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
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ABSTRACT |
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suppressor of cytokine signaling-3; signal transducer and activator of transcription 3
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.
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MATERIALS AND METHODS |
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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 = 23/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 = 67/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 manufacturers 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 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.
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RESULTS |
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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.
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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.
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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.
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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.
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DISCUSSION |
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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 leptins 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 leptins 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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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