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Increased plasma cholesteryl ester transfer activity in obese children
Atherosclerosis 129 (1997) 53 – 58
Increased plasma cholesteryl ester transfer activity in obese children Hidemasa Hayashibe*, Kohtaro Asayama, Takaya Nakane, Norihiko Uchida, Yasusuke Kawada, Shinpei Nakazawa Department of Pediatrics, Yamanashi Medical Uni6ersity, 1110 Shimokato, Tamahocho, Nakakoma, Yamanashi 409 -38, Japan Received 9 April 1996; revised 30 August 1996; accepted 7 November 1996
Abstract To determine whether enhanced activity of cholesteryl ester transfer protein (CETP) contributes to the development of atherogenic lipoprotein profiles in obese children, plasma CETP activity was assayed according to a micro-method, by co-incubating lipoprotein-deficient samples with exogenous donor and acceptor lipoproteins. The study subjects were 31 obese children (14 males and 17 females). Serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), TC:high-density lipoprotein (HDL)-C, LDL-C:HDL-C, apolipoprotein (apo) B, and apo B:apo A1 were increased in obese children. Thus they appeared to exhibit an atherogenic lipoprotein profile, with a relative decrease in cholesterol carried by HDL compared with the cholesterol in the other lipoprotein fractions. The mean fasting plasma insulin level was also increased. CETP activity was significantly higher in the obese children than in nonobese control children, and was correlated with LDL-C, TC:HDL-C, LDL-C:HDL-C, and apo B:apo A1. These results suggest that an increase in plasma CETP activity results in atherogenic change in lipoprotein metabolism in obese children. The increase in CETP may be due to the adiposity or insulin resistance. Alternatively, dyslipidemia per se, physical inactivity or excessive fat intake, that are commonly found in obese children, may contribute to the increase in CETP activity. © 1997 Elsevier Science Ireland Ltd. Keywords: Cholesteryl ester transfer protein; Obesity; Lipoproteins; Atherosclerosis; Apolipoproteins; Child
1. Introduction The serum enzyme, cholesteryl ester transfer protein (CETP) [1], catalyzes the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to verylow-density lipoproteins (VLDL) and low density lipoproteins (LDL). CEPT also regulates cholesterol (C) content in HDL and modifies its composition. A genetic deficiency of CETP [2] has been described in certain numbers of Japanese families. The plasma HDL-cholesterol (HDL-C) level was markedly increased in those patients. Although plasma HDL-C is a negative coronary risk factor [3], it is uncertain whether the patients with CETP deficiency enjoy longevity. On
* Corresponding author. Tel.: + 81 552 731111; Fax: +81 552 736745; e-mail: [email protected]
the other hand, atherosclerosis is accelerated in the transgenic mice expressing CETP [4] and the results in this series of the transgenic mice suggest that CETP exacerbates atherosclerosis. However, in another series of hypertriglyceridemic CETP transgenic mice, CETP appeared to protect against the formation of atherogenic plaques [5]. Obesity is considered to be an independent risk factor for coronary heart disease [6], and is also known to induce an atherogenic lipoprotein profile in adults. Low levels of plasma HDL-C are commonly found in obese adults [7], which should further increase their risk for coronary heart disease [8]. In several previous studies, plasma CETP activity was increased in obese adults [9,10]. According to these reports, the enhanced CETP activity was assumed to contribute to the development of hyperlipidemia and low serum HDL-C (i.e. atherogenic lipoprotein profile) levels found in obese adults.
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H. Hayashibe et al. / Atherosclerosis 129 (1997) 53–58
To our knowledge, plasma CETP activity has not yet been previously reported in obese children. Obesity can be readily recognized even in small children and, in significant populations, that obesity tends to exacerbate throughout childhood and adolescence and terminates with more serious adult obesity. Since an abnormality in lipoprotein metabolism during childhood can contribute to the development of atherosclerosis [11], the impact of childhood obesity on atherogenic change in lipoprotein metabolism should be explored. In the present study, we measured CETP activity in the sera of obese children using a previously reported micromethod for radioisotopic assay [12]. The assay procedure has been optimized for small children and has been performed with less than 0.5 ml of plasma sample.
2. Materials and methods
2.1. Subjects The study subjects were 31 obese children, 14 boys and 17 girls with ages ranging from 6 to 15 (9.9 9 0.4; mean 9S.E.M.) years, who were outpatients of the Clinic for Obese Children in Yamanashi Medical University. A child was considered as obese when body weight exceeded 120% of the standard body weight for height. The standard body weight was defined as a mean body weight for the height obtained for each child according to gender and was based on the largescaled data of national statistics of growth survey for Japanese school children. None of the subjects were diagnosed as having endocrine, metabolic, kidney disease or medical problems other than obesity. They were instructed to visit the Clinic in the morning after an overnight fast, where blood was drawn. During the same visit they were subjected to anthropometric measurements of their height, body weight, three measurements of girth ( waist, hip, and thigh), and four measurements of skinfold thickness (biceps, triceps, subscapular, and suprailiac). The percent overweight, body-mass index (BMI), percent body fat, waist-to-hip ratio (WHR), and waist-to-thigh ratio (WTR) were calculated from these values. The detailed methods for anthropometric measurement have been described elsewhere [13]. Percent body fat was calculated according to Nagamine’s method [14,15]. The control group for measuring CETP activity consisted of 21 nonobese children, 12 boys and 9 girls with a mean age of 9.4 9 0.5 years that did not significantly differ from that of the obese children. Their blood samples were also drawn in the morning after an overnight fast. The clinical laboratory data from obese children were compared to the reference values for children in Yamanashi Medical University. The reference values were obtained from fasting blood biochem-
istry data from 131 nonobese children. This group consisted of 69 boys and 52 girls, with ages ranging from 6 to 15 (mean 10.0) years and no history of endocrine, metabolic, or renal diseases. There were no appreciable gender related differences among the clinical laboratory data in these children. This study was approved by the Human Study Committee of Yamanashi Medical University. Informed consent was obtained from each subject or from the parents as appropriate.
2.2. Assay method for plasma cholesteryl ester transfer acti6ity Preparative ultracentrifuge fractionation of human plasma was performed by the method of Havel et al. [16] using a Beckman SW-28 rotor (Beckman Instruments, Fullerton, CA). The dB1.063 fraction (VLDL+ LDL) and d\1.125 (HDL3) fractions were prepared using pooled plasma freshly obtained from healthy adult donors after an overnight fast. After ultracentrifugation the lipoprotein fractions were dialyzed against 10 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA (pH 7.4) and stored at 4°C. The details of the assay method have been described previously [12]. HDL3 was labeled in the cholesteryl ester (CE) moiety according to the method described by Albers et al. [17], and the specific activity of the [14C]CE–HDL3 was 1451 cpm/mg of CE. To remove total lipoproteins from the plasma sample, the plasma density was adjusted to d= 1.21 and the samples were ultracentrifuged in a Beckman Airfuge (Beckman Instruments) for 3.5 h at 165 000×g. The infranatant was collected and dialyzed against 10 mM Tris–HCl, 150 mM NaCl (pH 7.4). CETP activity was determined by measuring the decrease in 14C-labeled CE in HDL3 after incubation with the dB1.063 lipoprotein fraction in the presence of lipoprotein deficient (d\ 1.21) plasma samples [18]. The assay mixture consisted of 20 ml of the[14C]CEHDL3 (6.32 mg CE), 40 ml of the dB1.063 lipoprotein fraction (121 mg CE) and 20 ml of the lipoprotein-deficient (d\ 1.21) samples. The volume was adjusted to 180 ml by adding 10 mM Tris-HCl, 150 mM NaCl buffer (pH 7.4). After incubation for 8 h at 37°C, apo B-containing lipoproteins were removed by heparin– MnCl2 precipitation. CETP activity was determined as the loss of labeled CE from the supernatant. The assay without a sample was used as a blank. The activity paralleled the volume of the sample when up to 50 ml of lipoprotein-deficient plasma was added to the assay system [12]. Each sample and blank were assayed in duplicate. CETP activity was expressed as nmol of labeled CE transferred from the donor (HDL3) to the acceptor (VLDL or LDL)/h/ml of plasma sample. To avoid assay-to-assay variation, all samples were measured simultaneously in one assay.
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Table 1 Anthropometric data in obese and nonobese children
Age (years) Height (cm) Weight (kg) Overweight BMI (kg/m2) Body fat (%) WHR WTR
Obese children n= 14 boys, 17 girls
Control children n =12 boys, 9 girls
t-Test probability
10.09 0.4 142.0 9 2.2 50.0 9 2.4 43.8 9 2.4 24.4 9 0.5 34.0 91.1 0.939 0.01 1.939 0.02
9.4 90.5 133.2 94.2 31.6 92.7 2.4 91.3 17.0 9 0.4 —a 0.83 9 1.859 0.04
ns ns PB0.001 PB0.001 PB0.001 —a PB0.001 ns
Data are expressed as means 9 S.E.M. Statistical significance was estimated by Student’s t-test.ns, Not significant. a Skinfold thickness is not measured in control children.
2.3. Other methods Serum total cholesterol (TC), triglyceride (TG), HDL-C, apolipoproteins (apo) A1, A2, and B, and serum insulin (IRI) were measured in the clinical laboratory of Yamanashi Medical University Hospital. TC and TG were measured enzymatically with commercial kits (Determiner L TC, TG, Kyowa, Medex, Tokyo, Japan). HDL-C was measured by a dextran sulfate– Mg2 + precipitation method. Apo A1, A2 and B were measured by immunoturbidimetry method using commercial kits (Apolipoprotein A1, A2, B determination kits, Daiichi Chemicals, Tokyo, Japan). IRI was assayed by two-site immuno-enzymometric method using commercial kits (EIA-PACK IRI, Tosoh, Tokyo, Japan). LDL-C was calculated from the Friedewald equation (LDL-C=TC −HDL-C −TG/2.18) [19].
2.4. Statistics Data are presented as mean values 9S.E.M. To evaluate the degree of abnormality in clinical laboratory data of obese children, we also calculated the percentile values of the respective reference biochemical data that corresponded to the mean values for the obese children. Statistical analyses were performed using SYSTAT 5.2.1 software (Systat, Evanston, IL, USA). Pearson’s correlation coefficients were calculated by least-squares linear regression analysis. The statistical significance between means were estimated by an unpaired t-test. Differences were considered statistically significant at P B 0.05. Since there were no significant gender related differences in the biochemical data used in the present study, males and females were not divided into separate groups. 3. Results
3.1. Anthropometric data Anthropometric data for obese and control children
are shown in Table 1. Age and height were similar between the obese and control children. On the other hand, weight, % overweight, BMI and WHR were much higher in the obese children than in the controls. Between the obese boys and girls, the mean age, height, body weight, percent overweight, BMI, and percent body fat values were similar. On the other hand, WHR (0.959 0.01 versus 0.919 0.01, P = 0.03) and WTR (2.009 0.03 versus 1.889 0.03, P = 0.006) values were significantly greater in the obese boys than in the obese girls.
3.2. Blood biochemistry data Table 2 summarizes the biochemical data obtained from obese children and nonobese control children (i.e. reference values). The mean serum TG level of obese children was significantly higher than that of the reference value, and corresponded to the 79th percentile of the reference value. Similarly, TC was significantly increased in the obese children (70th percentile of the reference value), as was the LDL-C level (71st percentile). Although the mean HDL-C level was similar to the controls (57th percentile of the reference value), the ratios of TC:HDL-C (74th percentile)and LDL-C:HDL-C (69th percentile) were significantly increased in the obese children. The apo A1 (43rd percentile) and apo A2 (56th percentile) levels were similar to the controls, while the apo B (80th percentile) was significantly increased in the obese children. The ratio of apo A1:apo A2 showed a slight decrease, while the apo B:apo A1 (the atherogenic risk ratio) was markedly increased (84th percentile) in the obese children. The mean serum IRI level in obese children was markedly increased and was higher than the 90th percentile of the reference value (16.3 versus 16.0 mU/ml). The TG, apo-B, and IRI values were significantly correlated with age
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Table 2 Clinical laboratory data in obese and nonobese children
TG (mmol/l) TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) TC:HDL-C LDL-C:HDL-C Apo A1 (mg/dl) Apo A2 (mg/dl) Apo B (mg/dl) Apo A1:apo A2 Apo B:apo A1 Insulin (mU/ml)
Obese children n= 31
Control children (n = 131)
t-Test probability
1.039 0.11 4.56 9 0.15 2.729 0.12 1.379 0.05 3.469 0.15 2.08 9 0.12 12794 3491 8194 3.739 0.08 0.659 0.03 16.3 92.4
0.82 90.03 4.259 0.06 2.499 0.04 1.389 0.02 3.139 0.04 1.859 0.04 132 9 2 34 9 1 72 91 3.94 90.05 0.55 90.01 10.4 90.4
P =0.005 P =0.023 P =0.031 ns P =0.003 P =0.014 ns ns P =0.003 P =0.049 PB0.001 PB0.001
Data are expressed as means 9S.E.M. for obese children.
of the obese children, while the other clinical laboratory data were not (data not shown).
3.3. Plasma cholesteryl ester transfer acti6ity Plasma CETP activity was significantly higher in the obese children than in nonobese control children (Fig. 1). Table 3 lists the correlations between CETP activity and other biochemical data obtained from obese children. The CETP activity was significantly correlated
with LDL-C, TC:HDL-C, LDL-C:HDL-C, apo B, and apo B:apo A1, but not with TG, TC, HDL-C, apo A1 apo A2, apo A1:apo A2, nor IRI. The CETP activity was correlated with none of the anthropometric indices evaluated in the present study nor with age (data not shown).
4. Discussion In the present obese children, no gender difference was observed in body size, fat mass and biochemical data, although body fat distribution was somewhat different between genders. Thus the degree of obesity was similar in both boys and girls. Serum TC, LDL-C, TG and Apo B were increased in obese children compared to the respective reference values. Although the serum levels of HDL-C, apo A1 and apo A2 were within normal limits in the obese children, the biochemTable 3 Correlations between CETP activity and other biochemical data in obese children
CETP Activity
Fig. 1. Plasma CETP activity in obese and nonobese control children. Each symbol represents the CETP activity for an individual subject. Statical significance was estimated by Student’s t-test (unpaired).
Variables
Correlation
Probability
vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.
−0.014 0.257 0.465 −0.297 0.527 0.636 −0.200 −0.114 0.223 −0.190 0.365 −0.051
ns ns P = 0.008 ns P = 0.002 P = 0.001 ns ns ns ns P = 0.044 ns
TG TC LDL-C HDL-C TC:HDL-C LDL-C:HDL-C apo A1 apo A2 apo B apo A1:apo A2 apo B:apo A1 insulin
Data are Pearson’s correlation coefficients and the probabilities of 31 observations for each set of variables. ns; Not significant.
H. Hayashibe et al. / Atherosclerosis 129 (1997) 53–58
ical ratios of TC:HDL-C, LDL-C:HDL-C and apo B:apo A1 (i.e. atherogenic risk ratio) were increased, indicating the relative decrease in HDL mass compared with those of TG-rich lipoproteins. Thus, the present obese children appeared to exhibit an atherogenic change in lipoprotein metabolism, as has been reported previously in other studies of obese children [20–22]. Serum IRI was markedly increased in the obese children, suggesting that their obesity had induced a significant insulin resistance. It is well established that obesity induces hyperlipidemia and low HDL-C values in adults [7,8]. Although a similar abnormality in serum lipoprotein was found in previous studies of obese children [20 – 22], the decreased HDL-C level is more variable in children than in adults [20,21]. In ‘The Bogalusa Heart Study’, the effect of obesity on the levels of serum lipids and lipoproteins was examined in 3311 children and young adults aged 5–26 years. According to the results of this latter study, the serum HDL-C level was inversely correlated with obesity, and this negative association was more prominent in older age groups than in younger groups [23]. The lack of decrease in the absolute value of HDL-C in the obese children of the present study may be ascribed to the fact that they (mean age 10 years old) were not old enough to show the negative association between obesity and HDL-C. That their dyslipidemia was significant but mild was also considered to be due to the young ages. Two previous reports have documented the increased plasma CETP activity in adult obese subjects. Dullaart et al. [9] reported that plasma CETP activity was elevated in obese men by 35% when compared with nonobese men. They also noted that the CETP activity was positively correlated with BMI, fasting blood glucose, and plasma C-peptide. Arai et al. [10] reported that both activity and protein mass of CETP were increased in obese subjects, and that CETP activity and protein mass returned toward the normal range after weight reduction. Plasma CETP activity was increased in the obese children of the present study, as was the case in the previous adult studies. However, the CETP activity in the present study correlated with neither the percent of the children’s overweight nor their IRI values. This lack of correlation can be explained by our previous findings that glucose intolerance and insulin resistance are less prominent in obese children than in obese adults, and that the degree of obesity is less closely associated with metabolic derangement in children than in adults [13]. Recently, CETP has been reported to be synthesized in the liver, as well as the small intestines and adipose tissues. That the mRNA expression is high in the adipocytes [24] suggests that accumulation of fat tissues in obese subjects leads to the increased secretion of CETP from fat tissues to the circulation.
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Several other factors that are also closely related to obesity are known to modify plasma CETP activity. Insulin resistance is postulated to induce increased CETP activity in patients with non-insulin dependent diabetes mellitus [25] and insulin therapy restores the activity toward the normal range [26]. In patients with insulin-dependent diabetes mellitus, insulin decreases CETP activity when administered via the portal route, but on the contrary, increases it via the subcutaneous route, suggesting that peripheral hyperinsulinemia induces insulin resistance in the patients [27]. Thus insulin resistance may have contributed to the increased CETP activity in the obese children, although lack of correlation between the CETP activity and serum insulin level in this study does not support this concept. Hyperlipidemia per se can also modify the plasma CETP activity. In hypertriglyceridemia, the rate of cholesterol transfer from HDL to TG-rich lipoproteins can be retarded by the structural changes related to excessive TG in the acceptors, unless there is a compensatory increase in CETP. The reciprocal decrease in HDL-C occasionally found in the presence of hypertriglyceridemia may be due to an enhanced lipid transfer reaction [28]. Exercise decreases plasma CETP activity, and CETP activity is reported to be low in long-distance runners [29]. CETP activity also declines with exercise in obese subjects, regardless of the change in body weight [30]. Thus physical inactivity may have contributed to the increased CETP activity in the obese children. CETP synthesis is known to be up-regulated by dietary cholesterol [31]. The CETP level increases in association with enhanced peripheral cholesterol transport via LDL, b-VLDL, or chylomicron remnants. Accordingly, CETP activity increases in hypercholesterolemia regardless of the pathogenesis of the increase in cholesterol [32]. Further, hepatic CEPT gene expression is reported to be regulated by plasma cholesterol level in CETP transgenic mice [33]. LDL-C and apo B were increased, while HDL-C was decreased in the plasma of transgenic mice expressing CETP [4]. The excessive transfer of CE to TG-rich lipoproteins may retard the removal rate of their remnants, leading to an accumulation of apo B in the circulation. This mechanism would explain the increase in the apo B level in the present study. Similarly, the positive association between CETP and either LDL-C, TC:HDL-C, or LDL-C:HDL-C observed here, suggests that CETP modifies the level of both HDL-C and TG-rich lipoproteins. Thus, an increase in CETP activity may have exacerbated the atherogenic profiles of lipoproteins and apolipoproteins in the obese children studied here. The present results suggest that an increase in plasma CETP activity results in an atherogenic change in lipoprotein metabolism in obese children. The increase in
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CETP activity may be due to fat accumulation or insulin resistance. Alternatively, the increased CETP activity may be secondary to dyslipidemia per se, physical inactivity or excessive fat intake that are commonly found in obese children.
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[15] Nagamine S, Suzuki S. Anthropometry and body composition of Japanese young men and women. Hum Biol 1964;36:8–15. [16] Havel RJ, Eder HA, Bragdon, JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345 – 1353. [17] Albers JJ, Tollefson JH, Chen CH, Steinmetz A. Isolation and characterization of human plasma lipid transfer proteins. Arteriosclerosis 1984;4:49 – 58. [18] Yamashita S, Hui DY, Wetterau JR et al. Characterization of plasma lipoproteins in patients heterozygous for human plasma cholesteryl ester transfer protein (CETP) deficiency. Metabolism 1991;40:756 – 763. [19] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol without use of the preparative ultracentrifuge. Clin Chem 1972;18:499. [20] Freedmann DS, Burke GL, Harsha DW et al. Relationship of changes in obesity to serum lipid and lipoprotein changes in childhood and adolescence. JAMA 1985;254:515 – 520. [21] Zwiauer KF, Pakosta R, Mueller T, Widhalm K. Cardiovascular risk factors in obese children in relation to weight and body fat distribution. J Am Coll Nutr 1992;11:41S – 50S. [22] Knip M, Nuutinen O. Long-term effects of weight reduction on serum lipids and plasma insulin in obese children. Am J Clin Nutr 1993;57:490 – 493. [23] Wattigney WA, Harsha DW, Srinivasan SR, Webber LS, Berenson GS. Increasing impact of obesity on serum lipids and lipoproteins in young adults. The Bogalusa Heart Study Arch Intern Med 1991;151:2017– 2022. [24] Jiang XC, Moulin P, Quinet E et al. Mammalian adipose tissue and muscle are major sources of lipid transfer protein. J Biol Chem 1991;266:4631– 4639. [25] Bagdade JD, Lane JT, Subbaiah PV, Otto ME, Ritter MC. Accelerated cholesteryl ester transfer in noninsulin-dependent diabetes mellitus. Atherosclerosis 1993;104:69 – 77 [26] Sutherland WH, Walker RJ, Lewis-Barned NJ, Pratt H, Tillman HC. The effect of acute hyperinsulinemia on plasma cholesteryl ester transfer protein activity in patients with non-insulin-dependent diabetes mellitus and healthy subjects. Metabolism 1994;43:1362 – 1366. [27] Bagdade JD, Dunn FL, Eckel RH, Ritter MC. Intraperitoneal insulin therapy corrects abnormalities in cholesteryl ester transfer and lipoprotein lipase activities in insulin-dependent diabetes mellitus. Arterioscler Thromb 1994;14:1933 – 1939. [28] Mann CJ, Yen FT, Grant AM, Bihain BE. Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia. J Clin Invest 1991;88:2059 – 2066. [29] Serrat-Serrat J, Ordonez-Llanos J, Serra-Grima R et al. Marathon runners presented lower serum cholesteryl ester transfer activity than sedentary subjects. Atherosclerosis 1993;101:43– 49. [30] Seip RL, Moulin P, Cocke T et al. Exercise training decreases plasma cholesteryl ester transfer protein. Arterioscler Thromb 1993;13:1359 – 1367. [31] Quinet E, Tall A, Ramakrishnan R, Rudel L. Plasma lipid transfer protein as a determinant of the atherogenicity of monkey plasma lipoproteins. J Clin Invest 1991;87:1559 – 1566. [32] McPherson R, Mann CJ, Tall AR et al. Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia. Relation to cholesteryl ester transfer protein activity and other lipoprotein variables. Arterioscler Thromb 1991;11:797–804. [33] Masucci-Magoulas L, Plump A, Jiang XC, Walsh A, Breslow JL, Tall AR. Profound induction of hepatic cholesteryl ester transfer protein transgene expression in apolipoprotein E and low density lipoprotein receptor gene knockout mice. A novel mechanism signals changes in plasma cholesterol levels. J Clin Invest 1996;97:154 – 161.
Increased plasma cholesteryl ester transfer activity in obese children Hidemasa Hayashibe*, Kohtaro Asayama, Takaya Nakane, Norihiko Uchida, Yasusuke Kawada, Shinpei Nakazawa Department of Pediatrics, Yamanashi Medical Uni6ersity, 1110 Shimokato, Tamahocho, Nakakoma, Yamanashi 409 -38, Japan Received 9 April 1996; revised 30 August 1996; accepted 7 November 1996
Abstract To determine whether enhanced activity of cholesteryl ester transfer protein (CETP) contributes to the development of atherogenic lipoprotein profiles in obese children, plasma CETP activity was assayed according to a micro-method, by co-incubating lipoprotein-deficient samples with exogenous donor and acceptor lipoproteins. The study subjects were 31 obese children (14 males and 17 females). Serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), TC:high-density lipoprotein (HDL)-C, LDL-C:HDL-C, apolipoprotein (apo) B, and apo B:apo A1 were increased in obese children. Thus they appeared to exhibit an atherogenic lipoprotein profile, with a relative decrease in cholesterol carried by HDL compared with the cholesterol in the other lipoprotein fractions. The mean fasting plasma insulin level was also increased. CETP activity was significantly higher in the obese children than in nonobese control children, and was correlated with LDL-C, TC:HDL-C, LDL-C:HDL-C, and apo B:apo A1. These results suggest that an increase in plasma CETP activity results in atherogenic change in lipoprotein metabolism in obese children. The increase in CETP may be due to the adiposity or insulin resistance. Alternatively, dyslipidemia per se, physical inactivity or excessive fat intake, that are commonly found in obese children, may contribute to the increase in CETP activity. © 1997 Elsevier Science Ireland Ltd. Keywords: Cholesteryl ester transfer protein; Obesity; Lipoproteins; Atherosclerosis; Apolipoproteins; Child
1. Introduction The serum enzyme, cholesteryl ester transfer protein (CETP) [1], catalyzes the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to verylow-density lipoproteins (VLDL) and low density lipoproteins (LDL). CEPT also regulates cholesterol (C) content in HDL and modifies its composition. A genetic deficiency of CETP [2] has been described in certain numbers of Japanese families. The plasma HDL-cholesterol (HDL-C) level was markedly increased in those patients. Although plasma HDL-C is a negative coronary risk factor [3], it is uncertain whether the patients with CETP deficiency enjoy longevity. On
* Corresponding author. Tel.: + 81 552 731111; Fax: +81 552 736745; e-mail: [email protected]
the other hand, atherosclerosis is accelerated in the transgenic mice expressing CETP [4] and the results in this series of the transgenic mice suggest that CETP exacerbates atherosclerosis. However, in another series of hypertriglyceridemic CETP transgenic mice, CETP appeared to protect against the formation of atherogenic plaques [5]. Obesity is considered to be an independent risk factor for coronary heart disease [6], and is also known to induce an atherogenic lipoprotein profile in adults. Low levels of plasma HDL-C are commonly found in obese adults [7], which should further increase their risk for coronary heart disease [8]. In several previous studies, plasma CETP activity was increased in obese adults [9,10]. According to these reports, the enhanced CETP activity was assumed to contribute to the development of hyperlipidemia and low serum HDL-C (i.e. atherogenic lipoprotein profile) levels found in obese adults.
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H. Hayashibe et al. / Atherosclerosis 129 (1997) 53–58
To our knowledge, plasma CETP activity has not yet been previously reported in obese children. Obesity can be readily recognized even in small children and, in significant populations, that obesity tends to exacerbate throughout childhood and adolescence and terminates with more serious adult obesity. Since an abnormality in lipoprotein metabolism during childhood can contribute to the development of atherosclerosis [11], the impact of childhood obesity on atherogenic change in lipoprotein metabolism should be explored. In the present study, we measured CETP activity in the sera of obese children using a previously reported micromethod for radioisotopic assay [12]. The assay procedure has been optimized for small children and has been performed with less than 0.5 ml of plasma sample.
2. Materials and methods
2.1. Subjects The study subjects were 31 obese children, 14 boys and 17 girls with ages ranging from 6 to 15 (9.9 9 0.4; mean 9S.E.M.) years, who were outpatients of the Clinic for Obese Children in Yamanashi Medical University. A child was considered as obese when body weight exceeded 120% of the standard body weight for height. The standard body weight was defined as a mean body weight for the height obtained for each child according to gender and was based on the largescaled data of national statistics of growth survey for Japanese school children. None of the subjects were diagnosed as having endocrine, metabolic, kidney disease or medical problems other than obesity. They were instructed to visit the Clinic in the morning after an overnight fast, where blood was drawn. During the same visit they were subjected to anthropometric measurements of their height, body weight, three measurements of girth ( waist, hip, and thigh), and four measurements of skinfold thickness (biceps, triceps, subscapular, and suprailiac). The percent overweight, body-mass index (BMI), percent body fat, waist-to-hip ratio (WHR), and waist-to-thigh ratio (WTR) were calculated from these values. The detailed methods for anthropometric measurement have been described elsewhere [13]. Percent body fat was calculated according to Nagamine’s method [14,15]. The control group for measuring CETP activity consisted of 21 nonobese children, 12 boys and 9 girls with a mean age of 9.4 9 0.5 years that did not significantly differ from that of the obese children. Their blood samples were also drawn in the morning after an overnight fast. The clinical laboratory data from obese children were compared to the reference values for children in Yamanashi Medical University. The reference values were obtained from fasting blood biochem-
istry data from 131 nonobese children. This group consisted of 69 boys and 52 girls, with ages ranging from 6 to 15 (mean 10.0) years and no history of endocrine, metabolic, or renal diseases. There were no appreciable gender related differences among the clinical laboratory data in these children. This study was approved by the Human Study Committee of Yamanashi Medical University. Informed consent was obtained from each subject or from the parents as appropriate.
2.2. Assay method for plasma cholesteryl ester transfer acti6ity Preparative ultracentrifuge fractionation of human plasma was performed by the method of Havel et al. [16] using a Beckman SW-28 rotor (Beckman Instruments, Fullerton, CA). The dB1.063 fraction (VLDL+ LDL) and d\1.125 (HDL3) fractions were prepared using pooled plasma freshly obtained from healthy adult donors after an overnight fast. After ultracentrifugation the lipoprotein fractions were dialyzed against 10 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA (pH 7.4) and stored at 4°C. The details of the assay method have been described previously [12]. HDL3 was labeled in the cholesteryl ester (CE) moiety according to the method described by Albers et al. [17], and the specific activity of the [14C]CE–HDL3 was 1451 cpm/mg of CE. To remove total lipoproteins from the plasma sample, the plasma density was adjusted to d= 1.21 and the samples were ultracentrifuged in a Beckman Airfuge (Beckman Instruments) for 3.5 h at 165 000×g. The infranatant was collected and dialyzed against 10 mM Tris–HCl, 150 mM NaCl (pH 7.4). CETP activity was determined by measuring the decrease in 14C-labeled CE in HDL3 after incubation with the dB1.063 lipoprotein fraction in the presence of lipoprotein deficient (d\ 1.21) plasma samples [18]. The assay mixture consisted of 20 ml of the[14C]CEHDL3 (6.32 mg CE), 40 ml of the dB1.063 lipoprotein fraction (121 mg CE) and 20 ml of the lipoprotein-deficient (d\ 1.21) samples. The volume was adjusted to 180 ml by adding 10 mM Tris-HCl, 150 mM NaCl buffer (pH 7.4). After incubation for 8 h at 37°C, apo B-containing lipoproteins were removed by heparin– MnCl2 precipitation. CETP activity was determined as the loss of labeled CE from the supernatant. The assay without a sample was used as a blank. The activity paralleled the volume of the sample when up to 50 ml of lipoprotein-deficient plasma was added to the assay system [12]. Each sample and blank were assayed in duplicate. CETP activity was expressed as nmol of labeled CE transferred from the donor (HDL3) to the acceptor (VLDL or LDL)/h/ml of plasma sample. To avoid assay-to-assay variation, all samples were measured simultaneously in one assay.
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Table 1 Anthropometric data in obese and nonobese children
Age (years) Height (cm) Weight (kg) Overweight BMI (kg/m2) Body fat (%) WHR WTR
Obese children n= 14 boys, 17 girls
Control children n =12 boys, 9 girls
t-Test probability
10.09 0.4 142.0 9 2.2 50.0 9 2.4 43.8 9 2.4 24.4 9 0.5 34.0 91.1 0.939 0.01 1.939 0.02
9.4 90.5 133.2 94.2 31.6 92.7 2.4 91.3 17.0 9 0.4 —a 0.83 9 1.859 0.04
ns ns PB0.001 PB0.001 PB0.001 —a PB0.001 ns
Data are expressed as means 9 S.E.M. Statistical significance was estimated by Student’s t-test.ns, Not significant. a Skinfold thickness is not measured in control children.
2.3. Other methods Serum total cholesterol (TC), triglyceride (TG), HDL-C, apolipoproteins (apo) A1, A2, and B, and serum insulin (IRI) were measured in the clinical laboratory of Yamanashi Medical University Hospital. TC and TG were measured enzymatically with commercial kits (Determiner L TC, TG, Kyowa, Medex, Tokyo, Japan). HDL-C was measured by a dextran sulfate– Mg2 + precipitation method. Apo A1, A2 and B were measured by immunoturbidimetry method using commercial kits (Apolipoprotein A1, A2, B determination kits, Daiichi Chemicals, Tokyo, Japan). IRI was assayed by two-site immuno-enzymometric method using commercial kits (EIA-PACK IRI, Tosoh, Tokyo, Japan). LDL-C was calculated from the Friedewald equation (LDL-C=TC −HDL-C −TG/2.18) [19].
2.4. Statistics Data are presented as mean values 9S.E.M. To evaluate the degree of abnormality in clinical laboratory data of obese children, we also calculated the percentile values of the respective reference biochemical data that corresponded to the mean values for the obese children. Statistical analyses were performed using SYSTAT 5.2.1 software (Systat, Evanston, IL, USA). Pearson’s correlation coefficients were calculated by least-squares linear regression analysis. The statistical significance between means were estimated by an unpaired t-test. Differences were considered statistically significant at P B 0.05. Since there were no significant gender related differences in the biochemical data used in the present study, males and females were not divided into separate groups. 3. Results
3.1. Anthropometric data Anthropometric data for obese and control children
are shown in Table 1. Age and height were similar between the obese and control children. On the other hand, weight, % overweight, BMI and WHR were much higher in the obese children than in the controls. Between the obese boys and girls, the mean age, height, body weight, percent overweight, BMI, and percent body fat values were similar. On the other hand, WHR (0.959 0.01 versus 0.919 0.01, P = 0.03) and WTR (2.009 0.03 versus 1.889 0.03, P = 0.006) values were significantly greater in the obese boys than in the obese girls.
3.2. Blood biochemistry data Table 2 summarizes the biochemical data obtained from obese children and nonobese control children (i.e. reference values). The mean serum TG level of obese children was significantly higher than that of the reference value, and corresponded to the 79th percentile of the reference value. Similarly, TC was significantly increased in the obese children (70th percentile of the reference value), as was the LDL-C level (71st percentile). Although the mean HDL-C level was similar to the controls (57th percentile of the reference value), the ratios of TC:HDL-C (74th percentile)and LDL-C:HDL-C (69th percentile) were significantly increased in the obese children. The apo A1 (43rd percentile) and apo A2 (56th percentile) levels were similar to the controls, while the apo B (80th percentile) was significantly increased in the obese children. The ratio of apo A1:apo A2 showed a slight decrease, while the apo B:apo A1 (the atherogenic risk ratio) was markedly increased (84th percentile) in the obese children. The mean serum IRI level in obese children was markedly increased and was higher than the 90th percentile of the reference value (16.3 versus 16.0 mU/ml). The TG, apo-B, and IRI values were significantly correlated with age
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Table 2 Clinical laboratory data in obese and nonobese children
TG (mmol/l) TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) TC:HDL-C LDL-C:HDL-C Apo A1 (mg/dl) Apo A2 (mg/dl) Apo B (mg/dl) Apo A1:apo A2 Apo B:apo A1 Insulin (mU/ml)
Obese children n= 31
Control children (n = 131)
t-Test probability
1.039 0.11 4.56 9 0.15 2.729 0.12 1.379 0.05 3.469 0.15 2.08 9 0.12 12794 3491 8194 3.739 0.08 0.659 0.03 16.3 92.4
0.82 90.03 4.259 0.06 2.499 0.04 1.389 0.02 3.139 0.04 1.859 0.04 132 9 2 34 9 1 72 91 3.94 90.05 0.55 90.01 10.4 90.4
P =0.005 P =0.023 P =0.031 ns P =0.003 P =0.014 ns ns P =0.003 P =0.049 PB0.001 PB0.001
Data are expressed as means 9S.E.M. for obese children.
of the obese children, while the other clinical laboratory data were not (data not shown).
3.3. Plasma cholesteryl ester transfer acti6ity Plasma CETP activity was significantly higher in the obese children than in nonobese control children (Fig. 1). Table 3 lists the correlations between CETP activity and other biochemical data obtained from obese children. The CETP activity was significantly correlated
with LDL-C, TC:HDL-C, LDL-C:HDL-C, apo B, and apo B:apo A1, but not with TG, TC, HDL-C, apo A1 apo A2, apo A1:apo A2, nor IRI. The CETP activity was correlated with none of the anthropometric indices evaluated in the present study nor with age (data not shown).
4. Discussion In the present obese children, no gender difference was observed in body size, fat mass and biochemical data, although body fat distribution was somewhat different between genders. Thus the degree of obesity was similar in both boys and girls. Serum TC, LDL-C, TG and Apo B were increased in obese children compared to the respective reference values. Although the serum levels of HDL-C, apo A1 and apo A2 were within normal limits in the obese children, the biochemTable 3 Correlations between CETP activity and other biochemical data in obese children
CETP Activity
Fig. 1. Plasma CETP activity in obese and nonobese control children. Each symbol represents the CETP activity for an individual subject. Statical significance was estimated by Student’s t-test (unpaired).
Variables
Correlation
Probability
vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.
−0.014 0.257 0.465 −0.297 0.527 0.636 −0.200 −0.114 0.223 −0.190 0.365 −0.051
ns ns P = 0.008 ns P = 0.002 P = 0.001 ns ns ns ns P = 0.044 ns
TG TC LDL-C HDL-C TC:HDL-C LDL-C:HDL-C apo A1 apo A2 apo B apo A1:apo A2 apo B:apo A1 insulin
Data are Pearson’s correlation coefficients and the probabilities of 31 observations for each set of variables. ns; Not significant.
H. Hayashibe et al. / Atherosclerosis 129 (1997) 53–58
ical ratios of TC:HDL-C, LDL-C:HDL-C and apo B:apo A1 (i.e. atherogenic risk ratio) were increased, indicating the relative decrease in HDL mass compared with those of TG-rich lipoproteins. Thus, the present obese children appeared to exhibit an atherogenic change in lipoprotein metabolism, as has been reported previously in other studies of obese children [20–22]. Serum IRI was markedly increased in the obese children, suggesting that their obesity had induced a significant insulin resistance. It is well established that obesity induces hyperlipidemia and low HDL-C values in adults [7,8]. Although a similar abnormality in serum lipoprotein was found in previous studies of obese children [20 – 22], the decreased HDL-C level is more variable in children than in adults [20,21]. In ‘The Bogalusa Heart Study’, the effect of obesity on the levels of serum lipids and lipoproteins was examined in 3311 children and young adults aged 5–26 years. According to the results of this latter study, the serum HDL-C level was inversely correlated with obesity, and this negative association was more prominent in older age groups than in younger groups [23]. The lack of decrease in the absolute value of HDL-C in the obese children of the present study may be ascribed to the fact that they (mean age 10 years old) were not old enough to show the negative association between obesity and HDL-C. That their dyslipidemia was significant but mild was also considered to be due to the young ages. Two previous reports have documented the increased plasma CETP activity in adult obese subjects. Dullaart et al. [9] reported that plasma CETP activity was elevated in obese men by 35% when compared with nonobese men. They also noted that the CETP activity was positively correlated with BMI, fasting blood glucose, and plasma C-peptide. Arai et al. [10] reported that both activity and protein mass of CETP were increased in obese subjects, and that CETP activity and protein mass returned toward the normal range after weight reduction. Plasma CETP activity was increased in the obese children of the present study, as was the case in the previous adult studies. However, the CETP activity in the present study correlated with neither the percent of the children’s overweight nor their IRI values. This lack of correlation can be explained by our previous findings that glucose intolerance and insulin resistance are less prominent in obese children than in obese adults, and that the degree of obesity is less closely associated with metabolic derangement in children than in adults [13]. Recently, CETP has been reported to be synthesized in the liver, as well as the small intestines and adipose tissues. That the mRNA expression is high in the adipocytes [24] suggests that accumulation of fat tissues in obese subjects leads to the increased secretion of CETP from fat tissues to the circulation.
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Several other factors that are also closely related to obesity are known to modify plasma CETP activity. Insulin resistance is postulated to induce increased CETP activity in patients with non-insulin dependent diabetes mellitus [25] and insulin therapy restores the activity toward the normal range [26]. In patients with insulin-dependent diabetes mellitus, insulin decreases CETP activity when administered via the portal route, but on the contrary, increases it via the subcutaneous route, suggesting that peripheral hyperinsulinemia induces insulin resistance in the patients [27]. Thus insulin resistance may have contributed to the increased CETP activity in the obese children, although lack of correlation between the CETP activity and serum insulin level in this study does not support this concept. Hyperlipidemia per se can also modify the plasma CETP activity. In hypertriglyceridemia, the rate of cholesterol transfer from HDL to TG-rich lipoproteins can be retarded by the structural changes related to excessive TG in the acceptors, unless there is a compensatory increase in CETP. The reciprocal decrease in HDL-C occasionally found in the presence of hypertriglyceridemia may be due to an enhanced lipid transfer reaction [28]. Exercise decreases plasma CETP activity, and CETP activity is reported to be low in long-distance runners [29]. CETP activity also declines with exercise in obese subjects, regardless of the change in body weight [30]. Thus physical inactivity may have contributed to the increased CETP activity in the obese children. CETP synthesis is known to be up-regulated by dietary cholesterol [31]. The CETP level increases in association with enhanced peripheral cholesterol transport via LDL, b-VLDL, or chylomicron remnants. Accordingly, CETP activity increases in hypercholesterolemia regardless of the pathogenesis of the increase in cholesterol [32]. Further, hepatic CEPT gene expression is reported to be regulated by plasma cholesterol level in CETP transgenic mice [33]. LDL-C and apo B were increased, while HDL-C was decreased in the plasma of transgenic mice expressing CETP [4]. The excessive transfer of CE to TG-rich lipoproteins may retard the removal rate of their remnants, leading to an accumulation of apo B in the circulation. This mechanism would explain the increase in the apo B level in the present study. Similarly, the positive association between CETP and either LDL-C, TC:HDL-C, or LDL-C:HDL-C observed here, suggests that CETP modifies the level of both HDL-C and TG-rich lipoproteins. Thus, an increase in CETP activity may have exacerbated the atherogenic profiles of lipoproteins and apolipoproteins in the obese children studied here. The present results suggest that an increase in plasma CETP activity results in an atherogenic change in lipoprotein metabolism in obese children. The increase in
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CETP activity may be due to fat accumulation or insulin resistance. Alternatively, the increased CETP activity may be secondary to dyslipidemia per se, physical inactivity or excessive fat intake that are commonly found in obese children.
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