Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.     

Contraceptive steroids increase cholesterol in bile: mechanisms of action

Contraceptive steroids increase the risk of acquiring cholesterol gallstones. The factors responsible include an increase in cholesterol saturation of bile and an increase in rate of secretion of cholesterol into bile. The goal of this study was to investigate the mechanism(s) of these increases in biliary cholesterol. During the use of contraceptive steroids, cholesterol saturation of gallbladder bile and the amount of cholesterol secreted per mole of bile acid increased (P less than 0.05 and P less than 0.02, respectively). Cholesterol absorption, cholesterol synthesis, chylomicron remnant clearance, and the concentration of plasma and lipoprotein lipids were not altered by contraceptive steroids. Despite this apparent lack of effect, important correlations were present during steroid use. LDL (low density lipoprotein) cholesterol increased as dietary cholesterol increased (r = 0.58, P less than 0.025). Cholesterol synthesis correlated directly with VLDL cholesterol concentration (r = 0.64, P less than 0.01), biliary cholesterol secretion (r = 0.68, P less than 0.01) and with molar percent cholesterol in bile (r = 0.49, P = 0.06). Chylomicron remnant clearance also correlated with cholesterol secretion (r = 0.85, P less than 0.001). As either remnant uptake or synthesis increased, the effect of the other source of hepatic cholesterol on biliary cholesterol secretion diminished. These relationships were not observed in the same subjects when they were not taking the hormones. The findings suggest that both newly synthesized and dietary cholesterol contribute to the cholesterol secreted in bile. This is consistent with the hypothesis that cholesterol for secretion into bile and VLDL is derived from a common metabolic pool of free cholesterol. It is proposed that contraceptive steroids exert their effect on biliary cholesterol by increasing cholesterol entering the pool and/or by inhibiting hepatic ACAT (acylcoenzyme A:cholesterol acyltransferase) activity, a known effect of progesterone, so that an increase in free cholesterol entering the pool leads to an increase in output.     

Synthesis and secretion of VLDL by rat hepatocytes--modulation by cholesterol and phospholipids.

The synthesis and secretion of apoB, the major protein component of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), were studied using rat hepatocytes maintained in primary culture. Supplementation of hepatocytes with rat serum VLDL and LDL increased the production of apoB while delipidated lipoproteins had no significant effect, suggesting a role for lipids in the production of apoB. Addition of cholesterol to the culture medium also increased the production of apoB in a concentration-dependent manner. Pulse labelling followed by chase in presence of cholesterol indicated enhancement in apoB secretion. Mevinolin which inhibits cholesterol synthesis significantly reduced the secretion of apoB. The presence of phosphatidylcholine and phosphatidylethanolamine in the culture medium also increased the secretion of apoB into the medium. These data suggest that availability of lipids, particularly cholesterol, is an important determinant of apoB synthesis and secretion as VLDL.     

Metabolism of very low density lipoproteins--effect of sardine oil.

The effect of feeding fish oil on the metabolism of lipoproteins was studied in rats. Rats were fed diet containing 10% sardine or groundnut oil for 6 weeks. There was a significant decrease in the total cholesterol, phospholipids and triglycerides as well as the amount of the lipids associated with VLDL and LDL in serum in fish oil-fed rats. The synthesis and secretion of lipoproteins particularly apoB containing lipoproteins by primary cultures of hepatocytes from these rats were studied by 14(C)-acetate or 3(H)-leucine labelling. Primary cultures of hepatocytes derived from sardine oil-fed rats showed reduced incorporation of 3(H)-leucine into apoB containing lipoproteins secreted into the medium when compared to those fed groundnut oil, indicating a decreased synthesis and secretion of apoB. This was further confirmed by significantly lower incorporation of 14(C)-radioactivity into total and individual lipids of VLDL secreted into the medium, as well as that associated with different lipids in cell layer. The activity of lipoprotein lipase in adipose tissue and aorta was significantly higher in rats fed sardine oil which may cause an increased clearance of triglyceride-rich lipoproteins from circulation. These results indicate that the fish oil exerts hypolipidemic effect particularly by decreasing the synthesis and secretion of VLDL by liver and possibly by an increased clearance of triglyceride-rich lipoproteins from circulation.     

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.     

Fatty acids alter time dependent loss of apolipoprotein E expression by primary cultures of rat hepatocytes

Although the phenomenon of intracellular apolipoprotein E (apoE) degradation has been reported in other cell types, the fate of newly synthesized apoE in the liver is not well understood. In the present study, we examined the expression (the balance of synthesis, secretion, and degradation) of apoE in primary cultures of rat hepatocytes and compared it with albumin, a typical secretory protein. Synthesis and secretion of [(35)S]apoE was diminished in primary hepatocytes cultured for more than 2 days, in agreement with an observed decrease in apoE mRNA. Cells cultured for 1 day and labeled for up to 4 hours secreted total protein, apoE, and albumin, linearly. The apparent rates of synthesis for apoE and albumin were similar (1,158 vs. 1,334 dpm/mg/min) but rates of their secretion differed significantly (225 vs. 1,159 dpm/mg/min). Pulse-chase experiments indicated that cell-associated [(35)S]albumin was secreted without degradation, whereas significant quantities of newly synthesized apoE were degraded. The overall synthesis and secretion of total proteins, including secretion of apoE, was enhanced by oleic acid (1 mmol/L). However, this effect may not be limited to oleic acid because other fatty acids showed a similar effect on apoE mRNA abundance. In control cells, apoE was found to associate with high density lipoproteins predominantly, although the fraction associated with very low density lipoprotein was increased in hepatocytes incubated with oleic acid. Overall, the findings from this study suggest that the level of apoE expression by primary hepatocytes is dependent on the age of the culture. The study also indicates that the phenomenon of apoE degradation occurs in primary hepatocytes.     

Secretion of serum amyloid protein and assembly of serum amyloid protein-rich high density lipoprotein in primary mouse hepatocyte culture

The serum amyloid protein (apo-SAA) is a unique high density lipoprotein apoprotein exhibiting dramatic increases in plasma concentration following host injury. The events involved in the secretion of apo-SAA and assembly of apo-SAA-rich lipoprotein particles were studied in primary, serum-free culture of adult BALB/c mouse hepatocytes harvested 3 h following administration of the potent apo-SAA inducer, bacterial endotoxin (50 micrograms of intraperitoneally administered Salmonella typhosa lipopolysaccharide). An approximately 3.5-fold increase in the initial rate of apo-SAA secretion was observed over that of hepatocytes isolated from control mice, whereas the rate of apo-A-I secretion was unchanged by endotoxin administration. Sodium dodecyl sulfate-gel electrophoresis and autoradiography of [35S]methionine-labeled cell products indicated the synthesis of both major mouse apo-SAA isotypes by hepatocytes. Essentially all of the secreted apo-SAA chromatographed in Sephadex G-150 with an elution volume corresponding to a molecular weight of approximately 12,000. Approximately 90% of the secreted apo-SAA was recovered in fractions (d greater than 1.21 g/ml) following ultracentrifugal fractionation. In media supplemented with human lipoproteins (100 micrograms/ml), approximately 50% of the secreted apo-SAA was recovered in the high density lipoprotein fraction. These results suggest that mouse apo-SAA is secreted in monomeric form and becomes associated with lipoproteins in the intravascular compartment.     

The molecular mechanism for the assembly and secretion of ApoB-100-containing lipoproteins.

We have reviewed the literature on the intracellular transport of ApoB-100 and the assembly of the ApoB-100-containing lipoproteins. ApoB-100 is a large molecule (4536 aa) that requires some 15 min to be completed. During the synthesis, the protein could take one of two pathways: a degradational pathway and a pathway that leads to secretion of the protein on mature lipoproteins. The degradational pathway starts with a cotranslational incorporation of ApoB-100 into the membrane of the endoplasmic reticulum in such a way that a relatively large portion of the sequence is exposed on the cytoplasmic surface of this membrane. The membrane bound ApoB-100 is retained in the ER and will eventually undergo intracellular degradation. To enter the pathway that leads to lipoprotein formation, ApoB-100 has to be cotranslationally translocated to the lumen of the ER. ApoB-100 will interact with the lipids during this translation-translocation process and the mature lipoprotein is released into the lumen of the secretory pathway when ApoB-100 is completed and leaves the ribosome. In addition to the mature lipoproteins, the secretory pathway contains an ApoB-100-containing lipoprotein with the density of a HDL particle. This particle is not secreted from the cells but is retained and eventually degraded. Of importance for the retention are sequences present in the C-terminal half of the protein. The mature lipoproteins rapidly leave the ER lumen and are transported to the Golgi apparatus, through which transfer takes considerably longer. The assembly process is a potential site for the regulation of the secretion of the ApoB-100-containing lipoproteins. This process is dependent on active synthesis of phosphatidylcholine and it is also highly dependent on the rate of triacylglycerol synthesis. On the other hand, ApoB-100 appears to be constitutively expressed. An increase in the rate of lipoprotein assembly induced by an increased triacylglycerol synthesis gives rise to an increased recruitment of ApoB-100 nascent polypeptides to interact cotranslationally with lipids. ApoB-100 that is not used for lipoprotein assembly is cotranslationally bound to the ER membrane and sorted to degradation.     

An estrogen responsive primary amphibian liver cell culture system

Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [³H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [³H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th–5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited.Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1–2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation.The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.     

Lipid homeostasis and lipoprotein secretion in Niemann-Pick C1-deficient hepatocytes

Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1(-/-) hepatocytes was 5-fold higher than that of Npc1(+/+) hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1(-/-) hepatocytes was 50% lower than of Npc1(+/+) hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis.     

Plasma fibronectin is synthesized and secreted by hepatocytes

Primary cultures of hepatocytes of rats and hamsters were established and examined for the synthesis and secretion of fibronectin. Hepatocytes of both species secreted fibronectin as a soluble dimeric protein which could be purified by its affinity for gelatin and using specific antisera. Plasma and cellular fibronectins could be clearly resolved on two-dimensional gels. In both species, the majority of the fibronectin secreted by hepatocytes was of the plasma type, as shown by analyses on one- and two-dimensional gels. The secretion of plasma fibronectin increased with time in culture, both in absolute terms and relative to the secretion of albumin. Even during the first day of culture, the secretion of fibronectin relative to that of albumin appeared to be sufficient to account for the relative levels of these two proteins in plasma. Hepatocytes of both species secreted preferentially the chain of plasma fibronectin with higher apparent molecular weight, although the faster migrating chain was also secreted. In addition, hamster hepatocytes cultured for 2 or more days appeared to secrete a cellular form of fibronectin. Possible origins for the different chain types of cellular and plasma fibronectins are discussed.     

Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [14C]acetic and [14C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [14C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [14C]acetic acid. Similarly, [14C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [14C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of bile secretion by calcium signaling in health and disease

Calcium (Ca²⁺) signaling controls secretion in many types of cells and tissues. In the liver, Ca²⁺ regulates secretion in both hepatocytes, which are responsible for primary formation of bile, and cholangiocytes, which line the biliary tree and further condition the bile before it is secreted. Cholestatic liver diseases, which are characterized by impaired bile secretion, may result from impaired Ca²⁺ signaling mechanisms in either hepatocytes or cholangiocytes. This review will discuss the Ca²⁺ signaling machinery and mechanisms responsible for regulation of secretion in both hepatocytes and cholangiocytes, and the pathophysiological changes in Ca²⁺ signaling that can occur in each of these cell types to result in cholestasis.     

Ferrylmyoglobin impairs secretion of VLDL triacylglycerols from stored intracellular pools: involvement of lipid peroxidation

Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [(3)H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with alpha-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.     

Serum apolipoprotein AI synthesis in rat hepatocytes and its secretion as proform

Rat hepatocytes in monolayer or suspension culture synthesize serum lipoprotein AI. It is secreted into the serum-free culture medium. Synthesis and secretion processes were studied in the presence of radiolabelled amino acids. The synthesis product of the hepatocytes and the secretion product from the medium were isolated by immunoprecipitation with a mono-specific rabbit antiserum against rat apolipoprotein AI. The intracellular and secreted products were homogeneous and identical in polyacrylamide gel electrophoresis but had reduced electrophoretic mobility as compared to native apolipoprotein AI. They were submitted to automated Edman degradation. They were present in their proform, the N-terminus of which is extended by a hexapeptide. In the presence of rat serum the proform is proteolytically transformed into the mature form of apolipoprotein AI.     

Characterization of lipoprotein secreted by cultured eel hepatocytes and its comparison with serum lipoproteins.

The lipoprotein secreted by cultured eel hepatocytes was fractionated by density gradient ultracentrifugation and compared with eel serum lipoproteins. Eel hepatocytes were cultured for 7 to 10 days as a monolayer in Williams' medium E containing 5% fetal bovine serum and 0.16 microM insulin on a dish precoated with fibronectin of horse serum. The only lipoprotein secreted by eel hepatocytes was a very-low-density lipoprotein like one which consisted of 69% triglyceride, 15% phospholipid, 4% cholesterol, and 12% protein. On the other hand, very-low-density lipoprotein and high density lipoprotein were found in eel serum, in which high density lipoprotein was a main lipoprotein. The secreted lipoprotein contained apo B and apo A as the main protein components. Furthermore, the lipoprotein contained proapo A-I in addition to apo A-I, which was proved by comparing the amino acid composition of both proteins. In our discussion, we noted that the lipoprotein secreted by eel hepatocytes was a good material for the study of high-density lipoprotein formation.     

Regulation of apolipoprotein E synthesis and mRNA by diet and hormones

Rats were fasted or fasted and refed simple purified diets so the effects of individual carbohydrates or fats could be studied. Freshly isolated hepatocytes from these animals were used to measure both apoE synthesis and mRNA levels so any changes in apoE synthesis that might occur without changes in its mRNA could be detected. Some of these experiments were done with both sexes. Both fasting and fasting and refeeding a 60% glucose fat-free diet significantly increased spoE synthesis. However, cyclic AMP is not likely to rapidly mediate the effect of fasting since dibutyryl cAMP slightly lowered (rather than increased) apoE synthesis and mRNA when injected into rats for 4.5 h. Dietary fat had no effect either in the absence of carbohydrate or when consumption of carbohydrate was constant in pair-fed rats. ApoE mRNA levels remained normal for 4 days in primary hepatocytes cultured in medium that had only amino acids as an energy source. Added hormones or fructose had no significant effect. Thus, only fasting and fasting and refeeding glucose were able to significantly change apoE synthesis or mRNA levels. Synthesis of apoE may be regulated to increase when apoE is secreted with very low density lipoprotein or when apoE in secreted high density lipoprotein is needed to acquire cholesteryl esters for the synthesis of bile salts and acids by liver.     

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.     

The biosynthesis of phosphatidylcholine by methylation of phosphatidylethanolamine derived from ethanolamine is not required for lipoprotein secretion by cultured rat hepatocytes

The role that phosphatidylcholine biosynthesis plays in the assembly and secretion of lipoproteins has been investigated in rat hepatocytes, since phosphatidylcholine is the major phospholipid in all serum lipoproteins. Phosphatidylcholine in rat hepatocytes can be made via the CDPcholine pathway or by the methylation of phosphatidylethanolamine. A specific inhibitor of cellular transmethylation, 3-deazaadenosine (10 microM), has been incubated with rat hepatocytes, and we have shown that the biosynthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine derived from ethanolamine was inhibited by greater than 95%. However, incubation of 3-deazaadenosine with cultured rat hepatocytes for up to 18 h did not affect the secretion of any of the apoproteins into VLDL, LDL, HDL fractions or a fraction with density greater than 1.18 g/ml (albumin was the major protein). Nor was there any effect by 3-deazaadenosine on the amount of phosphatidylcholine secreted into the culture medium or into VLDL or HDL. After 18 h the amount of phosphatidylethanolamine that accumulated in the cells was doubled by treatment with 3-deazaadenosine, and the amount of phosphatidylethanolamine secreted into the medium was increased by approximately 70%. It is thus apparent that the synthesis of phosphatidylcholine from ethanolamine is not required for lipoprotein secretion by rat hepatocytes.