Serum leptin concentration is associated with total body fat mass, but not abdominal fat distribution

International Journal of Obesity (1997) 21, 536±541 ß 1997 Stockton Press All rights reserved 0307±0565/97 $12.00 Serum leptin concentration is associated with total body fat mass, but not abdominal fat distribution H Shimizu 1, Y Shimomura 2, R Hayashi 2, K Ohtani 1, N Sato 1, T Futawatari 2 and M Mori 1 1 The First Department of Internal Medicine, Gunma University School of Medicine, and 2 Gunma Prefectural College of Medical Sciences, Gunma, Japan OBJECTIVE: The obese (ob) gene encodes leptin which inhibits appetite and stimulates thermogenesis. Serum leptin concentrations are determined by total body fat mass, but the in uence of visceral fat accumulation and other metabolic factors have not been clinically determined. METHODS: We determined the correlations between serum leptin concentrations and the total body fat mass, abdominal fat mass, abdominal fat distribution (estimated by ultrasound), and circulating metabolic factors in 104 Japanese healthy subjects (11 men and 93 women). In addition, the effect of food restriction (30 kcal/kg desired body weight/day) for four weeks on serum leptin concentrations were also examined in 30 women. RESULTS: There was a signi cant correlation between serum leptin concentrations and total body fat mass (r ˆ 0.708, P < 0.0001), the percentage of body fat (r ˆ 0.561, P < 0.001), and the body mass index (BMI, r ˆ 0.630, P < 0.001). Serum leptin concentrations were correlated with the abdominal wall preperitoneal and subcutaneous, but not the abdominal wall (AFI). Serum leptin concentrations were also correlated with serum immunoreactive insulin (IRI), but not glucose, or free fatty acid (FFA) concentrations. The weight loss after food restriction for four weeks signi cantly (P ˆ 0.016) reduced the serum leptin concentrations with a signi cant reduction of body fat mass, serum glucose, IRI and FFA concentrations. However, there was no signi cant correlation of the percentage change in serum leptin concentrations to that in body fat mass after food restriction. CONCLUSION: Serum leptin concentrations are well correlated with the total body fat mass in healthy subjects. Differences in abdominal fat distribution do not appear to be related to a difference in the in vivo leptin production from adipose tissue. Keywords: leptin; body fat; fat distribution; insulin; food restriction. Introduction The obese (ob) gene was recently identi ed as being involved in the regulation of energy balance in genetically obese mice. 1 The ob gene encodes the protein, leptin, that plays an important role in the regulation of food consumption and body weight. Since leptin inhibits appetite and stimulates thermogenesis in rodents, 2±4 this peptide is believed to act as a satiety factor in the hypothalamus. Recently, evidence has been accumulated that the ob gene expression is modi ed by various kinds of humoral factors such as insulin, glucocorticoids, estrogen, and cytokines which involve the regulation of food intake. 5±10 However, an interrelation between the ob gene expression and circulating metabolic factors has not been clinically determined. Correspondence: Dr H Shimizu, 1st Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371, Japan. Received 6 November 1996; revised 7 March 1997; accepted 12 March 1997 In human studies, serum leptin concentrations have recently been reported to be correlated with the percentage of body fat, suggesting that most obese people may be insensitive to increased levels of the anorexigenic peptide, leptin which was endogenously overproduced from adipose tissues. 11 On the other hand, visceral fat mass is related to the elevation of circulating metabolic factors such as insulin, 12 which may affect leptin production from the adipocytes. 5,7,8 It is possible that there may be a difference in the leptin production between the visceral and subcutaneous adipose tissues. However, whether the regional differences of visceral fat mass may affect serum leptin concentrations is still unknown in humans. In the present study, we determined correlations between serum leptin concentrations and metabolic parameters of body fat adiposity, abdominal fat distribution, and circulating metabolic factors in healthy Japanese subjects who have no metabolic disorders. In addition, the effect of body weight loss after food restriction on serum leptin concentrations was investigated.

Subjects and methods Subjects The present study included 104 healthy Japanese subjects (11 men and 93 women, mean age: 49.7 1.0 y old, and Body Mass Index (BMI): 23.9 0.3 kg/m 2 ). No subject was receiving medications for any metabolic disorders. All subjects were fully informed as to the nature of the study and provided consent. The study was conducted in accordance with the provisions of the Declaration of Helsinki, as amended in Tokyo in 1975 and Venice 1989. The study was approved by the Institutional Review Board of the Department of Medicine, Gunma University, School of Medicine (Maebashi, Japan). In the second study which examined the effect of food restriction for four weeks on serum leptin concentrations, 30 women were randomly included from all subjects in the rst study, and gave their informed consent to undergo food restriction. Study protocols All subjects included in the rst study were not requested to follow any particular diet prior to blood sampling. Blood specimens were collected from the cubital vein in the early morning after overnight fasting. Following immediate centrifugation, the obtained sera were stored at 7 70 C for the measurement of serum leptin, immunoreactive insulin (IRI), glucose and free fatty acid (FFA) concentrations. Total body fat mass and the percentage of body weight (% body fat) were measured by bioelectric impedance analysis method (TBF-101, Tanita Inc., Tokyo, Japan). wall subcutaneous and preperitoneal was estimated by using ultrasonography (SDL-310, Shimazu Industrial Ltd., Tokyo, Japan), according to the method previously reported by Suzuki et al. 13 The abdominal wall (AFI), which has been reported to be well correlated with visceral fat mass, 13 was calculated by dividing the maximum of abdominal wall preperitoneal fat by the minimum of abdominal wall subcutaneous. To study the effect of body weight loss due to food restriction, thirty healthy women (the average of age: 48.4 1.8 y old, the average of the Body Mass Index (BMI): 25.1 0.5 kg/m 2 ) received food restriction therapy (30 kcal/kg desired body weight/day) for four weeks. The desired body weight was calculated according to the modi cation of Broca's calculation method (The Desired Body Weight (kg) ˆ (Height (cm) 7 100) 6 0.9 (kg). The diet composition was not changed during the whole observation period after the start of food restriction. Changes in body fat mass, abdominal fat distribution, and circulating metabolic factors including serum leptin concentration were measured before and at four weeks after the start of food restriction, as described above. Assays Circulating glucose and FFA concentrations were measured with an automatic analyzer using a glucose oxidase method and an enzymatic method, respectively. Serum leptin concentration was assayed with a radioimmunoassay (RIA) kit, obtained from Linco Research, Inc. (St. Charles, Mo, USA). 2,12 The limit of sensitivity for human leptin assay was 0.5 ng/ml. Serum IRI concentration was also assayed with a commercially available RIA kit (Phadeceph Insulin Kit, Pharmacia Japan, Tokyo, Japan). Statistical analysis All data were expressed as means s.e. The correlation was evaluated using the linear regression analysis. The values before and after food restriction were compared by means of paired t-test. A value of P < 0.05 was considered to be statistically signi cant. Results Tables 1 and 2 show the simple correlation coef cients and statistical signi cance among serum leptin concentrations, body fat mass, % body fat, body mass index (BMI), abdominal wall, fasting glucose, serum IRI, FFA concentrations, and the P-values, respectively. As shown in Figure 1, there were signi cant positive correlations between the serum leptin concentration and the BMI (r ˆ 0.630, P < 0.001; Figure 1a), % body fat (r ˆ 0.561, P < 0.001; Figure 1b) and total body fat mass (r ˆ 0.708, P < 0.001; Figure 1c), respectively. The strongest correlation was between total body fat mass and serum leptin concentration. With regard to the abdominal wall fat distribution, serum leptin concentrations were signi cantly (P < 0.001) correlated with both abdominal wall subcutaneous (Figure 2a) and preperitoneal (Figure 2b), but not the AFI (Figure 2c). Thus, serum leptin concentration may not be associated with regional difference of abdominal fat distribution assessed with the AFI. Serum IRI concentrations were signi cantly correlated with serum leptin, and glucose concentrations, total body fat mass, BMI, preperitoneal, and AFI, but not % body fat, or subcutaneous (Tables 1 and 2). Serum FFA concentration was not correlated with any of the factors measured in this study. Table 3 shows the changes of all metabolic factors measured before and at four weeks after the start of food restriction in the second study. The food restriction for four weeks signi cantly (P < 0.001) decreased the total body fat mass by 1.8 kg. As shown in Figure 3, food restriction signi cantly (P ˆ 0.016) reduced serum leptin concentration by 79% of its initial value. However, the % change of total body fat mass by food restriction was not 537

538 Table 1 Relationships between parameters of serum leptin concentration, the total body fat mass, the percentage of body fat (% body fat), the body mass index (BMI), abdominal wall, fasting glucose (FG), immunoreactive insulin (IRI) and free fatty acid (FFA) concentrations in 104 Japanese healthy subjects Leptin Body fat mass % body fat BMI Preperitoneal fat pad Subcutaneous fat pad FG IRI FFA Leptin 1.000 0.708 0.561 0.630 0.451 0.525 0.076 0.038 0.291 70.074 Body fat mass 1.000 0.818 0.883 0.563 0.595 0.080 0.217 0.330 70.058 % Body fat 1.000 0.760 0.466 0.526 0.005 0.133 0.043 70.015 BMI 1.000 0.520 0.537 0.083 0.189 0.350 70.020 Preperitoneal Subcutaneous 1.000 70.435 0.013 0.105 70.124 1.000 0.327 0.651 0.065 0.313 70.105 1.000 0.129 0.241 0.021 FG 1.000 0.260 0.128 IRI 1.000 0.006 FFA 1.000 Table 2 Statistical signi cance of correlation coef cients between parameters of serum leptin concentration, the total body fat mass, the percentage of body fat (% body fat), the body mass index (BMI), abdominal wall, fasting glucose (FG), immunoreactive insulin (IRI) and free fatty acid (FFA) concentrations in 104 Japanese healthy subjects Body fat mass % body fat BMI Preperitoneal fat pad Subcutaneous fat pad FG IRI FFA Leptin P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 N.S. N.S. 0.0026 N.S. Body fat mass P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 N.S. 0.0264 0.0006 N.S. % body fat P < 0.0001 P < 0.0001 P < 0.0001 N.S. N.S. N.S. N.S. BMI P < 0.0001 P < 0.0001 N.S. N.S. 0.0002 N.S. Preperitoneal Subcutaneous 0.0006 P < 0.0001 N.S. 0.0011 N.S. P < 0.0001 N.S. N.S. N.S. N.S. 0.0135 N.S. FG 0.0075 N.S. IRI N.S. Figure 1 Correlations between serum leptin concentration and (a) body mass index (BMI); (b) percentage of body fat (% body fat); (c) total body fat mass in 104 healthy Japanese subjects. The closed circle represents each male value and the open circle represent one in female.

539 Figure 2 Correlations among serum leptin concentration and (a) abdominal subcutaneous; (b) preperitoneal fat ; (c) the abdominal (AFI) in 104 healthy Japanese subjects. Closed circle represents each male value and the open circle represent one in female. Table 3 Effects of food restriction on serum leptin concentration, the total body fat mass, the percentage of body fat (% body fat), the body mass index (BMI), abdominal wall, fasting glucose (FG), immunoreactive insulin (IRI) and free fatty acid (FFA) concentrations in 30 Japanese healthy subjects Before food restriction Four weeks after the start of diet restriction P-value Leptin (ng/ml) 12.48 1.48 9.85 1.21 0.0160 Total body fat mass (kg) 20.88 1.04 19.08 0.98 < 0.0001 % body fat (%) 35.08 1.10 33.33 1.02 < 0.0001 BMI (kg/m 2 ) 25.09 0.53 24.34 0.54 < 0.0001 Preperitoneal (mm) 9.30 0.53 9.11 0.59 0.9068 Subcutaneous (mm) 16.50 0.75 16.48 0.76 0.7873 0.58 0.03 0.55 0.78 0.7130 FG (mg/dl) 92.73 2.23 87.30 1.71 < 0.0001 IRI (mu/ml) 8.78 1.12 6.50 0.43 0.0308 FFA (meq/l) 0.90 0.06 0.72 0.04 0.0032 signi cantly correlated with that of serum leptin concentration (r ˆ 0.131). Fasting glucose, IRI and FFA concentrations were signi cantly reduced after food restriction (Table 3). However, neither abdominal wall preperitoneal, subcutaneous nor the AFI was signi cantly reduced by food restriction for four weeks. Discussion In the present study, we found strong correlations between serum leptin concentrations and the total body fat mass, % body fat, and BMI in healthy Japanese subjects who were of normal weight (23.9 0.3 kg/m 2 ). These results are consistent with previous observations by several investigators. 10,14 However, since the present studies did not include obese subjects, the extent to which the results obtained herein apply to obese individuals is unclear. In addition, we found that a reduction of 1.8 kg (9% of initial value) in total body fat mass by food restriction for four weeks was associated with a reduction of serum leptin concentrations. These data indicate that an increase of serum leptin concentration should re ect the greater production of leptin from increased body fat mass. However, it is possible that the reduction of serum leptin concentration after food restriction may be in uenced by other effects of recent dietary manipulation, since there was no obvious correlation between the % changes in total body fat mass and serum leptin concentration. The present study demonstrated that serum leptin concentrations was not associated with a regional difference of abdominal wall fat mass, evaluated by the ultrasonography method. Although it has been reported that the AFI is a good indicator of visceral fat deposition, 13 the present study demonstrated that

540 Figure 3 Changes in the serum leptin concentrations of 30 healthy Japanese subjects before and after the weight loss by food restriction (30 kcal/kg desired body weight/day) for four weeks. Open circles and horizontal bars represent mean s.e. before and after food restriction for four weeks. Closed circles represent serum leptin concentration before and after food restriction for four weeks in each subject. both preperitoneal and subcutaneous were signi cantly correlated with serum leptin concentration, while the AFI was not at all. This observation indicated that the absolute value ( ) should re ect abdominal fat mass more than the AFI which has been reported to be well correlated with the V/S ratio, calculated from the CT scanning image. We would like to emphasize the contrast between the strong association of the AFI to serum insulin concentration and the absence of any association between the AFI and serum leptin concentration. This suggests that whereas intra-abdominal fat is a determinant of the fasting insulin concentration, it may not be a determinant of serum leptin concentration. It has recently been reported that ob mrna expression varies from region to region in adipose tissues of rodents and humans. 15±17 The epididymal and perirenal s had higher ob mrna levels than subcutaneous fat in the rat. 15 Insulin infusion increased ob mrna expression in epididymal and perirenal fat pads, but not in the subcutaneous fat of the rat. 16 In humans, the ob mrna expression increased in omental fat cells from massively obese humans. 14 In contrast, another human study demonstrated that the ob mrna level in the subcutaneous adipose tissue was higher than those in the omental, retroperitoneal, and mesenteric adipose tissue. 17 The exact interrelation between leptin production and regional fat distribution has not been clinically established in the abdomen. In the present data, serum leptin concentration was correlated with both subcutaneous and preperitoneal, but not the AFI, which is thought to be well related to visceral fat mass. In the second study, the reduction of body fat mass by food restriction for four weeks signi cantly reduced serum leptin concentration, although the abdominal wall and the AFI were not changed. From those data, it was suggested that serum leptin concentration may be associated with the total body fat mass, rather than the increase of visceral fat mass in Japanese healthy subjects who are normal weight. The present data show that there was a strong correlation between circulating leptin concentration and total body fat mass, indicating that the increased fat mass is able to produce large amounts of leptin in obese subjects. Thus, there is no evidence that the metabolic, anorexigenic signal from adipose tissues to the hypothalamus (an increase of circulating leptin), which should regulate food intake, is de cient even in massively obese subjects. Recently, it was demonstrated that, in rodents, exogenously administered leptin acts on the hypothalamus to affect neuropeptide Y mrna expression, inhibiting food intake. 18 There is a possibility that the long-term, excessive production of leptin from an increased fat mass may result in a reduction of the sensitivity to circulating leptin in the feeding center of the hypothalamus in obese subjects, for example down regulation of leptin receptors. Another possibility is the existence of a genetic defect of leptin receptor function. 19,20 In contrast, the recent study which examined the cerebrospinal uid : plasma leptin ratio in obese subjects indicated that the reduced ef ciency of brain leptin delivery among obese individuals with high plasma leptin levels results in apparent leptin resistance. 21 However, it is unlikely that this observation can fully explain the discrepancy between high leptin concentration and hyperphagia in obese subjects, and further studies should be necessary to clarify this issue in humans. Conclusion Serum leptin concentration well re ects the total body fat mass in human subjects. Differences in the abdominal fat distribution are not related to any differences in the in vivo leptin production from adipose tissues. References 1 Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425±432. 2 Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effect of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269: 540±543. 3 Halaas JL, Gajawala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone R, Burley SK, Friedman JM. Weightreducing effects of the plasma protein encoded by the obese gene. Science 1995; 269: 543±546. 4 Weigle DS, Bukowski TR, Foster DC, Holderman S, Kramer JM, Lasser G, Loften-Day CE, Prunkard DE, Raymond C, Kujiper JL. Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest 1995; 96: 2065±2070.

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