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For the last 30 years fibrates have been widely prescribed to treat human dyslipidemia. However, the primary mechanism by which they lower plasma lipid levels is still unknown. Studies with transgenic mice have suggested that changes in apoC-III expression levels have a dramatic influence on plasma triglyceride levels. These results suggested that fibrates could reduce lipid levels by lowering apoC-III gene expression. In the current studies, we sought to determine whether the selected fibrates, bezafibrate, clofibrate, fenofibrate, and gemfibrozil, could reduce hepatic apoC-III mRNA and plasma apoC-III levels. Chow-fed rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of each of the fibrates for 1 week and in addition with gemfibrozil for 2 weeks. Bezafibrate and fenofibrate lowered plasma triglyceride by approximately half and dramatically reduced hepatic apoC-III mRNA and plasma apoC-III levels. In contrast, clofibrate did not reduce plasma triglyceride levels and only partially reduced apoC-III mRNA and plasma protein levels. Gemfibrozil strongly reduced plasma triglyceride levels and had an intermediate but significant effect on apoC-III mRNA and plasma apoC-III levels. Some of the fibrates, especially gemfibrozil also reduced plasma apoC-II levels, an effect that could contribute to the observed triglyceride-lowering effect. In addition, the ratio of plasma apoE to plasma apoC-II plus apoC-III was strongly and inversely correlated with plasma triglyceride levels. As plasma apoE levels were not reduced in gemfibrozil-treated animals, this could also have contributed to the triglyceride-lowering effect of this fibrate. Fibrate-mediated triglyceride lowering was not the result of a decreased apoB or VLDL production and, therefore, suggested an enhanced VLDL remnant catabolism. Our results suggest that the mechanism by which fibrates lower plasma triglycerides is by reducing the level of hepatic apoC-III expression.

The apparent protective effect of high density lipoprotein cholesterol (HDL) with respect to coronary heart disease (CHD) is generally thought to reside in its ability to transport cholesterol from peripheral cells to the liver for excretion from the body. Knozon as reverse cholesterol transport (RCT), this process involves many key steps and lipoprotein interconversions, and there is no consensus as to which step is most suitable for possible drug intervention. The membrane proteins, scavenger receptor class B, type 1 (SR-B1) and the ATP-binding cassette 1 (ABC1), have been strongly implicated as being important in cholesterol efflux; the former as a bona fide receptor for HDL and the latter as a lipid transporter. Lecithin:cholesterol acyltransferase (LCAT) then esterifies the effluxed cholesterol to form cholesteryl esters (Step 2), which are then transferred to apoB-containing lipoproteins by cholesteryl ester transfer protein (CETP, Step 3). Despite the complexities and uncertainties, drugs should be developed which impact all of the above steps, and short-term endpoints need to be defined for a cautious, systematic approach to clinical evaluation.

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.

For the last 30 years fibrates have been widely prescribed to treat human dyslipidemia. However, the primary mechanism by which they lower plasma lipid levels is still unknown. Studies with transgenic mice have suggested that changes in apoC-III expression levels have a dramatic influence on plasma triglyceride levels. These results suggested that fibrates could reduce lipid levels by lowering apoC-III gene expression. In the current studies, we sought to determine whether the selected fibrates, bezafibrate, clofibrate, fenofibrate, and gemfibrozil, could reduce hepatic apoC-III mRNA and plasma apoC-III levels. Chow-fed rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of each of the fibrates for 1 week and in addition with gemfibrozil for 2 weeks. Bezafibrate and fenofibrate lowered plasma triglyceride by approximately half and dramatically reduced hepatic apoC-III mRNA and plasma apoC-III levels. In contrast, clofibrate did not reduce plasma triglyceride levels and only partially reduced apoC-III mRNA and plasma protein levels. Gemfibrozil strongly reduced plasma triglyceride levels and had an intermediate but significant effect on apoC-III mRNA and plasma apoC-III levels. Some of the fibrates, especially gemfibrozil also reduced plasma apoC-II levels, an effect that could contribute to the observed triglyceride-lowering effect. In addition, the ratio of plasma apoE to plasma apoC-II plus apoC-III was strongly and inversely correlated with plasma triglyceride levels. As plasma apoE levels were not reduced in gemfibrozil-treated animals, this could also have contributed to the triglyceride-lowering effect of this fibrate. Fibrate-mediated triglyceride lowering was not the result of a decreased apoB or VLDL production and, therefore, suggested an enhanced VLDL remnant catabolism. Our results suggest that the mechanism by which fibrates lower plasma triglycerides is by reducing the level of hepatic apoC-III expression.

The in vitro potencies and hypocholesterolemic properties of CL 277,082 and PD 132301-2, two urea inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) were compared. PD 132301-2 was several-fold more potent at inhibiting ACAT in microsomes from rat and rabbit tissues and in cultured cells (murine macrophages and the human HepG2 cell line). This disubstituted urea was also relatively specific for ACAT as other cholesterol esterifying enzymes (e.g., lecithin:cholesterol acyltransferase, pancreatic cholesterol ester hydrolase), as well as intestinal diglyceride synthesis (acyl-CoA:monoglyceride acyltransferase), were unaffected in vitro at relevant concentrations. In normal chow-fed rats, both compounds reduced plasma triglycerides at doses50 mg/kg, but only PD 132301-2 reduced plasma cholesterol. In rat and rabbit models of hypercholesterolemia the greater in vitro potency of PD 132301-2 translated into greater in vivo efficacy (i.e., ED50 values 2- to 3-fold higher for CL 277,082 in both acute and chronic rate models). Of particular note was the greater elevation of high-density lipoprotein-cholesterol and parenteral activity of PD 132301-2 compared to CL 277,082 in the chronic rat model. Inhibition of cholesterol absorption in rats was also greater with PD 132301-2. In guinea pigs, in which 77% of plasma cholesterol was transported in low-density lipoprotein, PD 132301-2 potently reduced plasma total cholesterol (lowest significant dose = 1 mg/kg) as well as plasma triglycerides. CL 277,082 only reduced cholesterol at doses100 mg/kg in this low-density lipoprotein model. In a canine model of hypercholesterolemia CL 277,082 was inactive at doses up to 50 mg/kg, but PD 132301-2 was active at 3 mg/kg. Moreover, efficacy in dogs with PD 132301-2 was positively correlated with plasma drug concentration, an observation not previously demonstrated for other hypolipidemic drugs. The combined data illustrate that pharmacologic activities can vary widely among ACAT inhibitors of the same general class. In addition, the unique observation of proportionality between efficacy and blood drug levels in nonrodent animal models may not only help to simplify early stages in drug development but also may help to predict or monitor a direct action of the drug on vascular disease.

CI-976, a new trimethoxy fatty acid anilide, is a potent and specific inhibitor of liver and intestinal acyl coenzyme A:cholesterol acyltransferase (ACAT) in vitro. Several in vivo approaches were used to determine the efficacy and sites of action of this compound in rats. CI-976 decreased non-high density lipoprotein (HDL)-cholesterol and increased HDL-cholesterol in rats with pre-established dyslipidemia. High performance gel chromatographic separation of plasma lipoproteins also revealed that CI-976, but not CL 277,082, lowered low density lipoprotein (LDL)-cholesterol and elevated HDL-cholesterol. Bay o 2752, octimibate, melinamide, and SaH 58-035 were all less potent in vivo compared to CI-976 and CL 277,082, and CI-976 produced the greatest decrease in liver cholesteryl esters. Subcutaneous (SC) administration of CI-976 was also efficacious in cholesterol-fed animals. In sucrose-fed rats, oral and SC CI-976 administration potently lowered plasma triglycerides. Hepatic cholesteryl ester accumulation in the ethinyl estradiol-treated rat was also diminished by orally administered CI-976. ACAT activity and cholesteryl ester mass were dose-dependently decreased in the livers from cholesterol-fed rats treated with CI-976, suggesting a direct effect on the liver. In both hypercholesterolemic and hypertriglyceridemic models, CI-976 also decreased plasma apoB concentrations. In other experiments radiolabeled CI-976 accumulated in the liver after multiple doses. Time-dependent changes in biliary lipid and bile acid secretion suggested that free cholesterol did not accumulate in the liver but instead was excreted as such or as bile acid. Finally, inhibition of endogenous and exogenous intestinal cholesterol absorption was demonstrated using several in vivo techniques. The combined data strongly supports the hypothesis that orally administered CI-976 inhibits both intestinal and hepatic ACAT, and that both of these enzymes may be determinants of plasma lipid concentrations in the rat.

Rat mesenteric lymph contains all serum apolipoproteins. However, it is uncertain whether some of these apolipoproteins are derived from intestinal synthesis or are transferred from plasma. We compared lymph apolipoprotein composition, concentrations, and transport rates in normal rats and in rats treated with pharmacologic doses of ethinyl estradiol which have negligible concentrations of serum lipids and apolipoproteins. Lymph apolipoproteins were examined before and after duodenal lipid infusion. Lymph d less than 1.006 and 1.006-1.21 g/ml lipoproteins were isolated and SDS-electrophoresis was performed using 10 and 3.5% polyacrylamide. During lipid absorption, lymph flow increased in control but not in treated rats. Control lymph contained all major apolipoproteins, but lymph from ethinyl estradiol-treated rats contained only apoB, A-I, and A-IV. Two apoB bands were noted on 3.5% gels in control lymph, but only the lower molecular weight protein was found in lymph from ethinyl estradiol-treated rats. In control rats, transport rates for apoA-I, A-IV, E, and C proteins increased during lipid absorption, but only in the case of A-IV was this a reflection of increased apolipoprotein concentration and not the enhanced lymph flow. In ethinyl estradiol-treated rats only the A-IV transport rate increased due to lipid infusion. It is concluded that in the ethinyl estradiol-treated rat 1) the intestine does not synthesize apoE, C, or the high molecular weight apoB; 2) lymphatic output of A-IV is predominantly increased during lipid absorption; and 3) since plasma apolipoprotein concentrations are negligible, lymph lipoproteins from ethinyl estradiol-treated rats may represent a close approximation to nascent particles of intestinal origin.

Prenodal peripheral lymph was used as a model of interstitial fluid to obtain information on the composition of lipoproteins and apolipoproteins which are in direct contact with peripheral cells. Lipoproteins resembling plasma lipoproteins in size and electrophoretic mobility were present in the prenodal peripheral lymph of control as well as cholesterol-fed dogs. Most of the lipoproteins in control dogs were high density lipoproteins, both in the plasma and in the lymph. Cholesterol feeding resulted in an increased concentration of lipoprotein particles with decreased electrophoretic mobility (beta-VLDL and, in plasma, HDLc) and decreased concentration of HDL, both in plasma and in lymph. Size distribution of lipoproteins was also markedly altered by cholesterol feeding; most of the lipoproteins were present as IDL and VLDL both in plasma and in lymph. Judged by agarose gel chromatography, the size of the lymph HDL as consistently larger than plasma HDL in both groups of dogs. Furthermore, it appears that cholesterol feeding increased the size of an HDL subfraction, partially resolved by agarose chromatography, both in lymph and plasma. All apolipoproteins present in plasma were also present in lymph. Cholesterol feeding resulted in 3-10-fold increases in plasma apo B, E, and A-IV while apo A-I was drastically decreased. These changes were reflected in lymph to different degrees depending on the size of the lipoprotein fraction containing the individual apolipoproteins. Our findings provide direct evidence that the large, cholesterol-rich, 'atherogenic' lipoproteins found in the plasma of cholesterol-fed dogs (beta-VLDL) are also present in the interstitial fluid and presumably interact with peripheral cells. Our studies furthermore suggest modification of plasma HDL by peripheral cells and/or de novo assembly of an HDL subfraction. The utilization of this animal model may thus provide a direct approach to the study of the interaction of lipoproteins with peripheral cells.

The distribution, chemical, and apoprotein composition of plasma and peripheral lymph lipoproteins were compared in control and cholesterol-fed dogs. In both groups of animals, the agarose electrophoretic patterns of plasma and lymph lipoproteins were similar. In hypercholesterolemic dogs, beta-very low density lipoprotein, beta-migrating intermediate density lipoprotein, and HDLc were major components both in plasma and lymph, providing evidence for a potential interaction of these atherogenic particles with macrophages and other peripheral cells. The chemical composition and physical appearance of peripheral lymph HDL was markedly different from that of plasma HDL (high density lipoprotein), especially in the cholesterol-fed animals. Lymph HDL had a higher cholesterol to protein ratio and a markedly increased free cholesterol content (free cholesterol to cholesteryl ester ratio of 1.7 as opposed to 0.2 in plasma HDL in cholesterol-fed animals). The phospholipid content of lymph HDL was higher than that of plasma HDL, while the protein content was lower. A significant proportion of lymph HDL obtained from cholesterol-fed dogs was in the form of disc-shaped particles stacked in rouleau structures. Changes in plasma apolipoprotein concentrations due to cholesterol feeding were reflected in peripheral lymph to different degrees, depending largely on the relative size of the lipoproteins containing the individual lipoproteins. A considerable enrichment of lymph HDL with apoE and apoA-IV was observed by both immunochemical and electrophoretic methods. In lymph HDL from control and cholesterol-fed dogs, the apoE/apoA-I and apoA-IV/apoA-I ratios were several-fold elevated, compared to those of plasma HDL. It is concluded, therefore, that during cholesterol feeding a substantial portion of interstitial HDL is assembled de novo in the periphery as a crucial stage of reverse cholesterol transport to the liver. It is likely that further modification occurs upon entry to plasma and exposure to lecithin:cholesterol acyltransferase, possibly leading to generation of HDLc. Alternatively, these particles may be directly and rapidly removed by the liver.

An instrument has been designed and built to permit the measurement of the magnitude and direction of the magnetic dipole moment in a one-inch cubical rock specimen while the specimen is in a furnace. The measurement is obtained by giving the specimen a linear vibration at 40 c.p.s. and surrounding the furnace with a specially designed array of coils. Each of the three mutually perpendicular components of the dipole moment induces a voltage in the coils. By rearranging the interconnection of the coils, outputs proportional to any component may be obtained. This apparatus has a theoretical sensitivity to magnetization of about 4 × 10−5 e.m.u. (per cc). The actual sensitivity approached this but was limited by external electrical interference.

HDL cholesterol (HDL-C) was increased by gemfibrozil (+3.6-fold), fenofibrate (+1.3-fold) and ciprofibrate (+1.2-fold) but not clofibrate or bezafibrate when dosed PO at 50 mg/kg for 2 weeks in cholesterol-fed rats. Cholesterol in apo B-containing lipoproteins decreased with gemfibrozil (-76%), clofibrate (-12%) and ciprofibrate (-12%). Plasma apo B decreased to the greatest extent with gemfibrozil (-86%) followed by ciprofibrate (-47%), fenofibrate (-40%), clofibrate (-24%) and bezafibrate (-20%). Only gemfibrozil increased plasma apo E levels which are characteristically low in this rat model. Gemfibrozil, fenofibrate and ciprofibrate increased apo A-I concentrations. It is concluded that plasma lipid regulators which elevate HDL in this model might do so by altering the metabolism and hence plasma concentration of apoAI (fenofibrate, ciprofibrate) or both apo E and A-I (gemfibrozil). It is hypothesized that drugs which alter the metabolism of both HDL peptides result in the greatest HDL-C elevation in the rat.

Low-density lipoprotein-cholesterol (LDL-C) is currently estimated clinically by using the Friedewald formula, when plasma triglycerides are < 4000 mg/L, or as the difference between infranatant and high-density lipoprotein-cholesterol (HDL-C) values after ultracentrifugation of plasma at native density, when plasma triglycerides are > or = 4000 mg/L (beta quantification). HDL-C is measured by selective precipitation of apolipoprotein B-containing lipoproteins from whole plasma or from the density > 1.006 kg/L infranatant. We compared these conventional methods for LDL-C and HDL-C with "high-performance" gel chromatography (HPGC), a method that directly and simultaneously measures both LDL-C and HDL-C in a single, microliter volume of plasma. Not surprisingly, we found that the results by all these methods were highly correlated. However, LDL-C values were significantly higher and HDL-C values significantly lower by the direct HPGC method than by the conventional methods (paired t-test). In addition, both Bland-Altman plots and concordance correlation analyses indicated lack of agreement between the methods' results in the majority of patients' subgroups.