A phosphatidylinositol 3-kinase–phosphodiesterase3B–cyclic AMP pathway inhypothalamic action ofleptin on feedingAllan Z. Zhao, Jjing-Ning Huan, Sandhya Gupta, Rekha Pal and Abhiram Sahu

Department of Cell Biology and Physiology, University of Pittsburgh School ofMedicine, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA

Correspondence should be addressed to A.S. (asahu@pitt.edu)

Published online: 8 July 2002, doi:10.1038/nn885

Using male Sprague-Dawley rats implanted with third intracere-broventricular (ICV) cannulae, we found that cilostamide, a phos-phodiesterase 3 (PDE3) inhibitor, (i) reversed the establishedeffects of leptin on food intake and body weight, (ii) blocked, atthe hypothalamic level, the leptin-induced tyrosine phosphoryla-tion of signal transducer and activator of transcription 3 (Stat3)and (iii) blocked the DNA binding of p-Stat3. Additionally, ICVadministration of leptin increased hypothalamic phosphatidyli-nositol 3-kinase (PI3K) and PDE3B activities and decreased cyclicAMP (cAMP) concentration. These results indicate that aPI3K–PDE3B–cAMP pathway interacting with the Janus kinase 2(Jak2)–Stat3 pathway constitutes a critical component of leptinsignaling in the hypothalamus.

Leptin, an adipocyte-derived circulating hormone, is centrallyinvolved in energy homeostasis through its actions in the hypo-thalamus1,2, but the intracellular mechanisms underlying thisenergy regulation are not clearly understood. Activation of theJak–Stat pathway has been identified as one of the mechanismsof leptin signaling in the hypothalamus1. Studies in peripheraltissues3, however, have demonstrated an alternative leptin sig-naling pathway involving PI3K-dependent activation of PDE3Band reduction of cAMP. As PDE3B is localized in various brainregions, including the hypothalamus4 (A.S., unpub. data), andcentral injection of cAMP analogs or agents that increase endoge-nous cAMP induces feeding in satiated rats5, we tested thehypothesis that the PDE3B–cAMP pathway mediates leptin actionin the hypothalamus.

To investigate whether leptin’s inhibitory effect on feedingis mediated by an induction of PDE3 activity, we usedcilostamide (a specific PDE3 inhibitor)6 to block hypothalam-ic PDE3 activity. ICV injection of cilostamide blocked theinhibitory effect of leptin on food intake (Fig. 1a and b). Incontrast, RO-20-1724 (a specific PDE4 inhibitor)6 did notmodify this effect of leptin (Fig. 1c and d). In addition,cilostamide reversed the effect of daily administration of leptinon body weight (Fig. 2). Moreover, within 45 minutes of ICVleptin (4 µg) injection to rats that had fasted for 24 hours,hypothalamic PDE3B activity was increased by ∼ 3.3 times (con-trol, 84 ± 23 pmol/min/unit (mean ± s.e.m.), n = 5; leptin, 282± 41 pmol/min/unit, n = 6; P = 0.01) and cAMP concentrationwas reduced by ∼ 22% (control, 100 ± 8.7 relative %; leptin, 78± 3.9 relative %; n = 3 for each group; P = 0.03).

In various non-neuronal tissues, PI3K, in association with theinsulin receptor substrate IRS1/2, is an upstream regulator ofPDE3B3,7,8. We therefore examined the effect of ICV leptin onhypothalamic PI3K in fasted rats. Leptin stimulated the activa-tion of IRS1-associated PI3K by ∼ 3.7 times (leptin, 372 ± 50 rel-ative %; control, 100 ± 31 relative %; n = 3 for each group, P =0.009; Supplementary Fig. 1 online). This suggests that a stim-ulation of PI3K activity by leptin in rat hypothalamus is thebeginning of a signaling cascade that regulates cAMP.

Leptin induces Jak2 and Stat3 phosphorylation, and stimu-lates DNA binding activity of Stat3 in the hypothalamus1, andthus it is conceivable that the PDE3B–cAMP pathway mightinteract with the Jak2-Stat3 pathway in this brain region. Totest this, we examined the effects of PDE3 inhibition bycilostamide on leptin-induced Stat3 phosphorylation in thehypothalamus. While ICV leptin administration stimulated thetyrosyl-phosphorylation on Stat3 proteins as expected, priorICV treatment with cilostamide eliminated this effect (Fig. 3a).In addition, cilostamide attenuated the effect of leptin on theDNA binding activity of p-Stat3 in the hypothalamus (Fig. 3b).These findings suggest that the stimulation of PDE3B activityis a prerequisite step to lower the local cAMP threshold so thatthe Jak–Stat pathway can be activated.

We suggest that activation of PDE3B and the resultantdecrease in cAMP is one of the primary mechanisms of leptinsignaling in the hypothalamus. As PI3K is an upstream regula-tor of PDE3B3,7,8, an increase in both PI3K and PDE3B follow-ing leptin administration suggests that PI3K may be an upstreamregulator of PDE3B during leptin signaling in the hypothalamus.

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Fig. 1. Cilostamide, a PDE3 inhibitor, reverses the satiety action of lep-tin in male rats that are fed ad libitum, but RO-20-1724, a PDE4 inhibitor,does not. Cilostamide and RO-20-1724 were from BIOMOL ResearchLaboratories (Plymouth Meeting, Pennsylvania). Rats were first injectedICV into the third ventricle with artificial cerebrospinal fluid (aCSF) or 4 µg of leptin (provided by A. F. Parlow) and dimethyl sulfoxide (DMSO)or one of the PDE inhibitors; then were injected 1 h later with DMSOor the inhibitors. The cumulative food intakes were measured for 4 and24 h after the second injection. (a) 10 µg cilostamide, (b) 2 µgcilostamide, (c) 10 µg RO-20-1724 (RO-20), (d) 50 µg RO-20. *P < 0.05,relative to others except the groups with * or ** (one-way ANOVA andNewman-Keuls multiple test). The Institutional Animal Care and UseCommittee of the University of Pittsburgh approved all experiments.

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Central administration of a cAMP analog, or agents that increaseendogenous cAMP, stimulates feeding5, and central administra-tion of a cAMP analog induces gene expression of hypothalamicneuropeptide Y (NPY)9, a potent endogenous orexigenic (foodintake stimulating) signal10. Therefore, it is very likely that adecrease in hypothalamic cAMP by leptin initiates a chain ofevents that result in decreased food intake. Consistent with thisnotion are findings showing (i) an increased phosphorylation ofcAMP response element (CRE)–binding protein (CREB) andCRE-binding activity in the hypothalamus4,11 of fasted animalswith decreased circulating leptin levels12 and (ii) an alteration inCRE-mediated gene expression including that of NPY neuronsin the hypothalamic arcuate nucleus by leptin4. In addition, lep-tin not only modifies the gene expression of various hypothalamicpeptides, including NPY, but it also modifies the action of thesepeptides after being secreted13,14. NPY’s action on feeding is medi-ated by the cAMP–CREB pathway11, and central administration

of a cAMP analog increases NPY expression in the hypothala-mus9; it will be interesting to investigate whether the reductionof cAMP levels in the hypothalamus by leptin is responsible formodifying NPY gene expression and NPY’s action on feeding.

It is also plausible that cilostamide affects a different class ofneurons which, in turn, modulate the activity of leptin-sensitiveneurons. We do not yet know which cells in particular arecilostamide-responsive, and we do not know which cells showaltered cAMP levels in response to leptin: the cellular site of actionof cilostamide remains to be determined. Our observation thatinhibition of PDE3 by cilostamide reversed the effect of leptin onStat3 leads us to propose that a direct and/or indirect interactionbetween the PDE3B–cAMP and Jak2–Stat3 signaling pathways iscritical in transducing leptin action in the hypothalamus. In sum,we have identified the PI3K–PDE3B–cAMP pathway as a novelmechanism of leptin signaling in the hypothalamus for the reg-ulation of feeding. We speculate that this signaling mechanismmay mediate leptin’s action in the hypothalamus in general. Afurther understanding of this signal transduction pathway wouldtherefore be critical to unraveling the molecular mechanism ofhypothalamic leptin action in normal states and during the devel-opment of leptin resistance seen in obesity and related disorders.

Note: Supplementary information is available on the Nature Neuroscience website.

AcknowledgmentsThis work was supported by a National Institutes of Health grant DK 52844 (to

A.S.) and a Career & Development Award from the American Diabetes

Association (to A.Z.).

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 9 APRIL; ACCEPTED 4 JUNE 2002

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Nature 404, 661–671 (2000).3. Zhao, A. Z. et al. J. Biol. Chem. 275, 11348–11354 (2000).4. Shimizu-Albergine, M., Ippolito, D. L. & Beavo, J. A. J. Neurosci. 21,

1238–1246 (2001).5. Gillard, E. R. et al. J. Neurosci. 18, 2646–2652 (1998).6. Beavo, J. A. & Reifsnyder, D. H. Trends Pharmacol. Sci. 11, 150–155 (1990).7. Rahn, T. et al. FEBS Lett. 350, 314–318 (1994).8. Ahmad, M., Flatt, P. R., Furman, B. L. & Pyne, N. J. Cell. Signal. 12, 541–548

(2000).9. Akabayashi, A. et al. Brain Res. 660, 323–328 (1994).10. Sahu, A. & Kalra, S. P. Trends in Endocr. Metab. 4, 217–224 (1993).11. Sheriff, S., Chance, W. T., Fischer, J. E. & Balasubramaniam, A. Mol.

Pharmacol. 51, 597–604 (1997).12. Ahima, R. S., Saper, C. B., Flier, J. S. & Elmquist, J. K. Front. Neuroendocrinol.

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brief communications

Fig. 3. Cilostamide reverses the effect of leptin on Stat3 activation in thehypothalamus. Fasted (24 h) rats were injected ICV with DMSO orcilostamide (10 µg) followed 30 min later by leptin (4 µg) or aCSF. (a) Top,western blot of Stat3 and p-Stat3 in the mediobasal hypothalamic (MBH)extracts. Bottom, densitometric analysis of the immunoreactive bands forp-Stat3 and expressed as relative (%) to vehicle group (DMSO + aCSF). (b) Top, DNA binding activity of Stat3 in the MBH (in a separate study) asdetermined by an electrophoretic mobility shift assay using a 32P-labeledM67-SIE oligonucleotide probe. Top right, DNA binding activity is specificto p-Stat3 because a ‘supershift’ did not occur in the presence of anti-Socs3 (suppressor of cytokine signaling 3) antibody. Bottom, resultsobtained by phosphor imaging and expressed as relative (%) to vehicle. *P < 0.05 as compared to all other groups.

Fig. 2. Cilostamide reversed the effect of leptin on body weight. Oneach day, the rats were injected ICV with cilostamide (10 µg/µl DMSO)or leptin (4 µg/2 µl saline), alone or in combination. This was followed1 h later by an injection of DMSO or cilostamide. This protocol wasrepeated daily for three days. *P < 0.05 relative to other groups.

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