An assay is described for ace@-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: (a) The hepatocytes are made permeable by digitonin. 64 pg of digitonin per mg of celbdar protein were mosf effective in exposing enzyme activity without a significant effect on mitochondrial permeability. (b) Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: (i) rapidity, (ii) no need to prepare cell extracts, (iii) absence of product inhibition, (iv) no interference by mitochondrial enzymes, (v) useful in systems with bicarbonate buffers, and (vi) simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.

Periportal and perivenous hepatocytes were isolated from rats subjected to different treatments that induce (starvation, cold exposure) or depress (refeeding after starvation) hepatic fatty acid oxidation. These experiments were designed to determine factors that may be involved in creating and maintaining the asymmetrical distribution of this metabolic pathway in the acinus of the liver. The uneven distribution of mitochondrial [14C]-palmitate oxidation within the acinus (i) was very flexible and changed markedly with the physiological status of the animal (periportal/perivenous ratio: 1.5, 2.0, 1.0 and 0.4 for fed, starved, refed and cold-exposed animals respectively), (ii) coincided with a similar zonation of carnitine palmitoyltransferase I activity in fed as well as in cold-exposed animals, (iii) was paralleled by a comparable zonation of mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase activity in starved animals, and (iv) was not determined by zonal differences in any of the following parameters: sensitivity of carnitine palmitoyltransferase I to malonyl-CoA, intracellular concentration of malonyl-CoA, fatty acid synthesizing capacity, acetyl-CoA carboxylase activity, fatty acid synthase activity or relative content of the two hepatic acetyl-CoA carboxylase isoforms. Unlike mitochondrial oxidation, peroxisomal [14C]palmitate oxidation was always zonated towards the perivenous zone of the liver irrespective of the physiological status of the animal. The data presented show that changes in the acinar distribution of mitochondrial long-chain fatty acid oxidation involve specific long-term mechanisms under different physiological conditions.

Rats were fed a high-fat, liquid diet containing either 36% of total calories as ethanol or an isocaloric amount of sucrose, for a period up to 35 days. At different time intervals we measured the effects of ethanol administration on the activities of a number of key enzymes involved in hepatic lipid synthesis. At the start of the experimental period the activities of acetyl-CoA carboxylase and fatty acid synthase, measured in liver homogenates, increased in the control as well as in the ethanol-fed group. After 35 days these enzyme activities were still elevated but there were no significant differences between the two groups. In hepatocytes isolated from controls as well as from ethanol-fed rats, short-term incubations with ethanol induced an increase in the rate of fatty acid synthesis and in the activities of acetyl-CoA carboxylase and fatty acid synthase. However, no alterations in the regulation of these enzymes by short-term modulators of lipogenesis were apparent in hepatocytes isolated from alcohol-treated animals. The results do not indicate a major role for the enzymes of de ncruo fatty acid synthesis in the development of the alcoholic fatty liver. The amount of liver triacylglycerols increased in ethanol-fed rats during the entire treatment period, whereas the hepatic levels of phosphatidylcholine and phosphatidylethanolamine were not affected by ethanol ingestion. Ethanol administration for less than 2 weeks increased the activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and microsomal phosphocholine cytidylyltransferase, whereas the cytosolic activity of phosphocholine cytidylyltransferase was slightly decreased. Upon prolonged ethanol administration the activities of these enzymes were slowly restored to control values after 35 days, suggesting development of some kind of adaptation. It is interesting that, although the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase were restored to the levels found in the control rats, this effect was not accompanied by a stabilization or decrease of the concentration of hepatic triacylglycerols. 0 1988 Academic press, inc.

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Since its original discovery as a soluble enzyme (4) AAC The present work was performed to identify the sub-has been assumed to be located in the cytoplasm. Earcellular localization of hepatic acetyl-CoA carboxylase lier work concluded that the activity of the enzyme was (ACC). Cellular organelles involved in fatty acid oxida-not associated with subcellular particles (5), but later tion that contain a malonyl-CoA sensitive carnitine reports indicated that activity of ACC could be detected palmitoyltransferase (CPT) activity or that are linked in high speed precipitates of rat-liver homogenates (6-to the control of this activity were analysed for the 8) and in a so-called mitochondrial fraction of such hopresence of ACC. No significant amount of ACC was mogenates (9,10). Furthermore, permeabilization of is subject to modifi cation of that activity by several factors (12-15), and the avidin binding method is sensitive to interference by other biotin-containing proteins competing for labelled avidin (16). Therefore, the deterDepending on the physiological state of the animal, mination of subcellular distribution on the basis of avithe liver is a tissue that can either exhibit high rates din binding or enzyme activity is difficult to interpret. of fatty acid biosynthesis or high rates of fatty acidThe availability of ACC antibodies (17) and the techoxidation. Control of the activity of acetyl-CoA carbox-nique of permeabilizing isolated hepa tocytes with digylase (ACC) is of special interest in this respect because itonin (15) has permitted to re-address the issue of lothe product of ACC, malonyl-CoA, is not only a sub-calization of ACC and to cir cumvent some of the methstrate for the cytosolic process of long-chain fatty acid odological problems. synthesis but is also an inhibitor of the activity of carnitine palmitoyltransferase (CPT), an important pace-MATERIALS AND METHODS setting enzyme of long-chain fatty acid oxidation (1). As a matter of fact, malonyl-CoA sensitive CPT activity Male Wistar rats (250-300 g) which had free access to food and is present both in mitochondrial outer membranes water were used throughout in this study. Hepatocytes were isolated (CPT-I) (2) and in the peroxisomal matrix (3). and incubated as described in (18). Acetyl-CoA carboxylase activity, Given the functions of malonyl-CoA in or on different isoform distribution and mass were determined in isolated hepatic organelles of the cell, the enzyme responsible for its mitochondria, isolated hepatic peroxisomes or in digitonin-permeabilized hepatocytes. For isolation of mitochondria from liver tissue, production, ACC, may be compartmentalized as well.the procedure described by was followed. Mitochondria from hepatocytes were isolated by homogenizing cells (4-6 mg of protein) with a loose-fitting Dounce homogenizer in a low

Galectins, beta-galactoside binding proteins, expressed selectively in human breast carcinoma are attractive targets to employ lectin-aimed therapeutics. We examined beta-galactoside binding potency of neoplastic cells using fluorescein-labelled synthetic glycoconjugates as probes for flow cytometry. As a result, surface beta-galactoside binding proteins/galectins were discovered on mouse mammary carcinoma cells in vitro and in vivo unlike non-malignant cells from the several tissues; and asialo-GM1 ganglioside carbohydrate part--containing probe was the most specific one. However, in liver and lung metastatic cells galectins seem to be expressed within cytoplasm and/or nuclei. Galectin expression correlated directly with aggressive tumour potential in the A/Sn transplantable model similar to findings in several human breast carcinoma cell lines. However, galectin expression was reduced during tumour progression in more aggressive forms of spontaneous BLRB mammary carcinomas like it was shown for human breast carcinoma specimens. Analysis of the histopathological data led, however, to the conclusion that galectin expression hardly might be a suitable marker of aggressiveness of heterogeneous mammary carcinomas as the observed level of galectin expression is influenced by the amount of the stroma in a tumour sample and/or probably, galectin expression inversely correlates with tumour aggressiveness during the initial and advanced steps of mammary tumour progression. We conclude that surface beta-galactoside binding proteins/galectins that are selectively expressed during mouse mammary carcinoma progression, similarly to human breast carcinomas, seem to be proper targets for asialo-GM1-vectored cytotoxics and our mouse model system might be a relevant instrument to further test novel modes of anti-breast cancer therapy.

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