Endotoxin induces structure-function alterations of rat liver peroxisomes: Kupffer cells released factors as possible modulators.
ABSTRACT We report that endotoxin treatment results in decreased amounts of peroxisomes as well as changes in structure and function of peroxisomal membranes. Peroxisomes isolated from the liver of control and treated animals showed a marked decrease in total protein, but no significant alteration in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein profile. However, the Western blot study of the peroxisomal beta-oxidation enzymes and catalase showed an increase in those enzymes in the peroxisomal peak of normal density in endotoxin-treated rats. Disintegration of peroxisomal membranes by carbonate treatment from endotoxin-treated liver and change in the fluidity of peroxisomal membranes suggests alterations in peroxisomal membrane structure. No such alterations were found in mitochondrial or microsomal membranes of endotoxin-treated livers. The lipid analysis of these organelles showed that the only organelle affected was the peroxisome, with a significant decrease in the phospholipid and cholesterol concentrations. To understand the mechanism of endotoxin-mediated alterations in peroxisomes, we studied the possible role of Kupffer cell secreted soluble factors (tumor necrosis factor alpha [TNF-alpha]) on the peroxisomal structure/function. Inactivation/elimination of Kupffer cells by gadolinium chloride before endotoxin treatment did not normalize the overall peroxisomal protein amount and the lipid composition of isolated peroxisomes. However, the levels of individual protein amount in remaining peroxisomes were normalized. Endotoxin also decreased peroxisomal beta-oxidation, and this was partially restored with gadolinium treatment. These results clearly show that peroxisomes are severely affected by endotoxin treatment and suggest that the damage to this organelle may contribute, at least in part, to endotoxin-induced hepatic cytotoxicity.
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ABSTRACT: Very long chain fatty acids (lignoceric acid) are oxidized in peroxisomes and pathognomonic amounts of these fatty acids accumulate in X-adrenoleukodystrophy (X-ALD) due to a defect in their oxidation. However, in cellular homogenates from X-ALD cells, lignoceric acid is oxidized at a rate of 38% of control cells. Therefore, to identify the source of this residual activity we raised antibody to palmitoyl-CoA ligase and examined its effect on the activation and oxidation of palmitic and lignoceric acids in isolated peroxisomes from control and X-ALD fibroblasts. The normalization of peroxisomal lignoceric acid oxidation in the presence of exogenously added acyl-CoA ligases and along with the complete inhibition of activation and oxidation of palmitic and lignoceric acids in peroxisomes from X-ALD by antibody to palmitoyl-CoA ligase provides direct evidence that lignoceroyl-CoA ligase is deficient in X-ALD and demonstrates that the residual activity for the oxidation of lignoceric acid was derived from the activation of lignoceric acid by peroxisomal palmitoyl-CoA ligase. This antibody inhibited the activation and oxidation of palmitic acid but had little effect on these activities for lignoceric acid in peroxisomes from control cells. Furthermore, these data provide evidence that peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases are two different enzymes.Archives of Biochemistry and Biophysics 06/1989; 270(2):722-8. · 3.37 Impact Factor
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ABSTRACT: According to Poole et al. (1970, J. Cell Biol. 45:408-415), newly synthesized peroxisomal proteins are incorporated uniformly into peroxisomes (PO) of different size classes, suggesting that rat hepatic PO form a homogeneous population. There is however increasing cytochemical and biochemical evidence that PO in rat liver are heterogenous, undergoing significant modulations in shape and size in process of PO morphogenesis (Yamamoto and Fahimi, 1987. J. Cell Biol. 105:713-722). In the present study, the kinetics of incorporation of newly synthesized proteins into distinct PO-subpopulations have been studied using short-term in vivo labeling (5-90 min). Two distinct "heavy" and "light" crude PO fractions were prepared by differential pelleting from normal and regenerating liver, and highly purified PO were subsequently isolated by density-dependent metrizamide gradient centrifugation according to Völkl and Fahimi (1985. Eur. J. Biochem. 149:257-265). The peroxisomal fractions banded at 1.20 and 1.24 g x cm-3. They differed in their mean diameters and form-factors and particularly in respect to the activity of beta-oxidation enzymes which was higher in the "light" PO. Whereas the "light" PO exhibited a single immunoreactive band with the antibody to the 70-kD peroxisomal membrane protein the "heavy" PO contained an additional (68 kD) band. In pulse-labeling experiments "light" PO showed clearly a higher initial rate of incorporation than the "heavy" PO. The relative specific activity in the "heavy" PO fraction, however increased progressively reaching that of "light" PO by 90 min. These observations provide evidence for the existence of different PO populations in rat liver which differ in their morphological and biochemical properties as well as in their rates of incorporation of new proteins.The Journal of Cell Biology 07/1993; 121(6):1271-80. · 10.82 Impact Factor
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ABSTRACT: Exposure to a sublethal dose of endotoxin offers protection against subsequent oxidative stresses. The cellular mechanisms involved in generating this effect are not well understood. We evaluated the effect of endotoxin on antioxidant enzymes in liver peroxisomes. Peroxisomes have recently been shown to contain superoxide dismutase (SOD) and glutathione peroxidase (GPX) in addition to catalase. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment increased the specific activity of SOD and GPX in peroxisomes to 208% and 175% of control activity, respectively. These findings correlated with increases in peroxisomal SOD and GPX proteins observed by immunoblot. Although the quantity of catalase protein was increased when assessed by immunoblot analysis, the specific activity of catalase was decreased to 68% of control activity. Activation of catalase with ethanol only restored catalase activity to control levels suggesting that catalase had undergone irreversible inactivation. The observed increase in GPX activity may represent a compensatory mechanism triggered by accumulating H2O2. The data presented here suggest for the first time that mammalian peroxisomal antioxidant enzymes are altered during the oxidative injury of endotoxin treatment.Molecular and Cellular Biochemistry 10/1993; 126(1):25-35. · 2.33 Impact Factor