Ghrelin Induces Leptin Resistance by Activation of Suppressor of Cytokine Signaling 3 Expression in Male Rats: Implications in Satiety Regulation

NEUROENDOCRINOLOGY Ghrelin Induces Leptin Resistance by Activation of Suppressor of Cytokine Signaling 3 Expression in Male Rats: Implications in Satiety Regulation Andrea Heldsinger, Gintautas Grabauskas, Xiaoyin Wu, ShiYi Zhou, Yuanxu Lu, Il Song, and Chung Owyang Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48019 The anorexigenic adipocyte-derived hormone leptin and the orexigenic hormone ghrelin act in opposition to regulate feeding behavior via the vagal afferent pathways. The mechanisms by which ghrelin exerts its inhibitory effects on leptin are unknown. We hypothesized that ghrelin activates the exchange protein activated by cAMP (Epac), inducing increased SOCS3 expression, which negatively affects leptin signal transduction and neuronal firing in nodose ganglia (NG) neurons. We showed that 91 ⫾ 3% of leptin receptor (LRb) – bearing neurons contained ghrelin receptors (GHS-R1a) and that ghrelin significantly inhibited leptin-stimulated STAT3 phosphorylation in rat NG neurons. Studies of the signaling cascades used by ghrelin showed that ghrelin caused a significant increase in Epac and suppressor of cytokine signaling 3 (SOCS3) expression in cultured rat NG neurons. Transient transfection of cultured NG neurons to silence SOCS3 and Epac genes reversed the inhibitory effects of ghrelin on leptin-stimulated STAT3 phosphorylation. Patch-clamp studies and recordings of single neuronal discharges of vagal primary afferent neurons showed that ghrelin markedly inhibited leptin-stimulated neuronal firing, an action abolished by silencing SOCS3 expression in NG. Plasma ghrelin levels increased significantly during fasting. This was accompanied by enhanced SOCS3 expression in the NG and prevented by treatment with a ghrelin antagonist. Feeding studies showed that silencing SOCS3 expression in the NG reduced food intake evoked by endogenous leptin. We conclude that ghrelin exerts its inhibitory effects on leptinstimulated neuronal firing by increasing SOCS3 expression. The SOCS3 signaling pathway plays a pivotal role in ghrelin’s inhibitory effect on STAT3 phosphorylation, neuronal firing, and feeding behavior. (Endocrinology 155: 3956 –3969, 2014) G hrelin, a peptide primarily produced in the endocrine cells of the gastric mucosa, acts as an orexigenic hormone to stimulate feeding and growth hormone (GH) secretion by binding to the GH secretagogue receptor (GHS-R) (1–3). Conversely, leptin, an adipocyte-derived hormone, is a negative regulator of feeding and energy metabolism (4 – 6). Circulating leptin enters the brain by way of the blood-brain barrier (7) and exerts its effects by binding to the long form of the leptin receptor (LRb) (8). LRb receptors are expressed in several regions of the brain, mainly the arcuate nucleus, paraventricular nucleus, and the ventromedial, dorsomedial, and lateral hypothalamus (9). Ghrelin receptors are also expressed in various regions of the brain, including the hypothalamus and nonhypothalamus sites (6), indicating a central role for ghrelin (2). Research has shown that ghrelin and leptin act in opposition to regulate feeding behavior (2, 10). An intracerebroventricular injection of ghrelin blocks leptininduced inhibition of food intake (11). In GHS-R-null mice, ghrelin fails to affect the anorexigenic action of leptin, indicating that GHS-R activation directly mediates ghrelin activity (12). ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received November 27, 2013. Accepted July 17, 2014. First Published Online July 25, 2014 Abbreviations: Epac, exchange protein activated by cAMP; GAPDH, glyceraldehyde-3phosphate dehydrogenase; CCK, cholecystokinin; GHS-R, growth hormone secretagogue receptor; GHS-R1a, ghrelin receptor; JAK2, Janus activated kinase 2; LRb, leptin receptor; NG, nodose ganglia; NIH, National Institutes of Health; PKA, protein kinase A; siRNA, Small interfering RNA; SOCS3, suppressor of cytokine signaling 3. 3956 endo.endojournals.org Endocrinology, October 2014, 155(10):3956 –3969 doi: 10.1210/en.2013-2095 doi: 10.1210/en.2013-2095 In addition to the hypothalamus, LRb receptors are also expressed in vagal afferent neurons (13, 14). The binding of leptin to LRb receptors stimulates autophosphorylation of Janus activated kinase 2 (JAK2), which in turn activates STAT3 (14 –17). Similar to the LRb receptor, the functional ghrelin receptor GHS-R1a is synthesized in the nodose ganglia (NG) and transmitted to the vagal afferent terminals (18). Ghrelin secreted from the stomach may interact with GHS-R1a expressed in these terminals and the resulting signals may be transmitted to the hypothalamus by way of the nucleus of the solitary tract (19). Feeding studies suggest that ghrelin (2) and leptin (20) act by way of vagal afferent pathways to modulate satiety, whereas their receptors in the hypothalamus and other central nonhypothalamic sites are more likely to be involved in regulating long-term feeding behavior and energy metabolism (3, 9). The mechanisms by which ghrelin exerts inhibitory effects to regulate leptin’s action on short-term satiety are unclear. The reported intracellular signaling pathways for ghrelin are complex. Ghrelin has been shown to activate the adenylate cyclase-cAMP-protein kinase A (PKA) signaling pathways in endothelial and smooth muscle cells (21–23). Alternatively, ghrelin may also activate phospholipase C–PKC cascades in dopaminergic neurons (24). Other studies have shown that suppressor of cytokine signaling 3 (SOCS3) plays a pivotal role in the modulation of leptin signaling by inhibiting leptin-activated JAK2 and downstream STAT3 phosphorylation (25). In this manner, SOCS3 negatively regulates hypothalamic leptin signaling (26, 27) and plays an important role in leptin resistance (26). In hypothalamic propiomelanocortin neurons, overexpression of SOCS3 prevented an increase in leptin-stimulated insulin receptor substrate 1/PI3K activity (28) and phospho-STAT3 signaling (29). In contrast, neuronal SOCS3 deficiency enhanced hypothalamic leptin-dependent PI3K signaling (28). It is conceivable that ghrelin acts by way of SOCS3 pathways to induce leptin resistance. We hypothesize that, in the NG, ghrelin activates the exchange protein activated by cAMP (Epac) by way of a cAMP-dependent pathway, increasing SOCS3 expression, which negatively affects leptin signal transduction and neuronal firing. Using a multilayered approach that included Western blots, patch-clamp electrophysiological studies, and gene silencing techniques in cultured rat NG neurons, as well as in vivo single-cell electrical recordings and feeding studies, we show that ghrelin’s inhibitory actions on leptin-stimulated STAT3 phosphorylation and neuronal firing are mediated by way of an Epac–SOCS3 pathway, which contributes to leptin resistance and affects feeding behavior in rats. endo.endojournals. (...truncated)