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Biomedical subjects

Arno K Kumagai

Publications and source records attributed to Arno K Kumagai.

7 recordsLinked to original sources
PubMed2005

GLUT4 facilitative glucose transporter specifically and differentially contributes to agonist-induced vascular reactivity in mouse aorta.

OBJECTIVE: We hypothesized that GLUT4 is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs), and GLUT4 is necessary for agonist-induced VSMC contraction. METHODS AND RESULTS: Glucose deprivation and indinavir, a GLUT4 antagonist, were used to assess the role of GLUT4 and non-GLUT4 transporters in vascular reactivity. In isolated endothelium-denuded mouse aorta, approximately 50% of basal glucose uptake was GLUT4-dependent. Norepinephrine-mediated contractions were dependent on both GLUT4 and non-GLUT4 transporters, serotonin (5-HT)-mediated contractions were mainly GLUT4-dependent, and prostaglandin (PG) F(2alpha)-mediated contractions were dependent on non-GLUT4 transporters, whereas indinavir had no effect in GLUT4 knockout vessels. We also observed a 46% decrease in GLUT4 expression in aortas from angiotensin II hypertensive mice. Indinavir caused a less profound attenuation of maximal 5-HT-mediated contraction in these vessels, corresponding to the lower GLUT4 levels in the hypertensive aortas. Finally, and somewhat surprisingly, chronic GLUT4 knockout was associated with increased vascular reactivity compared with that in wild-type animals, suggesting that chronic absence or reduction of GLUT4 expression in VSMCs leads to opposite effects observed with acute inhibition of GLUT4. CONCLUSIONS: Thus, we conclude that GLUT4 is constitutively expressed in large arteries and likely participates in basal glucose uptake. In addition, GLUT4, as well as other non-GLUT4 facilitative glucose transporters, are necessary for agonist-induced contraction, but each transporter participates in VSMC contraction selectively, depending on the agonist, and changes in GLUT4 expression may account for some of the functional changes associated with vascular diseases like hypertension.

Angiotensin II↗
PubMed2005

Increased JNK phosphorylation and oxidative stress in response to increased glucose flux through increased GLUT1 expression in rat retinal endothelial cells.

PURPOSE: To investigate whether increased glucose flux through increased glucose transporter1 (GLUT1) expression results in increased oxidative stress and increased c-jun N-terminal kinase (JNK) phosphorylation. METHODS: GLUT1-overexpressing cells were established using a rat retinal endothelial cell line. The intracellular reactive oxygen species was detected by the oxidation of 5- (and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA). Western blot was performed to determine JNK phosphorylation and lipid peroxidation. Differentially expressed genes were detected by cDNA microarray analysis and confirmed by Northern blot analysis. RESULTS: Clones overexpressing GLUT1 showed an approximate four- to eightfold increase in GLUT1 expression and a 44% increase in intracellular glucose concentrations. GLUT1-overexpressing cells had a 80% increase in DCF fluorescence and increased lipid peroxidation, as well as increased JNK phosphorylation. Analysis of differentially expressed genes in GLUT1-overexpressing cells showed increased expression of JNK interacting protein (JIP)-1, a scaffold protein necessary for JNK activation. Northern blot analysis confirmed upregulation of JIP-1. Immunoprecipitation showed that phosphorylated JNK, but not total JNK, coimmunoprecipitated with JIP-1 protein. At the cellular level, JIP-1 was predominantly localized in cytoplasm, especially in the perinuclear area in retinal endothelial cells. CONCLUSIONS: GLUT1 overexpression and increased glucose flux result in increased oxidative stress and JNK phosphorylation in immortalized rat retinal endothelial cells. Further studies are needed to understand molecular events after increased glucose flux in retinal endothelial cells and the relation between increased oxidative stress and JNK phosphorylation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Inner blood-retinal barrier GLUT1 in long-term diabetic rats: an immunogold electron microscopic study.

PURPOSE: The GLUT1 glucose transporter mediates glucose entry into the endothelial cells of the inner blood-retinal barrier (BRB). In many cell types, exposure to high glucose concentrations or diabetes downregulates GLUT1. To determine whether long-standing diabetes alters the expression and distribution of inner BRB GLUT1, changes in immunoreactive retinal endothelial cell GLUT1 were studied in Goto-Kakizaki (GK) rats, an animal model of type 2 diabetes. METHODS: Immunogold staining for GLUT1 was performed on ultrathin sections of retinal specimens obtained from 1-year-old GK rats and age-matched Wistar controls. Retinal capillary endothelial cells were visualized by transmission electron microscopy, and GLUT1 immunogold was quantified on the luminal and abluminal membranes of endothelial cells from digital microphotographs of individual vessels by computer. RESULTS: Forty-one microvessels from six diabetic rats and 43 microvessels from six nondiabetic Wistar control rats were analyzed. Densitometric quantification revealed an asymmetry of GLUT1 distribution between luminal and abluminal membranes of both diabetic and nondiabetic rats, with a luminal-to-abluminal ratio of approximately 1 to 3. The distribution pattern and density of retinal endothelial GLUT1 immunoreactivity were not significantly different between the diabetic and control rats. CONCLUSIONS: As determined by GLUT1 immunogold distribution, there is no compensatory downregulation of GLUT1 on the inner BRB in an animal model of long-standing diabetes.

Animals↗
PubMed2003

Aldose reductase inhibitor fidarestat prevents retinal oxidative stress and vascular endothelial growth factor overexpression in streptozotocin-diabetic rats.

The study addressed the role for aldose reductase (AR) in 1) retinal oxidative stress and vascular endothelial growth factor (VEGF) overexpression in early diabetes, and 2) high glucose-induced oxidative stress in retinal endothelial cells. In vivo experiments were performed on control rats and diabetic rats treated with or without low or high dose of the AR inhibitor (ARI) fidarestat (2 or 16 mg. kg(-1). day(-1)). In vitro studies were performed on bovine retinal endothelial cells (BREC) cultured in either 5 or 30 mmol/l glucose with or without 1 micro mol/l fidarestat. Intracellular reactive oxygen species were assessed using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) probe and flow cytometry. Both low and high doses of fidarestat (i.e., the doses that partially and completely inhibited sorbitol pathway hyperactivity) arrested diabetes-induced retinal lipid peroxidation. This was achieved due to upregulation of the key antioxidative defense enzyme activities rather than changes in reduced glutathione, oxidized glutathione, ascorbate and dehydroascorbate concentrations, and the glutathione and ascorbate redox states. Diabetes-associated 2.1-fold VEGF protein overexpression (enzyme-linked immunosorbent assay; ELISA) was dose-dependently prevented by fidarestat, whereas total VEGF mRNA and VEGF-164 mRNA (RT-PCR) abundance were not affected by either diabetes or the ARI. In BREC, fidarestat corrected hyperglycemia-induced increase in H(2)DCFDA fluorescence but not oxidative stress caused by three different pro-oxidants in normoglycemic conditions. In conclusion, increased AR activity contributes to retinal oxidative stress and VEGF protein overexpression in early diabetes. The findings justify the rationale for evaluation of fidarestat on diabetic retinopathy.

Aldehyde Reductase↗
PubMed2002

Glucose uptake and adenoviral mediated GLUT1 infection decrease hypoxia-induced HIF-1alpha levels in cardiac myocytes.

Hypoxia causes a large array of adaptive and physiological responses in all cells including cardiac myocytes. In order to elucidate the molecular effects of increased glucose flux on hypoxic cardiac myocytes we focused on the basic helix-loop-helix transcription factor, hypoxia inducible factor 1 alpha (HIF-1alpha), which is rapidly upregulated in hypoxic cells and elicits a number of responses including augmentation of glucose uptake. Primary cultures of neonatal rat cardiac myocytes as well as embryonic rat heart-derived myogenic H9c2 cells demonstrated a significant upregulation of HIF-1alpha when subjected to hypoxia of 6-8h in the absence of glucose. Re-addition of extracellular glucose to the medium resulted in a decrease of HIF-1alpha levels by almost 50%. This glucose effect was blocked by addition of glycolytic inhibitors. In addition, glucose uptake and glycolysis resulted in substantial decreased levels of p53, which is regulated by HIF-1alpha. Adenoviral infection of cultures of cardiac myocytes with the facilitative glucose transporter, GLUT1 followed by hypoxia of 24h also resulted in a significant reduction in the protein expression of HIF-1alpha compared to control vector-infected cultures. GLUT1 infected cultures also demonstrated fewer apoptotic cells and a reduction in the release of cytochrome c after hypoxia. Inhibition of the ubiquitin-proteasomal pathway by a variety of 26S proteasomal inhibitors increased HIF-1alpha to similar levels under both normoxic and hypoxic conditions and in the presence or absence of glucose. This result suggested that glucose induces HIF-1alpha degradation via a proteasomal pathway. This conclusion was substantiated by immunoprecipitation experiments of total cell extracts, which demonstrated an increase of ubiquitinated HIF-1alpha relative to total HIF-1alpha in the presence of glucose during hypoxia. Thus, glucose as well as GLUT1 overexpression diminishes hypoxia-induced HIF-1alpha protein via an ubiquitin-proteasomal pathway in hypoxic cardiac myocytes. This represents a novel feedback mechanism that may play an important role in adaptation of cardiac myocytes to hypoxia and ischemia.

Adenoviridae↗
PubMed2002

Insulin-like growth factor-1 effects on bovine retinal endothelial cell glucose transport: role of MAP kinase.

In order to maintain normal metabolism, the neuroretina is completely dependent on the constant delivery of glucose across the retinal microvascular endothelial cells comprising the inner blood-retinal barrier. Glucose uptake into these cells is influenced by various stimuli, including hypoxia and growth factors. Recently, insulin-like growth factor-1 (IGF-1) was shown to enhance retinal endothelial glucose transport in a process that is dependent on protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3 kinase). In the current study, the role of mitogen-activated protein kinase (MAP kinase) in regulating IGF-1 effects on retinal endothelial cell glucose transport was investigated in a bovine retinal endothelial cell (BREC) culture model. IGF-1 (25 ng/mL) caused a rapid increase in MAP-kinase activity and ERK phosphorylation. Inhibition of MAP kinase with PD98059 (100 microm) blocked IGF-1 enhancement of 2-deoxyglucose uptake. In order to clarify the relationship between PKC, PI3 kinase and MAP kinase in IGF-1 signaling in retinal endothelial cells, the effects of selective inhibitors of MAP kinase (PD98059), PKC (GF109203X), and PI3 kinase (wortmannin, LY294002) on signal transduction by IGF-1 were studied. Inhibition of MAP kinase abolished IGF-1 stimulation of PKC but had no effect on PI3 kinase activity, whereas inhibition of either PKC and PI3 kinase had no effect on MAP kinase phosphorylation or activity in IGF-1-treated cells. Taken together, these data demonstrate that IGF-1 stimulation of BREC glucose transport requires activation of MAP kinase and that MAP kinase is upstream from PKC but is independent of PI3 kinase in mediating the actions of IGF-1 on retinal endothelial cells.

Animals↗