Search PubMed⌕ Search

Biomedical subjects

Yoshitomo Oka

Publications and source records attributed to Yoshitomo Oka.

At least 73 records · Page 4Linked to original sources
PubMed2002

Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes.

TNFalpha, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest.

3T3 Cells↗
PubMed2002

Resistin is regulated by C/EBPs, PPARs, and signal-transducing molecules.

Expression of the adipocyte-derived protein resistin, which is thought to play a key role in the development of insulin resistance in vivo, is regulated by a variety of hormones and mediators, including insulin and TNFalpha. Here we describe our use of adenovirus-mediated gene transfer to determine which transcription factors and signaling pathways affect resistin expression in 3T3-L1 adipocytes. We found that resistin expression was enhanced by overexpression of C/EBPalpha and suppressed by C/EBPzeta, a negative regulator of C/EBPalpha. Additionally, C/EBPalpha induced resistin even in L6 myocytes. Overexpression of PPARgamma markedly reduced resistin expression, whereas PPARalpha had no significant effect. Resistin expression was markedly suppressed by overexpression of the PI3-kinase p110alpha catalytic subunit and by Akt. Finally, overexpression of MEK1, MKK6, or MKK7 suppressed resistin expression. These findings indicate that resistin expression is regulated by C/EBPalpha and PPARgamma, partly via modulation of signal transduction in the PI3-kinase and MAP kinase pathways.

3T3 Cells↗
PubMed2002

MEK kinase 1 interacts with focal adhesion kinase and regulates insulin receptor substrate-1 expression.

MEK kinase 1 (MEKK1) has been shown to contribute to the regulation of cell migration, whereas focal adhesion kinase (FAK) is a major player involved in both cell migration and integrin signaling. Here we show that MEKK1 and FAK are co-immunoprecipitated from mouse fibroblasts. Moreover, the association between MEKK1 and FAK appears to be physiologically relevant, as it is enhanced by treatment with epidermal growth factor (EGF). Targeting FAK to the membrane also enhanced its association with MEKK1, indicating that MEKK1 is localized to a membrane-related subcellular domain, perhaps focal adhesions. Interestingly, the expression of insulin receptor substrate-1 (IRS-1) was diminished in MEKK1-deficient fibroblasts, which is similar to an earlier finding in FAK-deficient fibroblasts. Insulin-like growth factor 1 (IGF-1)-induced ERK activation was diminished in MEKK1-deficient cells, but phosphatidylinositol 3-kinase/Akt activation was not. Although integrin reportedly regulates the transcription of the IRS-1 gene via FAK-mediated JNK activation, no impairment of fibronectin-stimulated activation of FAK, ERK, or JNK was observed in MEKK1-deficient cells. Reconstitution of MEKK1 expression restored IRS-1 expression as well as IGF-1-induced ERK activation. Taken together, these findings indicate that MEKK1 interacts with FAK in focal adhesions and regulates IRS-1 expression.

Animals↗
PubMed2002

Association of upregulated activity of K(ATP) channels with impaired insulin secretion in UCP1-expressing insulinoma cells.

Insulin-secreting MIN6 cells overexpressing uncoupling protein-1 (UCP1) were studied regarding insulin secretion in response to various secretagogues. Overexpression of UCP1 prevented an increase of cytosolic ATP levels induced by glucose. In contrast, glucose utilization was not affected, nor was glycerol phosphate flux. The UCP1-expressing cells showed an inability to increase cytosolic Ca(2+) concentration ([Ca(2+)](i)) in response to glucose or alpha ketoisocaproate and this resulted in less insulin secretion, whereas initial reduction in [Ca(2+)](i) occurring upon either nutrient addition was not affected. Moreover, the effectiveness of tolbutamide on [Ca(2+)](i) increase was reduced and the dose-response relations for insulin secretion induced by the agent was shifted toward the right in the UCP1-expressing cells. The resting membrane potential of the UCP1-expressing cells was significantly hyperpolarized by 6.2 mV compared with control cells. In the perforated and conventional whole-cell patch-clamp configurations, the conductance density of ATP-sensitive K(+) (K(ATP)) channels of the UCP1-expressing cells was 6-fold and 1.7-fold greater than that of the control cells, respectively. The sensitivity of K(ATP) channels for tolbutamide was not different between two groups, indicating that in intact cells more than 6-fold higher concentrations of tolbutamide were required to reduce the K(ATP) channel currents of UCP1-expressing cells to the same levels as of the control cells. The current density of the voltage-dependent Ca(2+) channels was not influenced. In conclusion, UCP1-expressing cells showed a refractoriness to respond to tolbutamide as well as nutrients. An upregulated activity of K(ATP) channels was associated with unresponsiveness to the agent in the cells with impaired mitochondrial function.

Adenosine Triphosphate↗
PubMed2002

Atypical muscle pathology and a survey of cis-mutations in deaf patients harboring a 1555 A-to-G point mutation in the mitochondrial ribosomal RNA gene.

We investigated three families with maternally inherited deafness associated with a 1555 A-to-G substitution in the 12S ribosomal RNA gene. Probands in these families developed deafness following streptomycin treatment, whereas several family members who did not receive aminoglycoside showed onset of deafness in middle age. One proband had a non-synonymous A14062G mutation in the ND5 gene and the other had a non-synonymous G15221A mutation in the cytochrome b gene and a T1391C mutation in the 12S ribosomal RNA gene, whose importance in disease expression remains to be clarified. Two muscle biopsies from the patients with and without streptomycin treatment, showed similar findings; a moth-eaten appearance with decreased cytochrome c oxidase activity and abnormal mitochondrial morphology. These findings suggest that even without exposure to aminoglycoside the A1555G mutation may impair mitochondrial function and that the mitochondrial abnormalities associated with the A1555G mutation may be expressed in tissues other than those of the auditory system.

Adult↗
PubMed2002

Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes.

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKalpha2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKalpha2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKalpha2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.

3T3 Cells↗
PubMed2002

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC.

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

3T3 Cells↗
PubMed2002

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes.

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Acyl-coenzyme A dehydrogenases are localized on GLUT4-containing vesicles via association with insulin-regulated aminopeptidase in a manner dependent on its dileucine motif.

Insulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55-82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55-82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.

3-Mercaptopropionic Acid↗
PubMed2002

Unregulated elevation of glutamate dehydrogenase activity induces glutamine-stimulated insulin secretion: identification and characterization of a GLUD1 gene mutation and insulin secretion studies with MIN6 cells overexpressing the mutant glutamate dehydrogenase.

Glutamate dehydrogenase (GDH) is important in normal glucose homeostasis. Mutations of GDH result in hyperinsulinism/hyperammonemia syndrome. Using PCR/single-strand conformation polymorphism analysis of the gene encoding GDH in 12 Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we found a mutation (Y266C) in one PHHI patient. This mutation was not found in any of the control or type 2 diabetic subjects. The activity of the mutant GDH (GDH266C), expressed in COS-7 cells, was constitutively elevated, and allosteric regulations by ADP and GTP were severely impaired. The effect of the unregulated increase in GDH activity on insulin secretion was examined by overexpressing GDH266C in an insulinoma cell line, MIN6. Although glutamine alone did not stimulate insulin secretion from control MIN6-lacZ, it remarkably stimulated insulin secretion from MIN6-GDH266C. This finding suggests that constitutively activated GDH enhances oxidation of glutamate, which is intracellularly converted from glutamine to alpha-ketoglutarate, a tricarboxylic acid cycle substrate, which thereby stimulates insulin secretion. Interestingly, insulin secretion is also exaggerated significantly at low glucose concentrations (2 and 5 mmol/l) but not at higher glucose concentrations (8--25 mmol/l). Our results directly illustrate the importance of GDH in the regulation of insulin secretion from pancreatic beta-cells.

Adenosine Diphosphate↗
PubMed2002

Humoral regulation of resistin expression in 3T3-L1 and mouse adipose cells.

Resistin is a hormone secreted by adipocytes that acts on skeletal muscle myocytes, hepatocytes, and adipocytes themselves, reducing their sensitivity to insulin. In the present study, we investigated how the expression of resistin is affected by glucose and by mediators known to affect insulin sensitivity, including insulin, dexamethasone, tumor necrosis factor-alpha (TNF-alpha), epinephrine, and somatropin. We found that resistin expression in 3T3-L1 adipocytes was significantly upregulated by high glucose concentrations and was suppressed by insulin. Dexamethasone increased expression of both resistin mRNA and protein 2.5- to 3.5-fold in 3T3-L1 adipocytes and by approximately 70% in white adipose tissue from mice. In contrast, treatment with troglitazone, a thiazolidinedione antihyperglycemic agent, or TNF-alpha suppressed resistin expression by approximately 80%. Epinephrine and somatropin were both moderately inhibitory, reducing expression of both the transcript and the protein by 30-50% in 3T3-L1 adipocytes. Taken together, these data make it clear that resistin expression is regulated by a variety of hormones and that cytokines are related to glucose metabolism. Furthermore, they suggest that these factors affect insulin sensitivity and fat tissue mass in part by altering the expression and eventual secretion of resistin from adipose cells.

3T3 Cells↗