Biomedical subjects

K Tsukuda

Publications and source records attributed to K Tsukuda.

At least 55 records · Page 3Linked to original sources

PubMed1994

Successful treatment of cytophagic histiocytic panniculitis with modified CHOP-E. Cyclophosphamide, adriamycin, vincristine, predonisone, and etoposide.

A 27-year-old woman developed generalized subcutaneous painful nodules, fever, abnormal liver function, a bleeding tendency, and pancytopenia. Skin biopsies revealed the lobular panniculitis with a morphologically benign histiocytic infiltration with prominent phagocytosis. Leukophagocytosis and erythrophagocytosis were also present in the bone marrow. The diagnosis of cytophagic histiocytic panniculitis was made. The patient received polychemotherapy with cyclophosphamide, Adriamycin, and vincristine on day 1, prednisone on day 1-5 (modified CHOP), with the addition of etoposide (E). This regimen was repeated 8 times every 3 weeks. The patient obtained a complete clinical remission that has lasted almost 2 years after the completion of chemotherapy. Thus we suggest modified CHOP-E chemotherapy for an effective treatment for the aggressive form of cytophagic histiocytic panniculitis.

Adult↗

PubMed1993

Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin.

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.

3-O-Methylglucose↗

PubMed1993

Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets.

Glucose-stimulated insulin secretion, glucose transport, glucose phosphorylation and glucose utilization have been characterized in the insulinoma cell line MIN6, which is derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells. Glucose-stimulated insulin secretion occurred progressively from 5 mmol/l glucose, reached the maximal level approximately seven-fold above the basal level at 25 mmol/l, and remained at this level up to 50 mmol/l. Glucose transport was very rapid with the half-maximal uptake of 3-O-methyl-D-glucose being reached within 15 s at 22 degrees C. Glucose phosphorylating activity in the cell homogenate was due mainly to glucokinase; the Vmax value of glucokinase activity was estimated to be 255 +/- 37 nmol.h-1.mg protein-1, constituting approximately 80% of total phosphorylating activity, whereas hexokinase activity constituted less than 20%. MIN6 cells exhibited mainly the high Km component of glucose utilization with a Vmax of 289 +/- 18 nmol.h-1.mg protein-1. Thus, glucose utilization quantitatively and qualitatively reflected glucose phosphorylation in MIN6 cells. In contrast, MIN7 cells, which exhibited only a small increase in insulin secretion in response to glucose, had 4.7-fold greater hexokinase activity than MIN6 cells with a comparable activity of glucokinase. These characteristics of MIN6 cells are very similar to those of isolated islets, indicating that this cell line is an appropriate model for studying the mechanism of glucose-stimulated insulin secretion in pancreatic beta cells.

3-O-Methylglucose↗

PubMed1993

Expression of GLUT-4 glucose transporter in unweighted soleus muscle of normal and STZ-induced diabetic rats.

Effects of 6 days of hindlimb suspension on expression of glucose transporters were studied in the skeletal muscle of nondiabetic and streptozotocin-induced diabetic rats. Although total membrane protein recovered from soleus muscles tended to decrease with suspension, GLUT-4 protein concentration (amount per gram membrane protein) was increased by 66 and 91% compared with weight-bearing control in nondiabetic and diabetic rats, respectively. Therefore, the amount of GLUT-4 protein in whole soleus muscle did not decrease with the hindlimb suspension in normal and diabetic rats. In contrast, hindlimb suspension decreased GLUT-4 mRNA amount in whole soleus muscle by 47 and 27% in nondiabetic and diabetic rats, respectively. Thus the GLUT-4 protein-to-GLUT-4 mRNA ratio was increased 2.1-fold in nondiabetic and 1.4-fold in diabetic rats. The extensor digitorum longus muscle, which generally shows little response to unweighting, exhibited no such changes. These results suggest that the amount of GLUT-4 glucose transporter in the unweighted soleus muscle was maintained via a translational and/or posttranslational mechanism in nondiabetic rats as well as in streptozotocin-induced diabetic rats under the condition of reduced weight-bearing activity.

Animals↗

PubMed1993

Cloning and increased expression with fructose feeding of rat jejunal GLUT5.

We have isolated a clone from the rat jejunum cDNA library using a fragment of human GLUT5 cDNA as a probe. The coding region of this clone shares 80% nucleotide and 81% amino acid identity with human GLUT5 and is thus termed rat GLUT5 cDNA. Rat GLUT5 mRNA exhibited a tissue distribution very similar to that of human GLUT5, with the highest levels in the jejunum, but was not detected in fat, muscle, or testis on Northern blot analysis. The antipeptide antibody raised against the C-terminal domain of rat GLUT5 protein specifically recognized a rat jejunal protein with an apparent mol wt of 60,000 on immunoblots. The amount of GLUT5 mRNA and protein in the jejuni of rats fed a fructose-enriched diet (50%, wt/wt) for 3 days were increased 2.5- and 6-fold, respectively, compared to those of rats fed standard rat chow, whereas those of rats fed a starch-enriched diet (50%, wt/wt) were not altered. Similarly, GLUT5 mRNA and protein in the jejunum were increased 5- and 8-fold, respectively, after 15 days of fructose feeding. Thus, an increase in fructose absorption up-regulates GLUT5 expression in the jejunum. These results are consistent with the notion that GLUT5 plays a major role in fructose absorption in the small intestine.

Amino Acid Sequence↗

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

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↗

PubMed1992

[THP-adriamycin-based combination chemotherapy in the treatment of non-Hodgkin's lymphoma].

Thirty patients with non-Hodgkin's lymphoma were treated with THP-adriamycin-based combination chemotherapy. Complete remission was obtained in eighteen (72%) of 25 evaluable patients. Nine of 18 patients are still alive in complete remission lasting 9 to 34 months (median 24 mo). Severe side effects such as bone marrow suppression, gastrointestinal symptoms and cardiotoxicity were rare. It was concluded that THP-adriamycin-based combination chemotherapy is relatively mild and safe, so this regimen is useful for elderly patients or patients with complications.

Adult↗

PubMed1992

[Second marrow transplantation following high dose busulfan, etoposide, and Ara-C after testicular relapse in a patient with AML].

Prognosis of second marrow transplantation after leukemia relapse is usually gloomy. We report a patient with AML who was successfully treated by the second marrow transplant following high dose busulfan, etoposide, and Ara-C for the testicular relapse after the first marrow transplantation. A 24-year-old man was diagnosed as having acute myeloid leukemia (AML) in September, 1988. In December of 1989 when he was in early relapse after his 2nd remission, he received the first allogeneic BMT from his HLA identical brother after high dose busulfan and cyclophosphamide conditioning. His posttransplant course was uneventful and graft versus host disease was not observed. Three months after BMT, he noticed swelling on right testicle. Leukemic cell infiltration was confirmed by aspiration cytology. The testicular relapse was followed by marrow relapse. After successful remission induction chemotherapy, he received 17.5 Gy testicular irradiation and second marrow transplantation using high dose busulfan, etoposide, and Ara-C conditioning. Although his posttransplant period was complicated by severe mucositis, high fever and bronchopneumonia, hematologic recovery was obtained by 3 weeks after the second transplant. He is now continuing in complete remission 18 months after the second BMT. This case report suggests that the combination of high dose busulfan, etoposide, and Ara-C could be a choice as a conditioning regimen for resistant AML relapsing after BMT.

Adult↗

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↗