Remodeling of the HDL in NIDDM: a fundamental role for cholesteryl ester transfer protein.

When the Ay gene is expressed in KK mice, the yellow offspring (KKAy mice) become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP). To test the hypothesis that it is the absence of CETP that allows these hypertriglyceridemic mice to maintain high plasma HDL levels, simian CETP was expressed in the KKAy mouse. The KKAy-CETP mice retained the principal characteristics of KKAy mice except that their plasma HDL levels were reduced (from 159 +/- 25 to 25 +/- 6 mg/dl) and their free apolipoprotein A-I concentrations increased (from 7 +/- 3 to 22 +/- 6 mg/dl). These changes appeared to result from a CETP-induced enrichment of the HDL with triglyceride (from 6 +/- 2 to 60 +/- 18 mol of triglyceride/mol of HDL), an alteration that renders HDL susceptible to destruction by lipases. These data support the premise that CETP-mediated remodeling of the HDL is responsible for the low levels of that lipoprotein that accompany hypertriglyceridemic non-insulin-dependent diabetes mellitus.

Centripetal cholesterol flux from extrahepatic organs to the liver is independent of the concentration of high density lipoprotein-cholesterol in plasma.

High density lipoproteins (HDLs) play a role in two processes that include the amelioration of atheroma formation and the centripetal flow of cholesterol from the extrahepatic organs to the liver. This study tests the hypothesis that the flow of sterol from the peripheral organs to the liver is dependent upon circulating HDL concentrations. Transgenic C57BL/6 mice were used that expressed variable amounts of simian cholesteryl ester-transfer protein (CETP). The rate of centripetal cholesterol flux was quantitated as the sum of the rates of cholesterol synthesis and low density lipoprotein-cholesterol uptake in the extrahepatic tissues. Steady-state concentrations of cholesterol carried in HDL (HDL-C) varied from 59 to 15 mg/dl and those of apolipoprotein AI from 138 to 65 mg/dl between the control mice (CETPc) and those maximally expressing the transfer protein (CETP+). There was no difference in the size of the extrahepatic cholesterol pools in the CETPc and CETP+ animals. Similarly, the rates of cholesterol synthesis (83 and 80 mg/day per kg, respectively) and cholesterol carried in low density lipoprotein uptake (4 and 3 mg/day per kg, respectively) were virtually identical in the two groups. Thus, under circumstances where the steady-state concentration of HDL-C varied 4-fold, the centripetal flux of cholesterol from the peripheral organs to the liver was essentially constant at approximately 87 mg/day per kg. These studies demonstrate that neither the concentration of HDL-C or apolipoprotein AI nor the level of CETP activity dictates the magnitude of centripetal cholesterol flux from the extrahepatic organs to the liver, at least in the mouse.

Kinetics and inhibition of lipid exchange catalyzed by plasma cholesteryl ester transfer protein (lipid transfer protein).

The cholesteryl ester transfer protein-catalyzed cholesteryl ester transfer is inhibited by two compounds identified by a large-scale screening of cholesterol backbone-containing molecules. Kinetic analysis shows that U-95,594, an amino steroid, inhibits competitively the cholesteryl ester transfer protein-catalyzed transfer of both cholesteryl esters and triglycerides, as well from high-density lipoproteins as from synthetic microemulsions. In contrast, U-617, an organomercurial derivative of cholesterol, inhibits competitively the transfer of cholesteryl ester from either donor but is without any effect on triglyceride transfer. In addition to the rapid, competitive inhibition of cholesteryl ester transfer, U-617 also slowly and reversibly reacts with cholesteryl ester transfer protein to produce an additional 10-fold decrease in cholesteryl ester transfer activity but, again, without effect on triglyceride transfer.

Evidence that cynomolgus monkey cholesteryl ester transfer protein has two neutral lipid binding sites.

Two inhibitors of cynomolgus monkey cholesteryl ester transfer protein were evaluated. One, a monoclonal antibody made against purified cynomolgus monkey cholesteryl ester transfer protein, was capable of severely inhibiting triglyceride transfer, but had a variable effect on cholesteryl ester transfer. At low antibody to antigen ratios, there was what appeared to be a stoichiometric inhibition of cholesteryl ester transfer, but at high antibody to antigen ratios the inhibition of cholesteryl ester transfer was completely relieved, even though triglyceride transfer remained blocked. Fab fragments of the antibody had no effect whatsoever on cholesteryl ester transfer, but were capable of completely blocking triglyceride transfer. The other inhibitor, 6-chloromecuric cholesterol, severely inhibited cholesteryl ester transfer with minimal inhibition of triglyceride transfer. When both inhibitors were added to the assay, both cholesteryl ester and triglyceride transfer were inhibited; an indication that the inhibitors did not compete for the same binding site on cholesteryl ester transfer protein. When the antibody was given subcutaneously to cynomolgus monkeys at a dose which inhibited triglyceride transfer in the plasma by more than 90%, there was no detectable effect on the high density lipoprotein (HDL) cholesterol level, but the HDL triglyceride levels decreased from 13 +/- 2 to 1 +/- 0 mol/mol of HDL (mean +/- S.D.); an indication that the antibody uncoupled cholesteryl ester and triglyceride transfer in vivo. The 6-chloromecuric cholesterol could not be evaluated in vivo because it is a potent lecithin:cholesterol acyltransferase inhibitor. The fact that cholesteryl ester transfer can be inhibited without effect on triglyceride transfer and, conversely, that triglyceride transfer can be inhibited without effect on cholesteryl ester transfer indicates that these two lipids are not transferred by a single, non-discriminatory process.

Method for measuring the activities of cholesteryl ester transfer protein (lipid transfer protein).

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within < 30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --> exchange --> desorption --> diffusion.

The effect of population density on the development of experimental atherosclerosis in female mice.

The effect of cage population density on plasma lipids and the development of atherosclerosis was examined in female C57BL/6 mice. Mice were housed at a density of one, two or five animals per cage and fed an atherogenic diet for 28 weeks. Subsequently, the animals were bled, sacrificed, the hearts removed and the extent of fatty lesion development in the aorta examined and quantified. As the population density increased, there was a statistically significant increase in total cholesterol levels, VLDL+LDL cholesterol levels, the VLDL+LDL/HDL ratio and lesion severity. These differences are due to the psychosocial stress associated with living within a confined space with high population density over an extended period of time.

Cholesteryl ester transfer protein's role in high-density lipoprotein metabolism Insights from studies with transgenic mice.

A substantial percentage of people who develop coronary artery atherosclerosis have plasma cholesterol levels in the "desirable" range. The principal lipid abnormality in most of these individuals is a low plasma high-density lipoprotein (HDL) level (HDL cholesterol levels of 35 mg/dL or less). As a result, low HDL levels are not only recognized as a risk factor for the disease, but are considered the single best predictor of an individual's likelihood of developing coronary heart disease. Yet we are only now beginning to understand what regulates plasma HDL levels and why they are low in some individuals. Cholesteryl ester transfer protein (CETP), a plasma protein that shuttles neutral lipids (cholesteryl esters and triglycerides) back and forth between lipoproteins in the circulation, appears to play a key role in HDL metabolism, and recent studies using transgenic mice expressing that protein have broadened our understanding of the metabolic pathways that control plasma HDL levels. In this article, we review some of the key observations regarding CETP's role in HDL metabolism, with special emphasis on the discoveries made using transgenic mice, and we discuss these observations in the context of a model linking plasma triglyceride metabolism with low HDL levels.

Co-expression of cholesteryl ester transfer protein and defective apolipoprotein E in transgenic mice alters plasma cholesterol distribution. Implications for the pathogenesis of type III hyperlipoproteinemia.

Despite the definite etiologic link between apolipoprotein (apo) E mutations and type III hyperlipoproteinemia (HLP), it is not clear what additional factors are involved in the development of florid hyperlipidemia and how to explain the wide variability in the expression of the hyperlipidemic phenotype in carriers of receptor binding-defective apoE variants. The present study was designed to determine whether the overexpression of cholesteryl ester transfer protein (CETP), a plasma protein that transfers cholesteryl esters from the high density lipoproteins (HDL) to the very low density lipoproteins (VLDL) and whose activity is increased in hyperlipidemic states, plays a role in the development of hyperlipidemia and beta-VLDL accumulation in type III HLP. We produced double-transgenic mice that co-expressed high levels of simian CETP and either high or low levels of a human receptor binding-defective apoE variant, apoE(Cys-142). We previously reported that apoE(Cys-142) high-expresser mice showed spontaneous hyperlipidemia and accumulation of beta-VLDL, whereas the low-expresser mice showed only a modest increase in VLDL cholesterol. Co-expression of CETP induced a massive transfer of cholesteryl esters from the HDL to the VLDL in both lines of double-transgenic mice. As a result, HDL cholesterol and apoA-I levels were reduced to about 50% of normal, VLDL cholesterol increased 2.5-fold, and the cholesteryl ester content of VLDL reached values similar to those observed in human beta-VLDL. The ratio of defective to normal apoE in VLDL was unaffected by CETP co-expression and was higher in animals expressing high apoE levels. Finally, in spite of an increased accumulation of beta-VLDL in the high-expresser mice, the VLDL of the low-expresser mice maintained pre-beta mobility upon co-expression of CETP. The results of this study demonstrate that the ratio of defective to normal apoE on the VLDL, rather than the cholesteryl ester content of VLDL, is the major factor determining the development of severe hyperlipidemia and the formation and accumulation of beta-VLDL in type III HLP.

The development of fatty liver is accelerated in transgenic mice expressing cynomolgus monkey cholesteryl ester transfer protein.

Expression of cynomolgus monkey cholesteryl ester transfer protein (CETP) in C57BL/6 mice has been shown to have a profound effect on the lipoprotein profile in those animals. The objective of this study was to examine the effect of CETP expression on the hepatic lipids of the CETP transgenic mice. The triglyceride, cholesterol and phospholipid composition of livers from 6- and 12-month-old transgenic mice were evaluated and compared with those of age-matched C57BL/6 mice. Statistical analysis indicated that fatty liver was more severe in CETP transgenic mice than C57BL/6 controls (p < 0.01); progressed with age (p < 0.01); and developed more rapidly in males than females (p < 0.01). The lipid that accumulated was triglyceride. These data indicate that CETP expression accelerates the development of fatty liver in the C57BL/6 mouse and raise the possibility that CETP may also contribute to the process of hepatic steatosis in man.

Purification of human cholesteryl ester transfer protein by affinity chromatography on immobilized triazine dyes.

Human cholesteryl ester transfer protein was purified from lipoprotein-depleted serum or plasma in a three-step procedure utilizing commercially available triazine dyes immobilized on agarose. The method used consisted of successive chromatography steps on Reactive Red 120 agarose (Procion Red H-E3B, Cibachron Brilliant Red 4G-E), CM Sepharose, and Reactive Yellow 86 agarose (Procion Yellow M-8G). Upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resultant protein preparation displayed two bands of variable intensity. The two components had apparent molecular weights of approximately 64,000 and approximately 65,000, respectively. Both bands reacted strongly to a monoclonal antibody directed against an epitope consisting of the last eight amino acids at the carboxy terminus of human CETP. Yields of cholesteryl ester transfer activity are 10-40% of the activity present in lipoprotein-depleted serum. The activity is approximately 50,000- to 100,000-fold purified relative to the starting material.

Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into the mechanisms responsible for low plasma high density lipoprotein levels.

Expression of simian cholesteryl ester transfer protein (CETP) in C57BL/6 mice causes the animals' high density lipoprotein (HDL) levels to decrease. The purpose of these studies was to determine how CETP expression caused that reduction. Chemical analysis showed that the HDL of the CETP transgenic mice had about twice as much triglyceride and only about 60% as much cholesteryl ester as the HDL from the C57BL/6 mice. Both strains of mouse had high levels of a circulating lipase. When plasma from the mice was incubated at 37 degrees C for 5 h, the triglycerides in the HDL were hydrolyzed, and apoA-I was shed from the particle. However, apoA-I was shed from the CETP HDL more rapidly than it was shed from the C57BL/6 HDL. Because "free" apoA-I is rapidly cleared by the kidney, increased production of free apoA-I would be expected to shorten the average life span of apoA-I in the mouse. Kinetic analyses indicated that the life span of apoA-I was significantly reduced in the CETP transgenic mice. It was concluded that CETP expression enriched the core of the HDL with triglyceride, which rendered it vulnerable to lipolysis, causing apoA-I to be shed from the particle. That shortened the life span of apoA-I in the CETP mice, which led to lower plasma levels of the protein.

Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) is a plasma protein that mediates the exchange of neutral lipids among the lipoprotein. Because the principal core lipid of very-low-density lipoprotein (VLDL) is triglyceride and that of high-density lipoprotein (HDL) is cholesterol ester, CETP mediates a 'heteroexchange' of cholesterol ester for triglyceride between those lipoproteins. As a result, animals that express CETP tend to have higher VLDL and low-density lipoprotein (LDL) cholesterol levels, whereas those with no CETP activity tend to have high HDL cholesterol levels. Because VLDL and LDL are associated with the progression of atherosclerosis, and HDL are considered anti-atherogenic, CETP could be an 'atherogenic' protein, that is, given the other conditions required for atherosclerosis to develop, expression of CETP would accelerate the rate at which the arterial lesions progress. We report here that transgenic mice expressing CETP had much worse atherosclerosis than did non-expressing controls, and we suggest that the increase in lesion severity was due largely to CETP-induced alterations in the lipoprotein profile.

Lipoprotein profile characterization of the KKA(y) mouse, a rodent model of type II diabetes, before and after treatment with the insulin-sensitizing agent pioglitazone.

The purpose of this study was to characterize the lipoprotein profile in the KKA(y) mouse, a rodent model of type II diabetes, before and after treatment with the insulin-sensitizing drug pioglitazone. Analysis of the plasma from untreated KKA(y) mice showed that they were severely hyperglycemic, severely hypertriglyceridemic, and moderately hypercholesterolemic. Agarose column chromatographic analysis showed that essentially all of the triglyceride eluted with very low density lipoprotein, and the majority of the cholesterol eluted with high density lipoprotein. Thus, both the very low density lipoprotein and high density lipoprotein levels were markedly elevated in KKA(y) mice. Analysis of the lipoproteins by agarose electrophoresis-immunoblotting showed that apoprotein A-I and apoprotein B had aberrant electrophoretic behavior, typical of apoproteins that have been modified by nonenzymatic glycosylation. Treatment of KKA(y) mice with pioglitazone for 8 days caused a marked reduction in blood glucose and plasma triglyceride concentrations but had no significant effect on plasma cholesterol concentration or distribution. The aberrant electrophoretic behavior of the apoproteins was corrected to normal by drug treatment. These data show that the KKAy mouse has a severe dyslipoproteinemia that is probably secondary to its insulin resistance, but that its lipoprotein profile differs significantly from that of the insulin-resistant human in that the majority of the plasma cholesterol is carried in high density lipoprotein, and those high density lipoprotein levels are very high.

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.

The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Insights from studies with transgenic mice.

To assess the effects of cholesteryl ester transfer protein (CETP) on the primate lipoprotein profile, a transgenic mouse expressing cynomolgus monkey CETP was developed. The C57BL/6 mouse was used, and four lines expressing the primate CETP were established. The level of CETP activity in the plasma of the transgenic mice ranged from values similar to those obtained for the monkey to levels approximately sixfold higher than that in the normal monkey. When all of the lines were taken into consideration, there was a strong (r = -0.81 or higher, p less than 0.01) negative correlation between plasma CETP activity and total plasma cholesterol, plasma apolipoprotein (apo) A-I levels, and plasma apo A-I to apo B ratio. There was a strong positive correlation (r = 0.77) between plasma CETP activity and plasma apo B levels. The size of the apo A-I-containing lipoproteins was significantly reduced in mice with high plasma CETP activity, and that reduction in size was due to the absence of the larger (HDL1 and HDL2) apo A-I-containing particles in the plasma. When the transgenic mice were fed a high-fat, high-cholesterol diet, the effects of the diet on lipoprotein profile were more prominent in the CETP transgenic mice than the controls. The CETP transgenic mice had, for example, substantially higher plasma cholesterol and plasma apo B levels (p less than 0.01), and the apo B-containing lipoproteins were generally larger than those in the nontransgenic C57BL/6 mice consuming the same diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Pioglitazone hydrochloride inhibits cholesterol absorption and lowers plasma cholesterol concentrations in cholesterol-fed rats.

Diabetes is associated with altered cholesterol metabolism that may contribute to cardiovascular complications. Treatment of rats with pioglitazone hydrochloride, a novel antidiabetic compound that improves the general response of target cells to insulin, significantly lowered cholesterol levels in rats fed a hypercholesterolemic diet and produced a significant reduction in cholesterol absorption. Drug treatment was ineffective in rats that were not given dietary cholesterol. To determine whether these effects of pioglitazone hydrochloride might be related to the known ability of this compound to improve the response to circulating insulin, similar studies were conducted in streptozocin-induced diabetic rats with and without insulin replacement. Diabetic rats absorbed a greater percentage of dietary cholesterol than control rats. Treatment of insulin-deficient diabetic rats with pioglitazone alone did not affect cholesterol absorption; however, the combination of insulin and pioglitazone was synergistic to lower absorption of cholesterol and circulating cholesterol and triglycerides. Treatment of either normal rats or diabetic rats receiving insulin with pioglitazone hydrochloride produced a twofold decrease in the ratio of total cholesterol to high-density lipoprotein cholesterol. These results suggest that treatments that improve insulin sensitivity may also have a positive impact on coronary artery disease associated with diabetes.

mRNA quantitation by a simple and sensitive RNAse protection assay.

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.

Molecular cloning, sequence, and expression of cynomolgus monkey cholesteryl ester transfer protein. Inverse correlation between hepatic cholesteryl ester transfer protein mRNA levels and plasma high density lipoprotein levels.

A cDNA clone containing the coding region for cynomolgus monkey cholesteryl ester transfer protein (CETP) was isolated by the polymerase chain reaction with primers based on the human CETP cDNA sequence and cDNA synthesized from liver poly (A+) RNA. Analysis of that cDNA indicated that the nucleotide and amino acid sequences of cynomolgus monkey CETP were greater than 95% homologous with the human sequences. A fragment of the cDNA was used to develop an internal-standard/RNAse protection assay that allowed precise quantification of CETP mRNA levels. Analysis of total RNA from various tissues with this assay revealed that the liver and thoracic aorta expressed high levels of CETP mRNA; the mesenteric fat, adrenal gland, spleen, and abdominal aorta had low but detectable levels of the mRNA; and the brain, kidney, intestine, and skeletal muscle had undetectable levels of that mRNA. When the monkeys were made hypercholesterolemic by a high-fat, high-cholesterol (HFHC) diet, hepatic levels of CETP mRNA increased from 1.6 +/- 0.4 pg/micrograms total RNA (mean +/- SEM) to 4.1 +/- 0.8 pg/micrograms (p less than 0.005); mesenteric fat CETP mRNA increased from 0.4 +/- 0.1 pg/micrograms total RNA to 5.3 +/- 2.2 pg/micrograms (p less than 0.05); and plasma CET activity increased approximately fourfold. The CETP mRNA levels in the thoracic and abdominal aortas were not significantly increased in monkeys fed the HFHC diet, even though those animals had gross atherosclerosis. The apoprotein E mRNA levels, however, were markedly increased in the aortas of monkeys with atherosclerosis, with the largest increase occurring in the abdominal aorta. Taken together, these data suggest that lipid deposition in the artery was not accompanied by increased expression of the CETP gene in that tissue. Statistical analysis showed that a strong, negative correlation existed between hepatic CETP mRNA levels and both high density lipoprotein cholesterol (r = -0.85, p less than 0.001) and apoprotein A-I (r = -0.84, p less than 0.001). These data suggest that HFHC diet-induced changes in high density lipoprotein metabolism may be linked to altered expression of a function CETP gene.

Apo B metabolism in the cynomolgus monkey: evidence for post-transcriptional regulation.

Previous studies have shown that hepatic apo B mRNA levels do not increase in animals fed high cholesterol diets, even though plasma apo B concentrations increase markedly. As a result, it has been suggested that the diet-induced increase in plasma apo B levels was due solely to an inhibited clearance of those lipoproteins. The present study was undertaken to test that hypothesis. Hepatic apo B mRNA levels were measured in liver biopsies taken from five male cynomolgus monkeys before and twice after, they began to consume a high cholesterol diet. The diet had no effect on hepatic apo B mRNA levels, even though it caused a 7-fold increase in the plasma apo B levels. However, measurements of the apo B secretion rate in eight separate monkeys (four chow-fed and four cholesterol-fed) by isotope dilution showed that apo B secretion by the liver was increased 4-fold in the cholesterol-fed monkeys. These data, taken together, indicate that apo B secretion is not regulated by the rate at which the apo B gene is transcribed, but at some point further along in the secretion pathway.

Evidence that the nonparenchymal cells of the liver are the principal source of cholesteryl ester transfer protein in primates.

Previous studies showed that 90% or more of the cholesteryl ester transfer protein (CETP) mRNA is contained in the liver of cynomolgus monkeys. The purpose of this study was to determine if the parenchymal cells (hepatocytes) were the hepatic cell type that contained that mRNA. The parenchymal and nonparenchymal cells were separated by standard methods, and the CETP, apoA-I, apoB, and apoE mRNA content of the preparation determined at each step in the purification process. ApoA-I and apoB are produced only in the parenchymal cells; apoE is produced by both cell types. The mRNA measurements showed that the CETP mRNA: apoA-I mRNA and the CETP mRNA: apoB mRNA ratios were more than 2500-fold greater in the nonparenchymal cell preparation than in the starting material, and that the purified parenchymal cell fraction was virtually devoid of CETP mRNA. In situ hybridization studies showed that, whereas the apoA-I mRNA signal was evenly distributed over the tissue section, the CETP mRNA signal was associated with the hepatic sinusoids, suggesting that it was the hepatic sinusoidal cells that were principally responsible for the high CETP mRNA levels in the liver. We conclude that the nonparenchymal cells are the principal source of CETP in the cynomolgus monkey.