Biomedical subjects

H Katagiri

Publications and source records attributed to H Katagiri.

At least 19 recordsLinked to original sources

PubMed1992

Nonsense mutation of glucokinase gene in late-onset non-insulin-dependent diabetes mellitus.

A nonsense mutation at codon 186 in exon 5 of the gene for glucokinase, an enzyme important for glucose-induced insulin secretion, was identified in a Japanese patient with late-onset non-insulin-dependent diabetes mellitus (NIDDM). All affected members of her family were heterozygous for the mutation and had late-onset NIDDM or impaired glucose tolerance, whereas unaffected members showed normal glucose tolerance. The early insulin response to oral glucose was impaired in affected relatives, but was normal in those unaffected. These findings suggest that the glucokinase mutation raises the set-point of pancreatic beta cells for glucose-induced insulin secretion, leading to abnormal glucose tolerance in some patients with late-onset NIDDM.

Adolescent↗

PubMed1992

Characterization of GLUT3 protein expressed in Chinese hamster ovary cells.

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.

3-O-Methylglucose↗

PubMed1992

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.

Amino Acid Sequence↗

PubMed1992

Domains responsible for the differential targeting of glucose transporter isoforms.

Facilitative glucose transporter isoforms, GLUT1 and GLUT4, have different intracellular distributions despite their very similar structure. In insulin-responsive tissues such as adipose tissues and muscle, GLUT4 protein resides mainly in the intracellular region in a basal condition and is translocated to the plasma membrane upon stimulation of insulin. In contrast, GLUT1 protein was distributed about equally between plasma membranes and low density microsomal membranes in 3T3-L1 adipocytes. Furthermore, GLUT1 and GLUT4 were reported to be differentially targeted to the plasma membrane and intracellular region, respectively, when expressed in Chinese hamster ovary cells and HepG2 cells. To elucidate the differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1 have been stably expressed in Chinese hamster ovary cells. Immunofluorescence and immunoelectron microscopy as well as measurement of glucose transport activity revealed that two domains of GLUT4, which are not the NH2- or COOH-terminal domain, determine its targeting to the intracellular vesicles. The first domain contains the consensus sequence of the leucine zipper structure, suggesting that a dimer-forming structure of the glucose transporter might be required for its proper targeting. The other domain contains 28 amino acids, nine of which are different between GLUT1 and GLUT4. Immunoelectron microscopy revealed that the chimeric transporters containing both of these two domains of GLUT1, only the first domain of GLUT1, and none of the domains, exhibited a different cellular distribution with approximately 65, 30, and 15% of the transporters apparently on the plasma membrane, respectively. The addition of insulin did not alter the apparent cellular distributions of these chimeric transporters. These domains would be specifically recognized by intracellular targeting mechanisms in Chinese hamster ovary cells.

Animals↗

PubMed1992

Deletion of C-terminal 12 amino acids of GLUT1 protein does not abolish the transport activity.

We engineered the GLUT1 cDNA to delete C-terminal 12 amino acids of encoded GLUT1 protein. This mutated GLUT1 protein expressed in CHO cells by transfection of its cDNA was demonstrated to reside on the plasma membrane by cell surface labeling technique, and retain the transport activity, similar to that of the wild-type GLUT1. In addition, metabolic labeling of the intact cells with 35S indicated that the half-life of the mutated GLUT1 was not significantly different from that of the wild-type GLUT1. These results suggest that C-terminal 12 amino acids of GLUT1 are not important for the transport activity and the stability of the protein. Taken together with our previous results on the mutant without C-terminal 37 amino acids, the amino acids between the 37th and the 13th from the C-terminus appear to be essential for the transport activity.

Animals↗

PubMed1992

Glucose binding enhances the papain susceptibility of the intracellular loop of the GLUT1 glucose transporter.

Digestion of human GLUT1 protein in erythrocytes with 5 micrograms/ml papain for 5 min yielded several fragments. By using several site-specific antibodies, two of these fragments containing the intracellular loop domain between M6 and M7 were demonstrated to be further digested by a prolonged incubation with papain. The addition of 0.2 M D-glucose enhanced this digestion between M6 and M7 by approximately 3.5-fold, while the addition of 0.2 M D-sorbitol exhibited no effects. These results strongly suggest that D-glucose binding induces the conformational change of the intracellular loop domain between M6 and M7 of GLUT1 protein. Since the homology of the amino acid sequence was low in this intracellular domain among the five facilitative glucose transporter isoforms, this intracellular loop might contribute to the difference in their Km and Vmax values for glucose uptake.

Erythrocyte Membrane↗

PubMed1992

Two glucose transporter isoforms are sorted differentially and are expressed in distinct cellular compartments.

Rat GLUT4 (adipocyte/muscle-type glucose transporter) was expressed in two fibroblastic cell lines, Chinese hamster ovary (CHO) cells and 3T3-L1 fibroblasts, under the control of the methallothionein I promoter. Although immunoblotting with a GLUT4-specific anti-peptide antibody demonstrated that the amount of GLUT4 expressed was comparable with that in 3T3-L1 adipocytes and rat adipose tissues, no increase in 2-deoxy-D-glucose uptake was observed in the basal state in fibroblasts. Immunocytochemical studies showed that the expressed GLUT4 appeared to be localized in a specific region in the cytoplasm. These results were in marked contrast to those obtained in CHO cells expressing GLUT1 (HepG2/erythrocyte-type glucose transporter) using the same expression vector. In this case the expressed GLUT1 protein appeared to reside mainly on the plasma membranes, and a significant increase in glucose uptake was observed. Although insulin increased glucose uptake in CHO cells and 3T3-L1 fibroblasts as well as in the cells expressing rat GLUT4, an increment due to insulin above basal values was small, at most 2-fold, and no significant differences were observed in insulin-stimulated glucose uptake between transfected and parental cells. In addition, no apparent differences in the subcellular distribution of expressed GLUT4 were observed between the insulin-stimulated and the basal state. These results indicate that in fibroblastic cell lines GLUT1 and GLUT4 proteins are sorted in a different fashion, and the expression of GLUT4 protein per se is not enough to produce a large insulin-induced increase in glucose transport activity such as that observed in rat adipocytes and 3T3-L1 adipocytes. Thus unidentified aspects of the cellular environment which are present in the adipocytes but not in fibroblastic cell lines may be required for a large insulin-induced increase in glucose transport activity to be observed.

Adipose Tissue↗

PubMed1992

Development of a chemical form discrimination system for 14C species in gaseous waste.

A new system was developed for discriminating the chemical form of 14C species in gaseous waste from nuclear facilities. The system consists of an air sample reservoir, a concentration apparatus, a radio-gas chromatograph, and microcomputers. An air sample, which is part of the gaseous effluent released from a stack of a facility, is automatically collected in the tank by trigger signals of stack monitors. After passing the air through a silica gel column, carbon species contained in it are recovered in an activated carbon-supported palladium trap held at -120 degrees C. The recovered species are swept with helium gas and analyzed by the radiogas chromatograph using an activated carbon column and gas-flow counters. Output signals of the gas-flow counters are continuously recorded by the microcomputer. By using this system, the chemical forms of 14C, such as CO, CO2, CH4, C2H4, C2H6, and C3H8 are identified and quantified. A detectable concentration of 0.3 mBq cm-3 for 14CO2 can be obtained. The system has been used to monitor the gaseous waste released from the radioisotope production laboratory and the Japan Research Reactor 2 at Japan Atomic Energy Research Institute.

Air Pollutants, Radioactive↗

PubMed1992

Expression of glucose transporter isoforms with aging.

To elucidate the cellular mechanisms for impairment of glucose metabolism associated with aging, the facilitative glucose transporter protein and mRNA were studied in various tissues of young (7-week-old) and aged (20-month-old) rats. GLUT4 glucose transporter protein, a major glucose transporter isoform in the insulin-responsive tissues, was selectively decreased in the epididymal fat tissues of the aged rats compared with the young rats. This decrease is likely to be due to a decrease in protein synthesis rather than in protein stability, since GLUT4 mRNA per unit cellular total RNA was also decreased. GLUT4 mRNA in the skeletal muscle was rather increased in spite of the decreased level of GLUT4 protein in the aged rats, suggesting that the translational efficiency and/or stability of GLUT4 protein is decreased in the skeletal muscle of the aged rats compared with the young rats. In contrast to these alterations in GLUT4 expression, no apparent decrease in the GLUT1 protein amount was observed in the fat tissues, skeletal muscle and brain of the aged rats compared with the young rats. Thus, the tissue and isoform-specific alterations in glucose transporter expression are associated with aging and may contribute to impairment of glucose metabolism observed with aging.

Adipose Tissue↗

PubMed1992

Upregulation of GLUT2 mRNA by glucose, mannose, and fructose in isolated rat hepatocytes.

Previously, demonstrated that GLUT2 mRNA and protein are increased in liver of streptozocin-induced diabetic rats. To examine the mechanisms whereby GLUT2 mRNA is regulated, we cultured isolated hepatocytes in the absence and presence of various concentrations of glucose. Culture of hepatocytes in high glucose concentration (27.8 mM) for 20 h induced a 3.2-fold increase in GLUT2 mRNA levels compared with hepatocytes cultured without D-glucose. Interestingly, D-mannose and D-fructose could substitute for D-glucose to elevate the GLUT2 mRNA level, whereas 3-O-methyl-D-glucose, 2-deoxy-D-glucose, and sucrose, which were not metabolized or taken up by the cells, were without effect. Insulin had no significant effect on GLUT2 mRNA levels in hepatocytes in the presence or absence of D-glucose. Therefore, the regulation of the GLUT2 gene by D-glucose in hepatocytes is contrary to that reported for GLUT1 and GLUT4 genes, which are downregulated by D-glucose. These results also suggest that the elevated GLUT2 mRNA level observed in diabetic rat liver is due to the high blood glucose concentration rather than to insulin deficiency.

3-O-Methylglucose↗

PubMed1991

The role of N-glycosylation of GLUT1 for glucose transport activity.

To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.

Affinity Labels↗

PubMed1991

Expression of the GLUT1 glucose transporter increases thymidine uptake in Chinese hamster ovary cells at low glucose concentrations.

An increase in expression of the GLUT1 glucose transporter gene has been observed to be associated with an increase in glucose transport activity upon oncogenic transformation of the cells. Increased expression of this glucose transporter isoform has been also observed in fetal tissues. To investigate the consequences of this phenomenon on cellular metabolism and cell growth, an expression vector containing the GLUT1 glucose transporter complementary DNA was transfected into Chinese hamster ovary cells. Overexpression of this glucose transporter isoform resulted in an increase in not only glucose uptake and utilization but also thymidine uptake when cells were exposed to glucose-deficient conditions. This increase in glucose metabolism and DNA synthesis may play an important role on the growth and/or survival of cancer cells and fetal tissues.

3-O-Methylglucose↗

PubMed1991

The glucose transport activity of GLUT1 is markedly decreased by substitution of a single amino acid with a different charge at residue 415.

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of aspartic acid for asparagine 415, which is conserved among all facilitative glucose transporter isoforms. Although a significant amount of the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells by transfection with expression vector, almost no increase in glucose transport activity was observed. Analysis of glucose uptake with Lineweaver-Burk plot depicts that the mutation induced a marked decrease (more than 5-fold) in turnover number and a slight increase (1.5-fold) in Km compared with the wild-type GLUT1. Results obtained with cytochalasin B and ethylidene glucose suggested that the inner but not outer glucose binding site was modulated. These results suggest that asparagine 415 is located close to the inner glucose binding site and the putative inner gate of GLUT1 glucose transporter and that an ionic charge in this domain might play an important role in the rate of conformational change between an inward-facing form and an outward-facing form of glucose transporter.

Animals↗

PubMed1991

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter.

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.

Affinity Labels↗

PubMed1991

Altered expression of glucose transporter isoforms with aging in rats--selective decrease in GluT4 in the fat tissue and skeletal muscle.

To elucidate the cellular mechanisms of glucose intolerance associated with aging, both the protein and mRNA levels of glucose transporter isoforms were studied in the various tissues of young (7-week-old) and aged (20-month-old) rats. GluT4 (adipose/muscle-type glucose transporter) protein, which is specifically expressed in insulin-responsive tissues, was selectively decreased per milligram of cellular membrane protein in both the epididymal fat tissues and the gastrocnemius muscle of the aged rats compared with the young rats. When the changes in total cellular membranes per gram of tissue are taken into account, a further decrease in GluT4 protein per gram of tissue was observed in the tissues of the aged rats compared with the young rats. The decreased amount of GluT4 protein in the fat tissues of the aged rats is probably due to the decreased protein synthesis rather than the stability, since GluT4 mRNA/micrograms of cellular total RNA was also decreased. In contrast, GluT4 mRNA in the gastrocnemius muscle was rather increased and a ratio of GluT4 protein/GluT4 mRNA was decreased by 70% in the aged rats, suggesting that the translational efficiency and/or stability of GluT4 protein is decreased in the skeletal muscle of the aged rats compared with the young rats. GluT2 (liver-type glucose transporter) protein and mRNA in the liver were also decreased in the aged rats, while no apparent decrease in GluT1 (HepG2/brain-type glucose transporter) protein/mg of cellular membrane protein was observed in the skeletal muscle and fat tissues of the aged rats compared with the young rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1991

Impact of supersonic versus propeller wind data on dispersion estimates for nuclear plants.

Statistical analyses and comparisons of the wind data obtained by the propeller and supersonic anemometers at the Japan Atomic Energy Research Institute (JAERI) were performed. For wind speeds less than 1 ms-1, the propeller readings are inaccurate and are generally 0.5 ms-1 less than the supersonic readings. Thus, the number of calms recorded differs. These differences affect the sum of the inverse of wind speed, frequency distribution of wind direction, and the annual percentage frequency distribution of stability categories. The resultant average air concentrations and ground-level gamma exposure rates due to the normal operational releases from a nuclear plant are overestimated when calculated using the propeller wind data.

Air Pollutants, Radioactive↗

PubMed1991

Peptide-based radioimmunoassay specific for GLUT1 glucose transporter.

A radioimmunoassay for the GLUT1 glucose transporter was developed with a synthesized peptide based on the sequence of the cDNA for GLUT1. A peptide corresponding to the COOH-terminal domain of the GLUT1 glucose transporter (Thr-Pro-Glu-Glu-Leu-Phe-His-Pro-Leu-Gly-Ala-Asp-Ser-Gln-Val) was synthesized and conjugated to keyhole limpet hemocyanin through the NH2-terminal of the peptide. An antibody was raised against this complex and affinity purified with the immobilized peptide. A second peptide, with tyrosine residue added to the NH2-terminal of the above peptide, was synthesized and used as a standard and iodinated for preparation of the radioactive ligand. The assay is highly reproducible, sensitive, and specific for the COOH-terminal domain of the GLUT1 glucose transporter. It has no cross-reactivity with the other glucose-transporter isoforms GLUT2 and GLUT4. Furthermore, the results obtained with this radioimmunoassay on the number of glucose transporters in human erythrocytes were in good agreement with previous studies based on cytochalasin B binding, suggesting that this radioimmunoassay is able to quantify the number of glucose transporters. The assay is completed within 4 h and can be used for simultaneous measurement of GLUT1 in many samples. In addition, it can be applied to the measurement of GLUT1 in several types of tissue.

Amino Acid Sequence↗

PubMed1991

[Study on cases of D dimer values were dissociated from FDP-E].

Determination of FDP D-dimer (D-dimer) has been recently developed for the diagnosis of thrombotic diseases with secondary fibrinolysis. We have studied the correlation between D-dimer and FDP-E concentrations in plasma from 282 patients with 630 samples. A linear correlation (r = 0.9269) was observed between the values of FDP-E and D-dimer. However, 13 out of 282 cases revealed an apparent dissociation of D-dimer concentrations from FDP-E values. Among them, 4 of these 13 cases (Group A) have shown to possess higher level of D-dimer when compared with the expected values from FDP-E, while 9 of 13 cases (Group B) revealed lower levels of D-dimer than that expected from FDP-E. All of Group A patients have been diagnosed as disseminated intravascular coagulation (DIC). On the other hand, in Group B patients, 6 of 9 were shown to have a widespread metastasis of cancer and 2 of them were under treatment with urokinase. To study whether Group B patients were under hypercoagulable or hyper-fibrinolytic state, we have examined ratios of AT III/alpha 2 PI and PIC/TAT in these cases. It has been shown that 4 of 9 patients in Group B have higher ratios of both AT III/alpha 2 PI and PIC/TAT if compared with other patients than Group B. This suggests that patients in Group B have been under hyper-fibrinolytic states.

Adult↗