Comparison of the effects of cyclic AMP analogues on cholesterol metabolism in cultured rat and hamster hepatocytes.

The effects of two cell-permeable cyclic AMP analogues, 8-chloro cyclic AMP (8-Cl cAMP) and 8-(4-chlorophenylthio) cyclic AMP (8-CPT cAMP), on cholesterol esterification, cholesteryl ester hydrolysis and bile acid synthesis were compared in cultured rat and hamster hepatocytes. Cholesterol esterification, as measured by the incorporation of [3H]oleate into cholesteryl ester, was increased by 58-88% by the analogues in rat hepatocytes and by 33-43% in hamster cells. The response in rat hepatocytes, however, was observed after a relatively short incubation time (28% increase after 1 hr), whereas that in hamster cells required a longer period (36% after 12 hr) to become apparent. The activity of the cytosolic neutral cholesteryl ester hydrolase in rat hepatocytes was also stimulated by both cyclic AMP analogues (31-37%, but the microsomal activity was unaffected. In hamster hepatocytes, however, microsomal cholesteryl ester hydrolase activity was increased (47-80%) in the presence of 8-Cl cAMP or 8-CPT cAMP. Bile acid synthesis was increased by 8-CPT cyclic AMP in rat cells (approximately 25%) but was unchanged by both analogues in hamster hepatocytes. These results indicate significant differences in the way in which cholesterol metabolism responds to cyclic AMP in cultured rat and hamster hepatocytes.

Evaluation of cultured hamster hepatocytes as an experimental model for the study of very low density lipoprotein secretion.

The secretion of triacylglycerol, cholesterol and cholesteryl ester in very low density lipoprotein (VLDL) by cultured hamster hepatocytes was studied, and the results compared with those obtained previously using cultured rat hepatocytes and the human hepatoma cell line HepG2. The hamster cells secreted apolipoprotein B and VLDL triacylglycerol, cholesterol and cholesteryl ester linearly during 24 h in culture, and this time period was used in all experiments. Addition of oleate (1 mM) to the culture medium resulted in increased secretion of triacylglycerol, but cholesterol ester output were unchanged. Triacylglycerol secretion was also increased in the presence of lipogenic substrates (10 mM lactate + 1 mM pyruvate) plus dexamethasone (1 microM), but not with either of these agents alone. Inhibition of cholesterol synthesis in the hamster cells by incubation with mevinolin (2 micrograms/ml) did not change VLDL lipid secretion, but stimulation using mevalonate lactone resulted in decreased triacylglycerol output. Manipulation of the rate of cholesterol esterification in the hepatocytes by inhibiting or stimulating the activity of acyl coenzyme A cholesterol:acyl transferase using the inhibitor Dup128 (25 microM) and 25-hydroxycholesterol (50 microM), respectively, had no effect on the secretion of VLDL lipid. In the presence of 1 mM oleate plus 25-hydroxycholesterol, however, a rise in the output of triacylglycerol and cholesteryl ester was observed. Hepatocytes prepared from hamsters fed 2% cholestyramine secreted significantly less triacylglycerol than those from animals given the control diet, but cholesterol and cholesteryl ester output were unchanged, despite a decrease of about 40% in the total cholesterol content of the cells. These results show that the secretion of lipid in VLDL in hamster hepatocytes differs from that in rat and human liver in its response to dietary cholestyramine, and from rat hepatocytes and HepG2 cells in its response to changes in the rate of lipogenesis and cholesterol synthesis and esterification. Overall, hamster hepatocytes appear to be less susceptible to modification the rate of hepatic VLDL secretion, and should provide a useful additional tool for the investigation of this process.

Bile acid synthesis in hamster hepatocytes in primary culture: sources of cholesterol and comparison with other species.

The synthesis of bile acids by primary hamster hepatocytes in culture has been studied. Measurable rates of bile acid synthesis were obtained from cells prepared from livers of animals fed 2% w/w cholestyramine to induce the synthesis of bile acids through the rate-limiting enzyme cholesterol 7 alpha-hydroxylase. The effects of various sources of substrate for bile acid synthesis in these cultured cells were examined over a period of 24 h and the results compared with published or parallel studies in primary rat hepatocytes or in the human hepatoma cell line, HepG2. In all the cells, bile acid synthesis was stimulated by the addition of 7 alpha-hydroxycholesterol, indicating the rate-limiting role of the cholesterol 7 alpha-hydroxylase. Bile acid synthesis in the hamster hepatocytes was also stimulated by a variety of sources of cholesterol as substrate, mevalonic acid (increasing the production of newly-synthesised cholesterol in the cell), and as an exogenous source, hamster LDL. Similarly, if cholesterol was diverted from intracellular esterification using the ACAT inhibitor Dup128, a further increase in bile acid synthesis could be demonstrated. These results show that hepatocytes obtained from cholestyramine-treated hamsters are deficient in substrate cholesterol for bile acid synthesis. A similar conclusion can be drawn from the published work with rat hepatocytes and is further supported by experiments on the regulation of cholesterol 7 alpha-hydroxylase activity at the mRNA and the protein level, although some in vivo studies in animals and studies in man have led authors to suggest that cholesterol 7 alpha-hydroxylase is saturated with substrate.

The effect of cyclic AMP analogues and glucagon on cholesteryl ester synthesis and hydrolysis in cultured hamster hepatocytes.

Two cyclic AMP analogues, 8-chloro cyclic AMP and 8-(4 chlorophenylthio) cyclic AMP, were found to increase the incorporation of [3H]oleate into cholesteryl ester in cultured hamster hepatocytes (30-40%), while incorporation into triacylglycerol was unaffected. An increase of a similar magnitude was observed in the presence of glucagon and the phosphodiesterase inhibitor, theophylline. The cyclic AMP analogues also stimulated the activity of neutral cholesteryl ester hydrolase in the cells, and this effect was mimicked by glucagon and theophylline. These results show that cyclic AMP can affect the cholesteryl ester cycle in hamster hepatocytes, and support the idea that the enzymes involved may be co-ordinately regulated.

Adenylate kinase isoenzymes in Aspergillus nidulans.

Adenylate kinase isoenzymes localised in the mitochondria and in the cytosol have been detected in extracts of glucose-grown Aspergillus nidulans using specific staining after electrophoresis on cellulose acetate. The isoenzymes have similar Km values for AMP, ADP and MgATP2- but may differ in the mechanism used for internucleotide phosphate transfer.