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Endocrinology Vol. 140, No. 11 5233-5238
Copyright © 1999 by The Endocrine Society

ARTICLES

Developmental Changes in Long-Form Leptin Receptor Expression and Localization in Rat Brain

Junko Matsuda, Ichiro Yokota, Yoshihiro Tsuruo1, Takashi Murakami, Kazunori Ishimura, Kenji Shima and Yasuhiro Kuroda

Department of Pediatrics (J.M., I.Y., Y.K.), Department of Anatomy (Y.T., K.I.), and Department of Laboratory Medicine (T.M., K.S.), School of Medicine, University of Tokushima, Tokushima 770-8503, Japan

Address all correspondence and requests for reprints to: Ichiro Yokota, M.D., Department of Pediatrics, School of Medicine, University of Tokushima, Kuramoto-cho 3, Tokushima 770-8503, Japan. E-mail: yichiro{at}clin.med.tokushima-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression and localization of long-form leptin receptor (OB-Rb) were studied immunocytochemically in the brain of fetal and adult rats using a polyclonal antibody that specifically recognized OB-Rb. At 14 days of gestation, immunoreactive cells were observed in the ventricular layer, which contains premature neuronal cells. At 18 days of gestation, they were weakly stained but obvious in the paraventricular nucleus (PVN), and ependymal cells also showed immunoreactivity. At birth, the immunoreactivity of OB-Rb in the PVN seemed to be much lower than that in adult rats and remained low during the suckling period. Considering the presence of neuroendocrine and structural neuronal abnormalities in Lepob/Lepob mice with genetic leptin deficiency, these results suggest that the expression of OB-Rb in premature neuronal cells may have some function, and that the regulation of energy balance by leptin through hypothalamic regions, such as PVN, may not yet be developed in the perinatal period.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE adipocyte-secreted circulating hormone leptin interacts with specific long-form leptin receptors (OB-Rb) in the hypothalamus to regulate appetite and energy expenditure in humans and rodents (1, 2, 3). In adult rats, OB-Rb is apparently expressed in the arcuate nucleus (ARC), paraventricular nucleus (PVN), and supraoptic nucleus (SON) of the hypothalamus (4, 5, 6, 7, 8). However, it is still unclear when leptin effectively begins to regulate the energy balance through hypothalamic nuclei. In addition, recent studies have demonstrated the expression of OB-R in various fetal tissues, the production of leptin by the human placenta, and relatively high concentrations of leptin during the fetal periods in humans and rodents, and it has been suggested that leptin may play some other important roles in fertility, pregnancy, and fetal growth (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Detailed studies in both humans and rodents carrying mutations in the leptin or leptin receptor gene also have indicated that leptin is an important physiological regulator of several endocrine functions (22, 23, 24, 25, 26, 27) and may play a role in brain development, as suggested by the presence of neuroendocrine and structural neuronal abnormalities in Lepob/Lepob mice with genetic leptin deficiency (28, 29, 30). These observations suggest that leptin plays some other role(s) during the perinatal period in addition to regulating the appetite in the later period.

In this study, we immunocytochemically examined the developmental changes in OB-Rb expression and localization in the rat brain to estimate when leptin begins to regulate the energy balance through a hypothalamic pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Fetal, neonatal, and adult rats (Japan SLC Inc., Shizuoka, Japan) were used in this study. The day on which spermatozoa were found in a vaginal smear was designated as embryonic day 0 (E0), and the day of birth, usually at E22, was designated as postnatal day 0 (P0). The newborn pups suckled ad libitum, and handling was limited to cage cleaning. They were weaned on regular laboratory chow at day 21. The animals were maintained on a 12-h light-dark cycle at 20 C and fed laboratory chow and water ad libitum. The study protocol was approved by the Committee on Animal Research, The University of Tokushima.

Tissue preparation
At various gestational days from E14 through E21, pregnant rats were killed by cervical dislocation and the fetuses were removed. The brains of the embryos were immediately immersed in ice-cold fixative consisting of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) and kept overnight at 4 C. The brains of newborns at early postnatal ages (P0-P6) were dissected out under anesthesia by the ip injection of pentobarbital (50 mg/kg body weight) and immersed in the fixative overnight at 4 C. Older neonates (P7 or later) and adult rats (P70 or later) were perfused through the ascending aorta with warm (37 C) heparinized 0.1 M PB (pH 7.4), followed by the fixative under anesthesia (pentobarbital 50 mg/kg body weight, ip). The excised brains were immersed in the same fixative overnight at 4 C. After fixation, the brains were sliced and successively soaked in 10%, 15%, and 20% sucrose in 0.01 M PBS, pH 7.4, until they sank. The specimens were frozen and cut into 10 µm-thick frontal sections using a cryostat. Frozen sections were thaw mounted on glass slides coated with chrome alum gelatin and air dried for approximately 1 h.

Partial purification of the secreted form of rat OB-R (OB-Re)
The Chinese hamster ovary (CHO) cell clone, which stably expresses rat OB-Re (31), was incubated with serum-free medium S-Clone SF-02 (Sanko Junyaku, Co. Ltd., Tokyo, Japan). After dialysis against buffer A (25 mM HEPES, pH 7.4, 100 mM NaCl), the conditioned media were twice passed through a Wheat Germ Lectin Sepharose 6MB column (Amersham Pharmacia Biotech, Ltd., Buckinghamshire, UK). The column was washed with buffer A and then eluted with 0.3 M N-acetyl-D-glucosamine in the buffer. The eluate was concentrated and washed using buffer A with Centricon Plus-20 (100 kDa molecular mass cut off) (Millipore Corp., Bedford, MA).

Rat OB-Re consisted of the extracellular region common to OB-R (up to amino acid 796) and nine additional amino acids in an OB-Re-specific region and was used in the peptide competition test.

Preparation of antirat OB-Rb antiserum
A portion of the intracellular domain (from amino acid 981 to 1130) (24) of rat OB-Rb was produced in Escherichia coli using QIA expressionist (Qiagen GmbH, Hilden, Germany) with NH2-terminal fusion to the His-tag sequence. This protein was purified and refolded from inclusion bodies according to the manufacturer’s recommended protocols and finally dialyzed against PBS. This protein was used as the antigen to immunize Hartley guinea pigs (obtained from Japan SLC, Inc., Shizuoka, Japan). The animals were injected with a 1:1 emulsion of the antigen solution and complete Freund’s adjuvant followed by several booster immunizations of a 1:1 emulsion in incomplete Freund’s adjuvant, which were given every 2 weeks. The antisera were collected a week after the last booster and stored at -80 C. The antiserum characteristics were verified using sections from adult rat brain. Rat OB-Re and a peptide of the intracellular domain of OB-Ra (sc-1834p, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (6, 7) were used as controls in the peptide competition test.

Western blot analysis
The establishment of CHO cell clones that stably expressed OB-Ra or OB-Rb, referred to as CHO-OBRa or CHO-OBRb, respectively, has been previously described (32, 33). CHO-OBRa cells (clone 15), CHO-OBRb cells (clone 13), or parent CHO cells were individually cultured in 100-mm dishes containing Ham’s F-12 nutrient mixture (F-12; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (HyClone Laboratories, Inc. Logan, UT). Each dish of subconfluent cells was washed with ice-cold PBS and lysed with 1.0 ml of the buffer [50 mM HEPES (pH 7.5), 150 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mg/ml bacitracin, 20 mM sodium fluoride, 1% Triton X-100, 20 µM (p-amidino-phenyl) methanesulfonyl fluoride hydrochloride]. After centrifugation at 15,000 x g for 5 min at 4 C, the supernatants were separated by 2–12% gradient SDS-PAGE and transferred to a nitrocellulose membrane (Amersham International, Buckinghamshire, UK). The membrane was blocked by incubation for 1 h at room temperature in blocking buffer [20 mM Tris (pH 7.5), 150 mM sodium chloride, 5% skim milk, 0.1% Tween 20] and then incubated overnight at 4 C in the same buffer containing the antiserum (final 1:1,000 dilution). The membrane was washed five times for 10 min each in a washing buffer [20 mM Tris (pH 7.5), 150 mM sodium chloride, 0.1% Tween 20] and then incubated for 1 h at room temperature in the blocking buffer that contained the peroxidase-conjugated donkey antiguinea pig IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), followed by five washes of 10 min each with the washing buffer. The bound antibody was visualized using the ECL chemiluminescent Western blotting detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and exposed to x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Immunocytochemical procedure
The sections were rinsed with PBS for 20 min. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide in distilled water for 20 min at room temperature. They were then rinsed with PBS for 10 min, and endogenous avidin/biotin binding was blocked with avidin D, followed by biotin (Vectastain, Vector Laboratories, Inc., Burlingame, CA). The sections were then incubated with the antiserum against rat OB-Rb diluted 1:10,000 or with normal preimmune guinea pig serum diluted 1:10,000 in PBS containing 1% normal goat serum (NGS) overnight at 4 C, followed by biotinylated goat antiguinea pig IgG diluted at 1:100 in PBS containing 1% NGS for 1 h at 32 C, and then with Elite ABC solution (50 µl of avidin and biotin peroxidase in 5 ml of 0.01 M PBS containing 1% NGS) for 1 h at 32 C. The immunoreaction was visualized in 50 mM Tris-HCl buffer, pH 7.6, containing 0.01% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide, and examined by light microscopy. These immunocytochemical procedures were performed at the same time on the sections of fetuses and adults. These experiments were repeated using three different samples. The specificity of the antigenic signals was confirmed with preimmune guinea pig serum and by peptide competition experiments.

Measurement of serum leptin concentration
Truncal blood was obtained from rats at different gestational days and postnatal ages. Serum concentrations of rat leptin were measured in duplicate by enzyme immunoassay kits according to the manufacturer’s protocol (Immuno Biological Laboratories, Gunma, Japan). The minimal detectable concentration of rat leptin was 0.2 ng/ml. The intra- and interassay coefficients of variation (CVs) in our assay were 7.8% and 5.6%, respectively.

Statistical analysis
All quantitative data are presented as the mean ± SD. Differences between groups were evaluated by the Mann-Whitney U test. A value of P < 0.05 was considered significant. All analyses were conducted with SPSS software (version 6.1 for Macintosh, SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Western blotting
Cell extracts of CHO cells and CHO cells that stably expressed OB-Ra (CHO-OBRa cells) or OB-Rb (CHO-OBRb cells) were subjected to Western blot analysis using the specific anti-OB-Rb antiserum. As shown in Fig. 1Go, two common bands (~70 kDa and 110 kDa, respectively) were observed in extracts of both CHO cells and CHO-OBRa cells. In the extract of CHO-OBRb cells, two smeared bands (~140 kDa and 190 kDa) were observed in addition to the two common bands.



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  		Figure 1. Western blot analysis of CHO cells expressing OB-R. Cell extracts of CHO cells and CHO cells stably expressing OB-Ra (CHO-OBRa cells) or OB-Rb (CHO-OBRb cells) were subjected to Western blot analysis using anti-OB-Rb antiserum. Positions of molecular mass markers are indicated.

 
Developmental changes in immunocytochemical localization and immunoreactivity for OB-Rb in the rat brain
In adult rats, strong OB-Rb immunoreactivity was detected in the PVN, SON, and ARC of the hypothalamus. Ependymal cells also showed strong immunoreactivity for OB-Rb (Fig. 2Go). Immunoreaction seemed to be located on cell membranes, especially at high magnification (Fig. 4eGo). In the peptide competition test, this immunoreaction was not inhibited after the preabsorption of OB-Ra peptide or OB-Re. However, no immunoreactivity was observed after the preabsorption of OB-Rb immunogen (Fig. 3Go).



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  		Figure 2. Immunostaining of OB-Rb in the adult rat. Immunoreactive cells located in the PVN, the ependyma (a), and the SON (b) are shown (magnification, 150x). The immunoreaction was localized around the cell membrane. V, Third ventricle; OT, optic tract. Bar, 100 µm.

 


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  		Figure 4. Developmental changes in the immunostaining of OB-Rb. The immunostaining of OB-Rb was observed in the rat brain at E14, E18, P0, P14, and P70. At E14, many immunoreactive cells were localized in the ventricular layer (a), but they were not observed in the hypothalamic regions. Immunoreactive cells were weakly stained but obvious in the PVN at E18 (b). At birth (P0) (c), the intensity of the immunoreaction in the PVN was very weak compared with that at P70 (e) and was still weak at P14 (d). The representative immunostained cells are indicated by arrowheads (magnification, 300x). Bar, 50 µm.

 


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  		Figure 3. Characterization of antirat-OB-Rb antiserum by a peptide competition test. OB-Rb immunostaining in adjacent sections of the SON in the rat hypothalamus at 10 weeks of age (magnification, 100x). Many immunoreactive cells in the SON were observed with anti-OB-Rb antiserum (a), but no positive immunoreaction was seen with the antiserum preabsorbed with the immunogen of 5 µM OB-Rb (b). The immunoreaction was present after preabsorption of the antiserum with OB-Ra peptide (c) or OB-Re (d) in the same concentration. OT, Optic tract; V, blood vessel. Bar, 100 µm.

 
At E14 in fetuses, the immunoreaction was observed in many immature neuronal cells in the cerebral wall, especially in the ventricular layer (Fig. 4aGo), but not in the hypothalamic region. Immunoreactive cells were weakly stained but obvious in the PVN at E18 (Fig. 4bGo). The ependymal cells also showed immunoreactivity. The intensity of the immunoreaction in the PVN of newborns (P0) (Fig. 4cGo) was faint compared with that in P70 (Fig. 4eGo) and remained weak throughout the suckling period (Fig. 4dGo). The number and staining intensity of OB-Rb-immunoreactive cells in the PVN increased with aging. These observations were confirmed by three independent series of samples prepared from each brain.

Developmental changes in the serum leptin concentration
Serum leptin concentrations at E14 were higher than those in adults (mean 22.8 ± 9.5 ng/ml) and decreased toward the late stage of gestation (E21; mean 9.7 ± 2.2 ng/ml). After birth, the leptin concentrations in newborns declined rapidly to extremely low levels within a day (at 9 h after birth; mean, 0.09 ± 0.15 ng/ml), followed by a rapid increase to the fetal level (at 3 days of life; mean, 9.3 ± 3.6 ng/ml). A distinct surge was observed at 21 days after birth, but the level then declined after weaning. Postweaning, the serum leptin concentrations remained lower than those in adults but tended to increase gradually to the adult level (mean 13.2 ± 2.1 ng/ml) (Fig. 5Go).



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  		Figure 5. Developmental changes in serum leptin concentrations in rat. The closed circles and error bars indicate the mean ± SD of serum leptin concentrations. The closed and open squares and error bars indicate the mean ± SD of body weight in males and females, respectively. Serum leptin concentrations (ng/ml) were as follows: E14 (n = 4); 22.8 ± 9.5 (10.7–31.7), E21 (n = 8); 9.3 ± 2.2 (6.0–12.8), P0 (~3 h after birth) (n = 10); 4.9 ± 1.9 (2.7–8.0), P0 (~12 h after birth) (n = 8); 0.09 ± 0.15 (0.00–0.42), P1 (n = 9); 3.8 ± 1.7 (2.0–7.2), P3 (n = 9); 9.3 ± 3.6 (3.2–16.1), P7 (n = 10); 3.6 ± 2.3 (1.3–8.7), P14 (n = 10); 4.7 ± 5.8 (0.2–14.8), P21 (n = 7); 15.2 ± 3.6 (11.8–23.1), P29 (n = 14); 4.1 ± 1.9 (1.9–8.5), P72 (n = 4); 5.8 ± 0.7 (5.1- 6.5), adult (n = 7); 13.2 ± 2.1 (8.6–14.8). The male-female ratio was almost the same in each sample. *, P = 0.13 vs. adult; P = 0.017 vs. embryo at 21 days. §, P < 0.001 vs. embryo at 21 days; P < 0.001 vs. 3 days after birth.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In adult rat brain, strong OB-Rb immunoreactivity was detected in the ARC, PVN, and SON of the hypothalamus, regions that are thought to be primary sites for the satiating effect of leptin in rats, as previously described (4, 5, 6, 7, 8). This immunoreactivity was not blocked by OB-Ra peptide or OB-Re, but completely blocked by OB-Rb immunogen in the peptide competition test. These results confirmed the specificity of our antiserum against OB-Rb. Using this antiserum, the immunoreactivity of OB-Rb was immunocytochemically detected at E14 in the ventricular layer, which contains premature neuronal cells. Considering the dysfunction of the neuroendocrine system, the structural abnormalities in the central nervous system, and the impaired myelination in Lepob/Lepob mice with genetic leptin deficiency (28, 29, 30), the expression of OB-Rb in premature neuronal cells in embryos may suggest that leptin contributes in part to the development of the central nervous system during the fetal period.

In this study, we found that the PVN showed immunoreactivity for OB-Rb at E18. However, this immunoreactivity for OB-Rb in the fetal PVN was very weak compared with that in adults and remained at a low level during the suckling period. This observation suggests that the system for regulating satiety and energy balance mediated by circulating leptin through the hypothalamic nuclei is still immature and may not work effectively during the neonatal period in rats, although a considerable concentration of leptin already exists in the blood during this period. This fact itself is reasonable, since extra energy intake is necessary for growth in this period. Furthermore, this may well explain the indistinguishable levels of milk intake during the suckling period between lean and Leprfa/Leprfa rats that have impaired OB-Rb functions (34).

If the leptin during the perinatal period does not act as a satiety signal, what is the meaning of the significant rise in serum leptin during this period? It is possible that the decreased sensitivity of hypothalamic nuclei may cause a considerable rise in the leptin concentration without controlling the energy balance through a hypothalamic pathway. However, it has been reported that Leprfa/Leprfa rats have a defect in thermogenesis at the first week after birth, and excess fat deposition is apparent during the first week of life (34, 35). In addition, lean suckling-age rats treated with recombinant leptin can reduce fat storage solely by increasing their energy expenditure (36). These observations suggest that leptin already plays a crucial role in heat production and energy expenditure in the neonatal period. The weak OB-Rb expression in the hypothalamic nuclei during the perinatal period requires some interpretations to explain the mechanism of the regulation of energy expenditure by leptin in this period. One probable explanation is that the weak expression of OB-Rb in the hypothalamic nuclei is enough to transduce sympathetic signals to peripheral tissues, especially to brown adipose tissue. On the other hand, it has been reported that OB-Rb is expressed in brown and white adipose tissues and that leptin has a direct action on energy metabolism in these tissues (37). These peripheral actions, especially regarding heat production, may be an important physiological action of leptin during the neonatal period. We have also found that OB-Rb is expressed in brown adipose tissue at birth by the RT-PCR method (our unpublished observation).

In summary, this study provides new information on the ontogenetic changes in OB-Rb expression and localization in the rat brain. The expression of OB-Rb in premature neuronal cells of fetal rat brain and the significantly weak expression of OB-Rb in the hypothalamic nuclei at birth throughout the suckling period were observed. These results suggest the possibility that leptin may have other important role(s) during the perinatal period, apart from its regulatory role as a satiety signal through a hypothalamic pathway in adulthood.


   Acknowledgments
 
We thank Drs. M. Ito and E. Naito for their helpful suggestions. We also thank Drs. T. Yamashita and I. Sato for their excellent technical assistance.


   Footnotes
 
1 Current address: Department of Anatomy, Wakayama Medical College, 811–1 Kimiidera, Wakayama 641-0012, Japan. Back

Received February 19, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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E. C. Cottrell, R. L. Cripps, J. S. Duncan, P. Barrett, J. G. Mercer, A. Herwig, and S. E. Ozanne
Developmental changes in hypothalamic leptin receptor: relationship with the postnatal leptin surge and energy balance neuropeptides in the postnatal rat
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M P Di Yorio, M G Bilbao, M C Pustovrh, J P Prestifilippo, and A G Faletti
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J. Endocrinol., August 1, 2008; 198(2): 355 - 366.
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W. Pan, H. Hsuchou, H. Tu, and A. J. Kastin
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Endocrinology, March 1, 2008; 149(3): 877 - 885.
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A G Ricci, M P Di Yorio, and A G Faletti
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R. Malcher-Lopes, S. Di, V. S. Marcheselli, F.-J. Weng, C. T. Stuart, N. G. Bazan, and J. G. Tasker
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A. Valerio, V. Ghisi, M. Dossena, C. Tonello, A. Giordano, A. Frontini, M. Ferrario, M. Pizzi, P. Spano, M. O. Carruba, et al.
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S. G. Bouret and R. B. Simerly
Minireview: Leptin and Development of Hypothalamic Feeding Circuits
Endocrinology, June 1, 2004; 145(6): 2621 - 2626.
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J.-N. Huan, J. Li, Y. Han, K. Chen, N. Wu, and A. Z. Zhao
Adipocyte-selective Reduction of the Leptin Receptors Induced by Antisense RNA Leads to Increased Adiposity, Dyslipidemia, and Insulin Resistance
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S. Kitamura, I. Yokota, H. Hosoda, Y. Kotani, J. Matsuda, E. Naito, M. Ito, K. Kangawa, and Y. Kuroda
Ghrelin Concentration in Cord and Neonatal Blood: Relation to Fetal Growth and Energy Balance
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N. K. Ryan, K. H. Van der Hoek, S. A. Robertson, and R. J. Norman
Leptin and Leptin Receptor Expression in the Rat Ovary
Endocrinology, November 1, 2003; 144(11): 5006 - 5013.
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J. T. Smith and B. J. Waddell
Leptin Distribution and Metabolism in the Pregnant Rat: Transplacental Leptin Passage Increases in Late Gestation but Is Reduced by Excess Glucocorticoids
Endocrinology, July 1, 2003; 144(7): 3024 - 3030.
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M. Caprio, E. Fabbrini, G. Ricci, S. Basciani, L. Gnessi, M. Arizzi, A. R. Carta, M. U. De Martino, A. M. Isidori, G. V. Frajese, et al.
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G. E. Truett, J. A. Walker, and R. B. S. Harris
A developmental switch affecting growth of fatty rats
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2000; 279(6): R1956 - R1963.
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