2-deoxy-d-glucose Ameliorates Animal Models of Dermatitis.
ABSTRACT: Glucose metabolism is a key metabolic pathway that orchestrates cellular homeostasis by generating ATP, nucleotides, and amino acids. Abnormal glucose signaling has been found in many diseases including cancers and inflammatory diseases. According to recent report, glycolysis contributes to pathogenesis of psoriasis and ablation of Glut1 attenuates animal models of psoriasis. While we were screening a molecular target for atopic dermatitis, we found the levels of glucose transporters including Glut1 (SLC2a1) and Glut3 (SLC2a3) are highly expressed in skin biopsies of dermatitis patients from multiple datasets. We demonstrated that administration of 2-deoxy-d-glucose (2DG) ameliorates animal models of 12-o-tetradecanoylphorbol-13-acetate (TPA) and oxazolone induced dermatitis using morphological and histological analysis. These results suggest that inhibition of glucose metabolism ameliorates dermatitis in animal models.
Project description:Despite evidences linking methylation changes in the cancer tissues, little is known about the methylation modification in the peripheral blood. With the current study, we identified differential methylation regions (DMRs) across human genome by collecting the blood samples of colorectal cancer (CRC) patients compared to that of their blood-related family who shared genetic inheritance and environmental influences, and unrelated obese and non-obese controls by accessing publicly available Gene Expression Omnibus data. We performed genome-wide analyses using the reduced representation bisulfite sequencing (RRBS) method covering about 25% of CpGs for whole human genome of the four groups (n = 5 each). In comparison to the non-obese controls, we observed significant DMRs in CRC for genes involved in tumorigenesis including MLH3, MSH2, MSH6, SEPT9, GNAS; and glucose transporter genes associated with obesity and diabetes including SLC2A1/GLUT1, and SLC2A3/GLUT3 that were reported on methylation being modified in cancer tissues. In addition, we observed significant DMRs in CRC for genes involved in the methylation pathways including PEMT, ALDH1L1, and DNMT3A. CRC and family members shared significant DMRs for genes of tumorigenesis including MSH2, SEPT9, GNAS, SLC2A1/GLUT1 and SLC2A3/GLUT3); and CAMK1, GLUT1/SLC2A1 and GLUT3/SLC2A3 genes involved in glucose and insulin metabolism that played vital role in development of obesity and diabetes. Our study provided evidences that these differentially methylated genes in the blood could potentially serve as candidate biomarkers for CRC diagnostic and may provide further understanding on CRC progression. Further studies are warranted to validate these methylation changes for diagnostic and prevention of CRC.
Project description:Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) owing to preferential BTIC survival and to adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3, and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, tumor initiating cells may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may maintain the tumor hierarchy and portend poor prognosis.
Project description:The facilitative glucose transporter-1 (GLUT1) deficiency or de Vivo syndrome is a rare neuropediatric disorder characterized by drug-resistant epilepsy, acquired microcephaly, delayed psychomotor development, intermittent ataxia, and other paroxysmal neurological disorders due to the presence of dominant mutations in the SLC2A1 gene. Alternating hemiplegia of childhood (AHC) is another rare neuropediatric disorder characterized by episodes of hemiplegia developing during the first 1.5 years of life. Before the recent finding of the gene ATP1A3 as the major cause of AHC, a heterozygous missense mutation in the SLC2A1 gene encoding GLUT1 was described in one child with atypical AHC, suggesting some clinical overlap between AHC and GLUT1 deficiency syndrome (GLUT1DS1). Half of patients with symptoms evocative of GLUT1DS1 with hypoglycorrhachia and up to 25 % of patients with AHC remain molecularly undiagnosed. We investigated whether mutations in SLC2A3 encoding GLUT3, another glucose transporter predominant in the neuronal cell, may account the case of a cohort of 75 SLC2A1 negative GLUTDS1-like patients and seven patients with AHC who were negative for ATP1A3 and SLC2A1 mutations. Automated Sanger sequencing and qPCR analyses failed to detect any mutation of SLC2A3 in the patients analyzed, excluding this gene as frequently mutated in patients with GLUT1DS1 like or AHC.
Project description:Huntington's disease (HD) is a devastating neurodegenerative disorder which is inherited in an autosomal dominant manner. HD is caused by a trinucleotide CAG repeat expansion that encodes a polyglutamine stretch in the huntingtin (HTT) protein. Mutant HTT expression leads to a myriad of cellular dysfunctions culminating in neuronal loss and consequent motor, cognitive and psychiatric disturbances in HD patients. The length of the CAG repeat is inversely correlated with age of onset (AO) in HD patients, while environmental and genetic factors can further modulate this parameter. Here, we explored whether the recently described copy-number variation (CNV) of the gene SLC2A3-which encodes the neuronal glucose transporter GLUT3-could modulate AO in HD. Strikingly, we found that increased dosage of SLC2A3 delayed AO in an HD cohort of 987 individuals, and that this correlated with increased levels of GLUT3 in HD patient cells. To our knowledge this is the first time that CNV of a candidate gene has been found to modulate HD pathogenesis. Furthermore, we found that increasing dosage of Glut1-the Drosophila melanogaster homologue of this glucose transporter-ameliorated HD-relevant phenotypes in fruit flies, including neurodegeneration and life expectancy. As alterations in glucose metabolism have been implicated in HD pathogenesis, this study may have important therapeutic relevance for HD.
Project description:Objectives:The gene encoding glucose transporter 3 (GLUT3, SLC2A3) is present in the human population at variable copy number. An overt disease phenotype of SLC2A3 copy number variants has not been reported; however, deletion of SLC2A3 has been previously reported to protect carriers from rheumatoid arthritis, implicating GLUT3 as a therapeutic target in rheumatoid arthritis. Here we aim to perform functional analysis of GLUT3 copy number variants in immune cells, and test the reported protective association of the GLUT3 copy number variants for rheumatoid arthritis in a genetic replication study. Methods:Cells from genotyped healthy controls were analyzed for SLC2A3/GLUT3 expression and glycolysis capacity. We genotyped the SLC2A3 copy number variant in four independent cohorts of rheumatoid arthritis and controls and one cohort of multiple sclerosis and controls. Results:Heterozygous deletion of SLC2A3 correlates directly with expression levels of GLUT3 and influences glycolysis rates in the human immune system. The frequency of the SLC2A3 copy number variant is not different between rheumatoid arthritis, multiple sclerosis and control groups. Conclusions:Despite a robust SLC2A3 gene copy number dependent phenotype, our study of large groups of rheumatoid arthritis cases and controls provides no evidence for rheumatoid arthritis disease protection in deletion carriers. These data emphasize the importance of well powered replication studies to confirm or refute genetic associations, particularly for relatively rare variants.
Project description:Vertebrate cells that are transformed by oncogenes such as v-src or are stimulated by mitogens have increased rates of glucose uptake. In rodent cells, the mechanisms whereby glucose transport is up-regulated are well understood. Stimulation of glucose transport involves an elevation in mRNA encoding the GLUT1 glucose transporter that is controlled at the levels of both transcription and mRNA stability. Cloning and sequencing of chicken GLUT1 cDNA showed that it shares 95% amino acid sequence similarity to mammalian GLUT1s. Nevertheless, unlike mammalian GLUT1 mRNA, it was not induced by v-src, serum addition, or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in chicken embryo fibroblasts. Rather, the induction of glucose transport in chicken embryo fibroblasts by v-src, serum, and 12-O-tetradecanoylphorbol 13-acetate was associated with induction of GLUT3 mRNA level and GLUT3 transcription. Rat fibroblasts were also found to express both GLUT1 and GLUT3 isoforms, but v-src induced GLUT1 and not GLUT3. This suggests that animal cells require both a basal and an upregulatable glucose transporter and that these functions have been subsumed by different GLUT isoforms in avian and mammalian cells.
Project description:L6 muscle cells survive long-term (18 h) disruption of oxidative phosphorylation by the mitochondrial uncoupler 2,4-dinitrophenol (DNP) because, in response to this metabolic stress, they increase their rate of glucose transport. This response is associated with an elevation of the protein content of glucose transporter isoforms GLUT3 and GLUT1, but not GLUT4. Previously we have reported that the rise in GLUT1 expression is likely to be a result of de novo biosynthesis of the transporter, since the uncoupler increases GLUT1 mRNA levels. Unlike GLUT1, very little is known about how interfering with mitochondrial ATP production regulates GLUT3 protein expression. Here we examine the mechanisms employed by DNP to increase GLUT3 protein content and glucose uptake in L6 muscle cells. We report that, in contrast with GLUT1, continuous exposure to DNP had no effect on GLUT3 mRNA levels. DNP-stimulated glucose transport was unaffected by the protein-synthesis inhibitor cycloheximide. The increase in GLUT3 protein mediated by DNP was also insensitive to cycloheximide, paralleling the response of glucose uptake, whereas the rise in GLUT1 protein levels was blocked by the inhibitor. The GLUT3 glucose transporter may therefore provide the majority of the glucose transport stimulation by DNP, despite elevated levels of GLUT1 protein. The half-lives of GLUT3 and GLUT1 proteins in L6 myotubes were determined to be about 15 h and 6 h respectively. DNP prolonged the half-life of both proteins. After 24 h of DNP treatment, 88% of GLUT3 protein and 57% of GLUT1 protein had not turned over, compared with 25% in untreated cells. We conclude that the long-term stimulation of glucose transport by DNP arises from an elevation of GLUT3 protein content associated with an increase in GLUT3 protein half-life. These findings suggest that disruption of the oxidative chain of L6 muscle cells leads to an adaptive response of glucose transport that is distinct from the insulin response, involving specific glucose transporter isoforms that are regulated by different mechanisms.
Project description:Cerebellar granule neurons in primary culture express increasing levels of two glucose transporter isoforms, GLUT1 and GLUT3, as they differentiate in vitro. We have determined the relative abundance of GLUT1 and GLUT3 in these neurons by three different labelling methods. (1) Photoaffinity cell surface labelling of neurons with an impermeant bis-mannose photolabel revealed 6-10-fold more GLUT3 than GLUT1 and dissociation constants (Kd) for the photolabel of 55-68 microM (GLUT3) and 146-169 microM (GLUT1). Binding to both transporters was inhibited by cytochalasin B. (2) Photoaffinity labelling of neuronal membranes with a permeant forskolin derivative showed 5.5-8-fold more GLUT3 than GLUT1, whereas in rat brain membranes containing both neuronal and glial membranes, GLUT3 and GLUT1 were detected in similar proportions. (3) Biosynthetic labelling of neurons with [35S]methionine and [35S]cysteine showed GLUT3 to be 6-10-fold more abundant than GLUT1. Thus GLUT3 is quantitatively the predominant glucose-transport isoform in cultured cerebellar granule neurons.
Project description:This study was designed to determine whether glucose regulates the gene expression of glucose transporter GLUT3 in neurons. We examined the regulation of GLUT3 mRNA by glucose in vivo in mouse brain and in vitro by using neuronal cultures from rat embryos. Hypoglycaemia (< 30 mg/dl), produced by 72 h of starvation, increased GLUT3 mRNA in mouse brain by 2-fold. Hybridization studies in situ demonstrated that hypoglycaemia-induced increases in GLUT3 mRNA expression were observed selectively in brain regions including the hippocampus, dentate gyrus, cerebral cortex and piriform cortex, but not the cerebellum. Primary neuronal cultures from rat embryos deprived of glucose for 48 h also showed an increase (4-fold over control) in GLUT3 mRNA, indicating that glucose can directly regulate expression of GLUT3 mRNA. In contrast with hypoglycaemia, hyperglycaemia produced by streptozotocin did not alter the expression of GLUT3 mRNA. We also confirmed previous findings that hypoglycaemia increases GLUT1 mRNA expression in brain. The increase in GLUT1 expression was probably limited to the blood-brain barrier in vivo, since GLUT1 mRNA could not be detected in neurons of the mouse cerebrum. Thus we conclude that up-regulation of neuronal GLUT3 in response to glucose starvation represents a protective mechanism against energy depletion in neurons.
Project description:We have expressed GLUT3 protein, an isoform of a facilitative glucose transporter, in Chinese hamster ovary cells by transfection of its cDNA using an expression vector. The expressed GLUT3 protein was detected by Western-blot analysis as a broad band of 45-65 kDa, indicating intensive glycosylation of the protein. The expressed GLUT3 protein was observed, by immunofluorescence staining, to be located mainly at the plasma membrane, and its expression was associated with a marked increase in glucose-transport activity. Kinetic analysis revealed that the Km value of GLUT3 protein for 3-O-methylglucose uptake was approx. 35% of that of GLUT1 protein, whereas the Km value of GLUT3 protein for 2-deoxy-D-glucose uptake was very similar to that of GLUT1 protein. The Vmax. value of GLUT3 protein for 3-O-methylglucose and 2-deoxyglucose uptake was approx. 20-50% of that of GLUT1 protein. GLUT3 protein was well photolabelled with [3H]cytochalasin B or a mannose derivative, 2-N-4-[3H](1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos -4-yloxy)-2- propylamine. Thus GLUT3 protein has very similar characteristics to GLUT1 protein including its subcellular localization, but exhibits lower Km and Vmax. values for 3-O-methylglucose uptake.