that during supplemental thiamine conditions its expression may be exploited by tumor cells for a redox advantage contributing to tumor progression.

The bacterial pyridine nucleotide uptake family of transporters mediates the uptake of B-type vitamins, but their transport mechanism is unknown. Jaehme et al. show that the PnuT thiamine transporter utilizes a facilitated diffusion mechanism and supports metabolic trapping of phosphorylated thiamine., Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.

Thiamine (vitamin B1) and its phosphorylated precursors are necessary for decarboxylation reactions required in carbohydrate and branched chain amino acid metabolism. Due to its critical roles in central metabolism, thiamine is essential for human and animal hosts and their resident gut microbes. However, little is known about how thiamine availability shapes the composition of gut microbial communities and the physiology of individual species within those communities. Our previous work has implicated both thiamine biosynthesis and transport activities in the fitness ofBacteroidesspecies. To better understand thiamine-dependent gene regulation inBacteroides, we examined thiamine biosynthesis and transport genes in three representative species:Bacteroides thetaiotaomicron, Bacteroides uniformis, andBacteroides vulgatus. All three species possess thiamine biosynthetic operons controlled by highly conservedcis-acting thiamine pyrophosphate (TPP) riboswitches.B. thetaiotaomicronandB. uniformishave additional TPP riboswitch-controlled operons encoding thiamine transport functions. Transcriptome analyses showed that eachBacteroidesspecies had a distinct transcriptional response to exogenous thiamine. Analysis of transcript levels and translational fusions demonstrated that inB. thetaiotaomicron, the TPP riboswitch upstream of biosynthesis genes acts at the level of transcription, while TPP riboswitches upstream of transport operons work at the level of translation. InB. uniformisandB. vulgatus, TPP riboswitches work at the transcriptional level to control downstream operons. The varying responses to exogenous thiamine and use of varied regulatory mechanisms may play an important role in niche establishment by the Bacteroidetes in the complex and constantly shifting gut environment.ImportanceBacteroidesspecies are important and abundant members of human gut microbiome communities. Their activities in the gut are influenced by constant changes in nutrient availability. In this study, we investigated the genetic basis of thiamine (Vitamin B1) uptake and biosynthesis in three representativeBacteroidesspecies. We found species-specific differences in the response to exogenous thiamine, and distinct mechanisms for regulation of uptake and biosynthesis gene expression. Our work implies that gutBacteroideshave evolved distinct strategies for making or acquiring an essential nutrient. These mechanisms may play an important role in the success ofBacteroidesin establishing a niche within complex gut microbiome communities.

Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1-dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high-dose and low-dose (nutritional) intake, and heritability of thiamine and thiamine-phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR-2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research. peerReviewed

Energy-coupling factor (ECF) transporters are involved in the uptake of micronutrients in bacteria. The transporters capture the substrate by high-affinity binding proteins, the so-called S-components. Here, we present the analysis of two regions of the substrate-binding pocket of the thiamine-specific S-component in Lactococcus lactis, ThiT. First, interaction of the thiazolium ring of thiamine with residues Trp34, His125 and Glu84 by π-π-stacking and cation-π is studied, and second, the part of the binding pocket that extends from the hydroxyl group. We mutated either the transported ligand (chemically) or the protein (genetically). Surprisingly, modifications in the thiazolium ring by introducing substituents with opposite electronic effects had similar effects on the binding affinity. We hypothesize that the electronic effects are superseeded by steric effects of the added substituents, which renders the study of isolated interactions difficult. Amino acid substitutions in ThiT indicate that the electrostatic interaction facilitated by residue Glu84 of ThiT and thiamine is necessary for picomolar affinity. Deazathiamine derivatives that explore the subpocket of the binding site extending from the hydroxyl group of thiamine bind with high affinity to ThiT and may be developed into selective inhibitors of thiamine transport by ECF transporters. Molecular-dynamics simulations suggest that two of these derivatives may not only bind to ThiT, but could also be transported.

While the pathogenic role of dicarbonyl stress and accelerated formation of advanced glycation end products (AGEs) to glucose intolerance and to the development of diabetic complications is well established, little is known about these processes in gestational diabetes mellitus (GDM), a condition pathogenically quite similar to type 2 diabetes. The aims of the present study were (i) to determine plasma thiamine and erythrocyte thiamine diphosphate (TDP) and transketolase (TKT) activity in pregnant women with and without GDM, (ii) to assess relationships between thiamine metabolism parameters and selected clinical, biochemical and anthropometric characteristics and, finally, (iii) to analyse relationship between variability in the genes involved in the regulation of transmembrane thiamine transport (i.e. SLC19A2 and SLC19A3) and relevant parameters of thiamine metabolism. We found significantly lower plasma BMI adjusted thiamine in women with GDM (P = 0.002, Mann-Whitney) while levels of erythrocyte TDP (an active TKT cofactor) in mid-trimester were significantly higher in GDM compared to controls (P = 0.04, Mann-Whitney). However, mid-gestational TKT activity - reflecting pentose phosphate pathway activity - did not differ between the two groups (P > 0.05, Mann-Whitney). Furthermore, we ascertained significant associations of postpartum TKT activity with SNPs SLC19A2 rs6656822 and SLC19A3 rs7567984 (P = 0.03 and P = 0.007, resp., Kruskal-Wallis). Our findings of increased thiamine delivery to the cells without concomitant increase of TKT activity in women with GDM therefore indicate possible pathogenic role of thiamine mishandling in GDM. Further studies are needed to determine its contribution to maternal and/or neonatal morbidity.

Variation in the ability of gut microbes to transport, synthesize, and compete for vitamin B1 (thiamine) is expected to impact the structure and stability of the microbiota, and ultimately this variation may have both direct and indirect effects on human health. Our study identifies the diverse strategies employed by gut Bacteroidetes to acquire thiamine. We demonstrate how the presence or absence of thiamine biosynthesis or transport dramatically affects the abundance of B. thetaiotaomicron in a competitive environment. This study adds further evidence that altering the presence or concentrations of water-soluble vitamins such as thiamine may be an effective method for manipulating gut community composition. In turn, targeted thiamine delivery could be used therapeutically to alter dysbiotic communities linked to disease., Thiamine (vitamin B1) is an essential cofactor for all organisms. Humans primarily acquire thiamine through their diet, and thiamine deficiencies have adverse neurological effects. However, the role gut microbes play in modulating thiamine availability is poorly understood, and little is known about how thiamine impacts the stability of microbial gut communities. To investigate thiamine’s role in the gut, we utilized the model gut microbe Bacteroides thetaiotaomicron. Transcriptome sequencing (RNA-seq) revealed a global downregulation of thiamine and amino acid biosynthesis, glycolysis, and purine metabolism when thiamine was present. Using genetic mutants with thiamine biosynthesis and transport locus mutations, we determined both systems were critical for growth in thiamine-deficient medium. The defect in the double transport mutant suggests an uncharacterized feedback mechanism between thiamine transport and biosynthesis in B. thetaiotaomicron. Mutant phenotypes were recapitulated during pairwise competitions, reinforcing the importance of encoding versatile thiamine acquisition mechanisms when thiamine concentrations are variable. In addition, liquid chromatography-mass spectrometry (LC-MS) analyses corroborate that exogenous thiamine levels affect the internal thiamine pool of B. thetaiotaomicron. Furthermore, we computationally examined the ability of other gut microbes to acquire thiamine and identified lineage-specific differences in thiamine acquisition strategies. Among the Bacteroidetes, the capacities for both thiamine transport and biosynthesis are common. Together, these data show that thiamine acquisition mechanisms used by B. thetaiotaomicron not only are critical for its physiology and fitness but also provide the opportunity to model how other gut microbes may respond to the shifting availability of thiamine in the gut. IMPORTANCE Variation in the ability of gut microbes to transport, synthesize, and compete for vitamin B1 (thiamine) is expected to impact the structure and stability of the microbiota, and ultimately this variation may have both direct and indirect effects on human health. Our study identifies the diverse strategies employed by gut Bacteroidetes to acquire thiamine. We demonstrate how the presence or absence of thiamine biosynthesis or transport dramatically affects the abundance of B. thetaiotaomicron in a competitive environment. This study adds further evidence that altering the presence or concentrations of water-soluble vitamins such as thiamine may be an effective method for manipulating gut community composition. In turn, targeted thiamine delivery could be used therapeutically to alter dysbiotic communities linked to disease. Author Video: An author video summary of this article is available.

Thiamine plays a very important coenzymatic and non-coenzymatic role in the regulation of basic metabolism. Thiamine diphosphate is a coenzyme of many enzymes, most of which occur in prokaryotes. Pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes as well as transketolase are the examples of thiamine-dependent enzymes present in eukaryotes, including human. Therefore, thiamine is considered as drug or diet supplement which can support the treatment of many pathologies including neurodegenerative and vascular system diseases. On the other hand, thiamine antivitamins, which can interact with thiamine-dependent enzymes impeding their native functions, thiamine transport into the cells or a thiamine diphosphate synthesis, are good propose to drug design. The development of organic chemistry in the last century allowed the synthesis of various thiamine antimetabolites such as amprolium, pyrithiamine, oxythiamine, or 3-deazathiamine. Results of biochemical and theoretical chemistry research show that affinity to thiamine diphosphate-dependent enzymes of these synthetic molecules exceeds the affinity of native coenzyme. Therefore, some of them have already been used in the treatment of coccidiosis (amprolium), other are extensively studied as cytostatics in the treatment of cancer or fungal infections (oxythiamine and pyrithiamine). This review summarizes the current knowledge concerning the synthesis and mechanisms of action of selected thiamine antivitamins and indicates the potential of their practical use.

Wernicke's encephalopathy, a common neurological disease, is caused by thiamine (vitamin B1) deficiency. Neuropathy resulting from thiamine deficiency is a hallmark of Wernicke-Korsakoff syndrome in chronic alcohol users. The underlying mechanisms of this deficiency and progression of neuropathy remain to be understood. To uncover the unknown mechanisms of thiamine deficiency in alcohol abuse, we used chronic alcohol consumption or thiamine deficiency diet ingestion in animal models. Observations from animal models were validated in primary human neuronal culture for neurodegenerative process. We employed radio-labeled bio-distribution of thiamine, qualitative and quantitative analyses of the various biomarkers and neurodegenerative process. In the present studies, we established that disruption of thiamine transport across the intestinal gut blood-brain barrier axis as the cause of thiamine deficiency in the brain for neurodegeneration. We found that reduction in thiamine transport across these interfaces was the cause of reduction in the synthesis of thiamine pyrophosphate (TPP), an active cofactor for pyruvate dehydrogenase E1α (PDHE1α). Our findings revealed that decrease in the levels of PDHE1α cofactors switched on the activation of PD kinase (PDK) in the brain, thereby triggering the neuronal phosphorylation of PDHE1α (p-PDHE1α). Dysfunctional phosphorylated PDHE1α causes the reduction of mitochondrial aerobic respiration that led to neurodegeneration. We concluded that impairment of thiamine transport across the gut-BBB-axis that led to insufficient TPP synthesis was critical to Wernicke-neuropathy, which could be effectively prevented by stabilizing the thiamine transporters.

Alaskan Husky encephalopathy (AHE(1) ) is a fatal brain disease associated with a mutation in SLC19A3.1 (c.624insTTGC, c.625C>A). This gene encodes for a thiamine transporter 2 with a predominately (CNS) central nervous system distribution. Considering that brain is particularly vulnerable to thiamine deficiency because of its reliance on thiamine pyrophosphate (TPP)-dependent metabolic pathways involved in energy metabolism and neurotransmitter synthesis, we characterized the impact of this mutation on thiamine status, brain bioenergetics and the contribution of oxidative stress to this phenotype. In silico modeling of the mutated transporter indicated a significant loss of alpha-helices resulting in a more open protein structure suggesting an impaired thiamine transport ability. The cerebral cortex and thalamus of affected dogs were severely deficient in TPP-dependent enzymes accompanied by decreases in mitochondrial mass and oxidative phosphorylation (OXPHOS) capacity, and increases in oxidative stress. These results along with the behavioral and pathological findings indicate that the phenotype associated with AHE is consistent with a brain-specific thiamine deficiency, leading to brain mitochondrial dysfunction and increased oxidative stress. While some of the biochemical deficits, neurobehavior and affected brain areas in AHE were shared by Wernicke's and Korsakoff's syndromes, several differences were noted likely arising from a tissue-specific vs. that from a whole-body thiamine deficiency.

Inhibition of thiamine transporters has been proposed as a putative mechanism for the observation of Wernicke's encephalopathy and subsequent termination of clinical development of fedratinib, a Janus kinase inhibitor (JAKi). This study aimed to determine the potential for other JAKi to inhibit thiamine transport using human epithelial colorectal adenocarcinoma (Caco-2) and thiamine transporter (THTR) overexpressing cells and to better elucidate the structural basis for interacting with THTR. Only JAKi containing a 2,4-diaminopyrimidine were observed to inhibit thiamine transporters. Fedratinib inhibited thiamine uptake into Caco-2 cells (IC50 = 0.940 µM) and THTR-2 (IC50 = 1.36 µM) and, to a lesser extent, THTR-1 (IC50 = 7.10 µM) overexpressing cells. Two other JAKi containing this moiety, AZD1480 and cerdulatinib, were weaker inhibitors of the thiamine transporters. Other JAKi-including monoaminopyrimidines, such as momelotinib, and nonaminopyrimidines, such as filgotinib-did not have any inhibitory effects on thiamine transport. A pharmacophore model derived from the minimized structure of thiamine suggests that 2,4-diaminopyrimidine-containing compounds can adopt a conformation matching several key features of thiamine. Further studies with drugs containing a 2,4-diaminopyrimidine resulted in the discovery that the antibiotic trimethoprim also potently inhibits thiamine uptake mediated by THTR-1 (IC50 = 6.84 µM) and THTR-2 (IC50 = 5.56 µM). Fedratinib and trimethoprim were also found to be substrates for THTR, a finding with important implications for their disposition in the body. In summary, our results show that not all JAKi have the potential to inhibit thiamine transport and further establish the interaction of these transporters with xenobiotics.

Wernicke’s Encephalopathy is an acute neuro-psychiatric condition caused by an insufficient supply of thiamine (Vitamin B1) to the brain. If undiagnosed or inadequately treated, it is likely to proceed to Korsakoff’s Syndrome. Wernicke’s Encephalopathy can result from dietary deficiency alone and this form is usually successfully treated, with little chance of Korsakoff’s Syndrome supervening. On the other hand, thiamine deficiency associated with alcohol misuse/dependence may require up to 1 gram of thiamine IV in the first 24 hours to be treated successfully. The reasons for this difference in treatment will be discussed. Thiamine diphosphate acts as a co-factor for a number of thiamine-dependent enzymes. Thiamine deficiency leads to a reduction in the activity of these enzymes, and this leads to alterations in mitochondrial activity, impairment of oxidative metabolism, decreased energy status and eventually selective neuronal death. The damage caused by the combination of thiamine deficiency and alcohol metabolism probably interferes with adequate thiamine transport at a number of sites in the body, including the blood–brain barrier, as well as causing damage to the apoenzymes which then require higher concentrations of thiamine to work normally. The accumulated damage is likely to render the use of oral thiamine therapeutically inadequate since the body is unable to produce high enough concentrations of thiamine in the blood to traverse the blood–brain barrier. Some individuals are probably genetically predisposed to develop Wernicke’s. Long before individuals with alcohol misuse or dependence develop Wernicke’s Encephalopathy the neurons and other cells of the body are functioning sub-optimally because of the inadequate supply of thiamine and the neurotoxic effect of alcohol. This relative deficiency initiates a series of pathological changes which accumulate and further interfere with the supply of thiamine and its utilisation at a time when the requirements are increased. The best treatment for Korsakoff’s Syndrome is timely recognition of Wernicke’s Encephalopathy and appropriate intervention and prevention.

The review highlights metabolism and biological functions of vitamin B 1 (thiamine). It considers thiamine transport systems in various organisms enzymes of its biosynthesis and degradation, as well as molecular basis of thiamine-dependent hereditary pathologies. A special attention is paid to discussion of the role of thiamine triphosphate and adenylated thiamine triphosphate, a new thiamine derivative recently discovered in living cells.

Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B1). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer′s broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed thatListeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout ofthiD(lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate ofL. monocytogenesin epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation ofL. monocytogenesin mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDΔthiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs includingBacillusYuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated “ThiT.” Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy ofL. monocytogenesand possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.

In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes place primarily in the proximal part of the small intestine by means of a dual mechanism, which is saturable at low (physiological) concentrations and diffusive at higher. Thiamine undergoes intracellular phosphorylation mainly to thiamine pyrophosphate, while at the serosal side only free thiamine is present. Thiamine uptake is enhanced by thiamine deficiency, and reduced by thyroid hormone and diabetes. The entry of thiamine into the enterocyte, as evaluated in brush border membrane vesicles of rat small intestine in the absence of H+ gradient, is Na+- and biotransformation-independent, completely inhibited by thiamine analogs and reduced by ethanol administration and aging. The transport involves a saturable mechanism at low concentrations of vitamin and simple diffusion at higher. Outwardly oriented H+ gradients enhance thiamine transport, whose saturable component is a Na+-independent electroneutral uphill process utilizing energy supplied by the H+ gradient, and involving a thiamine/ H+ 1:1 stoichiometric exchange. The exit of thiamine from the enterocyte, as evaluated in basolateral membrane vesicles, is Na+-dependent, directly coupled to ATP hydrolysis by Na+-K+-ATPase, and inhibited by thiamine analogs. Transport of thiamine by renal brush border membrane vesicles is similar to the intestinal as far as both H+ gradient influence and specificity are concerned. In the erythrocyte thiamine transport is a Na+-independent, electroneutral process yet with two components: saturable, prevailing at low thiamine concentrations, and diffusive at higher. The saturable (specific) component is missing in patients of the rare disease known as thiamine-responsive megaloblastic anaemia (TRMA), producing a general disturbance of thiamine transport up to thiamine deficiency. The TRMA gene is located in chromosome 1q23.3. Recently, the thiamine transporter has been cloned: it is a protein of 497 amino acid residues with high homology with the reduced-folate transporter.

Retinal abnormality and visual disturbances occur in thiamine-responsive megaloblastic anaemia (TRMA), an autosomal recessive disorder caused by mutations in the human thiamine transporter-1 (hTHTR-1). Human retinal pigment epithelial cells play a pivotal role in supplying thiamine to the highly metabolically active retina but nothing is known about the mechanism, regulation or biological processes involved in thiamine transport in these cells. To address these issues, we used human-derived retinal pigment epithelial ARPE-19 cells to characterize the thiamine uptake process. Thiamine uptake is energy- and temperature-dependent, pH-sensitive, Na+-independent, saturable at both the nanomolar (apparent Km, 30 ± 5 nm) and the micromolar (apparent Km, 1.72 ± 0.3 μm) concentration ranges, specific for thiamine and sensitive to sulfhydryl group inhibition. The diuretic amiloride caused a concentration-dependent inhibition in thiamine uptake, whereas the anti-trypanosomal drug, melarsoprol, failed to affect the uptake process. Both hTHTR-1 and hTHTR-2 are expressed in ARPE-19 cells as well as in native human retinal tissue with expression of the former being significantly higher than that of the latter. Uptake of thiamine was adaptively regulated by extracellular substrate level via transcriptionally mediated mechanisms that involve both hTHTR-1 and hTHTR-2; it was also regulated by an intracellular Ca2+–calmodulin-mediated pathway. Confocal imaging of living ARPE-19 cells expressing TRMA-associated hTHTR-1 mutants (D93H, S143F and G172D) showed various expression phenotypes. These results demonstrate for the first time the existence of a specialized and regulated uptake process for thiamine in a cellular model of human retinal pigment epithelia that involves hTHTR-1 and hTHTR-2. Further, clinically relevant mutations in hTHTR-1 lead to impaired cell surface expression or function of the transporter in retinal epithelial ARPE-19 cells.

The water-soluble micronutrient thiamine is required for normal tissue growth and development in humans. Thiamine is accumulated into cells through the activity of two cell surface thiamine transporters (hTHTR1 and hTHTR2), which are differentially targeted in polarized tissues. Mutational dysfunction of hTHTR1 is associated with the clinical condition of thiamine-responsive megaloblastic anemia: the symptoms of which are alleviated by thiamine supplementation. Recently, two hTHTR2 mutants (G23V, T422A) have been discovered in clinical kindreds manifesting biotin-responsive basal ganglia disease (BBGD): the symptoms of which are alleviated by biotin administration. Why then does mutation of a specific thiamine transporter isoform precipitate a disorder correctable by exogenous biotin? To investigate the suggestion that hTHTR2 can physiologically function as a biotin transporter, we examined 1) the cell biological basis of hTHTR2 dysfunction associated with the G23V and T422A mutations and 2) the substrate specificity of hTHTR2 and these clinically relevant mutants. We show that the G23V and T422A mutants both abrogate thiamine transport activity rather than targeting of hTHTR2 to the cell surface. Furthermore, biotin accumulation was not detectable in cells overexpressing either the full length hTHTR2 or the clinically relevant hTHTR2 mutants, yet was demonstrable in the same assay using cells overexpressing the human sodium-dependent multivitamin transporter, a known biotin transporter. These results cast doubt on the most parsimonious explanation for the BBGD phenotype, namely that hTHTR2 is a physiological biotin transporter.

We isolated a strain carrying a recessive constitutive mutation (thi81) for the expression of thiamine metabolism in Saccharomyces cerevisiae. The thi81 mutant exhibits significant thiamine transport, thiamine-repressible acid phosphatase (T-rAPase) activities and significant activities of enzymes involved in thiamine biosynthesis which are repressed in the wild-type strain in medium supplemented with thiamine (2 x 10(-7) M). The thi81 mutant exhibited the same level of thiamine pyrophosphokinase activity and intracellular thiamine pyrophosphate concentration as the wild-type strain in medium supplemented with exogenous thiamine. The mutant strain constitutively produced PHO3 mRNA encoding T-rAPase in medium supplemented with thiamine. These results suggest that the thi81 mutant lacks a negative factor involved in the regulation of the genes encoding proteins involved in yeast thiamine metabolism.

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant ( P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.

The present study examined the intestinal uptake of thiamine (vitamin B1) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) Na+independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 μM and maximal velocity of 13.37 ± 0.94 pmol ⋅ mg protein−1⋅ 3 min−1; 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a Ca2+/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is Na+independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells.

In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon, thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli.

Outwardly oriented H+ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of adult Wistar rats. At a gradient pHin5:pHout7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of the gradient. Under the same conditions, the binding component of uptake accounted for only 10–13% of intravesicular transport. At the same gradient, the K m and J max values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 ± 1.4 μm and 14.9 ± 3 pmol · mg−1 protein · 6 sec−1 respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H+ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H+ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H+ antiport, and imipramine. Conversely, the guanidine/H+ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase in thiamine transport rate. In the absence of H+ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to 99% at pH 9.

Thiamine, in the form of thiamine pyrophosphate, is a cofactor for a number of enzymes which play important roles in energy metabolism. Although dietary thiamine deficiency states have long been recognised, it is only relatively recently that inherited defects in thiamine uptake, activation and the attachment of the active cofactor to target enzymes have been described, and the underlying genetic defects identified. Thiamine is transported into cells by two carriers, THTR1 and THTR2, and deficiency of these results in thiamine-responsive megaloblastic anaemia and biotin-responsive basal ganglia disease respectively. Defective synthesis of thiamine pyrophosphate has been found in a small number of patients with episodic ataxia, delayed development and dystonia, while impaired transport of thiamine pyrophosphate into the mitochondrion is associated with Amish lethal microcephaly in most cases. In addition to defects in thiamine uptake and metabolism, patients with pyruvate dehydrogenase deficiency and maple syrup urine disease have been described who have a significant clinical and/or biochemical response to thiamine supplementation. In these patients, an intrinsic structural defect in the target enzymes reduces binding of the cofactor and this can be overcome at high concentrations. In most cases, the clinical and biochemical abnormalities in these conditions are relatively non-specific, and the range of recognised presentations is increasing rapidly at present as new patients are identified, often by genome sequencing. These conditions highlight the value of a trial of thiamine supplementation in patients whose clinical presentation falls within the spectrum of documented cases.

An increased carbon flux and exploitation of metabolic pathways for the rapid generation of biosynthetic precursors is a common phenotype observed in breast cancer. To support this metabolic phenotype, cancer cells adaptively regulate the expression of glycolytic enzymes and nutrient transporters. However, activity of several enzymes involved in glucose metabolism requires an adequate supply of cofactors. In particular, vitamin B1 (thiamine) is utilized as an essential cofactor for metabolic enzymes that intersect at critical junctions within the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and thiamine pyrophosphokinase-1 (TPK-1). Therefore, the objective of this study was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. Employing cDNA arrays of breast cancer and normal breast epithelial tissues, SLC19A2, SLC25A19 and TPK-1 were found to be significantly up-regulated. Similarly, up-regulation was also observed in breast cancer cell lines compared to human mammary epithelial cells. Thiamine transport assays and quantitation of intracellular thiamine and thiamine pyrophosphate established a significantly greater extent of thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells. Overall, these findings demonstrate an adaptive response by breast cancer cells to increase cellular availability of thiamine.

Hepatic thiamine transport is thought to be a saturable, Na(+)- and energy-dependent process. However, the transport of this organic cation has not been examined in experimental models that allow direct characterization of carrier-mediated processes. Recently, a sinusoidal organic cation/H+ antiport was identified, using N1-methylnicotinamide as a marker. To determine whether thiamine is a substrate for this antiport, the characteristics of thiamine uptake were examined in rat liver basolateral membrane vesicles. An inwardly directed Na+ gradient had no effect on thiamine uptake as compared with an identical K+ gradient. An outwardly directed H+ gradient stimulated thiamine uptake as compared with pH-equilibrated conditions, and H(+)-dependent uptake was not the result of an H+ diffusion potential. Identical pH gradients stimulated uptake under voltage-clamped conditions, consistent with electroneutral thiamine/H+ exchange. Unlabeled intravesicular thiamine trans-stimulated [3H]thiamine uptake. Choline and imipramine cis-inhibited thiamine/H+ exchange; a series of other organic cations and thiamine analogues had no effect. Carrier-mediated [3H]thiamine uptake showed two saturable systems. In conclusion, a thiamine/H+ antiport is present on the sinusoidal membrane, distinct from Na+/H+ and NMN+/H+ exchange.

A 9-year study of thiamine metabolism and cellular transport was performed in two patients with thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and sensorineural deafness, in their relatives, and in age-matched controls from the same area. The ratios between the content of thiamine and that of its phosphoesters in erythrocytes were within the normal range, whereas the absolute values of thiamine and thiamine compounds were reduced by about 40% as compared to controls. Thiamine pyrophosphokinase activity was about 30% lower than in controls. Thiamine treatment restored the levels of thiamine and thiamine compounds to normal values, whereas kinase was unaffected. Both the saturable (specific, predominant at low, less than 2 mumol/L, physiological concentrations of thiamine) and the non-saturable component of thiamine transport were investigated. Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from thiamine-responsive megaloblastic anaemia patients contain low levels of thiamine compounds, probably due to their inability to take up and retain physiological concentrations of thiamine, as a result of the lack of the saturable, specific component of transport and reduced thiamine pyrophosphokinase.

We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.

We have investigated the thiamine metabolism in Schizosaccharomyces pombe and shown that: (1) Thiamine-repressible acid phosphate, coded for by the gene pho4, dephosphorylates thiamine phosphates indicating that the enzyme acts as a thiamine phosphate phosphatase. (2) In vivo synthesized thiamine is present intracellularly mainly as thiamine diphosphate. Starving cells for glucose decreases the intracellular thiamine pool. (3) The genes thi2, thi3 and thi4 control thiamine biosynthesis and probably code for thiamine biosynthetic enzymes. Thi3, which is involved in the synthesis of the pyrimidine moiety of the thiamine molecule, is allelic to the thiamine repressible gene nmt1. (4) Thiamine uptake is a thiamine regulated process, probably occurs by active transport and is controlled by the gene ptr1.

Bacampicillin was developed as a prodrug to improve the intestinal absorption of its metabolite ampicillin. This study was undertaken to characterize bacampicillin transport in Caco-2 cells. The uptake of bacampicillin in Caco-2 cells was significantly greater than those of ampicillin and pivampicillin. An Eadie-Hofstee plot obtained from 5-min uptake of 0.2-5 mM bacampicillin was linear, indicating the presence of a saturable transport system for bacampicillin with K(m) and V(max) of 3.6 mM and 23.9 nmol/mg protein/min, respectively. Hydrophilic organic cations such as choline, cimetidine, guanidine, nicotinamide, 1-methylnicotiamide, and tetraethylammonium failed to modulate bacampicillin uptake in Caco-2 cells whereas diphenhydramine, procainamide, and thiamine significantly depressed it. Moreover, when thiamine was preloaded in Caco-2 cells, bacampicillin uptake was significantly increased, indicating that this cationic vitamin was capable of trans-stimulating bacampicillin transport across the apical membrane of Caco-2 cells. However, trans-stimulated bacampicillin uptake was not observed in the presence of diphenhydramine. Bacampicillin uptake increased with elevation of the medium pH, and the known modulators of thiamine transport such as amiloride and oxythiamine significantly inhibited bacampicillin uptake. Thiamine also significantly decreased the apical-to-basolateral transport of bacampicillin across Caco-2 cell monolayers. However, thiamine did not exert any modulating effect on pivampicillin uptake and its apical-to-basolateral permeation in Caco-2 cells. These results suggest that bacampicillin is transported in Caco-2 cells, sharing a carrier-mediated system with thiamine.

The clinical development of a selective JAK2 inhibitor, fedratinib, was terminated due to cases of Wernicke's encephalopathy, a disease related to thiamine insufficiency. Furthermore, a recent publication reported potent inhibition of the thiamine transporter 2 (THTR2; SLC19A3) by fedratinib1. This study aimed to determine the potential for other JAK inhibitors to inhibit thiamine transport and to better elucidate the structural basis for inhibition. The effect of the aminopyrimidine containing JAK inhibitors fedratinib, AZD1480, momelotinib, and XL-019 on thiamine transport in Caco-2 cells and HEK293 cells transfected with human THTR1 (SLC19A2) and 2 was assessed. Consistent with the prior publication, fedratinib inhibited thiamine uptake in Caco-2 cells (IC50=0.94 ± 0.08 μM) and THTR2 transfected cells (IC50=1.6 ± 0.52 μM). We also observed inhibition of THTR1 by fedratinib (IC50=7.9 ± 0.97 μM). Another JAK inhibitor, AZD1480, also inhibited thiamine transport, but to a lesser degree (Caco-2, IC50=8.1 ± 0.1 μM). In contrast, momelotinib and XL-019 did not inhibit thiamine transport in any of the systems tested. A pharmacophore model derived from the minimized structure of thiamine suggests that the 2,4-diaminopyrimidine containing JAK inhibitors fedratinib and AZD1480 can adopt a conformation matching several key features of thiamine, while inhibitors lacking the 4-amino group and/or an additional hydrogen bond donor poorly align with thiamine. In summary, these results suggest that not all JAK inhibitors have the potential to inhibit thiamine transport. In particular, momelotinib, currently in Phase 3 clinical trials for the treatment of pancreatic cancer and myelofibrosis, showed no potential to affect thiamine levels. Disclosures Giacomini: Gilead Sciences: Employment. Hao:Gilead Sciences: Employment. Chandrasekhar:Gilead Sciences: Employment. Twelves:Gilead Sciences: Employment. Whitney:Gilead Sciences: Employment. Lepist:Gilead Sciences: Employment. Ray:Gilead Sciences: Employment.

Thiamine (T) analogues pyrithiamine, oxythiamine or amprolium in amounts 10-1000 times higher than labelled T, were i.p. injected into rats together with 14C-T (30 microg; 46.25 KBq). The radioactivity associated with T and its phosphoesters in the plasma and cerebral cortex, brainstem, cerebellum, and sciatic nerve were determined at time intervals from 0.25 to 240 h from injection. The experimental data obtained were processed with a mathematical compartmental model that calculated the fractional rate constants. These are the amount of content in a given compartment that is replaced in 1 h and expressed in per hour. The results showed that all three analogues inhibited thiamine entry from plasma. Instead, oxythiamine enhanced T phosphorylation to T pyrophosphate (TPP); amprolium and oxythiamine enhanced TPP dephosphorylation to monophosphate (TMP); pyrithiamine reduced TPP dephosphorylation and TMP formation, while none of the analogues modified TMP dephosphorylation to T. In conclusion, in living rats, the action of T analogues was much more complex than could be expected from their structure and action in vitro.

Fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA) syndrome with diabetes and deafness undergo apoptotic cell death in the absence of supplemental thiamine in their cultures. The basis of megaloblastosis in these patients has not been determined. Here we use the stable [1,2-13C2]glucose isotope-based dynamic metabolic profiling technique to demonstrate that defective high-affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via the nonoxidative branch of the pentose cycle. RNA ribose isolated from TRMA fibroblasts in thiamine-depleted cultures shows a time-dependent decrease in the fraction of ribose derived via transketolase, a thiamine-dependent enzyme in the pentose cycle. The fractional rate of de novo ribose synthesis from glucose is decreased several fold 2 to 4 days after removal of thiamine from the culture medium. No such metabolic changes are observed in wild-type fibroblasts or in TRMA mutant cells in thiamine-containing medium. Fluxes through glycolysis are similar in TRMA versus control fibroblasts in the pentose and TCA cycles. We conclude that reduced nucleic acid production through impaired transketolase catalysis is the underlying biochemical disturbance that likely induces cell cycle arrest or apoptosis in bone marrow cells and leads to the TRMA syndrome in patients with defective high-affinity thiamine transport. (Blood. 2003;102: 3556-3561)

Malignant cells exhibit alterations in metabolic enzymes requiring an adequate supply of cofactors to support enzymatic activity. In particular, Vitamin B1 (thiamine) is an important component of cellular growth and metabolism within all cells, functioning as an essential enzyme cofactor in key metabolic pathways within in the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and the conversion of thiamine to the active coenzyme, thiamine pyrophosphate (TPP) by thiamine pyrophosphokinase (TPK1). Therefore, the objective was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. This work will aid in linking the metabolic phenotype of cancer cells with the adaptive regulation of thiamine homeostasis. This may provide a molecular basis for dietary interventions and new therapeutic targets directed towards cancer cell metabolism. Methods: cDNA arrays of breast cancer and normal breast tissues were employed to determine expression of SLC19A2, SLC19A3, SLC25A19 and TPK-1. Western blotting was used to determine whole cell protein expression. To test the extent of cell surface localization of the transporters, cell surface proteins were biotinylated and run on Western Blot. Transport assays were used to quantify thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells (HMEC). Results: SLC19A2, SLC25A19 and TPK-1were found to be significantly up-regulated in clinical tissues and breast cancer cell lines 6.48 (P=0.0013), 5.97 (P=0.0003), and 2.33 (P=0.0063) folds, respectively. Additionally, thiamine transporters THTR1 and THTR2 exhibited different cellular localization and expression depending on the ER status of the cancer cell. The increase in thiamine homeostasis genes correlated with an increase in intracellular free thiamine levels but not with the coenzyme TPP. Conclusions: Overall, these findings demonstrate an adaptive response by breast cancer cells to bolster an increase in the cellular availability of thiamine. Citation Format: Jason Zastre, Brad Hanberry, Rebecca Sweet, Cary McGinnis, Kristen Venuti, Michael Bartlett, Raj Govindarajan. Up-regulation of vitamin B1 homeostasis genes in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1903. doi:10.1158/1538-7445.AM2013-1903

Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier (KM = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [14C]thiamine or [14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1-2 h. When preloading the cells with [14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism.

Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient.

Chronic alcoholism results in thiamine deficiency partly due to inadequate dietary intake of the vitamin. However, in addition, there is evidence to suggest that chronic alcohol consumption leads to impaired absorption of thiamine from the gastrointestinal tract (Thomson et al., 1970). Central nervous system neurons require a continuous supply of thiamine for metabolic and biosynthetic purposes. Transport of thiamine across the blood-brain barrier takes place by both a saturable process and (to a lesser extent) passive diffusion. Saturable uptake occurs at a rate of 0.3 μg/h/g tissue (Greenwood et al., 1982). This rate is surprisingly similar to the rate calculated for thiamine turnover in brain (Rindi et al., 1980), suggesting that thiamine transport across the blood-brain barrier may be just sufficient to meet brain requirements and that there might exist only a limited surplus capacity of this system. Brain structures such as cerebellum and brainstem have particularly high thiamine turnover rates (of the order of 0.3–0.5 μg/h/g tissue) (Rindi et al., 1980), which might explain the selective vulnerability of these brain structures to thiamine deficiency. Furthermore, ethanol ingestion appears to have direct effects on brain thiamine metabolism.

The rare autosomal recessive human disorder, TRMA syndrome (thiamine-responsive anemia with diabetes and deafness, OMIM 249270), is caused by mutations in the high affinity vitamin B1 transporter, SLC19A2. In human TRMA, ringed sideroblasts and megaloblastic changes are found in the marrow, suggesting potential defects in heme synthesis or mitochondrial function, and DNA synthesis, respectively. The mechanism whereby defective marrow thiamine transport causes these findings in the marrow was examined in a mouse model for TRMA. We previously demonstrated in fibroblasts from humans with TRMA a defect in the rate and pathway used for de novo ribose synthesis using Stable Isotope Dynamic Metabolic Profiling (SIDMaP analysis - Boros et al, Blood102: 3556, 2003). To examine whether this fibroblast defect accurately defined the marrow pathophysiology, we have now examined this pathway in detail in mice defective for the orthologous slc19a2 gene by targeted gene disruption (Fleming et al, Mol Genet Metab80:234, 2003). Slc19a2 mutant (−/−) or wildtype (+/+) mice were subjected to normal mouse chow (22 mg/kg thiamine), or thiamine deficient chow diet. Diets were confirmed thiamine deficient by chemical analysis. To examine heme biosynthesis, we used 55Fe-transferrin, ex vivo, to label marrow from +/+ or −/− mice maintained for 10 days in thiamine replete or deficient chow. Mutant mice had cell surface labeling indistinguishable from +/+ at 0°C (P>0.3), reflecting available transferrin receptors. Total cellular 55Fe uptake at 37 C was equal to or slightly greater than that of +/+ marrow in thiamine depleted state. However, incorporation of 55Fe into heme lagged in −/− cells (7.3 +/− 0.4 pmol/106cells/7 min) compared to +/+ (8.6 +/− 0.5 pmol/106 cells, P =0.018 in triplicate experiments). To examine deoxyribose (dRibose) synthesis, mice were maintained on thiamine replete or depleted chow for 10 days, then [1,2 13C] glucose was administered intraperitoneally overnight and 3 hours before sacrifice and marrow harvest. DNA from whole marrow and from sorted basophilic erythroblasts (CD71+/Ter119+) was isolated, hydrolized, derivatized, and dRibose analyzed by GC-MS. The initial rate of incorporation of 13C into DNA dRibose, reflecting de novo synthetic rate, was reduced 34% in basophilic erythroblasts of −/− mice after thiamine depletion (n=3 in duplicate experiments, P

A kinetic study of [14C]thiamine uptake over a concentration range from 0.1 microM to 4 mM was performed in isolated rat hepatocytes. The results showed that two processes contribute to the entry in rat hepatocytes: a low affinity process with a Kt of 34.1 microM and Vmax of 20.8 pmol/10(5) cells per 30 s and a high affinity process with a Kt of 1.26 microM and Vmax of 1.21 pmol/10(5) cells per 30 s. The uptake of thiamine by the high affinity process was concentrative and reduced in a betaine medium or K+ medium. Both ouabain and 2,4-dinitrophenol decreased the thiamine uptake by the high affinity process. These findings indicate that the transport of thiamine via a high affinity process is dependent on Na+ and biological energy. The uptake of thiamine was strongly inhibited by thiamine analogs such as dimethialium and chloroethylthiamine. Among quarternary ammonium compounds other than thiamine derivatives, choline and acetylcholine significantly inhibited thiamine uptake by rat liver cells, whereas betaine and carnitine did not. A kinetic study of thiamine uptake by rat hepatocytes preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, revealed that the biphasic property of thiamine uptake disappeared and a single carrier system for thiamine with a Kt of 40.5 microM, which was similar to the Kt value of the low affinity process, was retained. These results strongly suggest that thiamine transport system in rat liver cells is closely connected with thiamine pyrophosphokinase, which accelerates the uptake rat of thiamine by pyrophosphorylation at physiological concentrations of thiamine.

1. By measurement of the rate of disappearance of injected tracer thiamine from the bloodstream, a programme for the continuous injection of thiamine at a variable rate has been devized by which a steady raised level can be achieved rapidly and maintained in the circulation. By this means the flux of radioactive thiamine across the blood-brain barrier has been measured. 2. In separate experiments progressively higher levels of thiamine were maintained in the bloodstream. Evidence was obtained that the transport of thiamine across the blood-brain barrier is a carrier-mediated process which can be saturated by raised levels of thiamine. 3. The saturation of the transport process was incomplete: kinetic analysis showed that there was a non-saturable component of the transport which was probably due to passive diffusion. 4. The contribution of the non-saturable component was normally small and is probably insufficient to meet the needs of the brain for the vitamin unless the concentration of the vitamin in the blood is raised considerably above normal. 5. This two-component transport process had substantially similar kinetic parameters in different regions of the brain.

The characteristics of the uptake of the lipophilic cation tetraphenylphosphonium (TPP+) into Saccharomyces cerevisiae have been investigated in order to establish whether this compound can be used to monitor the membrane potential of this organism. Unlike dibenzyldimethylammonium, TPP+ is not translocated via the thiamine transport system, nor via another inducible translocation mechanism. On changing the experimental conditions the equilibrium potential of TPP+ varies according to expected changes of the membrane potential. TPP+ accumulation is higher in metabolizing cells than in non-metabolizing cells. In addition, decreasing the medium pH, addition of the proton conductor 2,4-dinitrophenol and addition of K+ all cause an apparent depolarization, whereas Ca2+ apparently hyperpolarizes the cell membrane. It is concluded that TPP+, if applied at low concentrations, can be used to measure the membrane potential of S. cerevisiae.

Age is an important factor in the process of thiamine resorption in the small intestine (jejunum-ileum) of rats. Thiamine transport rate through the intestinal wall is significantly lower in ageing rats (26 months old) as compared to young (3 and a half months old) animals. It has been determined that the alkaline phosphatase activity in the small intestine mucosa and the rate of thiamine resorption change unidirectionally. The phosphorus ester content of thiamine (TMP, TDP) and FT in the intestinal mucosa of ageing rats is lower, as compared to that of young rats. TTP is an exception, its content in the intestinal mucosa of ageing rats being higher. TMP, TDP, TTP and FT content in the liver tissue of young rats is higher than in ageing rats.