Effects of a DASH-like diet containing lean beef on vascular health.

A DASH (dietary approaches to stop hypertension) dietary pattern rich in fruits and vegetables and low-fat dairy products with increased dietary protein provided primarily from plant protein sources decreases blood pressure. No studies, however, have evaluated the effects of a DASH-like diet with increased dietary protein from lean beef on blood pressure and vascular health. The aim of this study was to study the effect of DASH-like diets that provided different amounts of protein from lean beef (DASH 28 g beef per day; beef in an optimal lean diet (BOLD) 113 g beef per day; beef in an optimal lean diet plus additional protein (BOLD+) 153 g beef per day) on blood pressure, endothelial function and vascular reactivity versus a healthy American diet (HAD). Using a randomized, crossover study design, 36 normotensive participants (systolic blood pressure (SBP), 116 ± 3.6 mm Hg) were fed four isocaloric diets,: HAD (33% total fat, 12% saturated fatty acids (SFA), 17% protein (PRO), 20 g beef per day), DASH (27% total fat, 6% SFA, 18% PRO, 28 g beef per day), BOLD (28% total fat, 6% SFA, 19% PRO, 113 g beef per day) and BOLD+ (28% total fat, 6% SFA, 27% PRO, 153 g beef per day), for 5 weeks. SBP decreased (P<0.05) in subjects on the BOLD+ diet (111.4 ± 1.9 mm Hg) versus HAD (115.7 ± 1.9). There were no significant effects of the DASH and BOLD diets on SBP. Augmentation index (AI) was significantly reduced in participants on the BOLD diet (-4.1%). There were no significant effects of the dietary treatments on diastolic blood pressure or endothelial function (as measured by peripheral arterial tonometry). A moderate protein DASH-like diet including lean beef decreased SBP in normotensive individuals. The inclusion of lean beef in a heart healthy diet also reduced peripheral vascular constriction.

Design, synthesis and structure-activity relationship studies of novel 4,4-bis(trifluoromethyl)imidazolines as acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors and antihypercholesterolemic agents.

Novel 4,4-bis(trifluoromethyl)imidazolines have been found to be the potent acyl-CoA cholesterol acyltransferase (ACAT) inhibitors. ACAT is responsible for cholesterol esterification in the intestine, liver, and the arterial wall. These novel imidazolines also inhibit cholesterol ester formation in the macrophage. Several compounds have shown potent serum cholesterol-lowering activity in several animal models. Para-substitution of the 2-phenyl is critical for in vitro and in vivo activity. The 4,4-bis(trifluoromethyl)imidazolines with a para-cyano group on 2-phenyl and a 4-alkylcyclohexyl amide as the side-chain at the 5-position possess the most potent inhibitory activity in this series. Based on biochemical studies, this series acts as a competitive inhibitor with respect to cholesterol binding at the enzyme, which distinguishes it from most of the ACAT inhibitors discovered to date. Preliminary biological studies supported by X-ray crystal structures, molecular modeling, and structure-activity relationship (SAR) studies suggest that this series may be a cholesterol mimic.

ACAT inhibitors derived from hetero-Diels-Alder cycloadducts of thioaldehydes.

Acyl-CoA:cholesterol acyltransferase (ACAT) is the enzyme largely responsible for intracellular cholesterol esterification. A systemic inhibitor of ACAT is believed to be able to slow or even reverse the atherosclerotic process. Towards that goal, a series of cyclic sulfides, derived from the hetero-Diels-Alder reaction of thioaldehydes with 1,3-dienes, and bearing carboxamide substituents, were prepared and evaluated for in vitro (in several tissues and species) and ex vivo ACAT inhibition. Minor changes in subsequent structure were found to have a significant effect in optimization of the biological activity of this series of compounds.

Gene expression of acyl-coenzyme-A:cholesterol-acyltransferase is upregulated in human monocytes during differentiation and foam cell formation.

The gene expression and enzyme kinetics of acyl coenzyme A:cholesterol acyltransferase (ACAT) were investigated in human monocytes, macrophages, and foam cells. Northern blot analysis using a 1.65-kb coding region of human ACAT cDNA as the probe showed that each of the cell types exhibited four mRNA transcripts. The levels of the 4.2- and 3.7-kb ACAT transcripts were three- and sixfold higher, respectively, in macrophages than monocytes. These transcripts were expressed at the same high levels after conversion of macrophages to foam cells. In contrast, the 6.3- and 4.4-kb transcripts for ACAT were expressed at a relatively constant level in all three cell types. The expression of mRNA for glyceraldehyde phosphate dehydrogenase, the control gene in this study, was also expressed at a constant level in each of the cell types. The increase in ACAT mRNA was accompanied by changes in the kinetic properties of the enzyme. Specifically, there was a 14-fold increase in Vmax and a 71% decrease in Km with respect to oleoyl coenzyme A. Although not definitive, the concomitant changes in mRNA and Vmax strongly suggest that the amount of ACAT protein increases upon conversion of monocytes to macrophages. The data show that ACAT in monocytes can be regulated by both substrate and gene expression.

Hepatic and adrenal changes in rabbits associated with hyperlipidemia caused by a semi-synthetic diet.

Several investigators have reported that feeding a semi-synthetic diet of casein and dextrose to New Zealand White (NZW) rabbits will increase total serum cholesterol concentration, principally through an increase in the beta-lipoprotein fractions, thereby creating a useful model for atherosclerosis research. Although there is evidence to suggest that the dextrose/casein diet alters low-density lipoprotein receptor and bile acid clearance of cholesterol, the underlying mechanism is not completely understood. The effects of the diet on the overall physiology of the rabbit have received little attention. In this study feeding a diet of casein and dextrose of male NZW rabbits for 4 weeks resulted in changes in the serum lipid concentrations. During that time the rabbits fed the dextrose/casein diet gained less weight than did control rabbits. In the test diet rabbits, liver aspartate and alanine transaminase activities were increased from baseline values of 27 +/- 2 U/L and 89 +/- 9 U/L respectively to 112 +/- 21 U/L and 281 +/- 34 U/L respectively, then returned to the high end of the reference range. Necropsy findings included hepatomegaly caused by vacuolar hepatopathy in 19 or 20 experimental rabbits; rabbits fed the control diet had no hepatic lesions. Ultrastructural analysis revealed that enlargement of the liver cells was due to glycogen deposition. Adrenal glands from animals fed the experimental diet had a minimal change in the size of the adrenocortical cells consisting of slight ballooning and rarefaction of the cytoplasm. In a second study the level of dietary fiber was doubled. This resulted in a three-fold increase in lipid concentrations, compared with the fivefold increase in the first study. The liver enzyme activities were increased to the same extent as in the first study. Histologic changes were comparable to those in the first study. The activity of hepatic cholesterol 7alpha-hydroxylase was 3.7 +/- 0.4 pmol/min/mg of protein, compared with the control value of 7.7 +/- 1.1 pmol/min/mg of protein (P < 0.05) in the second study. The improved rate of weight gain and the lesser increase in total serum cholesterol concentration in the second study with increased dietary fiber suggest that two separate activities may be involved. Although the level of dietary fiber may be related to weight gain and total serum cholesterol values, the relation to the decrease in liver transaminase activities in study 1 was probably coincidental. It appears that the dextrose/casein diet causes decreased activity of hepatic cholesterol 7alpha-hydroxylase, which could cause a decrease in the biliary excretion of cholesterol.

Design, synthesis, and structure--activity relationship studies for a new imidazole series of J774 macrophage specific acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors.

Acyl-CoA:cholesterol acyltransferase (ACAT) is the primary enzyme involved in intracellular cholesterol esterification. Arterial wall infiltration by macrophages and subsequent uncontrolled esterification of cholesterol leading to foam cell formation is believed to be an important process which leads to the development of fatty streaks. Inhibitors of the ACAT enzyme may retard this atherogenic process. We have recently discovered a series of imidazoles which are potent in vitro ACAT inhibitors in the J774 macrophage cell culture assay. This paper will describe the design, synthesis, and structure--activity relationship for this very potent series of compounds.

Acyl CoA:cholesterol acyltransferase (ACAT) inhibitors: synthesis and structure-activity relationship studies of a new series of trisubstituted imidazoles.

A series of 4,5-diaryl-2-(substituted thio)-1H-imidazoles has been synthesized and demonstrated to be potent inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). The design, synthesis, and structure-activity relationships for this series are reported herein. One of the compounds from this series, N'-(2,4-difluorophenyl)-N-[5-[(4,5-diaryl-1H-imidazol-2- yl)thio]pentyl]-N-heptylurea (DuP 128), was selected for development as an intestinally active ACAT inhibitor. DuP 128 is a potent ACAT inhibitor in vitro and in vivo, inhibiting ACAT in rat hepatic microsomes with an IC50 = 10 nM and possessing potent antihypercholesterolemic activity in vivo.

Inhibition of hepatic ACAT decreases ApoB secretion in miniature pigs fed a cholesterol-free diet.

To test the hypothesis that hepatic cholesteryl ester is involved in the regulation of apolipoprotein (apo) B secretion into plasma, apoB kinetic studies were performed in six control miniature pigs and in six pigs after a 21-day administration of the acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor DuP 128 (2.2 mg.kg-1.d-1 i.v.). Pigs were fed low-fat, cholesterol-free diets. Total plasma cholesterol, triglyceride, very-low-density lipoprotein (VLDL) triglyceride, and low-density lipoprotein (LDL) cholesterol decreased 18%, 29%, 40%, and 26% respectively (P < .03). 131I-VLDL and 125I-LDL were injected simultaneously into each animal, and apoB kinetics were analyzed by using multi-compartmental analysis (SAAM30). VLDL apoB pool size decreased significantly by 60% (0.32 versus 0.84 mg/kg), which was due to a 65% reduction in the VLDL apoB production or secretion rate (1.03 versus 2.94 mg.kg-1.h-1). The fractional catabolic rate was unchanged. LDL apoB pool size decreased nonsignificantly by 18% (5.61 versus 6.90 mg/kg) due entirely to a 24% decrease in production rate (0.26 versus 0.34 mg.kg-1.h-1). At necropsy, hepatic microsomal ACAT activity decreased by 68% (0.28 versus 0.88 nmol.min-1.mg-1; P < .0002). Although an increase in hepatic free cholesterol leading to a decreased LDL receptor expression might be expected, this did not occur. The concentration of hepatic cholesterol and the LDL apoB fractional catabolic rate were unaffected by DuP 128. In addition, the concentration of hepatic triglyceride and the activity of diacylglycerol acyltransferase were not altered by DuP 128, indicating a lack of effect of DuP 128 on hepatic triglyceride metabolism. We conclude that inhibition of hepatic cholesteryl ester synthesis in vivo decreases apoB secretion into plasma.

Effect of the acyl-CoA:cholesterol acyltransferase inhibitor DuP 128 on cholesterol absorption and serum cholesterol in humans.

Intestinal cholesterol esterification by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is a presumed prerequisite for cholesterol absorption. We evaluated the effect of a potent, poorly absorbed ACAT inhibitor (DuP 128: N'-(2,4-difluorophenyl)-N-[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pe ntyl]- N-heptylurea) on cholesterol absorption in a randomized trial. Thirty subjects received DuP 128 for 7 weeks, 10 each at 900 mg per day, 1800 mg per day, and 3600 mg per day; six subjects received placebo; and nine subjects received 1 gm neomycin twice a day. Cholesterol absorption determinations used a continuous dual isotope 14C-cholesterol and 3H-beta sitosterol method. DuP 128 (pooled doses) induced at 14.4% +/- 11.4% reduction in cholesterol absorption (p < 0.05 versus placebo): 17.6% +/- 8.4% at 900 mg, 9.1% +/- 11.4% at 1800 mg, and 17.1% +/- 12.9% at 3600 mg. Neomycin induced a 26.4% +/- 10.7% reduction (p < 0.01). After 6 weeks, neomycin reduced serum total and low-density lipoprotein cholesterol by 22.4% +/- 9.2% and 24.0% +/- 11.6%, respectively (p < 0.01 versus placebo). DuP 128 induced reductions of 3.9% +/- 11% (difference not significant) and 4.95% +/- 14.3% (p = 0.05). ACAT inhibitors limit cholesterol absorption in humans; however, the magnitude of the effect, as exemplified by DuP 128, is small.

Whole blood and plasma concentrations of 4-hydroxy-2-nonenal in Watanabe heritable hyperlipidemic versus New Zealand White rabbits.

Both plasma and whole blood concentrations of 4-hydroxy-2-nonenal (4HNE) were significantly elevated in a population (n = 6) of 2 year old Watanabe heritable hyperlipidemic rabbits relative to a population (n = 6) of New Zealand White rabbits. The plasma concentrations were 74 +/- 10 nmol/L for the Watanabe group and 47 +/- 6 nmol/L for the New Zealand White group. The whole blood concentrations were 364 +/- 55 nmol/L for the Watanabe group and 188 +/- 64 nmol/L for the New Zealand White group. These results indicate that 4HNE concentrations in blood can be elevated in individuals with atherosclerosis and demonstrate the potential link between the formation of 4HNE and the progression of atherosclerosis.

Regulation of ACAT activity by a cholesterol substrate pool during the progression and regression phases of atherosclerosis: implications for drug discovery.

The regulation of aortic ACAT by a cholesterol substrate pool (CSP) was investigated in a rabbit progression/regression model of dietary-induced atherosclerosis. ACAT activity increased 25-fold during the 10-week progression phase of the study. ACAT activity decreased 8-fold during the 24-week regression phase of the study, however, it was still 14-fold greater than in normal aortas. ACAT activity assayed in the absence vs. the presence of exogenous cholesterol was used as a qualitative measure of the amount of cholesterol in the CSP. The CSP was filled to 28% of capacity in normal aortas, this increased to 75% during the progression phase. By the end of the regression phase, the CSP was filled to 100% of capacity even though serum cholesterol levels had returned to normal. The data are discussed in terms of emerging concepts of intracellular cholesterol trafficking, ACAT inhibitors, and the types of atherosclerotic lesions which may be subject to amelioration by ACAT inhibitors.

Effect of lanthanum chloride on established atherosclerosis in the cholesterol-fed rabbit. Mitral valve as a site for assessment of treatment effects.

The ability of lanthanum chloride (LaCl3) to retard the progression of established atherosclerosis was investigated in cholesterol-fed rabbits. Rabbits were initially maintained on a high-fat plus cholesterol-supplemented diet for 10 weeks to induce lesions and were then changed to a low-fat diet or a low-fat diet supplemented with LaCl3 for an additional 24 weeks to permit their serum cholesterol levels to normalize. LaCl3 did not affect the rate at which serum cholesterol levels returned to normal. The dose of LaCl3 was approximately 30 mg/kg body weight/day. In comparison with controls, LaCl3-treated rabbits exhibited histologically less severe coronary artery and mitral valve atherosclerosis. Lesion severity in the carotid arteries was unaffected by LaCl3 treatment. Although statistically significant, the salutary effects of LaCl3 were relatively small. The data support the hypothesis that calcium antagonists can retard the progression of established atherosclerotic lesions. The data also illustrate the value of the mitral valve as a site to assess treatment effects on monocyte/macrophages in vivo.

Alterations in lipid metabolism and cellular morphology in the liver of female rats treated with ethmozine.

Alterations in lipid metabolism and cellular morphology in the liver were examined in female rats treated with 100 mg Ethmozine/kg/day for 7 days. The incorporation of either [3H]acetate with [methyl-14C]choline, or [methyl-14C]methionine was used to monitor the effect of the drug on neutral and phospholipid syntheses. Ethmozine (ETH) reduced the incorporation of choline into phosphatidylcholine (PC) by 50%, but the transmethylation of phosphatidylethanolamine to form PC was unaffected. The formation of lyso-PC was reduced by one-half irrespective of the donor radiolabel. An accumulation of both micro- and macrovesicles (fat) was found in the centri- and midlobular zones of the liver, which is likely the result of increased synthesis and decreased secretion of triacylglycerol (TAG). Incorporation of acetate into TAG was increased fivefold by ETH treatment, and to a lesser degree into cholesterol and cholesterylester/squalene.

Biochemical and morphological studies of ammonium perfluorooctanoate-induced hepatomegaly and peroxisome proliferation.

Ammonium perfluorooctanoate (APFO) is known to induce a striking hepatomegaly in rats. The purpose of these studies was to determine the causes of the hepatomegaly and compare the effect to other liver-enlarging compounds. Since the total hepatic DNA content was similar in control and APFO-treated rats, the hepatomegaly represented a hypertrophic rather than a hyperplastic response. The cytochrome P-450 content and activity of benzphetamine N-demethylase increased in the livers of APFO-treated rats, indicating the proliferation of the smooth endoplasmic reticulum. In contrast to the membrane-bound enzymes, the soluble enzymes glutathione S-transferase and UDPglucuronyltransferase were unaffected by APFO treatment. The activity of carnitine acetyltransferase was disproportionately increased relative to carnitine palmitoyltransferase in the livers of APFO vs that in control rats, confirming the predominant proliferation of peroxisomes vs that of mitochondria. Morphological studies confirmed the proliferation of the endoplasmic reticulum, mitochondria, and peroxisomes in the livers of APFO-treated rats. In contrast to many other peroxisome proliferating agents, APFO did not possess hypolipidemic activity.

Regulation of acyl-CoA:cholesterol acyltransferase activity in normal and atherosclerotic rabbit aortas: role of a cholesterol substrate pool.

A new substrate optimized assay for acyl-CoA:cholesterol acyltransferase (ACAT) was developed that permits the accurate measurement of ACAT activity in normal arterial microsomes. The apparent Km and Vmax of ACAT with respect to oleoyl-CoA were determined to be 3 microM and 17.7 pmole min-1 mg-1. While the Km value is similar to other values reported in the literature, the Vmax is 5- to 8-fold higher. The higher Vmax is attributable to the saturation of ACAT with not only oleoyl-CoA, but also cholesterol. The observation that exogenous cholesterol was necessary for the determination of maximal ACAT activity indicates that under normal conditions the endogenous level of microsomal cholesterol does not saturate ACAT. Assay of ACAT in the presence and absence of exogenous cholesterol permits a qualitative assessment of the amount of cholesterol in the cholesterol substrate pool of ACAT. Using this approach, it was found that hypercholesterolemia results in the expansion of the cholesterol substrate pool of ACAT. Of the 21-fold increase in ACAT activity in atherosclerotic aortas observed in this study. 80% of the increase was attributable to expansion of the cholesterol substrate pool, while 20% was attributable to more enzyme. Notably, the increase in the amount of ACAT was observed after only 2 weeks of hypercholesterolemia.

Pulmonary response and intrapulmonary lipids in rats exposed to bismuth orthovanadate dust by inhalation.

Rats were exposed to 0, 1.2, or 0.11 mg/liter of uncoated bismuth orthovanadate (BOV), 1.3 or 0.15 mg/liter of silica-coated BOV, or 1.9 mg/liter of silica-coated TiO2 for 2 weeks. Rats were killed 0, 1, 3, 6, and 12 months postexposure (PE). After the rats were exposed for 2 weeks their pulmonary response to silica-coated TiO2 was characterized by dust-laden macrophage (dust cell) response with hyperplasia of type II pneumocytes. The lung reaction to uncoated BOV or silica-coated BOV was both similar to that of silica-coated TiO2 and dose-related. After the rats were exposed for 3 months, foamy macrophage infiltration in silica-coated TiO2 was evident. Alveolar proteinosis, foamy macrophages with cholesterol clefts, and hyperplastic type II pneumocytes were observed in rats exposed to uncoated BOV or silica-coated BOV. By 6 months PE, the lung exposed to silica-coated TiO2 was restored to essentially normal architecture with removal of most dust cells. In the silica-coated BOV or uncoated BOV exposure groups, alveolar proteinosis and cholesterol granulomas became obvious with degenerative foamy macrophages. After 1 year PE, the lungs exposed to silica-coated TiO2 were almost normal with only a few dust cell aggregates remaining. The pulmonary lesions of silica-coated or uncoated BOV were reduced but alveolar proteinosis and cholesterol granulomas still persisted. Electron microscopy revealed massive accumulation of intraalveolar phospholipid material with hyperplastic type II pneumocytes showing overloaded myelin figures after 2 weeks exposure. The increase in phospholipid at 1 month PE and sterol content at 3 months PE in the lungs correlated with the accumulation of intraalveolar myelin figures and lamellar structure, foamy macrophage infiltration, and occurrence of cholesterol clefts.

Effects of hexafluoroacetone on Leydig cell steroidogenesis and spermatogenesis in the rat.

Steroidogenesis was investigated in Leydig cell-enriched fractions isolated from the testes of rats dosed dermally with 130 mg/kg/day hexafluoroacetone (HFA) for 14 days and from pair-fed control rats. Compared to controls, Leydig cells from HFA-treated rats exhibited decreased incorporation of [14C]acetate (78%) and [3H]mevalonate (41%) into sterols and steroids. Testosterone was decreased 50% in the testes of HFA-treated rats. Incubation of Leydig cells from untreated rats with 1.0 mM HFA did not affect steroidogenesis. HFA treatment led to the development of histopathological lesions in the testes within 24 hr after a single dose; with daily dosing the lesions became progressively more severe. Despite the development of severe lesions, HFA treatment did not affect the blood levels of luteinizing hormone or testosterone; however, follicle-stimulating hormone was slightly elevated (48%) after 14 days of treatment. The data indicate that steroidogenesis is inhibited in Leydig cells of HFA-treated rats; the inhibition is not due to a direct or immediate effect of HFA, nor does it appear to be hormonally mediated.

Toxicokinetics of [14C]hexafluoroacetone in the rat.

The disposition and metabolism of [14C]hexafluoroacetone (HFA) were studied in the rat as part of an investigation of the mechanism of HFA-induced testicular atrophy. After sc injection of 13 mg/kg [14C]HFA, radioactivity was eliminated in a biphasic manner from the blood; the half-life of the initial phase was 22.6 hr and that of the elimination phase 75.1 hr. Following injection of 130 mg/kg [14C]HFA, the elimination of radioactivity was initially zero order, but with time it became first order and biphasic with half-lives of the initial and terminal elimination phases being 23.0 and 59.9 hr, respectively. The primary route of elimination was via the urine and all of the [14C]HFA was excreted unmetabolized. [14C]HFA was uniformly distributed throughout the major organs of the body with the exception of the liver which contained disproportionately higher levels of [14C]HFA. The hepatic binding of [14C]HFA was noncovalent and capacity limited. Notably, the testes, the target organ of HFA-induced toxicity, did not exhibit any unusual accumulation or retention of [14C]HFA.

Ultrastructural alterations in hexafluoroacetone-induced testicular atrophy in the rat.

Testicular atrophy was induced in rats by dermal application of hexafluoroacetone (HFA) at 39 or 130 mg/kg/day for 14 days, but not at a dosage of 13 mg/kg/day. Affected germ cells were mostly spermatids and to a much lesser extent spermatocytes; spermatogonia were unaffected. Late spermatids were retained in Sertoli cells and showed degenerative changes. Sertoli cells exhibited cytoplasmic vacuolation, distended endoplasmic reticulum, and a marked increase in lipid droplets. Leydig cells exhibited a slight increase in lipid droplets, fewer mitochondria, and diminution and segregation of the agranular endoplasmic reticulum from mitochondria. A correlation between ultrastructural and biochemical changes in HFA-induced testicular atrophy is presented.

Effects of hexafluoroacetone on testicular morphology and lipid metabolism in the rat.

Rats dosed dermally with 39 or 130 mg/kg/day hexafluoroacetone sesquihydrate (HFA) for 14 days developed moderate or severe testicular atrophy, respectively; rats dosed with 13 mg/kg/day HFA for 14 days did not. Histologic evaluation of the testes revealed that spermatids, followed by spermatocytes, were the germ cells most affected by HFA; spermatogonia and Sertoli cells appeared to be less vulnerable. Lipogenesis from [3H]acetate and[14C]glucose was investigated in vitro in testes from HFA-treated and pair-fed control rats. Triacylglycerol and phospholipid synthesis was increased whereas sterol synthesis was decreased in testes from HFA-treated rats. Vitamin A and zinc were measured in the testes of control and HFA-treated rats; no differences in the levels of these nutrients were observed between the two groups. The data support the hypothesis that altered lipid metabolism, in particular sterol metabolism, is associated with the development of HFA-induced testicular atrophy.