Insulin-treated Zucker diabetic fatty rats retain the hypertriglyceridemia associated with obesity.

Lipoprotein and apolipoprotein changes were evaluated in 10-week-old Zucker diabetic fatty (ZDF) male rats following 12 weeks of insulin treatment, which normalized blood glucose and maintained weight gaining characteristic of nondiabetic Zucker fatty rats. Compared with untreated ZDF rats (saline-injected), insulin treatment resulted in increased very-low-density lipoprotein (VLDL; d < 1.006 g/mL) and decreased alpha lipoprotein on agarose gel electrophoresis. These findings were consistent with an observed increase in VLDL triglyceride and cholesterol, and decreased high-density lipoprotein (HDL) cholesterol with insulin treatment in isolated lipoproteins. B100 levels were unchanged by insulin treatment, but B48 levels were significantly increased in the VLDL fraction. Insulin treatment depressed apolipoprotein (apo) A-I levels in HDL, but had little effect on total apo E, apo A-IV, or apo C, although apo C was redistributed to the VLDL fraction. These results suggest that insulin treatment of ZDF rats normalizes hyperglycemia and prevents age-related changes in lipoprotein parameters associated with development of insulinopenic diabetes. Insulin therapy in ZDF rats thereby sustains the hyperlipidemic lipoprotein pattern associated with hyperinsulinemia and obesity.

Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinopenic hyperglycemia.

Lipoprotein and apolipoprotein parameters were studied in the male Zucker diabetic fatty (ZDF) rat at 10 and 20 weeks of age, corresponding to hyperinsulinemic and insulinopenic type 2 diabetes mellitus, respectively. At both ages, ZDF rats had elevated serum triglycerides, free fatty acids, and corticosterone, whereas 20-week ZDF rats had reduced thyroid hormones. At 10 weeks, the hyperlipidemia was confined to elevations in pre-beta triglyceride-rich (d < 1.006 g/mL) lipoproteins. By 20 weeks, all lipoprotein density fractions were increased compared with lean rats, with substantial increases in both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. In ZDF rats, there was a progressive increase in apolipoprotein B (apo B) from 1.9 times control at 10 weeks to three times control at 20 weeks. The increase in apo B was accompanied by a shift of apo B, particularly B100, from very-low-density lipoprotein (VLDL) into denser lipoproteins corresponding to intermediate-density lipoproteins plus LDLs (1.006 < d < 1.063 g/mL). In Zucker and 10-week ZDF rats, in the presence of hyperinsulinemia, the increase in serum apo B was predominantly apo B48 present in VLDL. By 20 weeks, when ZDF rats are insulinopenic, the mass ratio of B48:B100 shifted from 2.7 to 0.7. The shift was associated with a decrease in hepatic-edited apo B mRNA. Apo E increased in lean rats between 10 and 20 weeks of age. Although apo E also increased in ZDF rats, the increase by 20 weeks was less than that of lean rats. The molar ratio of apo E to B in VLDL was decreased in ZDF rats. In lean rats, greater than 50% of apo E was present in HDL, in contrast to ZDF rats, where less than 20% of apo E was present in HDL. VLDL apo E shifted to denser fractions by 20 weeks of age, similar to apo B. The apo C level was more than double compared with the level in lean rats and was redistributed from the HDL fraction to lipoprotein fractions containing apo B. Both apo A-I and apo A-IV levels more than doubled between 10 and 20 weeks in ZDF rats. The ZDF rat model may be useful in comparative studies of lipoproteins during diabetic progression from hyperinsulinemia to insulinopenia.

An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing.

A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.

Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes.

Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 micrograms/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarba moyl]-2- oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.

Insulin-mimetic effects of vanadate in primary cultures of rat hepatocytes.

To evaluate possible mechanisms by which insulin inhibits hepatic apolipoprotein B (apoB) secretion, we incubated primary cultures of rat hepatocytes with sodium orthovanadate, a phosphotyrosine phosphatase inhibitor and insulin-mimetic agent. Vanadate (10 microM) and insulin (10 nM) inhibited the medium accumulation of apoB (secretion) by 21 and 37%, respectively, without increasing intracellular apoB. The effects of insulin and vanadate together were not additive. Both insulin and vanadate enhanced intracellular glycogen accumulation by 82 and 37%, respectively. Unlike insulin, vanadate, at a concentration that inhibited apoB secretion (10 microM), had no effect on intracellular lipogenesis, inhibited the secretion of newly synthesized hepatic proteins, and had a delayed onset and termination of action on inhibition of apoB secretion. At higher concentrations (40 and 80 microM), vanadate stimulated intracellular lipogenesis. In conclusion, our data indicate that vanadate mimics insulin action in hepatocytes with regard to the inhibition of medium accumulation of apoB. These data are consistent with the hypothesis that inhibition of apoB secretion may be secondary to an increase in phosphotyrosine content at its site of synthesis. The kinases responsible for this effect have not been identified. Several effects of vanadate, however, are different from those of insulin, suggesting a differential sensitivity to vanadate, a divergence of the signal transfer by insulin and vanadate at the insulin-receptor or postreceptor level, or both.

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.

Insulin effects on apolipoprotein B lipoprotein synthesis and secretion by primary cultures of rat hepatocytes.

Lipoprotein synthesis and secretion were examined in primary cultures of rat hepatocytes cultured on collagen-coated plates and incubated with pharmacologic and physiologic concentrations of insulin. Media insulin concentration declined rapidly over the course of incubation indicating that hepatocytes rapidly degrade insulin. When insulin was present in the media, cellular triglyceride accumulated while lipid secretion declined. Insulin inhibited the incorporation of labeled amino acids into total secretory lipoprotein apoproteins and apolipoprotein B (apo B) as well as apo B mass as measured by monoclonal radioimmunoassay. The effect of insulin on apo B secretion occurred as early as three hours after the addition of insulin to the culture media and both apo B of higher molecular weight (apo BH) and apo B of lower molecular weight (apo BL) were affected. Cellular apo B did not accumulate within cells. The majority of secretory lipid radioactivity synthesized from acetate was in VLDL density lipoproteins. The composition of newly synthesized lipids as assessed by thin layer chromatography was not significantly altered with insulin. These studies support the finding that insulin inhibits VLDL secretion by hepatocytes while at the same time stimulating overall triglyceride synthesis. A suggested mechanism is that insulin uncouples triglyceride and apo B synthesis, which influences subsequent lipoprotein assembly and secretory pathways. These results are consistent with the concept that postprandial insulin release inhibits hepatic lipoprotein secretion while intestinal lipoprotein metabolic pathways are most active.

The production and utility of monoclonal antibodies to rat apolipoprotein B lipoproteins.

Murine hybridomas were prepared by fusion of azaguanine-resistant plasmacytoma cells and spleen cells from BALB/c mice immunized with intact rat lipoproteins (Lp). Seventeen stable hybridomas from 5 fusions producing antibody against rat apolipoprotein B (apo B)-containing Lp were prepared. The distribution of antigenic determinants of rat apo B variants defined by these antibodies was similar to that found for human apo B. Thirteen mAbs were reactive to epitopes found on both the higher (apo BH) and lower (apo BL) molecular weight forms of rat apo B; two were reactive to apo BH-specific epitopes and two were reactive to apolipoprotein E. No antibodies reactive to unique apo BL epitopes were found. One anti-rat apo B mAb (1.34.3) of high affinity and avidity was reactive to denatured and intact Lp-apo B. Reactivity towards Lp-apo B was independent of variant composition and density class of Lp. The mAb was subsequently used to develop a radioimmunoassay (RIA) which was sensitive down to 5 ng apo B. The assay was used successfully to measure total nascent Lp-apo B secreted by hepatocytes in culture.

A freezing method for cell fusions to distribute and reduce labor and permit more thorough early evaluation of hybridomas.

A method is described whereby cell fusions can be bulk-frozen shortly after the hybridization step. Recoveries are shown to be comparable to those obtained for control hybridomas cultured without freezing. Advantages are discussed in terms of labor distribution and antibody assay and evaluation strategies. It is further shown that peritoneal feeder cell preparations can be conveniently frozen as a means of workload reduction.