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Brain Stem Is a Direct Target for Leptin’s Action in the Central Nervous System

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan

Address all correspondence and requests for reprints to: Dr. Yasunobu Okuma, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan. E-mail: okumay{at}pharm.hokudai.ac.jp.

	Abstract

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Leptin is a circulating molecule for the regulation of food intake and body weight suggested to be mediated in the hypothalamus via Ob-Rb receptor, which activates Janus kinase-signal transducer and activator of transcription (STAT) pathways. Although leptin receptors exist in many regions of the brain, there have been few in vivo functional studies of leptin’s target site other than the hypothalamus. We report here that peripherally applied leptin increased STAT3 phosphorylation not only in the hypothalamus but also in the brain stem as assessed by Western blotting. Moreover, administration of leptin induced expression of the suppressor of cytokine signaling 3 mRNA, a negative feedback regulator of leptin signaling, in the brain stem as well as in the hypothalamus. Using immunohistochemistry, we observed phosphorylated STAT3-immunoreactive cells in the arcuate nucleus, ventromedial hypothalamus, lateral hypothalamic area of the hypothalamus, and the nucleus of the tractus solitarius, dorsal motor nucleus of the vagus nerve, lateral parabrachial nucleus, and central gray of the brain stem of leptin-injected mice. These findings represent physiologically functional leptin Ob-Rb receptor in the brain stem as well as in the hypothalamus. It is suggested that circulating leptin may directly act in the brain stem to elicit autonomic and neuroendocrine control of food intake and energy expenditure.

	Introduction

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LEPTIN, THE 16-kDa protein encoded by the ob gene (1), is an important regulator of energy balance through its actions in the brain on appetite and energy expenditure (2, 3). Leptin is mainly secreted by adipose tissue and released into the circulation to act in both the periphery and the brain. Leptin enters the brain via a saturable transport mechanism (4) and was suggested to activate the hypothalamic centers. Leptin receptors (Ob-R) are found in many tissues in several alternatively spliced forms (5, 6). One form of the receptor, Ob-Rb, has a consensus amino acid sequence involved in the activation of Janus kinase-signal transducer and activator of transcription (JAK-STAT) tyrosine kinases (7, 8) and activates STAT protein both in vitro (7, 9) and in vivo (10, 11, 12). The essential role of the Ob-Rb receptor was demonstrated by the finding that its absence results in extreme obesity in db/db mice (8, 13).

Of the central targets for leptin’s action that may contribute to food intake control, only those in the hypothalamus have been considered. However, Ob-Rb receptor has been demonstrated in various brain regions (14, 15, 16). Therefore, these observations led us to investigate whether physiologically circulating leptin could activate Ob-Rb receptor existing in various brain regions. It was demonstrated that the central nervous system circuitry was activated by systemic application of leptin using the expression of Fos protein as a marker of cellular activation (17, 18). Leptin-induced Fos-positive cells could reflect the direct activation by Ob-Rb and other isoforms of leptin receptors or the secondary activated neuronal circuit by leptin’s target neurons in the brain and periphery, such as the afferent vagus nerve (19, 20). Ob-Rb receptor is the major brain-intrinsic leptin receptor isoform capable of full activation of the JAK-STAT signal transduction. Leptin-induced STAT3 activation and expression of the suppressor of cytokine signaling 3 (SOCS3), a negative regulator for the JAK-STAT signaling pathway, could be functional evidence for the activation of Ob-Rb receptor. In the present study, therefore, we examined whether iv injected leptin could directly activate STAT3 and induce SOCS3 expression in various brain regions. Furthermore, we immunohistochemically identified the target neurons of the leptin-responsive site in the brain using phospho-specific STAT3 antibody.

	Materials and Methods

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Animals
C57BL mice were obtained at 7 wk of age from Charles River, Inc. (Yokohama, Japan). Mice were maintained in a room at 22–24 C under a constant day-night rhythm and were given food and water ad libitum. All animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and were approved by the animal care and use committee at Hokkaido University.

Leptin injection and sample preparation
Murine leptin (PeproTech, London, UK) was dissolved in saline, and all injections were administered iv via the tail vein and delivered at an injection volume of 5 ml/kg. Mice were killed by decapitation, and the brain was quickly removed. The hypothalamus, hippocampus, cortex, cerebellum, and brain stem were rapidly dissected out on an ice-cold plate. Then, the samples were snap-frozen in liquid nitrogen and stored at -80 C. For Western blotting, tissue samples were sonicated in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 21 mM Na₃VO₄, 10 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, and 1% Nonidet P-40 for 30 sec. The samples were centrifuged at 30,000 x g for 30 min at 4 C, and the supernatants were collected.

RT-PCR
Total RNA was isolated using Tri-Reagent (Sigma, St. Louis, MO). cDNA was synthesized from 2 µg total RNA by RT using 100 U Superscript reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) and oligo(deoxythymidine)_12–18 primer (Life Technologies, Inc.) in a 20-µl reaction containing 1x Superscript buffer (Life Technologies, Inc.), 1 mM deoxy-NTP mix, 10 mM dithiothreitol, and 40 U ribonuclease inhibitor. Total RNA and oligo(deoxythymidine)_12–18 primer were incubated at 70 C for 10 min before RT. After incubation for 1 h at 42 C, the reaction was terminated by a denaturing enzyme for 15 min at 70 C. For PCR amplification, 1.2 µl cDNA were added to 12 µl of a reaction mix containing 0.2 µM of each primer, 0.2 mM deoxy-NTP mix, 0.6 U Taq polymerase, and 1x reaction buffer. PCR was performed in a DNA thermal cycler (2400-R, Perkin-Elmer, Norwalk, CT). The primers used were as follows: Ob-Ra and Ob-Rb common upstream, 5'-aca ctg tta att tca cac cag ag-3'; Ob-Ra downstream, 5'-agt cat tca aac cat agt tta gg-3'; Ob-Rb downstream, 5'-tgg ata aac cct tgc tct tca-3'; SOCS3 upstream, 5'-acc agc gcc act tct tca cg-3'; SOCS3 downstream, 5'-gtg gag cat cat act gat cc-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) upstream, 5'-aaa ccc atc acc atc ttc cag-3'; and GAPDH downstream, 5'-agg ggc cat cca cag tct tct-3'. The PCR products (10 µl) were resolved by electrophoresis in an 8% polyacrylamide gel in 1x TBE buffer (Tris-borate, EDTA). The gel was stained with ethidium bromide, and the gels were photographed under UV light. Band densities were obtained using NIH Image 1.61 software.

cDNA for GAPDH and SOCS3 were amplified for 18 and 32 cycles, respectively, and these PCR reactions were run separately. These cycle numbers were chosen based on a preliminary study determining the linear range of amplification for each respective molecule. For details, the amount of each amplified product was integrated and plotted graphically against the number of PCR cycles to determine whether the increase in intensity of the amplified product was linear to the number of PCR cycles. Moreover, each cycle of PCR used in the present study produced a linear relation between the amount of input cDNA and the resulting PCR product. To compare the expression of mRNAs in the different experimental groups, the amount of mRNA in each structure studied was estimated as the ratio of Ob-Ra, Ob-Rb, or SOCS3 to GAPDH.

Western blotting
The samples were boiled with Laemmli buffer for 3 min, and total protein was fractionated by 8% SDS-PAGE and transferred to nitrocellulose membranes at 4 C. After blocking with 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween 20 (TBST) containing 5% skimmed milk for 3 h at room temperature, the membranes were incubated with phospho-specific STAT3 (Tyr⁷⁰⁵) antibody or STAT3 antibody (Cell Signaling; diluted to 1:1000 in TBST and 5% BSA) at 4 C overnight. The filter was then washed with TBST and incubated with antihorseradish peroxidase-linked antibody (Cell Signaling; diluted to 1:2000 in TBST and 5% skimmed milk) at room temperature for 1 h. After washing with TBST, horseradish peroxidase-labeled antibodies were detected by chemiluminescence (Cell Signaling).

Immunohistochemistry
Thirty-minutes after leptin administration (5 mg/kg, iv), mice were anesthetized with diethylether and perfused transcardially with ice-cold 4% paraformaldehyde for 20 min. The brains were removed, postfixed in the same fixative for 1 h, and stored in 30% sucrose at 4 C. The specimens were frozen and cut into 40-µm-thick free floating sections using a cryostat. The sections were collected serially in PBS and stored at 4 C until processing. The sections were rinsed in PBS for 30 min and pretreated with 3% H₂O₂ in methanol for 40 min. The tissue was rinsed again in PBS before immersion in 0.3% glycine PBS for 25 min. After a further rinse, sections were placed in 0.03% sodium dodecyl sulfate for 25 min. All sections were rinsed again and placed in 4% normal serum, 0.4% Triton X-100, and 1% BSA for 30 min before incubation with a rabbit polyclonal primary antibody for phosphorylated (Tyr⁷⁰⁵) STAT3 (Cell Signaling; 1:3000) overnight at 4 C in 1% normal serum, 0.4% Triton X-100, and 1% BSA. Then the sections were treated with biotinylated goat antirabbit secondary antibody (Histofine SAB-PO kit, Nichirei Co., Tokyo, Japan). Products of the avidin-biotin peroxidase complex (Histofine SAB-PO Kit, Nichirei Co.) were visualized with 3,3'-diaminobenzidine.

Statistics
Results were expressed as the mean ± SE. Statistical analysis was performed with t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leptin induced an increase in STAT3 activation in the brain stem
Consistent with previous findings (5, 15, 16), leptin receptors (Ob-Ra and Ob-Rb isoform) were expressed in the brain stem as well as in the hypothalamus, as assessed by RT-PCR (Fig. 1). We also observed Ob-Rb receptor in the cortex, hippocampus, and cerebellum (unpublished observations). To investigate the functional significance for these areas, such as for leptin’s target site, we performed Western blotting analysis using phospho (Tyr⁷⁰⁵)-specific antibody for STAT3. As circulating leptin was suggested to be transported by the blood-brain barrier, we injected leptin via an iv route to investigate leptin’s physiological site of action in the brain. Thirty-minutes after leptin (5 mg/kg, iv) administration, we observed an increase in STAT3 phosphorylation in the hypothalamus and brain stem (7- and 2-fold, respectively; Fig. 2). On the other hand, we could not detect an increase in STAT3 phosphorylation in other areas, such as the hippocampus, cortex, and cerebellum (Fig. 2).

View larger version (42K): [in this window] [in a new window]   Figure 1. mRNA expression of leptin receptors (Ob-Ra and Ob-Rb) in the hypothalamus (Hypo) and brain stem (BS). A, The brain regions were dissected, and RT-PCR was performed. B, The amounts of leptin receptors (Ob-Ra and Ob-Rb) are expressed as ratios of densitometric measurements of the samples to the corresponding GAPDH internal standard. n = 3 per group.

View larger version (54K): [in this window] [in a new window]   Figure 2. Leptin activates STAT3 protein in the brain stem. A, The 1) hypothalamus, 2) hippocampus, 3) cortex, 4) cerebellum, and 5) brain stem were obtained 30 min after the administration of leptin (L; 5 mg/kg, iv) or saline (S), and Western blotting was performed. B, Densitometric analysis of the hypothalamus and brain stem using image analyzing software. Values are presented as the mean ± SE (n = 3–4/group). **, P < 0.01 (statistically significant difference from saline-injected mice).

View larger version (31K): [in this window] [in a new window]   Figure 3. Leptin induces SOCS3 mRNA expression in the brain stem. A, The hypothalamus and brain stem were obtained 2 h after the administration of leptin (5 mg/kg, iv), and RT-PCR was performed. B, The amounts of SOCS3 mRNA are expressed as ratios of the densitometric measurements of the samples relative to the corresponding GAPDH internal standard. Values are presented as the mean ± SE (n = 5/group). **, P < 0.01; ***, P < 0.001 (statistically significant difference from saline-injected mice).

View larger version (63K): [in this window] [in a new window]   Figure 4. Effect of leptin on phospho-STAT3 immunoreactivity in the mouse hypothalamus. Phospho-STAT3 immunoreactivity in hypothalamic sections are shown under basal saline-injected conditions (A) and leptin-injected (5 mg/kg, iv, 30 min) conditions (B). 3V, Third ventricle. Scale bars, 200 µm.

View larger version (174K): [in this window] [in a new window]   Figure 5. Effect of leptin on phospho-STAT3 immunoreactivity in the mouse brain stem. Phospho-STAT3 immunoreactivity in the brain stem sections are shown under basal saline-injected conditions (A, C, and E), and leptin-injected (5 mg/kg, iv, 30 min) conditions (B, D, and F). CC, Central canal; scp, superior cerebellar peduncle; Aq, aqueduct. Scale bars, 200 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies in mice pointed to the brain as a potential target for leptin’s action on the basis of the greater potency of leptin to reduce food intake when administered intracerebroventriculary vs. peripherally (2). Leptin enters the brain across the blood-brain barrier (4, 28), and it was postulated that such a transport system may occur through the Ob-Ra receptor (29). It was reported recently that intracerebroventricularly applied leptin induced a nuclear translocation of immunoreactive STAT3 in hypothalamic nuclei (10). In the present study leptin was administrated iv to mimic the physiological condition, because circulating leptin is transported into specific target areas of the brain. The localization of leptin-induced phospho-STAT3 immunoreactive cells in the Arc, VMH, and LHA, all of which are known to regulate feeding behavior, adds further evidence in support of hypothalamic sites of leptin’s action. We observed potent induction of phospho-STAT3-immunoreactive cells in the Arc. It has been shown that the highest concentration of Ob-Rb mRNA is in the Arc (5, 15), and the Arc overlies the median eminence, a candidate site at which leptin may enter the brain (4). Thus, the Arc may respond well to circulating leptin.

Although leptin receptor is expressed in many regions of the brain, the physiological role(s) of leptin in nonhypothalamic regions is unknown. In the present study we observed that iv injected leptin induced STAT3 activation and SOCS3 induction in the brain stem as well as in the hypothalamus. Therefore, the brain stem may be a physiological target site in response to circulating leptin. Indeed, a previous study reported the colocalization of leptin receptor immunoreactivity with tyrosine hydroxylase and serotonin in the NTS and Raphe nuclei (30). Furthermore, the brain stem localization of Ob-Rb receptor expression, described previously (16, 31), matched the phospho-STAT3 immunoreactivity detected in the NTS, LPB, and CG in the present study. Therefore, these findings support our arguments regarding brain stem leptin signaling. However, in contrast to these structures that matched the phospho-STAT3 immunoreactivity and Ob-Rb expression, discrepant cases also exist. Although Ob-Rb leptin receptor expression was found in the area postrema (16), we could not detect leptin-induced phospho-STAT3 immunoreactivity in this nucleus. Ob-Rb receptors were also expressed in the cortex, hippocampus, and cerebellum. However, we could not detect an increase in STAT3 phosphorylation by Western blotting analysis or phospho-STAT3 immunoreactive cells in these brain regions after the iv administration of leptin. These results suggest that the brain regions where Ob-Rb receptors exist did not always respond to physiologically circulating leptin.

Increasing evidence has suggested that that cholecystokinin (CCK) and leptin interact in multiple ways. Bado et al. (32) reported that leptin immunoreactivity was present in the stomach, and administration of CCK-8 decreased leptin immunoreactivity in the fundic epithelium, with a concomitant increase in the concentration of leptin in plasma. Moreover, the existence of a functional synergistic interaction between leptin and CCK to reduce short-term food intake has been reported (33). CCK is known to produce satiety by interacting with vagal afferent fibers (34), which is an important component for transmitting peripheral immune or satiety signals to the brain (35, 36, 37). Leptin receptor is expressed in the vagus nerve (38), and leptin has been shown to activate vagal afferent nerves (19, 20). Therefore, these observations suggest that leptin and CCK may interact with the vagus nerve to elicit satiety responses. In the present study, we observed that peripheral leptin increased phosphorylated STAT3-immunoreactive cells in the NTS and LPB. NTS is the predominant termination site of the afferent vagus nerve. A number of forebrain regions, such as the hypothalamus, receive information of visceral origin either directly from the NTS or via the LPB (39). Interestingly, neurons in the LPB contain CCK-immunoreactive neurons and project to the VMH (40). Therefore, the present results suggest that leptin may directly act in the NTS and LPB to elicit satiety responses in concert with CCK.

Anatomical evidence suggests that MSH produced by proopiomelanocortin-containing neurons is found in the NTS as well as in hypothalamic structures, such as the Arc (41). MSH inhibits feeding by acting on central melanocortin receptors (42), and leptin regulates the levels of proopiomelanocortin mRNA in the Arc (43, 44). Moreover, application of melanocortin receptor ligands into the brain stem produced long-lasting reduction of feeding and body weight (45). The present finding that leptin increased phosphorylated STAT3-immunoreactive cells in the NTS suggests that leptin might affect feeding through the induction of MSH in the NTS.

The peripheral satiety signals, which activate the afferent vagus nerve, project via the NTS into the DMV that provides direct parasympathetic control to the viscera. The present demonstration that peripheral leptin increased phosphorylated STAT3-immunoreactive cells in the DMV suggests that there exists a functional receptor for leptin signaling and leptin in the DMV that may modulate visceral functions. Indeed, leptin injected centrally inhibited gastric emptying (46, 47).

The present observation of phospho-STAT3 immunostaining in brain stem nuclei suggests the direct action of leptin in these areas. The ability of circulating leptin to access the brain stem sites is not clear. However, the area postrema, a circumventricular organ that exists in proximity to the NTS might allow access to the nucleus. In comparison to that in the mouse, leptin receptor mRNA was present at very low levels in the brain stem of the rat (16). Elmquist et al. (31) reported that although leptin Ob-Rb receptors were hardly detected in the NTS or LPB in rats, systemic administration of leptin caused Fos expression in these cell groups (48). Therefore, it cannot be ruled out that leptin administration may activate a neurotransmitter system that subsequently activates the STAT3 signaling system in these neurons. Leptin receptors are present on the vagus nerve (38), and leptin can activate the vagal afferent nerves (19, 20). We observed an increase in phopho-STAT3 immunoreactivity in the NTS, the major termination site for the vagal afferent nerve. Therefore, there is a possibility that leptin activation of the vagus nerve could have caused the secondary increase in phospho-STAT3.

The present findings suggest that leptin may interact directly with physiologically functional Ob-Rb receptors in several brain stem nuclei as well as in the hypothalamus. Thus, leptin may have direct involvement in the regulation of neuroendocrine, autonomic, and behavioral systems via the central nervous system.

	Acknowledgments

 

	Footnotes

 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan (to Y.O. and Y.N.), and Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists (to T.H.).

Abbreviations: Arc, Arcuate nucleus; CCK, cholecystokinin; CG, central gray matter; DMV, dorsal motor nucleus of the vagus nerve; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JAK, Janus kinase; LHA, lateral hypothalamic area; LPB, lateral parabrachial nucleus; NTS, nucleus of the tractus solitarius; SOCS3, suppressor of cytokine signaling 3; STAT, signal transducer and activator of transcription; VMH, ventromedial hypothalamus.

Received January 22, 2002.

Accepted for publication May 16, 2002.

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