Thomas Riediger

Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus.

Leptin decreases food intake and increases energy expenditure in rodents by inhibiting neurones in the hypothalamic arcuate nucleus. The growth hormone secretagogue (GHS) ghrelin is known to stimulate food intake and to be the endogenous ligand for the GHS‐receptor, which is strongly expressed in the arcuate nucleus, like the leptin receptor (Ob‐R). In this study, we analysed the effect of systemic ghrelin administration on Fos expression in the arcuate nucleus on neurones expressing Ob‐R. Injection of ghrelin (0.2 mg/kg, i.p) significantly increased the number of neurones expressing Fos protein in the ventromedial arcuate nucleus. Fifty‐seven percent of all Fos‐positive cells in the ventromedial arcuate nucleus were also positive for Ob‐R staining. Furthermore, we investigated electrophysiologically the effect of ghrelin and leptin on the activity of arcuate neurones in an in‐vitro slice preparation. Ghrelin stimulated the electrical activity dose‐dependently in 80% of all cells tested (n=49) with a threshold concentration of 10−11 M; only 8% were inhibited and 12% did not respond. The effect of ghrelin (10−7 M) was weakly antagonized by the peptidic GHS‐receptor antagonist (D‐Lys3)‐GHRP‐6 (10−4 M), which also showed a much weaker affinity (IC50, 0.9 × 10−6 M) to the GHS‐receptor than ghrelin (IC50, 0.3 × 10−9 M). Ghrelin increased the electrical activity in 76% of all cells which were inhibited by leptin (n=17). These data show that ghrelin interacts with the leptin hypothalamic network in the arcuate nucleus. The opposite effect of leptin and ghrelin on neurones in the arcuate nucleus may serve as a neurophysiological correlate of the orexigenic and anorectic effects of ghrelin and leptin.

Site-specific effects of ghrelin on the neuronal activity in the hypothalamic arcuate nucleus

The recently discovered hormone ghrelin, which is secreted from the stomach during fasting and hypoglycemia opposes the homeostatic functions of leptin by increasing food intake and decreasing energy expenditure. The hypothalamic arcuate nucleus (Arc) mediates the effects of leptin and contains a high density of ghrelin receptors. The leptin- and ghrelin-responsive network involves the hypothalamic neuropeptide Y/alpha-melanocyte stimulating hormone (NPY/alpha-MSH) system. In the rat, neurons expressing the orexigenic peptide NPY are mainly located in the ventromedial Arc (ArcM), while pro-opiomelanocortin (POMC) neurons, synthesizing the anorectic peptide alpha-MSH, predominate in the ventrolateral Arc (ArcL). In extracellular single unit recordings from in vitro slice preparations of the Arc, superfusion of ghrelin (10(-8) M) exerted predominantly excitatory effects on ArcM neurons (73%, n=93), while a high number ArcL neurons were inhibited in response to ghrelin (42%, n=43). The excitatory effect of ghrelin on neuronal activity was postsynaptic since it was unaffected by synaptic blockade (low Ca(2+)/high Mg(2+) solution). In contrast, the inhibitory response in the ArcL was abolished by the blockade of synaptic interactions indicating a presynaptic mechanism. These results indicate that circulating ghrelin may oppose the actions of leptin by directly activating NPY-neurons of the ArcM and by indirectly inhibiting POMC neurons of the ArcL.

Peptide YY directly inhibits ghrelin-activated neurons of the arcuate nucleus and reverses fasting-induced c-Fos expression.

The hypothalamic arcuate nucleus (Arc) monitors and integrates hormonal and metabolic signals involved in the maintenance of energy homeostasis. The orexigenic peptide ghrelin is secreted from the stomach during negative status of energy intake and directly activates neurons of the medial arcuate nucleus (ArcM) in rats. In contrast to ghrelin, peptide YY (PYY) is released postprandially from the gut and reduces food intake when applied peripherally. Neurons in the ArcM express ghrelin receptors and neuropeptide Y receptors. Thus, PYY may inhibit feeding by acting on ghrelin-sensitive Arc neurons. Using extracellular recordings, we (1) characterized the effects of PYY on the electrical activity of ghrelin-sensitive neurons in the ArcM of rats. In order to correlate the effect of PYY on neuronal activity with the energy status, we (2) investigated the ability of PYY to reverse fasting-induced c-Fos expression in Arc neurons of mice. In addition, we (3) sought to confirm that PYY reduces food intake under our experimental conditions. Superfusion of PYY reversibly inhibited 94% of all ArcM neurons by a direct postsynaptic mechanism. The PYY-induced inhibition was dose-dependent and occurred at a threshold concentration of 10–8M. Consistent with the opposite effects of ghrelin and PYY on food intake, a high percentage (50%) of Arc neurons was activated by ghrelin and inhibited by PYY. In line with this inhibitory action, peripherally injected PYY partly reversed the fasting-induced c-Fos expression in Arc neurons of mice. Similarly, refeeding of food-deprived mice reversed the fasting-induced activation in the Arc. Furthermore, peripherally injected PYY reduced food intake in 12-hour fasted mice. Thus the activity of Arc neurons correlated with the feeding status and was not only reduced by feeding but also by administration of PYY in non-refed mice. In conclusion, our current observations suggest that PYY may contribute to signaling a positive status of energy intake by inhibiting Arc neurons, which are activated under a negative status of energy intake by signals such as ghrelin.

Infusion of the amylin antagonist AC 187 into the area postrema increases food intake in rats.

According to previous studies, the area postrema (AP) of the hindbrain may play an important role in mediating the anorectic effect of the pancreatic hormone amylin. Peripheral amylin has been suggested to directly act on AP neurons to bring about its anorectic effect. Cyclic GMP may act as second messenger in this regard. In the present study, we wanted to further delineate the role of the AP in amylins effect and to find out whether endogenous amylin might reduce feeding via the AP. Rats with chronic cannulas aiming at the AP were infused with various doses of amylin, its agonist salmon calcitonin (sCT) or a cyclic guanosine monophosphate (cGMP) analogue. Amylin and sCT inhibited food intake for about 2 h after food presentation, mainly by reducing meal size when infused into the AP [e.g., 1 h food intake after amylin (0.4 microg/rat) infusion in 12-h deprived rats: NaCl 4.0+/-0.5 vs. amylin 2.4+/-0.5, P<.05]. The effect was comparable in ad libitum fed and 12-h food-deprived rats with a minimal effective dose of 0.04 microg/rat. Similar to amylin and sCT, the cGMP analogue 8-Br-cGMP (200 nmol/rat) also reduced food intake and meal size. Infusion of the amylin antagonist AC 187 (30 microg) into the AP significantly reduced the anorectic effect induced by an intraperitoneal injection of amylin (5 microg/kg). Furthermore, AC 187 alone increased feeding when infused into the AP. This study is in line with previous work pointing to an important role of the AP in mediating the anorectic effect of amylin. Furthermore, we provide evidence for a physiological role of endogenous amylin to reduce food intake. This may also involve an action via the AP.

Peripheral amylin activates circumventricular organs expressing calcitonin receptor a/b subtypes and receptor-activity modifying proteins in the rat.

The pancreatic hormone amylin (AMY) and the AMY-receptor-agonist salmon-calcitonin (sCT) reduce short-term food-intake after binding to the area postrema (AP), a circumventricular organ (CVO) lacking blood-brain-barrier characteristics. AMY has also been proposed to induce drinking via another CVO, the subfornical organ (SFO). In cellular systems, AMY-binding is generated by interaction of calcitonin-receptor a/b (CT((a))/CT((b))) with receptor-activity modifying proteins (RAMPs). By using in situ hybridization, the codistribution of CT((a))/CT((b)) with RAMP1-3 and c-fos was mapped in CVOs of rats. AMY and sCT induced c-fos within the SFO which contained CT((a)) and/or CT((b)) and RAMP1/2 mRNA. AMY and sCT also activated AP neurons, which express the CT((a)), but not the CT((b)), receptor and RAMP2/3 mRNA. These data emphasize the important role of these structures as primary targets for circulating AMY.

Immunohistochemical mapping of calcitonin receptors in the adult rat brain.

Calcitonin receptors (CTR) have previously been identified in specific regions of the rat central nervous system using in situ hybridization or autoradiography with iodinated ligands. In this study, the results of immunohistochemical mapping of CTR in the adult rat brain are reported, using a potent and recently developed antibody that recognizes an intracellular epitope of the rat CTR, and high-resolution immunofluorescence techniques. Abundant expression was found in the brain, with highest densities in the nucleus accumbens, lateral arcuate nucleus, lateral substantia nigra, bed nucleus of the stria terminalis, locus coeruleus, area postrema, nucleus of the solitary tract, and some of the nuclei of the reticular formation. These results are in close correspondence with previous mapping studies. However, we detected CTR immunoreactivity in several additional brain areas, as the ventromedial, lateral and posterior hypothalamus, where CT binding has not yet been described. Our detailed mapping of the CTR in the rat brain has identified CTR-positive cells that will be important for subsequent characterization of behavioral functions associated with the actions of CT-related peptides.

Lesion of the lateral parabrachial nucleus attenuates the anorectic effect of peripheral amylin and CCK

Amylin and CCK activate the area postrema (AP)/nucleus of the solitary tract (NTS) - lateral parabrachial nucleus (LPBN) - central amygdala (CeA) pathway. However, except for the brainstem structures the role of these nuclei for the anorectic effect of these peptides is not yet well characterized. The current study investigated the role of the LPBN in mediating the inhibitory effect of peripheral amylin and CCK on feeding behavior. Rats with electrolytic lesions in the LPBN (LPBN-X) were used in behavioral as well as in immunohistological c-Fos studies. LPBN-X significantly reduced the anorectic effect of amylin (5 microg/kg, i.p.). The effect of a higher amylin dose (10 microg/kg, i.p.) was only slightly attenuated in the LPBN-X rats. In agreement with previous studies, LPBN lesions also reduced the inhibitory effect of CCK on food intake. In the immunohistological experiments, amylin and CCK induced c-Fos expression in the AP, NTS, LPBN and CeA in the SHAM rats. Both the amylin- and CCK-induced activation of the CeA was completely abolished in the animals with a LPBN lesion. These results clearly suggest that the signal transduction pathway between the AP/NTS and CeA has been disrupted by the LPBN ablation. We conclude that the LPBN is a crucial brain site mediating the anorectic effect of amylin and CCK. Furthermore, an intact LPBN seems to be essential for the c-Fos response in the CeA induced by these peptides.

Noradrenergic neurons of the area postrema mediate amylin's hypophagic action

Circulating amylin inhibits food intake via activation of the area postrema (AP). The aim of this study was to identify the neurochemical phenotype of the neurons mediating amylins hypophagic action by immunohistochemical and feeding studies in rats. Expression of c-Fos protein was used as a marker for neuronal activation and dopamine-beta-hydroxylase (DBH), the enzyme-catalyzing noradrenaline synthesis, as a marker for noradrenergic neurons. We found that approximately 50% of amylin-activated AP neurons are noradrenergic. To clarify the functional role of these neurons in amylins effect on eating, noradrenaline-containing neurons in the AP were lesioned using a saporin conjugated to an antibody against DBH. Amylin (5 or 20 microg/kg s.c.)-induced anorexia was observed in sham-lesioned rats with both amylin doses. Rats with a lesion of > 50% of the noradrenaline neurons were unresponsive to the low dose of amylin (5 microg/kg) and only displayed a reduction in food intake 60 min after injection of the high amylin dose (20 microg/kg). In a terminal experiment, the same rats received amylin (20 microg/kg) or saline. The AP and nucleus of the solitary tract (NTS) were stained for DBH to assess noradrenaline lesion success and for c-Fos expression to evaluate amylin-induced neuronal activation. In contrast to sham-lesioned animals, noradrenaline-lesioned rats did not show a significant increase in amylin-induced c-Fos expression in the AP and NTS. We conclude that the noradrenergic neurons in the AP mediate at least part of amylins hypophagic effect.

Inhibitory effects of lipopolysaccharide on hypothalamic nuclei implicated in the control of food intake

The arcuate nucleus (Arc) and the lateral hypothalamic area (LHA), two key hypothalamic nuclei regulating feeding behavior, express c-Fos, a marker of neuronal activation in fasted animals. This is reversed by refeeding. In the present study we tested whether an anorectic dose of lipopolysaccharide (LPS), the cell wall component of Gram-negative bacteria, also inhibits fasting-induced c-Fos expression in these hypothalamic nuclei. This would suggest that they are involved in anorexia during bacterial infections as well. We also studied whether LPS modulates the activity of orexin-A positive (OX+) LHA neurons. Food deprived BALB/c mice were injected with LPS or saline and were sacrificed 4 or 6h later. Four hours after injection, LPS reduced the number of c-Fos positive cells in the Arc and in the LHA, but had no effect on c-Fos in OX+ neurons. Six hours after injection, LPS reduced c-Fos expression in the LHA, both in the OX- and OX+ neurons, but not in the Arc. These results show that LPS modulates neuronal activity in the Arc and LHA similar to feeding-related stimuli, suggesting that the observed effects might contribute to the anorectic effect of LPS. Thus, physiological satiety signals released during refeeding and anorexia during bacterial infection seem to engage similar neuronal substrates.

Differential effects of amylin and salmon calcitonin on neuropeptide gene expression in the lateral hypothalamic area and the arcuate nucleus of the rat.

The pancreatic peptide hormone amylin (AMY) and the AMY receptor agonist salmon calcitonin (sCT) reduce short-term food intake in rats primarily by activating neurons located in the circumventricular area postrema. In the present study we analyzed the involvement of (an)orexigenic neuropeptides expressed in the lateral hypothalamic area (LHA) and in the arcuate nucleus in mediating the AMY and sCT-induced suppression of food intake. By using semiquantitative in situ hybridization 120 min after intraperitoneal injection of AMY or sCT (50 microgram/kg), orexin mRNA levels were decreased in LHA by AMY or sCT treatment. Moreover, sCT significantly suppressed the orexigenic melanin concentrating hormone in LHA, whereas mRNA levels of neuropeptide Y, cocaine and amphetamine regulated transcript, agouti-gene-related protein and proopiomelanocortin were unaffected by either treatment. In conclusion, the anorexigenic effect of AMY/sCT might be mediated by the observed reduced expression of orexigenic neuropeptides in the LHA.