Leptin modulation of peripheral controls of meal size

Physiology & Behavior 89 (2006) 511 – 516

Leptin modulation of peripheral controls of meal size Timothy H. Moran ⁎, Susan Aja, Ellen E. Ladenheim Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Ave., Baltimore, lMD 21205, United States Received 27 January 2006; received in revised form 17 April 2006; accepted 21 April 2006

Abstract Leptin reduces food intake through a specific effect on meal size. Investigations into how this within meal effect of leptin is mediated have demonstrated that leptin increases the ability of within meal inhibitory feedback signaling to limit intake and activate neurons within the nucleus of the solitary tract (NTS). Leptin's effects on neural activation can be demonstrated both as an increase in c-fos activation and as increase in electrophysiolgoical activity in response to peripheral stimuli. Leptin can exert these effects through interactions at hypothalamic sites and activation of a descending pathway. NPY has opposite effect suggesting a role for reduced NPY signaling in the actions of leptin. Forebrain ventricular administration of a melanocortin agonist does not mimic the actions of leptin. As well as modulating within meal signaling through a descending pathway leptin, NPY and melanocortins could work directly at hindbrain integrative sites suggesting the possibility of distributed controls of meal size by anorexigenic and orexigenic signaling. © 2006 Elsevier Inc. All rights reserved. Keywords: Leptin; NPY; Melanocortin; Nucleus of the solitary tract; Satiety

Our knowledge of the actions of central and peripheral peptides in food intake continues to expand. Even over the past few years, novel feeding actions of both peripheral and central peptide systems have been identified and our understanding of central pathways mediating the actions of peptide signals in energy balance has significantly increased. Recent work has suggested significant interactions between peripheral and brain peptide systems. For example, some gut peptides have been proposed to exert their actions on food intake by altering activity in hypothalamic peptide systems involved in energy balance. Conversely, leptin and its downstream hypothalamic mediators have been proposed to affect food intake by modifying neural responses to within meal peptide satiety signals. Although such interactions between gut and brain peptide systems have been proposed, the specific nature and overall extent of these interactions have yet to be identified. In this review, we focus on the identified actions of brain and gut feeding systems and, specifically, how modulation of the leptin

⁎ Corresponding author. Tel.: +1 410 955 2344; fax: +1 410 502 2769. E-mail address: [email protected] (T.H. Moran). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.04.020

signaling pathway alters the efficacy of within meal satiety signaling. The identification of leptin as the protein product of the ob gene by Friedman and colleagues in 1994 [1] provided the key to our present understanding of the organization of hypothalamic systems controlling energy balance. Leptin is released from adipose tissue in direct proportion to the overall fat mass, providing a signal of body energy stores [2]. Leptin receptors are widely distributed in the brain but a major site of leptin action appears to be the arcuate nucleus within the basal hypothalamus. The arcuate nucleus contains two distinct neural populations that directly respond to leptin [3,4]. The first is a population of neurons that expresses the orexigenic peptides neuropeptide Y (NPY) and agouti related peptide (AgRP). The second is a group of neurons that express the peptide precursor pro-opiomelanocortin (POMC). Leptin receptors are expressed in both of these neural populations. Leptin activates POMC containing neurons by both direct depolarization [5] and altering gene expression [4,6]. Leptin increases POMC expression leading to elevated levels of the anorexigenic peptide alpha-melanocyte stimulating hormone (α-MSH) [7]. In contrast, leptin inhibits NPY and AgRP mRNA expression

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[8] and hyperpolarizes these neurons thus reducing the release of these orexigenic stimuli [5]. Leptin inhibition of these neurons also reduces activity of a GABAergic inhibitory input onto POMC containing neurons [9]. These data suggest that elevated leptin levels at times of metabolic excess activate an anorexigenic pathway and reduce activity in orexigenic pathways. Low leptin levels, occurring at times of nutrient deficit, result in a reduction of inhibitory influences on NPY/AgRP neurons, a lack of activation of POMC containing neurons and an overall increase in orexigenic signaling. Arcuate NPY/AgRP and POMC containing neurons have direct projections to both the paraventricular nucleus (PVN) [10,11] and to the lateral hypothalamus [4], two nuclei involved in autonomic regulation. Lesions of the PVN result in hyperphagia and obesity [12] suggesting that this nucleus plays a major role in limiting food intake and body weight. The PVN contains multiple neuronal populations and is a major hypothalamic site with projections to the dorsal vagal complex [13], the brain site receiving neural input from the gastrointestinal tract. In contrast, LH lesions result in aphagia and profound weight loss suggesting an overall stimulatory role in food intake control [14]. The LH is the site for neurons containing two additional peptides that have been implicated in feeding control, orexin [15,16] and melanin concentrating hormone (MCH) [16]. Both of these peptides are orexigenic and have been proposed to be among the downstream targets of leptin signaling. Both of these LH substrates receive innervation from NPY/AgRP and POMC containing arcuate neurons [4]. NPY has been demonstrated to activate orexin and MCH neurons [17] and leptin has been demonstrated to inhibit orexin and MCH mRNA expression [17,18]. LH neurons directly innervate autonomic centers in the hindbrain including the dorsal vagal complex [19]. Shortly after its discovery, exogenous leptin was demonstrated to inhibit food intake [20]. However, the mode of action through which leptin inhibited feeding was not identified until 1998. Three groups of investigators reported that doses of peripheral and central leptin that inhibited food intake did so by reducing the size of spontaneous meals without affecting the number of meals consumed [21–23]. These data were consistent with earlier demonstrations that the hyperphagia that derived from various states of impaired leptin signaling was characterized by meal size specific alterations. Thus, the hyperphagia of ob/ob mice, lacking leptin, and Zucker rats, with deficits in leptin receptors, are both characterized by chronic increases in the size of spontaneous meals [24,25]. Leptin's action to reduce feeding through a specific action on meal size raised the possibility that leptin was affecting feeding by directly modulating how the brain responded to within meal feedback signaling. Such an action had previously been suggested for the feeding inhibitory effects of central insulin administration. Central insulin also reduces food intake and Reidy et al. [26] demonstrated that doses of insulin that by themselves were below threshold for inhibiting food intake increased the feeding inhibitory efficacy of a peripheral dose of the within meal satiety peptide cholecystokinin (CCK). They proposed that central insulin interacted with a CCK sensitive

pathway within the brain to produce this effect but left open where or how this interaction may occur. We and others investigated whether the feeding inhibitory actions of leptin may have similar effects on the efficacy of within meal satiety feedback signaling and focused on the dorsal hindbrain, the site at which such ascending signals are received by the brain, as a potential site of such interaction [27,28]. As shown in Fig. 1, consistent with the effects of insulin, leptin increased the efficacy with which peripheral CCK administration inhibited intake in short term tests and did so in a dose dependent fashion. Importantly, a similar potentiation could be demonstrated for the ability of CCK to activate neurons in the nucleus of the solitary tract (NTS) the hindbrain site that is the central termination for vagal afferent fibers. CCK has been demonstrated to affect food intake by activating vagal afferent fibers resulting in the activation of neurons in the NTS. Central leptin administration greatly enhanced the degree of NTS neural activation produced by CCK as measured by the induction of the immediate early gene c-Fos (Fig. 2). The ability of leptin to enhance the feeding inhibitory action of within meal feedback signaling is not limited to interactions with CCK. Gastric nutrients loads [29] and peripheral administration of the peptide bombesin [30] show similar interactions with leptin: leptin enhances the magnitude of feeding suppression produced by these treatments and results in greater c-fos activation with the NTS than the treatments alone.

Fig. 1. Leptin modulates the inhibition of intake by CCK. A: Intake in response to CCK and leptin given alone or in combination. ⁎ denotes significant difference from veh/veh. δ denotes significant difference from CCK/veh. B: Dose related effect of leptin on CCK induced feeding inhibition. ⁎ denotes significant difference from 0 dose. ⁎⁎ denotes significant difference from 3.5 μg dose (adapted from Emond et al., 1999 [28]).

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Fig. 2. Leptin modulates CCK induced c-fos activation. ⁎ denotes significant difference from CCK alone (adapted from Emond et al., 1999 [28]).

Leptin enhancement of NTS neuronal activation induced by the gastrointestinal nutrient factors can be demonstrated not only as a greater number of cells expressing c-Fos but also as increased activation of individual neurons. Using an electrophysiological approach, we identified single NTS neurons that were responsive to gastric balloon inflation. We assessed the change in neuronal activity produced by a range of intragastric balloon volumes both prior to and following icv leptin administration. As shown in Fig. 3, leptin increased the sensitivity of individual NTS neurons to gastric stimulation. The rate of firing in response to a 4 ml gastric load was significantly greater following than before leptin administration. This leptin induced response enhancement was found at all gastric volumes. The effect appeared to occur at the level of the individual NTS neuron since the vagal afferent response to gastric distention was not affected by the leptin administration. These data strongly suggest that leptin inhibits intake by sensitizing NTS neurons to inhibitory gastrointestinal signals [31]. The next questions were how and where this action of leptin was mediated. There are multiple possibilities. We had suggested that the effects of leptin on CCK satiety were mediated through hypothalamic sites and the excitation of descending pathways that then modified the responsivity of

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Fig. 4. NPY modulation of CCK induced NTS c-fos activation. ⁎ denotes significant difference from sal/sal. δ denotes significant difference from sal/CCK.

NTS neurons [32]. A hypothalamic site of action for leptin in modulating meal size is supported by recent work from Morton and colleagues [33]. They took advantage of the genetic defect in Koletsky rats that lack the leptin receptor and develop hyperphagia and severe obesity. They demonstrated that such rats had increased meal size and reduced response to exogenous CCK administration. Using an adenovirus expressing leptin receptors, they restored leptin signaling specifically to the hypothalamic arcuate nucleus. Restoration of arcuate leptin signaling reduced meal size and normalized the satiety response to exogenous CCK. Consistent with prior work demonstrating leptin/CCK interactions in NTS activation, rats with restored arcuate leptin signaling also had normalized NTS c-fos activation in response to CCK administration. These data demonstrate that leptin activation of the hypothalamic arcuate nucleus is sufficient for leptin mediated effects on meals size and modulation of within meal satiety signaling. As discussed above, leptin's actions in the arcuate nucleus include the activation of POMC containing neurons and the inhibition of NPY/AgRP containing neurons. Thus, reduced NPY or increased melanocortin signaling are candidates for mediating the meal related actions of leptin. McMinn and colleagues [34] assessed the potential for NPY mediation in

Fig. 3. Leptin increases gastric load induced activation of NTS neurons. Activity of single NTS load responsive neuron in response to a 4 ml intragastric saline load before (top tracing) and 60 min following (bottom tracing) icv leptin administration. Load infusion begins at the start of the line above the tracing and reaches the 4 ml volume at the arrow (adapted from Schwartz and Moran, 2002).

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Fig. 5. NPY decreases gastric load induced activation of NTS neurons. Activity of single NTS load responsive neuron in response to a 4 ml intragastric saline load before (top tracing) and 60 min following (bottom tracing) icv NPY administration. Load infusion begins at the start of the line above the tracing and reaches the 4 ml volume at the arrow (adapted from Schwartz and Moran, 2002).

these effects. They began by demonstrating that CCK satiety was blunted by fasting and this effect was reversed by leptin administration. They then assessed whether increased NPY could mediate the effect of fasting on CCK satiety. They demonstrated that NPY administered into the 3rd cerebral ventricle reduced the ability of a dose of CCK to inhibit food intake. Although interpretation of this result alone is complicated given that the two treatments have opposing effects on food intake [35], an examination of the effect of NPY on CCK induced NTS c-fos induction provided support for a potential role for increased NPY signaling in the effect of fasting. NPY reduced the amount of c-fos activation produced by peripheral CCK. We have produced similar results. As shown in Fig. 4, a low lateral ventricle dose of NPY that by itself did not induce NTS c-fos, reduced the degree of activation produced by peripheral CCK, and a higher dose which did induce NTS c-fos by itself still resulted in less CCK induced c-fos induction. It seems likely that NPY and CCK are activating different neuronal populations in these experiments and that NPY activates some cells while reducing the responsivity of others. Consistent with these data, we also found in electrophysiolog-

Fig. 6. MTII fails to modulate CCK-induced inhibition of food intake. Both CCK and MTII independently decrease intake. MTII does not increase the suppression produced by CCK. ⁎ denotes decrease relative to baseline level.

ical experiments that icv NPY inhibited the ability of gastric volumes to excite NTS neurons (Fig. 5) [31]. The other arcuate target for leptin is POMC neurons, resulting in increased melanocortin signaling. We investigated whether the lateral ventricle administration of the melanocortin agonist MTII mimicked the actions of leptin in modulating the behavioral and neural activational response to CCK. As shown in Fig. 6, although both MTII and CCK inhibited feeding, there was no positive interaction in combining peripheral CCK with central MTII. Similar results were found in the examinations of NTS c-fos activation (Fig. 7). At a dose of MTII that by itself did not induce c-fos action, there was no positive modulation of CCK-induced NTS c-fos. At a higher dose of MTII that alone did induce c-fos activation, there was not only no positive interaction between CCK and MTII, there was even a tendency for negative modulation. Again there was no positive interaction. These data are consistent with the findings of Azzara et al. [36] that intraventricular MTII failed to augment reductions in food intake produced by intraduodenal nutrient infusion. Together these results suggest that alterations in melanocortin signaling do not mediate the effect of leptin on within meal satiety signaling. However, recent results from Berthoud and colleagues suggest an alternate interpretation.

Fig. 7. MTII fails to modulate CCK induced NTS c-fos activation. ⁎ denotes significant difference from sal/sal.

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Rather than using forebrain ventricular administration, they examined the effects of 4th ventricular MTII and the melanocortin antagonist SHU9119 on meal patterns and the actions of CCK to induce NTS neuronal activation as measured by phosphorylation of ERK1/2. MTII decreased and SHU9119 increased food intake and both did so by altering meal size without affecting meal frequency [37]. Fourth ventricular administration of MTII dose dependently increased ERK1/2 phosphorylation and the effects of CCK/MTII combinations were additive [38]. Fourth ventricular administration of SHU9119 abolished the ability CCK to induce phosphorylation. These data suggest that specifically altering hindbrain melanocortin signaling does modulate the mediation of within meal satiety signaling and further suggest that the negative results with forebrain MTII administration may be due to activation of multiple pathways. The source of melanocortins for such hindbrain modulation is unclear. There are again multiple possibilities. The hindbrain does contain POMC expressing neurons providing the basis for a local action [39]. However, there is evidence for direct projections from arcuate POMC neurons to the NTS [37], providing a basis for mediation by a descending pathway and a role for melanocortin signaling in mediating the actions of forebrain leptin. Further support for leptin activating a descending pathway to modulate meal size comes from experiments examining the actions of paraventricular (PVN) oxytocin containing neurons. Third ventricular leptin has been demonstrated to induce c-fos in PVN oxytocin neurons that project to the NTS and central administration of an oxytocin antagonist attenuates the effect of leptin to reduce food intake and to modulate CCK induced NTS c-fos [40]. These findings have been interpreted to suggest that leptin's action in modulating within meal satiety signaling depend upon the activation of PVN oxytocin neurons and the activation of oxytocin receptors in the NTS. Other PVN neurons with different peptide phenotypes may also play a role in such modulation. Although such data strongly support a descending hypothalamic to hindbrain pathway in mediating the actions of leptin, there is also the possibility that leptin exerts direct effects at an NTS site of action. NTS neurons have been shown to contain leptin receptors and fourth ventricular or direct NTS leptin injections inhibit food intake [41]. Whether this effect is specific to meal size has yet to be assessed. Similarly, hindbrain injections of NPY increase food intake [42] providing the basis for both positive and negative modulation of within meal feedback signaling directly at this hindbrain site. Such distributed actions for peptides in feeding control have been suggested [43]. The caudal hindbrain does contain the sufficient neural substrates for responding to within meal satiety signaling. Decerebrate rats decrease food intake in response to CCK [44] and fail to show normal satiety in a sham feeding paradigm [45]. Leptin and NPY are only a subset of the peptides that can affect feeding at hindbrain sites. Interactions between forebrain and hindbrain feeding systems are multiple and the coordinated control evident in spontaneous meals reflects such multiple interactions. Although our understanding of how leptin can affect meal size has significantly advanced since the first

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