Lp Formation Research Articles

Carboxyl-terminal truncation of apolipoproteinB-100 inhibits lipoprotein(a) particle formation

Recombinant expression systems for both apo(a) and apoB were used to identify sequences in apoB which are required for Lp(a) formation. Incubation of a [ 35S]Cys-labelled 17-kringle form of apo(a) with supernatants from rat hepatoma (McA-RH7777) cells expressing apoB-88, apoB-94 and apoB-100 resulted in covalent r-Lp(a) formation only with apoB-100. Additionally, apoB-86 present in the LDL of a hypobetalipoproteinemic subject did not associate with a 12-kringle form of recombinant apo(a) to form r-Lp(a) complexes. Our data suggest that sequences within the C-terminal 6% of apoB-100 are essential for Lp(a) assembly.

Intracellular processing of apo(a) in primary baboon hepatocytes

We have developed a serum-free medium for the long-term culture of highly differentiated primary baboon hepatocytes. Hepatocytes isolated from animals with defined plasma Lp(a) levels and apo(a) glycoprotein phenotypes were used to study the assembly of Lp(a). A combination of steady-state and pulse-chase labeling studies and endoglycosidase digests demonstrated that apo(a) was synthesized as a lower molecular weight precursor. After a prolonged period of time in the endoplasmic reticulum, apo(a) was converted to a mature form and secreted. A proportion of mature apo(a) also had a prolonged residence time in the trans Golgi apparatus. In all experiments, apoB coimmunoprecipitated with apo(a) from the culture medium but not from the cell lysates, supporting an extracellular association of the proteins for the formation of Lp(a). Analysis of hepatic RNA from 29 ‘null’ Lp(a) phenotype baboons revealed that one-third of the animals had detectable apo(a) transcripts, whereas the remainder had no detectable apo(a) mRNA. The baboon hepatocyte system therefore represents a valuable model to examine the effect of allelic variation at the apo(a) locus on Lp(a) assembly.

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.

Age dependent changes of amrinone and milrinone effects on rat cardiac muscle: A mechanical and biochemical approach

The effects of various subtype-selective opioid agonists and antagonist on the phosphoinositide (Pl) turnover response were investigated in the rat brain. The κ-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other κ-agonists Dynorphin-A (1-1-13) amide, and its protected analog D[Ala]2-dynorphin-A (1–13) amide also produced a significant increase in the formation of [3H]-lP's, whereas the μ-selective agonists [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin and morphine and the δ-selective agonist [D-Pen2,5]-enkephalin were ineffective. The increase in lP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the κ-selective antagonists nor-binaltorphimine and MR 2266. The formation of lP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medulla. The results indicate that brain κ- but neither μ- nor δ-receptors are coupled to the Pl turnover response.

Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

The effects of various subtype-selective opioid agonists and antagonist on the phosphoinositide (Pl) turnover response were investigated in the rat brain. The κ-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other κ-agonists Dynorphin-A (1-1-13) amide, and its protected analog D[Ala] 2-dynorphin-A (1–13) amide also produced a significant increase in the formation of [ 3H]-lP's, whereas the μ-selective agonists [D-Ala 2-N-Me-Phe 4-Gly 5-ol]-enkephalin and morphine and the δ-selective agonist [D-Pen 2,5]-enkephalin were ineffective. The increase in lP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the κ-selective antagonists nor-binaltorphimine and MR 2266. The formation of lP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medulla. The results indicate that brain κ- but neither μ- nor δ-receptors are coupled to the Pl turnover response.

Intestinal lipoproteins in the rat with D-(+)-galactosamine hepatitis.

D-(+)-galactosamine (GalN) induces severe reversible hepatocellular injury in the rat accompanied by lecithin: cholesterol acyltransferase (LCAT) deficiency, defective chylomicron (CM) catabolism, and accumulation of abnormal plasma lipoproteins (Lps), including discoidal high density lipoproteins (HDL). These abnormalities are presumed to result from hepatic injury alone, but the effect of GalN on intestinal Lps has not been studied. To assess possible effects on intestinal Lp formation and secretion, mesenteric lymph fistula rats were injected with GalN or saline. Twenty-four hours later a 2-hr fasting lymph sample was collected; this was followed by an 8-hr duodenal infusion of a lipid emulsion containing 17.7 mM [3H]triolein at 3 ml/hr. Fasting lymph and fat-infused lymph flow rates, 3H, triglyceride, and cholesterol output, residual 3H in intestinal lumen and mucosa, total 3H recovery, and d less than 1.006 g/ml Lp size and lipid composition were unchanged by GalN treatment, but d less than 1.006 g/ml Lps were depleted of apoE and C. Fat-infused lymph phospholipid (PL) output was higher in GalN rats due to PL-enriched d greater than 1.006 g/ml Lps. Electron microscopy of lymph and plasma LDL and HDL revealed spherical Lps in all samples. GalN plasma, fasting lymph, and fat-infused lymph also contained large abnormal LDL and discoidal HDL. Control lymph LDL and HDL did not differ in size from control plasma LDL and HDL. Control lymph LDL contained both apoB240K and B335K. However, spherical LDL and discoidal HDL in fasting lymph from GalN rats differed significantly in size from the corresponding plasma particles and became closer in size to the plasma particles with fat infusion. GalN lymph LDL contained only apoB240K and had a lower PL/CE than GalN plasma LDL. GalN fasting lymph HDL, depleted of apoC and having a PL/CE of 5, became enriched in apoE and the PL/CE increased to 10 with fat infusion to closely resemble GalN plasma HDL. GalN reduces apoE and C (mainly of hepatic origin) in d less than 1.006 g/ml gut Lps, which may contribute to the CM catabolic defect in GalN rats. Lymph LDL and HDL, especially in fasting lymph, may be partially gut-derived with increased filtration of plasma Lps into lymph with fat infusion. GalN fat-infused lymph HDL is enriched in apoE, but unable to transfer apoE to d less than 1.006 g/ml intestinal Lps. We conclude that GalN hepatitis is a model that allows study of intestinal Lps with normal lipid digestion and absorption in the face of severe hepatic injury and LCAT deficiency.

The action of insulin on the composition of cells and medium during culture of chick heart explants.

SUMMARY 1. Cultures of heart tissue from 13-day chick embryos have been grown for periods of 6 days in a medium of embryo extract and serum and in the presence of varying concentrations of insulin. 2. Doses of 1–3 i.u. insulin/ml. medium greatly stimulate the formation of phospholipids (LP), ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and protein nitrogen (PN). This growth-promoting action of insulin is accompanied by a rise in the RNA : DNA ratio, and in the ratio of RNA to PN in the cells. 3. At doses of 1 × 10−2 i.u./ml. medium insulin increases the formation of LP, RNA and DNA, but there is no growth-promoting action with 1 × 10−4 i.u./ml. Insulins of British and Danish origin act similarly, and the presence of the hyperglycaemic factor associated with insulin (in a specially prepared extract) appears to have no action on the growing explants. 4. With higher concentrations of insulin, enhanced synthesis is accompanied by increased glucose utilization and by a pronounced fall in pyruvate content of the medium. The α-ketoglutaric acid level in the medium is unaffected by these changes. 5. It is concluded that insulin has a direct growth-promoting action on these embryonic cells, and that it stimulates increased energy production and general synthesis through its control over pyruvate metabolism.