The internalisation of [125I] insulin by isolated rat hepatocytes.

This work was undertaken to examine the relationship between the binding, internalisation and degradation of insulin by isolated rat hepatocytes. Internalisation of hormone reached a maximum after 5-7 minutes at 37 degrees C; at an insulin concentration of 0.1 nM, 25% of the specifically bound hormone was internalised. Internalisation and the degradation of internalised insulin were inhibited by the intralysosomal protease inhibitors chloroquine and methylamine but not by the thiol oxidant diamide. It is concluded that internalisation of insulin by rat hepatocytes is not a necessary step in the degradation of the hormone molecule.

125I-labelled insulin degradation by isolated rat hepatocytes: the roles of glutathione-insulin transhydrogenase and insulin-specific protease.

Isolated rat hepatocytes degraded 125I-insulin with a Km of 150 nmol/l. Degradation was stimulated by the addition of glutathione and dithiothreitol. In cells incubated with diamide, glutathione was oxidised to the disulphide. Regeneration of reduced glutathione commenced after a further 30 min incubation at 37 degrees C. Diamide (1 mmol/l) significantly inhibited insulin degradation by hepatocytes (p less than 0.001). The 'apparent Vmax' for insulin degradation was decreased tenfold and the Km decreased to 25 nmol/l. The diamide-insensitive degrading activity was cell-associated and produced an intermediate of hormone degradation that was apparently of a higher molecular weight than insulin A chain. The biological activity of the intermediate was 0.03% of that of insulin. The diamide-insensitive activity was not due to release of protease into the medium by cell lysis. We conclude that there are at least two pathways capable of degrading insulin existing in rat hepatocytes.

The induction of synthesis of L-type pyruvate kinase in cultured rat hepatocytes.

Hepatocytes were isolated from preweaned neonatal and adult rats and maintained in primary monolayer culture. Cells from preweaned newborns possessed no L-type pyruvate kinase, nor did they synthesize the enzyme. Incubation for 48-72 h in culture medium supplemented with 2 mM-fructose and 0.1 microM-insulin induced the synthesis of L-type pyruvate kinase, as judged by increased enzyme activity and the increased incorporation of [3H]leucine into immunoprecipitable L-type pyruvate kinase. Hepatocytes isolated from 48 h-starved adult rats incorporated less [3H]leucine into L-type pyruvate kinase than did cells isolated from high-carbohydrate-diet-fed rats. The rate of enzyme synthesis by cells from 48 h-starved rats was increased by the inclusion of fructose and insulin in the incubation medium, after a lag phase of 24-48 h. After 4 days in culture in the presence of fructose and insulin, hepatocytes from 48 h-starved rats synthesized L-type pyruvate kinase at similar rates to hepatocytes isolated from high-carbohydrate-diet-fed rats.

The turnover of L-type pyruvate kinase in cultured rat hepatocytes.

Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-(3)H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [(3)H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t((1/2)) (half-time) 4.9h and a duration of 8-10h was followed by a phase with t((1/2)) 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1mum-insulin to the culture medium increased the t((1/2)) of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.

The metabolism of 125I-labelled insulin by isolated Zucker rat hepatocytes.

The metabolism of 125I-labelled insulin by hepatocytes isolated from 48-h-starved Zucker lean and obese rats was studied. Hepatocytes from the lean animals bound significantly more 125I-labelled insulin and had a greater receptor number per cell than did cells from obese littermates. Hepatocytes from the lean animals degraded and internalized more hormone than did those from obese ones. Increased degradation and internalization correlated with the increased receptor number.

Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin.

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.