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Plasma palmitoleic acid content and obesity in children^1,2,3

¹ From the Department of Pediatrics, Nihon University School of Medicine, Tokyo, Japan

² Supported by a grant from the Japanese Study Groups for Obesity and Related Metabolism in Childhood and Adolescence.

³ Address reprint requests to T Okada, Department of Pediatrics, Nihon University School of Medicine, 30-1 Oyaguchi kamimachi Itabashi-ku, Tokyo 173-8610, Japan. E-mail: tomokada{at}med.nihon-u.ac.jp.

	ABSTRACT

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Background:Palmitoleic acid (16:1n–7) is a product of endogenous lipogenesis. In human obesity, 16:1n–7 is reported to correlate with indexes of adiposity and insulin concentrations.

Objective:We investigated the relation between adiposity, especially in the abdominal region, and plasma monounsaturated fatty acid (MUFA) profiles in obese children.

Design:A case-control study was performed. The study subjects were 59 obese children ( ± SD age: 11.8 ± 3.8 y) and 53 age- and sex-matched healthy, nonobese children (aged 12.5 ± 0.5 y). The study's variables included anthropometric measurements, serum lipids, leptin, and fatty acid composition in plasma.

Results:MUFA profiles of obese subjects showed a significantly higher content of 16:1n–7, 18:1n–9, and 20:1n–9 and significantly higher stearoyl-CoA desaturase (SCD) activity (ratio of 16:1n–7 to 16:0) than in nonobese controls. In a multiple regression analysis, percentage body fat, waist-to-height ratio, and waist-to-hip ratio (WHR) were significant determinants of 16:1n–7 content. SCD activity had a positive, significant correlation with leptin. However, in a multiple regression analysis that included percentage body fat, WHR, and leptin as independent determinants, WHR was the only determinant of SCD activity.

Conclusions:Plasma 16:1n–7 content has a significant relation with abdominal adiposity in obese children. This change in the MUFA profile may be caused by activation of SCD that is not sufficiently suppressed by leptin. Endogenous lipogenesis may be an important factor in the pathogenesis of obesity in children.

Key Words: Palmitoleic acid • obesity in children • stearoyl-CoA desaturase • abdominal adiposity • leptin

	INTRODUCTION

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Visceral obesity plays an important role in the development of metabolic syndrome (1). Even in children, abdominal adiposity predicts cardiovascular risks (2). In visceral obesity, the fatty acid composition of blood and tissues changes. The fatty acid composition of tissue lipids affects several important physiologic functions relating to the development of metabolic syndrome. Plasma and muscle arachidonic acid (20:4n–6) content has been shown to correlate with the development of insulin resistance associated with obesity (3, 4). Furthermore, disturbances in the ⁶- and ⁵-desaturase metabolic pathways may be a clinical manifestation of metabolic syndrome (5).

Palmitoleic acid (16:1n–7), one of the major monounsaturated fatty acids (MUFAs), is an important product of endogenous lipogenesis. The amount of palmitoleic acid in serum cholesterol ester reflects the hepatic lipid pool of carbon flux from carbohydrates to fatty acids (6). In rats rendered obese by sucrose administration, 16:1n–7 and oleic acid (18:1n–9) increase in plasma and liver, but hyperinsulinemia and hyperglycemia are not present in the early phase of obesity (7). Similarly, in human obesity, 16:1n–7 in serum cholesterol ester and adipose triacylglycerols correlates strongly with indexes of adiposity (8). Furthermore, MUFA concentrations in muscle phospholipids are positively correlated with fasting insulin concentrations (9). Taken together, these findings indicate that changes in MUFA composition may be central components of obesity and metabolic syndrome. In the present study, we investigated the relation between adiposity, especially in the abdominal region, and the plasma MUFA profile in obese children.

	SUBJECTS AND METHODS

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Subjects
Fifty-nine obese children from our outpatient clinic participated in the present study from 2000 to 2002. Informed consent was obtained from each child and his or her parents. The study was performed according to the guidelines of the ethical committee of clinical research at Nihon University Hospital. Fifty-three control children and their parents were recruited, and consent was obtained from them for participation. Obesity was defined as relative body weight >120% of predicted for sex, age, and height with the use of standards obtained from the Japanese Ministry of Health, Labor, and Welfare (10). The obese subjects included 20 girls (± SD age: 11.2 ± 3.0 y) whose relative body weight was 159.0 ± 17.8% of predicted and 39 boys (aged 12.2 ± 4.1 y) whose relative body weight was 158.0 ± 26.3% of predicted. The children were free of other known illnesses and were not dieting or participating in physical training. The control subjects were healthy schoolchildren (25 girls and 28 boys) aged 12.6 ± 0.5 y with a relative body weight <120% of predicted (97.0 ± 9.6% in boys, 94.6 ± 8.6% in girls). The control children were recruited through a screening and care program for lifestyle-related diseases in schoolchildren.

Percentage body fat (%BF) was measured a bioelectrical impedance method with an Impimeter III (Sekisui Co, Tokyo, Japan). Body mass index was calculated by dividing mass in kilograms by the square of height in meters (kg/m²). Waist circumference was measured at the level of the umbilicus, and hip circumference was measured at the point of maximal protrusion of the buttocks. Waist-to-hip ratios (WHRs) and waist-to-height ratios were calculated from these measurements.

Measurement of serum insulin and leptin concentrations
Blood samples were collected from the subjects after they had fasted overnight. Serum insulin was measured by the liquid phase method with a commercial kit (Insulin RIABEAD Kit, Dainabott, Tokyo, Japan). Serum leptin was measured by using a human leptin radioimmunoassay kit (LINCO Research Inc, St Charles, MO). Plasma total cholesterol, triacylglycerol, and HDL-cholesterol concentrations were measured with a Hitachi (Tokyo, Japan) 7450 automated analyzer and commercial kits. LDL-cholesterol concentrations were calculated according to the Friedewald equation (11). Plasma insulin and glucose concentrations were measured, and the homeostasis model of assessment ratio (HOMA-R) was obtained by using Matthews et al's (12) formula as an index of insulin resistance.

Lipid extraction and fatty acid analyses
Lipids were extracted from plasma with chloroform:methanol solution (2:1, by vol) by the method of Folch et al (13) in the presence of 0.01% butylated hydroxytoluene. Extracted plasma fatty acids were dried under nitrogen gas, resuspended with 1.0 mL benzene, and then trans-esterified in 3.0 mL BF₃ (Kanto Chemicals, Tokyo, Japan) at 80 °C for 90 min. The resulting methyl esters were extracted twice by adding 5.0 mL petroleum ether. The petroleum ether extract was evaporated under nitrogen gas, and the fatty acid methyl esters were dissolved in n-hexane. Then, 1 µL of the sample was used for gas chromatography analysis. Gas chromatography separation was performed with a PEG 20 M bonded capillary column (0.25 mm x 50 m; SGE, Austin, TX) in a Hitachi G-3000 gas chromatograph. Gas chromatography conditions were as follows: injector and detector port temperatures of 130 °C and 240 °C, respectively, and column temperature ranging from 130 °C to 180 °C with an increase of 8 °C/min, from 180 °C to 205 °C at 1.5 °C/min, from 205 °C to 230 °C at 8 °C/min, and a final hold of 15 min. Methyl esters were identified by comparing them with authentic standards of the methyl ester form of fatty acids (GL Sciences, Tokyo, Japan). Nonadecanoic acid (19:0) was added to each sample as an internal standard.

Enzyme activity index
Enzyme activity was estimated by relating the amount of the specific substrate to the corresponding product of the respective enzyme (4, 14). The ratios of 16:1n–7 to 16:0 and of 18:0 to 16:0 represent the activity of stearoyl-CoA desaturase (SCD) and elongase, respectively.

Statistical analyses
All data are expressed as means ± SDs. Group differences were assessed by using the Mann-Whitney U test. The correlation coefficients between 2 variables were determined by Spearman rank analysis. A P value < 0.05 was considered to indicate statistical significance. All statistical analyses were conducted by using the statistical package STATVIEW (version 4.5; Abacus Concepts, Berkeley, CA).

	RESULTS

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Characteristics of the subjects
The obese subjects had higher serum triacylglycerol (P < 0.01), leptin (P < 0.01), and insulin (P < 0.01) concentrations and lower HDL-cholesterol concentrations (P < 0.01) than did the controls. Serum total cholesterol and LDL cholesterol did not differ significantly between the groups (Table 1).

Table 2

Table 3

Table 4

	DISCUSSION

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The mechanism of this link between altered MUFA content and visceral obesity is not clear; however, some human studies have shown that 16:1n–7 content reflects the hepatic lipid pool, and elevated concentrations of 16:1n–7 in the serum represent a shift of carbon from carbohydrates to fatty acids that may underlie visceral obesity (6). The metabolic aberrations in humans with glycogen storage disease type 1 also support the idea that 16:1n–7 concentrations reflect the flux of carbon from carbohydrates to fatty acids. Glycogen storage disease type 1 is an inborn error of metabolism caused by deficiency of glucose-6-phosphatase, the enzyme catalyzing the conversion of glucose-6-phosphatase to glucose. Because the terminal step of glycogen breakdown is defective in this disease, carbon from carbohydrate is diverted to lipid, and synthesis of 16:0, the precursor of 16:1n–7, is increased 40-fold (15).

In the present study, the activity of SCD, the rate-limiting enzyme catalyzing the synthesis of 16:1n–7 from 16:0, was significantly higher in obese subjects, which suggests that SCD activity made an important contribution to the elevation in content of 16:1n–7. A similar increase in skeletal muscle SCD activity is seen in obese Pima Indians, and this increase is independently correlated with obesity in these subjects (16). Furthermore, recent studies showed that SCD is a key enzyme not only for the carbon shift described above but also for the regulation of lipogenesis and fatty acid oxidation. Ntambi and Miyazaki (17) investigated changes in the expression of several genes in the livers of mice lacking SCD-1 (Scdl^–/^–). They found that lipid oxidation genes and targets of peroxisome proliferator activated receptor , such as acyl-CoA oxydase, very-long-chain acyl-CoA dehydrogenase, carnitine palmitoyl-transferase 1, and fasting-induced adipocyte factor, were up-regulated. Conversely, lipid synthesis genes, such as sterol regulatory element binding protein 1, fatty acid synthase, and mitochondrial glycerol phosphate acyl transferase, were down-regulated (17). Elongase activity as estimated by the ratio of 18:0 to 16:0 in total plasma lipids was reduced in obese children. The ratio may reflect increased production of 16:0, rather than reduced elongase activity. We used the substrate-to-product ratios to estimate the enzyme activities. A direct measurement of enzyme activity with the use of hepatic cells or muscle is the best method for investigating endogenous lipogenesis, but fatty acid ratios are the best estimates of rate-limiting enzymes for clinical applications. Further studies are necessary to clarify the role of elongase in endogenous lipogenesis.

Many dietary, hormonal, and environmental factors regulate SCD expression (17). High-carbohydrate diets (18, 19), cholesterol, and insulin induce SCD-1 gene expression. Dietary n–6 and n–3 polyunsaturated fatty acids and leptin, however, inhibit SCD-1 mRNA transcription in the liver. In the study of dietary and genetic factors influencing serum fatty acid composition in obese identical twins, the high degree of intrapair similarity in 16:1n–7 content indicates that 16:1n–7 is a metabolic product under strong genetic control in humans (8). However, in that study, plasma 18:2n-6 content in the obese subjects was reduced as a result of dilution by the increased triacylglycerol content of the plasma rather than as a result of genetic influence. Future cohort studies should examine whether dietary intakes of carbohydrates, polyunsaturated fatty acids, and cholesterol are associated with visceral adiposity in children.

We showed that SCD activity correlates positively with leptin concentrations but not with serum insulin in obese children. In many animal studies, however, leptin inhibits SCD function. ob/ob mice lacking SCD-1 were reported to be less obese and have histologically normal livers with reduced triacylglycerol storage, which suggests that down-regulation of SCD-1 is an important component of the anti-obesity effect of leptin (20). Our findings here suggest that the same may not hold true in human obesity, even though leptin concentrations were elevated in the obese children. This difference may be induced by other strong regulators of SCD or may be due to leptin resistance. Further study is necessary to clarify the mechanism.

In the present study, the plasma MUFA profile was not associated with HOMA-R. In humans, MUFA concentrations in muscle phospholipids are positively correlated with fasting serum insulin (4). Furthermore, it has been reported that in skeletal muscle of obese Pima Indians, SCD activity correlates with insulin sensitivity and that elongase activity is reduced in insulin resistance (16). In the present study, we investigated the MUFA profile in plasma; this difference in sample (ie, tissue phospholipids versus mixed plasma lipids) may partly explain the negative association we found between HOMA-R and the MUFA profile. However, in rats rendered obese by feeding with sucrose, increases in 16:1n–7 and 18:1n–9 were observed in plasma and liver but not in muscle or adipose tissue during the early phase of obesity (which is marked by visceral fat deposition and increases in serum triacylglycerol concentrations and normal insulin concentrations) (7). These time- and tissue-dependent changes may also explain our results. Therefore, the MUFA profile in plasma may predict insulin resistance in later life.

In summary, our results suggest that the plasma 16:1n–7 content has a significant relation with abdominal adiposity in obese children. This change in the MUFA profile may be caused by activation of SCD that is not suppressed sufficiently by leptin. These results suggest that endogenous lipogenesis is an important factor in the pathogenesis of obesity in children.

	ACKNOWLEDGMENTS

 
TO and KH were responsible for the study design; TO, NF, YK, and MM were responsible for data collection; TO and FI were responsible for data analysis; and TO and KH were responsible for writing the manuscript. None of the authors had any financial or personal conflicts of interest.

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