- Email: [email protected]
Circulating concentrations of soluble leptin receptor: influence of menstrual cycle and diet therapy
APPLIED NUTRITIONAL INVESTIGATION
Circulating Concentrations of Soluble Leptin Receptor: Influence of Menstrual Cycle and Diet Therapy Hiroyuki Shimizu, MD, PhD, Kenju Shimomura, MD, Mayumi Negishi, MD, Miki Masunaga, MD, Yutaka Uehara, MD, PhD, Noriyuki Sato, MD, PhD, Yohnosuke Shimomura, MD, PhD, Kikuo Kasai, MD, PhD, and Masatomo Mori, MD, PhD From the First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan; the Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Shimotsuga, Tochigi, Japan; and the Gunma Prefectural College of Medical Sciences, Gunma, Japan Concentrations of the soluble leptin receptor (sOB-R) may be related to leptin resistance in obesity. We measured sOB-R concentrations in serum in 103 non-diabetic Japanese men and women. All subjects were grouped according to body mass index (BMI; in kg/m2). Serum sOB-R concentrations did not differ significantly between normal-weight (18.5 ⱕ BMI ⬍ 25.0) men and women, but were significantly higher in underweight subjects (BMI ⬍ 18.5) than in normal-weight subjects. In contrast, overweight (25 ⱕ BMI ⬍ 30) and obese (30 ⱕ BMI ⬍ 35.0, 35.0 ⱕ BMI ⬍ 40, and BMI ⱖ 40) subjects had significantly lower sOB-R concentrations than did normal-weight subjects. Serum sOB-R concentrations were inversely correlated with BMI and serum immunoreactive leptin concentrations. Very low-energy diet therapy for 4 wk significantly lowered serum immunoreactive leptin concentrations but did not significantly affect serum sOB-R concentrations. Serum sOB-R concentrations did not change significantly during the menstrual cycle. Our results showed that serum sOB-R concentrations decrease with increasing BMI and that sex hormones likely do not affect serum sOB-R concentrations in non-pregnant women. The reduction in serum sOB-R concentrations in overweight and obese persons may reflect downregulation of hypothalamic leptin receptor production as a result of an increase in circulating leptin and might be an important factor in leptin resistance. Nutrition 2002;18:309 –312. ©Elsevier Science Inc. 2002 KEY WORDS: soluble leptin receptor, leptin, obesity, body mass index
INTRODUCTION Leptin, the product of the ob gene, was first discovered in genetically obese (ob/ob) mice.1 Central and peripheral administrations of leptin inhibit food intake and enhance thermogenesis, resulting in loss of body weight.2,3 Circulating concentrations of this anorexigenic peptide have been well correlated with body fat weight4 but not with abdominal fat distribution.5 Increased circulating concentrations of leptin, however, do not inhibit appetite in obese persons. The mechanism of leptin resistance in humans is not fully understood. Peripheral administration of the soluble receptor of interleukin (IL)– 6, which belongs to the class II cytokine family, as does leptin, augments central IL-6 effects in vivo.6 The results of a recent study showed that soluble leptin receptor (sOB-R) is the major leptin binding protein in the blood in humans.7 Thus, changes in circulating concentrations of sOB-R may modify biological activities of leptin in vivo. Because leptin exists in the serum in a free form and in a form bound to a carrier protein, leptin’s effects on the hypothalamus may be determined according to the ratio of the free form to the bound one.8,9 Circulating concentrations of sOB-R may be involved in establishing central
leptin resistance in obesity. Little is known about circulating concentrations of sOB-R in obese persons. In the present study, we measured serum concentrations of sOB-R in non-diabetic Japanese men and women categorized as underweight, lean, or obese and compared sOB-R concentrations with changes in circulating leptin concentrations. We previously showed that serum leptin concentrations fluctuate during the menstrual cycle and that estrogen may play an important role in determining circulating leptin concentrations in rodents and humans.10 The placenta produces large amounts of the secretory isoform of leptin receptor messenger RNA, which encodes a soluble binding protein.11 It was also shown that trophoblast cells differentiate, express, and release sOB-R in vivo and in vitro.12 These previous observations suggest that sex hormones affect circulating sOB-R concentrations. However, changes in circulating concentrations of sOB-R during the menstrual cycle have not been reported in humans. Therefore, we also examined the influence of the menstrual cycle on serum sOB-R concentrations.
SUBJECTS AND METHODS Subjects and Study Design
Correspondence to: Hiroyuki Shimizu, MD, PhD, First Department of Internal Medicine, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi, Gunma 371-8511, Japan. E-mail: [email protected]. gunma-u.ac.jp Nutrition 18:309 –312, 2002 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved.
One hundred three persons (45 men and 58 women) were randomly included in this study. All subjects were free of metabolic diseases, including diabetes mellitus. Subjects were grouped according to body mass index (BMI; in kg/m2) as follows: underweight, BMI ⬍ 18.5; normal weight, 18.5 ⱕ BMI ⬍ 25; over0899-9007/02/$22.00 PII S0899-9007(01)00787-0
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Nutrition Volume 18, Number 4, 2002 TABLE I.
SERUM IMMUNOREACTIVE LEPTIN (IRL) AND SOLUBLE LEPTIN RECEPTOR (sOB-R) CONCENTRATIONS IN NORMALWEIGHT SUBJECTS (18.5 ⬍ BMI ⬍ 25 kg/m2)
Sex Male Female
BMI (kg/m2)
IRL (ng/ml)
sOB-R (U/ml)
22.14 ⫾ 0.44 20.34 ⫾ 0.77
3.88 ⫾ 0.40 11.45 ⫾ 2.74*
21.57 ⫾ 2.23 21.49 ⫾ 2.84
Data represent mean ⫾ SE. * P ⬍ 0.05 versus male.
weight, 25 ⱕ BMI ⬍ 30; and obese, 30 ⱕ BMI ⬍ 35 (class I), 35 ⱕ BMI ⬍ 40 (class II), and BMI ⱖ 40 (class III).13 Thirteen 20-y-old women who were not taking oral contraceptives were included to study the influence of the menstrual cycle on serum sOB-R concentrations. These women were asked to measure their basal body temperatures every morning for 28 d, and blood samples were collected in the middle of the luteal and follicular cycles. We calculated total body fat weight according to the subjects’ body weights and measured the percentage of body fat by bioelectric impedance analysis (TBF-101; Tanita Inc, Tokyo, Japan). Seven massively obese patients (three men and four women; aged 31.9 ⫾ 4.9 y; BMI ⫽ 46.0 ⫾ 5.2) were admitted to our hospital and prescribed a very low-energy diet (VLED) to be consumed orally. Initially, the patients received 1200 kcal/d (5.0 MJ/d); 1 wk later, total daily energy intake was reduced by 420 kcal/d (1.8 MJ/d) and VLED therapy was continued for 4 wk. After 4 wk, total daily energy intake was increased by 1200 kcal/d (5.0 MJ/d) and the patients were discharged 1 wk later. Serum samples were collected before the start of the VLED, during weeks 2 and 4 of the VLED, and 1 wk after the return to the 1200-kcal/d (5.0 MJ/d) diet. Assays Serum sOB-R concentrations were measured by using an enzymelinked immunosorbent assay kit (BioVender Laboratory Medicine,
FIG. 2. Correlation of serum sOB-R concentrations with BMI (A) and IRL (B) concentrations in all subjects. BMI, body mass index; IRL, immunoreactive leptin; sOB-R, soluble leptin receptor.
Inc, BMO, Czech Republic).14 In this assay, the antibodies are directed to the extracellular portion of the leptin receptor. Intraassay and interassay coefficients of variation were 2.08% to 4.62% and 1.36% to 7.4%, respectively, and the limit of detection was 0.4 U/mL. Serum immunoreactive leptin (IRL) concentrations were measured by radioimmunoassay with use of a commercially available kit (Linco Research, St. Charles, MO, USA). The leptin assay measured free and bound forms of leptin in serum. Statistics All data are reported as means ⫾ standard errors. Means were analyzed statistically by the use of one-way analysis of variance, followed by Student’s t test and Duncan’s multiple-range test for individual comparisons of means. Repeated-measures analysis of variance was used to analyze the effects of the menstrual cycle and the VLED on sOB-R concentrations. Correlations were evaluated by linear regression analysis. P ⬍0.05 was considered significant.
FIG. 1. Change in serum sOB-R concentrations by BMI in all subjects: underweight, BMI ⬍ 18.5; normal weight, 18.5 ⱕ BMI ⬍ 25; overweight, 25 ⱕ BMI ⬍ 30; and obese, 30 ⱕ BMI ⬍ 35 (class I), 35 ⱕ BMI ⬍ 40 (class II), and BMI ⱖ 40 (class III). Significantly different from normalweight subjects: *P ⬍ 0.05, **P ⬍ 0.01. BMI, body mass index; sOB-R, soluble leptin receptor.
RESULTS As shown in Table I, serum IRL concentrations were significantly higher in normal-weight women than in normal-weight men. In contrast, there was no significant difference in serum sOB-R
Nutrition Volume 18, Number 4, 2002
FIG. 3. Comparison of serum IRL concentrations and serum sOB-R concentrations in normal-weight (18.5 ⱕ BMI ⬍ 25) and overweight and obese (BMI ⱖ 25) subjects. Significantly different from normal-weight subjects: *P ⬍ 0.05, **P ⬍ 0.01. BMI, body mass index; IRL, immunoreactive leptin; sOB-R, soluble leptin receptor.
concentrations between normal-weight men and women. The relation between serum IRL and sOB-R concentrations differed in men and women. The relation between BMI and serum sOB-R concentrations in all subjects is shown in Figure 1. Serum sOB-R concentrations were significantly higher in underweight subjects than in normalweight subjects. In contrast, sOB-R concentrations were significantly lower in overweight and obese subjects than in normalweight subjects. There was no significant difference in sOB-R concentrations among the overweight and obese subjects. As shown in Figure 2A, serum sOB-R concentrations were significantly negatively correlated with BMI. Serum sOB-R concentrations also were significantly negatively correlated with serum IRL concentrations (Fig. 2B). In contrast with the observed changes in serum IRL concentrations with BMI (Fig. 3), serum sOB-R concentrations did not differ significantly among subjects with BMIs greater than 25. BMI decreased from 45.96 ⫾ 5.18 to 41.7 ⫾ 4.57 with VLED therapy for 4 wk. Figure 4 shows the changes in serum IRL and sOB-R concentrations during VLED therapy. VLED therapy significantly lowered serum IRL concentrations at 4 wk. In contrast, serum sOB-R concentrations tended to increase 2 wk after the start of VLED treatment (not significant). When daily energy intake was returned to 1200 kcal/d (5.0 MJ/d), serum IRL concentrations remained lower than concentrations at the start of treatment. Changes in the percentage of body fat, serum IRL concentrations, and sOB-R concentrations during the menstrual cycle are shown in Figure 5. Serum IRL concentrations were significantly higher in the luteal phase than in the follicular phase, whereas neither percentage of body fat nor sOB-R concentrations changed significantly.
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FIG. 4. Changes in serum IRL and sOB-R concentrations after VLED therapy for 4 wk; n ⫽ 7 in each period. Significantly different from before VLED, *P ⬍ 0.05. IRL, immunoreactive leptin; sOB-R, soluble leptin receptor; VLED, very low-energy diet.
jects were about 50% lower than those in normal-weight subjects. However, there was no significant difference in sOB-R concentrations by degree of obesity (BMI ⱖ 30). Serum IRL concentrations are higher in premenopausal women than in men and are higher during the luteal phase than during the follicular phase.10 In addition, we showed that ovariectomized female rats have reduced IRL concentrations that are restored with estrogen supplementation.10 Therefore, estrogen may be involved in the sex difference in serum IRL concentrations. In rats, transcript levels of total leptin receptor are lowest in proestrus in the choroids plexus, but levels of the full-length leptin receptor in the arcuate and ventromedial nuclei of the hypothalamus do not correspond with the levels of total leptin receptor.16 In contrast, the present study in humans showed no significant differences in serum sOB-R concentrations by menstrual cycle phase. Thus, although pregnancy may increase circulating concentrations
DISCUSSION Compared with concentrations in normal-weight subjects, serum sOB-R concentrations were lower in obese subjects and higher in lean subjects and serum sOB-R concentrations correlated inversely with BMI. These findings agree with the previous observation that most leptin circulates in the bound form in lean individuals, whereas most leptin is present in the free form in obese individuals.15 In the present study, sOB-R concentrations in obese sub-
FIG. 5. Changes in the percentage of body fat (A), serum IRL (B), and sOB-R (C) during the menstrual cycle; n ⫽ 13 in each period. IRL, immunoreactive leptin; sOB-R, soluble leptin receptor.
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of leptin and sOB-R in humans,17 sex hormones do not appear to affect circulating sOB-R concentrations in non-pregnant women. Lahlou et al.18 reported that individuals with a mutation in the leptin receptor have markedly elevated concentrations of sOB-R. Their results indicated that the truncated leptin receptor is secreted into the blood and binds most of the serum leptin in homozygous and heterozygous individuals. In the present study, we did not examine the possibility that any of the subjects had leptin receptor mutations, but no subject had markedly high serum IRL concentrations relative to BMI. In addition, serum sOB-R concentrations were obviously lower in obese subjects and there were no significant differences in serum IRL concentrations across the different classes of obese subjects. These findings suggest that none of the present subjects had leptin receptor mutations. Transcripts of the shortest splice variant of the leptin receptor, the secretory isoform of leptin receptor, which is expected to encode a soluble form of the receptor, are detected in relatively high amounts in many tissues in mice.19 In humans, the origin of sOB-R is not known. The sOB-R may be produced by proteolytic cleavage of membrane-associated leptin receptor (e.g., OB-Ra and OB-Rb). The relative expression of the long form (OB-Rl) and the shortest membrane-bound isoform (OB-Rs) in mononuclear cells was significantly lower in overweight (BMI ⬎ 26) than in lean (BMI ⬍ 25) subjects.20 If sOB-R is derived from the whole body including the hypothalamus, the reduction in sOB-R might reflect, at least in part, a downregulation of leptin receptor because the number of leptin receptors may be reduced in parallel to increased circulating leptin concentrations in obese subjects. The inhibition of leptin receptor production by hyperleptinemia may reach a maximum in subjects with BMIs greater than 30, and further increases in circulating leptin concentrations may fail to further increase leptin receptor production. The present study showed no significant change in circulating concentrations of sOB-R with VLED therapy but did show significant reductions in serum IRL concentrations. Sinha et al.21 reported that bound leptin concentrations do not decrease significantly with fasting in lean and obese subjects, in contrast with a pronounced decrease in free leptin in lean subjects. These observations suggest that circulating sOB-R concentrations should not change with a reduction in energy intake. Others have suggested that the soluble form of the cytokine receptor affects the actions of cytokines. A soluble form of the IL-6 receptor acted agonistically with IL-6 on cells expressing the signal transducer gp130.22 Further, the existence of a soluble form of the IL-6 receptor enhances the effect of IL-6 on feeding behavior.6 The weight-reducing effect of leptin was enhanced in C57Bl/6 ob/ob mice in which sOB-R is overexpressed.23 The sOB-R should be involved in determining the effects of leptin on food intake and body weight regulation. The sOB-R is thought to serve as a transport protein for leptin to fetal tissues through the placenta.24 Thus, sOB-R may contribute to the active transport of circulating leptin to the hypothalamus through the blood– brain barrier. Therefore, the observed reduction in circulating sOB-R concentrations in obese subjects may be attributable in part to the establishment of leptin resistance in these individuals.
Nutrition Volume 18, Number 4, 2002
REFERENCES 1. Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425 2. Halaas JL, Gajiwala KS, Maffei M, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995;269:543 3. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995;269:546 4. Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin levels in normal-weight and obese humans. N Engl J Med 1996;334:292 5. Shimizu H, Shimomura Y, Hayashi R, et al. Serum leptin concentration is associated with total body fat mass, but not abdominal fat distribution. Int J Obes Relat Metab Disord 1997;21:536 6. Schobitz B, Pezeshki G, Pohl T, et al. Soluble interleukin-6 (IL-6) receptor augments central effects of IL-6 in vivo. FASEB J 1995;9:659 7. Lammert A, Kiess W, Bottner A, Glasow A, Kratzsch J. Soluble leptin receptor represents the main leptin binding activity in human blood. Biochem Biophys Res Commun 2001;283:982 8. Sinha MD, Opentanova I, Ohannesian JP, et al. Evidence of free and bound leptin in human circulation. J Clin Invest 1996;98:1277 9. Houseknecht KI, Mantzoros CS, Kuliawat R, et al. Evidence for leptin binding to proteins in serum of rodents and humans: modulation with obesity. Diabetes 1996;45:1638 10. Shimizu H, Shimomura Y, Nakanishi Y, et al. Estrogen increases in vivo leptin production in rats and human subjects. J Endocrinol 1997;154:285 11. Gavrilova O, Barr V, Marcus-Samuels B, Reitman M. Hyperleptinemia of pregnancy associated with the appearance of a circulating form of the leptin receptor. J Biol Chem 1997;272:30546 12. Yamaguchi M, Murakami T, Yasui Y, et al. Mouse placental cells secrete soluble leptin receptor (sOB-R): camp inhibits sOB-R production. Biochem Biophys Res Commun 1998;252:363 13. Bray GA. Who are the obese? Body composition and prevalence. In: Bray GA, ed. Contemporary diagnosis and management of obesity. Newton, PA: Handbooks in Health Care, 1998:9 14. Stejskal D, Bartek J, Ruieka V, et al. Determination of leptin receptor in the serum and relations to laboratory and anthropological parameters in patients with atherosclerotic complications. Acta Univ Palacki Olomuc Facul Med 1999;142:89 15. Sinha MK, Caro JF. Clinical aspects of leptin. Vitam Horm 1998;54:1 16. Bennett PA, Lindell K, Wilson C, et al. Cyclical variations in the abundance of leptin receptors, but not in circulating leptin, correlate with NPY expression during the oestrous cycle. Neuroendocrinology 1999;69:417 17. Lewandowski K, Horn R, O’Callaghan CJ, et al. Free leptin, bond leptin, and soluble leptin receptor in normal and diabetic pregnancies. J Clin Endocrinol Metab 1999;84:300 18. Lahlou N, Clement K, Carel J-C, et al. Soluble leptin receptor in serum of subjects with complete resistance to leptin. Relation to fat mass. Diabetes 2000;49:1347 19. Lollmann B, Gruninger S, Stricker-Krongrad A, Chiesi M. Detection and quantification of the leptin receptor splice variants Ob-Ra, b, and, e in different mouse tissues. Biochem Biophys Res Commun 1997;238:648 20. Tsiotra PC, Pappa V, Raptis SA, Tsigos C. Expression of the long and short leptin receptor isoforms in peripheral blood mononuclear cells: implications for leptin’s actions. Metabolism 2000;49:1537 21. Sinha MK, Opentanova I, Ohannesian JP, et al. Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting. J Clin Invest 1996;98:1277 22. Graeve L, Korolenko TA, Hemmann U, et al. A complex of the soluble interleukin-6 receptor and interleukin-6 is internalized via the signal transducer gp 130. FEBS Lett 1996;399:131 23. Huang L, Wang Z, Li C. Modulation of circulating leptin levels by its soluble receptor. J Biol Chem 2000;276:6343 24. Schulz S, Hackel C, Weise W. Hormonal regulation of neonatal weight: placental leptin and leptin receptor. BJOG 2000;107:1486
Circulating Concentrations of Soluble Leptin Receptor: Influence of Menstrual Cycle and Diet Therapy Hiroyuki Shimizu, MD, PhD, Kenju Shimomura, MD, Mayumi Negishi, MD, Miki Masunaga, MD, Yutaka Uehara, MD, PhD, Noriyuki Sato, MD, PhD, Yohnosuke Shimomura, MD, PhD, Kikuo Kasai, MD, PhD, and Masatomo Mori, MD, PhD From the First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan; the Department of Endocrinology and Metabolism, Dokkyo University School of Medicine, Shimotsuga, Tochigi, Japan; and the Gunma Prefectural College of Medical Sciences, Gunma, Japan Concentrations of the soluble leptin receptor (sOB-R) may be related to leptin resistance in obesity. We measured sOB-R concentrations in serum in 103 non-diabetic Japanese men and women. All subjects were grouped according to body mass index (BMI; in kg/m2). Serum sOB-R concentrations did not differ significantly between normal-weight (18.5 ⱕ BMI ⬍ 25.0) men and women, but were significantly higher in underweight subjects (BMI ⬍ 18.5) than in normal-weight subjects. In contrast, overweight (25 ⱕ BMI ⬍ 30) and obese (30 ⱕ BMI ⬍ 35.0, 35.0 ⱕ BMI ⬍ 40, and BMI ⱖ 40) subjects had significantly lower sOB-R concentrations than did normal-weight subjects. Serum sOB-R concentrations were inversely correlated with BMI and serum immunoreactive leptin concentrations. Very low-energy diet therapy for 4 wk significantly lowered serum immunoreactive leptin concentrations but did not significantly affect serum sOB-R concentrations. Serum sOB-R concentrations did not change significantly during the menstrual cycle. Our results showed that serum sOB-R concentrations decrease with increasing BMI and that sex hormones likely do not affect serum sOB-R concentrations in non-pregnant women. The reduction in serum sOB-R concentrations in overweight and obese persons may reflect downregulation of hypothalamic leptin receptor production as a result of an increase in circulating leptin and might be an important factor in leptin resistance. Nutrition 2002;18:309 –312. ©Elsevier Science Inc. 2002 KEY WORDS: soluble leptin receptor, leptin, obesity, body mass index
INTRODUCTION Leptin, the product of the ob gene, was first discovered in genetically obese (ob/ob) mice.1 Central and peripheral administrations of leptin inhibit food intake and enhance thermogenesis, resulting in loss of body weight.2,3 Circulating concentrations of this anorexigenic peptide have been well correlated with body fat weight4 but not with abdominal fat distribution.5 Increased circulating concentrations of leptin, however, do not inhibit appetite in obese persons. The mechanism of leptin resistance in humans is not fully understood. Peripheral administration of the soluble receptor of interleukin (IL)– 6, which belongs to the class II cytokine family, as does leptin, augments central IL-6 effects in vivo.6 The results of a recent study showed that soluble leptin receptor (sOB-R) is the major leptin binding protein in the blood in humans.7 Thus, changes in circulating concentrations of sOB-R may modify biological activities of leptin in vivo. Because leptin exists in the serum in a free form and in a form bound to a carrier protein, leptin’s effects on the hypothalamus may be determined according to the ratio of the free form to the bound one.8,9 Circulating concentrations of sOB-R may be involved in establishing central
leptin resistance in obesity. Little is known about circulating concentrations of sOB-R in obese persons. In the present study, we measured serum concentrations of sOB-R in non-diabetic Japanese men and women categorized as underweight, lean, or obese and compared sOB-R concentrations with changes in circulating leptin concentrations. We previously showed that serum leptin concentrations fluctuate during the menstrual cycle and that estrogen may play an important role in determining circulating leptin concentrations in rodents and humans.10 The placenta produces large amounts of the secretory isoform of leptin receptor messenger RNA, which encodes a soluble binding protein.11 It was also shown that trophoblast cells differentiate, express, and release sOB-R in vivo and in vitro.12 These previous observations suggest that sex hormones affect circulating sOB-R concentrations. However, changes in circulating concentrations of sOB-R during the menstrual cycle have not been reported in humans. Therefore, we also examined the influence of the menstrual cycle on serum sOB-R concentrations.
SUBJECTS AND METHODS Subjects and Study Design
Correspondence to: Hiroyuki Shimizu, MD, PhD, First Department of Internal Medicine, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi, Gunma 371-8511, Japan. E-mail: [email protected]. gunma-u.ac.jp Nutrition 18:309 –312, 2002 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved.
One hundred three persons (45 men and 58 women) were randomly included in this study. All subjects were free of metabolic diseases, including diabetes mellitus. Subjects were grouped according to body mass index (BMI; in kg/m2) as follows: underweight, BMI ⬍ 18.5; normal weight, 18.5 ⱕ BMI ⬍ 25; over0899-9007/02/$22.00 PII S0899-9007(01)00787-0
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Nutrition Volume 18, Number 4, 2002 TABLE I.
SERUM IMMUNOREACTIVE LEPTIN (IRL) AND SOLUBLE LEPTIN RECEPTOR (sOB-R) CONCENTRATIONS IN NORMALWEIGHT SUBJECTS (18.5 ⬍ BMI ⬍ 25 kg/m2)
Sex Male Female
BMI (kg/m2)
IRL (ng/ml)
sOB-R (U/ml)
22.14 ⫾ 0.44 20.34 ⫾ 0.77
3.88 ⫾ 0.40 11.45 ⫾ 2.74*
21.57 ⫾ 2.23 21.49 ⫾ 2.84
Data represent mean ⫾ SE. * P ⬍ 0.05 versus male.
weight, 25 ⱕ BMI ⬍ 30; and obese, 30 ⱕ BMI ⬍ 35 (class I), 35 ⱕ BMI ⬍ 40 (class II), and BMI ⱖ 40 (class III).13 Thirteen 20-y-old women who were not taking oral contraceptives were included to study the influence of the menstrual cycle on serum sOB-R concentrations. These women were asked to measure their basal body temperatures every morning for 28 d, and blood samples were collected in the middle of the luteal and follicular cycles. We calculated total body fat weight according to the subjects’ body weights and measured the percentage of body fat by bioelectric impedance analysis (TBF-101; Tanita Inc, Tokyo, Japan). Seven massively obese patients (three men and four women; aged 31.9 ⫾ 4.9 y; BMI ⫽ 46.0 ⫾ 5.2) were admitted to our hospital and prescribed a very low-energy diet (VLED) to be consumed orally. Initially, the patients received 1200 kcal/d (5.0 MJ/d); 1 wk later, total daily energy intake was reduced by 420 kcal/d (1.8 MJ/d) and VLED therapy was continued for 4 wk. After 4 wk, total daily energy intake was increased by 1200 kcal/d (5.0 MJ/d) and the patients were discharged 1 wk later. Serum samples were collected before the start of the VLED, during weeks 2 and 4 of the VLED, and 1 wk after the return to the 1200-kcal/d (5.0 MJ/d) diet. Assays Serum sOB-R concentrations were measured by using an enzymelinked immunosorbent assay kit (BioVender Laboratory Medicine,
FIG. 2. Correlation of serum sOB-R concentrations with BMI (A) and IRL (B) concentrations in all subjects. BMI, body mass index; IRL, immunoreactive leptin; sOB-R, soluble leptin receptor.
Inc, BMO, Czech Republic).14 In this assay, the antibodies are directed to the extracellular portion of the leptin receptor. Intraassay and interassay coefficients of variation were 2.08% to 4.62% and 1.36% to 7.4%, respectively, and the limit of detection was 0.4 U/mL. Serum immunoreactive leptin (IRL) concentrations were measured by radioimmunoassay with use of a commercially available kit (Linco Research, St. Charles, MO, USA). The leptin assay measured free and bound forms of leptin in serum. Statistics All data are reported as means ⫾ standard errors. Means were analyzed statistically by the use of one-way analysis of variance, followed by Student’s t test and Duncan’s multiple-range test for individual comparisons of means. Repeated-measures analysis of variance was used to analyze the effects of the menstrual cycle and the VLED on sOB-R concentrations. Correlations were evaluated by linear regression analysis. P ⬍0.05 was considered significant.
FIG. 1. Change in serum sOB-R concentrations by BMI in all subjects: underweight, BMI ⬍ 18.5; normal weight, 18.5 ⱕ BMI ⬍ 25; overweight, 25 ⱕ BMI ⬍ 30; and obese, 30 ⱕ BMI ⬍ 35 (class I), 35 ⱕ BMI ⬍ 40 (class II), and BMI ⱖ 40 (class III). Significantly different from normalweight subjects: *P ⬍ 0.05, **P ⬍ 0.01. BMI, body mass index; sOB-R, soluble leptin receptor.
RESULTS As shown in Table I, serum IRL concentrations were significantly higher in normal-weight women than in normal-weight men. In contrast, there was no significant difference in serum sOB-R
Nutrition Volume 18, Number 4, 2002
FIG. 3. Comparison of serum IRL concentrations and serum sOB-R concentrations in normal-weight (18.5 ⱕ BMI ⬍ 25) and overweight and obese (BMI ⱖ 25) subjects. Significantly different from normal-weight subjects: *P ⬍ 0.05, **P ⬍ 0.01. BMI, body mass index; IRL, immunoreactive leptin; sOB-R, soluble leptin receptor.
concentrations between normal-weight men and women. The relation between serum IRL and sOB-R concentrations differed in men and women. The relation between BMI and serum sOB-R concentrations in all subjects is shown in Figure 1. Serum sOB-R concentrations were significantly higher in underweight subjects than in normalweight subjects. In contrast, sOB-R concentrations were significantly lower in overweight and obese subjects than in normalweight subjects. There was no significant difference in sOB-R concentrations among the overweight and obese subjects. As shown in Figure 2A, serum sOB-R concentrations were significantly negatively correlated with BMI. Serum sOB-R concentrations also were significantly negatively correlated with serum IRL concentrations (Fig. 2B). In contrast with the observed changes in serum IRL concentrations with BMI (Fig. 3), serum sOB-R concentrations did not differ significantly among subjects with BMIs greater than 25. BMI decreased from 45.96 ⫾ 5.18 to 41.7 ⫾ 4.57 with VLED therapy for 4 wk. Figure 4 shows the changes in serum IRL and sOB-R concentrations during VLED therapy. VLED therapy significantly lowered serum IRL concentrations at 4 wk. In contrast, serum sOB-R concentrations tended to increase 2 wk after the start of VLED treatment (not significant). When daily energy intake was returned to 1200 kcal/d (5.0 MJ/d), serum IRL concentrations remained lower than concentrations at the start of treatment. Changes in the percentage of body fat, serum IRL concentrations, and sOB-R concentrations during the menstrual cycle are shown in Figure 5. Serum IRL concentrations were significantly higher in the luteal phase than in the follicular phase, whereas neither percentage of body fat nor sOB-R concentrations changed significantly.
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FIG. 4. Changes in serum IRL and sOB-R concentrations after VLED therapy for 4 wk; n ⫽ 7 in each period. Significantly different from before VLED, *P ⬍ 0.05. IRL, immunoreactive leptin; sOB-R, soluble leptin receptor; VLED, very low-energy diet.
jects were about 50% lower than those in normal-weight subjects. However, there was no significant difference in sOB-R concentrations by degree of obesity (BMI ⱖ 30). Serum IRL concentrations are higher in premenopausal women than in men and are higher during the luteal phase than during the follicular phase.10 In addition, we showed that ovariectomized female rats have reduced IRL concentrations that are restored with estrogen supplementation.10 Therefore, estrogen may be involved in the sex difference in serum IRL concentrations. In rats, transcript levels of total leptin receptor are lowest in proestrus in the choroids plexus, but levels of the full-length leptin receptor in the arcuate and ventromedial nuclei of the hypothalamus do not correspond with the levels of total leptin receptor.16 In contrast, the present study in humans showed no significant differences in serum sOB-R concentrations by menstrual cycle phase. Thus, although pregnancy may increase circulating concentrations
DISCUSSION Compared with concentrations in normal-weight subjects, serum sOB-R concentrations were lower in obese subjects and higher in lean subjects and serum sOB-R concentrations correlated inversely with BMI. These findings agree with the previous observation that most leptin circulates in the bound form in lean individuals, whereas most leptin is present in the free form in obese individuals.15 In the present study, sOB-R concentrations in obese sub-
FIG. 5. Changes in the percentage of body fat (A), serum IRL (B), and sOB-R (C) during the menstrual cycle; n ⫽ 13 in each period. IRL, immunoreactive leptin; sOB-R, soluble leptin receptor.
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of leptin and sOB-R in humans,17 sex hormones do not appear to affect circulating sOB-R concentrations in non-pregnant women. Lahlou et al.18 reported that individuals with a mutation in the leptin receptor have markedly elevated concentrations of sOB-R. Their results indicated that the truncated leptin receptor is secreted into the blood and binds most of the serum leptin in homozygous and heterozygous individuals. In the present study, we did not examine the possibility that any of the subjects had leptin receptor mutations, but no subject had markedly high serum IRL concentrations relative to BMI. In addition, serum sOB-R concentrations were obviously lower in obese subjects and there were no significant differences in serum IRL concentrations across the different classes of obese subjects. These findings suggest that none of the present subjects had leptin receptor mutations. Transcripts of the shortest splice variant of the leptin receptor, the secretory isoform of leptin receptor, which is expected to encode a soluble form of the receptor, are detected in relatively high amounts in many tissues in mice.19 In humans, the origin of sOB-R is not known. The sOB-R may be produced by proteolytic cleavage of membrane-associated leptin receptor (e.g., OB-Ra and OB-Rb). The relative expression of the long form (OB-Rl) and the shortest membrane-bound isoform (OB-Rs) in mononuclear cells was significantly lower in overweight (BMI ⬎ 26) than in lean (BMI ⬍ 25) subjects.20 If sOB-R is derived from the whole body including the hypothalamus, the reduction in sOB-R might reflect, at least in part, a downregulation of leptin receptor because the number of leptin receptors may be reduced in parallel to increased circulating leptin concentrations in obese subjects. The inhibition of leptin receptor production by hyperleptinemia may reach a maximum in subjects with BMIs greater than 30, and further increases in circulating leptin concentrations may fail to further increase leptin receptor production. The present study showed no significant change in circulating concentrations of sOB-R with VLED therapy but did show significant reductions in serum IRL concentrations. Sinha et al.21 reported that bound leptin concentrations do not decrease significantly with fasting in lean and obese subjects, in contrast with a pronounced decrease in free leptin in lean subjects. These observations suggest that circulating sOB-R concentrations should not change with a reduction in energy intake. Others have suggested that the soluble form of the cytokine receptor affects the actions of cytokines. A soluble form of the IL-6 receptor acted agonistically with IL-6 on cells expressing the signal transducer gp130.22 Further, the existence of a soluble form of the IL-6 receptor enhances the effect of IL-6 on feeding behavior.6 The weight-reducing effect of leptin was enhanced in C57Bl/6 ob/ob mice in which sOB-R is overexpressed.23 The sOB-R should be involved in determining the effects of leptin on food intake and body weight regulation. The sOB-R is thought to serve as a transport protein for leptin to fetal tissues through the placenta.24 Thus, sOB-R may contribute to the active transport of circulating leptin to the hypothalamus through the blood– brain barrier. Therefore, the observed reduction in circulating sOB-R concentrations in obese subjects may be attributable in part to the establishment of leptin resistance in these individuals.
Nutrition Volume 18, Number 4, 2002
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