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The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Institute of Biochemistry, Food Science and Nutrition, Rehovot 76100, Israel
2To whom correspondence should be addressed. E-mail:Madar{at}agri.huji.ac.il.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (NF-B), one of the transcription factors involved in eNOS and iNOS transcriptional regulation, was decreased 15% in the nucleus by LE treatment. These results suggest that TG reduces nitrite production by elevating intracellular ROS levels (prolonged oxidative stress), and the downregulation of NOS enzymes may occur at least in part via the NFB pathway.
KEY WORDS: • liver • triglyceride • reactive oxygen species • nitric oxide
Chronic exposure to excess lipid is thought to be a risk factor for cardiovascular disease and diabetes (1). Overloading of white adipose tissue beyond its storage capacity leads to lipid disorders in nonadipose tissues such as skeletal and cardiac muscles, pancreas, and liver. This in turn may lead to tissue-specific disorders in insulin response, increased lipid deposition, and lipotoxicity coupled with abnormal plasma metabolic and (or) lipoprotein profiles (1,2). In humans, triacylglycerol (TG)3 and FFA accumulation in the liver is associated with nonalcoholic steatohepatitis (NASH), characterized by an inflammatory response with evidence of hepatocyte damage and fibrosis that can progress to cirrhosis (3). Long-term total parenteral nutrition (TPN) can frequently lead to the development of hepatic steatosis (4). NASH has also been described in obese individuals, in diabetics, and in patients with lipodystrophy (5). To better understand the mechanisms of hepatic lipotoxicity, many factors must be taken into consideration, including the role of nitric oxide (NO) and reactive oxygen species (ROS).
NO is an important factor in liver function. Impaired sinusoidal endothelial nitric oxide synthase (eNOS) activity was suggested to be responsible, at least in part, for the increased intrahepatic blood vessel resistance and portal hypertension, which is one of the characteristic dynamic changes of liver cirrhosis (6,7). The potentially protective role of endogenous NO in liver injury is supported indirectly by several studies demonstrating enhanced hepatocellular injury in postischemic animals given nonselective NOS inhibitors such as nitro-L-arginine methyl ester or N-monomethyl-L-arginine (8,9).
ROS act as signaling intermediates regulating multiple cellular processes. Exposure to lipids increases ROS production in endothelial cultures (10), in the blood of human subjects (11), and in pancreatic islets (12). This effect is thought to be an important regulator of cell death pathways and thus may play a role in lipid-induced lipotoxicity (13). However, very little information is available concerning the effects of exposure to high levels of lipids in the liver and which metabolic pathways may be activated or inhibited. In the case of TPN-associated hepatic steatosis, the exact pathogenesis is not fully understood. Studies suggest a major role for ROS in the etiology of NASH (14). We recently showed that exposing macrophages to lipid emulsion (LE) increased ROS production by the mitochondria, thus facilitating cellular necrosis (15).
Oxidative stress is also thought to play an important role in regulating the nuclear transcription factors responsible for NOS expression. The promoters of the inducible nitric oxide synthase (iNOS) and eNOS genes contain binding sites for transcription factors including binding sites for the transcription factors nuclear factor-B (NF-B) and activator protein-1 (AP-1) (16). Activity of these transcription factors is thought to be modified in the presence of ROS.
This study was designed to provide a better understanding of the metabolic effect of exposure to high levels of lipids in the liver, using a model of rat isolated hepatocytes exposed to soy oil–based LE as a source of TG. Effects on the NO system and intracellular redox balance were evaluated.
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MATERIALS AND METHODS |
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, TriReagent was purchased from Sigma, and ReddymixTM was obtained from ABgene. Lipid emulsion containing 63.8% saturated short- and medium-chain fatty acids [(6:0–12:0), 4% 16:0; 1.6% 18:0; 8.5% 18:1; 19.5% 18:2; 2% 18:3; 0.6% 20:4 20:5 22:6] (17) and vitamin E (200 mg/L; Lipofundin 20%) was a gift from Uri Kogan (Luxembourg Pharmaceuticals, Lod, Israel). All cell culture materials were purchased from Biological Industries and all other chemicals were purchased from Sigma. Hepatocyte isolation. Rat hepatocytes were isolated as described by Berry et al. (18). The cells were suspended at a concentration of 2 x 109 cells/L in DMEM containing 10% fetal calf serum, 100 mg/L penicillin, 100 mg/L streptomycin, and 100 mg/L gentamicin and plated onto 10-cm plates. The cultures were incubated at 37°C and used 3–4 h after plating. All experimental procedures using rats were approved by the Institutional Animal Care Committee of the Hebrew University of Jerusalem.
Cell culturing and treatment.
Isolated hepatocytes were seeded at 2 x 109 cells/L; after 4 h, the culture medium was replaced and cells were incubated for 48 h with 0.01–0.1% LE. Rotenone, S-nitroso-N-acetyl-penicillamine (SNAP), buthionine sulfoximine (BSO), antioxidants, or TNF- were added to the culture medium in specific experiments. At the end of the experiment, the medium was collected and the cells were harvested for further assays.
TLC for determination of FFAs. TG and FFAs were separated using TLC on DC-Plastikfolien 60, thickness 0.2 mm (Merck). TLC plates were loaded with standards of TG and FFAs along with medium samples and placed in a solvent system comprised of petroleum ether:diethyl ether:acetic acid (80:19:1, by vol) for 50–60 min (19). Visualization was performed by iodine staining.
Determination of cellular fatty acid content. The concentrations of FFAs were measured using GC (15). Briefly, cell lipid content was extracted with a mixture of chloroform:methanol (2:1). After the addition of C17 internal standard (0.1 mg for 106 cells) and 20 µL hydrolysis-methylation reagent (MetPREP) to each extract, the samples were suspended in 50 µL toluene. The samples were injected and analyzed by GC using a flame ionization detector.
Protein expression (Western blot analysis). Cells were scraped and lysed in 750 µL of lysis buffer (20 mmol/L Tris, pH 7.8; 0.1% Nonidet P-40; 100 mmol/L NaCl; 50 mmol/L NaF; 10% glycerol, 1 mmol/L sodium orthovanadate). Lysates were centrifuged at 8500 x g for 10 min. The supernatant was collected and used for the analysis of eNOS and iNOS. For the determination of p65 and p-c-Jun, nuclear extracts were prepared by suspending the cells in hypotonic buffer [20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.5 mmol/L dithiothreitol (DTT), 0.1% Triton X 100, 20% glycerol, 2 mmol/L phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail] followed by centrifugation at 1600 x g for 10 min. The supernatant was discarded. The pellets were suspended in hypertonic buffer (20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.5 mmol/L DTT, 0.1% Triton X 100, 20% glycerol, 2 mmol/L PMSF, 420 mmol/L NaCl, and protease inhibitor cocktail) and incubated on a shaker for 4 min at 4°C. Subsequently, the samples were centrifuged at 35,000 x g for 10 min and the supernatant was collected.
Protein concentration was determined by the Bradford method (20) using bovine serum albumin as a standard. Samples were boiled for 5 min with SDS sample buffer; 60 µg of protein per sample was loaded onto a 10% SDS-polyacrylamide gel. Electroblots were blocked in Tris buffer NaCl-Tween (TBST) containing 5% skim milk powder at room temperature. Western blot analysis with a specific antibody against iNOS (Biomol), eNOS, p65, and p-c-Jun antibodies (Santa Cruz Biotechnology) was carried out. The antibodies were diluted in TBST buffer + 5% skim milk and left overnight at 4°C. After a TBST washing procedure, the blots were incubated with horseradish-peroxidase labeled anti-rabbit antibody (Pierce) for 1 h at room temperature. The immune reaction was detected by enhanced chemiluminescence. Bands were quantified by scanning densitometry and expressed as arbitrary units.
Determination of nitrite concentrations in culture media (Griess reaction). Nitrite in culture media was measured by the Griess reaction (21). The values obtained were compared with standards of sodium nitrite dissolved in the cell culture media. Nitrite release was calculated and expressed in µmol/106 cells.
Total RNA isolation and reverse transcription PCR analysis. Total RNA was prepared using TriReagent. Analyses of mRNA levels of iNOS, eNOS, and gylceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using RT-PCR. The synthesis of cDNA was carried out using a Reverse-it (ABgene) first-strand synthesis kit. The reaction was conducted at 40–55°C for 50 min. Finally, the reaction was terminated by incubation at 70°C for 15 min. PCR amplification was performed in the Biometra T personal cycler instrument (Biometra). Cycles were performed at 94°C for 30 s followed by 57°C for 30 s and 72°C for 30 s. cDNA was incubating with ReddyMix and gene-specific PCR primers, designed using Primer 3 software (Whitehead Institute for Biomedical Research). The primers were synthesized by MBC: eNOS, forward primer, 5'-GAGCATACCCGCACTTCTGT-3', and reverse primer, 5'-GAAGATATCTCGGGCAGCAG-3'; iNOS, forward primer, 5'CAGCACAGAGGGCTCAAAGG-3', and reverse primer, 5'- TCGTCGGCCAGCTCTTTCT-3'. As a loading control, RNA was hybridized with a probe of the housekeeping gene, GAPDH, forward primer, 5'-TCCGCCCCTTCCGCTGATG-3', and reverse primer, 5'- CACGGAAGGCCATGCCAGTGA-3'. PCR products were electrophoresed on 0.1% agarose gel containing 5 µL ethidium bromide, and the gel image was quantified using Doc-it gel image analysis program (UVP). The number of cycles for all the genes was selected within the linear part of a standard curve.
Reduced glutathione (GSH) measurement. GSH was measured using HPLC (15). Hepatocytes were suspended in 4% metaphosphoric acid and analyzed in running buffer (50 mmol/L KH2PO4 and 2% acetonitrile, pH 2.7) in a Synergy 4-µm Polar-RP 80A column (Phenomenex) when the cell potential was 800 mV. Detection was made by an electrochemical detector. The results were adjusted to protein levels of the samples.
Cell viability, TG accumulation, and ROS. Cell membrane integrity (15), TG accumulation (22), and intracellular ROS levels (23,24) were detected by flow cytometer (FACScalibur BD). Hepatocytes were stained with 2 mg/L DNA-interchelating dye propidium iodide, which is excluded by viable cells; with 1 mg/L Nile red, which accumulates in intracellular lipid droplets; or with 50 mmol/L dichlorodihydrofluorescein diacetate (H2DCF-DA), a probe that has high reactivity to hydrogen peroxide, lipid hydroperoxide, and hydroxyl radicals and low reactivity to superoxide anions (23,24). Fluorescence settings were as follows: excitation at 488 nm and emission at 575 nm for propidium iodide and Nile red and 488 nm and recorded at 530 nm for H2DCF-DA. Data were collected from 10,000 cells.
Statistical analysis. The significance of the differences between means was determined by Student’s t test when a single comparison was performed. When multiple comparisons were carried out, the significance was tested using either 1- or 2-way ANOVA, depending on the number of factors considered. When necessary, data were log transformed to achieve stabilized variance. A post-hoc test (Tukey-Kramer) was performed when the interaction between treatments was significant. Differences were considered significant at P < 0.05. JMP version 3.1.6 (SAS Institute) was used for all analyses.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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90% was measured in control cultures collected after 12, 24, and 48 h of incubation. LE at concentrations ranging from 0.01 to 0.1% did not cause cell death (P > 0.1; data not shown)
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for 3–12 h. LE attenuated the TNF-–stimulative effect of nitrite production at 9 and 12 h after induction (Table 6).
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B.
LE treatment decreased 15% (P < 0.05; data not shown) the abundance of the p65 subunit of the of transcription factor NF-B in the nucleus. LE did not change the abundance of AP1 p-c-Jun subunit in the nucleus (data not shown). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The lipid content of the cells treated with the LE was 40% greater than that of untreated cells (Table 1), with a significant increase in the fatty acids 16:0, 18:0, 18:1, and 18:2. The total TG content was also increased in the hepatocytes (Table 2). TG accumulation in the cells supports the assumption that effects seen in this study can be attributed to increased lipid content of hepatocytes. We were unable to identify the presence of FFAs in the culture media after 48 h of incubation, indicating that the cells were exposed to intact TG. It is possible that part of the lipotoxic effect of impairment of the NO system could be due to an increase in FFAs in the cell (27).
The reduction in nitrite levels was further investigated to elucidate the mechanisms by which the LE modulates nitrite formation. Measurements of mRNA and protein levels of eNOS and iNOS showed a significant decrease in mRNA levels of both isoforms (Fig. 2A,B). This was accompanied by a reduction in iNOS and eNOS protein expression (Fig. 2C; data not shown). These results suggest that after exposure to LE, production of NO is downregulated at the level of transcription. Work carried out by Kim et al. (28) in rats reported that high-fat diets increased iNOS expression and NO production in the liver, probably via pathways involving cholesterol. However, in human studies, LE infusions reduced NO blood levels (26).
Lipid uptake (Table 2) led to increased intracellular ROS levels (Fig. 1). It was shown that exposure to lipids increases ROS production in endothelial cultures (10) and in the blood (29). The elevation in ROS levels was inversely correlated with nitrite levels, indicating that elevated ROS levels may regulate the decrease in NO production. Miralles et al. (30) found that inducing oxidative stress by exposing isolated hepatocytes to 95% oxygen increased iNOS transcription and expression. In contrast, induction of ROS production after exposure to lipid seemed to inhibit rather than induce NO production. In human studies, intralipid infusions led to a reduction in nitrite and nitrate production accompanied by an increase in ROS levels in the blood (11). To further support this viewpoint, it was found that in hepatocytes exposed to rotenone-induced oxidative stress (48 h treatment), NO production was significantly decreased (Table 4). To substantiate causality for the correlation between oxidative stress and inhibition of NO production, hepatocytes were treated with BSO to block GSH synthesis. BSO had an effect similar to that of LE and reduced nitrite levels (Table 5). GSH supports NO production by keeping the tetrahydrobiopterin in its reduced state (31). In addition, glutathione can increase iNOS expression (32). It was established that inhibition of GSH synthesis decreases iNOS transcription in hepatocytes (33,34). The transcription factors AP-1 and NF-B that activate e/iNOS expression are activated after transient exposure of cells to ROS (35,36). However, these nuclear factors have to function in a reducing environment to bind DNA response elements. Thus, lipid-mediated prolonged oxidative stress, which cannot be compensated for by the cellular antioxidant defense system, may be the primary reason for downregulation of NO production in hepatocytes.
Exposure of hepatocytes to elevated levels of TG was also able to reduce the induction of NO production by TNF- in the hepatocyte cultures (Table 6). A similar effect was found in rat islets of Langerhans (37) where reduction of GSH was able to inhibit interleukin-1ß induction of iNOS transcription. SNAP did not affect intracellular ROS levels in the presence or absence of LE (Table 3), implying that NO had little or no effect on ROS production.
As seen in this study, ascorbate, resveratrol, and NAC attenuated or prevented the inhibitory effect of LE on NO production (Fig. 3). NAC achieved partial recovery of the NOS isoform transcription (Fig. 2A,B) and NOS protein levels (Fig. 2C; data not shown). These protective effects of antioxidants further support the hypothesis that LE regulates NO production by altering the redox balance toward oxidation.
The abundance of p-c-Jun subunit of AP-1 in the nucleus (data not shown) was not affected by LE exposure. However, the p65 subunit of NF-B in the nucleus was reduced (data not shown; P < 0.05), demonstrating an effect on NF-B translocation to the nucleus. The 15% decrease in the translocation of p65 to the nucleus is probably only one factor affecting the inhibition of e/iNOS expression and may not explain the large reduction in their expression levels. One may postulate that lipid treatment may interfere with the DNA binding capacity of these transcription factors and their ability to activate gene expression via redox-dependent or -independent mechanisms that are beyond nuclear translocation.
We suggest that downregulation of NO, as reported here, may play a key role in liver lipotoxicity. Our results indicate that LE reduced nitrite production by elevating intracellular ROS levels in isolated hepatocytes, a phenomenon preventable by NAC, ascorbate, and resveratrol. The effect on NO production most likely involves interference with mRNA transcription, resulting in a reduction in the mRNA and protein levels of the NOS isoforms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AP-1, activator protein 1; BSO, buthionine sulfoximine; DTT, dithiothreitol; eNOS, endothelial nitric oxide synthase; GAPDH, gylceraldehyde-3-phosphate dehydrogenase; GSH, reduced glutathione; H2DCF-DA, dichlorodihydrofluorescein diacetate; iNOS inducible nitric oxide synthase; LE, lipid emulsion; NAC, N-acetyl-L-cysteine; NASH, nonalcoholic steatohepatitis; NF-B, nuclear factor-B; NO, nitric oxide; PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species; SNAP, S-nitroso-N-acetyl-penicillamine; TG, triacylglycerol; TNF, tumor necrosis factor; TPN, total parenteral nutrition.
Manuscript received 17 February 2005. Initial review completed 25 March 2005. Revision accepted 6 June 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Grundy, S. M., Abate, N. & Chandalia, M. (2002) Diet composition and the metabolic syndrome: what is the optimal fat intake?. Am. J. Med. 113(suppl. 9B):25S-29S.
2. Schaffer, J. E. (2003) Lipotoxicity: when tissues overeat. Curr. Opin. Lipidol. 14:281-287.[Medline]
3. Yu, A. S. & Keeffe, E. B. (2002) Nonalcoholic fatty liver disease. Rev. Gastroenterol. Disord. 2:11-19.[Medline]
4. Sharif, K., Beath, S. V., Kelly, D. A., McKiernan, P., van Mourik, I., Mirza, D., Mayer, A. D., Buckels, J. A. & de Ville de Goyet, J. (2003) New perspective for the management of near-total or total intestinal aganglionosis in infants. J. Pediatr. Surg. 38:25-28; discussion 25–28.[Medline]
5. Zafrani, E. S. (2004) Non-alcoholic fatty liver disease: an emerging pathological spectrum. Virchows Arch. 444:3-12.[Medline]
6. Rockey, D. C. & Chung, J. J. (1998) Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension. Gastroenterology 114:344-351.[Medline]
7. Shah, V., Toruner, M., Haddad, F., Cadelina, G., Papapetropoulos, A., Choo, K., Sessa, W. C. & Groszmann, R. J. (1999) Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology 117:1222-1228.[Medline]
8. Kobayashi, H., Nonami, T., Kurokawa, T., Takeuchi, Y., Harada, A., Nakao, A. & Takagi, H. (1995) Role of endogenous nitric oxide in ischemia-reperfusion injury in rat liver. J. Surg. Res. 59:772-779.[Medline]
9. Cottart, C. H., Do, L., Blanc, M. C., Vaubourdolle, M., Descamps, G., Durand, D., Galen, F. X. & Clot, J. P. (1999) Hepatoprotective effect of endogenous nitric oxide during ischemia-reperfusion in the rat. Hepatology 29:809-813.[Medline]
10. Kunsch, C. & Medford, R. M. (1999) Oxidative stress as a regulator of gene expression in the vasculature. Circ. Res. 85:753-766.
11. Lopes, H. F., Morrow, J. D., Stoijiljkovic, M. P., Goodfriend, T. L. & Egan, B. M. (2003) Acute hyperlipidemia increases oxidative stress more in African Americans than in white Americans. Am. J. Hypertens. 16:331-336.[Medline]
12. Carlsson, C., Borg, L. A. & Welsh, N. (1999) Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro. Endocrinology 140:3422-3428.
13. Gottlieb, E., Vander Heiden, M. G. & Thompson, C. B. (2000) Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis. Mol. Cell. Biol. 20:5680-5689.
14. Mehta, K., Van Thiel, D. H., Shah, N. & Mobarhan, S. (2002) Nonalcoholic fatty liver disease: pathogenesis and the role of antioxidants. Nutr. Rev. 60:289-293.[Medline]
15. Aronis, A. M., Madar, Z. & Tirosh, O. (2005) Mechanism underlying oxidative stress-mediated lipotoxicity: exposure of J774.2 macrophages to triacylglycerols facilitates mitochondrial ROS production and cellular necrosis. Free Radic. Biol. Med. 38:1221-1230.[Medline]
16. Kun, J. F. (2003) Regulation of nitrogen monoxide production in human malaria. Redox Rep. 8:289-291.[Medline]
17. Planas, M., Porta, I., Sagrista, M. L., Mora, M., Padro, J. B. & Pico, M. (1999) Fatty acid composition of platelet membrane lipids after administration of two different fat emulsions in critically ill patients. Intensive Care Med. 25:395-398.[Medline]
18. Berry, M. N. & Phillips, J. W. (2000) The isolated hepatocyte preparation: 30 years on. Biochem. Soc. Trans. 28:131-135.[Medline]
19. Skipski, V. P. & Barday, M. (1969) Thin-layer chromatography of lipids. Methods Enzymol. 14:530-598.
20. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[Medline]
21. Moreno, J. J., Carbonell, T., Sanchez, T., Miret, S. & Mitjavila, M. T. (2001) Olive oil decreases both oxidative stress and the production of arachidonic acid metabolites by the prostaglandin G/H synthase pathway in rat macrophages. J. Nutr. 131:2145-2149.
22. Sartor, C. I., Zhou, H., Kozlowska, E., Guttridge, K., Kawata, E., Caskey, L., Harrelson, J., Hynes, N., Ethier, S., Calvo, B. & Earp, H. S., 3rd (2001) Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells. Mol. Cell. Biol. 21:4265-4275.
23. Vanden Hoek, T. L., Shao, Z., Li, C., Schumacker, P. T. & Becker, L. B. (1997) Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J. Mol. Cell. Cardiol. 29:2441-2450.[Medline]
24. LeBel, C. P., Ischiropoulos, H. & Bondy, S. C. (1992) Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5:227-231.[Medline]
25. Wang, H., Gao, X., Fukumoto, S., Tademoto, S., Sato, K. & Hirai, K. (1998) Post-isolation inducible nitric oxide synthase gene expression due to collagenase buffer perfusion and characterization of the gene regulation in primary cultured murine hepatocytes. J. Biochem. 124:892-899.
26. Steinberg, H. O., Paradisi, G., Hook, G., Crowder, K., Cronin, J. & Baron, A. D. (2000) Free fatty acid elevation impairs insulin-mediated vasodilation and nitric oxide production. Diabetes 49:1231-1238.[Abstract]
27. Listenberger, L. L., Ory, D. S. & Schaffer, J. E. (2001) Palmitate-induced apoptosis can occur through a ceramide-independent pathway. J. Biol. Chem. 276:14890-14895.
28. Kim, J. W., Kang, K. W., Oh, G. T., Song, J., Kim, N. D. & Pak, Y. K. (2002) Induction of hepatic inducible nitric oxide synthase by cholesterol in vivo and in vitro. Exp. Mol. Med. 34:137-144.[Medline]
29. Lopes, H. F., Martin, K. L., Nashar, K., Morrow, J. D., Goodfriend, T. L. & Egan, B. M. (2003) DASH diet lowers blood pressure and lipid-induced oxidative stress in obesity. Hypertension 41:422-430.
30. Miralles, C., Busquets, X., Santos, C., Togores, B., Hussain, S., Rahman, I., MacNee, W. & Agusti, A. G. (2000) Regulation of iNOS expression and glutathione levels in rat liver by oxygen tension. FEBS Lett. 476:253-257.[Medline]
31. Stuehr, D. J., Kwon, N. S. & Nathan, C. F. (1990) FAD and GSH participate in macrophage synthesis of nitric oxide. Biochem. Biophys. Res. Commun. 168:558-565.[Medline]
32. Chen, G., Wang, S. H. & Warner, T. D. (2000) Regulation of iNOS mRNA levels in endothelial cells by glutathione, a double-edged sword. Free Radic. Res. 32:223-234.[Medline]
33. Harbrecht, B. G., Di Silvio, M., Chough, V., Kim, Y. M., Simmons, R. L. & Billiar, T. R. (1997) Glutathione regulates nitric oxide synthase in cultured hepatocytes. Ann. Surg. 225:76-87.[Medline]
34. Tirmenstein, M. A., Nicholls-Grzemski, F. A., Schmittgen, T. D., Zakrajsek, B. A. & Fariss, M. W. (2000) Glutathione-dependent regulation of nitric oxide production in isolated rat hepatocyte suspensions. Antioxid. Redox Signal. 2:767-777.[Medline]
35. Hoffmann, A., Gloe, T. & Pohl, U. (2001) Hypoxia-induced upregulation of eNOS gene expression is redox-sensitive: a comparison between hypoxia and inhibitors of cell metabolism. J. Cell. Physiol. 188:33-44.[Medline]
36. Sen, C. K., Roy, S. & Packer, L. (1996) Involvement of intracellular Ca2+ in oxidant-induced NF-kappa B activation. FEBS Lett. 385:58-62.[Medline]
37. Nikulina, M. A., Andersen, H. U., Karlsen, A. E., Darville, M. I., Eizirik, D. L. & Mandrup-Poulsen, T. (2000) Glutathione depletion inhibits IL-1 beta-stimulated nitric oxide production by reducing inducible nitric oxide synthase gene expression. Cytokine 12:1391-1394.[Medline]
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