Effects of nonketotic streptozotocin diabetes on apolipoprotein B synthesis and secretion by primary cultures of rat hepatocytes.

The effects of hypoinsulinemic nonketotic streptozotocin diabetes on hepatic apo B synthesis and secretion was studied in primary cultures of rat hepatocytes. Diabetic rats were characterized by their significantly elevated serum glucose, apo B, and triglyceride levels, while serum insulin levels were less than a third of normal. Serum transminase activities of diabetic rats were significantly elevated when compared with control rats, which was attributed to an increase in liver transaminase activity in diabetic rats. The pattern of enzyme activities of hepatocytes reflected that observed in livers of donor rats and the pattern was retained by primary cultures of hepatocytes over the culture period. Hepatocytes from diabetic rats secreted only one third of the apo B secreted by hepatocytes from control rats, which was determined by monoclonal immunoassay of rat total apo B. Decreases in secretion were confirmed by measurement of secretory [35S]methionine-labeled lipoprotein apo B radioactivity. The decreased apo B content of media of hepatocytes from diabetic rats was not due to increased apo B catabolism since hepatocytes from diabetic rats were shown to degrade less lipoprotein-apo B than hepatocytes from normal rats in control experiments. In addition, the apo B content of detergent-solubilized hepatocytes from diabetic rats was significantly less than that of hepatocytes from control rats. These results suggest that insulin is necessary for normal hepatic apo B synthesis and secretion and that the hyperlipidemia associated with hypoinsulinemia in vivo is primarily of intestinal origin.

Secretion of high and low molecular weight phosphorylated apolipoprotein B by hepatocytes from control and diabetic rats. Phosphorylation of APO BH and APO BL.

Apolipoprotein B (apo B) phosphorylation was examined in primary cultures of hepatocytes from control and nonketotic streptozotocin diabetic rats. Following a 5-h incubation with ortho[32P]phosphate, media lipoproteins (d less than 1.063 g/ml) were isolated, and delipidated apolipoproteins were separated by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGGE), and gels were heat fixed. Autoradiographic bands corresponding to high (apo BH) and low molecular weight apo B (apo BL) were observed in media lipoproteins isolated from control rats, and these bands were more prominent in media lipoproteins isolated from diabetic rats. Apo B-specific activity was estimated from aqueous alcohol-precipitated radioactivity and apo B monoclonal immunoassay of isolated media lipoproteins. In lipoproteins secreted by hepatocytes of diabetic rats, the calculated apo B specific activity was between 18- and 31-fold greater than that secreted by hepatocytes of control rats, consistent with the SDS-PAGGE gel data. The increase in secretory 32P-labeled apo B from hepatocytes of diabetic rats was due, at least in part, to an increase in labeled phospho-tyrosine as determined by phosphoamino acid analysis. These data demonstrate that apo BH may be secreted as a phosphorylated protein and that apo B phosphorylation occurs on tyrosine as well as serine residues.

Normal hepatic insulin receptor autophosphorylation in nonketotic diabetes mellitus.

Insulin receptor autophosphorylation is the earliest recognizable event in insulin action subsequent to insulin binding. To determine if the postbinding hepatic insulin resistance of nonketotic diabetes mellitus could reside in an inability of insulin to stimulate insulin receptor autophosphorylation, we evaluated the ability of insulin to stimulate 32P incorporation into the beta subunit of lectin-purified rat liver plasma membrane insulin receptors. The data indicate that both the absolute plasma membrane insulin receptor autophosphorylation in response to insulin as well as the insulin dose-response relationship for autophosphorylation are normal in diabetic animals when expressed per microgram of protein or per unit of binding activity. The previous data from our laboratory indicates that hepatic insulin resistance in non-ketotic streptozotocin-induced diabetes mellitus is present despite normal to increased insulin binding, is selective, is reversible with insulin treatment and involves an inability of insulin to stimulate the release of the putative mediator of insulin action. We conclude, therefore, that the hepatic insulin resistance of nonketotic diabetes mellitus resides distal to insulin receptor binding and autophosphorylation and is reflected in metabolic events at or near the plasma membrane which may include the generation or release of the putative mediator of insulin action.