Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

Stagg, Amy; Fleming, James E.; Baker, Meghan A; Sakamoto, Massayuki; Cohen, Nadine; Neufeld, Ellis J.

doi:10.1172/jci3895

Cited by 73 publications

(57 citation statements)

References 17 publications

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“…The TUNEL assay was performed as described (22) in paraffin-embedded sections of the pancreases by using an in situ cell death detection kit (Boehringer Mannheim). The endogenous perioxidase activity was blocked by immersing the sections in 0.3% H 2 O 2 in methanol for 30 min before cell permeablization.…”

Section: Methodsmentioning

confidence: 99%

Glucose stimulates protein modification by O -linked GlcNAc in pancreatic β cells: Linkage of O -linked GlcNAc to β cell death

Liu

Paterson

Chin

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

226

217

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The pancreatic ␤ cell can respond in the long term to hyperglycemia both with an increased capacity for insulin production and, in susceptible individuals, with apoptosis. When glucose-induced apoptosis offsets the increasing ␤ cell capacity, type 2 diabetes results. Here, we tested the idea that the pathway of glucose metabolism that leads to the modification of intracellular proteins with the O-linked monosaccharide N-acetylglucosamine (O-GlcNAc) is involved in the glucose-induced apoptosis. This idea is based on two recent observations. First, the ␤ cell expresses much more O-GlcNAc transferase than any other known cell, and second, that the ␤ cell-specific toxin, streptozotocin (STZ), itself a GlcNAc analog, specifically blocks the enzyme that cleaves O-GlcNAc from intracellular proteins. As a consequence, we now show that hyperglycemia leads to the rapid and reversible accumulation of O-GlcNAc specifically in ␤ cells in vivo. Animals pretreated with STZ also accumulate O-GlcNAc in their ␤ cells when hyperglycemic, but this change is sustained upon re-establishment of euglycemia. In concert with the idea that STZ toxicity results from the sustained accumulation of O-GlcNAc after a hyperglycemic episode, we established a low-dose STZ protocol in which the ␤ cells' toxicity of STZ was manifest only after glucose or glucosamine administration. Transgenic mice with impaired ␤ cell glucosamine synthesis treated with this protocol are resistant to the diabetogenic effect of STZ plus glucose yet succumb to STZ plus glucosamine. This study provides a causal link between apoptosis in ␤ cells and glucose metabolism through glucosamine to O-GlcNAc, implicating this pathway of glucose metabolism with ␤ cell glucose toxicity.T he pancreatic ␤ cell is the primary regulator of glucose flux into stored energy in vertebrates. To accomplish this function, ␤ cells must sense the plasma glucose concentration and secrete the appropriate amount of insulin to direct glucose uptake and storage of its chemical energy in the fat, muscle, and liver. Considerable progress has been made in the understanding of glucose sensing by the ␤ cell. Glucose enters the ␤ cell through the GLUT2 glucose transporter (1), and it is phosphorylated by glucokinase, which has a K m for glucose that allows substantial glucose phosphorylation to proceed only when plasma glucose concentrations exceed 5 mM (2). Once glucose is phosphorylated, it can enter a variety of metabolic pathways, including glycolysis. An increased ATP͞ADP from glucose metabolism generally is believed to regulate the immediate release of insulin (3, 4). The normal ␤ cell is also capable of adapting its capacity for insulin release depending on long-term nutritional status. For example, exposure to a higher than normal carbohydrate load can condition the ␤ cell to secrete even more insulin after exposure to the same load several hours later (5). Finally, the ␤ cell is capable of even more long-term adaptation by increasing the ␤ cell number through hyperplasia (6, 7). However, this hyp...

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Section: Methodsmentioning

confidence: 99%

Glucose stimulates protein modification by O -linked GlcNAc in pancreatic β cells: Linkage of O -linked GlcNAc to β cell death

Liu

Paterson

Chin

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

226

217

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“…4 Fibroblasts isolated from patients with TRMA show cycle arrest and increased apoptosis in low-thiamine-containing media. 5 Although thiamine deficiency has been known to cause apoptosis of cells in sensitive brain regions including the thalamus, mammillary, and medial geniculate nuclei, 6 the biochemical disturbance resulting from defective high-affinity thiamine transport in human cells is yet to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA)

Boros

Steinkamp

Fleming

et al. 2003

Blood

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Fibroblasts from patients with thiamineresponsive 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 [1,2-13 C 2 ]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 thiaminecontaining 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. IntroductionThe pentose cycle, or "pentose-monophosphate shunt," allows de novo synthesis of ribose from glucose by either or both of 2 possible routes: a nonoxidative pathway via transaldolase and transketolase, and an oxidative pathway, via glucose-6-phosphate (G6P) and 6-phosphogluconate dehydrogenase (G6PDH). The oxidative pathway also generates nicotinamide adenine dinucleotide phosphate-positive (NADPH ϩ ) reducing equivalents. Whereas the oxidative branch of the pathway predominates in circulating red blood cells, in many tissues the nonoxidative branch is quantitatively more important for ribose synthesis destined for nucleic acids. 1-3 Thiamine pyrophosphate is a cofactor for transketolase, a key rate-limiting enzyme in the nonoxidative pathway of the pentose cycle. Defective thiamine uptake in fibroblasts isolated from patients with thiamine-responsive megaloblastic anemia (TRMA) is associated with the mutation and loss of function of the high-affinity, low-capacity thiamine transporter. 4 Fibroblasts isolated from patients with TRMA show cycle arrest and increased apoptosis in low-thiamine-containing media. 5 Although thiamine deficiency has been known to cause apoptosis of cells in sensitive brain regions including the thalamus, mammillary, and medial geniculate nuclei, 6 the biochemical disturbance resulting from defective high-affinity thiamine transport in human cells is yet to be elucidated.Metabolic pathway flux changes appear to be critical factors in cell differentiation, in apoptosis, and in mediating the antiproliferative effect of imatinib mesylate in Bcr-Abl-positive myeloid leukemia cells in culture. 7...

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“…TRMA is an autosomal recessive disorder characterized by megaloblastic anemia, pancytopenia, diabetes mellitus, and sensory neuronal deafness (15). In vitro experiments with epithelial cells from TRMA patients have shown that these cells have decreased thiamine-dependent enzymatic activity and undergo apoptosis when passaged at physiological levels of thiamine, which do not affect growth and survival of normal epithelial cells (16).…”

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confidence: 99%

Disruption of Thiamine Uptake and Growth of Cells by Feline Leukemia Virus Subgroup A

Mendoza

Miller

Overbaugh

2013

J Virol

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Feline leukemia virus (FeLV) is still a major cause of morbidity and mortality in domestic cats and some wild cats despite the availability of relatively effective vaccines against the virus. FeLV subgroup A (FeLV-A) is transmitted in natural infections, and FeLV subgroups B, C, and T can evolve directly from FeLV-A by mutation and/or recombination with endogenous retroviruses in domestic cats, resulting in a variety of pathogenic outcomes. The cell surface entry receptor for FeLV-A is a putative thiamine transporter (THTR1). Here, we have addressed whether FeLV-A infection might disrupt thiamine uptake into cells and, because thiamine is an essential nutrient, whether this disruption might have pathological consequences. First, we cloned the cat ortholog of the other of the two known thiamine transporters in mammals, THTR2, and we show that feline THTR1 (feTHTR1) and feTHTR2 both mediate thiamine uptake, but feTHTR2 does not function as a receptor for FeLV-A. We found that feTHTR1 is widely expressed in cat tissues and in cell lines, while expression of feTHTR2 is restricted. Thiamine uptake mediated by feTHTR1 was indeed blocked by FeLV-A infection, and in feline fibroblasts that naturally express feTHTR1 and not feTHTR2, this blockade resulted in a growth arrest at physiological concentrations of extracellular thiamine. The growth arrest was reversed at high extracellular concentrations of thiamine. Our results show that FeLV-A infection can indeed disrupt thiamine uptake with pathological consequences. A prediction of these experiments is that raising the plasma levels of thiamine in FeLVinfected cats may ameliorate the pathogenic effects of infection.

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Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

Cited by 73 publications

References 17 publications

Glucose stimulates protein modification by O -linked GlcNAc in pancreatic β cells: Linkage of O -linked GlcNAc to β cell death

Glucose stimulates protein modification by O -linked GlcNAc in pancreatic β cells: Linkage of O -linked GlcNAc to β cell death

Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA)

Disruption of Thiamine Uptake and Growth of Cells by Feline Leukemia Virus Subgroup A

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