These findings suggest that the increased morbidity to sepsis in obesity may result from exaggerated microvascular inflammatory and thrombogenic responses that include the activation of endothelial cells with subsequent expression of adhesion molecules, such as P-selectin.

In this study we investigated the effect of glucose on GSK3β and β-catenin expression and the involvement of the N-linked glycosylation and hexosamine pathways in the Wnt canonical pathway in response to in vitro conditions resembling normoglycemia (5  mmol) and hyperglycemia (20  mmol) in the metastatic breast cancer-derived cell line MDA-MB-231. We also investigated the relationship between this circuitry and the thioredoxin-interacting protein (TXNIP) regulation that seems to be related. MDA-MB-231 cells were grown either in 5 or 20  mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5  mM glucose and shifted to 20  mM at time 0. Both protein and mRNA levels for GSK3β but only the protein expression for β-catenin, were increased in response to high glucose. Furthermore, we assessed the response of GSK3β, β-catenin, and TXNIP to inhibition of the N-linked glycosylation, hexosamine, and Wnt pathways. Wnt signaling pathway activation was validated by specific reporter assay. We show that high levels of glucose regulate mRNA and protein expression of GSK3β, and consequently higher levels of activated β-catenin protein, which locates to the nucleus and is associated with increased levels of cyclin D1 expression. This event coincides with increased level of N-terminal Ser 9 phosphorylation of GSK3β protein. The inhibition of both the hexosamine pathway and N-linked glycosylation along with Wnt signaling pathway by sFRP1 and DKK1 is associated with significant decrease of the protein levels of GSK3β, β-catenin, and TXNIP RNA. Our work illuminates a novel and never described before function of this signaling pathway that relates glucose metabolism with redox regulation mechanism.

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Hyperglycemia represents one of the early events in the development of diabetes type 2. Susceptibility to this disease has been linked to Wnt/β-catenin signaling pathway that also controls breast cancer progression. We had previously analyzed the effects of hyperglycemia on gene expression profile (GEP) by Affimetrix analysis in breast cancer derived MDA-MB-231 cells (Turturro F. et al., BMC Cancer 2007, 7:96). Wnt/βcatenin signaling pathway had emerged as one of the most prominent signaling pathway regulated by increased glucose in culture media (5 mM versus 20 mM). The aim of the present study was to validate the GEP data by assessing the mRNA levels of both GSK-3b and Wnt1 by RT-PCR in response to hyperglycemia as previously described. We analyzed the levels of RT-PCR products of the target genes relative to the control mRNA of β-actin in MDA-MB-231 cells, cultured in the same conditions as previously illustrated (Turturro F. et al., BMC Cancer 2007, 7:96). The values were expressed as densitometric readings. Relative levels of GSK-3b mRNA raised from 0.14 + 0.053 for 5 mM glucose to 0.308 + 0.0476 for 5/20 shift (p< 0.05 at 5 vs. 5/20 mM) and to 0.432 + 0.0321 for 20 mM (p< 0.05 at 5 vs. 20 mM) resulting in an overall 3 fold increase. We also examined the effect of hyperglycemia on gene expression of Wnt-1 which inhibits the expression and activity of GSK-3b. The relative levels of mRNA of Wnt-1 assessed by RT-PCR, were decreased from 0.163 + 0.0322 for 5 mM glucose to 0.068 + 0.001 for 20 mM glucose, with an overall 2.7 fold reduction (p<.05 at 5 vs. 20 mM). These data confirmed the GEP results in terms of relationship between hyperglycemia and gene expression of the Wnt signaling pathway in this particular breast cancer cell line. Our study supports further investigation in the area of diabetes and breast cancer invasiveness and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3141.

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