Dataset Information

Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance.

ABSTRACT: The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O-GlcNAc and insulin resistance by using a potent and selective inhibitor of O-GlcNAcase (NButGT (1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-D]-Delta 2'-thiazoline), 1200-fold selectivity). A comparison of the structures of a bacterial homologue of O-GlcNAcase in complex with PUGNAc or NButGT reveals that these inhibitors bind to the same region of the active site, underscoring the competitive nature of their inhibition of O-GlcNAcase and the molecular basis of selectivity. Treating 3T3-L1 adipocytes with NButGT induces rapid increases in global O-GlcNAc levels, but strikingly, NButGT treatment does not replicate the insulin desensitizing effects of the non-selective O-GlcNAcase inhibitor PUGNAc. Consistent with these observations, NButGT also does not recapitulate the impaired insulin-mediated phosphorylation of Akt that is induced by treatment with PUGNAc. Collectively, these results suggest that increases in global levels of O-GlcNAc-modified proteins of cultured adipocytes do not, on their own, cause insulin resistance.

SUBMITTER: Macauley MS

PROVIDER: S-EPMC3259902 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance.

Macauley Matthew S MS Bubb Abigail K AK Martinez-Fleites Carlos C Davies Gideon J GJ Vocadlo David J DJ

The Journal of biological chemistry 20081008 50

The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O-GlcNAc and insulin resistance by usi ...[more]

PMID: 18842583

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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.

Dataset Information

Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance.

Publications

Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance.

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