Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling

Christopher D. Morrison,1,2 Gregory J. Morton,1 Kevin D. Niswender,1,3 Richard W. Gelling,1 and Michael W. Schwartz1

1University of Washington and Harborview Medical Center, Seattle, Washington; 2Pennington Biomedical Research Center, Baton Rouge, Louisiana; and 3Vanderbilt University, Nashville, Tennessee

Submitted 2 March 2005 ; accepted in final form 22 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phosphatidylinositol 3-OH-kinase (PI3K) and STAT3 are signal transduction molecules activated by leptin in brain areas controlling food intake. To investigate their role in leptin-mediated inhibition of hypothalamic neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression, male Sprague-Dawley rats (n = 5/group) were either fed ad libitum or subjected to a 52-h fast. At 12-h intervals, the PI3K inhibitor LY-294002 (LY, 1 nmol) or vehicle was injected intracerebroventricularly (ICV) as a pretreatment, followed 1 h later by leptin (3 µg icv) or vehicle. Fasting increased hypothalamic Npy and Agrp mRNA levels (P < 0.05), and ICV leptin administration prevented this increase. As predicted, LY pretreatment blocked this inhibitory effect of leptin, such that Npy and Agrp levels in LY-leptin-treated animals were similar to fasted controls. By comparison, leptin-mediated activation of hypothalamic STAT3 signaling, as measured by induction of both phospho-STAT3 immunohistochemistry and suppressor of cytokine signaling-3 (Socs3) mRNA, was not significantly attenuated by ICV LY pretreatment. Because NPY/AgRP neurons project to the hypothalamic paraventricular nucleus (PVN), we next investigated whether leptin activation of PVN neurons is similarly PI3K dependent. Compared with vehicle, leptin increased the number of c-Fos positive cells within the parvocellular PVN (P = 0.001), and LY pretreatment attenuated this effect by 35% (P = 0.043). We conclude that leptin requires intact PI3K signaling both to inhibit hypothalamic Npy and Agrp gene expression and activate neurons within the PVN. In addition, these data suggest that leptin activation of STAT3 is insufficient to inhibit expression of Npy or Agrp in the absence of PI3K signaling.

suppressor of cytokine signaling-3; signal transducer and activator of transcription 3


LEPTIN IS A KEY REGULATOR of feeding and long-term energy homeostasis that acts on discrete neuronal pathways to reduce food intake and body fat content. Among the key targets for leptin action are neurons within the hypothalamic arcuate nucleus (ARC) that coexpress the orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AgRP). These NPY/AgRP neurons express leptin receptors (17) and are inhibited by leptin (28) via a cellular mechanism that is poorly understood. Because obesity is frequently associated with leptin resistance (15), elucidating the cellular mechanisms underlying leptin action on neuronal systems involved in energy homeostasis is an important priority.

One mechanism implicated in leptin regulation of cellular function is the activation of Janus-activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling (1, 31), and recent data implicate this pathway in leptin-mediated stimulation of ARC proopiomelanocortin (POMC) neurons (22). This conclusion stems in part from studies of mice in which the endogenous leptin receptor was replaced by one incapable of activating STAT3 (3). In these animals, regulation of Pomc gene expression was impaired, suggesting a key role for leptin-mediated STAT3 activation in this process. However, Npy and Agrp mRNA levels, which are markedly increased in mice lacking leptin receptors, were not dramatically affected by the loss of leptin-stimulated STAT3 signaling. Therefore, additional STAT3-independent signaling mechanisms appear to be involved in leptin regulation of NPY/AgRP neurons.

The phosphatidylinositol 3-OH-kinase (PI3K) signaling pathway warrants consideration as a STAT3-independent mediator of leptin inhibition of NPY/AgRP neurons. Leptin activates PI3K signaling within the mediobasal hypothalamus (24) and other tissues (13) and regulates the activity of glucose-responsive ARC neurons via a PI3K-dependent mechanism in vitro (18). Furthermore, intracerebroventricular (ICV) pretreatment with a PI3K inhibitor prevents leptin-mediated reductions of food intake (24). We therefore sought to determine whether pretreatment with a PI3K inhibitor blocks leptin inhibition of hypothalamic Npy and Agrp gene expression and, if so, whether this effect occurs despite intact leptin-mediated activation of STAT3. In addition, we investigated whether actions of leptin on the PVN, a key hypothalamic area downstream of ARC NPY/AgRP neurons, are also dependent on PI3K signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All procedures were performed in accordance with National Institutes of Health guidelines for the care and use of animals and were approved by the Animal Care Committees of the University of Washington and the Pennington Biomedical Research Center. Animals were housed singly and maintained on a 12:12-h light-dark cycle with ad libitum access to standard rat chow and water unless otherwise noted.

Effect of PI3K Inhibitor on Leptin Regulation of Hypothalamic NPY, AgRP, and Socs3 mRNA Expression

Four groups (n = 5/group) of male Sprague-Dawley rats (Charles River Labs, Wilmington, MA) averaging 367 ± 6.9 g in weight were studied ≥10 days after stereotaxic implantation of a third cerebroventricular cannula (24). One group was fed ad libitum, and the other three were subjected to a 52-h fast. Fasting reduces basal leptin levels, allowing for clear and robust changes in hypothalamic gene expression in response to exogenous leptin. At 12-h intervals, the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one [LY294002 (LY, 1 nmol; Calbiochem, La Jolla, CA)] or its vehicle (1 µl) was injected ICV as a pretreatment, followed 1 h later by ICV administration of recombinant murine leptin (3 µg; Dr. A. F. Parlow, National Hormone and Peptide Program) or its vehicle in a volume of 1.5 µl.

The injection regimen was repeated every 12 h throughout the 52-h study period, resulting in four treatment groups: vehicle-vehicle fed, vehicle-vehicle fasted, vehicle-leptin fasted, and LY-leptin fasted. At the end of this 52-h period, rats were euthanized, brains were removed and snap-frozen, and hypothalami (defined caudally by the mammillary bodies, rostrally by the optic chiasm, laterally by the optic tract, and superiorly by the apex of the third ventricle) were excised and stored at –80°C for subsequent determination of Npy, Agrp, and Socs3 mRNA content.

Effect of PI3K Inhibitor Alone on NPY and AgRP mRNA Expression

To control for possible effects of LY independent of leptin treatment, male Long-Evans rats (n = 5/group; Harlan, Indianapolis, IN) were fasted for 52 h and randomly assigned to receive ICV injections of either vehicle or LY, followed 1 h later by injection of vehicle at 12-h intervals during a 52-h fast. Hypothalami were collected and processed as in the previous study. This dose of ICV LY had no effect in either Sprague-Dawley or Long Evans rats when given alone but blocked the effects of leptin and insulin on food intake in both strains (23, 24).

Effect of PI3K Inhibitor on Leptin Activation of STAT3 Within the ARC

At least 10 days after the third ventricular cannulation, three groups (n = 2–3/group) of male Sprague-Dawley rats were fasted for 24 h. Each animal subsequently received an ICV injection of LY (1 nmol) or its vehicle, followed 1 h later by leptin (3 µg icv) or its vehicle. Twenty minutes after the leptin injection, animals were perfused via cardiac puncture with 60 ml of ice cold 0.1 M PBS, followed by 4% paraformaldehyde in 0.1 M PBS. Perfused brains were removed and immersion fixed overnight in 4% paraformaldehyde in 0.1 M PBS followed by 25% sucrose in 0.1 M PBS. Brains were then snap-frozen in isopentane cooled with liquid nitrogen and stored at –80°C until sectioning at 14 µm with a cryostat. Sections were the mounted onto slides and stored at –80°C until immunostained for phospho-STAT3.

Effect of PI3K Inhibitor on Leptin-Stimulated c-Fos Induction in the PVN

At least 10 days after the third ventricular cannulation, three groups (n = 6–7/group) of male Long Evans rats weighing 339 ± 5.2 g were fasted for 24 h, and each subsequently received an ICV injection of LY (1 nmol) or its vehicle, followed 1 h later by leptin (3 µg icv) or its vehicle. Two hours after the second injection, animals were perfused via cardiac puncture with 60 ml of ice cold 0.1 M PBS followed by 4% paraformaldehyde in 0.1 M PBS. Perfused brains were removed and immersion fixed overnight in 4% paraformaldehyde in 0.1 M PBS followed by 25% sucrose in 0.1 M PBS. Brains were then snap-frozen in isopentane cooled with liquid nitrogen and stored at –80°C until sectioning at 14 µm with a cryostat until immunostaining to detect c-Fos, a marker of neuronal activation.

RNA Extraction and Real-Time PCR Quantification

Total hypothalamic RNA was extracted using TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions and processed for real-time PCR as previously described (20), using the following primer sets: Npy: foward 5'-accaggcagagatatggcaaga-3', reverse 5'-ggacattttctgtgctttctctcatta-3'; Agrp: forward 5'-agggcatcagaaggcctgaccagg-3', reverse 5'-cattgaagaagcggcagtagcacgt-3'; Gapdh: forward 5'-aacgaccccttcattgac-3', reverse 5'-tccacgacatactcagcac-3'; Socs3: forward 5'-gagtacccccaagagagcttacta-3', reverse 5'-ctctttaaagtggagcatcatactg-3'. mRNA expression levels for Npy, Agrp, and Socs3 were quantified using the {Delta}CT method and normalized to Gapdh mRNA content.

Immunohistochemistry

Phospho-STAT3. Phospho-STAT3 (pSTAT3) immunostaining was carried out as described by Levin et al. (15). Free-floating sections were washed in potassium phosphate buffered saline (KPBS) at room temperature and were subsequently incubated for 20 min in freshly prepared 1% NaOH and 1% H2O2 in KPBS, washed, incubated in 0.3% glycine in KPBS for 10 min, washed again, and then placed in freshly prepared 0.15% SDS in KPBS for 10 min. Sections were then washed and blocked for ~2 h with 4% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) in KPBS, containing 0.4% Triton X-100, and subsequently incubated in this blocking solution plus rabbit anti-pSTAT3 antibody (1:1,000; Sigma-Aldrich, St. Louis, MO) overnight at 4°C. Sections were then washed and incubated for 2 h in blocking solution containing biotinylated donkey anti-rabbit antibody (1:250; Jackson Immunoresearch Laboratories), washed again, and incubated with Vector avidin-biotin complex (ABC) reagent (Vector Laboratories, Burlingame, CA) and developed with diaminobenzidine (DAB) plus nickel (Vector). Stained sections were mounted onto slides, dehydrated, cleared in xylene and coverslipped with Permount, using standard procedures. Fourteen-micrometer coronal sections were cut on a cryostat and anatomically matched. Paired sections were taken at 140-µm intervals through the rostral ARC. To determine the number of pSTAT3-positive cell nuclei within the rostral ARC, positive nuclei were counted manually on a Nikon Eclipse E800 microscope fitted with a grid reticule, with the investigator blinded to study conditions. Approximately six sections were counted per animal (2 ARCs per section), and the mean value derived from these six sections was used to generate a single observation per animal for statistical analysis.

c-Fos. Hypothalamic sections containing PVN were stained for c-Fos using standard immunohistochemical procedures (16). Sections were blocked in 5% normal donkey serum (Jackson Immunoresearch Laboratories) and 1% bovine serum albumin (Sigma) in 0.1 M PBS, and all antibodies were diluted in this blocking buffer. Rabbit anti c-Fos antibody (Ab5, 1:5,000; Oncogene, San Diego, CA) was used overnight at 4°C, donkey-anti-rabbit IgG SP Biotin (Jackson Immunoresearch Laboratories, 1:200) was used for 2 h at room temperature, and sections were then incubated for 30 min with Vectastain Elite ABC reagent (Vector Laboratories) and developed using the DAB substrate kit with nickel chloride (Vector Laboratories). Images were captured using a Nikon Eclipse E600 upright microscope equipped with a Diagnostic Instruments Spot RT Color digital camera. Relative c-Fos expression was determined using the Scion Image Software Package (Scion, Frederick, MD). Images were thresholded to background, and pixel area above background within a predefined and constant area over the parvocellular PVN was determined as total c-Fos-positive area. To compute the number of c-Fos positive cell nuclei, total c-Fos area was divided by the average size of individual c-Fos-positive nuclei. After this method was validated by comparison with c-Fos-positive cells counted by hand using a visual approach (data not shown), paired coronal sections were taken at 140-µm intervals through the PVN, and the mean value derived from an average of eight sections was used to generate a single observation per animal for statistical analysis. Anatomical matching of sections was performed by an investigator blinded to study conditions.

Statistical Analysis

All data are expressed as means ± SE. For mRNA levels, group mean values are expressed as a percentage of ad libitum-fed, vehicle-treated controls. Comparisons between multiple groups were made using a one-way ANOVA [Statistical Package for the Social Sciences (SPSS), version 10.1; SPSS, Fullerton, CA], with the least significant difference post hoc test to detect significant differences between group means. P < 0.05 was interpreted as a statistically significant difference between group means.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PI3K Inhibitor on Leptin Regulation of NPY and AgRP mRNA Expression

To test the hypothesis that intact PI3K signaling is required for leptin regulation of NPY/AgRP neurons, we determined whether ICV infusion of a PI3K inhibitor (LY) prevents leptin-mediated reductions of Npy and Agrp mRNA expression in the hypothalamus of fasted rats. Fasting for 52 h increased hypothalamic Npy mRNA levels in vehicle-treated rats (by 52% vs. vehicle-treated fed rats, P = 0.007, Fig. 1A), and ICV administration of leptin at 12-h intervals during the fast prevented this increase (P = 0.012). This inhibitory effect of leptin was completely prevented by ICV administration of the PI3K inhibitor LY 1 h before each leptin injection, such that Npy mRNA levels in the LY-leptin group were higher than in either vehicle-treated fed (P = 0.02) or leptin-treated fasted rats (P = 0.03). Similarly, fasting increased hypothalamic levels of Agrp mRNA (by 51% vs. vehicle-treated fed controls, P = 0.013; Fig. 1B), and leptin administration prevented this increase (P = 0.04). As was the case for Npy mRNA, ICV pretreatment with LY completely blocked the inhibitory effect of leptin, yielding Agrp mRNA levels that were higher than vehicle-leptin fasted (P = 0.06) or vehicle-vehicle fed rats (P = 0.02). In summary, ICV leptin reversed the effect of fasting to increase hypothalamic Npy and Agrp mRNA, and this leptin effect was blocked by ICV pretreatment with a PI3K inhibitor.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Effect of phosphatidylinositol 3-OH-kinase (PI3K) inhibitor on leptin inhibition of hypothalamic neuropeptide Y (Npy; A) and agouti-related peptide (Agrp; B) gene expression. At 12-h intervals, ad libitum-fed or 52-h-fasted rats were administered either the PI3K inhibitor LY-294002 (LY, 1 nmol) or vehicle (V) ICV, followed 1 h later by ICV leptin (L, 3 µg) or vehicle beginning at the onset of the fast. *P < 0.05 vs. V-V-treated fed rats.

 
To exclude the possibility that LY infusion blocked leptin action indirectly via an independent, stimulatory effect on hypothalamic Npy and Agrp mRNA expression, we used an identical protocol to administer LY or its vehicle as a pretreatment, followed by treatment with ICV vehicle (rather than leptin) at 12-h intervals during a 52-h fast in a separate group of rats. As expected, ICV administration of LY had no effect on either Npy or Agrp mRNA expression relative to vehicle (P > 0.25; Fig. 2).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Effect of ICV LY alone on Npy or Agrp gene expression in fasted rats. Rats (n = 5/group) were fasted for 52 h and administered LY (1 nmol) or vehicle ICV at 12-h intervals beginning at the onset of the fast. P > 0.10 for V-V vs. LY-V.

 
Effect of PI3K Inhibitor on Leptin Stimulation of Socs3 Expression

To determine whether leptin activation of STAT3-mediated gene transcription is sensitive to PI3K inhibition, we tested whether leptin induction of Socs3 gene expression is affected by ICV LY injection. Socs3 is a direct target of STAT3 transcriptional activity, and its expression has been validated as a marker of STAT3 activity (1, 9). As expected, ICV leptin treatment induced a robust increase of Socs3 mRNA levels in fasted rats (by 286% vs. vehicle-treated fasted controls, P < 0.04; Fig. 3), but unlike expression of Npy and Agrp, this response was not affected by ICV pretreatment with LY.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Effect of PI3K inhibitor on leptin induction of hypothalamic Socs3 gene expression, a marker for leptin activation of STAT3 signaling. Ad libitum-fed or 52-h-fasted rats (n = 5/group) were administered either LY (1 nmol) or vehicle ICV, followed 1 h later by ICV leptin (3 µg) or vehicle at 12-h intervals beginning at the onset of the fast. *P < 0.05 vs. V-V treated fed rats.

 
Effect of PI3K Inhibitor on Leptin Activation of STAT3 Within the ARC

To provide a second test of the hypothesis that leptin activation of STAT3 signaling is insensitive to PI3K inhibition, we determined whether leptin-mediated increases of STAT3 phosphorylation within the ARC are altered by pretreatment with a PI3K inhibitor. As expected, a pronounced, fivefold increase in the number of ARC neurons immunopositive for pSTAT3 was detected 20 min after leptin administration (P = 0.005; Fig. 4). However, pretreatment with the PI3K inhibitor LY did not influence leptin activation of STAT3 within this brain area.



View larger version (75K):
[in this window]
[in a new window]
 
Fig. 4. Effect of PI3K inhibitor on leptin activation of STAT3 phosphorylation within the arcuate nucleus (ARC). Twenty-four-hour-fasted rats (n = 2–3/group) were administered either LY (1 nmol) or vehicle ICV, followed 1 h later by ICV leptin (3 µg) or vehicle. Twenty minutes after injection of leptin, rats were perfused, and brains were collected and processed for phospho-STAT3 (pSTAT3) immunohistochemistry. *P < 0.05 vs. vehicle-vehicle treated rats. 3V, third ventricle.

 
Effect of a PI3K Inhibitor on Leptin-Induced c-Fos Within the PVN

The hypothalamic PVN is a key target of leptin action and is densely innervated by ARC NPY/AgRP neurons. To investigate whether leptin-mediated activation of PVN neurons requires PI3K signaling, we determined the effects of ICV pretreatment with LY on leptin-induced c-Fos immunopositive neurons within this brain area. As expected, ICV leptin increased the number of c-Fos positive cells within the parvocellular PVN (vehicle-leptin 74 ± 9 vs. vehicle-vehicle 15.5 ± 8 cells/PVN, P < 0.001; Fig. 5). This response was partially attenuated by ICV LY pretreatment, such that the number of c-Fos positive cells in the PVN was significantly lower in the LY-leptin group than the vehicle-leptin group (LY-leptin 53.5 ± 8 vs. vehicle-leptin 74 ± 9 cells/PVN, P = 0.043) but remained significantly greater than vehicle-vehicle controls (vehicle-vehicle 15.5 ± 8 vs. LY-leptin 53.5 ± 8 cells/PVN, P = 0.007). When expressed as the incremental increase over basal, the effect of leptin to increase c-Fos was attenuated by 35% in animals pretreated with ICV LY.



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 5. Effect of PI3K inhibitor on leptin induction of c-Fos within the hypothalamic paraventicular nucleus (PVN). After a 24-h fast, rats (n = 6–7/group) received a single ICV injection of LY (1 nmol) or vehicle, followed 1 h later by leptin (3 µg icv) or vehicle. Two hours after leptin injection, brains were collected and processed for c-Fos immunohistochemistry. *P < 0.05 vs. vehicle-vehicle treated fed rats, {dagger}P < 0.05 vs. vehicle-leptin.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leptin administration increases PI3K activity within the mediobasal hypothalamus (24, 33), and ICV pretreatment with inhibitors of PI3K signaling blocks leptin’s anorectic effects (24, 26). Together, these observations implicate PI3K as a physiological mediator of hypothalamic leptin action, but whether PI3K signaling is necessary for leptin regulation of specific neuronal subsets is not clearly understood. Because NPY/AgRP neurons are key targets of leptin action, we hypothesized that intact PI3K signaling is necessary for leptin-mediated inhibition of Npy and Agrp gene expression. Consistent with previous reports in rats (5, 14), we found that fasting increased Npy and Agrp mRNA, and that ICV leptin reduces hypothalamic Npy and Agrp mRNA content in fasted rats (19). Moreover, we report that, like leptin’s effect on food intake, its inhibitory effect on the expression of these orexigenic neuropeptides can be blocked by ICV pretreatment with the PI3K inhibitor LY. These observations support a model in which leptin-induced activation of PI3K signaling is required for leptin-dependent inhibition of Npy and Agrp expression. Whether the PI3K-dependent inhibition of NPY/AgRP neurons by leptin reflects a direct mechanism was not investigated in our studies, and it is alternatively possible that leptin activates PI3K in distinct neuronal subsets that project to NPY/AgRP neurons, thereby regulating them via an indirect mechanism. Indeed, recent work by Xu et al. (32) indicates that leptin-dependent regulation of PI3K signaling within NPY/AgRP neurons involves an indirect pathway that requires synaptic transmission.

Neuronal leptin action also involves signal transduction via the transcription factor STAT3. For example, STAT3 is implicated in leptin regulation of Pomc gene expression (6, 22) and is critical for hypothalamic control of energy homeostasis (3, 11). On the basis of published work suggesting that leptin-mediated activation of STAT3 involves PI3K signaling (33), pretreatment with a PI3K inhibitor could potentially inhibit leptin activation of STAT3, with reduced STAT3 signaling contributing to leptin’s inability to inhibit hypothalamic Npy and Agrp expression following pretreatment with a PI3K inhibitor. To investigate this hypothesis, we employed two different strategies to determine whether inhibition of PI3K signaling affects leptin-induced STAT3 activation within the hypothalamus. The first of these involved determination of hypothalamic Socs3 mRNA expression, a measure of STAT3 transcriptional activity. Although the time course over which leptin induces Socs3 gene expression differs substantially from leptin regulation of neuropeptide expression, we nonetheless found that leptin administration robustly increased hypothalamic Socs3 mRNA levels, which is indicative of leptin-mediated STAT3 activation (2, 9). Moreover, whereas ICV LY pretreatment blocked leptin inhibition of Npy and Agrp expression, this intervention had no effect on induction of Socs3 by leptin, suggesting that leptin-induced STAT3 transcriptional activity was not attenuated by pretreatment with a PI3K inhibitor. In addition, the effect of ICV LY pretreatment appears to have been relatively specific to leptin inhibition of Npy and Agrp expression, because LY pretreatment did not induce a generalized defect in leptin-mediated gene expression.

Because leptin-mediated induction of Socs3 expression occurs over a time course that is different from its effect on Npy and Agrp expression, we sought to more directly assess the hypothesis that leptin activation of hypothalamic STAT3 signaling is independent of PI3K. To accomplish this goal, we performed a second experiment, in which immunohistochemical staining was used to determine whether leptin induction of pSTAT3, which occurs in ARC neurons within minutes of leptin administration, is sensitive to pretreatment with ICV LY. The use of immunohistochemistry to identify and quantify pSTAT3-positive neurons has been previously validated as a measure of hypothalamic leptin action (12, 15, 21), and, relative to biochemical techniques such as Western immunoblotting, has the added advantage of localizing the leptin effect to specific brain areas. This anatomical specificity allowed us to detect a fivefold increase in STAT3 phosphorylation specifically within the ARC following leptin administration, despite using a relatively small sample size. As predicted, we found that pretreatment with a PI3K inhibitor at a dose that effectively blocks leptin regulation of feeding (24) did not alter leptin induction of STAT3 phosphorylation within ARC neurons. These results collectively establish that ICV pretreatment with a PI3K inhibitor does not alter leptin activation of STAT3 signaling in the hypothalamus.

Because most pharmacological enzyme inhibitors are not absolutely specific for a particular enzyme, it is conceivable that the effects we observed after pretreatment with LY-294002 were due to inhibition of an enzyme other than PI3K. However, previous studies suggest that this is an unlikely possibility; we and others (23, 24) have shown that leptin and insulin activate PI3K signaling both in vivo and in vitro. In addition, the ability of either hormone to reduce food intake is acutely blocked by ICV pretreatment with either of two PI3K inhibitors, LY294002 or wortmannin, that block PI3K activity via distinct mechanisms (8). Moreover, ICV pretreatment with LY does not block anorexia induced by increased melanocortin signaling, an effect mediated by protein kinase A rather than PI3K. Considering the evidence that LY does not impair leptin activation of the JAK/STAT pathway, we favor the conclusion that our findings using this drug are attributable to PI3K inhibition.

The observation that leptin is incapable of inhibiting Npy and Agrp in the presence of a PI3K inhibitor, despite intact STAT3 signaling, suggests that STAT3 activation alone is insufficient to mediate leptin-dependent inhibition of NPY/AgRP neurons. We emphasize that this interpretation does not imply that STAT3 is unnecessary for leptin inhibition of Npy and Agrp gene expression, only that it is insufficient to mediate this leptin effect in the absence of PI3K signaling. This interpretation is consistent with studies using mice, in which both leptin receptor alleles are replaced by a mutant allele that is incapable of activating STAT3 (3). Despite the absence of leptin-induced STAT3 signaling in these mice, Npy and Agrp gene expression remained relatively intact, especially compared with the marked increase of these hypothalamic neuropeptides seen in db/db mice that lack all signaling via the long form of the leptin receptor (3). A STAT3-independent mechanism is therefore implicated in the inhibitory effect of leptin on hypothalamic NPY/AgRP neurons, and our current findings suggest that this mechanism involves PI3K signaling.

In addition to its effects on hypothalamic neuropeptide gene expression, leptin also exerts rapid and potent effects on the membrane potential and firing rate of neurons in the ARC (7, 30), and these effects impinge upon downstream neurons within areas such as the hypothalamic PVN. Because NPY/AgRP and POMC neurons innervate the PVN, leptin-induced activation of PVN neurons (10, 25) is thought to arise, at least in part, as a consequence of leptin signaling within ARC neurons (4, 27, 29). We therefore reasoned that if PI3K signaling is required for leptin inhibition of NPY/AgRP neurons, as suggested by our data, then local inhibition of PI3K may also attenuate the ability of leptin to activate downstream neurons in the PVN. In support of this hypothesis, we demonstrated that the ability of ICV leptin to increase the number of c-Fos positive neurons in the PVN (10, 29) was significantly attenuated by ICV pretreatment with LY. However, unlike the effects of LY pretreatment on NPY and AgRP expression, LY pretreatment only inhibited leptin induction of c-Fos within the PVN by 32%. This discrepancy is not surprising because regulation of Npy and Agrp gene expression addresses an effect of leptin on a single subset of neurons, whereas the activation of PVN neurons involves a heterogenous cell population. On the basis of evidence that leptin-dependent activation of the PVN is mediated, in part, by leptin signaling in the ARC (4, 27, 29), it is tempting to speculate that our findings reflect a PI3K-dependent action of leptin on a subpopulation of neurons that project to the PVN (e.g., NPY/AgRP neurons). However, it is alternatively possible that leptin directly activates a subpopulation of PVN neurons via a PI3K-dependent mechanism or that leptin-sensitive neurons residing outside the ARC also impinge on the PVN and are inhibited by LY. In summary, these data indicate that leptin activation of a subgroup of PVN neurons is sensitive to PI3K inhibition, although the majority of PVN neurons are activiated by leptin via a mechanism that is PI3K-independent.

Intact leptin signaling is a prerequisite for maintenance of normal body weight, and growing evidence suggests that hypothalamic leptin resistance is a feature of common forms of obesity. Delineating the signal transduction mechanisms that mediate the various actions of leptin in the hypothalamus is therefore critical for an improved understanding of both the pathogenesis and consequences of leptin resistance. Here we report that both inhibition of hypothalamic Npy and Agrp gene expression and activation of PVN neurons by leptin requires intact PI3K signaling, a finding that provides mechanistic insight into the necessity for PI3K signaling in leptin’s anorectic effects (24). Because leptin induction of hypothalamic STAT3 signaling remains intact after ICV pretreatment with a PI3K inhibitor, we conclude that leptin inhibition of hypothalamic Npy and Agrp gene expression requires intact PI3K signaling and that an increase of STAT3 signaling is insufficient to inhibit the expression of these orexigenic neuropeptides in the absence of a PI3K signal.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
C. D. Morrison was supported by National Institute of Diabetes and Digestive and Kidney Diseases Training Grant T32 DK-07247. Further support was provided by National Institute of Neurological Disorders and Stroke Grant NS-32273 and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-68384 and DK-52989 to M. W. Schwartz by the Diabetes and Endocrinology Research Center and Clinical Nutrition Research Center of the University of Washington, and by a mentor-based fellowship award by the American Diabetes Association.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the skilled technical efforts of H. Nguyen, T. Huon, and L. Nguyen.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Michael Schwartz, Harborview Medical Center, Box 359757, 325 Ninth Ave., Seattle, WA 98108 (e-mail: mschwart{at}u.washington.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Banks AS, Davis SM, Bates SH, and Myers MG Jr. Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275: 14563–14572, 2000.[Abstract/Free Full Text]
  2. Baskin DG, Breininger JF, and Schwartz MW. SOCS-3 expression in leptin-sensitive neurons of the hypothalamus of fed and fasted rats. Regul Pept 92: 9–15, 2000.[CrossRef][ISI][Medline]
  3. Bates SH, Stearns WH, Dundon TA, Schubert M, Tso AW, Wang Y, Banks AS, Lavery HJ, Haq AK, Maratos-Flier E, Neel BG, Schwartz MW, and Myers MG Jr. STAT3 signaling is required for leptin regulation of energy balance but not reproduction. Nature 421: 856–859, 2003.[CrossRef][ISI][Medline]
  4. Bell ME, Bhatnagar S, Akana SF, Choi S, and Dallman MF. Disruption of arcuate/paraventricular nucleus connections changes body energy balance and response to acute stress. J Neurosci 20: 6707–6713, 2000.[Abstract/Free Full Text]
  5. Bi S, Robinson BM, and Moran TH. Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression. Am J Physiol Regul Integr Comp Physiol 285: R1030–R1036, 2003.[Abstract/Free Full Text]
  6. Bjorbaek C, Uotani S, da Silva B, and Flier JS. Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 272: 32686–32695, 1997.[Abstract/Free Full Text]
  7. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, Cone RD, and Low MJ. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411: 480–484, 2001.[CrossRef][ISI][Medline]
  8. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][ISI][Medline]
  9. Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C, Flier JS, Saper CB, and Elmquist JK. Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 23: 775–786, 1999.[CrossRef][ISI][Medline]
  10. Elias CF, Kelly JF, Lee CE, Ahima RS, Drucker DJ, Saper CB, and Elmquist JK. Chemical characterization of leptin-activated neurons in the rat brain. J Comp Neurol 423: 261–281, 2000.[CrossRef][ISI][Medline]
  11. Gao Q, Wolfgang MJ, Neschen S, Morino K, Horvath TL, Shulman GI, and Fu XY. Disruption of neural signal transducer and activator of transcription 3 causes obesity, diabetes, infertility, and thermal dysregulation. Proc Natl Acad Sci USA 101: 4661–4666, 2004.[Abstract/Free Full Text]
  12. Huo L, Munzberg H, Nillni EA, and Bjorbaek C. Role of signal transducer and activator of transcription 3 in regulation of hypothalamic trh gene expression by leptin. Endocrinology 145: 2516–2523, 2004.[Abstract/Free Full Text]
  13. Kellerer M, Koch M, Metzinger E, Mushack J, Capp E, and Haring HU. Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways. Diabetologia 40: 1358–1362, 1997.[CrossRef][ISI][Medline]
  14. Korner J, Wardlaw SL, Liu SM, Conwell IM, Leibel RL, and Chua SC Jr. Effects of leptin receptor mutation on AgRP gene expression in fed and fasted lean and obese (LA/N-faf) rats. Endocrinology 141: 2465–2471, 2000.[Abstract/Free Full Text]
  15. Levin BE, Dunn-Meynell AA, and Banks WA. Obesity-prone rats have normal blood-brain barrier transport but defective central leptin signaling before obesity onset. Am J Physiol Regul Integr Comp Physiol 286: R143–R150, 2004.[Abstract/Free Full Text]
  16. McMinn JE, Wilkinson CW, Havel PJ, Woods SC, and Schwartz MW. Effect of intracerebroventricular {alpha}-MSH on food intake, adiposity, c-Fos induction, and neuropeptide expression. Am J Physiol Regul Integr Comp Physiol 279: R695–R703, 2000.[Abstract/Free Full Text]
  17. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Morgan PJ, and Trayhurn P. Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 8: 733–735, 1996.[CrossRef][ISI][Medline]
  18. Mirshamsi S, Laidlaw HA, Ning K, Anderson E, Burgess LA, Gray A, Sutherland C, and Ashford ML. Leptin and insulin stimulation of signaling pathways in arcuate nucleus neurones: PI3K dependent actin reorganization and KATP channel activation. BMC Neurosci 5: 54, 2004.[CrossRef][Medline]
  19. Mizuno TM and Mobbs CV. Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology 140: 814–817, 1999.[Abstract/Free Full Text]
  20. Morton GJ, Niswender KD, Rhodes CJ, Myers MG Jr, Blevins JE, Baskin DG, and Schwartz MW. Arcuate nucleus-specific leptin receptor gene therapy attenuates the obesity phenotype of Koletsky (fa(k)/fa(k)) rats. Endocrinology 144: 2016–2024, 2003.[Abstract/Free Full Text]
  21. Munzberg H, Flier JS, and Bjorbaek C. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology 145: 4880–4889, 2004.[Abstract/Free Full Text]
  22. Munzberg H, Huo L, Nillni EA, Hollenberg AN, and Bjorbaek C. Role of signal transducer and activator of transcription 3 in regulation of hypothalamic proopiomelanocortin gene expression by leptin. Endocrinology 144: 2121–2131, 2003.[Abstract/Free Full Text]
  23. Niswender KD, Morrison CD, Clegg DJ, Olson R, Baskin DG, Myers MG Jr, Seeley RJ, and Schwartz MW. Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus: a key mediator of insulin-induced anorexia. Diabetes 52: 227–231, 2003.[Abstract/Free Full Text]
  24. Niswender KD, Morton GJ, Stearns WH, Rhodes CJ, Myers MG Jr, and Schwartz MW. Intracellular signaling. Key enzyme in leptin-induced anorexia. Nature 413: 794–795, 2001.[CrossRef]
  25. Powis JE, Bains JS, and Ferguson AV. Leptin depolarizes rat hypothalamic paraventricular nucleus neurons. Am J Physiol Regul Integr Comp Physiol 274: R1468–R1472, 1998.[Abstract/Free Full Text]
  26. Rahmouni K, Haynes WG, Morgan DA, and Mark AL. Intracellular mechanisms involved in leptin regulation of sympathetic outflow. Hypertension 41: 763–767, 2003.[Abstract/Free Full Text]
  27. Sarkar S and Lechan RM. Central administration of neuropeptide Y reduces alpha-melanocyte-stimulating hormone-induced cyclic adenosine 5'-monophosphate response element binding protein (CREB) phosphorylation in pro-thyrotropin-releasing hormone neurons and increases CREB phosphorylation in corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology 144: 281–291, 2003.[Abstract/Free Full Text]
  28. Schwartz MW, Seeley RJ, Campfield LA, Burn P, and Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98: 1101–1106, 1996.[Abstract/Free Full Text]
  29. Seeley RJ, Yagaloff KA, Fisher SL, Burn P, Thiele TE, van Dijk G, Baskin DG, and Schwartz MW. Melanocortin receptors in leptin effects. Nature 390: 349, 1997.[CrossRef][ISI][Medline]
  30. Spanswick D, Smith MA, Groppi VE, Logan SD, and Ashford ML. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 390: 521–525, 1997.[CrossRef][ISI][Medline]
  31. Tartaglia LA. The leptin receptor. J Biol Chem 272: 6093–6096, 1997.[Free Full Text]
  32. Xu AW, Kaelin CB, Takeda K, Akira S, Schwartz MW, and Barsh GS. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J Clin Invest 115: 951–958, 2005.[Abstract/Free Full Text]
  33. Zhao AZ, Huan JN, Gupta S, Pal R, and Sahu A. A phosphatidylinositol 3-kinase phosphodiesterase 3B-cyclic AMP pathway in hypothalamic action of leptin on feeding. Nat Neurosci 5: 727–728, 2002.[ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/6/E1051    most recent
00094.2005v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Morrison, C. D.
Articles by Schwartz, M. W.
PubMed
PubMed Citation
Articles by Morrison, C. D.
Articles by Schwartz, M. W.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.