Simon M. Luckman

Glucose-sensing neurons of the hypothalamus

Specialized subgroups of hypothalamic neurons exhibit specific excitatory or inhibitory electrical responses to changes in extracellular levels of glucose. Glucose-excited neurons were traditionally assumed to employ a ‘β-cell’ glucose-sensing strategy, where glucose elevates cytosolic ATP, which closes KATP channels containing Kir6.2 subunits, causing depolarization and increased excitability. Recent findings indicate that although elements of this canonical model are functional in some hypothalamic cells, this pathway is not universally essential for excitation of glucose-sensing neurons by glucose. Thus glucose-induced excitation of arcuate nucleus neurons was recently reported in mice lacking Kir6.2, and no significant increases in cytosolic ATP levels could be detected in hypothalamic neurons after changes in extracellular glucose. Possible alternative glucose-sensing strategies include electrogenic glucose entry, glucose-induced release of glial lactate, and extracellular glucose receptors. Glucose-induced electrical inhibition is much less understood than excitation, and has been proposed to involve reduction in the depolarizing activity of the Na+/K+ pump, or activation of a hyperpolarizing Cl− current. Investigations of neurotransmitter identities of glucose-sensing neurons are beginning to provide detailed information about their physiological roles. In the mouse lateral hypothalamus, orexin/hypocretin neurons (which promote wakefulness, locomotor activity and foraging) are glucose-inhibited, whereas melanin-concentrating hormone neurons (which promote sleep and energy conservation) are glucose-excited. In the hypothalamic arcuate nucleus, excitatory actions of glucose on anorexigenic POMC neurons in mice have been reported, while the appetite-promoting NPY neurons may be directly inhibited by glucose. These results stress the fundamental importance of hypothalamic glucose-sensing neurons in orchestrating sleep-wake cycles, energy expenditure and feeding behaviour.

Alternative role for prolactin-releasing peptide in the regulation of food intake.

Prolactin-releasing peptide (PrRP) is a peptide ligand for the human orphan G-protein-coupled receptor hGR3/GPR10 and causes the secretion of prolactin from anterior pituitary cells. However, the lack of immunoreactive staining for PrRP in the external layer of the median eminence seems to rule out this peptide as a classical hypophysiotropic hormone and, furthermore, PrRP is less effective than another inducer of prolactin secretion, thyrotropin-releasing hormone, both in vitro and in vivo. Here we show a reduction in the expression of PrRP mRNA during lactation and fasting and an acute effect of PrRP on food intake and body weight, supporting the hypothesis of an alternative role for the peptide.

Set points, settling points and some alternative models: Theoretical options to understand how genes and environments combine to regulate body adiposity

The close correspondence between energy intake and expenditure over prolonged time periods, coupled with an apparent protection of the level of body adiposity in the face of perturbations of energy balance, has led to the idea that body fatness is regulated via mechanisms that control intake and energy expenditure. Two models have dominated the discussion of how this regulation might take place. The set point model is rooted in physiology, genetics and molecular biology, and suggests that there is an active feedback mechanism linking adipose tissue (stored energy) to intake and expenditure via a set point, presumably encoded in the brain. This model is consistent with many of the biological aspects of energy balance, but struggles to explain the many significant environmental and social influences on obesity, food intake and physical activity. More importantly, the set point model does not effectively explain the ‘obesity epidemic’ – the large increase in body weight and adiposity of a large proportion of individuals in many countries since the 1980s. An alternative model, called the settling point model, is based on the idea that there is passive feedback between the size of the body stores and aspects of expenditure. This model accommodates many of the social and environmental characteristics of energy balance, but struggles to explain some of the biological and genetic aspects. The shortcomings of these two models reflect their failure to address the gene-by-environment interactions that dominate the regulation of body weight. We discuss two additional models – the general intake model and the dual intervention point model – that address this issue and might offer better ways to understand how body fatness is controlled.

Involvement of the noradrenergic afferents from the nucleus tractus solitarii to the supraoptic nucleus in oxytocin release after peripheral cholecystokinin octapeptide in the rat

Activation of abdominal vagal afferents by peripheral injection of cholecystokinin octapeptide induces oxytocin release into the circulation. To test the hypothesis that cholecystokinin increases oxytocin release via activation of noradrenergic afferents from the brainstem, we injected rats with 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine, a selective neurotoxin to noradrenergic fibres, into a lateral cerebral ventricle. The neurotoxin treatment reduced the noradrenaline content in the hypothalamus by 75% and reduced the oxytocin secretion in response to cholecystokinin by over 90%. In separate experiments, the neurotoxin was injected unilaterally in the vicinity of the supraoptic nucleus to test whether direct noradrenergic afferents to the supraoptic nucleus are involved in the response to cholecystokinin. The injection reduced the immunoreactivity for dopamine beta-hydroxylase in the supraoptic nucleus and significantly decreased the number of the supraoptic neurons expressing Fos-like protein after cholecystokinin but not after hypertonic saline. In further experiments, rhodamine-conjugated latex microspheres were injected into the supraoptic nucleus to retrogradely label afferent neurons, and the brains were processed with double-immunohistochemistry for tyrosine hydroxylase and Fos-like protein. In the C2/A2 but not the C1/A1 region of the brainstem, cholecystokinin increased the expression of Fos-like protein in the population of retrogradely-labelled catecholaminergic cells. In the C2/A2 region, the majority of retrogradely labelled cells expressing Fos-like protein after cholecystokinin were catecholaminergic. We conclude that noradrenergic afferents from the A2 but not from the A1 region of the brainstem to the hypothalamus mediate, at least in part, oxytocin release following cholecystokinin.

Centrally administered galanin-like peptide modifies food intake in the rat: a comparison with galanin.

Galanin‐like peptide (GALP) is a recently identified neuropeptide that shares sequence homology with the orexigenic neuropeptide, galanin. In contrast to galanin, GALP is reported to bind preferentially to the galanin receptor 2 subtype (GalR2) compared to GalR1. The aim of this study was to determine the effect of GALP on feeding, body weight and core body temperature after central administration in rats compared to the effects of galanin. Intracerebroventricular (i.c.v.) injection of GALP (1 µg−10 µg) significantly stimulated feeding at 1 h in both satiated and fasted Sprague‐Dawley rats. However, 24 h after GALP injection, body weight gain was significantly reduced and food intake was also usually decreased. In addition, i.c.v. GALP caused a dose‐related increase in core body temperature, which lasted until 6–8 h after injection, and was reduced by peripheral administration of the cyclooxygenase inhibitor, flurbiprofen (1 mg/kg). Similar to GALP, i.c.v. injection of galanin (5 µg) significantly increased feeding at 1 h in satiated rats. However, there was no difference in food intake and body weight at 24 h, and galanin only caused a transient rise in body temperature. Thus, similar to galanin, GALP has an acute orexigenic effect on feeding. However, GALP also has an anorectic action, which is apparent at a later time. Therefore, GALP has complex opposing actions on energy homeostasis.

Fos-Like Immunoreactivity in the Brainstem of the Rat Following Peripheral Administration of Cholecystokinin

Ninety min after intraperitoneal (ip) injection of Cholecystokinin (CCK, 50 μg/kg body wt) Fos‐like protein was expressed in cells throughout the nucleus of the tractus solitarii (NTS) and area postrema, and also in scattered cells in the lateral reticular area of the brainstem. Using dual fluorescent immunocytochemistry for Fos‐like protein and tyrosine hydroxylase (TH), catecholaminergic cells in the A2 region of the NTS and the A1 region of the lateral reticular area were shown to be activated.

The role of RFamide peptides in feeding

In the three decades since FMRFamide was isolated from the clam Macrocallista nimbosa, the list of RFamide peptides has been steadily growing. These peptides occur widely across the animal kingdom, including five groups of RFamide peptides identified in mammals. Although there is tremendous diversity in structure and biological activity in the RFamides, the involvement of these peptides in the regulation of energy balance and feeding behaviour appears consistently through evolution. Even so, questions remain as to whether feeding-related actions represent a primary function of the RFamides, especially within mammals. However, as we will discuss here, the study of RFamide function is rapidly expanding and with it so is our understanding of how these peptides can influence food intake directly as well as related aspects of feeding behaviour and energy expenditure.

Presynaptic actions of morphine: blockade of cholecystokinin-induced noradrenaline release in the rat supraoptic nucleus.

1. This study aimed to establish the site at which morphine acts to inhibit oxytocin release in response to peripheral administration of cholecystokinin (CCK). 2. Conscious rats were given morphine or vehicle followed by CCK or vehicle (I.V.). Fos immunoreactivity was apparent 90 min after CCK injection in the supraoptic nucleus of vehicle‐ but not morphine‐pretreated animals. 3. In the dorsomedial (C2/A2) and the ventrolateral (C1/A1) regions of the brainstem, about half of the cells immunoreactive for tyrosine hydroxylase (TH) expressed Fos‐like protein after CCK injection. In the C2/A2 region, 20% of the Fos‐positive cells also showed TH immunoreactivity, whereas in the C1/A1 region 68% did so. Morphine treatment did not significantly change the number of cells expressing Fos immunoreactivity, or the percentage of TH‐positive cells expressing Fos‐like protein. 4. Amine release was measured in the supraoptic nucleus of urethane‐anaesthetized rats using a microdialysis probe. An I.V. injection of CCK increased the concentrations in the dialysate of noradrenaline and serotonin, but not of either adrenaline or dopamine. Pretreatment with morphine (I.V.) blocked the effects of CCK in a naloxone‐reversible manner. 5. Inclusion of morphine in the dialysate also blocked the increase in noradrenaline and serotonin in response to CCK in a naloxone‐reversible manner. 6. These observations indicate that morphine acts near or within the supraoptic nucleus to block CCK‐evoked noradrenaline release presynaptically. This presynaptic action of morphine may be a cause of the blockade of oxytocin release after CCK.

The Maintenance of Normal Parturition in the Rat Requires Neurohypophysial Oxytocin

The neuropeptide oxytocin has long been known as a potent contractor of the uterus. However, it has remained difficult to attribute a definite role for neurohypophysial oxytocin in either the initiation or continuation of labour (1). Most recently, Lefebvre and colleagues (2) have suggested that oxytocin produced in the uterus, rather than in the hypothalamus, may be more important in parturition since at term the uterus of the rat contains 70‐fold more mRNA for oxytocin than the hypothalamus, and this disappears at about the time of parturition. Despite the high levels of mRNA the uterus contains only nanogram quantities of immunoreactive oxytocin per gram wet weight at term (2), compared to microgram quantities present in the pituitary (3,4). Here we show that activation of the neurohypophysial oxytocin system occurs, as reflected by expression of immunoreactivity for Fos in the hypothalamic supraoptic nucleus, and that this activation is indeed critical for normal parturition, since its inhibition results in a significant prolongation of parturition. In addition, we present evidence that pulsatile delivery of oxytocin into the circulation is important for the efficient progress of parturition, indicating that a major role of the neuronal circuits regulating oxytocin secretion for parturition, as is already known for suckling, is to produce an appropriately patterned hormonal output for efficient biological action.

Intravenous Peptide YY3-36 and Y2 Receptor Antagonism in the Rat: Effects on Feeding Behaviour

Systemic injection of peptide YY3‐36 reduces food intake in rodents and humans, although some groups have reported a lack of response. PYY3‐36 is thought to act via the Y2 receptor to presynaptically inhibit the release of neuropeptide Y and GABA from hypothalamic arcuate neurones. Due to the controversy surrounding its action in rodents, we tested the peptide intravenously on feeding behaviour in rats and attempted to block its actions with the Y2 receptor antagonist BIIE0246. PYY3‐36 significantly decreased food intake during the first hour in male Sprague‐Dawley rats fasted overnight and then re‐fed. BIIE0246 had no effect alone on re‐feeding, but completely blocked the action of PYY3‐36. In a second experiment of similar design, the behavioural satiety sequence (BSS) was studied. Normal rats eat, drink, explore and groom before entering rest. PYY3‐36 significantly reduced food eaten maintaining the normal BSS, although shifting it to the left as expected for a natural satiety factor. The latency to rest occurred earlier for animals given PYY3‐36 alone and PYY3‐36 tended to increase the total time in rest compared with controls. These behavioural effects of PYY3‐36 were blocked by BIIE0246, and BIIE0246 alone did not have an effect on the BSS. These results support the role of PYY3‐36 as a natural satiety factor acting through Y2 receptors.