Lipid-lowering efficacy of 3,4-di(OH)-phenylpropionic L-leucine in high-cholesterol fed rats

A preliminary study revealed that 3,4-di(OH)-hydrocinnamate (HC), a polyphenolic compound, lowered the plasma lipids in high-cholesterol fed rats. Accordingly, this study was designed to test the lipid-lowering efficacy of a synthetic derivative, 3,4-di(OH)-phenylpropionic (L-leucine) amide (PPLA), in rats fed a high-cholesterol (1%, wt/wt) diet. As such, HC or PPLA was given as supplement to a high-cholesterol diet for 6 weeks at a dose of 0.137 mmol/100 g diet. The supplementation of HC and PPLA significantly lowered the plasma and hepatic cholesterol and triglyceride levels compared to the control group. The activities of hepatic HMG-CoA reductase (164 +/- 9.12 and 124.74 +/- 17.09 pmol/min/mg protein vs. 245.41 +/- 13.01 pmol/min/mg protein, p < 0.05) and ACAT (411.49 +/- 11.48 and 334.35 +/- 17.68 pmol/min/mg protein vs. 490.41 +/- 16.69 pmol/min/mg protein, p < 0.05) were significantly lower in the HC- and PPLA-supplemented groups than in the control group. However, PPLA was more effective in inhibiting the enzyme activities than HC. The excretion of neutral sterol was significantly higher in HC- and PPLA-supplemented groups than in the control group. Therefore, these results indicate that PPLA, a leucine-attached version of HC, exhibited a similar significant hypocholesterolemic effect to HC in rats fed a high-cholesterol diet.     

Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm-acclimated carp. Based on current concepts of cholesterol metabolism, it is concluded that the cold-enhanced expression of hepatic HMG-CoA reductase activity is a homeostatic response directed against and compensating for a cold-induced but not yet characterized deficiency in hepatic cholesterol availability.     

Evaluation of hesperetin 7-O-lauryl ether as lipid-lowering agent in high-cholesterol-fed rats

The lipid-lowering efficacy of hesperetin was revealed in preliminary studies on experimental animals. As such, the current study compared the effect of hesperetin 7-O-lauryl ether, with that of hesperetin and lovastatin on the lipid profile and cholesterol-regulating mechanism in high-cholesterol-fed rats. Male rats were fed a high-cholesterol diet (1%, wt/wt) or high-cholesterol diet supplemented with lovastatin (1, 0.02%, wt/wt), hesperetin (2, 0.02%, wt/wt), or hesperetin 7-O-lauryl ether (3, 0.031%, wt/wt) for six weeks. The supplemental amount of 3 was 0.066mmol/100g diet as an equivalent to the supplemental amount of 2. The plasma total cholesterol and triglyceride levels were significantly lowered by the 2 and 3 supplements compared with the control or 1-supplemented group. The hepatic HMG-CoA reductase activities were also significantly lower in all the supplemented groups compared with the control group, and the hepatic ACAT activity was significantly lower in the 2- and 3-supplemented groups. The supplementation of 3 resulted in a higher excretion of total neutral sterol and total fecal sterol compared with the control or 1-supplemented group. Accordingly, overall, compound 3, exhibited a more potent plasma lipid-lowering effect than compound 1 based on inhibiting cholesterol biosynthesis and esterification, while also increasing the fecal sterol excretion.     

Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

Naringin alters the cholesterol biosynthesis and antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition

The purpose of the current study was to evaluate the lipid lowering and antioxidant capacity of naringin in LDL receptor knockout (LDLR-KO) mice fed a cholesterol (0.1 g/100 g) diet. As such, naringin or lovastatin (0.02 g/100 g) was supplemented in a cholesterol diet for 6 weeks. The naringin and lovastatin supplementation significantly lowered the plasma total cholesterol level compared to the control group. The plasma and hepatic triglyceride level was only lowered by the lovastatin supplement, while the hepatic cholesterol content was lowered by both the naringin and lovastatin supplements compared to the control group. The hepatic HMG-CoA reductase activity was significantly lower in the naringin and lovastatin supplemented groups than in the control group, whereas the ACAT activity was unaffected. The excretion of total sterol was significantly higher in the naringin and lovastatin groups compared to the control group due to significant changes in the acidic and neutral sterol, respectively. When comparing the hepatic antioxidant enzyme activities, the superoxide dismutase, catalase, and glutathione reductase activities were all significantly higher in the naringin-supplemented group than in the control group, while only the lovastatin supplement increased the glutathione reductase activity. Accordingly, the current results confirmed that naringin lowers the plasma cholesterol level via the inhibition of hepatic HMG-CoA reductase activity and increases the excretion of fecal sterol. Naringin was also found to improve the activities of hepatic antioxidant enzymes against oxidative stress in a hypercholesterolemic animal model, i.e. cholesterol-fed LDLR-KO mice.     

Hypercholesterolaemia: simvastatin and pravastatin alter cholesterol metabolism by different mechanisms

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin, reduced low-density-lipoprotein (LDL) cholesterol in hypercholesterolaemic patients by 40% (P less than 0.001). The reduction in LDL cholesterol was accompanied by a significant decrease in the esterified/free cholesterol ratio of the patients' LDL from 2.51 +/- 0.13 to 2.06 +/- 0.14 (P less than 0.01). This change led to a significant increase (P less than 0.05) in the capacity of the LDL to suppress [14C]acetate incorporation into cholesterol in mononuclear leucocytes. Furthermore, [14C]acetate incorporation into the patients mononuclear leucocytes was significantly lower (P less than 0.02) following drug treatment (117 +/- 22 vs. 162 +/- 29 nmol/mg cell protein). Comparison of simvastatin with another HMG-CoA reductase inhibitor pravastatin, showed similar reduction in LDL cholesterol. Pravastatin treatment however, did not result in a reduction in the LDL esterified/free cholesterol ratio or in the changes in cellular cholesterol synthesis and its regulation by LDL which accompanied simvastatin treatment. The activity of the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT) in patients' mononuclear cells remained unchanged after treatment with either drug. Results of the study show that while the drugs are equally effective in lowering LDL cholesterol, simvastatin has additional compositional effects on LDL which increase its capacity to regulate mononuclear leucocyte cholesterologenesis.     

Homeostatic restoration of microsomal lipids and enzyme changes in HMG-CoA reductase and Acyl-CoA : cholesterol acyltransferase in chick liver

We have studied the correlation between changes in the lipid composition in chick liver microsomes and the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acyl-CoA : cholesterol acyltransferase (ACAT) by in vivo and in vitro experiments with 21-day-old chicks. A 5% cholesterol diet for 3 hr produced an increase in the microsomal and plasmatic cholesterol content, a decrease in HMG-CoA reductase activity and a concomitant increase in ACAT activity. The effect produced by the short-term treatment virtually disappeared 27 hr after ending the cholesterol diet. In vitro experiments were carried out by using vesicles constituted by phosphatidycholine/cholesterol and phosphatidylcholine.     

Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits

The anti-atherogenic effects of the citrus flavonoids, naringin and naringenin, were evaluated in high cholesterol-fed rabbits. At 3 months of age, 30 male New Zealand White (NZW) rabbits were divided into three groups (n = 10 per group). The rabbits were fed a 1% cholesterol diet alone (control group) or a diet supplemented with either 0.1% naringin or 0.05% naringenin for 8 weeks. The plasma lipoprotein levels, total cholesterol, triglyceride, and high-density lipoprotein showed no significant differences in the control and experimental groups. Hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity was slightly low in naringin (5.0%)- and naringenin (15.0%)-fed rabbits, compared to control group. The aortic fatty streak areas were significantly lower in both the naringin (19.2 +/- 5.6%)- and naringenin (18.1 +/- 6.5%)-supplemented groups than in the control group (60.4 +/- 14.0%). The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), by semiquantitative RT-PCR analysis of the thoracic aorta, were significantly lower in the flavonoids supplemented groups than in the control group. These results suggest that the anti-atherogenic effect of the citrus flavonoids, naringin and naringenin, is involved with a decreased hepatic ACAT activity and with the downregulation of VCAM-1 and MCP-1 gene expression.     

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Short-term very low calorie diet reduces oxidative stress in obese type 2 diabetic patients

Oxidative stress is higher in obese diabetic than in non-diabetic subjects. This pilot study evaluates oxidative stress during short-term administration of a very low calorie diet in obese persons. Nine obese Type 2 diabetic patients (age 55+/-5 years, BMI 35.9+/-1.9 kg/m2) and nine obese non-diabetic control subjects (age 52+/-6 years, BMI 37.3+/-2.1 kg/m2) were treated by a very low calorie diet (600 kcal daily) during 8 days stay in the hospital. Serum cholesterol, triglycerides, non-esterified fatty acids (NEFA), beta-hydroxybutyrate (B-HB), ascorbic acid (AA), alpha-tocopherol (AT), plasma malondialdehyde (MDA) and superoxide dismutase (SOD) activity in erythrocytes were measured before and on day 3 and 8 of very low calorie diet administration. A decrease of serum cholesterol and triglyceride concentrations on day 8 was associated with a significant increase of NEFA (0.30+/-0.13 vs. 0.47+/-0.11 micromol/l, p<0.001) and B-HB (0.36+/-.13 vs. 2.23+/-1.00 mmol/l, p<0.001) in controls but only of B-HB (1.11+/-0.72 vs. 3.02+/-1.95 mmol/l, p<0.001) in diabetic patients. A significant decrease of plasma MDA and serum AT together with an increase of SOD activity and AA concentration (p<0.01) was observed in control persons, whereas an increase of SOD activity (p<0.01) was only found in diabetic patients after one week of the very low calorie diet. There was a significant correlation between NEFA or B-HB and SOD activity (p<0.01). We conclude that one week of a very low calorie diet administration decreases oxidative stress in obese non-diabetic but only partly in diabetic persons. Diabetes mellitus causes a greater resistance to the effects of a low calorie diet on oxidative stress.     

Regulation of cholesterol biosynthesis and esterification by 25-hydroxycholesterol in a macrophage-like cell line: uncoupling by progesterone

The coordinated control of cholesterol biosynthesis and esterification by 25-hydroxycholesterol was studied in the macrophage-like cell line P388D1. Since 25-hydroxycholesterol rapidly stimulated incorporation of [3H]oleate into the cholesteryl ester fraction of these cells, we have tested the possibility that the well-known inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) by 25-hydroxycholesterol might be the indirect consequence of an increased cholesterol esterification rather than a direct effect on HMG-CoA reductase. The experimental results show that progesterone, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), when added together with 25-hydroxycholesterol, abolished the increased cholesterol esterification without affecting the inhibition of HMG-CoA reductase by 25-hydroxycholesterol. Thus, uncoupling cholesterol esterification had no effect on 25-hydroxycholesterol's ability to inhibit HMG-CoA reductase. Unexpectedly, pretreatment of P388D1 cells with 25-hydroxycholesterol resulted in no elevation of ACAT activity as measured in broken cell preparations. Therefore, the possibility that 25-hydroxycholesterol stimulated cholesteryl ester formation by increasing the amount of cholesterol available for esterification, rather than by acting directly on ACAT activity, was considered. Labeling experiments using [14C]-cholesterol have provided evidence for this assumption.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Supplementation of naringenin and its synthetic derivative alters antioxidant enzyme activities of erythrocyte and liver in high cholesterol-fed rats

The antioxidative effects of naringenin (1) and its synthetic derivative, naringenin 7-O-cetyl ether (2), were tested. Male rats were fed a 1 g/100 g high-cholesterol diet for 6 weeks with supplements of either 1 or 2 (0.073 mmol/100 g diet) to study the effects on the antioxidant enzyme activities in the erythrocyte and liver. The erythrocyte catalase (CAT) and superoxide dismutase (SOD) activities were significantly higher in the compounds 1 or 2 supplemented groups than in the control group, whereas the hepatic SOD and CAT activities were significantly lower in the compound 2 supplemented group. The compounds 1 and 2 supplements to a high cholesterol diet lowered or tended to lower the plasma TBARS levels, that is, lipid peroxide products, while enhancing the plasma paraoxonase activity. These results indicate that the supplementation of 1 and 2 was effective in improving the antioxidant capacity of the erythrocyte and liver, plus the synthetic functional compound 2 appeared to be as potent as 1 in enhancing the antioxidant defense system.     

The A-204C polymorphism in the cholesterol 7alpha-hydroxylase (CYP7A1) gene determines the cholesterolemia responsiveness to a high-fat diet

The aim of the study was to ascertain whether the A-204C polymorphism in the cholesterol 7 -hydroxylase (CYP7A1) gene plays any role in determining LDL-cholesterol (LDL-C) concentration responsiveness to a high-fat diet. The concentrations of total cholesterol and LDL-cholesterol were measured in eleven healthy men (age: 30.9+/-3.2 years; BMI: 24.9+/-2.7 kg/m(2);;) who were homozygous for either the -204A or -204C allele, after 3 weeks on a low-fat (LF) diet and 3 weeks on a high-fat (HF) diet. During both dietary regimens, the isocaloric amount of food was provided to volunteers; LF diet contained 22 % of energy as a fat and 2.2 mg of cholesterol/kg of body weight a day, HF diet 40 % of fat and 9.7 mg of cholesterol/kg of body weight a day. In six subjects homozygous for the -204C allele, the concentrations of cholesterol and LDL-cholesterol were significantly higher on HF than on LF diet (cholesterol: 4.62 vs. 4.00 mmol/l, p<0.05; LDL-C: 2.15 vs. 1.63 mmol/l, p<0.01, respectively); no significant change was observed in five subjects homozygous for the -204A allele. There were no other differences in lipid and lipoprotein-lipid concentrations. Therefore, the polymorphism in the cholesterol 7alpha-hydroxylase promotor region seems to be involved in the determination of cholesterol and LDL-C responsiveness to a dietary fat challenge.     

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes.

There is no consensus whether hepatic lipid regulatory enzymes play primary or secondary roles in cholesterol cholelithiasis. We have used inbred mice with Lith genes that determine cholesterol gallstone susceptibility to evaluate the question. We studied activities of regulatory enzymes in cholesterol biosynthesis (HMG-CoA reductase), cholesterol esterification (acyl-CoA:cholesterol acyltransferase) and the "neutral" (cholesterol 7alpha-hydroxylase) and "acidic" (sterol 27-hydroxylase) pathways of bile salt synthesis in strains C57L/J and SWR/J as well as recombinant inbred (AKXL-29) mice, all of which have susceptible Lith alleles, and compared them to AKR/J mice with resistant Lith alleles. We determined hepatic enzyme activities of male mice before and at frequent intervals during feeding a lithogenic diet (15% dairy fat, 1% cholesterol, 0.5% cholic acid) for 12 weeks. Basal activities on chow show significant genetic variations for HMG-CoA reductase, sterol 27-hydroxylase, and acyl-CoA: cholesterol acyltranferase, but not for cholesterol 7alpha-hydroxylase. In response to the lithogenic diet, activities of the regulatory enzymes in the two bile salt synthetic pathways are coordinately down-regulated and correlate inversely with prevalence rates of cholesterol crystals and gallstones. Compared with gallstone-resistant mice, significantly higher HMG-CoA reductase activities together with lower activities of both bile salt synthetic enzymes are hallmarks of the enzymatic phenotype in mice with susceptible Lith alleles. The most parsimonious explanation for the multiple enzymatic alterations is that the primary Lith phenotype induces secondary events to increase availability of cholesterol to supply the sterol to the hepatocyte canalicular membrane for hypersecretion into bile.     

Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 micro M) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation. We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.