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Paraquat-induced oxidative stress represses phosphatidylinositol 3-kinase activities leading to impaired glucose uptake in 3T3-L1 adipocytes.

ABSTRACT: Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3'-OH kinase (PI 3-kinase) p85 regulatory subunit or p110alpha catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulin-dependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110alpha and p110beta that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110alpha mutated at Cys(90) in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes.

SUBMITTER: Shibata M

PROVIDER: S-EPMC2898352 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Paraquat-induced oxidative stress represses phosphatidylinositol 3-kinase activities leading to impaired glucose uptake in 3T3-L1 adipocytes.

Shibata Michihiro M Hakuno Fumihiko F Yamanaka Daisuke D Okajima Hiroshi H Fukushima Toshiaki T Hasegawa Takashi T Ogata Tomomi T Toyoshima Yuka Y Chida Kazuhiro K Kimura Kumi K Sakoda Hideyuki H Takenaka Asako A Asano Tomoichiro T Takahashi Shin-Ichiro S

The Journal of biological chemistry 20100429 27

Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-d ...[more]

PMID: 20430890

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Project description:Diabetes mellitus (DM) is a metabolic disorder with chronic hyperglycemia featured by metabolic outcomes owing to insufficient insulin secretion and/or insulin effect defect. It is critical to investigate new therapeutic approaches for T2DM and alternative, natural agents that target molecules in potential signal pathways. Medicinal plants are significant resources in the research of alternative new drug active ingredients. Bolanthus spergulifolius (B. spergulifolius) is one of the genera of the family Caryophyllaceae. In this study, it was explored the potential anti-diabetic effects in vitro of B. spergulifolius extracts on 3T3-L1 adipocytes. The total phenolic contents (TPC) of methanolic (MeOH), ethyl acettate (EA) and aqueous extracts of B. spergulifolius were evaluated via Folin-Ciocateau. B. spergulifolius extracts showing highly TPC (Aqueous< MeOH< EA) and their different concentrations were carried out on preadipocytes differentiated in to mature 3T3-L1 adipocytes to investigate their half-maximal (50%) inhibitory concentration (IC50) value by using Thiazolyl blue tetrazolium bromide (MTT) assay. The IC50 of MeOH, EA and Aqueous extracts were observed as 305.7 ± 5.583 μg/mL, 567.4 ± 3.008 μg/mL, and 418.3 ± 4.390 μg/mL and used for further experiments. A live/dead assay further confirmed the cytotoxic effects of MeOH, EA and Aqueous extracts (respectively, 69.75 ± 1.70%, 61.75 ± 1.70%, 70 ± 4.24%, and for all p< 0.05). Also, effects of extracts on lipid accumulation in mature 3T3-L1 adipocytes were evaluated by Oil-Red O staining assay. The extracts effectively decreased lipid-accumulation compared to untreated adipocytes (for all p< 0.05). Moreover, effect of extracts on apoptosis regulated by the Bax and Bcl-2 was investigated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The extracts significantly induced apoptosis by up-regulating pro-apoptotic Bax expression but down-regulated anti-apoptotic Bcl-2 gene expression compared to untreated adipocytes (for all p< 0.05). The Glut-4 expression linked with insulin resistance was determined by qRT-PCR, Western-blot analysis, and immunofluorescence staining. In parallel, the expression of Glut-4 in adipocytes treated with extracts was significantly higher compared to untreated adipocytes (for all p< 0.05). Extracts significantly suppressed cell migration after 30 h of wounding in a scratch-assay (for all p< 0.05). Cell morphology and diameter were further evaluated by phase-contrast microscopy, scanning electron microscopy, Immunofluorescence with F-Actin and Giemsa staining. The adipocytes treated with extracts partially lost spherical morphology and showed smaller cell-diameter compared to untreated adipocytes (for all p< 0.05). In conclusion, these results suggest that extracts of B. spergulifolius cause to an induce apoptosis, decrease lipid-accumulation, wound healing, up-regulating Glut-4 level and might contribute to reducing of insulin-resistance in DM.

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Paraquat-induced oxidative stress represses phosphatidylinositol 3-kinase activities leading to impaired glucose uptake in 3T3-L1 adipocytes.

Publications

Paraquat-induced oxidative stress represses phosphatidylinositol 3-kinase activities leading to impaired glucose uptake in 3T3-L1 adipocytes.

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