Hideo Toyoshima

University of Tsukuba, Tsukuba, Ibaraki-ken, Japan

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Publications (33)196.92 Total impact

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    ABSTRACT: Neovascularization is an important event in proliferative diabetic retinopathy (PDR), where various secretory proteins including multiple growth factors are considered to be involved in this process. We searched for secretory proteins expressed in a surgical specimen obtained from the eyes of patients with PDR. We developed the oligo-cap signal sequence trap (SST) strategy which enables us to screen for secretory or membrane proteins from a minimal starting material. Using this method, we were able to screen a cDNA library constructed from a surgical specimen obtained from the eyes of the patients with PDR. Majority of the cloned cDNAs turned out to encode secreted protein acidic and rich in cystein (SPARC), strongly suggesting that SPARC is highly expressed in PDR. Analysis of vitreous fluid from various patients has shown that the concentration of SPARC protein is increased in patients with PDR. Furthermore, subretinal injection of recombinant SPARC adenovirus induced PDR-like changes in the rat eye. Our results strongly suggested that SPARC is involved in the development of diabetic retinopathy (DR).
    Journal of atherosclerosis and thrombosis 05/2009; 16(2):69-76. · 2.93 Impact Factor
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    ABSTRACT: Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor, is induced during adipocyte differentiation; however, its precise contribution to this process is unknown. Using both in vitro and in vivo systems, we show that p21 is crucial for maintaining adipocyte hypertrophy and obesity-induced insulin resistance. The absence of p21 in 3T3-L1 fibroblasts by RNA-mediated interference knockdown or in embryonic fibroblasts from p21(-/-) mice impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet, p21(-/-) mice fed high energy diets had reduced adipose tissue mass and adipocyte size accompanied by a marked improvement in insulin sensitivity. Knockdown of p21 in enlarged epididymal fat of diet-induced obese mice and also in fully differentiated 3T3-L1 adipocytes caused vigorous apoptosis by activating p53. Thus, p21 is involved in both adipocyte differentiation and in protecting hypertrophied adipocytes against apoptosis. Via both of these mechanisms, p21 promotes adipose tissue expansion during high fat diet feeding, leading to increased downstream pathophysiological consequences such as insulin resistance.
    Journal of Biological Chemistry 08/2008; 283(30):21220-9. · 4.65 Impact Factor
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    ABSTRACT: Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.
    Nature Medicine 11/2007; 13(10):1193-202. · 22.86 Impact Factor
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    ABSTRACT: Sterol regulatory element-binding protein (SREBP)-1a is a unique membrane-bound transcription factor highly expressed in actively growing cells and involved in the biosynthesis of cholesterol, fatty acids, and phospholipids. Because mammalian cells need to synthesize membrane lipids for cell replication, the functional relevance of SREBP-1a in cell proliferation has been considered a biological adaptation. However, the effect of this potent lipid-synthesis activator on cell growth has never been explored. Here, we show that induction of nuclear SREBP-1a, but not SREBP-2, completely inhibited cell growth in inducible Chinese hamster ovary (CHO) cell lines. Growth inhibition occurred through G(1) cell-cycle arrest, which is observed in various cell types with transient expression of nuclear SREBP-1a. SREBP-1a caused the accumulation of cyclin-dependent kinase (cdk) inhibitors such as p27, p21, and p16, leading to reduced cdk2 and cdk4 activities and hypophosphorylation of Rb protein. In contrast to transactivation of p21, SREBP-1a activated p27 by enhancing stabilization of the protein through inhibition of SKP2 and KPC1. In vivo, SREBP-1a-expressing livers of transgenic mice exhibited impaired regeneration after partial hepatectomy. SREBP-1-null mouse embryonic fibroblasts had a higher cell proliferation rate than wild-type cells. The unexpected cell growth-inhibitory role of SREBP-1a provides a new paradigm to link lipid synthesis and cell growth.
    FEBS Journal 10/2007; 274(17):4440-52. · 4.25 Impact Factor
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    ABSTRACT: A number of adipocytokines have been suggested to be involved in the disruption of glucose metabolism, and also in the development of various diabetic complications. We attempted to identify and analyze additional adipocytokines, to better understanding the roles of adipocytes and adipocytokines. An oligo-capping signal sequence trap, developed in our laboratory for screening the cDNAs of secretory proteins, was used to sreen cDNAs expressed in mouse white adipose tissue. Profiles of the genes identified in mice and cultured cells were further investigated by northern blotting and luciferase assay. A cDNA fragment of interferon-stimulated gene 12b (ISG12b) was obtained in the search. A northern blot analysis revealed ISG12b to be highly expressed in white adipose tissue. Interferon alpha (IFNalpha) was shown to induce ISG12b expression in the adipose tissue of BL6 mice in vivo, and also in a 3T3-L1 preadipocyte cell line in vitro. The level of ISG12b was higher in mature adipocytes than in preadipocytes. A promoter analysis demonstrated that the 369bp upstream from the transcription initiation site of ISG12b mRNA contain strong promoter activity, and the interferon-stimulated response elements (ISREs) were not present within the 5593bp upstream region. ISG12b is an additional candidate for a adipocytokine induced to express in adipose tissue by interferon.
    Journal of atherosclerosis and thrombosis 09/2007; 14(4):179-84. · 2.93 Impact Factor
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    ABSTRACT: Sterol regulatory element-binding protein (SREBP)-1c is a transcription factor that controls synthesis of fatty acids and triglycerides in the liver and is highly regulated by nutrition and hormones. In the current studies we show that protein kinase A (PKA), a mediator of glucagon/cAMP, a fasting signaling, suppresses SREBP-1c by modulating the activity of liver X receptor alpha (LXRalpha), a dominant activator of SREBP-1c expression. Activation of PKA repressed LXR-induced SREBP-1c expression both in rat primary hepatocytes and mouse livers. Promoter analyses revealed that the LXRalpha-binding site in the SREBP-1c promoter is responsible for PKA inhibitory effect on SREBP-1c transcription. In vitro and in vivo PKA directly phosphorylated LXRalpha, and the two consensus PKA target sites (195, 196 serines and 290, 291 serines) in its ligand binding/heterodimerization domain were crucial for the inhibition of LXR signaling. PKA phosphorylation of LXRalpha caused impaired DNA binding activity by preventing LXRalpha/RXR dimerization and decreased its transcription activity by inhibiting recruitment of coactivator SCR-1 and enhancing recruitment of corepressor NcoR1. These results indicate that LXRalpha is regulated not only by oxysterol derivatives but also by PKA-mediated phosphorylation, which suggests that nutritional regulation of SREBP-1c and lipogenesis could be regulated at least partially through modulation of LXR.
    Journal of Biological Chemistry 05/2007; 282(16):11687-95. · 4.65 Impact Factor
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    ABSTRACT: Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.
    Cell Metabolism 09/2006; 4(2):143-54. · 14.62 Impact Factor
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    ABSTRACT: The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21Cip1 and p27Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after gamma-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27-/- and p27+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.
    Cancer Research 05/2006; 66(8):4020-9. · 8.65 Impact Factor
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    ABSTRACT: Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinase 3beta and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1). These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver. Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3. Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes.
    Nature Medicine 02/2006; 12(1):107-13. · 22.86 Impact Factor
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    ABSTRACT: Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that regulate lipid synthetic genes. In contrast to SREBP-2, which regulates cellular cholesterol level in normal cells, SREBP-1a is highly expressed in actively growing cells and activates entire programs of genes involved in lipid synthesis such as cholesterol, fatty acids, triglycerides, and phospholipids. Previously, the physiological relevance of this potent activity of SREBP-1a has been thought to regulate the supply of membrane lipids in response to cell growth. Here we show that nuclear SREBP-1a and SREBP-2 bind directly to a novel SREBP binding site in the promoter of the p21(WAF1/CIP1) gene, the major cyclin-dependent kinase inhibitor, and strongly activate its promoter activity. Only the SREBP-1a isoform consistently causes induction of p21 at both the mRNA and protein levels. Colony formation assays and polyploidy of livers from transgenic mice suggest that activation of p21 by SREBP-1a could inhibit cell growth. Activation of endogenous SREBPs in lipid deprivation conditions was associated with induction of p21 mRNA and protein. Expression of p21 was reduced in SREBP-1 null mice. These data suggest a physiological role of SREBP-1a in p21 regulation. Identification of p21 as a new SREBP target might implicate a new paradigm in the link between lipid synthesis and cell growth.
    Molecular and Cellular Biology 11/2005; 25(20):8938-47. · 5.37 Impact Factor
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    ABSTRACT: Sterol regulatory element-binding protein-1 (SREBP-1) is a transcription factor which regulates genes involved in the synthesis of fatty acids and triglycerides. The overexpression of nuclear SREBP-1a in transgenic mice under the control of the PEPCK promoter (TgSREBP-1a) caused a massively enlarged fatty liver and disappearance of peripheral white adipose tissue. In the current study, we estimated the impact of this lipid transcription factor on plasma glucose/insulin metabolism in vivo. TgSREBP-1a exhibited mild peripheral insulin resistance as evidenced by hyperinsulinemia both at fasting and after intravenous glucose loading, and retarded glucose reduction after insulin injection due to decreased plasma leptin levels. Intriguingly, hyperinsulinemia in TgSREBP-1a mice was markedly exacerbated in a fed state and sustained after intravenous glucose loading, and paradoxically decreased after the portal injection of glucose. TgSREBP-1a mice consistently showed very small plasma glucose increases after portal glucose loading because of a large capacity for hepatic glucose uptake. These data suggested that hepatic insulin resistance emerges postprandially. In addition, pancreatic islets from TgSREBP-1a were enlarged. These data demonstrate that SREBP-1a activation in the liver has a strong impact on plasma insulin levels, implicating the potential role of SREBPs in hepatic insulin metabolism relating to insulin resistance.
    Biochimica et Biophysica Acta 07/2005; 1740(3):427-33. · 4.66 Impact Factor
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    ABSTRACT: Pericyte ghosts and acellular capillaries are well known as early histological changes resulting from diabetic retinopathy. These histological changes mean that the cell death of retinal microvessels has accelerated. It was reported that apoptosis of retinal microvascular cells (RMCs) was increased in diabetic patients. Therefore, we investigated apoptosis of RMCs in Goto-Kakizaki (GK) rats, a type 2 diabetic model, and involvement with antioxidants (a combination of vitamins C and E) or a novel inhibitor of advanced glycation, OPB-9195. GK rats were treated with the antioxidants combination or OPB-9195 for 36 weeks. We obtained isolated preparations of the vascular network from their retinas by trypsin digestion. Apoptosis of retinal vascular cells was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. We found that apoptosis of RMCs was increased in the diabetic GK rats. Furthermore, a combination of vitamins C and E and an advanced glycation end-products inhibitor mostly inhibited this increased apoptosis. We concluded that apoptosis of RMCs was a good marker that indicates the progression of diabetic retinopathy in GK rats. Both oxidative stress and the accumulation of advanced glycation end-products appears to promote the apoptosis of retinal microvascular cells, and antioxidants or advanced glycation end-products inhibitors might ameliorate diabetic retinopathy.
    Diabetes/Metabolism Research and Reviews 05/2005; 22(1):38-45. · 2.97 Impact Factor
  • Hideo Toyoshima, Nobuhiro Yamada
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    ABSTRACT: Atherosclerosis is considered as a chronic inflammatory condition. PPARs are also thought to be involved in the control of inflammatory response, which may suggest an additional role in the development of atherosclerosis.
    Nippon rinsho. Japanese journal of clinical medicine 05/2005; 63(4):657-8.
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    ABSTRACT: The medical criteria for initiating insulin therapy, based on clinical profiles of type 2 diabetic patients, have not yet been clearly established. We explored various parameters with 48 type 2 diabetic patients who were taking oral hypoglycemic medication. Among parameters, body mass index (BMI), the fasting plasma glucose level (FPG), and plasma chloride concentration were identified by forward-stepwise discriminant analysis as parameters that can discriminate between patients who were and those who were not undergoing insulin therapy. In combination, these parameters correctly diagnosed 86.4% of the patients who were undergoing insulin therapy, and 84.6% of those who were not undergoing insulin therapy. Further, we observed significant correlations between plasma chloride concentrations and either plasma sodium or organic acid concentrations, suggesting that impaired insulin action may reduce plasma chloride concentrations through changes in plasma sodium and organic acid metabolism. Our results suggest that plasma chloride concentration is a possible new indicator of insulin insufficiency.
    Diabetes Research and Clinical Practice 03/2005; 67(2):137-43. · 2.74 Impact Factor
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    ABSTRACT: Influx of excess fatty acids and the resultant accumulation of intracellular triglycerides are linked to impaired insulin secretion and action in the pathogenesis of type 2 diabetes. Sterol regulatory element-binding protein (SREBP)-1c is a transcription factor that controls cellular synthesis of fatty acids and triglycerides. SREBP-1c is highly expressed in high-energy and insulin-resistant states. To investigate effects of this synthetic lipid regulator on insulin secretion, we generated transgenic mice overexpressing nuclear SREBP-1c under the insulin promoter. beta-Cell-specific expression of SREBP-1c caused reduction in islet mass and impaired glucose-stimulated insulin secretion and was associated with accumulation of triglycerides, suppression of pancreas duodenal homeobox-1, and upregulation of uncoupling protein 2 gene expression. The mice presented with impaired glucose tolerance that was exacerbated by a high-energy diet. Taken together with enhanced insulin secretion from SREBP-1-null islets, these data suggest that SREBP-1c and endogenous lipogenesis could be involved in beta-cell dysfunction and diabetes.
    Diabetes 03/2005; 54(2):492-9. · 7.90 Impact Factor
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    ABSTRACT: Rho family GTPases regulate multiple cellular processes through their downstream effectors, where their activities are stimulated by the guanine nucleotide exchange factors. Here, we report a new member of RhoGEF, WGEF, which has the classical structure of DH-PH domain and a C-terminal SH3 domain. WGEF was shown to activate RhoA, Cdc42, and Rac1 by pulldown assay, and forced expression of WGEF resulted in marked rearrangement of the actin cytoskeleton, which is typically seen by the activation of RhoA, Cdc42, and Rac1. WGEF was highly expressed in intestine and also in liver, heart and kidney, which may suggest the involvement of WGEF in the development and functions of these organs. The expression pattern may also suggest the possible importance of WGEF in the understanding of diseases based on metabolic disorder.
    Biochemical and Biophysical Research Communications 12/2004; 324(3):1053-8. · 2.28 Impact Factor
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    ABSTRACT: The ATP-binding-cassette transporter A1 (ABCA1) plays an essential role in cellular cholesterol efflux and helps prevent macrophages from becoming foam cells. The statins are widely used as cholesterol-lowering agents and have other anti-atherogenic actions. We tested the effects of four different statins (fluvastatin, atorvastatin, simvastatin, and lovastatin) on ABCA1 expression in macrophages in vitro. The statins suppressed ABCA1 mRNA expression in RAW246.7 and THP-1 macrophage cell lines and in mouse peritoneal macrophages. The effect was time- and dose-dependent and was abolished by the addition of the post-reductase product, mevalonate. These findings imply that there is a possible modulation of the well-known beneficial effects of the statins on the reverse cholesterol transport pathway.
    Biochemical and Biophysical Research Communications 05/2004; 316(3):790-4. · 2.28 Impact Factor
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    ABSTRACT: Insulin receptor substrate 2 (IRS-2) is the main mediator of insulin signalling in the liver, controlling insulin sensitivity. Sterol regulatory element binding proteins (SREBPs) have been established as transcriptional regulators of lipid synthesis. Here, we show that SREBPs directly repress transcription of IRS-2 and inhibit hepatic insulin signalling. The IRS-2 promoter is activated by forkhead proteins through an insulin response element (IRE). Nuclear SREBPs effectively replace and interfere in the binding of these transactivators, resulting in inhibition of the downstream PI(3)K/Akt pathway, followed by decreased glycogen synthesis. These data suggest a molecular mechanism for the physiological switching from glycogen synthesis to lipogenesis and hepatic insulin resistance that is associated with hepatosteatosis.
    Nature Cell Biology 05/2004; 6(4):351-7. · 20.76 Impact Factor
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    ABSTRACT: Insulin and glucose together have been previously shown to regulate hepatic sterol regulatory element-binding protein (SREBP)-1c expression. We sought to explore the nutritional regulation of lipogenesis through SREBP-1c induction in a setting where effects of sugars versus insulin could be distinguished. To do so, mice were insulin depleted by streptozotocin (STZ) administration and subjected to a fasting-refeeding protocol with glucose, fructose, or sucrose. Unexpectedly, the insulin-depleted mice exhibited a marked induction of SREBP-1c on all sugars, and this increase in SREBP-1c was even more dramatic than in the non-STZ-administered controls. The time course of changes in SREBP-1 induction varied depending on the type of sugars in both control and STZ-administered mice. Glucose refeeding gave a peak of SREBP-1c induction, whereas fructose refeeding caused slow and gradual increments, and sucrose refeeding fell between these two responses. Expression of various lipogenic enzymes were also gradually increased over time, irrespective of the types of sugars, with greater intensities in STZ-administered than in nontreated mice. In contrast, induction of hepatic glucokinase and suppression of phoshoenolpyruvate carboxykinase were insulin dependent in an early refed state. These data clearly demonstrate that nutritional regulation of SREBP-1c and lipogenic genes may be completely independent of insulin as long as sufficient carbohydrates are available.
    Diabetes 04/2004; 53(3):560-9. · 7.90 Impact Factor
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    ABSTRACT: The hepatocyte nuclear factor-4alpha (HNF-4alpha)/PGC-1 pathway plays a crucial role in the transcriptional regulation of hepatic gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and Glc-6-Pase, genes that are activated at fasting and suppressed in a fed state. SREBP-1c dominates the nutritional regulation of lipogenic genes inverse to gluconeogenesis. Here we show the mechanism by which SREBP-1 suppresses expression of gluconeogenic genes. A series of luciferase reporter assays demonstrated that SREBP-1a and -1c effectively inhibited the PEPCK promoter activity that was induced by HNF-4alpha. The HNF-4alpha-binding site in the glucocorticoid-response unit was responsible for the SREBP-1 inhibition, although SREBP-1 did not bind to the PEPCK promoter as demonstrated by electrophoretic mobility shift assays. The inhibitory effect was more potent in the isoform of SREBP-1a than SREBP-1c and was eliminated by deletion of the amino-terminal transactivation domain of SREBP-1. Coimmunoprecipitation experiments demonstrated that these two transcription factors directly interact through the transactivation domain of SREBP-1 and the ligand binding/AF2 domains of HNF-4alpha. Estimation of coactivator recruitment using HNF-4alpha-Gal4DBD fusion assay showed that SREBP-1 competitively inhibited PGC-1 recruitment, a requirement for HNF-4alpha activation. Consistent with these results, hepatic PEPCK and Glc-6-Pase mRNA levels are suppressed by overexpression of SREBP-1a and -1c in the transgenic mice. Our data indicate that SREBP-1 has a novel role as negative regulator of gluconeogenic genes through a cross-talk with HNF-4alpha interference with PGC-1 recruitment.
    Journal of Biological Chemistry 04/2004; 279(13):12027-35. · 4.65 Impact Factor