Plasma Leptin in Obese Subjects and Diabetics

Endocrine Journal 1997, 44(5), 671-676 Human Plasma Leptin in Obese Subjects and Diabetics YosHIMASA TASAKA, KEIKD YANAGISAWA, AND YASUHIKO IWAMOTO Diabetes Center, Tokyo Women's Medical College, Tokyo 162, Japan Abstract. Leptin is the protein product of the ob gene, an adipocyte-specific gene, recently discovered in mice. Plasma leptin levels were determined in six normals, twenty-one subjects with impaired glucose tolerance, and forty-nine untreated NIDDM subjects. They increased with the augmentation of obesity (body mass index, BMI kg/m2) and were higher in females than in males: in BMI less than 25 kg/m2 the values of plasma leptin were 2.24 ± 0.25 ng/ml (n=29) in males and 3.01 ± 0.39 ng/ml (n=13) in females (P<0.054), respectively, in BMI between 25 kg/m2 and 30 kg/m2 they were 3.14 ± 0.31 ng/ml (n=10) in males and 10.66 ± 2.86 ng/ml (n=7) in females (P<0.0018) and in BMI higher than 30 kg/m2 their levels were 8.98 ± 1.5 ng/ml (n=11) and 11.74 ± 2.2 ng/ml (n=6) (P<0.23), respectively. The severity of diabetes mellitus judged from the fasting plasma glucose level had no influence on the plasma leptin levels during OGTT, but the leptin levels decreased significantly during a tolerance test (P<0.001), and similar results were also seen during a breakfast test. The fasting plasma leptin in the male with FBS less than 140 mg/dl had a significant correlation with the fasting plasma IRI level, but this correlation disappeared after taking obesity into consideration. Thus the plasma leptin was chiefly dependent on the body weight and gender and had no special relation to diabetic severity. Key words: Plasma leptin, Obesity, Diabetes mellitus, Gender (Endocrine Journal 44: 671-676,1997) LEPTIN is the protein product of the ob gene, an adipocyte-specific gene, recently discovered in mice [1, 2]. Mutations in the ob gene, which cause a lack of circulating leptin, lead to increased food intake, reduced energy expenditure, and obesity in the ob/ob mouse [3]. Circulating concentrations of leptin are reported to be several times higher in obese than in lean individuals [4-8]. Leptin mrna is expressed exclusively in adipose tissue and its level of expression has been correlated with the amount of body fat [1, 3, 9-11]. The hypothalamus has been identified as the site for leptin signaling in studies in which lesions of the hypothalamus produced by chemical agents increased both body food intake and concentrations of leptin mrna in Received: February 4, 1997 Accepted: July 14, 1997 Correspondence to: Dr. Yoshimasa TASAKA, Diabetes Center, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan adipose tissue of treated rodents [12] and leptin receptor mrna was densely concentrated in the arcuate nucleus, with lower levels present in the ventromedial and dorsomedial hypothalamic nuclei and other brain areas involved in energy balance [13, 14]. Circulating concentrations of leptin in human subjects have been reported already. Plasma leptin was reported to be significantly higher in obese subjects than in non-obese ones, especially in females [4, 7, 8, 15]. Recently a radioimmunoassay kit of human leptin became available. With this assay kit we investigated the plasma leptin level in obese and diabetic subjects. Materials and Methods Seventy-six subjects were studied. They were six normal, twenty-one impaired glucose tolerant and forty-nine untreated diabetic subjects. The mean age of the 50 males was 46.5 ± 1.9 yrs (M ±

672 TASAKA et al. SEM) and that of 26 females 48.3 ± 3.1 yrs. The body mass index (BMI) of the males was 25.6 ± 0.87 kg/m2 and that of the female was 26.5 ± 1.39 kg/m2. They all received 75 g or 50 g OGTT for the determination of glucose tolerance. The plasma immunoreactive leptin was determined with an immunoassay kit supplied by Linco Research, Inc. (St. Louis, USA) and the immunoreactive insulin (IRI) was determined by the double-antibody system of Morgan-Lazarow (16]. Both the leptin calibrators (0.5-100 ng/ml) and 1251-labeled leptin were prepared with recombinant human leptin and other details have already been reported [7]. Three amounts of leptin (2.85, 5.7and 14.3 ng) were added to two plasma samples and the leptin concentrations were determined in two duplicate analyses. Recovery ranged from 95.7 to 105%. Recoveries of leptin in two human plasma specimens (initial concentrations 22.69, and 14.69 ng/ml) serially diluted 2 and 4-fold ranged from 109 to 110%, showing a slightly higher dilution effect. Withinand between-assay variation was assessed by repeated analysis of two plasma samples containing 0.9-2.96 ng/ml leptin. CVs ranged from 1.44 to 2.44% within runs and from 4.0 to 5.1% between runs. The recovery of plasma leptin even after 5 repeated freezings and thawings was 104% in two plasma samples (88.6 and 120%). Fig. 1. Correlation of plasma leptin concentration with BMI for women (LI) and men ( ). Women, 0.526x -6.76 (r=0.605, n=24); men, 0.458x - 7.38 (r=0.788, n=48). Statistical analysis Data are shown as the mean ± SEM. Statistical analysis was carried out by the non-parametric Mann-Whitney U-test. Paired analysis by Student's t-test was also performed. A probability of 0.05 was considered to indicate significance. Results Plasma leptin concentrations and BMI Fasting plasma leptin concentrations were measured in normal, impaired and diabetic subjects (Fig. 1). Plasma leptin concentrations measured in samples from fasting subjects correlated well with the subject's BMI values (r=0.688, P<0.01). The distribution of leptin values vs. BMI for the combined study group clearly represented superimposion of largely distinct distribution for men and women. Regression analysis of leptin Fig. 2. Comparison of plasma leptin levels in males and females. The number in bracket shows patient's number. Significant difference was found only in the group with BMI 25-30. values in relation to BMI separated by gender yielded a higher slope for women than for men (slope for females 0.526 and 0.458 for males). The gender effect was studied in more detail according to the level of BMI (Fig. 2). BMI was divided into three groups: less than 25, from 25 to 30 and more than 30 kg/m2. As shown in Fig. 2 the mean fasting plasma leptin concentrations were higher in women

PLASMA LEPTIN IN OBESE SUBJECTS AND DIABETICS 673 than in men at all levels of BMI, especially high in the BMI 25-30 kg/m2 range (P<0.002, males: 3.14 ± 0.31, females: 10.66 ± 2.86 ng/ml). In BMI less than 25 kg/m2 they were 2.24 ± 0.25 in males and 3.01 ± 0.39 ng/ml in females (P<0.054, not significant difference) and in BMI higher than 30 kg/m2 their levels were 8.98 ± 1.5 in males and 11.47 ± 2.2 ng/ml (P<0.23, not significant difference) in females. Plasma leptin concentrations and diabetes Plasma leptin levels were compared in the untreated subjects with different fasting plasma glucose levels including diabetes mellitus. They were all subjected to a 50 g OGTT test. The subjects were divided into three groups according to the fasting plasma glucose level: 140<, 140-200 and >200 mg/dl. All of the BMIs in these subjects were less than 25 kg/m2 since the leptin levels varied greatly in cases with a BMI higher than 25 kg/m2, as shown in Fig. 2, and each group contained the same number of males and females in order to avoid any effect on the leptin levels. The plasma responses of glucose and IRI are shown in Fig. 3. As already reported in many publications, plasma responses of IRI decreased reciprocally to plasma glucose level. On the other hand, plasma levels of leptin were similar at any time during OGTT in these three groups (Fig. 4). There was no significant change in plasma leptin levels after OGTT test in these number of cases. Plasma leptin concentrations in OGTT and breakfast tests were measured (Fig. 5). The upper figure shows the results of the breakfast test and the lower one those of the 50 g OGTT. In the breakfast test the subjects happened to be obese people, and therefore had a higher leptin level than in OGTT. In both groups the plasma leptin concentration decreased significantly during these loading tests. In 50 g OGTT the plasma leptin level in the fasting state was 2.74 ± 0.31 ng/ml, then the value fell significantly to 2.36 ± 0.24 ng/ml after 2 h (P<0.001). Similarly in the breakfast test the plasma leptin concentration decreased from 5.56 ± 1.15 ng/ml at fasting to 5.09 ± 1.21 ng/ml at 2 h (P<0,05). The relation between fasting plasma IRI and IRL was investigated. The cases with fasting plasma glucose less than 140 mg/dl were selected since the subjects with fasting plasma glucose more than 140 mg/dl are real diabetics and low insulin Fig. 3. Plasma glucose and IRI responses during 50 g OGTT. Diabetics were divided into three groups according to the fasting plasma glucose level (FPG). C], FPG < 140 mg/dl; 0, FPG 140-200 mg/dl; /, FPG > 200 mg/dl. The BMI of all the subjects was less than 25 kg/m2 and each group contained the same number of males and females. **, P<0.01; ***, P<0.001 (vs. FPG < 140 mg/dl). The number in bracket shows patient's number. responders, and they were all males in this case. As shown in Fig. 6, a significant correlation was found between them (r=0.613, P<0.001), but when the same statistics was done in the subjects with a BMI less than 30 kg/m2 in order to avoid the influence of obesity, no significant relation was present (r=-0.616, n=8).

674 TASAKA et al. Fig. 4. Plasma leptin response during 50 g OGTT. Diabetics were divided into three groups according to the FPG level. The BMI of all the subjects was less than 25 kg/m2 and each group contained the same number of males and females. LI, FPG < 140 mg/dl; 0, FPG 140-200 mg/dl;, FPG > 200 mg/dl. The number in bracket shows patient's number. Fig. 5. Plasma leptin levels during 50 g OGTT and breakfast test. Upper figure shows the results of breakfast test and lower one does those of 50 g OGTT. The number in bracket shows patient's number. Discussion The RIA kit for plasma human le ptin p rovi ded Fig. 6. Correlation between fasting plasma IRI and leptin levels. All the subjects were males (n=28) and had FPG less than 140 mg/dl. Y=0.711x-0.206 (r=0.613, P<0.01). accurate and precise analysis of the recently discovered hormone. Both the lower detection limit (0.5 ng/ml) and the dynamic range of the assay proved to be optimal. Maffei et al. [5] found plasma leptin concentrations ranging between 1 and 200 ng/ml in a patient population that included obese subjects (BMI up to 62 kg/m2). Leptin concentrations reported by Considine et al. [4] were 7.5 ± 9.3 ng/ml in normal-weight subjects and 31.3 ± 24.1 ng/ml in obese subjects, and those by Zhongmin et al. [7] were 3.84 ± 1.79 ng/ml in males (BMI between 18 and 25 kg/m2) and 7.36 ± 3.73 ng/ml in females, respectively. In our study plasma leptin concentrations measured in samples from fasting subjects correlated well with their BMI and in each of three stages the leptin level was higher in females than in males, especially in BMIs between 25 and 30 kg/m2. The reason why the plasma leptin concentrations are higher in females than in males is not known [7, 8, 15, 17]. Rosenbaum et al. [15] mentioned that leptin was significantly higher in pre- and post-menopausal females than in males, even when leptin was corrected for differences in body composition (premenopausal females > post-menopausal females > males). Considine et al. [4] reported that gender had no significant effect on the human adipocyte content of mrna, while Lonnqvist et al. [18] found higher ob mrna content in adipocytes from obese

PLASMA LEPTIN IN OBESE SUBJECTS AND DIABETICS 675 females, but not in non-obese females compared to obese and non-obese males, respectively. Leptin was reported to be better correlated with absolute fat mass than BMI or percentage body fat at usual body weight [15] and the fat mass is richer in females than in males. More studies from these viewpoints will therefore be necessary to solve this problem. The relationship between the severity of diabetes and the plasma leptin level was studied. In consideration of the influence of both gender and body weight on the leptin level, the same numbers of males and females with a BMI less than 25 were selected. As shown in this study the severity of diabetes had no influence on the plasma leptin levels. Segal et al. [6] reported that marked alterations in plasma glucose and insulin concentrations induced by glucose and tolbutamide injection did not cause any change in plasma leptin levels. Moreover, the plasma leptin level decreased significantly during the OGTT. This decrease was found also not only during the breakfast test but also during a long fast until lunch (results not shown). Similar findings were reported previously [23]. Such a decrease was thought to be a diurnal variation in plasma leptin. Saladin et al. [20] showed that rat adipose tissue oh mrna levels were lowest during the light circle, increasing soon after the rats start eating, reached the maximum around 0400 h and thereafter oh mrna steadily decreased, reaching the minimum in the afternoon. Although insulin increased plasma leptin concentrations in normal subjects and patients with NIDDM [21], the plasma level of insulin caused by infused insulin was higher than that after OGTT or breakfast test (480 fmol/ml vs. 246 fmol/ml in our OGTT) and stimulated insulin by OGTT or acute insulin administration was reported not to affect the plasma leptin level [22]. Sinha et al. [19] found that the plasma leptin level is highest between midnight and the early morning and lowest from noon to mid-afternoon. In this study a significant correlation was found between fasting plasma IRI and leptin levels in male NIDDM patients with fasting plasma glucose less than 140 mg/ml, but it disappeared after adjusting for obesity. In Western Samoans serum leptin concentrations were strongly correlated with serum insulin concentrations even after adjusting for obesity in both sexes [8], and a possible role in insulin resistance or hyperinsulinemia was suggested. These differences might be due to the ethnic differences as seen in the pathogenesis of NIDDM. Plasma leptin is considered to be regulated chiefly by an adipose tissue-hypothalamus relationship, but another mechanism including sex hormone, corticosteroid [24] or other unknown factors would be associated with it. Further investigation will be necessary for its clarification. Acknowledgments The authors gratefully acknowledge Mitsue Tomioka, Yasutomi Tanaka, Miya Hasegawa, Masami Tsuchiya and Koji Kodama for their excellent technical assistance. References 1. Zhang Y, Droenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425-432. 2. Halaas JL, Gajiwala KS, Maffei M, Cohen SC, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM (1995) Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269: 543-546. 3. Weigle DS, Bukowski TR, Foster DC, Holderman S, Kramer JM, Lasser G, Lofton-Day CE, Prunkard DE, Raymond C, Kuijper JL (1995) Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest 96: 2065-2070. 4. Considine RV, Sinha MK, Heiman ML, Kriaciunas A, Stephens TW, Nyce MR, Ohannesin JP, Marco CC, McKee Lj, Bauer TL, Caro JF (1996) Serum immunoreactive-leptin concentrations in normalweight and obese humans. N Engl J Med 334: 292-295. 5. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H,Kim H, Lallone R, Ranganathan S, Kern PA, Friedman JM (1995) Leptin levels in human and rodent: Measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nature Medicine 1:1155-1161.

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