Role of apoproteins B and E in the receptor-mediated uptake of very-low-density lipoprotein remnants by the perfused rat liver

Studies involving the incubation of rat epididymal fatbodies in uitro have shown that insulin and glucocorticoids can promote a protein-synthesis-dependent increase in the activity of lipoprotein lipase in the tissue, and it has been proposed that these hormones are responsible for the increase in the activity of the enzyme which occurs in vivo after the onset of feeding (Ashby & Robinson, 1980). In order to investigate the mechanism of these hormonal effects, we have devised a novel method for measuring the synthesis of lipoprotein lipase in rat adipose tissue. Epididymal fat-bodies from 24 h-starved rats were incubated for 1 h at 37°C in Krebs-Henseleit bicarbonate buffer solution, supplemented with amino acids at the concentrations defined by Eagle (1955), glucose (lOmM), casein (2% w/v) and ~-[4,5-~H]leucine (lOpCi/ml). At the end of the incubation period, groups of four fat-bodies were removed from the incubation flasks, and homogenized in lOml of 2% (w/v) casein, pH 7.2. The homogenate was delipidated by extraction with acetone and diethyl ether as previously described (Ashby et al., 1978). Each delipidated tissue residue was homogenized in 15 ml of 5 mM-sodium barbital, pH7.5, containing 20% (w/v) glycerol, 0.1% Triton X-100 and 5OmM-NaC1, and the suspension was centrifuged at 60000g for 30min. Lipoprotein lipase was isolated from the resultant supernatant in a single step by affinity chromatography on heparin-Sepharose as described by Parkin et al. (1982). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of this 3H-labelled lipoprotein lipase preparation revealed a single sharp peak of radioactivity corresponding to a polypeptide of M , 56000. This polypeptide has previously been identified as lipoprotein lipase (Parkin et al., 1982). No other peaks of radioactivity were detected on the gel and losses of lipoprotein lipase during this procedure were minimal. Thus the 3H-labelled enzyme can be rapidly and quantitatively isolated free from other radioactive adipose tissue proteins. In the presence of insulin (2m-i.u./ml), the incorporation of [3H]leucine into total adipose tissue protein and into lipoprotein lipase was increased by 1.3k0.2 fold and 2.6 4 0.6 fold respectively (mean f s.D., n = 7), compared with controls. Thus the insulin-induced stimulation of lipoprotein lipase synthesis is partly due to a general increase in total protein synthesis in addition to a specific effect on the synthesis of the enzyme. Similar results have been reported by Vydelingum et al. (1983). The regulation of lipoprotein lipase by glucocorticoids was investigated by preincubating fat-bodies in the presence or absence of dexamethasone (40011~) for 3 h before the addition of [3H]leucine, in order to allow for any lag in the glucocorticoid-induced effects. [3H]Leucine (10pCi/ml) was then added and the incubation was continued for a further lh . Insulin (2m-i.u./ml) was present in all the incubations. The rate of synthesis of lipoprotein lipase was increased by 1.99f0.72 (meanf sn , n = 5 ) fold in the presence of dexamethasone plus insulin, compared with insulin alone. This effect represents a specific induction of lipoprotein lipase synthesis since total protein synthesis was not affected by dexamethasone. These observed effects of insulin and glucocorticoids on the synthesis of lipoprotein lipase are consistent with the changes in enzyme activity which occur under these hormonal conditions. It thus seems likely that both hormones regulate the activity of lipoprotein lipase at the level of enzyme synthesis. Although the effect of insulin is partly explained by a non-specific increase in general protein synthesis, the effect of glucocorticoids apparently represents a specific induction of the enzyme.