Search PubMed⌕ Search

Biomedical subjects

E Araki

Publications and source records attributed to E Araki.

At least 73 records · Page 4Linked to original sources

Cloning of the mouse insulin receptor substrate-1 (IRS-1) gene and complete sequence of mouse IRS-1.

The mouse IRS-1 gene has been cloned and its structure determined. Mouse IRS-1 differs from rat by the absence of the potential C-terminal nucleotide binding site. Otherwise, the predicted IRS-1 protein is highly conserved between mouse, rat and humans, especially in the possible phosphorylation sites. The highly conserved nature of IRS-1 suggests the importance of these domains in the function of IRS-1 or its association with other proteins.

Amino Acid Sequence↗

A riboflavin-responsive lipid storage myopathy due to multiple acyl-CoA dehydrogenase deficiency: an adult case.

A 62-year-old man was admitted to our hospital because of easy fatigability of the lower limbs during walking. The biopsied muscle specimen showed excessive lipid accumulation. The carnitine concentration in the muscle was at the lower level of the normal range. Organic acid urinalysis was consistent with the diagnosis of multiple acyl-CoA dehydrogenase deficiency or glutaric acidemia type II. In cultured lymphoblastoid cells from this patient there was impaired beta-oxidation, but the activities of acyl-CoA dehydrogenases were normal. Riboflavin therapy resulted in a dramatic improvement in both clinical and biochemical aspects. In this patient, the defect in coenzyme binding to electron transfer flavoprotein (ETF) or ETF-dehydrogenase was suspected. In the adult case of lipid storage myopathy, multiple acyl-CoA dehydrogenase deficiency should be suspected as one of its pathogenesis and riboflavin therapy should be considered.

Acyl-CoA Dehydrogenases↗

Regulation of insulin receptor, insulin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-F442A adipocytes. Effects of differentiation, insulin, and dexamethasone.

Insulin rapidly stimulates tyrosine kinase activity of its receptor resulting in phosphorylation of its cytosolic substrate insulin receptor substrate 1 (IRS-1), which in turn associates with and activates the enzyme phosphatidylinositol 3-kinase (PI 3-kinase). In the present study we have examined these three initial steps in insulin action during the differentiation of 3T3-F442A adipocytes and after treatment with dexamethasone or insulin. The differentiation of 3T3-F442A cells was characterized by a 13-fold increase in insulin receptor protein, a 9-fold increase in IRS-1, and a 10- and 4.5-fold increase in their insulin-stimulated phosphorylation, respectively. The mRNA expression of these two proteins showed a similar 8-fold increase during differentiation. In addition there was a 3.5-fold increase in PI 3-kinase protein [85 kilodalton (kDa) subunit] and a 16-fold increase in IRS-1-associated PI 3-kinase activity between day 0 and day 8 of differentiation. Dexamethasone (1 microM) treatment of differentiated cells induced a further 48% (P < 0.05) increase in insulin receptor level, but the autophosphorylation of the receptor was decreased by 31 +/- 1% (P < 0.02). At the same time there was a decrease by 56 +/- 4% (P < 0.005) in IRS-1 protein and by 31 +/- 1% (P < 0.001) in IRS-1 phosphorylation. The expression of insulin receptor mRNA was unchanged, but the expression of IRS-1 mRNA was decreased by approximately 75% after dexamethasone. By contrast, dexamethasone induced a 69% increase in the level of PI 3-kinase as determined by immunoblotting. The combined effect of decreased IRS-1 phosphorylation and increased PI 3-kinase protein was a minimal change (15% decrease) in the association/activation between IRS-1 and PI 3-kinase. Chronic treatment with 100 nM insulin induced a time- and dose-dependent decrease in insulin receptor and IRS-1 protein levels reaching a nadir of 34 +/- 5% (P < 0.005) and 39 +/- 5% (P < 0.01) of control levels after 24 h, respectively. There was an even more marked decrease in the phosphorylation level of these proteins. Chronic insulin treatment also produced a 30% decrease in PI 3-kinase protein levels and a approximately 50% decrease in the association/activation between IRS-1/PI 3-kinase. The expression of insulin receptor and IRS-1 mRNA was unchanged during chronic insulin treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

[Characterization and expression of the insulin receptor gene and its promoter].

Insulin initiates diverse biological effects by binding to the insulin receptor. The insulin receptor gene is located on the short arm of chromosome 19 and is more than 130 kb in length and contains 22 exons. Two species of mRNAs encoding insulin receptors that differ 12 amino acids in length exist by alternative splicing of the exon 11. The promoter region of the human insulin receptor gene is extremely GC rich and lacks typical TATA and CAAT boxes but contains multiple potential binding sites of Sp1, which are the characteristics of a "housekeeping" gene. Possible regulation of the expression of the insulin receptor gene by hormones such as insulin and dexamethasone and some transcription factors including Sp1 and C/EBP have been shown at the mRNA and transcription levels.

Animals↗

[Characterization of the IRS-1 (insulin receptor substrate-1) gene and its promoter].

IRS-1 (insulin receptor substrate-1) is a major substrate of the insulin receptor. Rat and human IRS-1 cDNAs, and human and mouse IRS-1 genes have been cloned so far. They show high homology in nucleic acids and amino acids levels, which indicate the high conservation of IRS-1 across the species. Interestingly, the entire coding region is contained in the 1st exon in the IRS-1 gene. The promoter of the mouse IRS-1 gene lacks TATA and CAAT boxes but contains 9 potential Spl binding sites, indicating that IRS-1 is a "housekeeping" gene. By deletion analysis, two positively and two negatively regulating fragments are identified in the promoter. In cultured adipocytes, insulin and dexamethasone down regulate IRS-1 expression by different mechanisms. Insulin down regulates at the post-translational level by shortening the protein half life, and dexamethasone down regulates at the post-transcriptional level mainly by shortening the mRNA half life.

Animals↗

Relationship between development of diarrhea and the concentration of SN-38, an active metabolite of CPT-11, in the intestine and the blood plasma of athymic mice following intraperitoneal administration of CPT-11.

Severe diarrhea occurred during daily intraperitoneal administration of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) at a dose of 50 mg/kg in athymic mouse. Serial determination of CPT-11 and 7-ethyl-10-hydroxycamptothecin (SN-38), with the use of an on-line solid extraction HPLC system, demonstrated that much higher levels of the compounds are retained in the intestine and the blood plasma after five consecutive daily injections than after a single injection. Histologic examination of the gastrointestinal tract showed hemorrhagic colitis on day 7 and later after five consecutive daily injections of CPT-11. The direct cause of diarrhea associated with CPT-11 administration is considered to be enterocolitis caused by high levels of SN-38 and/or CPT-11 retained for a long period in the intestine.

Animals↗

Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.

Amino Acid Sequence↗

Effects of antitumor agents on subcutaneous implants and hepatic metastases of colon carcinoma 26 in mice.

We investigated the responses of experimentally produced hepatic metastases of colon carcinoma 26 tumor and subcutaneously (SC) implanted colon carcinoma 26 tumor in mice to 17 clinically-used and one under-development antitumor agents using same dose regimen. In intravenous administrations on days 7 and 14, there were no significant differences in their responses to most of the tested agents. However, there were big differences in the responses to some of the agents. Nimustine more effectively prolonged the lifespan of SC implanted tumor-bearing mice than of mice bearing hepatic metastases. Mitomycin C was, however, considerably more effective on hepatic metastases than on SC implanted tumor. ME2303, a new fluorinated anthracycline derivative, showed a similar effect to doxorubicin on both tumors. However, administrations of ME2303 on days 7, 11 and 15 showed more marked antitumor effect only on hepatic metastases than administrations on days 7 and 14. Doxorubicin was less active against both tumors for administrations on days 7, 11 and 15 than for those on days 7 and 14. These results suggest the importance of the site of tumor growth for the action of some drugs. ME2303 may be active against hepatic metastases if it is administered by multiple injections.

Alanine Transaminase↗

Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

Animals↗

[Reproductive and developmental toxicity study of prednisolone farnesylate (PNF)--study by subcutaneous administration of PNF during the period of fetal organogenesis in rats].

A teratogenicity study of Prednisolone farnesylate (PNF), a newly synthesized corticosteroid, was conducted in Sprague-Dawley rats. This compound was administrated subcutaneously to female rats at dose levels of 0(control), 1, 5 and 25 mg/kg/day, once a day, for 11 days from day 7 to day 17 of pregnancy. In each dose group, 26 or 27 dams were killed on day 20 of pregnancy to examine their fetuses. The remaining 14 or 15 dams of each group were allowed to litter naturally, and observations were made on the postnatal growth and development of their offspring. 1. In the dams treated at doses of 1 mg/kg or more, decreased body weight gains and food consumption and retention of the substance at the injected site were noted. However, general signs, parturition, lactation and nursing behaviors were not affected by the administration of PNF. 2. In the F1 fetuses, no embryonic or fetal lethal effect, fetal retardation and teratogenic effect were noted. 3. In the F1 newborns, the postnatal growth, development, responses, behaviors, learning ability and reproductive ability were not influenced. Additionally, no embryonic or fetal abnormalities of their fetuses (F2) were detected. From these results, the no-effect dose levels of PNF on the parental general states, the parental reproductive ability and those of the F1 offspring are thought to be less than 1 mg/kg/day, 25 mg/kg/day and 25 mg/kg/day, respectively, under the experimental conditions of this study. Moreover, the F2 fetuses are not affected by doses up to 25 mg/kg/day of PNF.

Animals↗

[Reproductive and developmental toxicity study of prednisolone farnesylate (PNF)--study of subcutaneous administration of PNF during the perinatal and lactation periods in rats].

The effects of Prednisolone farnesylate (PNF), a newly synthesized corticosteroid, administrated during the perinatal and postnatal periods were studied in Sprague-Dawley rats. This compound was injected subcutaneously to female rats at dose levels of 0(control), 0.05, 0.5 and 5 mg/kg/day, once a day, for the period from day 17 of pregnancy to day 21 after parturition. Twenty-two to 25 dams in each dose group were allowed to litter naturally, and observations were made on the postnatal growth and development of their offspring. 1. In the dams treated at doses of 0.5 and 5 mg/kg, decreased body weight gains, atrophy of the thymus and retention of the substance at the injected site were noted. However, general signs, food consumption, parturition, lactation and nursing behaviors were not affected by the administration of PNF. 2. In the F1 newborns, the postnatal growth, development, responses, behaviors, learning ability and reproductive ability were not influenced. Additionally, no embryonic or fetal abnormalities of their fetuses (F2) were detected. From these results, the no-effect dose levels of PNF on the parental general states, the parental reproductive ability and those of the F1 offspring are thought to be 0.05 mg/kg/day, 5 mg/kg/day or more and 5 mg/kg/day or more, respectively, under the experimental conditions of this study. Moreover, the F2 fetuses are not affected by doses up to 5 mg/kg/day of PNF.

Animals↗

[Clinical assessment of urinary free L-fucose levels].

We measured urinary levels of free L-fucose in healthy subjects, patients with benign diseases, and patients with cancer using an automated analyzer and a newly isolated L-fucose dehydrogenase, and evaluated the clinical usefulness of the results. The values obtained were corrected for urinary creatinine as micromoles per gram of creatinine. The cutoff value, set at the mean + 2SD for the healthy subjects, was 250 mumol/g.Cr. Patients with gallbladder cancer, bile-duct cancer, liver cancer, pancreatic cancer, or cirrhosis of the liver had significantly higher levels of L-fucose than the healthy subjects. The diagnostic sensitivity for these five diseases, taken together, was 68% (144/213). Specificity for the detection of cancer was calculated by use of false positives for patients with cholelithiasis, hepatitis, and pancreatitis: it was 73% (76/104). Diagnostic accuracy for these seven diseases taken together was therefore 69% (220/317). We compared the positive ratio of the L-fucose level with that of the tumor markers AFD and CA19-9. The positive ratio of an L-fucose value above the cutoff was higher than the positive ratio of either marker in bile-duct cancer, gallbladder cancer, liver cancer, and pancreatic cancer. The results suggested that the urinary levels of free L-fucose reflected the metabolism of sugar chains of glycoconjugates, and may be usefully clinically as a tumor marker.

Biomarkers, Tumor↗

[Serum HDL-cholesterol in patients with breast carcinoma].

To extend our previous findings on lipid metabolism in breast carcinoma, serum levels of cholesterol and HDL-cholesterol in 144 patients with breast carcinoma, 64 patients with cervix carcinoma, 52 patients with benign breast diseases and 83 healthy females were determined. Blood samples, obtained from patients with carcinoma of the breast and the cervix, were taken prior to radical surgery and at the time of sampling, none of them had received chemotherapy and/or total intravenous nutritional therapy. No significance was observed in the serum levels of cholesterol between the patients with carcinoma, benign diseases and healthy females. The serum levels of HDL-cholesterol, measured by the heparin-calcium-nickel precipitation method were lower among the patients with breast carcinoma than among the patients with cervix carcinoma and healthy females. The mean values and S.D.s of serum HDL-cholesterol of the patients in stage I (23), II (35) and (7) were 42.0 +/- 14.7, 36. 4 +/- 13.1 and 28.7 +/- 9.5 mg/100 ml, respectively, exhibiting a tendency to decrease with the increase in malignancy.

Breast Neoplasms↗

Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.

Since the discovery of insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. Our discovery that insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signalling. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism.

Animals↗

A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene.

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.

Animals↗