Intestinal expression of human apolipoprotein A-IV in transgenic mice fails to influence dietary lipid absorption or feeding behavior.

Two transgenic mouse lines, expressing low or high amounts of human apo A-IV were created. In low and high expressor HuAIVTg mice on a chow diet, serum human apo A-IV levels were 6 and 25 times the normal human level and on a high fat diet, they were 12 and 77 times higher. Human apo A-IV was equally distributed between lipoprotein (mainly HDL) and lipid-free fractions. Intestinal absorption of radiolabeled cholesterol and triglycerides was unaffected in HuAIVTg mice. Vitamin A, carried exclusively in chylomicrons and their remnants, was catabolized normally. When an intragastric vitamin E bolus is given to the HuAIVTg mice, the initial absorption and appearance in triglyceride-rich lipoproteins was similar to that observed in normal mice. However, elevated amounts of vitamin E were subsequently observed in the VLDL of the HuAIVTg mice. Furthermore, in the fed state, serum VLDL triglycerides were markedly elevated in HuAIVTg mice. This effect was greater in high expressor mice. Serum total cholesterol was not elevated, but the distribution was altered in the HuAIVTg mice; VLDL-C was increased at the expense of VLDL-C. Kinetic studies suggested a delayed clearance of VLDL in HuAIVTg mice. Apo A-IV has been suggested to be a satiety factor, but no effect on feeding behavior or weight gain was observed in these HuAIVTg mice. In summary, our studies with HuAIVTg mice show that additional apo A-IV does not effect intestinal absorption of fat and fat-soluble vitamins, and at least chronic elevation of plasma apo A-IV does not effect feeding behavior in this model system.

Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences.

Opposite effects of bezafibrate and gemfibrozil in both normal and hypertriglyceridemic rats.

Chow and sucrose-fed rats were used as animal models to study the dose-responses of bezafibrate and gemfibrozil in normolipidemic and hypertriglyceridemic states, respectively. Although both drugs lowered plasma triglycerides (TG) to about the same extent in chow-fed rats, gemfibrozil lowered liver TG as well as plasma total and LDL-cholesterol (LDL-C), but elevated HDL-cholesterol (HDL-C) and plasma apo E concentrations. Bezafibrate produced opposite effects, namely, decreased HDL-C, apo E and liver TG, and tended to increase LDL-C. TG lowering for both drugs in chow-fed rats was not due to changes in TG secretion (production) in normal rats but was associated with enhanced LPL activity. In hypertriglyceridemic rats both drugs modestly reduced TG secretion rates about 40% at a dose producing maximal TG lowering, but again, gemfibrozil elevated and bezafibrate lowered HDL-C and apo E. Unlike gemfibrozil, bezafibrate induced the appearance of LDL-C in hypertriglyceridemic rats which was not detected in control animals, and also tended to increase rather than decrease plasma apo B levels. Finally, changes in liver TG concentration (mg/g) in hypertriglyceridemic rats were opposite for these drugs, resulting in significant drug-related differences in liver TG content (mg/organ). From these data we postulate that, although similar with regard to TG lowering activity and mechanisms thereof, gemfibrozil and bezafibrate produce fundamentally different effects on LDL, HDL and apolipoprotein metabolism (apo B and apo E) in rats which may relate to potential differential effects on reverse cholesterol transport and atherogenesis.

Synthesis and biological activity of modified peptide inhibitors of angiotensin-converting enzyme.

A series of non-sulfhydryl modified dipeptides related to CI-906, CI-907, and enalapril was prepared in which various isosteric moieties (O, S, SO, SO2) have been substituted for the amino group and in which the proline residue has been replaced with various hydrophobic amino acids. The compounds were evaluated in vitro for inhibition of angiotensin-converting enzyme and in vivo for antihypertensive activity. Compound 7c, the most potent member of this series, had an in vitro IC50 of 1.4 X 10(-8) M and showed modest oral antihypertensive activity at 30 mg/kg in conscious, two kidney, one clip Goldblatt hypertensive rats. Structure-activity relationships are discussed.

Angiotensin converting enzyme inhibitors: modifications of a tripeptide analogue.

Modified nonhydrolyzable tripeptide analogues of (S)-1-[5-(benzoylamino)-1,4-dioxo-6-phenylhexyl]-L-proline (1), designed to impart oral angiotensin converting enzyme (ACE) inhibitory activity, were made and evaluated in vivo and in vitro. The N-methyl and C5-methyl analogues of 1 were inactive. Insertion of heteroatoms (O, S, NH) into the C--C chain of 1 gave a series of compounds with high in vitro activity in the guinea pig serum ACE assay. The O-analogue was the most potent with an IC50 = 4.4 x 10(-9) M compared to 1 with an IC50 = 3.2 x 10(-9) M. The structure-activity relationships in this series of compounds lead one to speculate that the heteroatom provides an additional binding site to the surface of the enzyme; however, these compounds were inactive when tested for antihypertensive activity in the renal hypertensive rat at 30 mg/kg by the oral route (captopril is active at 1.0 mg/kg po).

Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 2. Modification of fatty acid anilide ACAT inhibitors: bioisosteric replacement of the amide bond.

In order to further define the structural features necessary for potent inhibition of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in vitro and cholesterol lowering in vivo, systematic study of bioisosteric replacements for the amide bond in our previously identified series of fatty acid anilide ACAT inhibitors was undertaken. Only replacement of amide bonds with isosterases having both hydrogen bond donor and acceptor functionalities yielded compounds retaining ACAT inhibitory activity. Replacement of the amide bond with the urea bioisostere yielded compounds that were potent ACAT inhibitors in vitro and efficacious hypocholesterolemic agents in vivo. Examination of the structure activity relationships in the phenyl ring and alkyl portion of the N-phenyl-N'-alkylureas revealed that 2,6-diisopropyl substitution was optimal in the phenyl ring. When the 2,6-diisopropyl moiety was kept constant, potency in vitro and in vivo was maintained with straight and branched alkyl groups from 6 to 18 carbons in length.

Derivatives of the potent angiotensin converting enzyme inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline: effect of changes at positions 2 and 5 of the hexanoic acid portion.

Several derivatives of the potent angiotensin converting enzyme inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline (1) were synthesized and tested for converting enzyme inhibition activity and blood pressure lowering effects in rats. One compound, 5(S)-benzamido-2(R)-methyl-4-oxo-6-phenylhexanoyl-L-proline (2a), had and I50 against angiotensin converting enzyme of 1.0 x 10(-9) M and is the most potent inhibitor prepared thus far in this class of compounds. Testing of 2a orally at 30 mg/kg for inhibition of the angiotensin I induced blood pressure increase in conscious normotensive rats gave 100% inhibition that required 143 min before the angiotensin I blood pressure response returned to 70% of the pretreatment control response. In the conscious renal hypertensive rat, 2a given orally at a dose of 3 mg/kg caused a lowering of blood pressure that reached its maximum of 40 mmHg 8 h following drug administration.

Inhibitors of acyl-Coa:cholesterol acyltransferase. 4. A novel series of urea ACAT inhibitors as potential hypocholesterolemic agents.

We have synthesized a series of N-phenyl-N'-aralkyl and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). This intracellular enzyme is thought to be responsible for the esterification of dietary cholesterol; hence inhibition of this enzyme could reduce diet-induced hypercholesterolemia. For this series of compounds, the in vitro ACAT inhibitory activity was improved by increasing the bulk of the 2,6-substituents on the phenyl ring. Additionally, we found that spacing of the aromatic rings was critical for ACAT inhibitory activity. A phenyl ring five atoms away from the requisite 2,6-diisopropylphenyl moiety was optimal for in vitro activity. Substitution alpha to the N'-phenyl moiety enhanced in vitro potency. In the case of phenylcycloalkyl ureas, ACAT inhibitory activity was independent of the size of the cycloalkyl ring. From this series of analogs, compound 25, which had excellent in vitro potency for inhibiting ACAT, was found to lower plasma cholesterol by 73% in vivo when administered in the diet at 50 mg/kg in an animal model of hypercholesterolemia. In this model, compound 25 lowered plasma cholesterol dose dependently and was as efficacious as the Lederle ACAT inhibitor CL 277082.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 5. Identification and structure-activity relationships of novel beta-ketoamides as hypocholesterolemic agents.

A study of structure-activity relationships of substituted beta-ketoamide ACAT inhibitors I and II was performed. The results of this study suggest that whereas the beta-keto group was tolerated with no loss in activity, beta-hydroxy and oxime moieties led to significantly reduced activity in vitro and in vivo. The most potent inhibitor from the acyclic series (I) (11, IC50 = 0.006 microM) contained a C-13 alkyl chain. This compound reduced plasma total cholesterol by 38% and 66% at 3 and 30 mg/kg, respectively, in cholesterol-fed rats. Dimethylation alpha to the anilide core (5) and subsequent N-methylation of the amide NH (6) decreased in vitro potency significantly. It was also found that high potency was retained with inhibitors which incorporated the carbonyl into a lactam ring (II).

Synthesis and angiotensin-converting enzyme inhibitory activity of 3-(Mercaptomethyl)-2-oxo-1-pyrrolidineacetic acids and 3-(Mercaptomethyl)-2-oxo-1-piperidineacetic acids.

A number of gamma- and delta-lactam derivatives were synthesized and their in vitro angiotensin-converting enzyme (ACE) inhibitory activities were compared. The structures of these compounds were designed to include many of the important features of captopril. The synthesis involved the preparation of a variety of novel 3-methylene-2-pyrrolidinones (3-5 and 16) and 3-methylene-2-piperidinones (3a-5a, 10-12, and 17). The key intermediate 3-methylenelactams 3 and 3a were obtained from 3-(hydroxymethyl)lactams 2 and 2a by a direct dehydration with dicyclohexylcarbodiimide using cuprous iodide as a catalyst. Introduction of the sulfhydryl group was accomplished by a Michael addition of these alpha, beta-unsaturated lactams. The compound with the highest in vitro activity was 3-(mercaptomethyl)-2-oxo-1-piperidineacetic acid (7a). The activity of the 7a both in vitro and in vivo (dog) was shown to be less than that of captopril by a factor of about 100.

Novel synthesis of (S)-1-[5-(benzoylamino)-1,4-dioxo-6-phenylhexyl]-L-proline and analogues: potent angiotensin converting enzyme inhibitors.

A new approach was developed for the synthesis of (S)-1-[5-(benzoylamino)-1,4-dioxo-6-phenylhexyl]-L-proline (1) and 23 analogues. The delta-(acylamino)-gamma-keto acid intermediates were obtained by a modified Dakin--West reaction using 3-carbomethoxypropionyl chloride. Acylation of L-proline and recrystallization of the mixture of diastereomers gave the optically pure title compound in three reaction steps. The in vitro angiotensin converting enzyme (ACE) inhibitory activity of 1 was confirmed. Some of the novel analogues (6, 11, 13, and 17) were also found to be potent inhibitors of ACE in vitro with an IC50 of 1.4-8.8 x 10(-9) M (IC50 for captopril = 0.9 x 10(-8) M). In vivo these compounds (6, 11, 17, and 18) were much less active than captopril, especially by the oral route. Against angiotensin I (AI) challenge in normotensive conscious rats, 1 and 6 produced less than 50% inhibition at 30 mg/kg po but 57 to 82% inhibition at 3 mg/kg iv. Inhibition by both routes lasted less than 1 h. In renal hypertensive rats, 1 and 15 of its analogues failed to produce significant blood pressure lowering effects, in contrast to the marked effects of captopril. Near maximum inhibition of AI was achieved by continuous intravenous infusions of 1 and 20, suggesting that limited oral activity may by due to degradation and/or clearance.

Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 7. Development of a series of substituted N-phenyl-N'-[(1-phenylcyclopentyl)methyl]ureas with enhanced hypocholesterolemic activity.

We recently described our initial structure-activity relationship (SAR) studies on a series of N-phenyl-N'-aralkyl- and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). From this series of analogs, compound 1 (PD 129337) was identified as a potent inhibitor of ACAT with an IC50 value of 17 nM. It was also shown to dose-dependently lower plasma cholesterol in cholesterol-fed rats. However, further investigation led to the suggestion that this compound was poorly absorbed, due to a lack of efficacy when administered by gavage in an aqueous vehicle. To overcome this deficiency, we continued our SAR study on this novel series of ACAT inhibitors using an acute in vivo screen in which the compounds are administered to rats in an aqueous, CMC/Tween suspension vehicle. Modification of the N'-phenyl moiety by incorporating functional groups which were amenable to forming salts and/or polar groups to reduce lipophilicity led to the identification of several inhibitors which displayed excellent efficacy employing this protocol. Overall, substitution on the phenyl ring in the ortho, meta, or para positions led to inhibitors with only a slight decrease in potency in vitro compared to the parent unsubstituted compound. Bulkier groups in the para position tended to lower the ACAT inhibitory activity in vitro. Polar groups, such as carboxyl (33,34), lowered in vitro activity significantly, suggesting that polar-ionic interactions are disfavored for the enzyme activity. From this series, compound 28 was evaluated further in secondary in vivo screens. In a chronic cholesterol-fed rat model of hypercholesterolemia, compound 28 dose-dependently reduced nonHDL cholesterol and significantly elevated HDL cholesterol. It showed significantly greater aqueous solubility than the parent compound 1. However, it was shown to cause adrenal toxicity in guinea pigs. This led us to design a series of homologs (44-51) with increased basicity and lower lipophilicity. Some of these compounds were more potent ACAT inhibitors in vitro and demonstrated excellent hypocholesterolemic activity in vivo. Interestingly, compound 45, unlike 28, did not produce adrenal toxicity in guinea pigs and demonstrated excellent lipid-modulating activity in the chronic model of preestablished dyslipidemia in rats.

Synthesis of novel angiotensin converting enzyme inhibitor quinapril and related compounds. A divergence of structure-activity relationships for non-sulfhydryl and sulfhydryl types.

The synthesis and angiotensin converting enzyme (ACE) inhibiting activities of quinapril (CI-906, 22), its active diacid (CI-928, 33), and its dimethoxy analogue (CI-925, 25) are reported. These tetrahydro-3-isoquinolinecarboxylic acid derivatives possess equivalent in vitro potency and in vivo efficacy to enalapril. Sulfhydryl analogues with the same structural variation are also highly potent. In contrast, tetrahydro-1-isoquinolinecarboxylic acid and homologous isoindoline-1-carboxylic acid analogues show a striking divergence in potency between the two types, sulfhydryl analogues being essentially inactive, while non-sulfhydryl analogues are equipotent with the proline prototype. This is the first evidence suggesting that alternate binding modes may exist for the two major structural classes of small molecule ACE inhibitors.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 1. Identification and structure-activity relationships of a novel series of fatty acid anilide hypocholesterolemic agents.

A series of fatty acid anilides was prepared, and compounds were tested for their ability to inhibit the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) in vitro and to lower plasma total cholesterol and elevate high-density lipoprotein cholesterol in cholesterol-fed rats in vivo. The compounds reported were found to fall into two subclasses with different anilide SAR. For nonbranched acyl analogues, inhibitory potency was found to be optimal with bulky 2,6-dialkyl substitution. For alpha-substituted acyl analogues, there was little dependence of in vitro potency on anilide substitution and 2,4,6-trimethoxy was uniquely preferred. Most of the potent inhibitors (IC50 less than 50 nM) were found to produce significant reductions in plasma total cholesterol in cholesterol-fed rats. Additionally, in vivo activity could be improved significantly by the introduction of alpha,alpha-disubstitution into the fatty acid portion of the molecule. A narrow group of alpha,alpha-disubstituted trimethoxyanilides, exemplified by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (39), was found to not only lower plasma total cholesterol (-60%) in cholesterol-fed rats but also elevate levels of high-density lipoprotein cholesterol (+94%) in this model at the screening dose of 0.05% in the diet (ca. 50 mg/kg).

Heterocyclic ureas: inhibitors of acyl-CoA:cholesterol O-acyltransferase as hypocholesterolemic agents.

A series of diaryl-substituted heterocyclic ureas was prepared, and their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in cholesterol-fed animal models in vivo was examined. N-(2,6-Diisopropylphenyl)-N'-tetrazole or isoxazole-substituted heterocyclic ureas proved optimal. A carbon chain of 11-14 carbons substituted 1,3 with respect to the amine provided the optimal side chain. Substitution of the alkyl chain generally lowered activity. Tetrazole urea 2i dosed at 3 mg/kg lowered plasma total cholesterol (TC) 67% in an acute, cholesterol-fed (C-fed) rat model of hypercholesterolemia and 47% in C-fed dogs. Tetrazole 2i, dosed at 10 mg/kg, also lowered TC 52% and raised HDL cholesterol 113% in rats with pre-established hypercholesterolemia.

Cholesteryl ester transfer protein inhibition by PD 140195.

The presence of plasma cholesteryl ester transfer protein (CETP) activity may be atherogenic, and, therefore, strategies to inhibit its activity or production may result in a beneficial effect on lipoprotein profiles and the disease process. The current report describes 4-phenyl-5-tridecyl-4H-1,2,4- triazole-3-thiol (PD 140195), a novel CETP inhibitor. The concentration-dependent inhibition of CETP by PD 140195 and the inhibitory monoclonal antibody (Mab) TP2 is demonstrated in a variety of in vitro assay systems. Molecular models of PD 140195 suggest a spatial mimicry of the cholesteryl ester structure. Despite the structural similarity, kinetic studies with a fluorescent cholesteryl ester analog suggest that the inhibition of transfer is not competitive. PD 140195 also selectively inhibited cholesteryl ester but not triglyceride transfer, while the Mab TP2 blocked CETP transfer of both. Studies were carried out to determine whether CETP inhibition observed in vitro could also be demonstrated in vivo. When PD 140195 was intravenously infused to anesthetized rabbits (up to 20 mg/kg), only transient CETP inhibition was observed. In vitro reconstitution studies in the presence of bovine serum albumin resulted in marked reduction of PD 140195 inhibitory activity. Thus, the low activity of PD 140195 in whole plasma probably results from binding to other plasma proteins.

Use of fluorescent cholesteryl ester microemulsions in cholesteryl ester transfer protein assays.

In the present report we describe a simple and practical method to assess CETP activity in a defined system by use of microemulsions containing a fluorescent cholesteryl ester analog. The microemulsions are stable, simple to prepare, and can be made to defined composition. Initial transfer rates are easily determined by monitoring changes in fluorescence. We have used the fluorescent cholesteryl ester analog, cholesteryl 4,4-difluoro-5,7-dimethyl-4-boro-3 alpha, 4 alpha-diaza-3-indacenedodecanoate (BODIPY-CE), to demonstrate the utility of this assay. The assay takes advantage of the concentration-dependent self-quenching of BODIPY-CE, when this analog is incorporated into microemulsions. We have used this new assay to demonstrate fluorescent lipid transfer facilitated by rabbit and human d > 1.21 g/ml plasma fraction and recombinant human CETP. A known inhibitory monoclonal antibody (Mab) to human CETP blocked BODIPY-CE transfer in a dose-dependent manner. We have also used BODIPY-CE microemulsions to measure CETP activity in whole plasma.

CI-906 and CI-907: new orally active nonsulfhydryl angiotensin-converting enzyme inhibitors.

CI-906, [3S-[2[R*(R*)]], 3R*]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]-amino]-1-oxopropyl] 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, monohydrochloride, and CI-907, [2S-[1[R*(R*)]], 2 alpha, 3a beta, 7a 7a beta]-1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino] 1-oxopropyl]octahydro-1H-indole-2-carboxylic acid, monohydrochloride, are two new nonsulfhydryl-type angiotensin-converting enzyme (ACE) inhibitors. Monoester (prodrug) and diacid forms produced concentration-related ACE inhibition in guinea pig serum (IC50 for CI-906 = 8.3 X 10(-9) M, diacid = 2.8 X 10(-9) M; CI-907 = 1.0 X 10(-7) M, diacid = 2.6 X 10(-9) M). In isolated rabbit aortic rings and in in vivo rat and dog autonomic studies, both compounds were highly specific in suppressing the contractile or pressor responses to angiotensin I. In two-kidney, one-clip Goldblatt (renin-dependent) hypertensive rats there was a good correlation between the inhibition of vascular converting enzyme and blood pressure lowering and a poor correlation between blood pressure lowering and plasma and brain converting enzyme inhibition. Cardiovascular, pulmonary, and central nervous system performance evaluations showed no side effects or gross toxicity. The preclinical profile shows CI-906 and CI-907 to be specific, potent, orally active ACE inhibitors. They are expected to have therapeutic utility in hypertension and in any other condition where converting enzyme inhibition would be useful.

Elevated low-density lipoprotein in Alzheimer's disease correlates with brain abeta 1-42 levels.

Sera obtained in the immediate postmortem from 100 individuals, 64 neuropathologically diagnosed Alzheimer's disease (AD) cases and 36 nondemented controls, were analyzed for cholesterol, lipoproteins, apolipoproteins (Apo), and triglycerides. All individuals were ApoE genotyped, and the amounts of Abeta (N-40 and N-42) in cerebral cortex of AD and control subjects were determined. When compared to controls, AD individuals had significantly higher LDL cholesterol (P = 0.006), ApoB (P = 0.018), Abeta N-40 (P = 0.024) and Abeta N-42 (P < 0.001), and significantly lower HDL cholesterol (P = 0.040). There were positive correlations between the levels of serum total cholesterol (r = 0.359, P = 0.004), LDL cholesterol (r = 0.328, P = 0.008), and ApoB (r = 0.395, P = 0.001) to the amount of Abeta N-42 in AD brains, but not to Abeta N-40. These correlations were independent of ApoE genotype and were not seen in the control group. The present results suggest for the first time that elevated serum cholesterol, especially in the form of LDL, influences the expression of AD-related pathology.

Expression of the mRNA encoding truncated PPAR alpha does not correlate with hepatic insensitivity to peroxisome proliferators.

Two alternatively spliced forms of human PPAR alpha mRNA, PPAR alpha1 and PPAR alpha2, have been identified. PPAR alpha1 mRNA gives rise to an active PPAR alpha protein while PPAR alpha2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR alpha2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR alpha1 and PPAR alpha2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR alpha2 mRNA abundance is approximately half that of PPAR alpha1 mRNA; a correlation analysis of PPAR alpha1 and PPAR alpha2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR alpha2/PPAR alpha1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR alpha2/PPAR alpha1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPAR alpha activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR alpha2/PPAR alpha1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR alpha2/PPAR alpha1 mRNA. These data suggest that selective PPAR alpha2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators.