Orchiectomy and response to testosterone in the development of obesity in young Otsuka- Long-Evans-Tokushima Fatty (OLETF) rats

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1 International Journal of Obesity (1998) 22, 318±324 ß 1998 Stockton Press All rights reserved 0307±0565/98 $12.00 Orchiectomy and response to testosterone in the development of obesity in young Otsuka- Long-Evans-Tokushima Fatty (OLETF) rats H Shimizu 1, K-I Ohtani 1, Y Uehara 1, Y Abe 2, H Takahashi 1, T Tsuchiya 1, N Sato 1, Y Ibuki 2 and M Mori 1 1 The First Department of Internal Medicine and 2 Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi, Japan OBJECTIVE: Withdrawal of testosterone prevents the development of hyperglycaemia in male Otsuka-Long-Evans- Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus (NIDDM), but the exact mechanism has not been established. The present studies were undertaken to examine a possible role of testosterone in the development of obesity in young OLETF rats who have not shown marked hyperphagia. METHODS: Body weight, food intake and circulating concentrations of metabolic factors including immunoreactive leptin (IRL) were measured at ve weeks of age in young male OLETF rats and their lean controls, Long-Evans- Tokushima-Otsuka (LETO) rats. At six weeks of age, both LETO and OLETF rats were bilaterally orchiectomized (Orchx) and half of each group implanted with a silastic tube containing testosterone. After a three week observation period, all animals were killed and circulating concentrations of metabolic factors and the ob gene expression in retroperitoneal white adipose tissues were measured. RESULTS: Body weight and 24 h food intake were already increased in OLETF rats at ve weeks of age. Serum testosterone concentrations were signi cantly lower in OLETF rats than in LETO rats. Expression of the ob gene was signi cantly decreased in the retroperitoneal white adipose tissue of OLETF rats, and their serum IRL concentrations were lower. Food intake and body weight gain for three weeks after the operation were signi cantly lower in the Orchx group of OLETF rats than in the sham-operated group. Hyperglycaemia, accompanied by hyperinsulinaemia, was attenuated by orchiectomy in OLETF rats. Circulating IRL concentrations were signi cantly higher in OLETF rats than in LETO rats and decreased by orchiectomy. Testosterone supplement reversed all of the changes caused by orchiectomy in OLETF rats. In contrast, the changes, which were observed after orchiectomy in OLETF rats, were not obvious in LETO rats. CONCLUSION: The present data indicate that testosterone plays a role in the development of obesity and NIDDM in young OLETF rats, but that changes of leptin production in white adipose tissue may not be important in the development of obesity in young OLETF rats. Keywords: leptin; obesity; Orchiectomy; Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats; testosterone Introduction The Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat is a model of non-insulin dependent diabetes mellitus (NIDDM), established by Kawano et al. 1 Peripheral insulin resistance precedes impairment of pancreatic b-cell function in OLETF rats, and insulin resistance seems closely related to fat deposition in the abdominal cavity of OLETF rats. 2 Caloric restriction and exercise training have been reported to be effective in preventing the development of NIDDM in OLETF rats, like other animal models of NIDDM. 3,4 On the other hand, testosterone has been found to reduce glucose intolerance. 5 Recent studies added new ndings that testosterone is involved in the Correspondence: Hiroyuki Shimizu, MD, PhD, 1st Department of Internal Medicine, Gunma University School of Medicine, Showa-machi, Maebashi Gunma 371, Japan. Received 17 July 1997; revised 14 November 1997; accepted 28 November 1997 glucose metabolism of rat skeletal muscle 6 and an increase in visceral fat accumulation. 7 Withdrawal of testosterone after orchiectomy has been reported to prevent the development of hyperglycaemia in OLETF rats, 8 indicating a possible role of testosterone in the establishment of NIDDM in this model. In contrast, ovariectomy increased the incidence of diabetes mellitus and estradiol supplement restored the incidence of diabetes mellitus in female OLETF rats. High incidences of diabetes mellitus in male rats have also been observed in Wistar Fatty rats (fa=fa) 9 and Zucker Fatty rats. 10 These data indicate that testosterone might be involved in the development of NIDDM in rodents. However, the exact mechanism by which testosterone promotes the development of hyperglycaemia has not been established in these animal models of NIDDM including OLETF rats. Recently, the ob gene has been cloned in genetically obese (ob=ob) mice, and a genetic abnormality in the ob gene resulting in low plasma leptin levels has been found to play a critical role in the development of obesity and NIDDM in this model. 11 In addition, a

2 mutation in the leptin receptor has been reported to be involved in the development of diabetes mellitus in genetically diabetic (db=db) mice and Fatty Zucker rats, 12,13 but the attempts to detect changes in ob gene expression and=or circulating leptin concentrations have not been made in OLETF rats. There is a gender difference in circulating leptin levels in humans, however. 14,15 We have recently demonstrated that estrogen increases the relative ob gene mrna levels and circulating leptin concentrations in ovariectomized Wistar rats, 16 and a signi cant difference between circulating leptin concentrations in males and postmenopausal women has been found in our study and another report, indicating the involvement of androgen in the gender difference of circulating leptin levels in humans. 15,16 ; Since testosterone is thought to be involved in the development of NIDDM in OLETF rats, there is a possibility that ob gene expression may be altered in these rats. The present studies were undertaken to examine a possible role of testosterone in the development of obesity in young OLETF rats which do not yet show clear hyperglycaemia. In addition, strain differences and the effects of orchiectomy and testosterone supplement on adipose tissue leptin production were investigated in this model of NIDDM. Materials and methods Animals Both OLETF rats and their lean controls, Long-Evans- Tokushima-Otsuka (LETO) rats, were kindly provided by Tokushima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). The LETO rats are lean controls which are obtained from the same colonies of Long-Evans rats, and therefore, have the same genetic backgrounds as OLETF rats. All animals were maintained in a temperature-controlled room (22 1 C). They were supplied with standard rat Purina chow pellets (Oriental Yeast, Osaka, Japan) and tap water ad libitum. Body weight and daily food intake were measured in young male LETO and OLETF rats at the age of ve weeks. After the measurement of daily food intake for three days, the animals were killed. Rightside subcutaneous, right-side whole retroperitoneal and total mesenteric white adipose tissues (SWAT, RWAT, MWAT) were immediately dissected according to the method of Krotkiewski and BjoÈrntorp. 17 The upper area of SWAT is the caudal border of the xiphoid process of the sternum and the lower, urological organ and side, the dorsal and ventral midline of the body. The weight of SWAT, RWAT, MWAT was measured after blood collection. A sample of fat mass was immediately dissected from right RWAT and used for the ob gene mrna determination, as Testosterone and obesity in young OLETF rats described below. After centrifugation, the obtained sera were frozen at 720 C until assayed. In the second experiment, both LETO and OLETF rats were sham-operated or bilaterally orchiectomized (Orchx) at six weeks of age and half of them were implanted with a silastic tube containing testosterone. Bilateral orchiectomy was performed through scrotal incision under light ether anaesthesia. In Orchx rats with testosterone supplement, 1 cm of the silastic tube (inner diameter 1.56 mm, outer diameter 3.15 mm; Dow Corning Corporation, Midland, MI, USA) containing 3 mg testosterone (Sigma Chemical Co, St Louis, MO, USA) was simultaneously implanted through a small incision in the skin over the back of anaesthetized rats, according to the method of Morin and Cummings. 18 Changes in body weight and daily food intake were observed for three weeks after the operation. Following three-week observation, all animals were killed and a piece of RWAT was immediately dissected and used for the determination of ob gene mrna expression. After centrifugation sera were also kept at 720 C before assay. Determination of ob gene mrna expression The fat mass sample was sonicated in 0.8 ml of Isogene (Nippon Gene, Tokyo, Japan) and centrifuged at rpm for 10 min. The supernatant containing total RNA was taken from each sample and total RNA was extracted according to the protocol supplied by the manufacturer. Then, ob gene and b-actin mrna expression were measured by the reverse transcription-polymerase chain reaction (RT-PCR) method, using the following primers for the ob gene: forward primer; 5 0 -TGTCGGTTCCTGTGGCTTTG-3 0, backward primer; 5 0 -AGGCTGGTGAGGACCTGTTGATAG- 3 0, according to the cdna sequence of rat ob gene. 19 First strand cdna was synthesized from the obtained total RNA as described in the Gene Amp EZ rtth RNA PCR kit (Perkin Elmer, Branchburg, NJ, USA) containing 1.0 U rtth DNA polymerase, 3 nmole of dctp, dgtp, dttt, datp, and 5 pmole of each primer in 10 ml volume overlaid with mineral oil. PCR was performed for 31 cycles using a 1-min denaturation step at 94 C, a 1-min annealing step at 50 C and a 1-min extension step at 72 C. An additional 7-min extension step at 72 C was added after 31 cycles which were selected according to our preliminary results con rming the linear increase of both ob gene and b-actin mrna. PCR products were electrophoresed on a 6% polyacrylamide gel and visualized by ultraviolet uorescence following ethidium bromide staining. The intensity of uorescence of the band was calculated by NIH image. Assays Serum immunoreactive leptin (IRL) concentrations were measured with a radioimmunoassay (RIA) kit 319

3 320 Testosterone and obesity in young OLETF rats for rat leptin (Linco Research; Inc, St Charles, MO, USA). 20 The limit of sensitivity for the rat IRL assay is 0.5 ng/ml. Within- and between-assay variations were 2.0±4.6%, 3.0±5.7%, respectively. Serum immunoreactive insulin (IRI) concentrations were assayed with a commercially available RIA kit (Phadeceph Insulin, Pharmacia Japan, Tokyo, Japan) using rat insulin (Novo Nordisk A=S, Gentofte, Denmark) as a standard. Serum glucose concentrations were analyzed with an automatic analyzer by the glucose oxidase method. Serum LH and FSH concentrations were assayed by RIA using immuno-reagents provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Serum estradiol (E 2 ) concentrations were measured by RIA using an Estradiol Cortoria kit (CIS Diagnostic, Inc, Chiba, Japan). Serum corticosterone concentrations were measured by RIA using anti-corticosterone serum (corticosterone-3-cmo-3- SA). 21 Serum testosterone concentration was assayed by RIA using a DPC Total Testosterone Kit (Nippon DPC Corp, Tokyo, Japan). but the differences were not statistically signi cant between them. There was no difference in the MWAT=SWAT (M=S) ratio, indicating that both fat compartments were increased by the same percentage. The weight of the testis was slightly, but not signi cantly lower in OLETF rats. Table 2 shows changes of humoral factors in both strains. Blood glucose and serum IRI concentrations were not signi cantly higher in OLETF rats than in LETO rats. Serum testosterone concentrations were signi cantly lower in OLETF rats than in LETO rats. In contrast, serum estradiol (E 2 ) levels in OLETF rats were not different from those of LETO rats, indicating that the conversion of testosterone to E 2 may be enhanced in white adipose tissues of OLETF rats. The ratio of ob gene mrna levels to b-actin mrna levels was signi cantly decreased in RWAT of OLETF rats (LETO rats: arbitrary units, OLETF rats: arbitrary units, P < 0.01). Subsequently, serum IRL concentrations tended to be lower in OLETF rats than in LETO rats, as shown in Table 2. Statistics All data are expressed as means s.e.m. Statistical analysis was performed by the analysis of variance (ANOVA), followed by Duncan's multiple range test for individual comparisons of the means. Results i) Body weight, food intake and various metabolic factors at ve weeks of age As shown in Table 1, body weight and food intake for 24 h were already increased in OLETF rats at ve weeks of age. The weight of each white adipose tissue tended to be heavier in OLETF rats than in LETO rats, ii) Effect of orchiectomy on body weight gain, daily food intake for three weeks and changes in various metabolic factors Figure 1 and Figure 2 show chronological changes in daily food intake and body weight gain for three weeks after the operation. OLETF rats showed signi cant increases in daily food intake and body weight gain for those three weeks, compared to LETO rats. Orchiectomy signi cantly inhibited hyperphagia of OLETF rats, and subsequently, decreased body weight gain for the three weeks. Testosterone supplement reversed the effects of orchiectomy on daily food intake and body weight gain of OLETF rats. In contrast, the effects of orchiectomy on daily food intake and body weight gain were not observed in LETO rats. Table 1 Changes in body weight, food intake, weight of white adipose tissue, testicular weight in Long-Evans-Tokushima-Otsuka (LETO) and Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats at the age of ve weeks n Food intake (g/d) Body weight (g) SWAT (mg) RWAT (mg) MWAT (mg) M/S Testis (mg) LETO OLETF Statistics P < 0.01 P < NS NS NS NS NS n ˆ number of rats in each group; SWAT ˆ subcutaneous white adipose tissue; RWAT ˆ retroperitoneal white adipose tissue; MWAT ˆ mesenteric white adipose tissue; M/S ˆ MWAT weight/swat weight; NS ˆ statistically not signi cant Table 2 Changes of humoral metabolic factor levels in Long-Evans-Tokushima-Otsuka (LETO) and Otsuka-Long-Evans-Tokushima- Fatty (OLETF) rats at ve weeks of age n BG (mmol/l) IRI (pmol/l) IRL (ng/ml) T (nmol/l) E 2 (pmol/l) E 2 /T Corticosterone (nmol/l) LETO OLETF Statistics NS NS NS P < 0.05 NS P < 0.01 NS n ˆ number of rats in each group; BG ˆ blood glucose; IRI ˆ immunorective insulin; IRL ˆ immunoreactive leptin; T ˆ testosterone; E 2 ˆ estradiol; NS ˆ statistically not signi cant

4 Testosterone and obesity in young OLETF rats 321 Figure 1 Chronological changes of daily food intake after orchiectomy and testosterone supplement in Long-Evans-Tokushima- Otsuka (LETO) and Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats. Sham: sham-operated. O: bilaterally orchiectomized, O T: testosterone supplemented with bilateral orchietomy. n ˆ 5 in each group. serum IRL levels in both strains. The relative ob gene mrna levels in RWAT of OLETF rats were no different from these in LETO rats at this age. Orchiectomy signi cantly decreased the relative ob gene mrna levels in OLETF rats and testosterone supplement reversed the reduction of the relative ob gene mrna levels by orchiectomy. Serum IRL levels were signi cantly higher in OLETF rats than in LETO rats. Serum IRL levels were signi cantly decreased by orchiectomy in OLETF rats and testosterone supplement completely reversed the reduction of serum IRL levels by orchiectomy. However, those changes on the relative ob gene mrna and serum IRL levels were not observed in LETO rats. Figure 2 Changes of body weight gain three weeks after orchietomy and testosterone supplement in Long-Evans-Tokushima- Otsuka (LETO) and Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats. Sham: sham-operated, Orchx: bilaterally orchiectomized, Orchx T: bilaterally orchiectomized with testosterone supplement. n ˆ 5 in each group. Changes of circulating hormonal and metabolic factor levels in both strains after orchiectomy are shown in Table 3. OLETF rats had higher levels of serum glucose, IRI, LH and FSH than LETO rats. Serum testosterone levels were signi cantly lower in OLETF rats, but serum E 2 levels were signi cantly higher, suggesting that the conversion of testosterone to E 2 was enhanced in OLETF rats even at this age. Orchiectomy signi cantly lowered the increase in blood glucose and serum IRI concentrations of OLETF rats, and testosterone supplement restored these effects, but any signi cant effects of orchiectomy on blood glucose or serum IRI concentrations were not observed in LETO rats. Figure 3 demonstrated changes in the relative ratio of the ob gene mrna levels to b-actin mrna and Discussion The origin of obesity and peripheral insulin resistance in male OLETF rats is still unknown. There is a sexual dimorphism in the development of the obesity and peripheral insulin resistance in OLETF rats. 2 It has been reported that withdrawal of estrogen by ovariectomy, induced obesity and increased the incidence of diabetes mellitus in this animal model. Therefore, it is supposed that the induction of obesity may be related to peripheral insulin resistance in OLETF rats and that sex hormone may involve the development of peripheral insulin resistance, through modulating food intake and body weight gain. The present studies demonstrated that withdrawal of testosterone by orchiectomy prevented the development of obesity and glucose intolerance in young OLETF rats, whereas it had no signi cant effects in the control LETO rats. In the rst experiment, serum testosterone concentrations were signi cantly lower in OLETF rats than in LETO rats, and the weight of the testis was lower at ve weeks of age in OLETF rats.

5 Testosterone and obesity in young OLETF rats 322 Table 3 Changes in humoral metabolic factors concentrations at three weeks after orchiectomy (Orchx) and testosterone (T) supplement in Long-Evans-Tokushima-Otsuka (LETO) and Otsuka- Long-Evans-Tokushima Fatty (OLETF) rats n BG(mmol/l) IRI(pmol/l) LH(ng/ml) FSH(ng/ml) T(nmol/l) E 2 (pmol/l) E 2 /T Corticosterone (ng/ml) LETO Sham Orchx **** **** ND ND Orchx T **** * OLETF Sham ** ** ** ** ** Orchx **** *** **** ND ND Orchx T *** n ˆ number in each group; ND ˆ not detectable; BG ˆ blood glucose; IRI ˆ immunoreactive insulin; T ˆ testosterone; E 2 ˆ estradiol. * P < 0.05, ** P < 0.01 vs LETO rats, *** P < 0.05, **** P < 0.01 vs sham-operated rats in each strain. However, serum corticosterone concentrations were higher in OLETF rats. Since the E 2 /testosterone ratio was signi cantly increased in OLETF rats, the aromatase activity appears to be enhanced in adipose tissues of this strain. Serum testosterone levels in OLETF rats gradually increased with growth at 10 weeks of age (sham-operated rats in the second experiment), but were still lower than in LETO rats, and the reduction of circulating testosterone was accompanied by an increase of serum LH, FSH levels in OLETF rats. These ndings are indicative of reduced gonadal testosterone synthesis and=or secretion in young OLETF rats. In the second experiment, orchiectomy improved both hyperglycaemia and hyperinsulinaemia, indicating improved peripheral insulin resistance by orchiectomy in OLETF rats, in spite of the preceding hypogonadism. In general, castration in male rats is followed by insulin resistance in muscle, alleviated by testosterone substitution. 22 Our previous data have shown that withdrawal of testosterone by bilateral orchiectomy failed to affect body weight gain, hyperglycaemia, and hyperinsulinaemia in genetically obese (ob=ob) mice. 23 Therefore, an improvement of obesity and peripheral insulin resistance by orchiectomy might be speci c to OLETF rats. Taking into consideration our observations, it is proposed that the sensitivity to testosterone, regarding the development of peripheral insulin resistance perhaps in skeletal muscles and adipose tissues, may be enhanced in these obese rats and the complete withdrawal of testosterone should be necessary to improve peripheral insulin resistance in these rats. Adipose tissue ob gene mrna levels were decreased in young OLETF rats at ve weeks of age and gradually increased to the same level as in LETO rats at 10 weeks of age. Circulating IRL concentrations were lower at the age of ve weeks in young OLETF rats, but signi cantly increased by 10 weeks of age, as a result of increases in ob gene expression and total body fat mass in OLETF rats. Orchiectomy signi cantly decreased both the relative ob gene mrna levels and serum IRL concentrations, and testosterone supplement restored the effects of orchiectomy on the ob gene mrna levels and serum IRL concentrations in OLETF rats. The concentrations of E 2, which is involved in ob gene expression, 16 were unaffected by orchiectomy and testosterone supplement in OLETF rats. These observations indicate that changes in ob gene expression of OLETF rats can be attributable to testosterone. Leptin, the ob gene product, is thought to act on the central feeding center to inhibit food intake in rats. 24 The abnormalities in expression of the ob gene and the ob receptor gene have been reported in various models of obesity and NIDDM, 11±13,25 but the involvement of leptin has not been investigated in this model of NIDDM. While OLETF rats showed a hyperphagia with a decrease in circulating leptin concentrations at ve weeks of age, daily food intake of these rats

6 Testosterone and obesity in young OLETF rats 323 Figure 3 Changes of the ratio of ob gene mrna expression to b-actin mrna expression in retroperitoneal white adipose tissue (a) and serum immunoreactive leptin (IRL) levels (b) in Long-Evans-Tokushima-Otsuka (LETO) and Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats at three weeks after orchiectomy (Orchx) and testosterone (T) supplement. n ˆ 5 in each group. was increased at the age of 10 weeks, with a signi cant increase in circulating leptin levels. In addition, orchiectomy, which improved hyperglycaemia and hyperinsulinaemia, decreased body weight in OLETF rats, and also both ob gene expression and serum IRL levels as a result. The reduction of body fat mass and the reduction of circulating leptin, leading to the diminished anorexigenic message, did not cause hyperphagia in OLETF rats. The changes observed in the relative ob gene mrna expression and circulating leptin levels did not parallel the changes of daily food consumption in OLETF rats. These observations indicate that the observed changes in ob gene expression and circulating leptin may not be important in the development of obesity in young OLETF rats. The data obtained herein indicate that testosterone plays an important role in the development of obesity and peripheral insulin resistance in young OLETF rats. However, changes in leptin production, from increased fat mass, may not be important in the development of obesity in this animal model of NIDDM. References 1 Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T. Spontaneous long-term hyperglycemia rats with diabetic complications: Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 1992; 41: 1422± Ishida K, Mizuno A, Min Z, Sano T, Shima K. Which is the primary etiologic event in Otsuka Long-Evans Tokushima Fatty rats, a model of spontaneous non-insulin-dependent diabetes mellitus, insulin resistance, or impaired insulin secretion? Metabolism 1995; 44: 940± Okauchi N, Mizuno A, Yoshimoto S, Zhu M, Sano T, Shima K. Is caloric restriction effective in preventing diabetes mellitus in the Otsuka Long Evans Tokushima Fatty rat, a model of spontaneous non-insulin-dependent diabetes mellitus? Diab Res Clin Pract 1995; 27: 97± Shima K, Shi K, Sano T, Iwami T, Mizumo A, Noma Y. Is exercise training effective in preventing diabetes mellitus in the Otsuka Long Evans Tokushima Fatty rat, a model of spontaneous non-insulin-dependent diabetes mellitus? Metabolism 1993; 42: 971± Shoupe D, Lobo RA. The in uence of androgens on insulin resistance. Fertil Steril 1984; 41: 385± Rincon J, Holmang A, WahlstroÈm EO, Lonnroth P, BjoÈrntorp P, Zielrath JR, Wallberg-Henriksson H. Mechanism behind insulin resistance in rat skeletal muscle after oophorectomy and additional testosterone treatment. Diabetes 1996; 45: 615± Russell JC, Amy RM, Graham S, Wenzel LM, Dolphin PJ. Effect of castration on hyperlipidemic, insulin resistant JCR: LA-corpulent rats. Artherosclerosis 1993; 100: 113± Shi K, Mizuno A, Sano T, Ishida K, Shima K. Sexual difference in the incidence of diabetes mellitus in Otsuka- Long-Evans-Tokushima-Fatty rats: effects of castration and sex hormone replacement on its incidence. Metabolism 1994; 43: 1214± Ikeda H, Shino A, Mastuo T, Iwatsuka H, Suzuoki Z. A new genetically obese- hyperglycemic rat (Wistar Fatty). Diabetes 1981; 30: 1045± Clark JB, Palmer CJ, Shaw WN. The diabetic Zucker Fatty rat. Proc Soc Exp Biol Med 1983; 173: 68± 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± Chua SC Jr, Chung WK, Wu-Peng XS, Zhang Y, Liu S-M, Tartaglia L, Leibel RL. Phenotypes of mouse diabetes and rat Fatty due to mutations in the OB (leptin) receptor. Science 1996; 271: 994± Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, Caskey CT, Hess JF. Leptin receptor missense mutation in the Fatty Zucker rat. Nature Gen 1996; 13: 18± Havel PJ, Kasim-Karakas S, Dubuc GR, Mueller W, Phinney SD. Gender differences in plasma leptin concentrations. Nature Med 1996; 2: 949± Rosenbaum M, Nicolson M, Hirsch J, Heyms eld SB, Gallagher D, Chu F, Leibel RL. Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab 1996; 81: 3424± Shimizu H, Shimomura Y, Nakanishi Y, Futawatari T, Ohtani K, Sato N, Mori M. Estrogen increases in vivo leptin production in rats and human subjects. J Endocr 1997; 154: 285±292.

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