Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [³⁵S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [³⁵S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B _h , E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes. This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of Health (HL 15062), the American Heart Association, and the Louis Block Fund.     

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes

Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.     

Production of apolipoproteins E and A-I by rat hepatocytes in primary culture

Hepatocytes were isolated from normal adult rat livers and cultured in a modified HI-WO/BA medium. A nearly confluent monolayer was established at the plating concentration employed. The hepatocytes synthesized ansd secreted albumin at rates similar to those observed in vivo. The cells secreted triacylglycerol in the absence of fatty acid substrate. Under these conditions the most abundant triacylglycerol molecular species contained 53 carbons. Incubation with oleic acid markedly increased triacylglycerol secretion predominantly in the form containing a total carbon number of 57. Approx. 80% of the secreted cholesterol was in the free form and this was unaffected by oleic acid. Employing monospecific antibodies constant rates of synthesis and secretion of apolipoproteins E and A-I were demonstrated by quantitative electroimmunoassay of the cell culture media. The rates of albumin, apolipoprotein E, and apolipoprotein A-I production were 1480, 170 and 60 microgram/h per g cell protein, respectively.     

Octanoate inhibits very low-density lipoprotein secretion in primary cultures of chicken hepatocytes

The effects of octanoate, a medium-chain fatty acid, on very low-density lipoprotein (VLDL) secretion in primary cultures of chicken hepatocytes were compared with those of palmitate. Palmitate added to the incubation media at concentrations up to 0.36 mM increased intracellular triacylglycerol (TG) accumulation and VLDL-TG secretion in a concentration-dependent manner, whereas the addition of octanoate alone (0.21-0.6 mM) did not change these parameters. VLDL-TG secretion from hepatocytes cultured in media to which 0.6 or 1.0 mM octanoate had been added in the presence of 0.21 mM palmitate was significantly lower than that obtained under control incubation conditions (0.21 mM palmitate only). The addition of 1.0 mM octanoate to the incubation media with or without 0.21 mM palmitate decreased VLDL apolipoprotein B (apoB) secretion. These results demonstrate that the addition of octanoate to primary cultures of chicken hepatocytes reduces VLDL secretion in respect of both TG and apoB secretion. It is suggested that medium-chain fatty acids are a factor modulating VLDL secretion, which plays a key role in fat deposition in chickens.     

Structure of lipoprotein (a) studied by spin-labeling

The potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate causes a 2-fold increase in 1,2-diacylglycerol levels within 15–30 min in cultured chick embryo differentiated myoblasts. The weak tumor promoter 12-O-decanoylphorbol 13-acetate was 250 times less effective and the non-promoter 4-α-phorbol 12,13 didecanoate was ineffective at producing this response. During subcellular fractionation, the stimulated portion of the diacylglycerol was distributed similarly to the plasma membrane fraction. Evidence is presented that this diacylglycerol originates from pre-existing lipid rather than from $\text{de}\text{novo}$ synthesis. Possible implications of these findings with regard to the inhibition of myoblast fusion by the tumor promoter are discussed.     

Intracellular mechanisms regulating apoB-containing lipoprotein assembly and secretion in primary hamster hepatocytes

We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o-phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post-translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB-containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates.     

Conversion of the lipoprotein secreted by cultured eel hepatocytes to high density lipoprotein

• 1.1. The lipoprotein, a VLDL-like lipoprotein, secreted by cultured eel hepatocytes was incubated with whole eel serum, serum HDL, or serum VLDL. No change in the VLDL-like lipoprotein was found.
• 2.2. The secreted lipoprotein was incubated with five kinds of liposomes and a HDL-like particle was formed in the presence of BSA only when l-α-dimyristoyl lecithin liposome was used.
• 3.3. In the presence of 3% BSA, apo AI, proapo AI, apo AII and apo C of the secreted lipoprotein were transferred to the l-α-dimyristoyl lecithin liposome and a HDL-like particle was formed.
• 4.4. The secreted lipoprotein was hydrolyzed by lipoprotein lipase and a HDL-like particle formed after hydrolysis contained no triglyceride and had phospholipid as its main lipid.

     

Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes.

Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at O or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4 degrees C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.     

Atrio-ventricular block in a baboon (Papio ursinus)

Atrio-ventricular (A-V) block occurring in an anesthetized chacma baboon (Papio ursinus) is analysed. Hemodynamic, 12-lead electrocardiographical (ECG), hematological and biochemical parameters were monitored and data before and after block are compared. Though biochemical and other abnormal values were encountered, no conclusive evidence of the cause of the block was found.     

Characterization of an unusual lipoprotein similar to human lipoprotein a isolated from the baboon, Papio sp

An unusual lipoprotein was detected and purified from the blood of some members of a large colony of baboons, Papio sp. This lipoprotein was found to be similar to human lipoprotein a in all respects and is therefore termed lipoprotein a. Baboon lipoprotein a had a density of 1.052 g/ml and was located between low- and high-density lipoproteins in a density gradient ultracentrifugation. However, despite its greater density, baboon lipoprotein a was larger than low-density lipoprotein, based on gradient gel electrophoresis and gel filtration. The lipoprotein contained a very large apolipoprotein (apolipoprotein-lipoprotein a) which was found to consist of an apolipoprotein B linked to another protein called apolipoprotein a by a disulfide bridge(s). In all these characteristics, baboon lipoprotein a was similar to human lipoprotein a.     

Parturition in a feral baboon (Papio anubis)

During a study of feral baboons at the Gombe Stream National Park, Tanzania, the premature birth of a dead, breechborn infant was observed and photographed. The placenta was immediately eaten and the dead infant carried by the mother for two days. The birth itself attracted little attention from other troop members, but the dead infant was investigated by several baboons, especially three females who had recently become dominant to the mother in a fight.     

Isolation of apolipoprotein(a) from lipoprotein(a)

An easy method was developed for the rapid and selective isolation of apo(a) from human plasma Lp(a). This procedure was applied to a "low density" Lp(a) subspecies (usually found in the density interval 1.050 to 1.070 g/ml) from a single individual whose apo(a) was of a size smaller than apoB-100. After reduction with 0.01 M dithiothreitol, apo(a) was separated from the Lp(a) particle by rate zonal centrifugation on a 7.5-30% NaBr density gradient. Two completely water-soluble products were recovered: apo(a), which contained less than 1% each of phospholipid and cholesterol, remained at the bottom of the gradient, and a lipid-rich floating LDL-like particle which contained apoB but not apo(a) and which we referred to as Lp(a-). The separation of these two components was also achieved by subjecting reduced Lp(a) to electrophoresis on 2.5-16% polyacrylamide gradient gels. However, dissociation of reduced Lp(a) could not be achieved by gel filtration in either low or high salt solutions. These observations indicate that apo(a) is associated to Lp(a) by non-covalent interactions in addition to its disulfide linkage to apoB. The latter is sensitive to chemical reduction whereas the former are broken through the action of a gravitational or electrical field.     

Knuckle walking by a baboon (Papio cynocephalus).

This paper describes the development of a knuckle walking mode of locomotion by a free-living yellow baboon (Papio cynocephalus). Some implications of this occurrence for theories of the evolution of knuckle walking are discussed.     

Gastric haemorrhage and perforation caused by a trichobezoar in a baboon (Papio hamadryas)

A captive-bred male baboon presented with severe evidence of blood loss and melaena. An upper gastrointestinal radiological study with Gastrografin showed a large trichobezoar. Laparotomy confirmed its presence and revealed gastric ulceration with perforation and generalized peritonitis. The condition was successfully treated by surgery.     

Tuberculosis (Mycobacterium tuberculosis) in a pregnant baboon (Papio cynocephalus)

BACKGROUND: Old World monkeys are considered more susceptible to tuberculosis (TB) than New World monkeys. Several cases of TB in baboons are described in the literature. The data regarding baboon reaction to the tuberculin skin test (TST) are controversial. Some authors described anergy in this species, while the others documented a positive reaction. CASE REPORT: An 8-year-old clinically healthy pregnant female baboon (Papio cynocephalus anubis) developed positive TST after 3 years of negative tests in captivity while not pregnant. Thoracic radiographs demonstrated three nodular densities in the lung. RESULTS: Histological examination of tracheobronchial lymph nodes revealed multiple coalescing pyogranulomas filled with caseonecrotic debris and mineralized foci with numerous large foreign body-type and Langhans-type multinucleated giant cells. The bacterial culture contained a slow growing Mycobacterium tuberculosis complex. CONCLUSIONS: We describe, to the best of our knowledge, the first case of a positive TST in a wild caught, pregnant baboon with latent infection after 3 years in captivity.     

Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3-methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids.     

Inhibitors of lipoprotein(a) assembly

Compounds of the general structure A and B were investigated for their activity as lipoprotein(a), [Lp(a)], assembly (coupling) inhibitors. SAR around the amino acid derivatives (structure A) gave compound 14-6 as a potent coupling inhibitor. Oral dosing of compound 14-6 to Lp(a) transgenic mice and cymologous monkeys resulted in a>30% decrease in plasma Lp(a) levels after 1-2 weeks of treatment at 100 mg/kg/day.