PubMed Health⌕ Search

Biomedical subjects

Toshiyasu Sasaoka

Publications and source records attributed to Toshiyasu Sasaoka.

At least 19 recordsLinked to original sources
PubMed2006

Novel G423S mutation of human alpha7 nicotinic receptor promotes agonist-induced desensitization by a protein kinase C-dependent mechanism.

The alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) gene harbors a high degree of polymorphism. In this study, we found a novel variant (1267 G to A) in exon 10 of the CHRNA7 gene in a Japanese population. This variant results in glycine-to-serine substitution at position 423 (G423S) located in the large cytoplasmic loop of the protein. To clarify the possibility that the G423S mutation alters the pharmacological properties of alpha7 receptors, acetylcholine (ACh)-elicited current through alpha7-G423S mutant receptors expressed in Xenopus laevis oocytes was measured using the two-electrode voltage-clamp technique. We found that the current elicited by ACh (1 mM, 5 s) through alpha7-G423S receptors, but not through alpha7 receptors, was significantly decreased by treatment with a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 10-30 nM). In addition, PMA (10 nM) selectively promoted a progressive decrease in alpha7-G423S current induced by repetitive application of ACh pulses (1 mM, 0.1 s, 0.17-0.33 Hz) compared with alpha7 current. PMA also enhanced the inactivation of alpha7-G423S mutant receptors induced by a prolonged application of choline (30 microM) without affecting alpha7 receptor responses. Western blot analysis showed that the treatment with PMA (30 nM) increased the serine phosphorylation level of the alpha7-G423S mutant receptors but not that of the wild-type receptors. These findings demonstrate that the G423S mutation promotes receptor desensitization by a protein kinase C-dependent mechanism. Thus, we provide the first evidence that a variant in the human CHRNA7 gene alters the function of alpha7 nicotinic receptors.

Adult↗
PubMed2006

Chronic nicotine exposure enhances insulin-induced mitogenic signaling via up-regulation of alpha7 nicotinic receptors in isolated rat aortic smooth muscle cells.

Insulin resistance and smoking are significant risk factors for cardiac and cerebral vascular diseases. Because vascular smooth muscle cells play a key role in the development and progression of atherosclerosis, we investigated the effect of nicotine on insulin-induced mitogenic signaling in aortic vascular smooth muscle cells isolated from Sprague Dawley rats. RT-PCR revealed the expression of alpha2-7, alpha10, beta1-3, delta, and epsilon subunits of the nicotinic acetylcholine receptor (nAChR) in the cells. Short-term nicotine treatment stimulated phosphorylation of p44/42-MAPK, p38-MAPK, and signal transducer and activator of transcription 3. However, an additive effect of nicotine pretreatment on insulin stimulation was only observed on p44/42-MAPK. The nicotine-induced phosphorylation of p44/42-MAPK and [methyl-(3)H]thymidine incorporation were effectively suppressed by a alpha7-nAChR-selective antagonist, methyllycaconitine, and the phosphorylation of p44/42-MAPK was stimulated by a alpha7-nAChR-specific agonist, GTS21. Furthermore, the phosphorylation was mediated via calmodulin kinase II, Src, and Shc. Interestingly, long-term (48-h) pretreatment with nicotine increased the amount of alpha7-AChR in the plasma membrane and insulin-induced phosphorylation of p44/42-MAPK. These results provide the first evidence that acute exposure to nicotine enhances insulin-induced mitogenesis predominantly by affecting the phosphorylation of p44/42-MAPK and that chronic exposure further augments the insulin signal via up-regulation of alpha7-nAChR, which may be crucial for the development and progression of atherosclerosis in large vessels.

Animals↗
PubMed2006

Lipid phosphatases as a possible therapeutic target in cases of type 2 diabetes and obesity.

Phosphatidyl inositol 3-kinase (PI3-kinase) functions as a lipid kinase to produce PI(3,4,5)P(3) from PI(4,5)P(2) in vivo. PI(3,4,5)P(3) is crucial as a lipid second messenger in various metabolic effects of insulin. Lipid phosphatases, src homology 2 domain containing inositol 5'-phosphatase 2 (SHIP2) and skeletal muscle and kidney-enriched inositol phosphatase (SKIP) hydrolyze PI(3,4,5)P(3) to PI(3,4)P(2) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) hydrolyzes PI(3,4,5)P(3) to PI(4,5)P(2). SHIP2 negatively regulates insulin signaling relatively specifically via its 5'-phosphatase activity. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity and conferred protection from obesity induced by a high-fat diet. Polymorphisms in the human SHIP2 gene are associated, at least in part, with the insulin resistance of type 2 diabetes. Importantly, inhibition of endogenous SHIP2 through the liver-specific expression of a dominant-negative SHIP2 improves glucose metabolism and insulin resistance in diabetic db/db mice. Overexpression of PTEN and SKIP also inhibited insulin-induced phosphorylation of Akt and the uptake of glucose in cultured cells. Although a homozygous disruption of the PTEN gene in mice results in embryonic lethality, either skeletal muscle or adipose tissue-specific disruption of PTEN ameliorated glucose metabolism without formation of tumors in animal models of diabetes. The role of SKIP in glucose metabolism remains to be further clarified in vivo. Taken together, inhibition of endogenous SHIP2 in the whole body appears to be effective at improving the insulin resistance associated with type 2 diabetes and/or obesity. Inhibition of PTEN in the tissues specifically targeted, including skeletal muscle and fat, may result in an amelioration of insulin resistance in type 2 diabetes, although caution against the formation of tumors is needed.

Animals↗
PubMed2005

Altered subcellular distribution of estrogen receptor alpha is implicated in estradiol-induced dual regulation of insulin signaling in 3T3-L1 adipocytes.

We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17beta-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10(-8) m, but inhibited these effects at 10(-5) m. A concentration of 10(-5) m E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser(307), which was abolished by treatment with a c-Jun NH(2)-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10(-8) m, but inhibited it at 10(-5) m. Furthermore, E2 decreased the amount of estrogen receptor alpha at the plasma membrane at 10(-8) m, but increased it at 10(-5) m. In contrast, the subcellular distribution of estrogen receptor beta was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser(307) via a c-Jun NH(2)-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor alpha in response to E2 may explain the dual effect of E2.

3T3 Cells↗
PubMed2005

Interleukin-1alpha inhibits insulin signaling with phosphorylating insulin receptor substrate-1 on serine residues in 3T3-L1 adipocytes.

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1alpha is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1alpha may also cause insulin resistance. Here, we investigated the effects of IL-1alpha treatment on insulin signaling in 3T3-L1 adipocytes. IL-1alpha treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1alpha stimulation, when several serine kinases, IkappaB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1alpha-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1alpha. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1alpha in the presence of IL-6. Taken together, short-term IL-1alpha treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1alpha may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.

3T3-L1 Cells↗
PubMed2005

Antiangiogenic activity of beta-eudesmol in vitro and in vivo.

Abnormal angiogenesis is implicated in various diseases including cancer and diabetic retinopathy. In this study, we examined the effect of beta-eudesmol, a sesquiterpenoid alcohol isolated from Atractylodes lancea rhizome, on angiogenesis in vitro and in vivo. Proliferation of porcine brain microvascular endothelial cells and human umbilical vein endothelial cells (HUVEC) was inhibited by beta-eudesmol (50-100 microM). It also inhibited the HUVEC migration stimulated by basic fibroblast growth factor (bFGF) and the tube formation by HUVEC in Matrigel. beta-eudesmol (100 microM) blocked the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 induced by bFGF or vascular endothelial growth factor. Furthermore, beta-eudesmol significantly inhibited angiogenesis in subcutaneously implanted Matrigel plugs in mice and in adjuvant-induced granuloma in mice. These results indicate that beta-eudesmol inhibits angiogenesis, at least in part, through the blockade of the ERK signaling pathway. We considered that beta-eudesmol may aid the development of drugs to treat angiogenic diseases.

Angiogenesis Inhibitors↗
PubMed2005

Impact of SRC homology 2-containing inositol 5'-phosphatase 2 gene polymorphisms detected in a Japanese population on insulin signaling.

Src homology 2-containing 5'-inositol phosphatase 2 (SHIP2) is known to be one of lipid phosphatases converting PI(3,4,5)P3 to PI(3,4)P2 in the negative regulation of insulin signaling with the fundamental impact on the state of insulin resistance. To clarify the possible involvement of SHIP2 in the pathogenesis of human type 2 diabetes, we examined the relation of human SHIP2 gene polymorphisms to type 2 diabetes in a Japanese population. We identified 10 polymorphisms including four missense mutations. Among them, single nucleotide polymorphism (SNP)3 (L632I) was located in the 5'-phosphatase catalytic region, and SNP5 (N982S) was adjacent to the phosphotyrosine binding domain binding consensus motif in the C terminus. SNP3 was found more frequently in control subjects than in type 2 diabetic patients, suggesting that this mutation might protect from insulin resistance. Transfection study showed that expression of SNP3-SHIP2 inhibited insulin-induced PI(3,4,5)P3 production and Akt2 phosphorylation less potently than expression of wild-type SHIP2 in CHO-IR cells. Insulin-induced tyrosine phosphorylation of SNP5-SHIP2 was decreased compared with that of wild-type SHIP2, resulting in increased Shc/Grb2 association and MAPK activation. These results indicate that the polymorphisms of SHIP2 are implicated, at least in part, in type 2 diabetes, possibly by affecting the metabolic and/or mitogenic insulin signaling in the Japanese population.

Aged↗
PubMed2005

Marine alkaloids (-)-pictamine and (-)-lepadin B block neuronal nicotinic acetylcholine receptors.

Ascidians (sea squirts) contain a wealth of alkaloids, but their influence over neuronal nicotinic acetylcholine receptors (nAChRs) has not been evaluated. In this study, we examined the effects of two synthetic compounds, (-)-pictamine, a quinolizidine alkaloid from Clavelina picta, and (-)-lepadin B, a decahydroquinoline alkaloid from Clavelina lepadiformis, on major types of neuronal nicotinic receptors (alpha4beta2 and alpha7) expressed in Xenopus oocytes. We found that these alkaloids are potent blockers at these receptors: acetylcholine-elicited currents through alpha4beta2 and alpha7 receptors were blocked by (-)-pictamine with IC(50) values of 1.5 microM and 1.3 microM, respectively, and by (-)-lepadin B with IC(50) values of 0.9 microM and 0.7 microM, respectively. Interestingly, no recovery was observed after the removal of (-)-pictamine in oocytes expressing alpha4beta2 receptors, whereas the inhibited alpha7 currents quickly recovered after the removal of (-)-pictamine. Since there are few compounds that elicit irreversible blocks of alpha4beta2 receptors, (-)-pictamine will be a novel, valuable tool to remove the alpha4beta2-nAChR action from neuronal activities mediated by these two major types of nAChRs.

Alkaloids↗
PubMed2005

Impact of the liver-specific expression of SHIP2 (SH2-containing inositol 5'-phosphatase 2) on insulin signaling and glucose metabolism in mice.

We investigated the role of hepatic SH2-containing inositol 5'-phosphatase 2 (SHIP2) in glucose metabolism in mice. Adenoviral vectors encoding wild-type SHIP2 (WT-SHIP2) and a dominant-negative SHIP2 (DeltaIP-SHIP2) were injected via the tail vein into db/+m and db/db mice, respectively. Four days later, amounts of hepatic SHIP2 protein were increased by fivefold. Insulin-induced phosphorylation of Akt in liver was impaired in WT-SHIP2-expressing db/+m mice, whereas the reduced phosphorylation was restored in DeltaIP-SHIP2-expressing db/db mice. The abundance of mRNA for glucose-6-phosphatase (G6Pase) and PEPCK was increased, that for glucokinase (GK) was unchanged, and that for sterol regulatory element-binding protein 1 (SREBP)-1 was decreased in hepatic WT-SHIP2-overexpressing db/+m mice. The increased expression of mRNA for G6Pase and PEPCK was partly suppressed, that for GK was further enhanced, and that for SREBP1 was unaltered by the expression of DeltaIP-SHIP2 in db/db mice. The hepatic expression did not affect insulin signaling in skeletal muscle and fat tissue in both mice. After oral glucose intake, blood glucose levels and plasma insulin concentrations were elevated in WT-SHIP2-expressing db/+m mice, while elevated values were decreased by the expression of DeltaIP-SHIP2 in db/db mice. These results indicate that hepatic SHIP2 has an impact in vivo on the glucose metabolism in both physiological and diabetic states possibly by regulating hepatic gene expression.

Animals↗
PubMed2004

Deletion of the PDGFR-beta gene affects key fibroblast functions important for wound healing.

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.

Animals↗
PubMed2004

Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans.

BACKGROUND: Green tea is widely consumed in Asian countries and is becoming increasingly popular in Western countries. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The present study was aimed at providing evidence of improvement in glucose metabolism in diabetic mice and healthy humans upon green tea consumption. RESULTS: Green tea promoted glucose metabolism in healthy human volunteers at 1.5 g/body in oral glucose tolerance tests. Green tea also lowered blood glucose levels in diabetic db+/db+ mice and streptozotocin-diabetic mice 2-6 h after administration at 300 mg/kg without affecting serum insulin level, whereas no effect was observed in control mice (+m/+m and normal ddY mice). The serum protein profiles of db+/db+ and +m/+m mice were analyzed for the first time by SELDI (surface-enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), and then compared to investigate any effects of oral green tea administration on serum proteins. The protein profiles in db+/db+ mice showed that the spectral peak intensities at the mass/charge ratios (m/z) of 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those of 13075, 17406, 17407, 17418, 17622, 18431 and 26100 were >3 times higher than respective peak intensities in +m/+m mice. When green tea was administered to db+/db+ mice, the peak intensities were markedly decreased at m/z 11651 and 11863, and slightly decreased at m/z 4212. The peak intensities at 7495, 7595, 7808, 14983, 15614, 31204 were markedly increased after the administration. CONCLUSION: The present study provides evidence that green tea has an antidiabetic effect. Although we could not find simple reversed effect of green tea on the diabetes-induced modifications of the levels of several serum proteins, we found that the 4211 (4212) Da protein level that was decreased in the diabetic state was further decreased after green tea administration. This is the first report demonstrating that a certain serum protein may be involved in the antihyperglycemic effect of green tea. The contribution of this protein should be further studied.

Animals↗
PubMed2004

Alkaloids indolizidine 235B', quinolizidine 1-epi-207I, and the tricyclic 205B are potent and selective noncompetitive inhibitors of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors are key molecules in cholinergic transmission in the nervous system. Because of their structural complexity, only a limited number of subtype-specific agonists and antagonists are available to study nicotinic receptor functions. To overcome this limitation, we used voltageclamp recordings to examine the effects of several frog skin alkaloids on acetylcholine-elicited currents in Xenopus laevis oocytes expressing major types of neuronal nicotinic receptors (alpha4beta2, alpha7, alpha3beta2, alpha3beta4, and alpha4beta4). We found that the 5,8-disubstituted indolizidine (-)-235B' acted as a potent noncompetitive blocker of alpha4beta2 nicotinic receptors (IC50 = 74 nM). This effect was highly selective for alpha4beta2 receptors compared with alpha3beta2, alpha3beta4, and alpha4beta4 receptors. The inhibition of alpha4beta2 currents by (-)-235B' was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 microM). Moreover, the blockade of alpha4beta2 currents by (-)-235B' was voltage-dependent (more pronounced at hyperpolarized potentials) and use-dependent, indicating that (-)-235B' behaves as an open-channel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((-)-223A and (+)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((+)-207I) were less potent than (-)-235B', and none showed selectivity for alpha4beta2 receptors. The quinolizidine (-)-1-epi-207I and the tricyclic (+)-205B had 8.7- and 5.4-fold higher sensitivity, respectively, for inhibition of the alpha7 nicotinic receptor than for inhibition of the alpha4beta2 receptor. These results show that frog alkaloids alter the function of nicotinic receptors in a subtype-selective manner, suggesting that an analysis of these alkaloids may aid in the development of selective drugs to alter nicotinic cholinergic functions.

Alkaloids↗
PubMed2004

Impact of Src homology 2-containing inositol 5'-phosphatase 2 on the regulation of insulin signaling leading to protein synthesis in 3T3-L1 adipocytes cultured with excess amino acids.

Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) possesses 5'-phosphatase activity to specifically hydrolyze the phosphatidylinositol 3-kinase product PI(3,4,5)P3 in the regulation of insulin signaling. In the present study, we examined the impact of SHIP2 on the regulation of insulin signaling leading to protein synthesis in 3T3-L1 adipocytes cultured with standard and excess concentrations of amino acids. Insulin-induced translocation of PDK1 to the plasma membrane, phosphorylation of Akt and p70S6-kinase and ribosomal protein S6, increase in the amount of 4E-BP1 gamma-form, association of eIF4E with eIF4G, and protein synthesis were decreased by overexpression of wild-type SHIP2 by adenovirus-mediated gene transfer. The effect of SHIP2 overexpression on the regulation of insulin-induced phosphorylation of Akt and p70S6-kinase was somewhat augmented by the incubation with 5-fold excess concentrations of amino acids for 30 min. In contrast, the impact of SHIP2 expression was diminished in insulin-induced phosphorylation of p70S6-kinase and S6, but not of Akt, after the incubation for 16 h. Interestingly, incubation with the excess concentrations of amino acids for 30 min induced activation of phosphatidylinositol 3-kinase and phosphorylation of Akt, whereas phosphorylation of p70S6-kinase and S6 was decreased. Furthermore, although the exposure for longer time periods up to 24 h did not elicit phosphorylation of Akt, it markedly induced phosphorylation of p70S6-kinase and S6. These results indicate that SHIP2 plays an important role in the negative regulation of insulin signaling for the protein synthesis and that the impact of SHIP2 is altered, dependent on the acute or chronic exposure of excess concentrations of amino acids in culture.

3T3-L1 Cells↗
PubMed2004

SH2-containing inositol phosphatase 2 predominantly regulates Akt2, and not Akt1, phosphorylation at the plasma membrane in response to insulin in 3T3-L1 adipocytes.

SH2-containing inositol phosphatase 2 (SHIP2) is a physiologically important negative regulator of insulin signaling by hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI 3,4,5-trisphosphate in the target tissues of insulin. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity without affecting biological systems other than insulin signaling. Therefore, we investigated the molecular mechanisms by which SHIP2 specifically regulates insulin-induced metabolic signaling in 3T3-L1 adipocytes. Insulin-induced phosphorylation of Akt, one of the molecules downstream of PI3-kinase, was inhibited by expression of wild-type SHIP2, whereas it was increased by expression of 5'-phosphatase-defective (DeltaIP) SHIP2 in whole cell lysates. The regulatory effect of SHIP2 was mainly seen in the plasma membrane (PM) and low density microsomes but not in the cytosol. In this regard, following insulin stimulation, a proportion of Akt2, and not Akt1, appeared to redistribute from the cytosol to the PM. Thus, insulin-induced phosphorylation of Akt2 at the PM was predominantly regulated by SHIP2, whereas the phosphorylation of Akt1 was only minimally affected. Interestingly, insulin also elicited a subcellular redistribution of both wild-type and DeltaIP-SHIP2 from the cytosol to the PM. The degree of this redistribution was inhibited in part by pretreatment with PI3-kinase inhibitor. Although the expression of a constitutively active form of PI3-kinase myr-p110 also elicited a subcellular redistribution of SHIP2 to the PM, expression of SHIP2 appeared to affect the myr-p110-induced phosphorylation, and not the translocation, of Akt2. Furthermore, insulin-induced phosphorylation of Akt was effectively regulated by SHIP2 in embryonic fibroblasts derived from knockout mice lacking either insulin receptor substrate-1 or insulin receptor substrate-2. These results indicate that insulin specifically stimulates the redistribution of SHIP2 from the cytosol to the PM independent of 5'-phosphatase activity, thereby regulating the insulin-induced translocation and phosphorylation of Akt2 at the PM.

3T3-L1 Cells↗
PubMed2004

Nicotinic enhancement of proliferation in bovine and porcine cerebral microvascular endothelial cells.

Nicotinic acetylcholine receptors are found in microvascular endothelial cells. To reveal the functional role in cerebral angiogenic processes, we studied the nicotinic modulation of proliferation activity in cultured bovine and porcine cerebral microvascular endothelial cells. The proliferation activity was determined by an increase in the number of cells present in culture dishes. When the bovine cerebral endothelial cells at different passages were cultured in the presence of nicotine (10 nM), the proliferation activities were significantly increased in the cells at passage 1 and passage 3, but not at passage 4. Reverse transcriptase-polymerase chain reaction studies demonstrated that the expression of mRNAs coding for alpha3 nicotinic receptor subunit was significantly reduced in the bovine cerebral endothelial cells at passage 4, compared with that at passage 1. The proliferation of porcine cerebral endothelial cells (passage 1) was enhanced by acetylcholine (10 nM-100 microM) in the presence of atropine, a muscarinic antagonist, and this enhancing effect was inhibited by hexamethonium (100 microM, a nicotinic antagonist). The stimulation by acetylcholine (1 microM, with atropine) or nicotine (10 nM) induced the phosphorylation of a mitogen-activated protein (MAP) kinase (extracellular-signal regulated kinase: ERK) in the serum-starved endothelial cells. In the presence of PD98059 (2 microM, a MAP kinase kinase inhibitor) and atropine, acetylcholine (1 microM) failed to enhance the proliferation of porcine cerebral endothelial cells. These results demonstrate that nicotinic stimulation promotes the proliferation of bovine and porcine cerebral microvascular endothelial cells, at least in part, through the MAP kinase activation.

Animals↗
PubMed2003

Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P(3) to PI(4,5)P(2). Since the PI3-kinase product, PI(3,4,5)P(3), is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non-insulin-dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non-diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position -9 (-9C-->G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild-type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin-induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild-type PTEN gene. These findings suggest that the change of C to G at position -9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product.

5' Untranslated Regions↗
PubMed2003

Active Src expression is induced after rat peripheral nerve injury.

The non-receptor-type Src tyrosine kinases are key components of intracellular signal transduction that are expressed at high levels in the nervous system. To improve understanding of the cascades of molecular events underlying peripheral nerve regeneration, we analyzed active Src expression in the crushed or cut rat sciatic nerves using a monoclonal antibody (clone 28) that recognizes the active form of Src tyrosine kinases, including c-Src and c-Fyn. Western blots showed that active Src expressed in the normal sciatic nerve transiently increased up to threefolds after both types of injury. Immunohistochemistry using clone 28 showed that axonal components are the primary sites of active Src expression in the normal sciatic nerve. Soon after both types of injury, active Src was abundantly expressed in Schwann cells of the segments distal to the injury site. The expression of active Src in the cells decreased with restoration of the axon-Schwann cell relationship and eventually became depleted to very low levels after crushing, but was sustained at high levels in the cut model until the end of the experiment. Regenerated axons consistently expressed active Src throughout nerve regeneration and these eventually became the major sites of active Src expression in the crushed nerve. Among the Src tyrosine kinases, active c-Src selectively increased after crushing according to immunoprecipitation and immunoblotting analyses. Due to its potent biological activity, the increased amounts of the active form of Src probably enhance axonal regrowth, the Schwann cell response, and axon-Schwann cell contact for peripheral nerve regeneration.

Animals↗