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

Christine K. Castle1, Susan L. Kuiper1, William L. Blake1, Beverly Paigen2, Keith R. Marotti1, and George W. Melchior1

1 Pharmacia and Upjohn, Inc., Kalamazoo, Michigan 49001; and 2 Jackson Laboratories, Bar Harbor, Maine 04609

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.

free apolipoprotein A-I; pre-beta apolipoprotein A-I; high-density lipoprotein lipase; mouse plasma lipase; KKAy mouse; mouse model of non-insulin-dependent diabetes mellitus

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

NON-INSULIN-DEPENDENT diabetes mellitus (NIDDM) is associated with obesity, hyperinsulinemia, hypertriglyceridemia, and low high-density lipoprotein (HDL) levels. The mechanism by which NIDDM leads to low HDL levels is not known, but it has often been linked to the hypertriglyceridemia. There are several rodent models of NIDDM (5, 7, 19, 23, 30), and all have one or more of the characteristics of the human disease with one key exception: none have low HDL levels, even when severely hypertriglyceridemic. It has been suggested (7, 29) that rodents are able to maintain high plasma HDL levels despite the severe hypertriglyceridemia because they lack the ability to redistribute cholesteryl esters among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP) (36). That hypothesis was based on the results of previous studies in which CETP was expressed in C57BL/6 mice (27) and on the premise that the hypertriglyceridemia would augment the enrichment of HDL with triglyceride and thereby render that lipoprotein more vulnerable to destruction by a lipase (3, 11, 12).

The purpose of the experiments described here was to test that hypothesis. To accomplish that, simian CETP was expressed in the KKAy mouse, a rodent model of NIDDM with extraordinarily high HDL levels. Simian CETP was chosen as the source of CETP for these studies because it is virtually identical in structure to human CETP (32) and because that genetic construct has been shown previously to work exceptionally well in mice (24, 25, 27, 29).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals and diets. KKAy-CETP mice were produced as follows: C57BL/6J mice expressing the lethal yellow gene (C57BL/6J-Ay) were crossed with C57BL/6 mice expressing simian CETP to produce C57BL/6J-Ay-CETP mice. These mice are referred to hereafter as "B6-Ay-CETP mice." The B6-Ay-CETP mice were produced at Jackson Laboratories with the use of C57BL/6J-Ay mice from their colony and UCTP-20 mice (25, 27) provided by Pharmacia and Upjohn. A colony of B6-Ay-CETP mice was subsequently established at Pharmacia and Upjohn. To produce KKAy-CETP mice, B6-Ay-CETP mice were crossed with KK mice (22) and yellow offspring expressing CETP were recrossed with KK mice until that process had been repeated for six generations. At that point, the mice were designated "KKAy-CETP." The mice used for these studies were obtained from that colony. All were between 5 and 6 mo of age at the time of death and had consumed a custom-made natural-products rodent diet (Upjohn Irradiated Laboratory Rodent Diet 5011-3; PMI Feeds, Richmond, IN) from the time that they were weaned. The diet contained (by weight) 20.7% protein, 6.5% fat, 52.0% carbohydrate, 4.6% crude fiber, and adequate vitamins and minerals. Fat made up 16.8% of calories, and the cholesterol content was 17.4 parts per million (0.002% by weight). The diet contained 3.13 cal of metabolizable energy per gram.

Fifty mice were used for these studies: 22 KKAy mice (13 females and 9 males) and 28 KKAy-CETP mice (16 females and 12 males). On the day of death, food was removed from the cages at 7:00 AM, and at 1:00 PM, the mice were weighed, anesthetized with ether, and exsanguinated. The blood was heparinized and pooled for analysis. Seven KKAy pools were created that consisted of blood from three females, four females, four females, two females, and three pools each containing the blood from three males. Likewise, eight KKAy-CETP pools were created that consisted of blood from four females, four females, five females, three females, and four pools each containing the blood from three males. All procedures in this study are in compliance with the Animal Welfare Act regulations and with the National Research Council Guide for the Care and Use of Laboratory Animals.

Analytic measurements. Mouse plasma has exceptionally high lipase activity (14, 15, 33). The lipase activity in KKAy and KKAy-CETP mouse plasma was measured using human HDL that contained [9,10-3H]triolein (Du Pont-NEN, Boston, MA) as substrate. The HDL was radiolabeled with [9,10-3H]triolein exactly as described previously (27). Ten microliters of the HDL solution (23 µg of HDL cholesterol, 4.03 × 105 dpm of [9,10-3H]triolein) and 27 µl of the mouse plasma were diluted to a final volume of 200 µl with PBS and incubated for 5 h at 37°C. The reaction was stopped, the free fatty acids were extracted as described by Belfrage and Vaughan (4), and the associated radioactivity was quantified by liquid scintillation counting. Less than 0.5% of the radioactivity was extractable from the HDL substrate in the absence of a source of lipase.

Blood glucose and plasma insulin, cholesterol, triglyceride, phospholipid, and apolipoprotein (apo) A-I levels were all measured as described previously (25, 27, 28), as was the CETP activity and the HDL composition. All samples were placed in ice immediately after they were obtained, and the plasma glycerol and total and HDL-associated triglyceride concentrations were determined within 1 h thereafter. The free glycerol concentrations of plasma were determined using an enzymatic assay (Sigma, St. Louis, MO). Those values averaged 32 ± 9 mg/dl in KKAy mice and 36 ± 2 mg/dl in KKAy-CETP mice and were subtracted from the total glycerol concentrations to obtain the triglyceride concentrations. The concentration of HDL-associated triglycerides and HDL-associated cholesterol in the plasma was determined after polyethylene glycol precipitation as described previously (7). The cholesterol-to-triglyceride ratio determined by polyethylene glycol precipitation was then used to correct for loss of HDL triglyceride that occurred before and during the isolation of HDL by ultracentrifugation.

HDL size. The Stokes diameter of some HDL was measured by nondenaturing gradient gel electrophoresis. A Hoefer SE600 gel casting assembly and electrophoresis unit (Hoefer Scientific, San Francisco, CA) was used to make the gel and to perform the electrophoresis. A concave, exponential gradient of polyacrylamide ranging from 5.4 to 36% was used to form the gel. To produce the gradient, stock acrylamide solutions identical to those described by Asztalos et al. (2) were used. Glass plates, 16 × 18 cm and separated by a 3-mm spacer, were used to cast the gel. The acrylamide was pumped into the space between the plates until it had reached a height of 14 cm and was then allowed to polymerize. A 5% acrylamide solution was then layered over the polymerized gel, a 5- or 10-well comb (3 mm thick) was inserted, and the upper gel was allowed to polymerize for at least 4 h. Just before the electrophoresis was started, the combs were removed and the samples (25-50 µg of HDL protein dissolved in electrophoresis buffer) or standards of known Stokes diameter (high-molecular-weight electrophoresis calibrators plus ovalbumin; Pharmacia Biotech, Piscataway, NJ) were added to the wells. The power supply was set at 220 V, and the electrophoresis was started by applying a 25-mA current for 30 min (to allow the protein to penetrate the top of the gel). The amperage was then increased until the voltage became the limiting factor, and those conditions were maintained for the duration of the run (40 h). The finished gels were removed from the apparatus and stained with Coomassie blue R-250 for 3 h. The mean Stokes diameter of a given HDL was determined by comparing its migration distance with that of the standards.

HDL structure. The dimensions of HDL were also determined from the chemical composition by use of the model of HDL structure proposed by Shen, Scanu, and Kézdy (SSK model; Ref. 35) as follows.

With the assumption of ideal mixing of the components, the density of a lipoprotein particle, d, is calculated from the weight percent composition data by the equation
<IT>d</IT> = <FR><NU>100</NU><DE><FR><NU>%<SUB>aa</SUB></NU><DE>1.373</DE></FR> + <FR><NU>%<SUB>pl</SUB></NU><DE>1.031</DE></FR> + <FR><NU>%<SUB>ch</SUB></NU><DE>1.033</DE></FR> + <FR><NU>%<SUB>tg</SUB></NU><DE>0.915</DE></FR> + <FR><NU>%<SUB>ce</SUB></NU><DE>0.958</DE></FR></DE></FR> (1)
where the symbols %aa, %pl, %ch, %tg, and %ce stand for the weight percents of protein, phospholipid, free cholesterol, triglyceride, and cholesteryl ester, respectively. The density of a spherical particle of molecular weight (mol wt) and radius (in cm) rcm is given by
<IT>d</IT> = <FR><NU>mol wt</NU><DE>molar vol</DE></FR> = <FR><NU>mol wt</NU><DE><IT>N</IT> × molecular vol</DE></FR> = <FR><NU>mol wt</NU><DE><IT>N</IT> <FR><NU>4&pgr;</NU><DE>3</DE></FR> <IT>r</IT><SUP>3</SUP><SUB>cm</SUB></DE></FR> (2)
where N is Avogadro's Number. By solving this equation for molecular weight and converting the radius from centimeters to ångströms (rÅ), we obtain
mol wt = <FR><NU>4&pgr;</NU><DE>3</DE></FR> × <IT>d</IT> × 0.6023 × <IT>r</IT> <SUP>3</SUP><SUB>Å</SUB> (3)
In accordance with the SSK model, the outer surface of the particle is defined solely by the number of amino acid residues, naa, and by the number of phospholipid residues, npl
4 &pgr; <IT>r</IT> <SUP>2</SUP><SUB>Å</SUB> = 15.6 × <IT>n</IT><SUB>aa</SUB> + 62.7 × <IT>n</IT><SUB>pl</SUB> (4)
The number of residues of amino acids is calculated from the molecular weight of the particle, the weight percentage of protein, and the average residue molecular weight (mol wtaa, estimated to be 112) by
<IT>n</IT><SUB>aa</SUB> = <FR><NU>%<SUB>aa</SUB></NU><DE>100</DE></FR> × <FR><NU>mol wt</NU><DE>112</DE></FR> (5)
The number of other component molecules per particle is calculated in the same manner, using the average molecular weights: 775 for phospholipids, 387 for cholesterol, 850 for triglycerides, and 650 for cholesteryl esters. Thus the number of phospholipid molecules per particle, npl, is
<IT>n</IT><SUB>pl</SUB> = <FR><NU>%<SUB>pl</SUB></NU><DE>100</DE></FR> × <FR><NU>mol wt</NU><DE>775</DE></FR> (6)
By substituting naa and npl into Eq. 4, solving it for molecular weight, and equating it to the expression of the molecular weight from Eq. 3, we obtain
<FR><NU>4&pgr;</NU><DE>3</DE></FR> 0.6023 <IT>d</IT> <IT>r</IT> <SUP>3</SUP><SUB>Å</SUB> = <FR><NU>4&pgr;100 <IT>r</IT> <SUP>2</SUP><SUB>Å</SUB></NU><DE>15.6 <FR><NU>%<SUB>aa</SUB></NU><DE>112</DE></FR> + 62.7 <FR><NU>%<SUB>pl</SUB></NU><DE>775</DE></FR></DE></FR> (7)
This equation then yields the radius of the particle as
<IT>r</IT><SUB>Å</SUB> = <FR><NU>300</NU><DE>0.6023 <IT>d</IT> <FENCE>15.6 <FR><NU>%<SUB>aa</SUB></NU><DE>112</DE></FR> + 62.7 <FR><NU>%<SUB>pl</SUB></NU><DE>775</DE></FR></FENCE></DE></FR> (8)
With this value of rÅ, the molecular weight is calculated from Eq. 3, and then the number of individual component molecules is calculated just as naa is calculated from Eq. 5.

The SSK model posits that the volume of the spherical core of the lipoprotein is the sum of the volumes of the constituent triglyceride and cholesteryl ester molecules. The radius of the core is then given by
<IT>r</IT><SUB>core</SUB> = <RAD><RCD><FR><NU>3</NU><DE>4&pgr;</DE></FR> (1556 ⋅ <IT>n</IT><SUB>tg</SUB> + 1068 ⋅ <IT>n</IT><SUB>ce</SUB>)</RCD><RDX>3</RDX></RAD> (9)
where ntg and nce stand for the number of triglycerides and cholesteryl esters, respectively. The thickness of the surface monomolecular layer is rÅ - rcore. Finally, if the surface of the core is covered exclusively by the hydrophobic tails of the phospholipid and cholesterol molecules, then the area occupied by one phospholipid tail is
tail area = <FR><NU>4 &pgr; <IT>r</IT><SUP>2</SUP><SUB>core</SUB> − 39.1 <IT>n</IT><SUB>ch</SUB></NU><DE><IT>n</IT><SUB>pl</SUB></DE></FR> (10)
where nch is the number of free cholesterols.

Statistics. Differences in the means were tested using two-way ANOVA, with the factors being CETP and gender. Individual group comparisons were tested for significance using the Student-Newman-Keuls multiple comparison method (17). The null hypothesis was rejected if the probability of obtaining a larger t value simply due to chance was <1 in 20.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Mice have high levels of a triglyceride lipase in their circulation (14, 15, 33). To confirm that that lipase was present in the plasma of KKAy mice and to gain some insight into which lipoproteins were substrates for that lipase, human HDL radiolabeled with [9,10-3H]triolein was incubated with human plasma, mouse plasma, mouse plasma supplemented with mouse very low density lipoprotein (VLDL), and mouse plasma supplemented with mouse HDL. The results of that experiment are shown in Table 1. Note that the production of [9,10-3H]oleic acid was markedly higher when the enzyme source was KKAy mouse plasma than when it was human plasma. This shows that KKAy mice also contain high levels of this lipase in their circulation. Note also that addition of VLDL had no effect on the rate of production of the radiolabeled oleic acid, whereas addition of mouse HDL reduced recovery of the labeled free fatty acid almost 20%. Thus HDL triglycerides appear to be substrates of the lipase in mouse plasma but not VLDL triglycerides. Consequently, we hereafter refer to that lipase as "HDL lipase."

                              
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Table 1.   HDL lipase activity in KKAy mouse plasma

Table 2 shows the mean body weight, blood glucose concentration, plasma insulin concentration, HDL lipase activity and cholesteryl ester transfer activity in C57BL/6 mice, KKAy mice, and KKAy-CETP mice. The KKAy mice are obese, hyperglycemic, and severely hyperinsulinemic relative to the C57BL/6 mice, and expression of CETP had no effect on those characteristics. Thus the KKAy-CETP mice do, as intended, express fully those genetic traits associated with NIDDM. Expression of CETP was, however, associated with a substantial lowering of the HDL lipase activity.

                              
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Table 2.   Body weights, blood glucose and plasma insulin, HDL lipase, and CETP activity of C57BL/6, KKAy, and KKAy-CETP transgenic mice

Table 3 shows a comparison of the plasma lipid concentrations between KKAy and KKAy-CETP mice. Both strains of mouse are hypertriglyceridemic relative to the C57BL/6 mouse, in which the plasma triglycerides average 46 ± 2 mg/dl in males and 36 ± 18 mg/dl in females, and, in agreement with our previous observations (7), the KKAy mice have extraordinarily high plasma HDL levels whether measured as HDL cholesterol, HDL phospholipid, or HDL protein (Table 4). Thus hypertriglyceridemia per se does not cause "low" HDL levels, at least not in the mouse. By contrast, expression of CETP in the KKAy mouse had a severe effect on the HDL levels: HDL cholesterol concentrations decreased by more than 6-fold, HDL phospholipids decreased by 3.7-fold, HDL protein decreased by 3.1-fold, and HDL triglyceride increased by 2.8-fold.

                              
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Table 3.   Plasma lipid concentrations in KKAy and KKAy-CETP transgenic mice

                              
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Table 4.   HDL protein and apo A-I concentrations in KKAy and KKAy-CETP transgenic mice

Table 4 shows a comparison of the HDL protein and apo A-I distribution in the two strains of mouse. There was a marked effect of CETP expression on both of those parameters. Total HDL protein levels were reduced by ~70% and HDL-associated apo A-I levels by ~50% in KKAy-CETP mice, relative to the KKAy mice. This decrease in HDL-associated apo A-I was accompanied by an increase in the levels of non-HDL-associated apo A-I (apo A-I that did not migrate with HDL during agarose electrophoresis) in the plasma from mice expressing CETP. Comparison of agarose electrophoresis immunoblots with agarose electrophoresis strips stained with oil red O (Fig. 1) showed that there was little or no lipid staining in the area of the non-alpha -migrating apo A-I. As a result, that apo A-I is referred to as "free" apo A-I in this report. Quantification of the free apo A-I by agarose electrophoresis immunoblotting (8, 26, 27) indicated that ~5% of the apo A-I in the circulation of either C57BL/6 or KKAy plasma is free, whereas ~25% of the apo A-I in the circulation of KKAy-CETP plasma is free. Note that free apo A-I has been reported to inhibit lipase activity (10). The fact that KKAy-CETP mice have such high levels of free apo A-I may account for the reduced lipase activity levels observed in these animals (Table 2).


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Fig. 1.   Agarose electrophoresis of plasma from a KKAy-cholesteryl ester transfer protein mouse pool. A: immunoblot stained with anti-mouse apolipoprotein A-I (apo A-I). B: same plasma stained with lipid stain oil red O. Arrow, what is referred to in text as "free" apo A-I. Note that there was little or no oil red O staining in region of free apo A-I.

The apo A-I content of KKAy-CETP HDL (calculated from the HDL-associated apo A-I concentration and the HDL molecular weight; Table 5) was significantly higher than that of KKAy HDL (3.0 ± 0.4 mol of apo A-I/mol of KKAy-CETP HDL vs. 2.1 ± 0.1 mol of apo A-I/mol of KKAy HDL; P = 0.0047). The higher apo A-I content of KKAy-CETP HDL was probably a result of the increased levels of free apo A-I in those mice.

                              
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Table 5.   Physical characteristics of the HDL from KKAy and KKAy-CETP mice calculated from composition

Table 5 compares selected physical characteristics of the HDL from KKAy mice and from KKAy-CETP mice. Those characteristics were calculated from the HDL composition using the SSK model. The SSK model envisages HDL as a spherical particle with a hydrophobic core surrounded by a monolayer of phospholipids and free cholesterol. Previous studies with C57BL/6 mice (25) showed that the HDLs were monodisperse with regard to size and that the Stokes radius calculated using the SSK model was in very good agreement with that measured by nondenaturing gradient gel electrophoresis. To confirm that the same was true of KKAy-CETP mice, HDLs isolated from two pools of KKAy-CETP plasma were analyzed by both methods. Nondenaturing gradient gel electrophoresis showed that the HDLs from the KKAy-CETP mice were monodisperse, with mean Stokes radii of 52.2 and 49.2 Å. The corresponding Stokes radii calculated using the SSK model were 52.2 and 50.7 Å. Thus the HDLs from KKAy-CETP mice are monodisperse, and the SSK model does, in fact, accurately predict its Stokes radius. That being the case, the data in Table 5 indicate that CETP expression did not have a significant effect on the density or size of the HDL but did affect the surface thickness and the area occupied by the nonpolar portion of the phospholipids (termed the phospholipid tail area). The data indicate that the hydrophobic fatty acyl chains of the phospholipids are spread apart to a greater extent in KKAy-CETP HDL than in KKAy HDL, an effect that is probably largely due to the absence of free cholesterol, i.e., the fatty acyl chains that are present have to cover a greater area than they would with free cholesterol present in that layer.

Table 6 shows the effect of CETP expression on the composition of the HDL. There was no difference in the amount of protein (expressed as amino acids) associated with the particles, but there was a statistically significant decrease in the number of molecules of phospholipid, free cholesterol (free cholesterol was not detected in the HDL of KKAy-CETP mice), and cholesteryl ester per molecule of HDL and a marked increase in the number of molecules of triglyceride per molecule of HDL. Thus CETP expression had a profound effect on the composition of the HDL core. If one assumes a mean molecular volume of 1,090 Å3 for cholesteryl esters and 1,600 Å3 for triglycerides, then triglycerides make up, on average, only 4.3% by volume of the KKAy HDL core, whereas they make up 48% by volume of the KKAy-CETP HDL core. In view of the higher triglyceride content and looser packing of the phospholipids at the core surface, the KKAy-CETP HDL should be substantially more vulnerable to the HDL lipase than KKAy HDL.

                              
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Table 6.   Composition of HDL from KKAy and KKAy-CETP mice

Although the purpose of these studies was to evaluate the effects of CETP expression in these spontaneously hypertriglyceridemic mice, some gender and gender X gene interactions are noteworthy. The plasma HDL levels of females tended to be higher than those of males, whether measured as HDL cholesterol, triglyceride, or phospholipid (Table 3). The differences in HDL-associated phospholipid and triglyceride levels in the plasma were statistically significant, and the difference for HDL-associated cholesterol was of borderline significance. This observation (that the HDL levels are higher in female KKAy mice) is in agreement with reports for other mouse strains (31). There were also gender differences in the composition of the HDL (Tables 4-6). Females had higher levels of both HDL-associated and free apo A-I (Table 4), their HDL tended to be larger and less dense than that of males (Table 5), and the phospholipid and cholesteryl ester contents of the HDL were higher than those of males (Table 6). Finally, there were five instances of significant gene × gender interactions: plasma HDL-associated triglyceride (P = 0.0410), plasma apo A-I levels (P = 0.0066), pre-beta apo A-I levels (P = 0.0118), HDL-associated apo A-I levels (P = 0.0210), and HDL phospholipid content (P = 0.0569).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The purpose of the studies described here was to test the hypothesis that hypertriglyceridemic KKAy mice are able to maintain exceptionally high HDL levels because they lack the ability to redistribute cholesteryl esters and triglycerides between VLDL and HDL. It was reasoned that the absence of a mechanism for the redistribution of those lipids between VLDL and HDL in the wild-type KKAy mice essentially segregated the HDL, thereby protecting those lipoproteins from any effects that expansion of the VLDL triglyceride pool might have, and that introduction of such a mechanism would reverse that segregation, allow the HDLs to accumulate triglycerides, and thereby render them susceptible to lipases. It was further reasoned that the larger the VLDL triglyceride pool, the more rapidly those triglycerides would equilibrate with the HDL, and thus the greater the effect on the HDL. The results support that hypothesis: expression of CETP in the KKAy mouse caused a 17-fold increase in the triglyceride-to-cholesteryl ester ratio in the HDL core and a severe reduction in the plasma HDL concentrations to levels typical of hypertriglyceridemic patients with NIDDM (Table 3). In addition, free apo A-I accumulated in the plasma of the KKAy-CETP mice, suggesting that the triglyceride-enriched HDLs were indeed vulnerable to the action of lipases. Comparable expression of simian CETP in C57BL/6 mice with normal triglyceride concentrations (46 ± 2 mg/dl) resulted in only a twofold decrease in HDL cholesterol levels (from 82 ± 4 to 39 ± 3 mg/dl) and no accumulation of free apo A-I in the plasma (27). Thus the hypertriglyceridemia appears to exacerbate the HDL-lowering effect of CETP by accelerating the enrichment of the HDL with a lipid that is susceptible to destruction by lipases at the expense of one that is not.

The levels of hypertriglyceridemia in the KKAy mice used in the present study are somewhat lower than those reported previously (7) and are significantly lower than those produced in KKAy-CETP mice (Table 3). That was entirely due to the low plasma triglyceride concentrations in the KKAy females, as the plasma triglyceride concentrations of the KKAy males were not significantly different from those of KKAy-CETP males when tested by the Student-Newman-Keuls multiple comparison method. We have no explanation for the low triglyceride levels in KKAy females other than that day-to-day fluctuations of the plasma triglyceride concentrations in these females can be quite large, and perhaps we unwittingly selected animals in which the plasma triglyceride concentration was lower than normal at the time that they were bled. As a result, the observation that CETP expression enhanced the severity of the hypertriglyceridemia (Table 3) may be fortuitous. To exclude the possibility that the changes observed in the HDL levels in this study were due to the more severe hypertriglyceridemia in KKAy-CETP mice rather than to the expression of CETP, we compared the levels and composition of HDL isolated from pools of plasma with the same triglyceride concentrations (650 ± 30 vs. 652 ± 211 mg/dl, KKAy vs. KKAy-CETP, respectively). The HDL cholesterol levels in these KKAy plasma pools averaged 139 ± 3 mg/dl; those in the KKAy-CETP plasma averaged 18 ± 3 mg/dl. The triglyceride-to-cholesteryl ester molar ratio in the core of these KKAy HDLs averaged 0.0459 ± 0.0262, that in the KKAy-CETP HDLs averaged 0.728 ± 0.217, a 16-fold difference. These results fully support the conclusion that CETP is the agent affecting HDL levels and that in the absence of CETP, KKAy mice can develop a severe hypertriglyceridemia with no detectable effect on the HDL levels.

Expression of CETP in mice lowers their plasma HDL level (1, 25, 27), and the observation that hypertriglyceridemia exacerbates that effect has been reported previously (18), although the changes observed by those investigators were much smaller than those reported here. In addition, the inferences regarding the mechanism by which the hypertriglyceridemia mediated that lowering are also different. The data presented here suggest that it is the remodeling of the HDL by CETP that accounts for its loss from the plasma. That remodeling consists of an enrichment of the HDL core with triglyceride and the loss of cholesteryl ester. The data further indicate that as the pool of triglycerides available for transfer increases, that process is stimulated and the effects on HDL are exacerbated. This interpretation is at variance with that of Hayek et al. (18), who found minimal change in the HDL triglyceride content but a marked reduction in the size of the HDL from human apo CIII-CETP transgenic mice and argued that it was a CETP-induced increase in the cholesteryl ester and apo A-I fractional catabolic rates that were responsible for the loss of HDL. How hypertriglyceridemia would accelerate the cholesteryl ester and apo A-I fractional catabolic rates, other than through the mechanism proposed above, is not clear. Rather, one could argue that the same process was operative in the human apo CIII-CETP mice used by Hayek et al. (18) as was operating in the KKAy-CETP mice described here and that the decrease in HDL size and the increased apo A-I and cholesteryl ester fractional catabolic rates observed by those investigators were secondary effects due to the enrichment with and subsequent hydrolysis of HDL core triglycerides.

The accumulation of relatively large amounts of free apo A-I in the plasma of the KKAy-CETP mice was somewhat unexpected, but two aspects of the metabolic state of that animal, HDL with an elevated triglyceride content and kidneys with thickened glomerular capillary membranes, working in conjunction could account for that occurrence. Note that triglycerides constitute ~50% by volume of the KKAy-CETP HDL core. We have shown previously (27), using mouse HDL in which triglyceride constituted only one-third of the core volume, that hydrolysis of those triglycerides in vitro resulted in collapse of the HDL particle and release of free apo A-I and other surface constituents into the plasma. That occurred because in vitro there is no source of cholesterol to replace the triglyceride lost from the HDL. The same process may occur in the KKAy-CETP mouse, and, although there is a source of cholesterol to replace the triglyceride, the rate of replacement is inadequate, i.e., the process of cholesterol uptake and esterification is too slow to compensate for triglyceride hydrolysis. Thus there is net loss of core component that results, with time, in collapse of the particle and release of free apo A-I. In addition, because the KKAy mice have thickened glomerular capillary basement membranes (13), plasma clearance of that free apo A-I is retarded. The net effect of these processes is a low HDL level, increased concentrations of free apo A-I, and apo A-I-rich HDL. The latter phenomenon is presumably a result of the high concentrations of free apo A-I, i.e., the high levels of the free protein saturate the HDL with apo A-I, probably by displacing other apoproteins, creating the apo A-I-rich particle.

It has been reported previously (10) that free apo A-I inhibits a lipase in humans that may be analogous to what we refer to as the HDL lipase in these mice. The KKAy-CETP mice clearly had increased levels of free apo A-I in their plasma, and the activity of the HDL lipase in that same plasma was severely reduced. That the lower lipase activity was due to the higher levels of free apo A-I in these mice cannot be determined from these experiments, but that is a reasonable deduction. Note, however, that this process tends to preserve rather than destroy the HDL, since hydrolysis of the triglycerides is the key step in destruction of the particle. That may explain why complete loss of HDL is prevented in these mice and why the HDLs that remain are triglyceride rich.

One additional observation of interest was the virtual absence of free cholesterol in the surface of the KKAy-CETP HDL. Normally, ~25% of the cholesterol molecules in the HDL of mice are nonesterified and are located at the surface of the lipoprotein (27). Once esterified, that cholesterol relocates to the core of the HDL, leaving space in the surface for additional free cholesterol. The rate-limiting step in this cholesterol uptake-esterification-relocation process appears to be esterification, for significant amounts of free cholesterol are normally present in the lipoprotein. The fact that the free cholesterol levels were so low in the KKAy-CETP HDL indicates that either the esterification rate is increased and therefore no longer rate limiting in these mice or uptake of free cholesterol by these apo A-I-rich, triglyceride-rich HDLs is impaired. We cannot at this point distinguish between these two possibilities, but regardless of which process has been altered, it has had a significant effect on the structure of the HDL. It remains to be determined what, if any, effect these alterations have on HDL function.

One noteworthy difference between the HDL of the KKAy-CETP mice and that of humans with NIDDM is the size range of those lipoproteins. The HDLs in humans with hypertriglyceridemic NIDDM are polydisperse with regard to size, and those HDLs are generally smaller than normal. That was not the case with KKAy-CETP HDL. Although the plasma levels of HDL in the KKAy-CETP mice were quite similar to those of humans with hypertriglyceridemic NIDDM, those HDLs were monodisperse with respect to size, and their Stokes diameters were not different from those of KKAy HDLs. What accounts for that difference in size range between the two species is not known, although it has been suggested that differences in the structure between mouse and human apo A-I are somehow responsible (9, 34). What is clear from the studies reported here is that CETP does not contribute to the size polydispersity, and thus this rodent model of human NIDDM is inexact in this regard.

The results of the studies reported here establish that the creation of a pathway for movement of cholesteryl esters and triglycerides between lipoproteins can, in the presence of a large pool of triglycerides, produce an NIDDM-like lipoprotein profile in the KKAy mouse and imply that it is the enrichment of the HDL core with triglyceride that accounts for the HDL-lowering effect. The question then becomes whether this is the mechanism responsible for the low HDL levels in hypertriglyceridemic humans. Three observations suggest that it is. First, individuals with NIDDM can have all of the components necessary for destruction of the HDL by this pathway: active CETP, triglyceride-rich HDL (6), and an active HDL lipase (16). Second, it has been shown that incubation of a mixture of human VLDL, HDL, CETP, and hepatic lipase in the test tube does, in fact, disrupt human HDL (11, 12). Finally, humans with severely reduced CETP levels have very high HDL concentrations, and the composition of that HDL is quite similar to the HDL of KKAy mice (20, 21, 37). Thus, by expressing the CETP gene in these mice, we have created a condition indistinguishable from that in hypertriglyceridemic humans: a pathway for neutral lipid redistribution in the plasma that, in the presence of a large pool of triglyceride-rich VLDLs, renders the HDLs vulnerable to premature destruction. If this conclusion is correct, then these data explain why hypertriglyceridemia is often associated with low HDL levels in humans but not in rodents and why drugs that lower the plasma triglyceride concentrations tend to raise the HDL levels in humans. Finally, these data strongly imply that inhibition of CETP activity in hypertriglyceridemic humans would increase their plasma HDL levels substantially, an effect that might well slow the progression of the vascular disease so prevalent in individuals with this disorder.

    ACKNOWLEDGEMENTS

We thank Ferenc J. Kézdy for critical discussions during both the course of these experiments and the preparation of the manuscript.

    FOOTNOTES

Address for reprint requests: G. W. Melchior, Cell and Molecular Biology, 7252-209-4, Pharmacia and Upjohn, Inc., Kalamazoo, MI 49001.

Received 20 October 1997; accepted in final form 3 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 274(6):E1091-E1098
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