Hypothalamic paraventricular 5-hydroxytryptamine inhibits the effects of ghrelin on eating and energy substrate utilization

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Abstract

Ghrelin microinjections into discrete regions of the hypothalamus, including the paraventricular nucleus (PVN), stimulate eating and promote carbohydrate oxidation, effects similar to PVN microinjection of neuropeptide Y (NPY). We have also reported that NPY's orexigenic and metabolic effects are antagonized by pretreatment with 5-hydroxytryptamine (5-HT) or 5-HT receptor agonists. In order to determine whether 5-HT also inhibits ghrelin's orexigenic and metabolic actions, the present study examined the effects of 5-HT pretreatment on ghrelin-induced alterations in eating and energy substrate utilization following direct injections into the hypothalamic PVN. Both 5-HT (5–20 nmol) and ghrelin (100 pmol) were administered at the onset of the dark cycle. Food intake was measured 2 h postinjection. A separate group of rats (n = 8) was injected with 5-HT paired with ghrelin and respiratory quotient (RQ; VCO2/VO2) was measured over 2 h using an open circuit calorimeter. PVN injections of ghrelin increased food intake and increased RQ, reflecting a shift in energy substrate utilization in favor of carbohydrate oxidation. 5-HT effectively blocked the effects of ghrelin on both food intake and RQ. We then administered the 5-HT2A/2C, receptor agonist, DOI, immediately prior to ghrelin. Similar to 5-HT, PVN DOI blocked ghrelin-induced eating and inhibited the peptide's effect on substrate utilization. These data are in agreement with other evidence suggesting that ghrelin functions as a gut-brain peptide in the control of food intake and energy metabolism, and indicate that 5-HT acts within the PVN to modulate ghrelin's orexigenic and metabolic signaling.

Introduction

Ghrelin is a 28 amino acid peptide transmitter with a structural homology highly conserved across mammalian species (El-Salhy, 2009, Kojima & Kangawa, 2005). In both rat and human ghrelin the hydroxyl group at serine 3 is modified by n-octanoic acid (Dehlin et al., 2008, Kangawa, 2006, Smith et al., 2001). It is this acylation and conferral of a fatty acid side chain that is a unique biochemical characteristic associated with the peptide's functional activity (Dehlin et al., 2008, Hosoda et al., 2000). Peripheral ghrelin is synthesized and released primarily by endocrine cells of the gastric oxyntic mucosa (Date et al., 2000, El-Salhy, 2009) and central production of ghrelin has been identified within the hypothalamus where ghrelin receptors have been localized (Cowley et al., 2003, Mondal et al., 2005).

Intracerebroventricular injections of ghrelin increase c-Fos expression in multiple hypothalamic nuclei (Lawrence et al., 2002). Paraventricular nucleus (PVN) injections of the peptide, at orexigenic doses, induce increased c-Fos immunoreactivity in the PVN as well as the arcuate and dorsomedial nuclei (Olszewski et al., 2003). The distribution of ghrelin-producing cell bodies, axons and receptors is consistent with a hypothalamic site of action. Ghrelin interacts with other peptidergic systems within the hypothalamus including neuropeptide Y/agouti-related peptide (NPY/AGRP) neurons (Cowley et al., 2003, Gil-Campos et al., 2006, Hori et al., 2008, Willesen et al., 1999). Hypothalamic PVN microinjection of ghrelin increases food intake and alters energy metabolism specifically by increasing respiratory quotient or carbohydrate oxidation (Currie et al., 2005, Melis et al., 2002, Szentirmai et al., 2007). Ghrelin's orexigenic and metabolic actions are comparable to NPY as both neuropeptides elicit similar effects on eating and nutrient partitioning (Currie et al., 2005, Currie, 2003). These findings are in agreement with the hypothesis that ghrelin, like NPY, alters appetite and energy metabolism by acting on hypothalamic circuits mediating positive energy balance.

We have previously reported that pretreatment with either 5-hydroxtryptamine (5-HT) or DOI, a 5-HT2A/2C receptor agonist, microinjected into the PVN, inhibits feeding stimulated by PVN administration of NPY as well as NPY induced alterations in energy substrate utilization (Currie, 2003, Currie et al., 2002, Currie & Coscina, 1998, Currie & Coscina, 1996). A similar effect was not found following DOI pretreatment in the perifornical or ventromedial hypothalamus (Currie & Coscina, 1997, Currie & Coscina, 1996) suggesting that NPY-5-HT interactions are specific to the PVN. In order to assess whether serotonergic mechanisms also alter the orexigenic and metabolic action of ghrelin, the present study investigated the effects of 5-HT or DOI pretreatment on ghrelin-stimulated eating and substrate utilization. The results of this study, described recently in preliminary form (Currie et al., 2009), indicate that both 5-HT and DOI, injected into the PVN at the onset of the dark cycle, inhibit the increases in eating and RQ elicited by ghrelin.

Section snippets

Animals

Adult male Sprague–Dawley rats were purchased from Harlan Laboratories and weighed 275–300 g at the time of surgery. Rats were housed individually in polypropylene cages with free access to standard rodent chow pellets and water. The animal colony room was maintained on a 12 h light/dark cycle (lights on at 0400 h) and at a temperature of 22 ± 2 °C. All procedures described below were approved by the Institutional Animal Care and Use Committee of Reed College.

Apparatus

Oxygen consumption (O2) and carbon

Results

Fig. 1 illustrates the effects of PVN ghrelin microinjection on 2-h food intake at the onset of the nocturnal cycle. One-way analysis of variance (ANOVA) indicated that ghrelin increased eating behavior at each dose tested (F (2,18) = 13.2, p < 0.0002). The effects of PVN administration of 5-HT on ghrelin-stimulated-eating are shown in Fig. 2. One-way ANOVA confirmed that PVN ghrelin elicited a significant increase in 2 h food intake and that pretreatment with 5-HT antagonized this effect (F (3,27) = 

Discussion

The PVN is one of several hypothalamic sites responsive to the feeding-stimulant effects of ghrelin (Currie et al., 2005, Melis et al., 2002, Szentirmai et al., 2007). This suggests that ghrelin-releasing neurons innervate multiple hypothalamic systems implicated in energy intake and metabolism. However, additional evidence indicates that the PVN, in comparison with other hypothalamic sites, may function uniquely to coordinate various aspects of energy metabolism and appetite control (Currie,

Acknowledgement

Supported by NIH 070496-01A1 to PJC.

References (34)

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