Glut-4 is translocated to both caveolae and non-caveolar lipid rafts, but is partially internalized through caveolae in insulin-stimulated adipocytes

Caveolae and non-caveolar lipid rafts are two types of membrane lipid microdomains that play important roles in insulin-stimulated glucose uptake in adipocytes. In order to ascertain their specific functions in this process, caveolae were ablated by caveolin-1 RNA interference. In Cav-1 RNAi adipocytes, neither insulin-stimulated glucose uptake nor Glut-4 （glucose transporter 4） translocation to membrane lipid microdomains was affected by the ablation of caveolae. With a modified sucrose density gradient, caveolae and non-caveolar lipid rafts could be separated. In the wild-type 3T3- L l adipocytes, Glut-4 was found to be translocated into both caveolae and non-caveolar lipid rafts. However, in Cav1 RNAi adipocytes, Glut-4 was localized predominantly in non-caveolar lipid rafts. After the removal of insulin, caveolaelocalized Glut-4 was internalized faster than non-caveolar lipid raft-associated Glut-4. The internalization of Glut-4 from plasma membrane was significantly decreased in Cav-1 RNAi adipocytes. These results suggest that insulin-stimulated Glut-4 translocation and glucose uptake are caveolae-independent events. Caveolae play a role in the internalization of Glut-4 from plasma membrane after the removal of insulin.     

Hemopoietic cell transformation is associated with failure to downregulate glucose uptake during the G2/M phase of the cell cycle

Growth factors and cytokines initiate multiple signal transduction pathways that lead to cell survival, cell cycle progression or differentiation. A common feature of these pathways is increased cellular metabolism and glucose uptake. Furthermore, the energy requirements of many cancers and transformed cell lines are met by constitutive upregulation of glucose uptake. Relationships among transforming events, glucose uptake and cell cycle progression are not well understood. Here we investigated the regulation of glucose transport during the cell cycle of growth factor-dependent 32D cells, primary T-cells, src-transformed 32D cells and Jurkat cells. Cells were enriched in the G1, S and G2/M phases of the cell cycle, and glucose transporter expression and 2-deoxyglucose uptake were measured. Glucose transporter expression increased with cell volume as cells progressed through the cell cycle. Growth factor-dependent 32D cells and T-lymphocytes were characterised by increased 2-deoxyglucose uptake from G1 to S and reduced uptake at G2/M, with the highest specific activity of transporters in the S phase. In contrast, src-transformed 32D cells and Jurkat cells showed increased 2-deoxyglucose uptake from S to G2/M, with the highest glucose transporter specific activity in G2/M. Our results show that glucose transport is regulated in a cell cycle-dependent manner and suggest that this regulation may be altered in transformed cells.     

X-linked mental retardation with seizures and carrier manifestations is caused by a mutation in the creatine-transporter gene (SLC6A8) located in Xq28

A family with X-linked mental retardation characterized by severe mental retardation, speech and behavioral abnormalities, and seizures in affected male patients has been found to have a G1141C transversion in the creatine-transporter gene SLC6A8. This mutation results in a glycine being replaced by an arginine (G381R) and alternative splicing, since the G-->C transversion occurs at the -1 position of the 5' splice junction of intron 7. Two female relatives who are heterozygous for the SLC6A8 mutation also exhibit mild mental retardation with behavior and learning problems. Male patients with the mutation have highly elevated creatine in their urine and have decreased creatine uptake in fibroblasts, which reflects the deficiency in creatine transport. The ability to measure elevated creatine in urine makes it possible to diagnose SLC6A8 deficiency in male patients with mental retardation of unknown etiology.     

Distinct temporospatial expression patterns of glycolysis-related proteins in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the sixth most frequent human cancer worldwide and is characterized by rapid progression as well as resistance to systemic chemotherapy. Recently, glycolysis has emerged as a potent driving force of tumor growth and therapy failure. The precise role of glycolysis for the pathogenesis of human HCC has not been elucidated thus far. Therefore, we have conducted a comprehensive analysis of the expression patterns of central glycolysis-related factors [glucose transporter-1 and -2 (Glut-1 and Glut-2), phosphoglycerate kinase-1 (PGK-1) and hypoxia-inducible factor-1α (HIF-1α)] in a large cohort of benign and malignant human liver samples. PGK-1 protein and gene expression was scant in normal liver, elevated in cirrhotic livers and most intense in HCC. Strong immunoreactivity of Glut-2 was noted in cirrhotic livers, whereas in HCC it was only expressed in 50% of examined cases. Strikingly, PGK-1 as well as Glut-2 protein expression was indicative of poor patient prognosis. Glut-1 protein was absent in neoplastic hepatocytes but prominent in tumor-associated endothelial cells. Specific nuclear staining of HIF-1α was noted in only 12% of HCC samples. Our data point toward a tumor-promoting function of glycolysis in HCC and establish PGK-1 as an independent prognostic parameter. Furthermore, the endothelial-specific expression of Glut-1 makes a special dependence of vessels on glucose reasonable to assume. In summary, we believe our analysis warrants the validation of glycolytic inhibitors as innovative treatment approaches of human HCC. Christoph Benckert and Thorsten Cramer have contributed equally to this work.     

Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter

Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.     

Defective Glucose Transport Across Brain Tissue Barriers: A Newly Recognized Neurological Syndrome

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-0-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent K_m was similar in all cases (2–4 mmol/L), but the apparent V_max in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/10⁶RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the V_max to 935 fmol/s/10⁶ RBC (p < 3 × 10^–7). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (V_max was 38% of that of the mother's), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.     

Bench meets bedside: a 10-year-old girl and amino acid residue glycine 75 of the facilitative glucose transporter GLUT1

In 2000, amino acid residue G75 of the facilitative glucose transporter GLUT1 was identified by mutagenesis as being essential for transport function [Olsowski, A., et al. (2000) Biochemistry 39, 2469-74]. In 2002, we identified a heterozygous missense mutation substituting glycine at residue 75 for tryptophan in a 10-year-old girl with intractable seizures and low glucose concentrations in the cerebrospinal fluid indicative of GLUT1 deficiency. Glucose uptake into erythrocytes of the patient was 36% of controls, and GLUT1-specific immunoreactivity was normal, indicating a functional GLUT1 defect. In silico three-dimensional modeling of the G75W mutant provided a smaller gyration radius for transmembrane segment 2 as the potential pathogenic mechanism in this patient. This case illustrates a GLUT1 mutation characterized in vitro and later confirmed by disease itself and highlights the potential of basic science and clinical medicine to collaborate for the benefit of patients.     

Identification, characterization and cloning of SLC6A8C, a novel splice variant of the creatine transporter gene

SLC6A8 deficiency is caused by mutations in the X-linked creatine transporter gene (SLC6A8), which leads to cerebral creatine deficiency, mental retardation, speech and language delay, autistic-like behaviour and epilepsy. Insight in the mechanism of how the transporter is regulated is largely unknown and it is of importance for the development of successful treatment strategies of cerebral creatine deficient syndromes. Our goal was to characterize CRT2 (SLC6A8B), a published splice variant of the creatine transporter. Surprisingly, using RT-PCR we found a novel splice variant, SLC6A8C, which is predominantly found in human tissues with a high energy requirement such as brain, kidney, heart, small intestines and skeletal muscle, where SLC6A8 transporter is most required. The 5' untranslated region (UTR) of the SLC6A8C mRNA was identified using the Smart Race cDNA amplification kit. The SLC6A8C mRNA contains intron 4 and exons 5 through 13 of SLC6A8, including part of the 3' UTR. An open reading frame was found, which predicts a truncated protein identical to the SLC6A8 transporter, comprising the five last C-terminal transmembrane domains of the SLC6A8 transporter. SLC6A8C open reading frame was cloned as a fusion protein with EGFP and the SLC6A8C protein expression was detected by Western Blot. RT-PCR and sequence analysis showed that this splice variant is conserved in evolution, since we also detected it in mouse. This study reveals the presence of a novel SLC6A8 splice variant, SLC6A8C in human and mouse.     

SLC2A9 is a high-capacity urate transporter in humans

Background

Serum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man.

Methods and Findings

We expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K _i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82).

Conclusions

This study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.     

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARγ in L6 myotubes

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.     

Cloning and characterization of the promoter regions from the parent and paralogous creatine transporter genes

Interconversion between phosphocreatine and creatine, catalyzed by creatine kinase is crucial in the supply of ATP to tissues with high energy demand. Creatine's importance has been established by its use as an ergogenic aid in sport, as well as the development of intellectual disability in patients with congenital creatine deficiency. Creatine biosynthesis is complemented by dietary creatine uptake. Intracellular transport of creatine is carried out by a creatine transporter protein (CT1/CRT/CRTR) encoded by the SLC6A8 gene. Most tissues express this gene, with highest levels detected in skeletal muscle and kidney. There are lower levels of the gene detected in colon, brain, heart, testis and prostate. The mechanism(s) by which this regulation occurs is still poorly understood. A duplicated unprocessed pseudogene of SLC6A8–SLC6A10P has been mapped to chromosome 16p11.2 (contains the entire SLC6A8 gene, plus 2293 bp of 5′flanking sequence and its entire 3′UTR). Expression of SLC6A10P has so far only been shown in human testis and brain. It is still unclear as to what is the function of SLC6A10P. In a patient with autism, a chromosomal breakpoint that intersects the 5′flanking region of SLC6A10P was identified; suggesting that SLC6A10P is a non-coding RNA involved in autism. Our aim was to investigate the presence of cis-acting factor(s) that regulate expression of the creatine transporter, as well as to determine if these factors are functionally conserved upstream of the creatine transporter pseudogene.     

An extract of Artemisia dracunculus L. enhances insulin receptor signaling and modulates gene expression in skeletal muscle in KK-A mice

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models, but the cellular mechanisms by which insulin action is enhanced in vivo are not precisely known. In this study, we evaluated the effects of PMI 5011 to modulate gene expression and cellular signaling through the insulin receptor in skeletal muscle of KK-A^y mice. Eighteen male KK-A^y mice were randomized to a diet (w/w) mixed with PMI 5011 (1%) or diet alone for 8 weeks. Food intake, adiposity, glucose and insulin were assessed over the study, and at study completion, vastus lateralis muscle was obtained to assess insulin signaling parameters and gene expression. Animals randomized to PMI 5011 were shown to have enhanced insulin sensitivity and increased insulin receptor signaling, i.e., IRS-associated PI-3 kinase activity, Akt-1 activity and Akt phosphorylation, in skeletal muscle when compared to control animals (P<.01, P<.01 and P<.001, respectively). Gene expression for insulin signaling proteins, i.e., IRS-1, PI-3 kinase and Glut-4, was not increased, although a relative increase in protein abundance was noted with PMI 5011 treatment. Gene expression for specific ubiquitin proteins and specific 20S proteasome activity, in addition to skeletal muscle phosphatase activity, i.e., PTP1B activity, was significantly decreased in mice randomized to PMI 5011 relative to control. Thus, the data demonstrate that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling in an animal model of insulin resistance. PMI 5011 may modulate skeletal muscle protein degradation and phosphatase activity as a possible mode of action.     

X-linked creatine-transporter gene (SLC6A8) defect: a new creatine-deficiency syndrome

We report the first X-linked creatine-deficiency syndrome caused by a defective creatine transporter. The male index patient presented with developmental delay and hypotonia. Proton magnetic-resonance spectroscopy of his brain revealed absence of the creatine signal. However, creatine in urine and plasma was increased, and guanidinoacetate levels were normal. In three female relatives of the index patient, mild biochemical abnormalities and learning disabilities were present, to various extents. Fibroblasts from the index patient contained a hemizygous nonsense mutation in the gene SLC6A8 and were defective in creatine uptake. The three female relatives were heterozygous for this mutation in SLC6A8, which has been mapped to Xq28.     

Cytokine Stimulation Promotes Increased Glucose Uptake Via Translocation at the Plasma Membrane of GLUT1 in HEK293 Cells

Interleukin‐3 (IL‐3) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) are two of the best‐characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL‐3 and GM‐CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL‐3 and GM‐CSF are present in several non‐hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL‐3 and GM‐CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL‐3 and GM‐CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3‐kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non‐insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non‐hematopoietic cells. J. Cell. Biochem. 110: 1471–1480, 2010. © 2010 Wiley‐Liss, Inc.     

Thiamine-responsive megaloblastic anemia syndrome: a novel mutation

The thiamine-responsive megaloblastic anemia syndrome (TRMA) is an autosomal recessive disorder characterized by diabetes mellitus, megaloblastic anemia and sensorineural hearing loss due to mutations in SLC 19A2 that encodes a thiamine transporter protein. The disease can manifest at any time between infancy and adolescence, and not all cardinal findings are present initially. The anemia typically improves significantly with pharmacological doses of thiamine. Variable improvement in diabetes is also noted. However, the hearing loss is apparently irreversible, although a delay in the onset of deafness may be possible. We present a 2-year old girl with non-autoimmune diabetes mellitus and anemia in whom we found a novelc.95T>A (leu32X) mutation in the SLC19A2 gene in this study.Our patient with this new mutation did not suffer from hearing loss.     

Glucose transporter gene expression in early mouse embryos.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic beta cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation development or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.     

Characterization and expression pattern analysis of the facilitative glucose transporter 10 gene (slc2a10) in Danio rerio

The SLC2A10 gene located on chromosome 20q13.1 encodes the facilitative glucose transporter 10 (GLUT10), a class III member of the SLC2A facilitative glucose transporter family. Mutations in the human SLC2A10 gene cause arterial tortuosity syndrome (ATS), a rare autosomal recessive connective tissue disorder. In this work, we report the characterization of the slc2a10 ortholog gene in zebrafish (Danio rerio) and its expression pattern during embryonic development and in adult tissues. The slc2a10 gene consists of 5 exons, spanning 8 kb and mapping to a region on chromosome 11 that exhibits conserved synteny with human chromosome 20. The gene encodes Glut10, a 513 amino acid protein that maintains the 12 transmembrane domain structure typical of the GLUTs family, and shares the specific functional motifs involved in sugar transport with the vertebrate GLUT10. RT-PCR analysis showed that two specific splice variants, both including the 5’-UTR region, were expressed during embryogenesis and in different adult zebrafish tissues and organs. In situ hybridization analyses demonstrated a maternal origin of the total slc2a10 mRNA and its ubiquitous distribution until the early somitogenesis stage. In later embryonic stages, slc2a10 mRNA was detected in the otic vesicles, hatching gland cells, pectoral fin, posterior tectum and swim bladder. Overall, these results suggest a wide role of slc2a10 during zebrafish development.     

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.