Major Glucose Transporter Research Articles

Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes

Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland, therefore, plays a critical role in milk synthesis. Facilitative glucose transporters (GLUT) mediate glucose uptake in the mammary gland. Glucose transporter 1 (GLUT1) is the major facilitative glucose transporter expressed in the bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. Glucose transporter 1 is, therefore, thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 (bGLUT1) was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B, and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-d-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a Michaelis constant (Km) of 9.8±3.0mM for 2-deoxy-d-glucose, similar to 11.7±3.7mM for human GLUT1. Transport by bGLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bGLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bGLUT1 in taking up glucose across mammary epithelial cells for milk synthesis.

F-18 FDG PET/CT of a Gastric Schwannoma.

Schwannomas, also known as neurilemmomas, are tumors originating from nervous tissue; they have Schwann cell sheaths. According to a recent classification, about 80% of gastrointestinal mesenchymal tumors are gastrointestinal stromal tumors (GISTs) [1]. Gastrointestinal (GI) schwannomas have been reported to represent only 3% of all GI mesenchymal tumors [2]. These tumors make up only 0.2% of all gastric neoplasms [3]. Schwannomas of the GI tract are distinctive from conventional schwannomas that arise in soft tissue or the central nervous system. GI schwannomas are hypothesized to arise from the myenteric plexus within the GI tract wall. These tumors are usually benign, slow-growing, and asymptomatic, and therefore most are discovered incidentally [3]. The differentiation of schwannomas from other submucosal tumors is very difficult. The main differential diagnosis for a mass arising in the wall of the gastointestinal tract is a GIST, which is a potentially malignant mesenchymal GI tumor that arises from the interstitial cells of Cajal, which help regulate peristalsis [4]. The diagnostic determination of schwannomas requires positive histological tests for S-100 protein and vimentin, but negative histological tests for smooth muscle actin and c-KIT. In contrast, GISTs are c-KIT positive and can be S-100 positive if they are located in the small bowel [5]. Because most patients with schwannomas have excellent prognoses, surgical removal is sufficient for treatment. Gastric schwannomas are normally benign, and malignant transformation is extremely rare [6]. However, the current case (Figs. 1 and ​and2)2) illustrates that these tumors may exhibit avid F-18 FDG uptake. It remains unclear why high F-18 FDG uptake is found in benign tumors such as schwannomas. F-18 FDG uptake in soft tissue and neural schwannomas is variable but is frequently high, possibly due to over-expression of the glucose transporter by tumor cells. In particular, glucose transporter type 3 is found in all human tissues and is the major glucose transporter on the neuronal surface [7, 8]. However, for diagnostic purposes, intense F-18 FDG uptake does not distinguish benign schwannomas from potentially malignant GISTs, which also have high F-18 FDG uptake. These data suggest that F-18 FDG PET is of limited value as a preoperative diagnostic technique for the assessment of schwannoma versus GIST. Fig. 1 A 58-year-old woman was admitted for the evaluation and treatment of a gastric mass that had previously been detected by cancer-screening endoscopy. Endoscopy revealed a submucosal tumor that had a central ulcer on its surface. Contrast-enhanced CT showed ... Fig. 2 On gross inspection, the tumor was an 8 × 7 × 6 cm sized well-encapsulated ovoid, firm mass in the stomach wall. A mucosal ulceration (2 × 1.5 cm) was noted. The cut surfaces ...

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential.Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1.These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo.

Plasmodial sugar transporters as anti-malarial drug targets and comparisons with other protozoa

Glucose is the primary source of energy and a key substrate for most cells. Inhibition of cellular glucose uptake (the first step in its utilization) has, therefore, received attention as a potential therapeutic strategy to treat various unrelated diseases including malaria and cancers. For malaria, blood forms of parasites rely almost entirely on glycolysis for energy production and, without energy stores, they are dependent on the constant uptake of glucose. Plasmodium falciparum is the most dangerous human malarial parasite and its hexose transporter has been identified as being the major glucose transporter. In this review, recent progress regarding the validation and development of the P. falciparum hexose transporter as a drug target is described, highlighting the importance of robust target validation through both chemical and genetic methods. Therapeutic targeting potential of hexose transporters of other protozoan pathogens is also reviewed and discussed.

Myo1c Regulates Glucose Uptake in Mouse Skeletal Muscle

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ∼2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.

The small RNA SgrS controls sugar–phosphate accumulation by regulating multiple PTS genes

A number of bacterial small RNAs (sRNAs) act as global regulators of stress responses by controlling expression of multiple genes. The sRNA SgrS is expressed in response to glucose–phosphate stress, a condition associated with disruption of glycolytic flux and accumulation of sugar–phosphates. SgrS has been shown to stimulate degradation of the ptsG mRNA, encoding the major glucose transporter. This study demonstrates that SgrS regulates the genes encoding the mannose and secondary glucose transporter, manXYZ. Analysis of manXYZ mRNA stability and translation in the presence and absence of SgrS indicate that manXYZ is regulated by SgrS under stress conditions and when SgrS is ectopically expressed. In vitro footprinting and in vivo mutational analyses showed that SgrS base pairs with manXYZ within the manX coding sequence to prevent manX translation. Regulation of manX did not require the RNase E degradosome complex, suggesting that the primary mechanism of regulation is translational. An Escherichia coli ptsG mutant strain that is manXYZ+ experiences stress when exposed to the glucose analogs α-methyl glucoside or 2-deoxyglucose. A ptsG manXYZ double mutant is resistant to the stress, indicating that PTS transporters encoded by both SgrS targets are involved in taking up substrates that cause stress.

Human Cytomegalovirus Activates Glucose Transporter 4 Expression To Increase Glucose Uptake during Infection

Glucose transport into mammalian cells is mediated by a group of glucose transporters (GLUTs) on the plasma membrane. Human cytomegalovirus (HCMV)-infected human fibroblasts (HFs) demonstrate significantly increased glucose consumption compared to mock-infected cells, suggesting a possible alteration in glucose transport during infection. Inhibition of GLUTs by using cytochalasin B indicated that infected cells utilize GLUT4, whereas normal HFs use GLUT1. Quantitative reverse transcription-PCR and Western analysis confirmed that GLUT4 levels are greatly increased in infected cells. In contrast, GLUT1 was eliminated by a mechanism involving the HCMV major immediate-early protein IE72. The HCMV-mediated induction of GLUT4 circumvents characterized controls of GLUT4 expression that involve serum stimulation, glucose concentration, and nuclear functions of ATP-citrate lyase (ACL). In infected cells the well-characterized Akt-mediated translocation of GLUT4 to the cell surface is also circumvented; GLUT4 localized on the surface of infected cells that were serum starved and had Akt activity inhibited. The significance of GLUT4 induction for the success of HCMV infection was indicated using indinavir, a drug that specifically inhibits glucose uptake by GLUT4. The addition of the drug inhibited glucose uptake in infected cells as well as viral production. Our data show that HCMV-specific mechanisms are used to replace GLUT1, the normal HF GLUT, with GLUT4, the major glucose transporter in adipose tissue, which has a 3-fold-higher glucose transport capacity.

The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA

Vibrio cholerae causes the severe diarrhoeal disease cholera. A cascade of regulators controls expression of virulence determinants in V. cholerae at both transcriptional and post-transcriptional levels. ToxT is the direct transcription activator of the major virulence genes in V. cholerae. Here we describe TarA, a highly conserved, small regulatory RNA, whose transcription is activated by ToxT from toxboxes present upstream of the ToxT-activated gene tcpI. TarA regulates ptsG, encoding a major glucose transporter in V. cholerae. Cells overexpressing TarA exhibit decreased steady-state levels of ptsG mRNA and grow poorly in glucose-minimal media. A mutant lacking the ubiquitous regulatory protein Hfq expresses diminished TarA levels, indicating that TarA likely interacts with Hfq to regulate gene expression. RNAhybrid analysis of TarA and the putative ptsG mRNA leader suggests potential productive base-pairing between these two RNA molecules. A V. cholerae mutant lacking TarA is compromised for infant mouse colonization in competition with wild type, suggesting a role in the in vivo fitness of V. cholerae. Although somewhat functionally analogous to SgrS of Escherichia coli, TarA does not encode a regulatory peptide, and its expression is activated by the virulence gene pathway in V. cholerae and not by glycolytic intermediates.

Decreased Glucose Transporter 1 in Brains With Ischemic Cerebrovascular Diseases

The entry of glucose from blood into the brain through the blood-brain barrier relies on glucose transporters (GLUT). GLUT1 is the major GLUT in the blood-brain barrier and one of the molecular markers of the endothelial cells of brain capillaries. It has been demonstrated that GLUT1 expression is increased after acute cerebral ischemia in animal models. GLUT1 level in brain tissue from 7 cases of chronic ischemic cerebrovascular diseases (ICVD) were determined and compared with that from 11 cases of the age- and postmortem interval- matched controls. It was found that GLUT1 level was decreased markedly in ICVD, and this decrease appeared to result from a down-regulation of hypoxia-inducible factor-1α(HIF-1α). It was also found that the level of protein O-GlcNAcylation was not altered in the ICVD brain. These results shed new light into the molecular mechanism of ICVD.

A minimal base-pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA

Escherichia coli SgrS is an Hfq-binding small RNA that is induced under glucose-phosphate stress to cause translational repression and RNase E-dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31-nt-long stretch in the 3' region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre-annealed with ptsG mRNA by a high-temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base-pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base-pairing region is sufficient to inhibit ptsG translation in the context of full-length SgrS in vivo. We conclude that SgrS 168-181 is a minimal base-pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high-temperature pre-treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.

Dysregulation of Insulin Signaling, Glucose Transporters, O-GlcNAcylation, and Phosphorylation of Tau and Neurofilaments in the Brain : Implication for Alzheimer’s Disease

Recent studies have suggested a possible role of insulin dysfunction in the pathogenesis of sporadic Alzheimer's disease (AD). In AD, brain glucose metabolism is impaired, and this impairment appears to precede the pathology and clinical symptoms of the disease. However, the exact contribution of impaired insulin signaling to AD is not known. In this study, by using a nontransgenic rat model of sporadic AD generated by intracerebroventricular administration of streptozotocin, we investigated insulin signaling, glucose transporters, protein O-GlcNAcylation, and phosphorylation of tau and neurofilaments in the brain. We found impaired insulin signaling, overactivation of glycogen synthase kinase-3beta, decreased levels of major brain glucose transporters, down- regulated protein O-GlcNAcylation, increased phosphorylation of tau and neurofilaments, and decreased microtubule-binding activity of tau in the brains of streptozotocin-treated rats. These results suggest that impaired brain insulin signaling may lead to overactivation of glycogen synthase kinase-3beta and down-regulation of O-GlcNAcylation, which, in turn, facilitate abnormal hyperphosphorylation of tau and neurofilaments and, consequently, neurofibrillary degeneration.

Brain glucose transporters, O‐GlcNAcylation and phosphorylation of tau in diabetes and Alzheimer’s disease

Type 2 diabetes mellitus (T2DM) increases the risk for Alzheimer's disease (AD), but the underlying mechanism is unknown. In this study, we determined the levels of major brain glucose transporters, O-GlcNAcylation and phosphorylation of tau in the postmortem brain tissue from frontal cortices of 7 controls, 11 T2DM subjects, 10 AD subjects and 8 additional subjects who had both T2DM and AD. We found that the neuronal glucose transporter 3 was decreased to a bigger extent in T2DM brain than in AD brain. The O-GlcNAcylation levels of global proteins and of tau were also decreased in T2DM brain as seen in AD brain. Phosphorylation of tau at some of the AD abnormal hyperphosphorylation sites was increased in T2DM brain. These results suggest that T2DM may contribute to the increased risk for AD by impairing brain glucose uptake/metabolism and, consequently, down-regulation of O-GlcNAcylation, which facilitates abnormal hyperphosphorylation of tau.

Capture in the metabolic arena: co-selection of gamma and deltaretrovirus envelope glycoproteins and their receptors

Although all infectious and replication-competent retroviruses harbor an env gene, the origin of env remains unknown except for that of a few invertebrate retroviruses. It is generally admitted that infectious retroviruses have evolved from endogenous viral retrotransposons by insertion of an ''external'' gene that encodes for a fusion glycoprotein (env capture). In 2000, Malik et al. provided the first formal evidence for the origin of env in several invertebrate retroviruses. For instance, they showed that env of Gypsy, an infectious Drosophila retrotransposon, has been ''captured'' from a fusion protein-encoding gene of a baculovirus, a totally unrelated large DNA enveloped virus. Vertebrate retroviruses cover 7 genera distinguished by their gag (capsid encoding) and pol (enzyme encoding) genes. The gammaretroviruses (murine leukemia viruses, MLV as a prototype) and deltaretroviruses (the human T cell leukemia virus, HTLV as a prototype) are the most distant genera according to their pol genes. However, we reported that HTLV env is most closely related to MLV env, sharing similar modular organization and motifs even within the receptor-binding domain (RBD), the Env most variable sequence. Accordingly, swapping the RBD-encoding regions between the two types of Env yielded functional chimeras. We argued that this common ancestry between the env of remotely related retroviruses is also in favor of an env capture scenario by vertebrate retroviruses. Nonetheless, the origin of these env, and more specifically their RBD, remains unknown. Based on the gammaretrovirus Env modular organization, we derived soluble RBD domains of different gamma and deltaretroviruses. This has been instrumental to the identification of Glut1, the major glucose transporter, as the cognate receptor of HTLV-1 and 2 Env. We further showed that Glut1 binds all known HTLV or S(imian)TLV types and that intracellular interaction of HTLV or STLV RBD with Glut1 modulates glucose transport and glycolysis. Of most interest is that, like Glut1, all of the receptors identified so far for gammaretrovirus Env belong to the family of multimembrane-spanning molecules and that those for which a function has been determined are all metabolite transporters (amino acids, inorganic phosphate, vitamins, etc.). The selection as Env receptors of metabolite transporters that shuttles from cytoplasmic pools to the cell surface and the cytoplasmic interaction between RBD and their cognate receptors have us to postulate that gamma and deltaretrovirus Env, and more particularly their RBD modules, are related to transporter chaperone molecules that modulate their cell surface translocation.

Frequency distribution of XbaIG > T and HaeIIIT > C GLUT1 polymorphisms among different Brazilian ethnic groups

GLUT is the major glucose transporter in mammalian cells. Single nucleotide polymorphisms (SNP) at GLUT1 promoter and regulatory regions have been associated to the risk of developing nephropathy in different type 1 and type 2 diabetic populations. It has been demonstrated that differences in allelic and genotypic frequencies of GLUT1 gene (SLC2A1) polymorphisms occur among different populations. Therefore, ethnic differences in distribution of GLUT1 gene polymorphisms may be an important factor in determining gene-disease association. In this study, we investigated the XbaIG > T and HaeIIIT > C polymorphisms in six different Brazilian populations: 102 individuals from Salvador population (Northern Brazil), 56 European descendants from Joinville (South Brazil), 85 Indians from Tiryió tribe (North Brazil) and 127 samples from Southern Brazil: 44 from European descendants, 42 from African descendants and 41 from Japanese descendants. Genotype frequencies from both sites did not differ significantly from those expected under the Hardy-Weinberg equilibrium. We verified that the allele frequencies of both polymorphisms were heterogeneous in these six Brazilian ethnic groups.

Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28 degrees C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The DeltaKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis.

Targeting of PED/PEA-15 Molecular Interaction with Phospholipase D1 Enhances Insulin Sensitivity in Skeletal Muscle Cells

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.

O2-05-08: Protein O-Glcnacylation: A novel mechanism involved in Alzheimer's disease

Neurofibrillary degeneration plays a pivotal role in the pathogenesis of Alzheimer disease (AD) and correlates directly with dementia of affected individuals. However, the molecular mechanism(s) leading to neurofibrillary degeneration is not well understood. Microtubule-associated protein tau and neurofilaments are the key proteins involved in neurofibrillary degeneration and are post-translationally modified by both O-GlcNAcylation (a unique type of O-glycosylation of protein with β-N-acetyl-glucosamine, GlcNAc) and phosphorylation. The role of O-GlcNAcylation in phosphorylation of tau and neurofilaments were studied in vitro, in cultured cells, in metabolically active rat brain slices, and in vivo. We found that O-GlcNAcylation of tau and neurofilaments regulates their phosphorylation inversely. In cell cultures and in metabolically active rat brain slices, elevation of O-GlcNAcylation induced decreased phosphorylation of tau and neurofilaments at multiple phosphorylation sites, whereas reduction of O-GlcNAcylation resulted in hyperphosphorylation of these proteins. In vivo, fasting-induced decrease in glucose uptake/metabolism caused down-regulation of O-GlcNAcylation and upregulation of phosphorylation of tau and neurofilaments in the mouse brain. Hippocampus was found to be more vulnerable than cerebral cortex to the fasting-induced alteration of tau O-GlcNAcylation and phosphorylation, which is consistent with the differential susceptibility of various brain regions in AD. Re-feeding of the mice reversed the dysregulation of both O-GlcNAcylation and phosphorylation of tau. More importantly, the level of O-GlcNAcylation of tau and neurofilaments was decreased in AD brain, and this decrease was correlated negatively to the level of phosphorylation of these two proteins. The major glucose transporters (GLUTs) responsible for glucose uptake into neurons from blood stream, GLUT1 and GLUT3, were decreased in AD brain. This decrease correlated to the decrease in protein O-GlcNAcylation, to the hyperphosphorylation of tau, and to the density of neurofibrillary tangles in human brains. On the basis of our findings, we propose a novel mechanism by which the impaired brain glucose uptake/metabolism causes a decrease in tau O-GlcNAcylation, leading to Alzheimer neurofibrillary degeneration by facilitating abnormal hyperphosphorylation of tau and neurofilaments.

Characterization of KlGRR1 and SMS1 genes, two new elements of the glucose signaling pathway of Kluyveromyces lactis.

The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Deltasms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Deltasms1 mutation.

Characterization of the E. coli glucose permease fused to the maltose‐binding protein

The ptsG gene that encodes the major glucose transporter of Escherichia coli, II Glc, was inserted into a pMALE-amp r expression vector down-stream of the malE gene which encodes the E. coli maltose-binding protein (MBP). II Glc-MBP in the 2 h high speed supernatant of cell lysates eluted from a gel filtration column showing two activity peaks. The glucose-6-phosphate-dependent transphosphorylation (TP) activity of the membrane bound oligomeric peak 1 showed substrate inhibition while that of the soluble monomeric peak 2 did not. Purification of peak 2 yielded activity with weak substrate inhibition, and further gel filtration analyses showed that upon purification, some of the monomeric II Glc-MBP associated to higher molecular size forms. Assays of the phosphoenolpyruvate-dependent and transphosphorylation reactions showed that the specific activity of the purified enzyme from peak 1 was approximately double that from peak 2. The results show that the monomeric II Glc-MBP exhibits no substrate inhibition although the oligomeric form does. Purification promotes subunit association, an increase in catalytic activity, and restoration of substrate inhibition.

Expression of glucose transporters and hexokinase II in cholangiocellular carcinoma compared using [18F]‐2‐fluro‐2‐deoxy‐d‐glucose positron emission tomography

Cholangiocellular carcinoma (CCC) has been reported to have a high glucose uptake; however, the mechanism of glucose entry into these cells is still unclear. We investigated the relationship between [(18)F]-2-fluro-2-deoxy-D-glucose ((18)F-FDG) uptake and the expression of facilitative glucose transporters (Glut) and hexokinase (HK) II, as well as the association between the expression of different histological types of CCC. The expression of Glut (1-5) and HK II was studied using immunohistochemistry of 26 patients with CCC who underwent whole-body (18)F-FDG positron emission tomography before surgery or biopsy. CCC expressed immunohistochemically detectable Glut 1 in 81%, Glut 2 in 54%, Glut 3 in 19%, and HK II in 77% of the total cases. Glut 1, Glut 2, Glut 3, and HK II were more often detected in moderately differentiated and poorly differentiated than in well-differentiated CCC. A significant correlation was observed between (18)F-FDG uptake and the staining scores of Glut 1 and HK II (P = 0.02, rho = 0.45 and P = 0.001, rho = 0.59). The staining scores of Glut 1 and HK II were also significantly correlated (P = 0.002, rho = 0.3). Multivariate regression analysis revealed that lymph-node metastasis was independently associated with (18)F-FDG uptake. Our study showed a significant association between the expression of Glut 1 and HK II with (18)F-FDG uptake, indicating that Glut 1 is a major glucose transporter expressed in CCC and that HK II contributes to the increased metabolism of glucose, especially in moderately and poorly differentiated CCC.