Side chain hydroxylations in bile acid biosynthesis catalyzed by CYP3A are markedly up-regulated in Cyp27-/- mice but not in cerebrotendinous xanthomatosis.

The accumulation of various 25-hydroxylated C(27)-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27(-/-) mice do not show CTX-related abnormalities. Microsomal activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6beta-hydroxylase, a marker enzyme for CYP3A, in Cyp27(-/-) mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases and 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6beta-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27(-/-) mice, CYP3A activity was not up-regulated despite marked accumulation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in CTX.

Key regulatory oxysterols in liver: analysis as delta4-3-ketone derivatives by HPLC and response to physiological perturbations.

A number of oxysterols have been implicated in metabolic regulation. Key among these are (24S),25-epoxycholesterol and (24S)-hydroxycholesterol, high affinity ligands for the nuclear transcription factor liver X receptor alpha; 27-hydroxycholesterol, a bile acid synthetic intermediate; and 25-hydroxycholesterol, which has been used to study regulation of lipid metabolism by the sterol regulatory element-binding protein family of transcription factors. Investigation of the physiological importance of these compounds in vivo has been hampered by lack of analytical methods to reproducibly and accurately determine their concentrations in tissues. This article describes a method designed to determine quantitatively the amounts of these important side-chain oxysterols by derivatization to the Delta4-3-ketones followed by high performance liquid chromatography. The method was validated with known standards and then was used to determine the concentrations of these oxysterols in rodent liver under various physiological conditions. All four oxysterols were present in the picogram per milligram protein range and have distinct subcellular distributions and responses to physiological perturbations in vivo.

Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27(-/-) mice and CTX.

Cerebrotendinous xanthomatosis (CTX) is a rare, recessively inherited lipid storage disease characterized by a markedly reduced production of chenodeoxycholic acid and an increased formation of 25-hydroxylated bile alcohols and cholestanol. Patients with this disease are known to have mutations in the sterol 27-hydroxylase (Cyp27) gene. However, one study showed that mice with a disrupted Cyp27 gene did not have any CTX-related clinical or biochemical abnormalities. To explore the reason, hepatic cholesterol, cholestanol, and 12 intermediates in bile acid biosynthetic pathways were quantified in 10 Cyp27(-/-) and 7 Cyp27(+/+) mice, two CTX patients (untreated and treated with chenodeoxycholic acid), and four human control subjects by high resolution gas chromatography-mass spectrometry. Mitochondrial 27-hydroxycholesterol and 5beta-cholestane-3alpha,7alpha,12alpha,27-tetrol were virtually absent in both Cyp27(-/-) mice and CTX patients. In Cyp27(-/-) mice, microsomal concentrations of intermediates in the early bile acid biosynthetic pathway (7alpha-hydroxycholesterol, 7alpha-hydroxy-4-cholesten-3-one, 7alpha,12alpha-dihydroxy-4-cholesten-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol), 25-hydroxylated bile alcohols (5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, 5beta-cholestane-3alpha,7alpha,12alpha,23R,25-pentol, and 5beta-cholestane-3alpha,7alpha,12alpha,24R, 25-pentol), and cholestanol were all significantly elevated compared with those in Cyp27(+/+) mice, although the levels were lower than those in untreated CTX patients. The intermediate levels in early bile acid biosynthesis were more elevated in male (16;-86% of CTX) than in female Cyp27(-/-) mice (7-30% of CTX). In contrast, 25-hydroxylated bile alcohol concentrations were not significantly different between male and female Cyp27(-/-) mice and were considerably lower (less than 14%) than those in CTX patients.These results suggest that 1) in Cyp27(-/-) mice, especially in females, classic bile acid biosynthesis via 7alpha-hydroxycholesterol is not stimulated as much as in CTX patients; and 2) formed 25-hydroxylated bile alcohols are more efficiently metabolized in Cyp27(-/-) mice than in CTX patients.

Regulation of pathways determining cholesterol availability in the baboon placenta with advancing gestation.

Low density lipoprotein (LDL) is accepted as the primary source of cholesterol for progesterone biosynthesis in the primate placental syncytiotrophoblast. We hypothesized that the syncytiotrophoblast may, however, derive significant amounts of cholesterol from sources in addition to the LDL pathway, especially during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. To test this, alternate cholesterol-providing pathways were assessed in placentae at early (Days 60-61), mid (Days 98-102), and late (Days 160-167) gestation in the baboon (Papio sp., term approximately 184 days). Expression of LDL receptor mRNA transcripts in an enriched fraction of syncytiotrophoblast cells was approximately 13-fold greater (P < 0.05) in mid and late gestation than in early pregnancy, although no differences were observed in whole villous tissue. The abundance of transcripts for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for de novo cholesterol synthesis, remained unchanged in syncytiotrophoblast cells; however, HMG-CoA reductase activity declined approximately 2-fold from early to late pregnancy (P < 0.01), with a commensurate decline in immunoreactive HMG-CoA reductase protein. Activities for acyl-coenzyme A:cholesterol acyl transferase (ACAT), a rate-limiting enzyme for cholesterol esterification, were greater (P < 0.05) at early and mid pregnancy in placental homogenates than in those from late pregnancy, while ACAT-1 mRNA concentrations and cholesterol ester hydrolase activity remained unchanged. These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.

A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum.

We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we obtained a RER fraction from rat liver and established its high degree of purity by quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liver, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containing approximately 110-125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.

Differential expression of apolipoprotein E messenger RNA within the rat liver lobule determined by in situ hybridization.

Apolipoprotein (Apo) E plays a key role in the metabolism of lipoproteins. It also modulates immunoregulation, cell growth and differentiation and the response to nerve injury. The liver is a major site of ApoE synthesis. Most of the circulating ApoE is thought to be of hepatic origin with most synthesized in hepatocytes. We showed that total liver ApoE messenger RNA (mRNA) levels were greater in normal adult female rats than in male and that gender-specific patterns of liver ApoE mRNA expression were present by in situ hybridization. In the male liver, the signal was strongest in the portal area, decreasing toward the central vein with the weakest signal in pericentral hepatocytes, resulting in a hepatic lobular gradient of expression. In female liver, a strong periportal signal also was observed that decreased in Zone 2, similar to that in males, but which then increased in pericentral hepatocytes resulting in a bowl-like distribution in marked contrast with that of the male. The results suggest that ApoE mRNA level is regulated differentially in hepatocytes within the liver plate and that the regulation is gender-dependent. Further, the results suggest that in males, hepatocytes in the portal area are the major contributors of ApoE to the plasma and/or sinusoidal pool, whereas in females, both portal and central area hepatocytes play an equal role.

Effect of pregnancy and lactation on lipoprotein and cholesterol metabolism in the rat.

Origins of hyperlipidemia and cholestasis that occur during pregnancy were investigated by examining expression of key elements related to plasma and hepatic cholesterol metabolism during pregnancy, lactation, and post-lactation in the rat model. Among major findings were: during pregnancy, the activities of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl coenzyme A:cholesterol acyltransferase, acyl coenzyme A:diacylglycerol acyltransferase, cholesterol 7alpha-hydroxylase, cholesterol ester hydrolases, low density lipoprotein receptors, LRP, and mdr2 were significantly lower or similar to non-pregnant controls while SR-B1 was elevated. Once lactation began, reductase, cholesterol acyltransferase, 7alpha-hydroxylase activities, low density lipoprotein receptors, and mdr2 increased while SR-B1 decreased. In later stages of lactation most hepatic elements returned to near control levels. Plasma cholesterol levels were higher than control at birth and during lactation with increase in LDL-size particles. By 24 h post-lactation, plasma triglycerides were 3.7-fold higher while cholesterol remained unchanged. Very large lipoproteins were present while LDL-size particles were now absent. Hepatic cholesterol acyltransferase had decreased to 27% of control while diacylglycerol acyltransferase increased 3-fold and low density lipoprotein receptors doubled. Most elements were normalized 3 weeks after weaning except for LRP and low density lipoprotein receptors which were elevated. These studies provide an integrated picture of expression of key elements of hepatic and plasma cholesterol metabolism during pregnancy and lactation and advance understanding of hyperlipidemia and cholestasis during these states.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Cholesterol 7alpha-hydroxylase (CYP7A): patterns of messenger RNA expression during rat liver development.

Cholesterol 7-hydroxylase is a rate-limiting enzyme in bile acid synthesis, a major pathway for cholesterol catabolism. It plays a crucial role in postnatal development and survival. In an adult liver, its activity and messenger RNA (mRNA) are heterogeneously distributed with concentration in the pericentral area. We defined how the pattern of cholesterol 7-hydroxylase mRNA evolves during rat liver development, correlated this with its total liver mRNA levels, and determined when its heterogeneous pattern of expression is established. Cholesterol 7-hydroxylase mRNA was undetectable in 18-day-old fetal livers by Northern blot. It was increased markedly in newborns with a homogeneous liver lobular distribution as determined by in situ hybridization. At postnatal day four, mRNA levels were markedly decreased with concomitant appearance of a lobular gradient: mRNA was detected only in a few hepatocytes located around efferent venules. At 22 days, the time of highest mRNA expression, a marked extension of the gradient towards the periportal area was observed, indicating that the increase in total liver cholesterol 7-hydroxylase mRNA level was a result of recruitment of hepatocytes upstream from the central vein area. By 28 days, the adult pattern was observed. Thus, expression of cholesterol 7-hydroxylase mRNA is tightly regulated during rat liver development, both temporally and spatially supporting its critical role in normal postnatal development.

ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.

The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a approximately 46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3beta-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

Hypolipidemic mechanisms of pectin and psyllium in guinea pigs fed high fat-sucrose diets: alterations on hepatic cholesterol metabolism.

Studies were conducted to determine whether pectin (PE) or psyllium (PSY) could reverse the high plasma cholesterol and triacylglycerol (TAG) concentrations induced by high fat (HF) or high sucrose (HS) diets and which are the mechanisms involved. Male guinea pigs were fed either a low fat (LF) or a HF diet with 80% of the carbohydrate energy derived from sucrose. Cellulose was used as control. Plasma LDL cholesterol, TAG, apolipoprotein B, and hepatic cholesteryl ester were lower in guinea pigs fed PE and PSY compared to the control group (P < 0.03). In addition, a 45% higher number of hepatic apoB/E receptors was observed by PE and PSY intake. Hepatic ACAT, HMG-CoA reductase, and cholesterol 7alpha-hydroxylase (C7H) activities were higher in the HF compared to the LF groups (P < 0.01). PSY intake with HF resulted in up-regulation of C7H and HMG-CoA reductase activities (P < 0.05). Additional studies measuring the effects of PE and PSY on low density lipoprotein (LDL) transport and very low density lipoprotein (VLDL) secretion were conducted in the HF groups. ApoB secretion was reduced by pectin and psyllium (P < 0.01) intake while LDL fractional catabolic rates were 100% faster in guinea pigs fed PE or PSY. In these studies the extent of the hypolipidemic response was specific to each fiber type and associated with the amount of sucrose. In addition, PSY altered the activity of hepatic enzymes of cholesterol homeostasis in the HF group. These additional effects of PSY might explain the more dramatic changes in plasma lipid levels associated with PSY consumption.

Tissue expression studies on the mouse acyl-CoA: cholesterol acyltransferase gene (Acact): findings supporting the existence of multiple cholesterol esterification enzymes in mice.

Cholesterol esterification is involved in the regulation of cellular cholesterol content and has been hypothesized to play a role in important physiologic processes including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Although initial studies of the mouse acyl CoA:cholesterol acyltransferase gene (Acact) suggested that its gene product was responsible for cholesterol esterification in most tissues, we observed recently that Acact-/- mice have only tissue-specific reductions in cholesterol esterification. To better understand the role of Acact in cholesterol esterification, we used in situ hybridization and immunoblotting to perform tissue expression studies in wild-type mice. We found high levels of Acact expression in steroidogenic tissues, sebaceous glands, and atherosclerotic lesions, but not in the liver or the small intestine. These data support the hypothesis that multiple cholesterol esterification enzymes exist in mammals and that another enzyme is likely to be responsible for cholesterol esterification activity in mouse liver and intestine.

Feasibility of an immunoassay for mevalonolactone.

Mevalonic acid is a key intermediate in a broad spectrum of cellular biological processes and their regulation. Availability of a rapid, sensitive and accurate method for its assay would be highly useful. Therefore, the feasibility of developing an immunoassay for mevalonic acid in biological samples was explored. The strategy employed was to synthesize several racemic haptens structurally resembling R-mevalonolactone, the cyclic form of mevalonic acid present at lower pH and presumed to be more antigenic. Two of these haptens were coupled to keyhole limpet hemocyanin, and the resulting conjugates were used successfully to generate antibodies in rabbits. The first antiserum bound to R,S-mevalonolactone much more effectively at pH 4.0 than at pH 6.0, consistent with the structural resemblance of the haptens to the lactone form. This antiserum also bound the free hapten from which it was generated and two others of different structure with comparable effectiveness; and slightly better than it bound R,S-mevalonolactone at pH 4.0. Similar results were obtained with the antiserum to the second hapten. The binding of either antiserum to the natural enantiomer, R-mevalonolactone, was 20 times weaker than to R,S-mevalonolactone, suggesting that the nonbiological enantiomer was more antigenic. Nevertheless, the results demonstrate that an immunochemical approach to accurate quantitation of mevalonic acid in biological samples is feasible.

Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.

In addition to its ability to remove cholesterol from cells, HDL also delivers cholesterol to cells through a poorly defined process in which cholesteryl esters are selectively transferred from HDL particles into the cell without the uptake and degradation of the lipoprotein particle. The HDL-cholesteryl ester selective uptake pathway is known to occur in human, rabbit, and rodent hepatocytes where it may contribute to the clearance of plasma cholesteryl ester. The selective uptake pathway has been studied most extensively in steroidogenic cells of rodents in which it accounts for 90% or more of the cholesterol destined for steroid production or cholesteryl ester accumulation. In this study we have used apo A-I-, apo A-II-, and apo E-deficient mice created by gene targeting in embryonic stem cells to test the importance of the three major HDL proteins in determining cholesteryl ester accumulation in steroidogenic cells of the adrenal gland, ovary, and testis. apo E and apo A-II deficiencies were found to have only modest effects on cholesteryl ester accumulation. In contrast, apo A-I deficiency caused an almost complete failure to accumulate cholesteryl ester in steroidogenic cells. These results suggest that apo A-I is essential for the selective uptake of HDL-cholesteryl esters. The lack of apo A-I has a major impact on adrenal gland physiology causing diminished basal corticosteroid production, a blunted steroidogenic response to stress, and increased expression of compensatory pathways to provide cholesterol substrate for steroid production.

Effects of bile salts on rat hepatic acyl CoA:cholesterol acyltransferase.

Acyl CoA:cholesterol acyltransferase (EC2.3.1.26, ACAT), responsible for intracellular esterification of cholesterol, may play an important role in cholesterol trafficking within the cell, and thus, in maintenance of cellular cholesterol homeostasis. Bile acids are potential regulators of cholesterol trafficking in the liver. Therefore, the effect of bile salts on hepatic ACAT activity was studied in the perfused rat liver. ACAT activity was increased after liver perfusion with either taurocholate or taurochenodeoxycholate. However, addition of these bile salts at physiological concentrations in vitro had little effect on microsomal ACAT activity. The increase in hepatic ACAT activity due to perfusion with bile salts was accompanied by reduced accumulation of very low density lipoprotein cholesterol in the perfusate, but there was no effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hepatic ACAT activity was decreased after bile diversion for four hours in the intact animal. This treatment had no statistically significant effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. These data suggest that bile salts induce changes in hepatic compartmentation and traffic of cholesterol within the hepatocyte accompanied by response of ACAT activity to maintain cellular cholesterol homeostasis.

Gender differences in response to dietary soluble fiber in guinea pigs: effects of pectin, guar gum, and psyllium.

Dietary soluble fiber significantly lowers plasma low density lipoprotein (LDL) cholesterol concentrations in humans and animals. In male guinea pigs, alterations in hepatic cholesterol homeostasis induced by dietary fiber in part account for the decrease in plasma LDL levels (Fernandez et al. 1994. Am. J. Clin. Nutr. 59: 869-878; 1995. 61: 127-134, and J. Lipid Res. 1995. 36: 1128-1138). To test whether dietary fiber elicited similar hypocholesterolemic responses in both genders, female guinea pigs were fed diets containing 12.5% pectin (PE), 12.5% guar gum (GG), 7.5% psyllium (PSY), or 12.5% cellulose (control diet). In addition, physiological (0.04%) (LC) or pharmacological (0.25%) (HC) amounts of cholesterol were tested with the fibers to determine whether dietary cholesterol altered the plasma cholesterol response. Significant reductions in plasma cholesterol were observed in females fed LC diets with PE, GG, or PSY (P < 0.01) while the responses to fiber with high cholesterol intake were more moderate. Hepatic cholesterol concentrations were reduced in the LC group (P < 0.001) with increased HMG-CoA reductase and cholesterol 7 alpha-hydroxylase and decreased acyl CoA:cholesterol acyltransferase (ACAT) activities accompanied by a reduction in hepatic cholesterol pools induced by fiber intake. In addition, plasma LDL lowering in animals fed the LC diets was associated with increases in hepatic LDL receptor Bmax values. Effects of fiber on hepatic cholesterol in animals fed HC diets were moderate and hepatic enzymes were not altered to the same extent as in the LC groups. For the LC groups there was no gender effect on the magnitude of plasma LDL lowering, depletion of hepatic cholesterol, or alterations in hepatic cholesterol metabolism, although hepatic HMG-CoA reductase and ACAT activities were lower in females compared to males (P < 0.01). In contrast, females fed the control HC diet had higher plasma LDL levels than males and dietary fiber did not reduce hepatic cholesterol concentrations nor alter hepatic enzyme activities as effectively as in males. These studies demonstrate that female, compared to male, guinea pigs are more responsive to a dietary cholesterol challenge and, that with this pharmacological perturbation, fiber effects are moderate compared to males. In contrast, with low cholesterol intakes, the cholesterol lowering effects of fiber are similar in both genders.

Psyllium reduces plasma LDL in guinea pigs by altering hepatic cholesterol homeostasis.

Male Hartley guinea pigs were fed semipurified diets containing various levels of psyllium and cholesterol to determine mechanisms by which psyllium lowers plasma low density lipoprotein (LDL) concentrations. Four diets were tested: control diets with 12.5% (w/w) cellulose, and psyllium diets in which cellulose was partially replaced with 7.5% (w/w) psyllium. Two levels of dietary cholesterol were used, either low (LC, 0.04%, w/w) or high (HC, 0.25%, w/w). Plasma LDL was reduced by 30 and 54% with psyllium intake in the LC and HC groups, respectively (P < 0.001), while plasma very low density lipoprotein (VLDL) was lowered only in the HC group (P < 0.001). Psyllium intake modified LDL composition and size compared to LDL from control animals with a lower proportion of cholesteryl ester and higher proportion of triacylglycerol, lower molecular weight, smaller diameter, and higher peak density (P < 0.001). Plasma VLDL from animals fed the psyllium-HC diet compared to the control-HC contained lower relative proportions of free and esterified cholesterol and a higher proportion of triacylglycerol, compositional characteristics similar to VLDL from animals fed LC diets. Hepatic free and esterified cholesterol concentrations were significantly reduced by psyllium an average of 25 and 55%, respectively, while hepatic HMG-CoA reductase activity was increased in both psyllium groups compared to the respective controls (P < 0.001). In addition, psyllium intake reduced hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity in both the LC and HC groups (P < 0.001) and increased hepatic membrane apoB/E receptor number (Bmax) by 17 and 52% for animals fed LC and HC diets, respectively (P < 0.005). Significant psyllium-induced increases in cholesterol 7 alpha-hydroxylase of 4- and 1.6-fold were also observed in animals fed the LC and HC diets respectively (P < 0.001). These results indicate that psyllium generates a negative cholesterol balance across the liver which results in induction of cholesterol 7 alpha-hydroxylase and HMG-CoA reductase and suppression of ACAT activities, upregulation of apoB/E receptors, and secretion of smaller VLDL particles, metabolic alterations that contribute to a lowering of plasma LDL cholesterol levels.

Developmental expression of elements of hepatic cholesterol metabolism in the rat.

The expression of several key enzymes and receptors of rat hepatic cholesterol metabolism was studied during development. Among major findings were: acyl coenzyme A:cholesterol acyltransferase, the cholesteryl ester hydrolases, cholesterol-7 alpha-hydroxylase and the alpha 2-macroglobulin receptor (LRP) were very low in fetal livers, but all were induced shortly before birth, suggesting that these elements are important for extrauterine life. Although the other elements continued to increase, by day 6 of postnatal life, cholesterol-7 alpha-hydroxylase had reached undetectable levels. It reappeared by day 12 of suckling, placing it in the group of late-appearing activities necessary for the fully mature hepatic phenotype. Changes in acyl coenzyme A:cholesterol acyltransferase activity appeared due predominantly to changes in amount of active protein. The cholesteryl ester hydrolase (CEH) activities all showed different developmental patterns, suggesting that each was a unique activity. The bile salt-dependent CEH activity was much higher in the suckling period than in the adult where it was almost undetectable, suggesting that this CEH may have its major importance in the suckling period of development. Low density lipoprotein receptors exhibited a pattern very different from that of the alpha 2-macroglobulin receptors and did not show consistent correlation with any other elements. At some developmental time points, the relationships amongst the elements differed significantly from the adult pattern. These studies provide for the first time an integrated picture of developmental expression of key elements of hepatic cholesterol metabolism and set the stage for further studies on their modes of regulation.

Functional sizes of hepatic enzymes of cholesteryl ester metabolism determined by radiation inactivation.

Cellular cholesteryl ester metabolism is regulated largely by the balance between intracellular esterification catalyzed by acyl-CoA:cholesterol acyltransferase and cholesteryl ester hydrolysis catalyzed by the cholesteryl ester hydrolases. The hydrolases include both cytosolic and membrane-associated activities; acidic and neutral activities have been described in both compartments. Esterification via the acyltransferase is membrane-associated. Neither the acyltransferase nor the membrane-associated hydrolases have been purified and characterized, and little is known about their genes. Thus, nothing is known about their sizes or structures. Radiation inactivation was used to determine the functional sizes in situ of acyl-coenzyme A:cholesterol acyltransferase, fatty acyl-CoA hydrolase, and acidic and neutral membrane-associated cholesteryl ester hydrolase activities. The functional M(r) +/- SD of the acyltransferase was 213 +/- 35 kD; for the acidic membrane-associated hydrolase, 48 +/- 2 kD; for the neutral membrane-associated hydrolase, 94 +/- 15 kD; and for the fatty acyl-CoA hydrolase, 65 +/- 15 kD. Monoexponential curves were observed in all cases using radiation exposures that inactivated enzyme activities to < or = 10% of control values. Substrate specificity and inhibition studies suggested that the active sites of the acyltransferase and fatty acyl-CoA hydrolase were different, supporting the concept that the hydrolase is not part of the functional unit required for cholesterol esterification.