Heterogeneity in the low density lipoproteins of cholesterol fed African green monkeys (Cercopithecus aethiops).

The low density lipoproteins of cholesterol-fed African green monkeys were isolated by either preparative ultracentrifugation or agarose column chromatography, then radioiodinated and fractionated by agarose column chromatography. A marked variation was noted in the specific radioactivity of the column fractions regardless of the method used to obtain low density lipoproteins. The radioactivity in each fraction migrated with the beta-globluins, but when the fractions were mixed with carrier plasma, alpha-migrating radioactivity was also detected which was associated with the apo-A-I and apo-C of the high density lipoproteins. The uptake of these apoproteins by the high density lipoproteins reduced the variation in specific radioactivity among the low density lipoprotein fractions. By successive injections of the various 125I-labeled low density lipoprotein fractions into the same recipient, it was also possible to compare the kinetic behavior of the fractions. Large and small low density lipoprotein particles were metabolized differently, indicating that the low density lipoproteins from cholesterol-fed African green monkeys are heterogeneous.

Apo A-I metabolism in cynomolgus monkeys: male-female differences.

Female cynomolgus monkeys have significantly higher plasma apo A-I concentrations than males (P = 0.04) and are able to maintain higher levels than the males even after consuming a high-cholesterol diet that severely depresses the apo A-I concentration in primates (P less than 0.05). The mechanism responsible for this difference was investigated by comparing apo A-I turnover (synthesis and catabolism) in males and females consuming monkey chow and in a separate group of males and females that had consumed the high-cholesterol diet for several weeks. The average length of time an apo A-I molecule remained in the plasma compartment of chow-fed monkeys was 2.62 days but decreased to 1.52 days (P less than 0.01) in animals fed the HC diet. There were no male-female differences in the residence times. The absolute turnover rate (mg/day) of apo A-I was not statistically affected by diet or sex; however, the females were substantially smaller than the males (3.8 vs. 4.8 kg; P less than 0.01) and their plasma volumes were significantly smaller than those of the males, even after correction for differences in body wt. (32.6 vs. 37.0 ml/kg, respectively; P less than 0.01). Taken together, the data indicate that females cynomolgus monkeys have higher apo A-I synthesis rates than males of comparable plasma volume (P = 0.03), which we would propose accounts for the higher plasma apo A-I concentrations evident in females.

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.

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.

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.

The primary structure of cynomolgus monkey apolipoprotein A-1 deduced from the cDNA sequence: comparison to the human sequence.

We have cloned and analyzed a cDNA containing the complete coding sequence for cynomolgus monkey apolipoprotein A-1 (apoA-1). This cDNA clone was found to share approx. 97% nucleotide sequence identity with the published human apoA-1 and encodes a protein of the same size as the human protein. Paired proline residues are present at positions 3 and 4 in the mature protein as has been reported for other primate species and the propeptide sequence is identical to the human propeptide. The amino acid content derived from the nucleotide sequence predicts a more basic protein than human apoA-1 and this was confirmed by isoelectric focusing analysis. In addition, we present evidence for two different transcriptional initiation sites for the cynomolgus monkey gene in contrast to only one for human.

Changes in plasma apo B, apo E, apo A-I, and apo A-IV concentrations in dogs consuming different atherogenic diets.

To define the relationship between apoprotein levels and plasma cholesterol concentration in dogs, we measured the cholesterol, apo B, apo E, apo A-IV, and apo A-I levels in 6 dogs fed a synthetic diet (Diet I), and in 5 dogs fed dog chow supplemented with lard, cholesterol, bile salts, and propylthiouracil (Diet II). The diet-induced hypercholesterolemia exceeded 900 mg/dl in dogs fed Diet I and was accompanied by a 12-fold increase in apo B, a 30-fold increase in apo E, an 8-fold increase in apo A-IV, and a 1 1/2-fold increase in apo A-I. By contrast, the hypercholesterolemia averaged 1300 mg/dl in dogs fed Diet II and was accompanied by a 12-fold increase in apo B, an 11-fold increase in apo E, a 3-fold increase in apo A-IV, and a 5-fold decrease in apo A-I levels. When 3 of the Diet I dogs were switched to dog chow, their plasma cholesterol, apo B, and apo E levels dropped to 30% of their peak value within 7 days. The change in apo B and apo E levels was found to be highly correlated with the change in plasma cholesterol concentrations in each of the Diet I animals (r2 ranged from 0.92 to 0.99 for both apoproteins). A strong linear relationship was also observed between apo E and apo B (r2 ranged from 0.94 to 0.98), indicating that the plasma apo E to apo B ratio remained constant in these animals as the hypercholesterolemia progressed or regressed.

Apo E levels and distribution in rhesus monkeys consuming an atherogenic diet.

The effect of an atherogenic diet on serum apo E levels and distribution among the lipoproteins of rhesus monkeys was studied. Animals able to maintain their serum cholesterol levels below 250 mg/dl (hyporesponders) showed no significant change in their serum apo E levels; however, in monkeys whose serum cholesterol concentrations ranged from 250 to 850 mg/dl, serum apo E levels appeared to have increased in direct proportion to plasma cholesterol concentration (r2 = 0.92) such that in monkeys whose serum cholesterol concentration exceeded 650 mg/dl (hyperresponders), the apo E levels had increased 5-6-fold. The majority of the apo E (60%) in hyporesponders consuming the atherogenic diet was associated with HDL, whereas only 10% of the serum apo E was associated with HDL in hypercholesterolemic hyperresponders. Nonetheless, the absolute amount of HDL-associated apo E was the same in both phenotypes. Thus, essentially all of the increase in serum apo E levels in hyperresponders was due to an increase in non-HDL-associated apo E. The mean density of the fraction showing the greatest increase in apo E, and accounting for the majority of the d less than 1.063 g/ml apo E in hyperresponders, was 1.010 g/ml. That fraction was distinct from the lipoproteins principally responsible for the increase in apo B and cholesterol levels in those animals. The latter were smaller in size and higher in density than the major apo E-rich fraction. Nonetheless, the d less than 1.063 g/ml apo E apparently circulates on apo B-containing particles, since it was retained on an anti-apo B immunoaffinity column. These data show that a diet-induced hypercholesterolemia is accompanied by a marked increase in serum apo E levels in rhesus monkeys, but that the lipoproteins principally responsible for the increase in apo E levels are distinct from those mainly responsible for the hypercholesterolemia. They also suggest that the levels of apo E-containing HDL in hypercholesterolemic hyperresponders are not significantly lowered by the diet, even though those animals' apo A-I levels were severely reduced. Thus, both the LDL and the HDL of the hypercholesterolemic primate contain apo E-rich subfractions which are metabolically distinct from the principal lipoprotein family in each fraction.

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.

The effect of polyunsaturated fats on bile acid metabolism and cholelithiasis in squirrel monkeys.

Squirrel monkeys (Saimiri sciureus) were fed diets containing safflower oil, butter, or coconut oil and 1 mg cholesterol/cal for 15--17 mo to examine the effect of type of fat on cholelithiasis and bile acid metabolism. Controls were fed low cholesterol diets containing an isocaloric mixture of the three fats. Cholic acid fractional catabolic rate, pool size, chenodeoxycholic acid pool size, and total bile acid pool size and excretion rate were estimated using a modification of Lindstedt's isotopic turnover procedure. The animals fed the safflower oil diet had the highest incidence of cholelithiasis (9/10) when compared to those fed butter (3/7) and coconut oil (1/7). Animals consuming the low cholesterol control diet did not develop gallstones. The butter- and coconut oil-fed groups had significantly (p less than 0.05) expanded bile acid pools when compared to controls, and the butter-fed group had a significantly increased (p less than 0.05) cholic acid fractional catabolic rate. The safflower oil group had the smallest mean bile acid pool and the highest mean lithogenic index of the cholesterol-fed groups. It was concluded that the safflower oil-fed animals had a higher incidence of cholelithiasis than the butter group because, unlike the latter group, they did not compensate for a high cholesterol intake by stimulating bile acid synthesis. The animals consuming coconut oil apparently did not absorb cholesterol to the extent of the other groups and as a result their bile did not become saturated with cholesterol.

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.

Apolipoprotein A-I metabolism in cynomolgus monkey. Identification and characterization of beta-migrating pools.

Fresh plasma from control (C) and hypercholesterolemic (HC) cynomolgus monkeys was analyzed by agarose electrophoresis-immunoblotting with antibody to cynomolgus monkey apolipoprotein (apo) A-I. Two bands were evident on the autoradiogram: an alpha-migrating band (high density lipoprotein) and a beta-migrating band that comigrated exactly with cynomolgus monkey low density lipoprotein (LDL). The presence of beta-migrating apo A-I in the plasma of these monkeys was confirmed by Geon-Pevikon preparative electrophoresis, crossed immunoelectrophoresis, and isotope dilution studies in which radiolabeled apo A-I was found to equilibrate also with alpha- and beta-migrating pools of apo A-I in the plasma. Subfractionation of C and HC plasma by agarose column chromatography (Bio-Gel A-0.5M and A-15M) followed by agarose electrophoresis-immunoblotting indicated that the beta-migrating apo A-I in C was relatively homogeneous and eluted with proteins of Mr approximately 50 kD [apo A-I(50 kD)], whereas two beta-migrating fractions were identified in HC, one that eluted with the 50-kD proteins, and the other that eluted in the LDL Mr range [apo A-I(LDL)]. The apo A-I(LDL) was precipitated by antibody to cynomolgus monkey apo B. The apo A-I(50 kD) accounted for 5 +/- 1% (mean +/- SD) of the plasma apo A-I in C plasma, and 15 +/- 7% in HC plasma. No apo A-I(LDL) was detected in C plasma, but that fraction accounted for 9 +/- 7% of the apo A-I in HC plasma. These data establish the presence of multiple pools of apo A-I in the cynomolgus monkey, which must be taken into consideration in any comprehensive model of apo A-I metabolism in this species.

Cholesterol absorption and turnover in hypercholesterolemic dogs.

Cholesterol absorption was measured in chronically hypercholesterolemic dogs by four methods: the fecal recovery method of Borgström (1969, J. Lipid Res. 10: 331-337), the dual isotope method of Zilversmit and Hughes (1974, J. Lipid Res. 15: 465-473), the recovery of cholesterol in thoracic duct lymph collected continuously for 16 hr after a meal, and the recovery of isotopic cholesterol from the liver and plasma 24 hr after the animals consumed an isotope-containing meal. The four methods showed excellent agreement and indicated that dogs fed a cholesterol-rich synthetic diet absorb 5.2 +/- 0.5 g (mean +/- SD) of cholesterol per day and that cholesterol absorption is reasonably constant from week to week in these animals. Separate estimates of cholesterol excretion indicated that these dogs excreted 4.7 +/- 0.5 g of cholesterol per day, and thus were at or near the steady-state with regard to cholesterol input-output. These data, taken together with a previous report (1981, J. Lipid Res. 22: 598-609), indicate that the canine liver can clear up to 300 mg of chylomicron cholesterol/hr, and support the concept that chylomicron remnants do not contribute significantly to the hypercholesterolemia in these animals.

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.

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.

Quantitative analysis of individual bile acids by gas-liquid chromatography: an improved method.

The quantitative analysis of individual bile acids by gas-liquid chromatography has been improved by column oven temperature programming and by a new liquid phase, SP-2401. The method is fast; bile acids are well resolved; retention times are reproducible; detector responses are linear and sensitive to 0.1 mug: and there is little adsorption onto the liquid phase. The method has been successfully used for bile, and it has the potential for use on serum.

Effect of serum and stirring on diffusive 125I-albumin and Evans blue dye uptake.

The diffusive in vitro uptake of homologous 125I-albumin (MA, nmol.cm-2) and Evans blue dye (EBD) (ME, nmol.cm-2) by the deendothelialized canine aorta from serum and from a simple albumin solution with and without EBD and with and without vigorous stirring was measured in 18 preparations. The results show that 1) MA and ME were significantly smaller from serum than from a simple albumin solution, 2) vigorous stirring of the liquid phase caused a slight decrease (approximately 5%) in MA and increase (approximately 9%) in ME, 3) MA was not influenced by the presence of EBD, and 4) at least 90% of the radioactivity in the tissue was free 125I-albumin with an electrophoretic mobility identical to its nonlabeled cohort molecules and albumin in the original reagent. These observations confirm the identity of the tissue radioactivity with the labeled protein in the reagent, show that less than 10% of the labeled protein is irreversibly bound in the tissue, indicate that significant concentration gradients do not occur in the reagent phase, and indicate that albumin appears to interact with other plasma components in the reagent phase.

Chylomicron metabolism during dietary-induced hypercholesterolemia in dogs.

The metabolism of [1-3H]retinol- and [4-14C]cholesterol-labeled chylomicrons was studied in normal and cholesterol-fed dogs in order to estimate the relative contribution of chylomicron remnant cholesterol to diet-induced hypercholesterolemia. The plasma t 1/2 of intravenously administered Sf greater than 400 chylomicrons, Sf 20-400 chylomicrons, and whole lymph doubly labeled with [1-3H]retinol and [4-14C]cholesterol was not significantly prolonged in hypercholesterolemic recipients. When Sf greater than 400 chylomicrons were administered intravenously, 90% of the radioactivity was cleared from the plasma of both normal and cholesterol-fed dogs within 1 hr and 68 +/- 18% appeared in the liver within approximately 2 hr in normal dogs and 4 hr in hypercholesterolemic dogs. The use of the retinol-labeling technique for intestinal lipoproteins provided evidence that some LDL, but essentially none of the HDLc, was derived from d greater than 1.006 g/ml lymph lipoproteins. The failure of significant radioactivity to accumulate in the plasma compartment of hypercholesterolemic dogs after intravenous administration of doubly labeled chylomicrons and the relatively efficient uptake of radioactivity by the liver indicate that the dietary-induced hypercholesterolemia in dogs is not the result of impaired hepatic removal of chylomicron remnants.

An improved method for precise quantitation of cellular and tissue apolipoprotein A-I mRNA levels by use of an internal standard.

We have developed a method for the quantitation of apolipoprotein A-I (apoA-I) mRNA by using a variation of traditional S1 nuclease analysis. This method uses an internal standard RNA that allows a level of precision not obtainable with traditional S1 nuclease analysis. The internal standard RNA is synthesized in vitro using the T7 promoter from the transcription vector, pApoAI, which contains the full length apoA-I cDNA. This RNA molecule is identical to authentic apoA-I mRNA except for the addition of 48 bases at the 5'-end which are derived from the vector. A labeled ssDNA probe is produced from pApoAI in such a way that solution hybridization of the probe to a mixture of total RNA and internal standard RNA followed by S1 nuclease digestion results in the protection of DNA fragments from authentic and internal standard RNA, which differ in size by 48 bases. The DNA fragments can be resolved by gel electrophoresis and quantitated. The addition of internal standard RNA to each hybridization reaction allows for correction of variations in hybridization and other sources of experimental error. Using this method we demonstrate a sevenfold increase in precision (the 90% confidence interval was reduced from +/- 186% to +/- 26% of the mean value) when compared to traditional S1 nuclease analysis of apoA-I mRNA in liver biopsies and hepatocytes in culture. The internal standard/S1 nuclease method can be adapted to the analysis of any mRNA.