Biomedical subjects

K Inukai

Publications and source records attributed to K Inukai.

At least 73 records · Page 4Linked to original sources

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

A multicenter randomized trial of high frequency oscillatory ventilation as compared with conventional mechanical ventilation in preterm infants with respiratory failure.

A multicenter randomised trial was conducted in nine neonatal centers in Japan to re-evaluate the safety and the efficacy of high frequency oscillatory ventilation using the piston type oscillator (Hummingbird) in the treatment of respiratory failure in preterm infants weighing between 750 and 2000 g at birth. A total of 92 infants were enrolled in the study. Forty-six infants were allocated to high frequency oscillatory ventilation and 46 infants to conventional mechanical ventilation. There were no differences in sex, birth weight, gestation and Apgar score between groups. The study was begun 2.0 +/- 1.6 h (mean +/- S.D.) after birth in the high frequency oscillation group and 1.7 +/- 1.5 h after birth in the conventional mechanical ventilation group. The absence of intraventricular hemorrhage was confirmed by echography in all cases before beginning ventilation. Mortality was similar in high frequency oscillatory ventilation and conventional mechanical ventilation (0 and 2%). The incidence of intraventricular hemorrhage was also similar in the high frequency and conventional mechanical ventilation groups (15 and 13% overall; 4 and 2% in grades III and IV, respectively). Nine percent of the infants in high frequency oscillatory ventilation and 13% in conventional mechanical ventilation developed bronchopulmonary dysplasia, but the difference was not significant. The frequency of air leaks was also equal in both groups. Periventricular leukomalacia was detected in 9% of infants on conventional mechanical ventilation and 2% on high frequency oscillation, but the difference was not significant. Mean airway pressure was significantly higher in the high frequency oscillatory ventilation group and the infants on high frequency oscillation showed a significantly higher arterial to alveolar oxygen tension ratio after 6 h of treatment. These results suggest that high frequency oscillatory ventilation does not increase the risk of severe complications such as air leaks, intraventricular hemorrhage or periventricular leukomalacia when it is used by experienced neonatologists. Indeed high frequency oscillatory ventilation helps provide better oxygenation with higher mean airway pressure without increasing the risk of bronchopulmonary dysplasia and severe complications such as air leaks and intraventricular hemorrhage.

Bronchopulmonary Dysplasia↗

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

Neonatal cerebral infarction: symptoms, CT findings and prognosis.

In a retrospective multi-center study, we investigated eighteen infants with unilateral cerebral infarctions confirmed by computed tomography (CT) scans. The initial symptoms were observed in all the patients between 0 and 3 days of age. Convulsions or apneic attacks were the initial symptoms in all but one. Only 4 patients had complicated obstetric histories and none showed polycythemia or electrolyte abnormalities. All of the initial CT scans revealed unilaterally localized hypodense areas. In 10, the initial CT scans were performed within 24 hours after the clinical onset. In 16, the lesions were within the territory of the middle cerebral artery, 9 of which also involved the cortico-spinal tract (CST). In the remaining 2 patients, the lesions were located within the territory of the posterior cerebral artery. None of the 9 patients without CST involvement developed hemiplegia, whereas 5 (56%) of the 9 with CST involvement had hemiplegia, which is a fairly low incidence compared with that in adult cases. This difference was thought to be related to neonatal brain plasticity.

Cerebral Infarction↗

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

[A case of Meige's syndrome associated with post head trauma].

The pathogenesis of Meige's syndrome (MS) is controversial and has yet to be determined up to today. We studied a case of MS associated with post head trauma. The patient was a 52-year-old female. At the age of 46, she began to suffer from oro-lingual dystonia after head trauma induced by a traffic accident and the brief administration of neuroleptics to the delusion deteriorated the dystonia. She showed a wry appearance after 1 year and 6 months of the trauma and began to exhibit blepharospasms, oro-mandibular dystonia and cervical dystonia after 2 years and 3 months. For these symptoms her daily life became difficult. These symptoms were resistant to various drug therapies, although trihexyphenidyl relieved the symptoms transiently. Laboratory examinations and cranial MRI findings were normal. By surface electromyogram of ocular orbicular muscles, bilateral continuous discharge was observed. This patient was diagnosed as MS by clinical symptoms and surface electromyogram findings. It was inferred that the head trauma was associated with the development of MS. We discussed the pathogenesis of MS in the present case and it was speculated that MS was presented by a minute lesion of the brain stem which was produced at the time of the head trauma.

Craniocerebral Trauma↗

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

Postnatal head growth in very premature infants with good outcome.

Postnatal head growth was examined retrospectively in 118 infants (male 60, female 58) born with a gestational age of from 24 to 29 weeks. Infants fulfilled the following criteria: (1) Those with hydrocephalus and small for dates (less than -1.5 standard deviation) were excluded. (2) Infants had normal intelligence and no cerebral palsy or epilepsy at 3 years of age or above. They were divided into four groups according to gestational ages: 24-26 wks (n = 21), 27 wks (n = 25), 28 wks (n = 41) and 29 wks (n = 31). The head circumference was measured at least twice monthly. The head growth curves in each group were obtained. After a transient decrease, head growth averaged 1.1-1.2 mm/day from day 21-30 to day 61-70 in all groups. From day 61-70 to 91-100 head growth averaged 1.2 mm/day at 24-26 wks and 0.8 mm/day at 27-29 wks, respectively.

Cephalometry↗

PubMed1991

Magnetic resonance imaging in children with spastic diplegia: correlation with the severity of their motor and mental abnormality.

Magnetic resonance imaging (MRI) findings for 34 children with spastic diplegia, examined between two and 10 years of age, were analysed. Dilatation of the trigone, atrophy of the peritrigonal white matter and prominent deep cortical sulci were seen. On T2-weighted images, periventricular high-intensity areas in the white matter adjacent to the trigones and bodies of the lateral ventricles were seen in many children. These MRI features may reflect the pathological changes of periventricular leukomalacia in children with spastic diplegia. Among the MRI findings, only the amount of white matter correlated with severity of disability: white matter reduction corresponded to the more severe motor disabilities.

Brain↗

PubMed1991

Leg movements in the supine position of infants with spastic diplegia.

Leg movements in the supine position of 49 infants with spastic diplegia (three to 11 months corrected age) were examined. Only simultaneous flexion and extension of the hips and knees were seen, with exceptional isolated hip movements; the simultaneous movements had synergic features. When the knees were flexed, the hips were flexed, abducted and externally rotated, and the ankles were dorsiflexed. When the knees were extended, the hips were extended, adducted and internally rotated and the ankles were plantar-flexed. Hip flexion combined with knee extension (leg elevation) and isolated knee movements were not seen in diplegic infants, but were seen in all control preterm infants with a good prognosis, after five and six months corrected age, respectively. The absence of these movements is a useful diagnostic item for spastic diplegia.

Cerebral Palsy↗