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Endocrinology Vol. 139, No. 11 4442-4447
Copyright © 1998 by The Endocrine Society

ARTICLES

Leptin Signaling in the Hypothalamus of Normal Rats in Vivo1

Karen C. McCowen2, Jesse C. Chow2 and Robert J. Smith

Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Jesse C. Chow, Ph.D., Eisai Research Institute, 4 Corporate Drive, Andover, Massachusetts 01810. E-mail: jesse_chow{at}eisai.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin has been shown to activate multiple signaling molecules in cultured cells, including Janus kinase-2, STAT (signal transducer and activator of transcription) proteins, and mitogen-activated protein kinase, and to stimulate the DNA-binding activity of STAT3 in mouse hypothalamus. In this study, the activation of candidate leptin signaling molecules in the hypothalamus of normal rats in vivo was investigated. Fasted male Sprague-Dawley rats were injected iv with recombinant murine leptin or vehicle. Plasma leptin concentrations were determined at defined time points, and the phosphorylation of signaling proteins was assessed in hypothalamic lysates. There was a marked increase in plasma leptin concentration at 2 min and a gradual decline by 45 min after leptin injection. Immunoblotting analysis of hypothalamic lysates with a phosphospecific STAT3 antibody demonstrated a time-dependent stimulation of STAT3 tyrosine phosphorylation. STAT3 phosphorylation was first evident at 5 min and was maximal at 30 min after leptin injection. By contrast, leptin did not increase the phosphorylation of Janus kinase proteins, mitogen-activated protein kinase, or STAT1 and -5 despite abundant expression of these signaling molecules in the hypothalamus. These results differ from findings in cultured cells and in vitro systems. It remains unclear how signaling is propagated downstream from the leptin receptor to STAT3, but this may involve novel signaling intermediates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY is one of the most prevalent and clinically important metabolic disorders. Mechanisms underlying the development of human obesity are complex and poorly defined. In various rodent models of obesity, abnormal production and action of the hormone leptin have been described (1). Leptin is synthesized in white adipose tissue and released to the circulation. In the fasted state in humans and rodents, leptin levels are low and rise with feeding (2, 3). Plasma leptin concentrations also rise with increasing adiposity and are elevated in obesity in proportion to fat mass (4). The hypothalamus is thought to be a major target for leptin. In the hypothalamus, leptin may act to decrease food intake and increase physical activity, thus providing a homeostatic mechanism that serves to maintain fat mass at a set-point (5). In the ob/ob mouse model of obesity, leptin is absent (6); in the db/db mouse and fa/fa rat there are mutations in the genes coding for the leptin receptor, and leptin action is therefore defective (7, 8). Whether secondary to abnormalities in the hormone or the receptor, the ultimate consequence of inadequate leptin action is obesity.

In the normal animal, the exact signaling pathways activated by leptin downstream of its receptor in the hypothalamus are not well defined (9). The leptin receptor exists as multiple splice variants (10). A long form (OB-RL) is located predominantly in paraventricular, arcuate, and ventromedial nuclei of the hypothalamus, where it is thought to mediate most of the neural signaling of leptin and consequently its weight-reducing actions (11, 12). The OB-RL hypothalamic receptor bears substantial homology to other members of the class I cytokine receptor family (13). Through signaling motifs in the intracytoplasmic region, members of this family of receptors can associate with the Janus family of tyrosine kinases (JAKs). After ligand binding, JAKs become activated, autophosphorylate, and catalyze tyrosine phosphorylation of various STAT (signal transducer and activator of transcription) proteins. Activated STATs dimerize and translocate to the nucleus, where specific gene responses are elicited (12). Leptin activation of its receptor in mouse hypothalamus has been shown to result in the appearance of STAT3 in the nucleus 15 min after its injection in a dose-dependent fashion, but no information is available on how the receptor, which is not a tyrosine kinase, communicates its signal to STAT3 (14). It is known that some cytokines can stimulate a molecular cascade coupled with Ras activation, and one study in a mouse embryonic cell line has demonstrated that leptin causes a dose- and time-dependent activation of mitogen-activated protein (MAP) kinase (15). Despite the existence of a few in vitro studies, primarily in cells transfected with OB-RL, showing that JAK2, STAT, or MAP kinase proteins may be mediating the downstream effects of leptin (16, 17, 18), there is no in vivo evidence for such pathways and no information about the signals that link leptin and its receptor to the production of nuclear STAT3. This study was designed to determine the nature of early signaling events when leptin is administered in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant murine leptin was purchased from PeproTech, Inc. (Rocky Hill, NJ). Antibodies to JAK1, Tyk2, STAT1, STAT3, and STAT5 were obtained from Transduction Laboratories, Inc. (Lexington, KY). Antibodies to JAK2 and JAK3 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to phosphospecific STAT1 and STAT3 were obtained from New England Biolabs (Beverly, MA). Polyclonal phosphotyrosine antibody was prepared as described previously (19). Sodium amobarbital was obtained from Eli Lilly & Co. (Indianapolis, IN). Nitrocellulose membranes (BA85, 0.2 µm) were purchased from Schleicher & Schuell, Inc. (Keene, NH). Protein A-Sepharose was purchased from Pharmacia Biotech (Piscataway, NJ). Sodium orthovanadate was obtained from Aldrich Chemical Co. (Milwaukee, WI). Enhanced chemiluminescence detection reagents were obtained from Amersham Chemical Co. (Arlington Heights, IL), and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals
Male Sprague-Dawley rats (150 g) were purchased from Taconic Farms, Inc. (Germantown, NY), acclimated in a light-controlled room (12-h light, 12-h dark cycle), and allowed free access to standard rat chow and water. Food was removed from the cages 16 h before the experiments. Animal protocols were in compliance with the Guide for the Care and Use of Laboratory Animals published by the NIH and approved by the Joslin Diabetes Center institutional animal care committee.

Experimental procedures
Rats were anesthetized with sodium amobarbital (100 mg/kg, ip injection). A laparotomy was performed, and a bolus of leptin (1 µg/g BW) or vehicle (Tris-HCl, pH 7.8) was injected via the inferior vena cava. This dose of leptin used was in the range that previously has been shown to be effective in decreasing food intake (20, 21) and in stimulating the DNA-binding activity of hypothalamic STAT3 in mice (14). At various time points after the injection of leptin (2, 5, 15, 30, or 45 min) or vehicle (0 min), the chest cavity was opened, and venous blood was obtained by cardiac puncture. Blood samples from 12 rats were used for each time point. Decapitation was performed, the cranium was opened, and the hypothalamus was excised, snap-frozen in liquid nitrogen, and stored at -80 C until analysis. Each hypothalamus was homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) for 30 sec at 4 C in 500 µl buffer containing 20 mM Tris (pH 7.6), 120 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 10 mM sodium pyrophosphate, 40 µg/µl leupeptin, and 100 mM NaF. The resulting homogenates were incubated on a rocking platform at 4 C for 30 min and subsequently centrifuged for 45 min at 13,000 x g. Supernatants were collected, and protein concentrations were determined by the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as a standard.

For the immunoprecipitation experiments, 2 mg hypothalamic lysate protein were incubated with a JAK1, JAK2, JAK3, TYK2, or STAT5 antibody overnight at 4 C on a rocking platform. The immunocomplexes then were adsorbed to protein A-Sepharose beads for 90 min at 4 C, pelleted by centrifugation, and washed three times at 4 C in buffer containing 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF. These samples and additional tissue extracts not treated with antibodies (200 µg protein) were boiled in Laemmli sample buffer containing 100 mM dithiothreitol and then subjected to SDS-PAGE. The resolved proteins were electroblotted onto nitrocellulose membranes in buffer containing 25 mM Tris, 190 mM glycine, and 20% (vol/vol) methanol. Nonspecific protein binding was prevented by incubating the blots with blocking buffer (5% BSA, 10 mM Tris (pH 7.4), 150 mM NaCl, and 0.01% sodium azide) at 37 C for 2 h followed by immunoblotting with the appropriate antibody and detection using the enhanced chemiluminescence method. Membranes were stripped of blotting antibody in buffer containing 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) for 30 min at 55 C and reblotted with an antibody specific for the protein of interest to confirm equal loading of samples and demonstrate expression of the signaling protein within the hypothalamus. Positive controls were also included to confirm that the immunoprecipitation and immunoblotting procedures were effective. Plasma samples were analyzed for leptin using a commercially available RIA kit (Linco Research, Inc., St. Charles, MO).

Statistical analysis
Quantitative data are presented as the mean ± SE, and statistical significance was evaluated by one-way ANOVA followed by Bonferroni’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma leptin concentrations
Basal plasma leptin concentrations were at or below the lower limit of detection for the assay (<1 ng/ml), as expected in rats fasted overnight. There was a dramatic increase by 2 min (12,000 ng/ml) after leptin treatment (Fig. 1Go). Peak leptin concentrations were supraphysiological and well above the concentrations required to induce weight loss in normal rodents (21). By 15 min, leptin concentrations had fallen significantly (25% of maximum), and the decline continued at 30 and 45 min after injection.



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  		Figure 1. Time course of elevation in plasma leptin concentrations after an iv injection of leptin in rats. Plasma leptin concentrations were determined by RIA. Each bar represents the mean ± SE for 12 rats, except at 45 min (n = 4). Means with different superscripts are significantly different from each other (a vs. b, P < 0.001; a vs. c and b vs. c, P < 0.05).

 
Effect of leptin treatment on JAK family tyrosine kinases
To determine whether a bolus injection of leptin activates members of the Janus family of tyrosine kinases in vivo, hypothalamic proteins were sequentially immunoprecipitated with JAK1, JAK2, JAK3, or Tyk2 antibody and immunoblotted with phosphotyrosine antibody. Figure 2Go, a–d, illustrates that leptin treatment does not stimulate the tyrosine phosphorylation of JAK1, JAK2, JAK3, or Tyk2 at any of the time points examined (5, 15, and 30 min). However, the presence of all three JAK proteins and Tyk2 in the immunoprecipitates was confirmed after reblotting the same nitrocellulose membranes with each respective immunoprecipitating antibody (Fig. 2Go, e–h). To exclude the possibility of a rapid and transient activation of JAK2, a separate set of experiments was performed using an earlier time point (2 min) after leptin administration; JAK2 tyrosine phosphorylation was not evident at the 2 min point (not shown).



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  		Figure 2. Leptin treatment and the JAK family of tyrosine kinases in the hypothalamus. Tissue lysates from rats injected with vehicle (time zero) or leptin (1 µg/kg) for 5, 15, or 30 min were prepared as described in Materials and Methods. Lysates were immunoprecipitated (IP) with JAK1 (A), JAK2 (B), JAK3 (C), or Tyk2 (D) antibodies; resolved by SDS-PAGE; transblotted onto nitrocellulose; and immunoblotted (IB) with phosphotyrosine antibody (PY). Membranes were stripped and reprobed with each respective immunoprecipitating antibody (e–h). These are representative immunoblots, with each lane corresponding to an individual animal (n >= 4 for each time point).

 
Effect of leptin on MAP kinase phosphorylation
To investigate whether leptin activates MAP kinase in vivo, hypothalamic lysates were immunoblotted with antibody specific for the phosphorylated forms of two MAP kinases, Erk1 and Erk2 (Fig. 3Go). In the basal state (vehicle-injected rats), tyrosine phosphorylation of both isoforms of MAP kinase was detected, but this level of tyrosine phosphorylation was not increased at 5, 15, or 30 min after leptin treatment.



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  		Figure 3. Leptin treatment and MAP kinase (Erk1 and Erk2) phosphorylation in the hypothalamus. Tissue lysates from rats injected with vehicle (time zero) or leptin (1 µg/kg) for 5, 15, or 30 min were prepared as described in Materials and Methods. Lysates (200 µg) were resolved by SDS-PAGE, transblotted onto nitrocellulose, and immunoblotted with a phosphospecific MAP kinase antibody. This is a representative immunoblot, with each lane corresponding to an individual animal (n = 4 for each time point).

 
Effect of leptin on tyrosine phosphorylation of STAT proteins
To measure leptin-induced activation of STAT1 and STAT3, hypothalamic lysates were immunoblotted with antibody specific for phosphorylated STAT1 or STAT3. STAT1 phosphorylation was not detected at 5, 15, or 30 min after leptin injection (Fig. 4aGo). This observation cannot be attributed to the lack of STAT1 protein in the hypothalamus, as STAT1 was readily identified in the same blots after reprobing with a STAT1 antibody (Fig. 4cGo). In contrast, there was a time-dependent increase in STAT3 phosphorylation after leptin injection (Fig. 5bGo). This activation was not evident at 2 min (data not shown). Densitometric analysis of the phosphorylated STAT3 bands demonstrates that there was a 2- to 3-fold increase in STAT3 phosphorylation between 5–15 min, reaching 6-fold above basal levels at 30 min after leptin administration (Fig. 5cGo). At 45 min (n = 4), STAT3 phosphorylation was 5.5-fold above basal and was not different from that at the 30 min point (1.0 ± 0.01 densitometric units vs. 0.9 ± 0.01). Stripping and reblotting the membranes with a STAT3 antibody verified that the phosphorylated 83-kDa protein in Fig. 5bGo is indeed STAT3 and that each lane contained similar amounts of STAT3 protein (Fig. 5aGo).



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  		Figure 4. Leptin treatment and STAT1 or -5 in the hypothalamus. Tissue lysates from rats injected with vehicle (time zero) or leptin (1 µg/kg) for 5, 15, or 30 min were prepared as described in Materials and Methods. Lysates (200 µg) were directly analyzed (a) or immunoprecipitated (IP) with STAT5 antibody (b) followed by SDS-PAGE, transblotted onto nitrocellulose, and immunoblotted (IB) with a phospho-STAT1 or phosphotyrosine antibody (PY), respectively. Membranes were stripped and reprobed with an antibody for the indicated STAT protein (c and d). These are representative immunoblots, with each lane corresponding to an individual animal (n = 4 for each time point).

 


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  		Figure 5. Time course of leptin-induced tyrosine phosphorylation of STAT3 in the hypothalamus. Tissue lysates from rats injected with vehicle (time zero) or leptin (1 µg/kg) for 5, 15, or 30 min were prepared; resolved by SDS-PAGE; transblotted onto nitrocellulose; and immunoblotted with a phosphospecific STAT3 antibody (a). Membranes were then stripped and reprobed with STAT3 antibody (b) as described in Materials and Methods. This is a representative immunoblot, with each lane corresponding to an individual animal. The intensities of bands corresponding to phosphorylated STAT3 were quantitated by densitometry and expressed as arbitrary units, with each bar representing the mean ± SEM for eight rats per time point (c). The mean for the 30 min time point is significantly different from the others (a, 30 min vs. 5 or 15 min, P < 0.05; b, 30 min vs. zero time, P < 0.001).

 
To investigate whether leptin activates STAT5, lysates were immunoprecipitated with STAT5 antibody and immunoblotted with phosphotyrosine antibody. This protocol has previously been used to demonstrate STAT5 tyrosine phosphorylation in tissue lysates of GH-stimulated rats (22). However, leptin did not stimulate STAT5 phosphorylation at any of the time points examined (Fig. 4bGo). When the same blots were stripped and reblotted with STAT5 antibody, the protein was detected, albeit weakly, compared with the other STAT proteins (Fig. 4dGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates for the first time that leptin causes tyrosine phosphorylation of STAT3 in the hypothalamus in vivo, a response that occurs as early as 5 min after an iv bolus injection of leptin at a dose of 1 µg/g BW. No other signaling intermediates were found to be stimulated across a range of time points despite the clear presence of several members of the JAK protein family in the hypothalamus. This work supports the existence of a mechanism for rapid activation of signaling in the hypothalamus in response to changes in peripheral leptin concentrations, although the specific pathway leading to STAT3 tyrosine phosphorylation is not known. A previous study by Banks et al. (23) has shown that leptin is transported intact from the blood to the brain by a unidirectional saturable transport system in mice, which provides a mechanism for blood-borne leptin to rapidly affect the brain. Our findings extend the initial observations of Vaisse et al., who reported the appearance of STAT3 dimers in nuclear extracts of mouse hypothalamus 15 min after an in vivo bolus of leptin (14). The data in our study indicate that this nuclear translocation probably occurs as a result of tyrosine phosphorylation, thus allowing the STAT proteins to dimerize and bind to DNA. The question of which signaling proteins lie immediately upstream of STAT3 remains unanswered.

Most members of the structurally related class I cytokine receptors stimulate tyrosine phosphorylation of STAT proteins by first activating JAK proteins, which are associated in a noncovalent manner with the intracellular portion of the receptor (24). The binding of ligand to the receptor induces a sequence of events involving receptor dimerization and JAK phosphorylation. Activated JAKs, in turn, phosphorylate the receptor components, and these phosphorylated regions serve as docking sites for the Src homology 2 domains found in all STAT proteins. The inability to detect activation of JAKs in this study might reflect difficulties in detecting small increases in phosphorylated JAK proteins. However, this is unlikely because we have detected JAK2 tyrosine phosphorylation in various tissues after GH administration using similar methods (22). Another possibility is that the activation of JAK proteins by leptin is very transient. The earliest time point examined in this study was 2 min, and it is possible, although unlikely in view of the need for leptin to cross the blood-brain barrier, that JAK2 has already been dephosphorylated by one of several phosphatases and has returned to basal levels of phosphorylation by this time. As several reports have suggested that MAP kinase might be important in the JAK/STAT signaling cascade at various steps, we examined MAP kinase activation with a phosphospecific antibody and excluded the possibility that the MAP kinase proteins lie upstream of STAT3 activation in response to leptin in vivo. An interesting alternative is the possibility that yet unidentified signaling proteins, perhaps novel tyrosine kinases specific for the hypothalamus, mediate the proximal steps in leptin action.

In cultured cell systems, many different signaling cascades have been shown to be activated by leptin, including STAT1 (human renal adenocarcinoma cell lines, hepatoma cell lines, and COS cells stably overexpressing the long form of the receptor), STAT3 (hepatoma and COS cell lines), STAT5 and -6 (COS cell lines), JAK2 (BaF3 cells transfected with OB-RL), and MAP kinase (mouse embryonic cell line and COS cells expressing HA-Erk1), but the relevance of these findings to the intact animal in vivo is uncertain (15, 17, 18, 25, 26, 27). In addition, a recent paper (27) demonstrated cross-talk with the insulin signaling pathway in Chinese hamster ovary cells transfected with leptin receptors, manifested as leptin activation of insulin receptor substrate-1. It has been shown repeatedly that signaling results obtained in cultured cells, which are often overexpressing either receptors or components of the signaling cascade of interest, cannot be reproduced in vivo. Whole animal signaling studies demonstrate that there may be greater specificity in hormone-mediated events than is found in an in vitro system. The study of Vaisse et al. is in agreement with ours, in that only STAT3 among this family of proteins is activated by leptin in vivo. We have demonstrated STAT3 phosphorylation in response to a supraphysiological dose of leptin, and ultimately, it will be important to determine whether similar leptin-induced STAT3 phosphorylation occurs in response to physiological stimuli, such as refeeding in fasted animals.

In Chinese hamster ovary cells stably expressing the leptin receptor (isoform OBRa or OBRb), leptin-dependent induction of c-fos, c-jun, and jun-B messenger RNAs has been shown to occur within 30 min (28). This effect of leptin is supported by two additional studies (29, 30) that demonstrate c-fos activation 2 and 3 h after peripheral leptin injection in normal rats and ob/ob mice, respectively. How leptin-induced STAT3 phosphorylation in the hypothalamus may diminish appetite, influence physical activity, and ultimately regulate body weight is unknown. Leptin has been shown to inhibit the expression of neuropeptide Y messenger RNA and protein in rat and mouse hypothalamus (31, 32). Intracerebroventricular administration of neuropeptide Y is known to promote obesity by increasing food intake and diminishing thermogenesis (33, 34) and is a factor that may be an important downstream link between leptin and the appetite and activity centers in the brain.

In human obesity, with rare exception (35), leptin levels are elevated in proportion to the excess adiposity, and some form of leptin resistance has been postulated. Whether the defect is impaired transport of leptin across the blood-brain barrier or a receptor or postreceptor signaling event has yet to be established. A more precise elucidation of the molecular mechanism of leptin action is necessary to begin to understand these human forms of leptin resistance. This study examined the signaling proteins activated by leptin in vivo in the rat hypothalamus after an iv injection of leptin. We have shown that STAT3 is tyrosine phosphorylated within 5 min of injection of hormone, but that leptin signaling differs from that of GH and other ligands that bind members of this class of receptor by failing to stimulate measurable responses in any of the JAK family of proteins. Novel techniques need to be applied in further examination of this signaling pathway in the intact animal, as cultured cell studies are likely to yield data that may not be reflective of signaling events in vivo.


   Footnotes
 
1 This work was supported in part by a Boston Obesity Nutrition Research Center Grant (to J.C.C.), NIH Grant DK-50411 (to R.J.S.), and NIH Diabetes and Endocrinology Center Grant DK-36836 (to R.J.S.). Back

2 These authors contributed equally to the work. They were supported by an American Diabetes Association Mentor-Based Fellowship Grant (to R.J.S.) and a Mary K. Iaccoca Foundation Postdoctoral Fellowship, respectively. Back

Received January 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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