Adrian K. Hewson

Systemic administration of ghrelin induces Fos and Egr-1 proteins in the hypothalamic arcuate nucleus of fasted and fed rats.

Ghrelin, a recently identified endogenous ligand for the growth hormone secretagogue (GHS) receptor, induces growth hormone (GH) secretion following systemic administration. We sought to determine whether systemic administration of ghrelin activates cells in the hypothalamic arcuate nucleus by examining the distribution of cells expressing Fos and Egr‐1 proteins. In normally fed rats, both ghrelin and GHRP‐6 (a synthetic GHS) significantly increased the number of cells expressing Fos and Egr‐1 in the arcuate nucleus. The effects of ghrelin and GHRP‐6 to induce Fos or Egr‐1 protein expression was significantly greater in fasted than in fed rats. Thus, we show that (i) ghrelin is a centrally active peptide; (ii) it acts in a similar manner to synthetic GHS; and (iii) its central actions are increased in fasting, presumably reflecting physiological changes that accompany altered food intake and/or nutritional state.

Intracerebroventricular injection of apelin-13 reduces food intake in the rat.

Apelin is a peptide recently identified as a ligand for the APJ receptor, a receptor located in tissues such as the small intestine and in the hypothalamus. Apelin has been detected in adipose tissue, gastrointestinal tract, heart and stomach. Recent reports suggest a role for apelin in the regulation of blood pressure and the control of body fluid homeostasis. The present studies examined the consequences on food intake of intravenous (IV) and intracerebroventricular (ICV) injection of apelin-13 in the Wistar rat. IV injection of 10 nmol of apelin-13 did not cause any change in food intake in either fed or 24-h fasted rats. ICV injection of 1 and 3 nmol of apelin-13 resulted in a reduction in food intake in both fed and fasted rats. The earliest reduction in food intake occurred at 4 h post-injection in fed rats. In fasted rats, food intake was reduced at 24-h post-injection only. These data provide evidence of a possible role for apelin-13 in the control of food intake.

Intracerebroventricular injection of neuropeptide FF, an opioid modulating neuropeptide, acutely reduces food intake and stimulates water intake in the rat.

Neuropeptide FF (NPFF) is a mammalian peptide that is found in high concentrations in the central nervous system (CNS) and has also been detected in plasma. Various functions have been attributed to this peptide although its main action in the CNS remains unclear. In this study we observed that intracerebroventricular (ICV) injection of human NPFF, at early light phase in fasted rats, acutely reduced food intake and caused a large increase in water intake compared with saline injected controls. This effect was independently observed in two separate studies yielding similar results. Thus the central effects of NPFF to decrease food intake may be largely attributable to increased water intake.

Differential effects of α-, β- and γ2-melanocyte-stimulating hormones on hypothalamic neuronal activation and feeding in the fasted rat

Abstract Hypothalamic pro-opiomelanocortin neurones have an established role in the control of feeding. While pro-opiomelanocortin is the precursor for at least three melanocortin peptides, α-, β- and γ-melanocyte-stimulating hormone (MSH), it has been widely assumed that α-MSH is the predominant ligand involved. We compared the effects of centrally administered α-, β- and γ2-MSH on hypothalamic neuronal activation and on food intake in rats fasted for 48 h. Significant reductions in food intake were seen with α-MSH (first hour) and γ2-MSH (second hour) but not with β-MSH. The pattern of neuronal activation, assessed by the detection of early growth response factor-1 protein, showed considerable overlap; all three melanocortins activated cells in the arcuate, ventromedial, paraventricular, periventricular and supraoptic nuclei, as well as the preoptic area. α-MSH and β-MSH produced activation in the dorsomedial nuclei while γ2-MSH was only weakly active here. Retrograde labelling by systemic Fluorogold injection revealed that many cells activated by MSH compounds in the arcuate, paraventricular, periventricular and supraoptic nuclei (but not dorsomedial or ventromedial) project outside the blood–brain barrier and are therefore likely to include neuroendocrine cells. Desacetyl-α-MSH, which has previously been reported to lack effects on feeding, produced no discernible neuronal activation in the hypothalamus. Our finding that both the pattern of neuronal activation and the distribution of neuroendocrine cells activated in response to these closely related peptides show only partial overlap suggests that, in addition to common pathways, there may exist distinct hypothalamic circuits activated by different pro-opiomelanocortin products. The slower time course of γ2-MSH- versus α-MSH-induced suppression of feeding provides further support for the notion that the biological responses to individual melanocortin peptides may involve distinct neuronal mechanisms.

Actions of leptin on growth hormone secretagogue-responsive neurones in the rat hypothalamic arcuate nucleus recorded in vitro.

In the arcuate nucleus, the growth hormone (GH) secretagogue (GHS)‐responsive cells include a subpopulation of the neuropeptide Y (NPY) neurones. It is not known whether these include the orexigenic NPY population that are inhibited by the satiety hormone, leptin. Thus we investigated whether (i) the arcuate nucleus cells electrically excited by GHS are inhibited by leptin and (ii) chronic central leptin infusion alters GHS‐induced Fos expression. Of 36 cells recorded from a trimmed hypothalamic slice containing arcuate nucleus, 13 cells were excited by the nonpeptide GHS, CP‐459,599. The predominant response of these cells to leptin was inhibitory: six inhibited, three excited and four unresponsive. Similar responses were observed in a population of arcuate cells recorded from a preparation in which synaptic transmission was blocked, suggesting that leptin acts directly on a subpopulation of GHS‐responsive neurones. Intracerebroventricular infusion of leptin for 1 week did not alter the number of cells expressing Fos following GHS administration. Thus, while leptin does not appear to influence the central actions of GHS to induce immediate early gene expression, it does act directly on a subpopulation of cells excited by GHS, eliciting mostly inhibitory but also some excitatory responses. It will be interesting to discover the consequences of leptins inhibitory effects on the hypothalamic circuits excited by GHS, particularly since leptin paradoxically has a stimulatory effect on GH secretion, presumed to reflect a suppression of central NPY pathways.

Central Responsiveness to a Ghrelin Mimetic (GHRP‐6) is Rapidly Altered by Acute Changes in Nutritional Status in Rats

The hypothalamus appears to be more responsive to ghrelin and growth hormone secretagogues (GHS) in fasting, as reflected by a two‐ to three‐fold increase in the number of cells detected that express Fos protein in the arcuate nucleus, in 48‐h fasted rats compared to fed controls. Moreover, this increased hypothalamic responsiveness to GHS in fasting is regulated by the central action of exogenous leptin and insulin, although it is unknown whether these hormones mediate the changes in hypothalamic responsiveness to GHS associated with the fasting/fed state. In the present study, we show that refeeding with normal rat chow for only 2 h at the end of a 48‐h fast reversed the potentiation of the Fos response to GHRP‐6 observed in fasted rats. Circulating leptin and insulin levels remained significantly lower in refed rats compared to ad lib‐fed rats, suggesting that the change in the hypothalamic sensitivity brought about by refeeding was independent of these hormones. By contrast, 2 h of chow refeeding at the end of a fast restored plasma glucose levels to those of the fed state. Refeeding with sugar alone for 2 h at the end of a 48‐h fast also reduced the potentiated Fos response in fasting, indicating that elevated blood glucose can influence the central responsiveness to ghrelin/GHS. By contrast, infusion of the ileal satiety factor, PYY3‐36 (known to increase postprandially) did not alter the central responsiveness to GHRP‐6, although it suppressed feeding and body weight as expected. This study highlights the importance of nutritional status in regulating the action of exogenous GHS (and presumably endogenous ghrelin) on the hypothalamic circuits controlling food intake.

Interactions of Growth Hormone Secretagogues with Leptin-Sensitive Brain Networks

The growth hormone secretagogues (GHS) and ghrelin, an endogenous ligand for the cloned GHS receptor, have recently been shown to induce adiposity in rodents. For some time now, we have been investigating the central site and mechanism of action of GHS, mostly in relation to their growth hormone-releasing action. However, it has become clear that these compounds can activate neuropeptide Y-containing neurones and interact with the hypothalamic circuits controlling body weight and appetite. GHS and ghrelin induce adiposity, although the CNS mechanism underlying this effect may not be fully understood. Hypothalamic circuits regulating body weight can be readily identified as they are responsive to circulating satiety factors such as leptin and insulin. Thus, in electrophysiological studies in vitro we have shown that cells excited by GHS tend to be inhibited by leptin administration. Moreover, GHS-responsive cells appear to be direct targets for leptin, as these responses to GHS and leptin can be observed in a preparation in which synaptic transmission is blocked. Using Fos protein expression to quantify neuronal activation, we have also shown that the hypothalamus is more responsive to GHS/ghrelin in 48-hour fasted rats. This increased responsiveness can be reversed by chronic central infusion of either leptin or insulin. Also refeeding for only two hours at the end of a 48-hour fast reverses the increased responsiveness to GHS, suggesting that central responsiveness to GHS can be regulated acutely, perhaps via some aspect of the feeding process or by absorption of nutrients. We conclude that 1) GHS (and ghrelin) interact with hypothalamic circuits controlling body weight, including leptin- and insulin-sensitive circuits and 2) central responsiveness to GHS is altered by circulating satiety factors and by nutritional state.