Decrease in scavenger receptor expression in human monocyte-derived macrophages treated with granulocyte macrophage colony-stimulating factor.

To determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL; one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF-treated macrophages. Treatment of human monocyte-derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated approximately 50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte-derived macrophages, which might have implications for foam cell formation.

Effects of simvastatin on plasma lipids and apolipoproteins in familial hypercholesterolemic swine.

Familial hypercholesterolemia (FHC) in swine, which resembles human familial combined hyperlipidemia, is a complex lipid and lipoprotein disorder associated with the development of severe coronary lesions similar to those occurring in advanced human coronary disease. The disorder is characterized by elevated plasma total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C), apolipoproteins (apo) B, C-III, and E, and by decreased levels of HDL-cholesterol (HDL-C), apoA-I, and lecithin:cholesterol acyltransferase (LCAT) activity. A dose-response study with simvastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was conducted in four treatment groups of FHC animals, exhibiting TC > or = 250 mg/dL. The animals were fed 0, 80, 200, or 400 mg simvastatin daily for 3 weeks. The measured serum parameters included the levels of TC, VLDL-C, LDL-C, HDL-C, TG, lathosterol, apoA-I, B, C-III, and E, as well as LCAT activity. Simvastatin at 200 mg/d significantly decreased the levels of TC (-25%), LDL-C (-27%), lathosterol (-40%), apoB (-22%), apoC-III (-37%), and apoE (-24%) and modestly decreased the levels of HDL-C (-12%) and apoA-I (-11%) (percent relative to the average pretreatment and posttreatment baseline values) but did not affect the levels of TG, VLDL-C, the lathosterol/TC ratio, or LCAT activity. The levels of TC, LDL-C, apoB, and E were also lowered by simvastatin at 80 or 400 mg/d, but to a lesser extent than at 200 mg/d, while the other parameters were not influenced at these doses. The simvastatin-induced decreases of LDL-C, HDL-C, and apoA-I, B, C-III, and E were significantly correlated among each other. These results show that the trend of responses in TC, LDL-C, apoB, apoC-III, and apoE to simvastatin in the FHC swine is similar to that observed in humans, although the drugs is less potent and efficacious in swine, while the results are different from those in humans with regard to the remaining parameters.

Genetic analysis of sex and generation differences in plasma lipid, lipoprotein, and apolipoprotein levels in adolescent twins and their parents.

In a sample of Dutch families consisting of parents aged 35-65 years and their twin offspring aged 14-21 years, a significant difference between generations was observed in phenotypic variances and in genetic heritabilities for plasma levels of total cholesterol, triglycerides, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, and apolipoproteins (apo) A1, A2, B, and E. For all traits parents were more variable than their offspring. This increase in phenotypic variance was best explained by a genetic model in which individual specific environmental variance increased with increasing age. Genetic variance was the same across generations for nearly all traits except triglycerides and apoE, for which a decrease in genetic variance was observed. This model led to large intergenerational differences in genetic heritabilities. Heritabilities for children were between 65 and 87%, while heritabilities for their parents were between 10 and 50%. No evidence was found for effects of a shared family environment.

Secretion of apolipoproteins A-I and B by HepG2 cells: regulation by substrates and metabolic inhibitors.

It was the aim of this study to i) compare the effects of glucose and other hexoses with that of oleate on secretion of apolipoproteins (apos) A-I and B by HepG2 cells, and ii) document the effect of various metabolic inhibitors on the secretion of these apos in the absence or presence of extra glucose/oleate. i) The addition of 10 mM glucose increased secretion of apoA-I and apoB, as measured by enzyme immunoassay, by about 60% when cells were incubated for 48 h in DMEM + 10% fetal calf serum. The addition of extra glucose also increased the mRNA levels for these apos. Increased radioactivity was also found in these apolipoproteins by immunoprecipitation after metabolic labeling with [35S]methionine for 48 h. However, in a pulse-chase experiment (15 min labeling, 2 h chase), glucose was found to increase apoA-I synthesis but not apoB synthesis. More labeled apoB appeared in the medium during the chase because glucose inhibited its intracellular degradation. The effect of glucose on secretion of these apos could be mimicked by fructose and mannose but not by 6-deoxyglucose, showing that the hexoses must enter the cells and be phosphorylated. In contrast, the addition of 0.5 mM oleate had a weak inhibitory effect on secretion of apoA-I whereas it increased the secretion of apoB by more than twofold. The combination of 10 mM glucose and 0.5 mM oleate had no greater effect than glucose alone on apoA-I secretion but increased apoB secretion by fourfold. ii) Inhibiting glycolysis (by glucosamine) lowered secretion of both apoA-I and apoB, while inhibiting lipogenesis (using 8-Br-cyclic AMP or 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA)) did not affect apoA-I secretion but clearly decreased that of apoB. However, the inhibitory effect of TOFA on apoB secretion was much smaller in the presence of 0.5 mM oleate instead of extra glucose. Actinomycin-D and cycloheximide strongly suppressed the stimulatory effect of glucose on secretion of both apolipoproteins. Actinomycin-D also suppressed basal secretion of apoA-I but surprisingly stimulated that of apoB. These observations indicate that in HepG2 cells secretion of apoA-I is strongly dependent on ongoing protein synthesis and can be boosted by glucose, whereas that of apoB is primarily driven by internal (via lipogenesis from glucose) or external supply of fatty acyl-residues.

Effects of stigmastanyl-phosphocholine (Ro 16-6532) and lovastatin on lipid and lipoprotein levels and lipoprotein metabolism in the hamster on different diets.

Previous studies from our laboratory have shown that oral administration of stigmastanyl-phosphocholine (Ro 16-6532) reduces plasma cholesterol levels in experimental animals on diets free of added cholesterol. In the present study, effects of Ro 16-6532 and lovastatin on lipoprotein levels and metabolism were investigated in male golden Syrian hamsters. In hamsters fed a standard diet, Ro 16-6532 (1 mmol/kg/day) lowered cholesterol in all lipoprotein fractions, as well as apoB-100 and apoA-I. In contrast, lovastatin (25 mumol/kg/day) lowered high density lipoprotein (HDL)-cholesterol but had no effect on low density lipoprotein (LDL)-cholesterol or on apoB-100 or apoA-I while triglycerides and very low density lipoprotein (VLDL)-cholesterol increased. In hamsters fed a coconut fat-supplemented diet, Ro 16-6532 reduced all lipoproteins, with a stronger effect on VLDL- and LDL- than on HDL-cholesterol. Also apoB-100 was reduced. Lovastatin (50 mumol/kg/day) reduced LDL-cholesterol, HDL-cholesterol, and apoA-I while triglycerides and VLDL-cholesterol increased. The drop in LDL-cholesterol seen with both drugs in hamsters fed the diet supplemented with coconut fat occurred without any effect on the plasma removal rate of homologous LDL, or on the content of hepatic LDL-receptors. In contrast, the first phase of removal of homologous radioiodinated VLDL from plasma was markedly increased by both compounds, paralleled with an increased uptake of label in the liver and a decreased appearance of labeled apoB-100 in the LDL-fraction. Furthermore, retinyl ester-labeled chylomicrons were also cleared more rapidly in hamsters treated with Ro 16-6532. Hepatic uptake of label from VLDL and chylomicrons was strongly decreased by pre-injection of lactoferrin. In addition, Ro 16-6532 slightly decreased the secretion rate of VLDL in hamsters fed the coconut fat-supplemented diet. Taken together, these results indicate that the reduction of LDL-cholesterol after treatment with Ro 16-6532 and lovastatin observed in the hamster is mainly due to decreased conversion of VLDL into LDL, consequent to an increased hepatic removal of VLDL remnants. Ro 16-6532 also increased the liver uptake of chylomicron remnants. The hepatic uptake system implicated in this remnant removal can be completely blocked by lactoferrin. The nature of this uptake system is still unknown.

Regulation of human apolipoprotein A-I expression in Caco-2 and HepG2 cells by all-trans and 9-cis retinoic acids.

Retinoids are reported to stimulate apolipoprotein (apo) A-I gene promoter activity (Rottman et al. 1991. Mol. Cell. Biol. 11: 3814-3820) and apoA-I protein secretion by monkey hepatocytes (Kaptein et al. 1993. Arterioscler. Thromb. 13: 1505-1514). In this study we have assessed the effects of retinoids on parameters of apoA-I biosynthesis in human cell lines. Caco-2 and HepG2 cells (human intestinal and hepatoma cell lines, respectively, both known to express and secrete apoA-I) were stably transfected with a reporter gene construct containing 1.3 kb of the 5-'flanking region of the human apoA-I gene linked to the firefly luciferase coding region. These cells were incubated for 48 h with 10 microM all-trans retinoic acid (RA) or 9-cis RA. The cells were then assayed for luciferase activity, for apoA-I mRNA level, and for secretion of apoA-I protein in the medium. Secretion of apoB was monitored as well. In Caco-2 cells, all-trans and 9-cis RA increased luciferase activity, mRNA content, and protein secretion by 40% to 80% above control. Strikingly, in HepG2 cells all-trans and 9-cis RA caused a more marked stimulation of luciferase activity (by 100-150%) but a weaker increase of mRNA content and protein secretion (by 25-30%). In contrast, apoB secretion was inhibited by the two retinoids in Caco-2 cells and not changed in HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of the apolipoprotein E phenotype on plasma lipids is not influenced by environmental variability: results of a Dutch twin study.

We tested the influence of the apolipoprotein E (apoE) polymorphism on the intrapair differences in the levels of plasma cholesterol, plasma triglycerides, low density lipoprotein-cholesterol, apoB and apoE in monozygotic (MZ) twins, and estimated whether or not there was a interaction between the apoE polymorphism and environmental factors. In 65 MZ twin pairs, the intrapair differences in the measured lipoprotein parameters were similar in the different apoE phenotype classes. This indicates that the effect of the apoE polymorphism is not influenced by environmental variability between the MZ pair members and accordingly identifies the APOE gene as a "level" gene.

Lipoprotein(a): relation to other risk factors and genetic heritability. Results from a Dutch parent-twin study.

We measured plasma levels of lipoprotein(a) (Lp(a)) in a sample of 152 Dutch adolescent mono- and dizygotic twin pairs and their parents. The distribution of Lp(a) levels was skewed, with the highest frequencies at low levels and was similar for adult men and women and their children. The relationship of Lp(a) concentrations with other lipoprotein and apolipoprotein risk factors for coronary heart disease and with lathosterol, an indicator of whole-body cholesterol synthesis, was studied dependent on sex and generation. In mothers and children there was a small positive correlation between Lp(a) levels and plasma cholesterol and apolipoprotein (apo) B. In mothers and daughters there also was a correlation between Lp(a) and LDL cholesterol levels. No correlation was found between Lp(a) levels and plasma lathosterol, suggesting that there is no relationship between Lp(a) levels and cholesterol synthesis. Associations among family members, i.e. between monozygotic and dizygotic twins and between parents and offspring were used to study familial transmission of Lp(a) levels. Results showed that almost all of the variance in Lp(a) concentrations was accounted for by genetic heritability. A small, but significant, sex difference in heritability was observed, but heritabilities were the same in parents and offspring. Heritability estimates were 93% for females and 98% for males. No evidence was found for assortative mating or for the influence of a shared family environment. These results indicate that nearly all variance in Lp(a) concentrations that is not accounted for by the apo(a) size polymorphism, is also under genetic control.

A monogalactosylated cholesterol derivative that specifically induces uptake of LDL by the liver.

We described earlier the effect of tris-gal-chol (a triantennary galactose structure coupled to cholesterol) on the fate of low density lipoprotein (LDL) and high density lipoprotein (HDL). Tris-gal-chol-loaded LDL and HDL are both efficiently cleared from blood by hepatic galactose-specific receptors. Thus, tris-gal-chol combines a beneficial LDL-reducing effect with an equally effective but undesirable HDL-lowering effect. We recently synthesized a cholesterol derivative with a single terminal galactose residue, denoted mono-gal-chol. In the present study we show that this compound, which incorporates readily into both LDL and HDL, induces rapid association of LDL and HDL to the liver. The mono-gal-chol-stimulated hepatic association of HDL, however, was about fivefold lower than that of LDL. In the liver, Kupffer cells were mainly (90%) responsible for the liver uptake of mono-gal-chol-loaded LDL, whereas the complex of mono-gal-chol with HDL was predominantly (95%) taken up by parenchymal cells. Uptake by both cell types proceeded via galactose-specific receptors and was followed by degradation of the apolipoproteins in the lysosomes. Thus, compared with tris-gal-chol, mono-gal-chol is equally effective in the induction of galactose-specific uptake of LDL by Kupffer cells. However, the galactose-specific receptor on parenchymal cells recognizes mono-gal-chol-loaded HDL less efficiently than tris-gal-chol-containing HDL. These results indicate that mono-gal-chol might be used to specifically lower LDL levels in patients with a high LDL cholesterol level.

Evaluation of the use of serum lathosterol concentration to assess whole-body cholesterol synthesis in rabbits.

Serum lathosterol concentration in rabbits was assessed as a possible indicator of whole-body cholesterol synthesis. In random-bred New Zealand White (NZW) rabbits fed a control diet or a diet containing either cholesterol, simvastatin, or cholestyramine, neither serum lathosterol concentration nor the serum lathosterol:total cholesterol ratio systematically corresponded with the anticipated rate of cholesterol synthesis. In control rabbits and those fed simvastatin or cholestyramine, whole-body cholesterol synthesis, which was calculated from the sterol balance, was correlated with serum lathosterol concentration when expressed relative to cholesterol in very low, intermediate, and low density lipoproteins (VLDL + IDL + LDL) (r = 0.61; n = 23; P = 0.002). The low correlation coefficient indicates that the predictive value of the lathosterol: (VLDL + IDL + LDL) cholesterol ratio is limited when applied to individual rabbits. Cholesterol and simvastatin feeding reduced the group mean serum lathosterol:(VLDL + IDL + LDL) cholesterol ratio, whereas cholestyramine in the diet raised the group mean ratio in the NZW rabbits. We conclude that the serum lathosterol:(VLDL + IDL + LDL) cholesterol ratio may be an indicator of group mean rates of whole-body cholesterol synthesis in rabbits but may not yield reliable information on individual rabbits. The lathosterol:(VLDL + IDL + LDL) cholesterol ratio predicted that in hyperresponsive inbred rabbits, showing an excessive hypercholesterolemia after cholesterol feeding, baseline whole-body cholesterol synthesis is lower than in hyporesponsive rabbits. Addition of cholesterol to the diet caused a reduction of predicted cholesterol synthesis in hypo- but not in hyper-responsive rabbits.

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.

Plasma levels of lathosterol and phytosterols in relation to age, sex, anthropometric parameters, plasma lipids, and apolipoprotein E phenotype, in 160 Dutch families.

In this study, the relation of plasma levels of lathosterol (an indicator of whole body cholesterol synthesis) and plant sterols (indicator of cholesterol absorption) with age, sex, weight, height, plasma lipids, and lipoproteins, and with apolipoprotein (apo) E phenotype, was investigated in a group of 160 nuclear families consisting of twins living with their parents. Lathosterol was higher in fathers than in mothers, but not different between boys and girls. In each of these four groups, there was a strong correlation with plasma and low-density lipoprotein (LDL)-cholesterol and -triglyceride, as well as with body weight, but not with height or high-density lipoprotein (HDL)-cholesterol. In adults, lathosterol was inversely correlated with plant sterols. Lathosterol was higher in children with E4/3 phenotype than in those with E3/3 or E3/2; in adults, lathosterol did not differ among the various E phenotypes. The plasma levels of the two plant sterols, campesterol and beta-sitosterol, were highly correlated with each other, and also with plasma or LDL-cholesterol, in each of the four groups. Plant sterols were higher in adults or children with E4/3 phenotype as compared with those with other phenotypes. In multivariate analysis (performed separately for two groups of adults and children) plasma cholesterol, plasma plant sterols, plasma triglycerides, and weight were found to make significant contributions to the variation of lathosterol in all groups, and E phenotype and sex only in one group, while age did not contribute in any group.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of rat liver parenchyma in cholesterol 7 alpha-hydroxylase and bile acid synthesis.

Periportal and perivenous hepatocytes were isolated from rat liver by digitonin/collagenase perfusion for investigating the acinar distribution of bile acid synthesis. The specific activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) was 7.9-fold higher in perivenous cells than in periportal hepatocytes. Mass production of bile acids differed 4.4-fold between cultured perivenous and periportal hepatocytes. In contrast, the levels of free cholesterol in homogenates and microsomes derived from both subfractions were similar. Feeding of rats with the bile-acid-sequestering anion-exchange resin colestid resulted in a pronounced stimulation of cholesterol 7 alpha-hydroxylase activity and bile acid mass production, but decreased the perivenous/periportal ratio of both parameters. These results demonstrate that bile acid mass production, but decreased the perivenous hepatocytes, possibly owing to feedback suppression by bile acids from the enterohepatic circulation. Furthermore, the opposite acinar localization of cholesterol and bile acid biosynthesis provides an interesting alternative to current views of the regulation of their metabolic pathways.

Lathosterol level in plasma is elevated in type III hyperlipoproteinemia, but not in non-type III subjects with apolipoprotein E2/2 phenotype, nor in type IIa or IIb hyperlipoproteinemia.

We measured the serum lathosterol level, a reflection of the rate of whole body cholesterol synthesis, in 15 patients with manifest type III hyperlipoproteinemia (HLP), in 20 subjects with apolipoprotein (apo) E2/2 phenotype, but without type III HLP, in 21 patients with type IIA and 10 patients with type IIB HLP. A group of 100 subjects with apo E3/3 phenotype served as reference. Using ANCOVA, lathosterol was adjusted for serum cholesterol and triglyceride concentrations, since these parameters were found to independently correlate with lathosterol. The adjusted means (+/- SEM), in mumol/L, in these groups were 12.9 +/- 1.1, 8.2 +/- 1.1, 4.8 +/- 0.9, 9.8 +/- 1.4, and 7.8 +/- 0.4, respectively. Type III HLP patients had significantly higher lathosterol levels than all other groups except type IIB HLP. In addition, lathosterol was significantly lower in type IIA patients than in all other groups. The serum levels of plant sterols, used as a reflection of cholesterol absorption, did not differ among the various groups after adjustment for serum cholesterol. These findings suggest that an overproduction of cholesterol is one factor discriminating E2/2 homozygotes with type III HLP from those without the disease.

Water-soluble cholesteryl-containing phosphorothioate monogalactosides: synthesis, properties, and use in lowering blood cholesterol by directing plasma lipoproteins to the liver.

The synthesis of several monogalactoside-terminated phosphorothiolated cholesteryl derivatives is described. Monogalactosyl derivatives are coupled by phosphorothiolation to cholesterol by using ethylene glycol units as hydrophilic spacer moieties. The resulting compounds are easily soluble in water. Upon addition of such solutions to human serum (to 2 mM final concentration) the compounds are readily incorporated into lipoproteins. Isolated low-density lipoprotein (LDL) and high-density lipoprotein (HDL), preloaded with the compounds, are rapidly cleared from the circulation by the liver. The hepatic association is blocked by N-acetylgalactosamine, which indicates that galactose-specific recognition sites are responsible for the increased liver uptake. The plasma clearance and hepatic uptake of LDL loaded with the compounds is substantially higher (about 2-fold) than clearance and uptake of HDL containing the compounds. The selectivity of the effects of monogalactoside-terminated phosphorothiolated cholesteryl derivatives on the in vivo behavior of LDL as compared to that of HDL indicates that these compounds might be used to lower specifically LDL levels in patients with a high LDL-cholesterol level.

Vastatins inhibit cholesterol ester accumulation in human monocyte-derived macrophages.

Human monocyte-derived macrophages were incubated for 48 hours in Medium 199 with 1% human serum albumin, and with 100 micrograms acetyl low density lipoprotein (LDL) or beta-very low density lipoprotein (beta-VLDL), with or without various concentrations of compactin, lovastatin, simvastatin, or pravastatin. The mass of free (FC) and esterified (CE) cholesterol was determined, as well as the incorporation of [1-14C]acetate in sterols, that of [1-14C]oleate in CE, and that of [methyl-14C]choline in phospholipids. Moreover, we assessed the high-affinity association and degradation of 125I-labeled acetyl LDL. Compactin markedly decreased the cellular accumulation of CE induced by acetyl LDL or beta-VLDL and increased the content of FC. Compactin also decreased the incorporation of [1-14C]oleate in CE (by 70-90%) in incubations with or without added lipoproteins. The half-maximal inhibitory concentration for this effect of compactin was 30 nM. Lovastatin and simvastatin were more potent, but pravastatin was about 100-fold less potent. Although compactin also caused a clear inhibition of cholesterol synthesis in the presence of acetyl LDL, the effect on CE formation did not seem to be related to decreased cholesterol synthesis, since this was already very low in the presence of acetyl LDL. Compactin did not affect the association and degradation of labeled acetyl LDL and also had no effect on the rate of cholesterol loss after preloading the cells with CE by incubation with acetyl LDL. However, compactin had a slight stimulatory effect on the synthesis of phosphatidylcholine and sphingomyelin when compactin was added to incubations in the presence of acetyl LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial characterization of low density lipoprotein preparations isolated from fresh and frozen plasma after radiolabeling by seven different methods.

Four 99mTc and three 123I labeling methods were evaluated for their suitability to label low density lipoproteins (LDL) for the purpose of scintigraphic biodistribution studies. For 99mTc these methods were: direct incorporation in LDL of 99mTcO4- using sodium dithionite (dithionite method); a method using first N,N-dimethylformamide to prepare a 99mTc-complex reacting with LDL in a subsequent step (DMF method); a technique in which 99mTcO4- is first coupled to a diamide dithiolate derivative of pentanoic acid by reduction with dithionite, followed by coupling of this ligand to LDL (N2S2 method); and a method using sodium borohydride and stannous chloride as reducing agents (borohydride method). The iodination techniques were based on oxidation of I(-)----I+, using iodine monochloride (ICl method), 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (Iodogen method), and N-bromosuccinimide (NBS method) as oxidants. We studied labeling yields, modification of LDL caused by the labeling procedures using agarose-gel electrophoresis, and radiochemical stability of the labeled LDL complex upon incubation in plasma at 37 degrees C for 15 h. We used Sepharose CL6B chromatography to separate LDL from other plasma proteins. We also examined whether LDL isolated from frozen plasma (Pool-LDL) gave results similar to LDL obtained from freshly prepared plasma (Fresh-LDL). Pool-LDL radiolabeled by the dithionite, DMF, NBS, and Iodogen methods lost its label upon incubation with plasma. This also happened with Fresh-LDL when the DMF, NBS and Iodogen methods were used. Upon agarose-gel electrophoresis, no modification of LDL was observed with all methods when the radionuclide/LDL ratio was kept low.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile acids exert negative feedback control on bile acid synthesis in cultured pig hepatocytes by suppression of cholesterol 7 alpha-hydroxylase activity.

Feedback regulation of bile acid synthesis by its end products was studied in cultured hepatocytes of young weaned pigs. We previously showed that conversion of exogenous [14C] cholesterol into bile acids was suppressed by addition of bile acids to the culture medium. In the present study, the effects of bile acids on bile acid mass production and cholesterol 7 alpha-hydroxylase activity were examined. Mass production of bile acids was strongly inhibited by addition of taurocholic acid (50 and 100 mumol/L) to the culture medium. The inhibitory action was exerted specifically on activity of cholesterol 7 alpha-hydroxylase because conversion of [14C] 7 alpha-hydroxycholesterol to bile acids by pig hepatocytes was not affected. Suppression of cholesterol 7 alpha-hydroxylase activity after incubation of the hepatocytes with taurocholic acid was concentration- and time-dependent. Maximum suppression (-80%) was achieved after a 20 to 30 hr incubation of hepatocytes with 100 mumol/L of this bile acid. Decline of enzyme activity caused by 100 mumol/L taurocholic acid followed first-order kinetics with a half-life of 10 hr. Taurocholic acid had no direct effect on cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes as assessed by addition of the bile acid to the assay mixture. The effects of several other bile acids in a concentration of 100 mumol/L on cholesterol 7 alpha-hydroxylase activity were examined in 48 hr incubations. Glycochenodeoxycholic and glycohyodeoxycholic acids, which are the major bile acids in pig bile, their unconjugated forms and also deoxycholic and cholic acid pronouncedly inhibited activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol 7 alpha-hydroxylase activity and bile acid synthesis in hepatocytes of unweaned and weaned pigs in monolayer culture.

Activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7 alpha-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7 alpha-hydroxylase activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [14C]cholesterol, among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7 alpha-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7 alpha-hydroxylase activities and bile acid production rates.

Postnatal developmental profile of 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthetase and cholesterol 7 alpha-hydroxylase activities in the liver of domestic swine.

Activities of 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthetase and cholesterol 7 alpha-hydroxylase, measured in liver microsomal preparations from domestic swine between birth and adolescence, correlated strongly in individual animals. A synchronous increase was observed between 4 and 6 weeks after birth, i.e., immediately after weaning. Rise in activity was highest for HMG-CoA reductase (30-fold), and smallest for squalene synthetase (5-fold). In pubertal pigs (16 to 30 weeks old), activities of these enzymes had the same low values as in suckling piglets. The increase of both HMG-CoA reductase and squalene synthetase activities may be caused by the shift from high-cholesterol milk intake to a chow diet with low-cholesterol content. The rise in cholesterol 7 alpha-hydroxylase activity might be due to other dietary or hormonal factors.