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Ethanol inhibition of insulin signaling in hepatocellular carcinoma cells
K Banerjee1, L Mohr, J R Wands
1MGH East Cancer Center, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, USA.
Abstract:
Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyl phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such cells were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyl phosphorylation of the insulin receptor beta-subunit, tyrosyl phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyl phosphorylation of Shc. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on Shc phosphorylation, which activates p21ras through an IRS-1 independent pathway.
Insights
Chronic ethanol toxicity inhibits insulin signaling and growth-associated gene expression in liver cancer cells overexpressing insulin receptor substrate-1 (IRS-1). These cells show resistance to epidermal growth factor (EGF) signaling, suggesting a complex response to alcohol.
Area of Science:
- Hepatology
- Molecular Biology
- Cancer Research
Background:
- Chronic ethanol toxicity is known to impair liver regeneration and DNA synthesis.
- Ethanol's adverse effects are linked to the inhibition of insulin receptor and insulin receptor substrate-1 (IRS-1) tyrosyl phosphorylation.
- Overexpression of IRS-1 can lead to increased DNA synthesis and cellular transformation via mitogen-activated protein (MAP) kinase activation.
Purpose of the Study:
- To investigate the effects of ethanol on insulin signaling pathways in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1.
- To determine if IRS-1-overexpressing liver cancer cells are resistant to the inhibitory effects of ethanol on insulin signaling.
Main Methods:
- Treatment of FOCUS hepatocellular carcinoma cells with ethanol (100 mM).
- Assessment of insulin-stimulated tyrosyl phosphorylation of the insulin receptor beta-subunit and IRS-1.
- Measurement of MAP kinase and phosphatidylinositol-3 kinase activities and their association with IRS-1.
- Analysis of epidermal growth factor (EGF)-stimulated Shc phosphorylation.
- Quantification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA) gene expression.
Main Results:
- Ethanol treatment reduced insulin-stimulated tyrosyl phosphorylation of the insulin receptor and IRS-1 by 30-50%.
- Ethanol inhibited MAP kinase and phosphatidylinositol-3 kinase activities and their association with IRS-1.
- Ethanol did not affect EGF-stimulated Shc phosphorylation, indicating an IRS-1 independent pathway.
- Ethanol treatment decreased the expression of insulin-responsive genes GAPDH and PCNA by 30-50%.
Conclusions:
- In FOCUS hepatocellular carcinoma cells overexpressing IRS-1, ethanol substantially inhibits IRS-1 and MAP kinase signaling.
- Ethanol also reduces growth-associated gene expression in these cells.
- The findings suggest that while ethanol impacts insulin signaling, EGF-mediated p21ras activation through Shc phosphorylation remains unaffected, highlighting a potential pathway for cellular transformation independent of IRS-1.
