5'-nucelotidase and glucose-6-phosphatase are liver plasma and microsomal membranes markers and their respective activities were determined. In the liver homogenate, the activities of 5'-nucleotidase were 0.58 +/- 0.08 and 0.29 +/- 0.07 micromols/mg protein/10min in the control and 3'-methyl-4-dimethyl aminoazobenzene (3'-Me DAB) groups respectively. The enzyme activities m the partially purified plasma membranes were 2.15 +/- 0.25 and 1.31 +/- 0.23 micromols/mg protein/10min in the control and 3'-Me DAB groups respectively. The glucose-6-phosphatase activities in the homogenates of the control and 3'-Me DAB groups were 0.23 +/- 0.10, and 0.45 +/- 0.25 micromols/mg protein/10min, and in the microsomal fraction, 1.14 +/- 0.32, and 0.63 +/- 0.11 micromols/mg protein/10min, respectively, The concentrations of glucose carrier in the plasma membranes from the control and 3'-Me DAB group were 25, and 35 pmols/mg membrane protein, respectively, and the Ka values for cytochalsin B in each group were 5.20 X 109. and 5.14 X 109ml/mol, respectively. However in the microsomal fraction, no significant differences of glucose carrier were found between the control and 3'-Me DAB groups from the DEAE Sephadex A-50 ion exchange chromatography, fractions I and ll were obtained. Electrophoretic analysis of fraction I revealed a major protein band with a molecular weight of 45,000 and minor bands with MWs of 50,000, 55,000 and 15,000. Following AcA 34 gel filtration, a major protein band with a MW of 45,000 was obtained. From these results, it can be concluded that the glucose carrier protein was increased on plasma membrane of hepatoma induced by 3'-Me DAB, and the carrier protein showed similar molecular weight to other glucose carrier found in the RBC, muscle cells and adipocyte.
Animal ; Cell Membrane/enzymology ; Cell Membrane/metabolism ; Liver Neoplasms, Experimental/metabolism* ; Male ; Methyldimethylaminoazobenzene* ; Microsomes, Liver/enzymology ; Microsomes, Liver/metabolism ; Monosaccharide Transport Proteins/isolation & purification ; Monosaccharide Transport Proteins/metabolism* ; Rats ; Rats, Inbred Strains ; p-Dimethylaminoazobenzene*/analogs & derivatives

OBJECTIVE: To analyze the clinical phenotype and variant of SLC2A1 gene in a Chinese pedigree affected with glucose transporter type 1 deficiency syndrome (GLUT1-DS). METHODS: Clinical data of a child who was treated due to delayed motor and language development and his family members were collected. DNA was extracted from peripheral blood samples and subjected to high-throughput medical exome sequencing. Candidate variant was verified by Sanger sequencing of his parents and sister. The genotype-phenotype correlation was explored. RESULTS: The child, his mother and sister had common manifestations such as delayed mental and motor development, poor exercise tolerance, easy fatigue and paroxysmal dystonia, but the difference was that the child and his mother had microcephaly and seizures, while his sister did not. A heterozygous missense SLC2A1 c.191T>C (p.L64P) variant was identified in all affected members, which was unreported previously. CONCLUSION The missense SLC2A1 c.191T>C (p.L64P) variant probably underlay the disease in the proband and his mother and sister. Variability of the clinical phenotypes has reflected the genetic and phenotypic diversity of GLUT1-DS. Detection of the novel variant has enriched the spectrum of GLUT1-DS mutations.
Carbohydrate Metabolism, Inborn Errors ; China ; Glucose Transporter Type 1/genetics* ; Humans ; Monosaccharide Transport Proteins/deficiency* ; Mutation ; Pedigree ; Phenotype

OBJECTIVETo assess the relationship between the overexpression of facilitative glucose transporter-1 (Glut1) and fluorine-18 fluorodeoxyglucose (FDG) uptake in patients with primary lung squamous cell carcinoma.

METHODSFrom April 1999 to March 2001, 23 patients with lung squamous cell carcinoma were imaged using FDG positron emission tomography (PET) before surgery. Their maximum and mean standard uptake values (SUVmax and SUVmean) of tumor and SUV of the normal lung (SUVlung) were measured. The expression of Glut1 of all the 23 cases was analysed in paraffin sections using SP immunohistochemistry.

RESULTSAll the 23 tumors tested were Glut1 positive (69 +/- 18)% of tumor cell area was positive and staining intensity was 4.6 +/- 0.7. All tumors of the patients could be detected by FDG-PET. FDG uptake of tumor was higher than that of normal lung (P < 0.01). SUVmax, SUVmean and SUVlung were 8.33 +/- 4.14, 6.10 +/- 3.00 and 0.38 +/- 0.13 respectively. Correlations were found among Glut1 expression and FDG uptake and tumor size (P < 0.01).

CONCLUSIONS(1) Glut1 overexpression is universal in the lung squamous cell carcinoma. (2) SUV was higher in the lung squamous cell carcinoma than that of the normal lung tissue. (3) Glut1 expression and FDG uptake and tumor size appear to be correlated with each other in patients with lung squamous cell carcinoma.

Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; Fluorodeoxyglucose F18 ; metabolism ; Glucose Transporter Type 1 ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; Male ; Middle Aged ; Monosaccharide Transport Proteins ; analysis

D-Mannitol has wide applications in food, pharmaceutical, and chemical industries. In this study, we constructed a genetically stable Escherichia coli strain for D-mannitol production by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC 12291 into chromosome of E. coli ATCC 8739 and inactivating other fermentation pathways (including pyruvate formate-lyase, lactate dehydrogenase, fumarate reductase, alcohol dehydrogenase, methylglyoxal synthase and pyruvate oxidase). Using mineral salts medium with glucose and fructose as carbon sources, the engineered strain could produce 1.2 mmol/L D-mannitol after anaerobic fermentation for 6 days. Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for 80 generations, D-mannitol titer increased 2.6-fold and mannitol dehydrogenase activity increased 2.8-fold. Genetically stable strains constructed in this work could ferment sugars to produce D-mannitol without the addition of antibiotics, inducers and formate, which was favorable for industrial production.
Escherichia coli ; genetics ; metabolism ; Fermentation ; Industrial Microbiology ; methods ; Leuconostoc ; enzymology ; Mannitol ; metabolism ; Mannitol Dehydrogenases ; genetics ; Metabolic Engineering ; methods ; Monosaccharide Transport Proteins ; genetics

OBJECTIVETo investigate the clinical features, diagnosis and treatment of glucose transporter 1 deficiency syndrome (GLUT1-DS), as well as the diagnostic value of movement disorders.

METHODSThe clinical data of four children with GLUT1-DS were collected, and their clinical features, treatment, and follow-up results were analyzed.

RESULTSThere were two boys and two girls, with an age of onset of 2-15 months. Clinical manifestations included movement disorders, seizures, and developmental retardation. Seizures were the cause of the first consultation in all cases. The four children all had persistent ataxia, dystonia, and dysarthria; two had persistent tremor, two had paroxysmal limb paralysis, and two had eye movement disorders. Paroxysmal symptoms tended to occur in fatigue state. All four children had reductions in the level of cerebrospinal fluid glucose and its ratio to blood glucose, as well as SLC2A1 gene mutations. The four children were given a ketogenic diet, at a ketogenic ratio of 2:1 to 3:1, and achieved complete remission of paroxysmal symptoms within 5 weeks.

CONCLUSIONSGLUT1-DS should be considered for epileptic children with mental retardation and motor developmental delay complicated by various types of movement disorders. The ketogenic diet is effective at a ketogenic ratio of 2:1 to 3:1 for the treatment of GLUT1-DS.

Carbohydrate Metabolism, Inborn Errors ; diagnosis ; genetics ; therapy ; Child ; Child, Preschool ; Female ; Humans ; Male ; Monosaccharide Transport Proteins ; deficiency ; genetics ; Movement Disorders ; diagnosis ; genetics ; therapy

DNase I footprinting assay using liver nuclear extracts revealed six protected regions between nucleotide -600 and +110 and hence named Box I-VI. Upstream promoter element (UPE), a DNA element playing crucial role in transcriptional control of the tissue specific expression of pancreatic beta-cell, has been detected within the proximal region of rat GLUT2 promoter. This region is included in Box VI. The protein-DNA interaction in this region (Box VI) was confirmed by mobility shift assay using liver nuclear extracts. Deletion of the region between -585 bp and -146 bp resulted in dramatic changes in promoter activity when they were expressed in liver and beta-cell derived cell line. When -585/-146 construct was expressed in liver, the activity was decreased to 46%, whereas the activity in beta-cell line, HIT-T15 cell, was increased by 84% when compared to -146/+190 construct. These opposing phenomena can be explained by the fact that beta-cell specifically expresses the UPE binding protein. Assuming that there may be Box VI-binding protein playing negative roles both in hepatocyte and beta-cell, and that the protein acts as a negative regulator of GLUT2 gene, the UPE binding protein in the beta-cell may overcome the inhibition by binding to the protein.
Animal ; Binding Sites ; Cell Line ; Comparative Study ; DNA Footprinting ; Deoxyribonuclease I ; Gene Expression Regulation ; Islets of Langerhans/metabolism* ; Islets of Langerhans/cytology ; Liver/metabolism* ; Liver/cytology ; Monosaccharide Transport Proteins/genetics ; Monosaccharide Transport Proteins/biosynthesis* ; Promoter Regions (Genetics)* ; Protein Binding ; Rats ; Transcription Factor AP-1

OBJECTIVETo explore the effect of puerarin injection on the amount of GLUT4 protein at the plasma membrane in insulin-resistant rat skeletal muscle.

METHODThe rat model of insulin resistance (IR) was made by being fed with high-fat diet. The animals were divided into three groups (ten in each group): group I: controls; group II: Insulin-resistant rats; group III: Insulin-resistant rats + Puerarin treatment. Insulin-resistant rats were injected with 100 mg puerarin injection per kg body weight through abdominal cavity once a day for 4 weeks. Fasting blood glucose and fasting serum insulin levels were measured before and after Puerarin treatment, respectively. Insulin treatment was achieved by intraperitoneal injection of insulin (1 unit insulin per kg body weight.) 15 minute before killing the animals. The right hindlimb skeletal muscle was rapidly dissected. Then the expression of GLUT4 protein at the plasma membrane in all the animals was assessed with Western blot method.

RESULTThe GLUT4 content at the plasma membrane in insulin-resistant rats skeletal muscle was significantly lower (about 31%) than that of controls (P < 0.01). Puerarin Injection partly corrected fasting blood glucose (from 6.17 +/- 0.67 mmol x L(-1) to 5.54 +/- 0.35 mmol x L(-1)) and fasting serum insulin levels (from 17.09 +/- 2.02 mU x L(-1) to 11.86 +/- 1.35 mU x L(-1)) and increased the GLUT4 content at the plasma membrane by 1.18-fold in insulin-resistant rats skeletal muscle.

CONCLUSIONPuerarin Injection can ameliorate IR, and the mechanism may be involved in increasing cell-surface level of GLUT4 through decreasing fasting blood glucose and fasting serum insulin levels, improving GLUT4 trafficking and intracellular insulin signaling.

Animals ; Cell Membrane ; metabolism ; Glucose Transporter Type 4 ; Injections ; Insulin Resistance ; Isoflavones ; administration & dosage ; isolation & purification ; pharmacology ; Male ; Monosaccharide Transport Proteins ; metabolism ; Muscle Proteins ; metabolism ; Muscle, Skeletal ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Pueraria ; chemistry ; Rats ; Rats, Sprague-Dawley

An antisense approach was attempted to investigate the role of antisense GLUT1 RNA in suppressing tumor cell phenotypes using N-ras-transformed NIH 3T3 cells. The established cell line transformed by ras showed typical biological characteristics of cancer cells, such as increased glucose transport, GLUT1 mRNA contents, and the ability to form colonies on the soft agar. In this system, the plasmids (pMAM-GLUT1(rev)) which can transcribe the antisense GLUT1 RNA were transfected and the accompanying changes in the phenotypes of the ras-transformed cells were observed. The expression of antisense GLUT1 RNA by induction with dexamethasone reduced the glucose transport by 30% (1.97 +/- 0.13 nmoles) after 4 min incubation when compared to the non-induction group of transformed cell (2.85 +/- 0.19 nmoles). Also, the number of colonies sized over 50 microns on the soft agar was reduced significantly in the antisense RNA expressing group compared to non-induction group. These results suggest that the expression of antisense GLUT1 RNA reduced the glucose transport and transforming potential in soft agar possibly by hybridization with GLUT1 mRNA in N-ras-transformed NIH 3T3 cells.
3T3 Cells/metabolism ; Animal ; Base Sequence ; Cell Line, Transformed ; Cell Transformation, Neoplastic/metabolism/*pathology ; *Genes, ras ; Human ; Mice ; Molecular Sequence Data ; Monosaccharide Transport Proteins/*genetics ; Phenotype ; RNA, Antisense/*metabolism ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured/metabolism/pathology

OBJECTIVETo investigate the effect of the escharectomy during burn shock stage on expression of glucose translator-4 (GLUT4) mRNA in skeletal muscle and adipose tissue.

METHODS30% TBSA scalded rats were employed. Escharectomy were conducted at 8 h, 24 h, 168 h after burns respectively. Insulin, glucagon, cortisol and glucose levels in serum were analyzed. RT-PCR were employed to analyze GLUT4 mRNA expression in skeletal muscle and adipose tissue.

RESULTSGlucagon, cortisol and glucose levels in serum were declined in groups which escharectomy were conducted during burn shock stage. GLUT4 mRNA expression in both skeletal muscle and adipose tissue were downregulated after burns and escharectomy conducted during burn shock stage made it restored to near normal.

CONCLUSIONGLUT4 mRNA expression will declined after major burns in skeletal muscle and adipose tissue. Escharectomy during shock stage could make it upregulated, which will be helpful to improve glucose metabolism and hypermetabolism after major burns.

Adipose Tissue ; metabolism ; Animals ; Blood Glucose ; Burns ; physiopathology ; surgery ; Gene Expression ; Glucagon ; blood ; Hydrocortisone ; blood ; Insulin ; blood ; Male ; Monosaccharide Transport Proteins ; genetics ; Muscle, Skeletal ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Shock, Traumatic ; physiopathology

The 5'- and 3'-side half of liver type glucose transporter (GLUT2) cDNA was amplified from total RNA or mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified 5'-side fragment of GLUT2 cDNA was inserted into pGEM4Z and named pGLGT1, and the 3'-side fragment of GLUT2 cDNA was inserted into the HindIII site of pGLGT1 to construct pGLGT2 which contains an entire open reading frame of GLUT2 cDNA. The GLUT2 cDNA in pGLGT2 was transferred to an eukaryotic expression vector (pMAM) to construct pMLGT, which was expressed in the insulin-sensitive Chinese hamster ovary (CHO) cells. Western blot analysis showed that the GLUT2 gene in pMLGT was expressed in the transfected CHO cells successfully. The GLUT2 content in the plasma membrane fraction of insulin-treated CHO cells expressing GLUT2 increased 3.8-fold compared to that of the control group. This result suggests that GLUT2, which is not subjected to translocation by insulin in the cells of its major distribution, can be translocated if it is expressed in the suitable cells sensitive to insulin action.
Animal ; Base Sequence ; CHO Cells ; *Cloning, Molecular ; Hamsters ; Insulin/*pharmacology ; Liver/*metabolism ; Molecular Sequence Data ; Monosaccharide Transport Proteins/*genetics/*metabolism ; Oligonucleotide Probes/genetics ; Support, Non-U.S. Gov't ; *Translocation (Genetics)