cytochrome-c-t and Choline-Deficiency

Alcoholic liver disease is associated with nutritional deficiency and it may aggravate within the context of fatty liver. We investigated the relationship between alcohol intake (whiskey binge drinking) and a choline-deficient diet (CD) and assessed whether stellate cells could contribute to liver injury in this model.. Rats fed the CD diet plus whiskey showed increased liver damage compared to rats fed the CD diet, as demonstrated by H&E staining, elevated transaminases, steatosis, TNF-alpha levels, enhanced CYP2E1 activity, impaired antioxidant defense, elevated lipid peroxidation, and protein carbonyls. The combined treatment triggered an apoptotic response as determined by elevated Bax, caspase-3 activity, cytochrome-c release, and decreased Bcl-2 and Bcl-XL. Stellate cells were activated as increased expression of alpha-Sma was observed over that by the CD diet alone. The combined treatment shifted extracellular matrix remodeling towards a pro-fibrogenic response due to up-regulation of collagen I, TIMP1, and Hsp47 proteins, along with down-regulation of MMP13, MMP2, and MMP9 expression, proteases which degrade collagen I. These events were accompanied by increased phosphorylation of p38, a kinase that elevates collagen I.. Repeated alcohol binges in the context of mild steatosis may promote activation of stellate cells and contribute to liver injury.

Topics: Alcoholic Beverages; Animals; Antioxidants; Apoptosis Regulatory Proteins; Choline; Choline Deficiency; Collagenases; Cytochrome P-450 CYP2E1; Cytochromes c; Diet; Lipid Peroxidation; Liver; Liver Diseases, Alcoholic; Male; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Inbred Lew; Tumor Necrosis Factor-alpha

Previous studies show that acute choline deficiency (CD) triggers apoptosis in cultured rat hepatocytes (CWSV-1 cells). We demonstrate that prolonged EGF stimulation (10 ng/mL x 48 hrs) restores cell proliferation, as assessed by BrdU labeling, and protects cells from CD-induced apoptosis, as assessed by TUNEL labeling and cleavage of poly(ADP-ribose) polymerase. However, EGF rescue was not accompanied by restoration of depleted intracellular concentrations of choline, glycerphosphocholine, phosphocholine, or phosphatidylcholine. In contrast, we show that EGF stimulation blocks apoptosis by restoring mitochondrial membrane potential (Delta Psi(m)), as determined using the potential-sensitive dye chloromethyl-X-rosamine, and by preventing the release and nuclear localization of cytochrome c. We investigated whether EGF rescue involves EGF receptor phosphorylation and activation of the down-stream cell survival factor Akt. Compared to cells in control medium (CT, 70 micromol choline x 48 hrs), cells in CD medium (5 micromol choline) were less sensitive to EGF-induced (0-300 ng/mL x 5 min) receptor tyrosine phosphorylation. Compared to cells in CT medium, cells in CD medium treated with EGF (10 ng/mL x 5 min) exhibited higher levels of phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of AktSer473. Inactivation of PI3K was sufficient to block EGF-stimulated activation of Akt, restoration of mitochondrial Delta Psi(m), and prevention of cytochrome c release. These studies indicate that stimulation with EGF activates a cell survival response against CD-apoptosis by restoring mitochondrial membrane potential and preventing cytochrome c release and nuclear translocation which are mediated by activation of Akt in hepatocytes.

Topics: Animals; Apoptosis; Cell Line; Cell Proliferation; Choline; Choline Deficiency; Cytochromes c; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Hepatocytes; Male; Mitochondria; Phosphorylation; Rats; Rats, Inbred F344; Transcription Factors