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.

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.

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.

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.

Chronic peripheral lymphatic cannulation in the dog.

Peripheral lymph collected acutely has been commonly sampled as representative of non-visceral interstitial fluid. By developing a prenodal lymphatic-lymphatic (L-L) shunt, we were able to collect peripheral lymph for 3-5 days in unanesthetized dogs. The L-L shunt was constructed entirely of medical grade silicone rubber tubing designed with a slip of coupling which allowed the shunt to be disconnected for lymph collection and reconnected at night. Average peripheral lymph flow (4.9 ml/hr leg) in unanesthetized dogs was almost twice the flow rate previously observed in anesthetized dogs. The average lymph/plasma total protein concentration ratio (0.16), however, was similar to that previously found in anesthetized dogs. Lymph protein concentration fell with the collection during the day and became more concentrated at night. Lymph flow did not change greatly during daytime collection. Average peripheral lymph collection volume was greater than 200 ml/dog. The L-L shunt allows collection of prenodal-lymph in experiments where unanesthetized dogs are required (e.g., feeding studies). They also are useful when multiple protocols are conducted on the same dog or when large volumes of peripheral lymph are required.

Comparison of the lipid and apolipoprotein composition of skeletal muscle and peripheral lymph in control dogs and in dogs fed a high fat, high cholesterol, hypothyroid-inducing diet.

Most studies of peripheral interstitial fluid lipoprotein composition have been made on interstitial fluid-derived from skin and connective tissue. We developed techniques which allowed simultaneous comparison of lymph (a model of interstitial fluid) from skeletal muscle and skin in control (C) and cholesterol-fed (CF) dogs. Lipoprotein fractions were separated by ultracentrifugation. Skeletal muscle interstitial fluid HDL concentrations were approximately twice those of skin. However, the concentration of VLDL-LDL particles was similar in both interstitial spaces. HDL particles from both microvascular beds showed evidence of extensive remodelling when compared to plasma HDL from the same animal. Relative to apo A-I, skeletal muscle HDL was enriched in free cholesterol and apo E (C and CF dogs) and apo A-IV (CF dogs). Skin-derived HDL was consistently enriched in free cholesterol, apo E and A-IV in both C and CF dogs. These studies indicate that similar remodeling of plasma HDL occurs in widely different tissues which together constitute approximately 70% of the total interstitial space. The relatively high concentration of plasma-derived and remodeled HDL within the interstitial space of skeletal muscle is consistent with that tissue's importance in reverse cholesterol transport.

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.

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)

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.

Secretion of pre-beta-migrating apoA-I by cynomolgus monkey hepatocytes in culture.

Cynomolgus monkey hepatocytes that had been stored frozen were thawed, established in culture, and used to study apoA-I secretion. Protein synthetic activity was low at first, but increased with time, approaching what appeared to be the constitutive levels of the intact liver by day 7. During the first week, cellular RNA levels increased from 5.3 +/- 0.3 to 18.6 +/- 1.0 micrograms/10(6) cells; albumin secretion rates increased from undetectable to 55.4 micrograms/10(6) cells per day; apoA-I mRNA levels increased from 174 +/- 12 to 564 +/- 145 ng/10(6) cells; and apoA-I secretion rates increased from undetectable to 2.11 +/- 0.27 micrograms/10(6) cells per day. Analysis of day 7-conditioned media by agarose electrophoresis, gradient gel electrophoresis-immunoblotting, and column chromatography, showed that the apoA-I produced by the cells was present in three distinct forms. One had an apparent molecular mass greater than 1 million Da, migrated pre-beta, and accounted for 11 +/- 3 (mean +/- SD)% of the total; one had an apparent molecular mass of 104 kDa, had alpha migration, and accounted for 27 +/- 2% of the total; and one had an apparent molecular mass of 50 kDa, migrated pre-beta, and accounted for 46 +/- 9% of the total. These data support the proposition that the pre-beta-migrating, 50 kDa, apoA-I-containing particles identified in the plasma of cynomolgus monkeys are nascent hepatic HDL.

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.

Purification, cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-II: comparison to the human sequence.

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-II. Sequence analysis indicated that the monkey apo C-II cDNA was 200 bp longer than the human and the difference in size was all in the 5 degrees untranslated region of mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-II cDNA sequence encoding a preprotein of 101 amino acids - identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-II amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-II and this is confirmed by isoelectric focusing and immunoblotting.

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.

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state.

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.

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.