Liraglutide: short-lived effect on gastric emptying -- long lasting effects on body weight.

AIM: Previous studies with the novel once daily glucagon-like peptide-1 (GLP-1) analogue liraglutide and the GLP-1 receptor agonist exenatide have revealed profound insulinotrophic and antidiabetic effects, but also potent effects on gastric emptying (GE) and long-term and lasting reductions in body weight. In this study, we examined the acute and chronic effects of two different GLP-1 analogues with different pharmacokinetic profiles on GE, food intake and body weight. METHODS: On the basis of a series of dose-finding studies, the doses of exenatide and liraglutide with similar acute anorectic effects were identified. GE was assessed using a standard acetaminophen release assay. After the acute test, rats were dosed bi-daily for 14 days in which period food intake and body weight was monitored. On day 14, the GE rate was reassessed. RESULTS: While both compounds exerted robust acute reductions in GE, the effect was markedly diminished following 14 days of dosing with liraglutide. In contrast, exenatide-treated rats still displayed a profound reduction in GE at the 14-day time-point. Both compounds exerted similar effects on body weight. CONCLUSION: The data suggest that the 'gastric inhibitory' GLP-1 receptors in rats are subject to desensitization/tachyphylaxis but that this effect is dependent on full 24-h exposure as obtained by liraglutide. The body weight-lowering effects of GLP-1 receptor stimulation are not subject to desensitization. These data indicate that regulation of appetite signals in the brain, and not GE, is the main mechanism for liraglutide-induced weight loss.

Identification of cannabinoid type 1 receptor expressing cocaine amphetamine-regulated transcript neurons in the rat hypothalamus and brainstem using in situ hybridization and immunohistochemistry.

Recent data have indicated that the neuropeptide cocaine amphetamine-regulated transcript (CART) may be a downstream mediator of the effect of CB1 receptor antagonist on appetite regulation. In order to identify possible interactions between CART and central CB1R expressing neurons, a detailed mapping of CART and CB1R expression and immunoreactivity in the brain was initiated. Single radioactive in situ hybridizations revealed a predominant overlap between CART and CB1R mRNA in hypothalamic and lower brainstem nuclei. Using double in situ hybridization, co-localization between CART and CB1R mRNA expressing neurons was observed to be most pronounced in the retrochiasmatic and lateral hypothalamic areas, as well as in all parts of the dorsal vagal complex. Further attempts to immunohistochemically characterize the distribution of CB1R were, however, deemed impossible as any of eight commercially available antibodies/antisera gave rise to non-specific staining patterns. Furthermore, the staining pattern obtained was not discriminate between CB1R knockout mice and wild type mice. Collectively, we demonstrate at the messenger level that CB1R expressing perikarya colocalize with CART expressing neurons in hypothalamic and brainstem areas known to be important in appetite control, whereas interactions at the protein level necessitate a demand for cautious interpretations of immunohistochemical results.

Combination of the insulin sensitizer, pioglitazone, and the long-acting GLP-1 human analog, liraglutide, exerts potent synergistic glucose-lowering efficacy in severely diabetic ZDF rats.

OBJECTIVE: Severe insulin resistance and impaired pancreatic beta-cell function are pathophysiological contributors to type 2 diabetes, and ideally, antihyperglycaemic strategies should address both. RESEARCH DESIGN AND METHODS: Therapeutic benefits of combining the long-acting human glucagon-like peptide-1 (GLP-1) analog, liraglutide (0.4 mg/kg/day), with insulin sensitizer, pioglitazone (10 mg/kg/day), were assessed in severely diabetic Zucker diabetic fatty rats for 42 days. Impact on glycaemic control was assessed by glycated haemoglobin (HbA(1C)) at day 28 and by oral glucose tolerance test at day 42. RESULTS: Liraglutide and pioglitazone synergistically improved glycaemic control as reflected by a marked decrease in HbA(1C) (liraglutide + pioglitazone: 4.8 +/- 0.3%; liraglutide: 8.8 +/- 0.6%; pioglitazone: 7.9 +/- 0.4%; vehicle: 9.7 +/- 0.3%) and improved oral glucose tolerance at day 42 (area under the curve; liraglutide + pioglitazone: 4244 +/- 445 mmol/l x min; liraglutide: 7164 +/- 187 mmol/l x min; pioglitazone: 7430 +/- 446 mmol/l x min; vehicle: 8093 +/- 139 mmol/l x min). A 24-h plasma glucose profile at day 38 was significantly decreased only in the liraglutide + pioglitazone group. In addition, 24-h insulin profile was significantly elevated only in the liraglutide + pioglitazone group. Liraglutide significantly decreased food intake alone and in combination with pioglitazone, while pioglitazone alone increased cumulated food intake. As a result, rats on liraglutide alone gained significantly less weight than vehicle-treated rats, whereas rats on pioglitazone alone gained significantly more body weight than vehicle-treated rats. However, combination therapy with liraglutide and pioglitazone caused the largest weight gain, probably reflecting marked improvement of energy balance because of reduction of glucosuria. CONCLUSIONS: Combination therapy with insulinotropic GLP-1 agonist liraglutide and insulin sensitizer, pioglitazone, improves glycaemic control above and beyond what would be expected from additive effects of the two antidiabetic agents.

Chronic 5-HT6 receptor modulation by E-6837 induces hypophagia and sustained weight loss in diet-induced obese rats.

E-6837 is a novel, selective and high-affinity 5-HT(6) receptor ligand (pK(i): 9.13) which in vitro demonstrates partial agonism at a presumably silent rat 5-HT(6) receptor and full agonism at a constitutively active human 5-HT(6) receptor by monitoring the cAMP signaling pathway.The effects of chronic treatment with E-6837 were determined in diet-induced obese (DIO)-rats on changes in body weight, food and water intake, plasma indices of comorbid risk factors, and weight regain on compound withdrawal. The centrally acting antiobesity drug, sibutramine, was used as the reference comparator. Sustained body weight loss and decreased cumulative food intake of DIO-rats was observed with E-6837 (30 mg kg(-1), p.o., twice a day) during the 4-week treatment period. The onset of the E-6837 effect on body weight was slower than that of sibutramine (5 mg kg(-1), p.o.), while its maximal effect was greater, that is -15.7 versus -11.0%.E-6837-induced weight loss was exclusively mediated by a decrease (31.7%) in fat mass, with a concomitant reduction (49.6%) in plasma leptin. Reduced obesity was also reflected in improved glycemic control. Although weight regain occurred after withdrawal from either compound, the body weights after E-6837 (-6.6%) remained lower than after sibutramine (-3.8%) indicating that the greater efficacy of the former did not result in profound rebound hyperphagia/weight gain. These results show that the 5-HT(6) receptor partial agonist, E-6837, is a promising new approach to the management of obesity with the potential to produce greater sustained weight loss than sibutramine.

Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit.

Cocaine- and amphetamine-regulated transcript (CART) is present in a number of hypothalamic nuclei. Besides actions in circuits regulating feeding behaviour and stress responses, the hypothalamic functions of CART are largely unknown. We report that CART immunoreactivity is present in hypothalamic neuroendocrine neurones. Adult male rats received a systemic injection of the neuronal tracer Fluorogold (FG) 2 days before fixation, and subsequent double- and triple-labelling immunoflourescence analysis demonstrated that neuroendocrine CART-containing neurones were present in the anteroventral periventricular, supraoptic, paraventricular (PVN) and periventricular nuclei of the hypothalamus. In the PVN, CART-positive neuroendocrine neurones were found in all of cytoarchitectonically identified nuclei. In the periventricular nucleus, approximately one-third of somatostatin cells were also CART-immunoreactive. In the medial parvicellular subnucleus of the PVN, CART and FG coexisted with thyrotrophin-releasing hormone, whereas very few of the corticotrophin-releasing hormone containing cells were CART-immunoreactive. In the arcuate nucleus, CART was extensively colocalized with pro-opiomelanocortin in the ventrolateral part, but completely absent from neuroendocrine neurones of the dorsomedial part. To assess the possible role of CART as a hypothalamic-releasing factor, immunoreactive CART was measured in blood samples from the long portal vessels connecting the median eminence with the anterior pituitary gland. Adult male rats were anaesthetized and the infundibular stalk exposed via a transpharyngeal approach. The long portal vessels were transected and blood collected in 30-min periods (one prestimulatory and three poststimulatory periods). Compared to systemic venous plasma samples, baseline concentrations of immunoreactive CART were elevated in portal plasma. Exposure to sodium nitroprusside hypotension triggered a two-fold elevation of portal CART42-89 immunoreactivity throughout the 90-min stimulation period. In contrast, the concentration of portal plasma CART immunoreactivity dropped in the vehicle infused rats. The current study provides further evidence that CART is a neuroendocrine-releasing factor with a possible impact on anterior pituitary function during states of haemodynamic stress.

Effects of leptin on arcuate pro-opiomelanocortin and cocaine-amphetamine-regulated transcript expression are independent of circulating levels of corticosterone.

In the hypothalamic arcuate nucleus, neurones that coexpress cocaine-amphetamine-regulated transcript (CART) and alpha-melanocyte-stimulating hormone [alpha-MSH; pro-opiomelanocortin (POMC) derived] peptides exert catabolic actions and are stimulated by leptin. However, leptin treatment also affects other circulating factors that influence hypothalamic gene expression. Notably, the hypercorticosteronaemia of ob/ob mice is lowered by leptin treatment. To examine the interaction between glucocorticoids and leptin on POMC/CART mRNA expression, an experiment combining leptin and adrenalectomy (ADX) in leptin deficient ob/ob mice was carried out. Obese ob/ob and lean littermate Ob/? mice were ADX or sham-operated. ADX mice received a pellet containing 25% corticosterone subcutaneously. Seven days postoperatively, mice were injected intraperitoneally for 5 days with either recombinant human leptin or vehicle. On the sixth day, the mice were decapitated and the brains removed and trunk blood was collected for corticosterone analysis. Plasma concentrations of corticosterone were elevated in all ob/ob groups compared to Ob/?. For both ob/ob and Ob/? groups, corticosterone concentrations exhibited a decline across groups: vehicle-sham>leptin-sham>ADX-vehicle>ADX-leptin. Leptin inhibited food intake and bodyweight in ob/ob-sham and ob/ob-ADX to a similar extent, whereas no effect of leptin was observed in Ob/? mice. Similarly, leptin caused an identical increase in arcuate POMC and CART mRNA expression in ob/ob-sham and ob/ob-ADX compared to vehicle. The present data support the view that leptin influences arcuate POMC and CART mRNA expression directly, and that the effect is not modulated by corticosterone across a wide range of circulating corticosterone concentrations.

Light-dependent induction of cFos during subjective day and night in PACAP-containing ganglion cells of the retinohypothalamic tract.

Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.

[Glucagon-like peptide 2, a neurotransmitter with a newly discovered role in the regulation of food ingestion]. / Glucagonlignende peptid-2, en neurotransmitter med en nyopdaget rolle i reguleringen af fødeindtagelsen.

We report here that glucagon-like peptide 2(GLP-2) and its receptor constitute a distinct projection system connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus (DMH). The DMH contains a dense plexus of GLP-2 immunoreactive fibres and is the only hypothalamic nucleus expressing GLP-2 receptor mRNA. Consistent with this, central application of GLP-2 activates the expression of neurones solely in the DMH. Furthermore, central administration of GLP-2 causes a dose-related, a pharmacologically and behaviourally specific inhibition of food intake in rats. Surprisingly, the alleged GLP-1 receptor antagonist, Exending (9-39), proved a functional antagonist of centrally applied GLP-2. These data implicate GLP-2 as an important neurotransmitter in the regulation of food intake and likely bodyweight. Our data therefore point to the DMH as a crossroad for endocrine and visceral information affecting feeding behaviour.

Glucagon-like peptide containing pathways in the regulation of feeding behaviour.

The pre-proglucagon derived peptides, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are both involved in a wide variety of peripheral functions, such as glucose homeostasis, gastric emptying, intestinal growth, insulin secretion as well as the regulation of food intake. Pre-proglucagon is also found in the brainstem in a small population of nerve cells in the nucleus of the solitary tract (NTS) that process the pre-propeptide as in the gut to yield GLP-1 and GLP-2. GLP-1 containing nerve fibres and the GLP-1 receptor are found predominantly in hypothalamic midline nuclei. GLP-1 given centrally to naive rats results in a marked induction of c-Fos protein in the supraoptic nucleus, paraventricular nucleus of the hypothalamus (PVN) and central nucleus of the amygdala, but only a moderate increase in the arcuate nucleus. The pattern of c-Fos activation is compatible with the appetite suppressing effects of GLP-1. This anorectic effect of GLP-1 appears to be mediated by the PVN, as direct injections of GLP-1 into this nucleus cause anorexia without concomitant taste aversion, suggesting a specific action upon neuronal circuits involved in the regulation of feeding. Recent experiments have also shown that GLP-1 is implicated in mediating signals from the gastrointestinal tract pertaining to discomfort and malaise. The distribution of the co-localised peptide, GLP-2, displays a perfect overlap with GLP-1 in the CNS with the highest concentration in the diffuse ventral part of the dorsomedial nucleus (DMHv). In contrast to the widely distributed GLP-1 receptor mRNA, GLP-2 receptor mRNA is exclusively expressed in the compact part of the DMH (DMHc). Interestingly, the DMHc is also the only nucleus responding to central administration of GLP-2 with a significant increase in the number of c-Fos positive cells. When injected into the lateral ventricle, GLP-2 has a marked inhibitory effect on feeding. The effect of GLP-2 on feeding is both behaviourally and pharmacologically specific. Future experiments will elucidate whether or not GLP-1 and GLP-2 are involved in the long-term or short-term regulation of feeding behaviour and hence have an impact on bodyweight.

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake.

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9-39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.

Central administration of cocaine-amphetamine-regulated transcript activates hypothalamic neuroendocrine neurons in the rat.

We have recently shown that intracerebroventricular (i.c.v.) administration of the hypothalamic neuropeptide cocaine-amphetamine-regulated transcript (CART) inhibits food intake and induces the expression of c-fos in several nuclei involved in the regulation of food intake. A high number of CART-induced c-Fos-positive nuclei in the paraventricular nucleus of the hypothalamus prompted us to examine the effect of i.c.v. recombinant CART-(42-89) on activation of CRH-, oxytocin-, and vasopressin-synthesizing neuroendocrine cells in the paraventricular nucleus (PVN). In addition, plasma levels of glucose were examined after central administration of CART-(42-89). Seventy-six male Wistar rats were fitted with i.c.v. cannulas and singly housed under 12-h light, 12-h dark conditions. One week postsurgery the animals were injected i.c.v. in the morning with 0.5 microg recombinant CART-(42-89) or saline. Trunk blood was collected by decapitation at 0 (baseline), 10, 20, 40, 60, 120, or 240 min. CART caused a strong increase in circulating corticosterone that was significantly different from saline at 20, 40, 60, and 120 min postinjection (P<0.05). Furthermore, CART caused a transient rise in plasma oxytocin levels (P<0.05 at 10 and 20 min postinjection), whereas plasma vasopressin levels were unaffected by i.c.v. CART. Animals injected i.c.v. with CART showed a rise in blood glucose levels 10 min postinjection (P<0.05). To examine whether the stimulatory effect of i.c.v. CART on corticosterone and oxytocin secretion is caused by activation of paraventricular nucleus/supraoptic nucleus (PVN/SON) neuroendocrine neurons, we used c-Fos as a marker of neuronal activity. Animals injected with CART showed a strong increase in c-Fos-immunoreactive nuclei in the PVN. Double immunohistochemistry revealed that a high (89+/-0.4%) number of CRH-immunoreactive neurons in the PVN contained c-Fos after CART i.c.v.. c-Fos expression was also observed in oxytocinergic cells (in both magnocellular and parvicellular PVN neurons as well as in the supraoptic nuclei) 120 min after CART administration, whereas none of the vasopressinergic neurons contained c-Fos. Triple immunofluorescence microscopy revealed that CART-immunoreactive fibers closely apposed c-Fos-positive CRH neurons, suggestive of a direct action of CART on PVN CRH neurons. In summary, i.c.v. CART activates central CRH neurons as well as both magnocellular (presumably neurohypophysial) and parvicellular (presumably descending) oxytocinergic neurons of the PVN. The effect of CART on CRH neurons most likely leads to corticosterone secretion from the adrenal gland, which may contribute to the inhibitory effects of CART on feeding behavior.

Chronic intracerebroventricular administration of recombinant CART(42-89) peptide inhibits and causes weight loss in lean and obese Zucker (fa/fa) rats.

OBJECTIVE: Hypothalamic neuropeptide CART (cocaine-amphetamine-regulated transcript) is a leptin-dependent endogenous satiety factor in the rat, and single central injections of recombinant CART(42-89) lowers food intake in rats and mice. To assess the potential role of CART as a long-term regulator of food intake, we investigated the effects of continuous infusion of recombinant CART(42-89) on food consumption and body weight. RESEARCH METHODS AND PROCEDURES: Two doses of CART(42-89) were tested: 12 or 4.8 microg/d. Adult male, both lean (+/?) and Zucker (fa/fa) obese, rats were equipped with intracerebroventricular cannulae in the right lateral ventricle. The cannulae were connected to subcutaneously placed osmotic mini-pumps. Pumps were filled with either CART(42-89) or vehicle (50 mM phosphate-buffered saline, pH 7.4). The pumps delivered a continuous infusion of CART(42-89) or vehicle, and food intake and body weight were followed for 10 days (12 microg/d) or 7 days (4.8 microg/d). Animals given the low dose had the pump removed on Day 7, and from half of the group, trunk blood was collected after decapitation, whereas the other half of the group had their mini-pumps removed and were followed for another 7 days before being decapitated. RESULTS: Animals receiving the high doses displayed overt motor disturbances, whereas the low dose was devoid of such behavioral side effects. Both doses significantly lowered food intake with maximal effect on days 3 to 5 of the infusion period. The high dose of CART decreased body weight of normal animals to 85% of initial weight at days 3 to 5, whereas the weight of Zucker (fa/fa) obese rats dropped to 95% of the initial weight. In animals receiving 4.8 microg/d, moderate effects on body weight were seen between days 4 and 6 of the treatment period, but soon after termination of the treatment animals regained lost weight. To assess the biological activity of the contents of the osmotic mini-pumps, the pumps were removed from the subcutaneous implantation site, and 5 microL of their contents were injected intracerebroventricularly to naive animals kept on a restricted feeding schedule. The content of pumps from animals receiving 4.8 microg/d of CART(42-89) potently inhibited food intake, confirming full biological activity despite being kept for 7 days at body temperature. DISCUSSION: Due to obvious effects on motor behavior, it is impossible with certainty to conclude that the observed effects on feeding and body weight are primary interference with satiety centers or secondary to effects on locomotor pathways. Also, the present experiments suggest that hypothalamic appetite-regulating neurons are subject to pharmacological desensitization upon prolonged exposure to CART peptide. The underlying mechanism of such desensitization is as yet unknown.

Enhanced masking response to light in hamsters with IGL lesions.

Syrian hamsters with intergeniculate leaflet or sham lesions were given tests with a series of light pulses of gradually decreasing intensities. The light pulses were given early in the night, at zeitgeber time 14-15. The amount of wheel running during the pulses was compared to that in the same hour on a night with no light pulses. Hamsters with intergeniculate leaflet lesions showed a significantly greater suppression of their wheel running in response to light than the sham-lesioned animals. The lesioned animals also had larger negative phase angles of entrainment to the 14:10-h light-dark cycle than sham-operated controls. However, phase shifting in response to light pulses at either zeitgeber time 14 or 18 was not significantly altered by the lesions. Preferences for spending more time in a dark than a light area were not abolished by the lesions. It is concluded that the intergeniculate leaflet in the Syrian hamster cannot be of paramount importance for masking of locomotor activity by light but may play a modulating role.

The arcuate nucleus is pivotal in mediating the anorectic effects of centrally administered leptin.

The adipose tissue hormone leptin, which is secreted to the general circulation and transported into the brain in a facilitated manner, possibly acts via hypothalamic neurones to reduce food intake and increase energy expenditure. To evaluate the involvement of importance of the arcuate nucleus in leptin induced anorexia, groups of rats treated neonatally with monosodium-glutamate (MSG; arcuate lesioned) and littermate controls were injected centrally with 5 microg recombinant leptin or saline daily for three consecutive days. Leptin significantly inhibited food intake and caused weight-loss in non-MSG rats (-14.5+/-3.0 g vs. 10.2+/-4.3 g; mean +/-s.e.m.; leptin vs. vehicle) whereas MSG-treated rats were unresponsive to leptin treatment (5.0+/-2.2 g vs. 0.8+/-3.8 g; leptin vs. vehicle). The present data indicate that an intact arcuate nucleus is necessary for leptins actions on food intake and body weight.

Neurochemical characterization of hypothalamic cocaine- amphetamine-regulated transcript neurons.

The novel neuropeptide cocaine-amphetamine-regulated transcript (CART) is expressed in several hypothalamic regions and has recently been shown to be involved in the central control of food intake. To characterize the hypothalamic CART neurons and understand the physiological functions they might serve, we undertook an in situ hybridization and immunohistochemical study to examine distribution and neurochemical phenotype of these neurons. In situ hybridization studies showed abundant CART mRNA in the periventricular nucleus (PeV), the paraventricular nucleus of the hypothalamus (PVN), the supraoptic nucleus (SON), the arcuate nucleus (Arc), the zona incerta, and the lateral hypothalamic area. The distribution of CART-immunoreactive neurons as revealed by a monoclonal antibody raised against CART(41-89) displayed complete overlap with CART mRNA. Double immunohistochemistry showed co-existence of CART immunoreactivity (CART-IR) and somatostatin in some neurons of the PeV. In the magnocellular division of the PVN as well as the SON, CART-IR was demonstrated in both oxytocinergic and vasopressinergic perikarya. In the medial parvicellular region of the PVN a few CART-IR neurons co-localized galanin, but none was found to co-localize corticotropin-releasing hormone. In the Arc, almost all pro-opiomelanocortinergic neurons were shown to contain CART, whereas no co-localization of CART with NPY was found. In the lateral hypothalamic area nearly all CART neurons were found to contain melanin-concentrating hormone. The present data support a role for CART in neuroendocrine regulation. Most interestingly, CART is co-stored with neurotransmitters having both positive (melanin-concentrating hormone) as well as a negative (pro-opiomelanocortin) effect on food intake and energy balance.

Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour.

We have recently shown that the hypothalamic neuropeptide CART (cocaine-amphetamine-regulated-transcript) is a leptin dependent endogenous satiety factor in the rat. In the present study we confirm and extend our previous observations by showing that intracerebroventricular (i.c.v.) administered CART(42-89) dose-dependently inhibits 3-h food intake in food restricted rats with a lowest effective dose of 0.5 microgram. CART also potently inhibits NPY-induced food intake in satiated rats as well as nighttime food intake in free feeding animals. To identify brain areas potentially involved in mediating the anorectic effects of CART, the temporal expression pattern of the immediate early gene c-fos was examined in the central nervous system by immunohistochemistry in rats receiving recombinant CART. Compared to vehicle, CART induced c-Fos expression in several hypothalamic and brainstem structures implicated in the central control of food intake. In the hypothalamus, high numbers of c-Fos immunoreactive (-ir) cells were observed in the medial parvocellular part of the paraventricular nucleus and in the posterior part of the dorsomedial nucleus. Lower numbers of c-Fos positive nuclei were found in the supraoptic and arcuate nuclei. A relatively high number of c-Fos-ir cells was found in the central nucleus of the amygdala. In the brainstem, c-Fos-positive nuclei were found in the parabrachial nucleus, and in the nucleus of the solitary tract. Notably both the area postrema and the dorsal motor nucleus of the vagus were virtually devoid of c-Fos-ir cells. The present experiments suggest that CART peptide exerts its inhibitory effects on appetite by activating hypothalamic and brainstem neurones implicated in the central control of feeding behaviour and metabolism.

Neuronal projections from the mesencephalic raphe nuclear complex to the suprachiasmatic nucleus and the deep pineal gland of the golden hamster (Mesocricetus auratus).

Neuronal projections from the mesencephalic raphe system to the suprachiasmatic nucleus and the pineal complex were mapped in this study of the golden hamster, by use of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and the retrograde tracer cholera toxin subunit B. From the median raphe nucleus, a rostral projection ascended in the ventral part of the mesencephalon to continue in the medial forebrain bundle of the forebrain. Nerve fibres from this bundle innervated the ventral and medial parts of the suprachiasmatic nucleus. At the level of the interpeduncular nucleus of the mesencephalon, fibres of the ventral bundle bent dorsally to reach the epithalamic area and to continue in the forebrain in a periventricular position. Some of these fibres innervated the dorsal tip of the suprachiasmatic nucleus. The dorsal raphe nucleus was the origin of a nerve fibre bundle, located in the periaqueductal gray of the mesencephalon, innervating the deep pineal gland and pineal stalk. Injection of cholera toxin B into the suprachiasmatic nucleus labelled cells in the median raphe. Combination of the retrograde tracing from the suprachiasmatic nucleus and serotonin transmitter immunohistochemistry showed that some of the cholera toxin B-immunoreactive nerve cells also contained serotonin. Thus, this study of the golden hamster shows a serotonergic projection from the median raphe nucleus to the suprachiasmatic nucleus and a projection from the dorsal raphe nucleus to the deep pineal gland supporting physiological indications of an influence of serotonin on the photoreceptive circadian system of the brain.

Hypothalamic CART is a new anorectic peptide regulated by leptin.

The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

Revolutionary science: an improved running wheel for hamsters.

Golden hamsters, Mesocricetus auratus, ran more in wheels with the floor covered by a plastic mesh than in wheels with the usual rods. This preference was evident both in tests with a single wheel and in tests when the animals were offered a choice between two wheels. Phase shifts following a 3h confinement to a novel wheel were greater if the novel wheel had the plastic cover.