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Biomedical subjects

Kazuhiro Eto

Publications and source records attributed to Kazuhiro Eto.

32 records · Page 2Linked to original sources
PubMed2003

Exposure of neonatal rats to diethylstilbestrol affects the expression of genes involved in ovarian differentiation.

Exposing neonatal rats with the synthetic estrogen, diethylstilbestrol (DES), induces morphological and functional abnormalities in the adult ovary. We examined the events that lead to this condition using female rats that were exposed to DES for the first five days after birth. The expression of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), which are both required for steroidogenesis in the theca/interstitial region was markedly reduced. The expression of Mullerian inhibiting substance (MIS) was transiently increased in small growing follicles in the ovary of DES-treated rats at postnatal day 7 (P7), and the expression profile in the ovary differed between DES- and vehicle oil-treated rats at P14 and P21. The expression of the transcription factor, steroidogenic factor-1 (SF-1), reduced in theca/interstitial cells, but increased in granulosa cells of primary follicles. These results indicate that altered steroidogenesis and MIS production are mechanisms through which DES induces abnormal ovarian development, and support the notion that androgens and MIS are both critical factors in regulating early ovarian differentiation.

Analysis of Variance↗
PubMed2002

Impact of genetic background and ablation of insulin receptor substrate (IRS)-3 on IRS-2 knock-out mice.

Although we and others have generated IRS-2 knock-out (IRS-2(-/-)) mice, significant differences were seen between the two lines of IRS-2(-/-) mice in the severity of diabetes and alterations of beta-cell mass. It has been reported that although IRS-1 and IRS-3 knock-out mice showed normal blood glucose levels, IRS-1/IRS-3 double knock-out mice exhibited marked hyperglycemia. Thus, IRS-1 and IRS-3 compensate each other's functions in maintaining glucose homeostasis. To assess the effect of genetic background and also ablation of IRS-3 on IRS-2(-/-), we generated IRS-2/IRS-3 double knock-out (IRS-2(-/-)IRS-3(-/-)) mice by crossing IRS-3(-/-) mice (129/Sv and C57Bl/6 background) with our IRS-2(-/-) mice (CBA and C57Bl/6 background). Intercrosses of IRS-2(+/-)IRS-3(+/-) mice yielded nine genotypes, and all of them including IRS-2(-/-)IRS-3(-/-) mice were apparently healthy and showed normal growth. However, at 10-20 weeks of age, 20-30% mice carrying a null mutation for the IRS-2 gene, irrespective of the IRS-3 genotype, developed diabetes. When mice with diabetes were excluded from the analysis of glucose and insulin tolerance test, IRS-2(-/-)IRS-3(-/-) showed a degree of glucose intolerance and insulin resistance similar to those of IRS-2(-/-) mice. Both IRS-2(-/-) and IRS-2(-/-)IRS-3(-/-) mice had moderately reduced beta-cell mass despite having insulin resistance. Insulin-positive beta-cells were decreased to nearly zero in IRS-2(-/-) mice with diabetes. Although Pdx1 and glucose transporter 2 expressions were essentially unaltered in islets from IRS-2(-/-) mice without diabetes, they were dramatically decreased in IRS-2(-/-) mice with diabetes. Taken together, these observations indicate that IRS-3 does not play a role compensating for the loss of IRS-2 in maintaining glucose homeostasis and that the severity of diabetes in IRS-2(-/-) mice depends upon genetic background, suggesting the existence of modifier gene(s) for diabetes in mice of the 129/Sv genetic strain.

Animals↗
PubMed2002

Mouse GLI3 regulates Fgf8 expression and apoptosis in the developing neural tube, face, and limb bud.

The zinc finger transcription factor GLI3 is considered a repressor of vertebrate Hedgehog (Hh) signaling. In humans, the absence of GLI3 function causes Greig cephalopolysyndactyly syndrome, affecting the development of the brain, eye, face, and limb. Because the etiology of these malformations is not well understood, we examined the phenotype of mouse Gli3-/- mutants as a model to investigate this. We observed an up-regulation of Fgf8 in the anterior neural ridge, isthmus, eye, facial primordia, and limb buds of mutant embryos, sites coinciding with the human disease. Intriguingly, endogenous apoptosis was reduced in Fgf8-positive areas in Gli3-/- mutants. Since SHH is thought to be involved in Fgf8 regulation, we compared Fgf8 expression in Shh-/- and Gli3-/-;Shh-/- mutant embryos. Whereas Fgf8 expression was almost absent in Shh-/- mutants, it was up-regulated in Gli3-/-;Shh-/- double mutants, suggesting that SHH is not required for Fgf8 induction, and that GLI3 normally represses Fgf8 independently of SHH. In the limb bud, we provide evidence that ectopic expression of Gremlin in Gli3-/- mutants might contribute to a decrease in apoptosis. Together, our data reveal that GLI3 limits Fgf8-expression domains in multiple tissues, through a mechanism that may include the induction or maintenance of apoptosis.

Animals↗
PubMed2002

Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis.

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.

Adiponectin↗
PubMed2002

Switch to anaerobic glucose metabolism with NADH accumulation in the beta-cell model of mitochondrial diabetes. Characteristics of betaHC9 cells deficient in mitochondrial DNA transcription.

To elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 microg/ml ethidium bromide (EtBr) to the murine pancreatic beta cell line betaHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of glucose-stimulated insulin release (Hayakawa, T., Noda, M., Yasuda, K., Yorifuji, H., Taniguchi, S., Miwa, I., Sakura, H., Terauchi, Y., Hayashi, J.-I., Sharp, G. W. G., Kanazawa, Y., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998) J. Biol. Chem. 273, 20300-20307). To elucidate fully the metabolism-secretion coupling in these cells, we measured glucose oxidation, utilization, and lactate production. We also evaluated NADH autofluorescence in betaHC9 cells using two-photon excitation laser microscopy. In addition, we recorded the membrane potential and determined the ATP and ADP contents of the cells. The results indicated 22.2 mm glucose oxidation to be severely decreased by EtBr treatment compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01). By contrast, glucose utilization was only marginally decreased. Lactate production under 22.2 mm glucose was increased by 2.9- and 3.5-fold by EtBr treatment on days 4 and 6, respectively (both p < 0.01). Cellular NADH at 2.8 mm glucose was increased by 35 and 43% by EtBr on days 4 and 6 (both p < 0.01). These data suggest that reduced expression of the mitochondrial electron transport system causes NADH accumulation in beta cells, thereby halting the tricarboxylic acid cycle on one hand, and on the other hand facilitating anaerobic glucose metabolism. Glucose-induced insulin secretion was lost rapidly along with the EtBr treatment with concomitant losses of membrane potential depolarization and the [Ca(2+)](i) increase, whereas glibenclamide-induced changes persisted. This is the first report to demonstrate the connection between metabolic alteration of electron transport system and that of tricarboxylic acid cycle and its impact on insulin secretion.

Anaerobiosis↗
PubMed2002

Disruption of adiponectin causes insulin resistance and neointimal formation.

The adipocyte-derived hormone adiponectin has been proposed to play important roles in the regulation of energy homeostasis and insulin sensitivity, and it has been reported to exhibit putative antiatherogenic properties in vitro. In this study we generated adiponectin-deficient mice to directly investigate whether adiponectin has a physiological protective role against diabetes and atherosclerosis in vivo. Heterozygous adiponectin-deficient (adipo(+/-)) mice showed mild insulin resistance, while homozygous adiponectin-deficient (adipo(-/-)) mice showed moderate insulin resistance with glucose intolerance despite body weight gain similar to that of wild-type mice. Moreover, adipo(-/-) mice showed 2-fold more neointimal formation in response to external vascular cuff injury than wild-type mice (p = 0.01). This study provides the first direct evidence that adiponectin plays a protective role against insulin resistance and atherosclerosis in vivo.

Adiponectin↗
PubMed2002

Role of mitochondrial NADH shuttle system in acute amylase secretion by acetylcholine from mouse pancreatic acinar cells.

Using the mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate limiting enzyme of the glycerol-phosphate NADH shuttle, we investigated the role of the NADH shuttle system in amylase secretion in response to acetylcholine (ACh) in pancreatic acinar cells. The pancreatic acinar cells of mGPDH-deficient mice were not different in histology and immunohistochemistry from those of wild-type mice. In both types of pancreatic acinar cells from wild-type and mGPDH-deficient mice, ACh similarly potentiated amylase secretion, measured in 30 minutes after the ACh stimulation. A 30 minutes pre-treatment of wild-type cells with aminooxyacetate (AOA), an inhibitor of aspartate aminotransferases of the malate-aspartate NADH shuttle, did not change the rate of ACh-induced amylase secretion, measured in the following 30 minutes. In also mGPDH-deficient cells treated with AOA, thus in this situation all mitochondrial NADH shuttles being dysfunctioning, ACh induced amylase release in a similar amount to that in AOA-untreated cells. The basal levels of intracellular Ca2+ concentration ([Ca2+]i), the ACh-stimulated levels of [Ca2+]i and Ca2+ oscillation patterns in response to ACh were similar in wild-type and mGPDH-deficient cells, and the AOA-treatment did not affect these [Ca2+]i responses. The levels of intracellular concentration of ATP before and during stimulation with ACh were similar in wild-type and mGPDH-defficient cells. In only AOA-treated mGPDH-deficient cells, the level of ATP decreased after the ACh stimulation. These results suggest that acute response of amylase secretion to ACh from mouse pancreatic acinar cells does not require simultaneous functioning of the mitochondrial NADH shuttle system, although the supply of intracellular ATP decreases during the ACh stimulation.

Acetylcholine↗
PubMed2002

Phosphatidylinositol 3-kinase suppresses glucose-stimulated insulin secretion by affecting post-cytosolic [Ca(2+)] elevation signals.

The role of phosphatidylinositol (PI) 3-kinase in the regulation of pancreatic beta-cell function was investigated. PI 3-kinase activity in p85 alpha regulatory subunit-deficient (p85 alpha(-/-)) islets was decreased to approximately 20% of that in wild-type controls. Insulin content and mass of rough endoplasmic reticula were decreased in beta-cells from p85 alpha(-/-) mice with increased insulin sensitivity. However, p85 alpha(-/-) beta-cells exhibited a marked increase in the insulin secretory response to higher concentrations of glucose. When PI 3-kinase in wild-type islets was suppressed by wortmannin or LY294002, the secretion was also substantially potentiated. Wortmannin's potentiating effect was not due to augmentation in glucose metabolism or cytosolic [Ca(2+)] elevation. Results of p85 alpha(-/-) islets and wortmannin-treated wild-type islets stimulated with diazoxide and KCl showed that inhibition of PI 3-kinase activity exerted its effect on secretion, at least in part, distal to a cytosolic [Ca(2+)] elevation. These results suggest that PI 3-kinase activity normally plays a crucial role in the suppression of glucose-stimulated insulin secretion.

Androstadienes↗
PubMed2002

Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population.

An adipocyte-derived peptide, adiponectin (also known as GBP28), is decreased in subjects with type 2 diabetes. Recent genome-wide scans have mapped a diabetes susceptibility locus to chromosome 3q27, where the adiponectin gene (APM1) is located. Herein, we present evidence of an association between frequent single nucleotide polymorphisms at positions 45 and 276 in the adiponectin gene and type 2 diabetes (P = 0.003 and P = 0.002, respectively). Subjects with the G/G genotype at position 45 or the G/G genotype at position 276 had a significantly increased risk of type 2 diabetes (odds ratio 1.70 [95% CI 1.09-2.65] and 2.16 [1.22-3.95], respectively) compared with those having the T/T genotype at positions 45 and 276, respectively. In addition, the subjects with the G/G genotype at position 276 had a higher insulin resistance index than those with the T/T genotype (1.61 +/- 0.05 vs. 1.19 +/- 0.12, P = 0.001). The G allele at position 276 was linearly associated with lower plasma adiponectin levels (G/G: 10.4 +/- 0.85 microg/ml, G/T: 13.7 +/- 0.87 microg/ml, T/T: 16.6 +/- 2.24 microg/ml, P = 0.01) in subjects with higher BMIs. Based on these findings together with the observation that adiponectin improves insulin sensitivity in animal models, we conclude that the adiponectin gene may be a susceptibility gene for type 2 diabetes.

Adiponectin↗
PubMed2002

Genetic manipulations of fatty acid metabolism in beta-cells are associated with dysregulated insulin secretion.

Triacylglyceride (TG) accumulation in pancreatic beta-cells is associated with impaired insulin secretion, which is called lipotoxicity. To gain a better understanding of the pathophysiology of lipotoxicity, we generated three models of dysregulated fatty acid metabolism in beta-cells. The overexpression of sterol regulatory element binding protein-1c induced lipogenic genes and TG accumulation. Under these conditions, we observed a decrease in glucose oxidation and upregulation of uncoupling protein-2, which might be causally related to the decreased glucose-stimulated insulin secretion. The overexpression of AMP-activated protein kinase was accompanied by decreased lipogenesis, increased fatty acid oxidation, and decreased glucose oxidation; insulin secretions to glucose and depolarization stimuli were decreased, probably because of the decrease in glucose oxidation and cellular insulin content. It was notable that the secretory response to palmitate was blunted, which would suggest a role of the fatty acid synthesis pathway, but not its oxidative pathway in palmitate-stimulated insulin secretion. Finally, we studied islets of PPAR-gamma(+/-) mice that had increased insulin sensitivity and low TG content in white adipose tissue, skeletal muscle, and liver. On a high-fat diet, glucose-stimulated insulin secretion was decreased in association with increased TG content in the islets, which might be mediated through the elevated serum free fatty acid levels and their passive transport into beta-cells. These results revealed some aspects about the mechanisms by which alterations of fatty acid metabolism affect beta-cell functions.

Adenylate Kinase↗
PubMed2002

[Glucokinase].

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Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

The role of PPARgamma in high-fat diet-induced obesity and insulin resistance.

It has been well demonstrated that insulin resistance plays an important role in the clustering of coronary risk factors through the progression of atherosclerosis in animal models of insulin resistance. In humans, a high-fat diet is the major cause of obesity and insulin resistance. In this study, we investigated the role of peroxisome proliferator-activated receptor gamma (PPARgamma) in high-fat diet induced-obesity and insulin resistance by gene targeting and case-control study using the common PPARgamma2 polymorphism in human subjects. Homozygous PPARgamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet and the phenotypes were abrogated by PPARgamma agonist treatment. Heterozygous PPARgamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPARgamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPARgamma. A Pro12Ala polymorphism has been detected in the human PPARgamma2 gene. Since this amino acid substitution may cause a reduction in the transcriptional activity of PPARgamma, this polymorphism may be associated with decreased insulin resistance and decreased risk of Type 2 diabetes. To investigate this hypothesis, we performed a case-control study of the Pro12Ala PPARgamma2 polymorphism. In an obese group, subjects with Ala12 were more insulin sensitive than those without. The frequency of Ala12 was significantly lower in the diabetic group, suggesting that this polymorphism protects against Type 2 diabetes. These results revealed that both in mice and humans, PPARgamma is a thrifty gene mediating Type 2 diabetes.

Amino Acid Substitution↗
PubMedDate not supplied

In situ tissue engineering of periodontal tissues by seeding with periodontal ligament-derived cells.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

Alkaline Phosphatase↗