Keishi Yamauchi

Selective Attenuation of Metabolic Branch of Insulin Receptor Down-signaling by High Glucose in a Hepatoma Cell Line, HepG2 Cells

The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15–33 mm) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulin-induced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKCε and PKCδ from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKCε and PKCδ directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKCε and PKCδ are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.

CDK5-dependent Phosphorylation of the Rho Family GTPase TC10α Regulates Insulin-stimulated GLUT4 Translocation

Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10α on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10α was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10α) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10α but not T197A-TC10α and in parallel, T197D-TC10α but not T197A-TC10α depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10α in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10α through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10α cycling between lipid raft and non-lipid raft compartments.

Diminution of early insulin response to glucose in subjects with normal but minimally elevated fasting plasma glucose. Evidence for early beta-cell dysfunction

Aim Systematic analysis of β‐cell function in Japanese health examinees.

Suppression of insulin‐induced AP‐1 transactivation by menin accompanies inhibition of c‐Fos induction

The translation product of the MEN1 gene, menin, has been reported to suppress JunD‐mediated activator protein‐1 (AP‐1) transactivation and inhibit Ras‐mediated tumor formation, but its molecular mechanisms and physiologic significance have been poorly elucidated. To better understand the function of menin as a tumor suppressor, we examined the effect of menin on physiologically induced AP‐1 activity. Overexpression of menin strongly suppressed insulin‐induced AP‐1 activity in CHO‐IR cells, which express high levels of insulin receptor. We found that menin suppressed c‐Fos induction at the transcriptional level, although that cannot explain the entire mechanism of AP‐1 suppression by menin. Menin did not alter the expression levels of AP‐1 proteins except c‐Fos, phosphorylation of c‐Jun and JunD and DNA binding properties of AP‐1 proteins. Suppression of AP‐1 activation by menin may be exerted through 2 independent mechanisms, direct inhibition on AP‐1‐mediated transcription and suppression of c‐Fos induction. The molecular mechanism of inhibition of AP‐1 function by menin needs further elucidation.

Vascular Endothelial Cell Growth Factor Attenuates Actions of Transforming Growth Factor-β in Human Endothelial Cells

Because vascular endothelial cell growth factor (VEGF) and transforming growth factor-β (TGF-β) are both involved in cellular growth and differentiation, we examined whether VEGF modifies TGF-β signaling cascade in human umbilical cord vein endothelial cells (HUVEC). Production of plasminogen activator inhibitor-1 (PAI-1), which is under the specific control of TGF-β, was strongly enhanced (3.5-fold) by TGF-β treatment. Remarkably, physiological concentration of VEGF (30 nm) profoundly (by 60%) attenuated the TGF-β stimulation of PAI-1 production without an effect on the basal PAI-1 production. In HUVECs transiently transfected with an expression construct containing a PAI-1 promoter fused to luciferase reporter gene, TGF-β-stimulation of transcription of PAI-1 was clearly (by 60%) inhibited by VEGF. TGF-β phosphorylation of Smad2/3, an obligatory step of intracellular TGF-β signaling, was also suppressed by VEGF. VEGF attenuation of TGF-β action was also demonstrated in two other endothelial cell lines. In conclusion, VEGF attenuates TGF-β action in the human endothelial cell, specifically at the level of transcription of PAI-1 gene and Smad2/3 phosphorylation.

An Early Insulin Intervention Accelerates Pancreatic β-Cell Dysfunction in Young Goto-Kakizaki Rats, a Model of Naturally Occurring Noninsulin-Dependent Diabetes1

This study was designed to delineate the nature of β-cell dysfunction in a model of genetically determined nonobese diabetes, the Goto-Kakizaki (GK) rat. Pancreatic β-cell function was analyzed immediately after weaning and 5 weeks thereafter, comparing animals with or without insulin treatment during the interval. In 3.5-week-old GK rats, fasting plasma glucose was mildly elevated with normoinsulinemia, and the islet insulin content was reduced by 33%. When incubated with 3–30 mm glucose in vitro, the GK rat islets showed reduced glucose sensitivity, i.e. the EC50 values were 19.5 and 15.9 mm, and the Hill constants for the positive cooperativity 2.1 and 4.2, in the islets of GK and the control rats, respectively. On the other hand, the maximum response to glucose was not attenuated when reduced islet insulin content was considered. In 8.5-week-old GK rats, hyperglycemia worsened and glucose-stimulated insulin release by the islets more severely impaired. A daily insulin injection from the 3.5–8.5 weeks ...

Size-related and size-unrelated functional heterogeneity among pancreatic islets

Functional heterogeneity of pancreatic islets was systematically analyzed for the first time using freshly isolated single rat pancreatic islets. First, 60 islets were sequentially exposed to 3, 9.4, 15.6, and 24.1 mM glucose for 30 min each in incubation experiments: 36 (60%) responded in a concentration-dependent and 19 (32%) in an all-or-none manner, and 5 (8%) islets did not respond to high glucose. As a group, the larger the islet, the higher the beta cell glucose sensitivity. However, glucose-stimulated elevation of [Ca2+]i in the beta cell. insulin/glucagon ratio in the islet, and expression of glucose transporter 2, glucokinase, and pancreatic duodenal homeobox factor-1 in the beta cell were not significantly related to islet size. Second, 50 islets were stimulated with 16.7 mM glucose in perifusion. A biphasic insulin release was found in 39 (78%), and no or little first phase response in 11 (22%) islets, irrespective of the islet size. Nevertheless, when the response was plotted as a group, it was clearly biphasic. Islet size, insulin content and the amount of insulin release were positively correlated with each other. In conclusion, there are size-related and size-unrelated functional diversity among pancreatic islets. The reason for such heterogeneity remained to be determined.

RS3PE in Association With Dipeptidyl Peptidase-4 Inhibitor: Report of Two Cases

Remitting seronegative symmetrical synovitis with pitting edema (RS3PE), first described by McCarty (1), is characterized by symmetrical pitting edema of the hands and feet; tenderness, swelling, and/or limited motion of the joints; fatigue; and fever (1,2). It commonly occurs in elderly people (1,2). We have recently encountered two cases of RS3PE that developed shortly after the initiation of a dipeptidyl peptidase-4 (DPP4) inhibitor and resolved markedly upon cessation of it. Case 1 was a 74-year-old woman with a 30-year history of type 2 diabetes who presented with edema of the hands, low grade fever, and malaise, which developed 5 weeks after switching from 20 mg gliclazide to 50 mg sitagliptin. Physical examination revealed severe pitting edema …

d-Glucose and insulin stimulate migration and tubular formation of human endothelial cells in vitro

Effects of high D-glucose and insulin on the endothelial cell migration and tubular formation were investigated with the use of ECV304 cells, a clonal human umbilical cord endothelial cell line. Exposure of the cells to high D-glucose resulted in a marked increase in the migration, which was blocked by inhibitors of protein kinase C such as H7 (10 microM) and GF109203X (200 nM). Furthermore, a protein kinase C agonist, phorbol 12-myristate 13-acetate, had an effect similar to that of glucose on ECV304 cells. Glucose stimulation of the migration was additively enhanced by 100 nM insulin, and the insulin effect was found to be unaffected by either PD-98059 or wortmannin, a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, respectively. Neither did H7 inhibit insulin stimulation of the migration. In contrast, a combination of high D-glucose and insulin, rather than either one alone, promoted tubular formation, which was inhibited by addition of 10 microM PD-98059. Stimulation of ECV304 cells by the combination of high D-glucose and insulin also caused an activation of MAPK, which was again obliterated by the same concentration of PD-98059. In conclusion, human endothelial cell migration and tubular formation are stimulated by high D-glucose and insulin in different ways. In the former reaction, either is effective, a combination of the two results in an additive effect, and activation of protein kinase C is involved. In contrast, tubular formation will only occur in the presence of a combination of high D-glucose and insulin, and MAPK plays an essential role.Effects of highd-glucose and insulin on the endothelial cell migration and tubular formation were investigated with the use of ECV304 cells, a clonal human umbilical cord endothelial cell line. Exposure of the cells to highd-glucose resulted in a marked increase in the migration, which was blocked by inhibitors of protein kinase C such as H7 (10 μM) and GF109203X (200 nM). Furthermore, a protein kinase C agonist, phorbol 12-myristate 13-acetate, had an effect similar to that of glucose on ECV304 cells. Glucose stimulation of the migration was additively enhanced by 100 nM insulin, and the insulin effect was found to be unaffected by either PD-98059 or wortmannin, a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, respectively. Neither did H7 inhibit insulin stimulation of the migration. In contrast, a combination of high d-glucose and insulin, rather than either one alone, promoted tubular formation, which was inhibited by addition of 10 μM PD-98059. Stimulation of ECV304 cells by the combination of highd-glucose and insulin also caused an activation of MAPK, which was again obliterated by the same concentration of PD-98059. In conclusion, human endothelial cell migration and tubular formation are stimulated by highd-glucose and insulin in different ways. In the former reaction, either is effective, a combination of the two results in an additive effect, and activation of protein kinase C is involved. In contrast, tubular formation will only occur in the presence of a combination of highd-glucose and insulin, and MAPK plays an essential role.

Evidence for the existence of protein inhibitors for S-adenosyl-L-methionine-mediated methylation of phosphatidylethanolamine in rat liver cytosol

The effect of rat liver cytosol on phosphatidylcholine biosynthesis via the N-methylation of phosphatidylethanolamine has been studied. The purified rat liver plasma membrane was used as an enzyme source. The rat liver 105,000 x g supernatant(cytosol) contained two different inhibitors for S-adenosyl-L-methionine-mediated methylation of phosphatidylethanolamine to phosphatidyl-N-mono-methylethanolamine. The inhibitors were inactivated by pretreatment with trypsin or heating at 96 degrees C, but were not inactivated by RNase. The inhibitors did not inhibit the synthesis of phosphatidylcholine from phosphatidyl-N-mono-methylethanolamine. The results indicate that two different protein inhibitors for methylation are present in rat liver cytosol fraction and that the inhibition by these proteins may have a role for the regulation of phosphatidylcholine synthesis in the plasma membrane.