Cocaine- And Amphetamine-regulated Transcript Immunoreactivity Research Articles

Mate calling alters expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain of male frog Microhyla nilphamariensis

Croaking is a unique component of reproductive behaviour in amphibians which plays a key role in intraspecies communication and mate evaluation. While gonadal hormones are known to induce croaking, central regulation of sound production is less studied. Croaking is a dramatic, transient activity that sets apart an animal from non-croaking individuals. Herein, we aim at examining the profile of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in actively croaking and non-croaking frog Microhyla nilphamariensis. In anurans, this peptide is widely expressed in the areas inclusive of acoustical nuclei as well as areas relevant to reproduction. CART immunoreactivity was far more in the preoptic area (POA), anteroventral tegmentum (AV), ventral hypothalamus (vHy), pineal (P) and pituitary gland of croaking frog compared to non-croaking animals. On similar lines, tissue fragments collected from the mid region of the brain inclusive of POA, vHy, AV, pineal and pituitary gland of croaking frog showed upregulation of CART mRNA. However, CART immunoreactivity in the neuronal perikarya of raphe (Ra) was completely abolished during croaking activity. The data suggest that CART signaling in the brain may be an important player in mediating croaking behaviour in the frog.

Reproductive phase related variations in the expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain and pituitary gland of adult male Microhyla ornata

Cocaine- and amphetamine-regulated transcript (CART) peptide is a multifaceted neuropeptide involved in several physiological functions including appetite and reproduction. While studies in mammals, aves and fishes suggest evolutionary conserved role of CART, the information in amphibian is scanty. We have investigated the reproductive phase related variations of CART in the brain of adult male Microhyla ornata. Seasonal changes in the expression of CART peptide were noticed in the brain and pituitary of M. ornata. Significant differences were observed in the nucleus infundibularis ventralis (NIV), epiphysis (E), anteroventral tegmental region (AV), raphe nucleus (Ra) of the brain and pars intermedia (PI), pars distalis (PD) of the pituitary. Compared to the pre-breeding and post-breeding seasons, increase in CART immunoreactivity was seen in E, NIV, AV, Ra of brain and PI, PD of pituitary gland of animals collected during breeding season. Similarly, highest mRNA levels of CART were also observed in the breeding season in the middle region of brain that includes hypothalamus and pituitary gland. Variation in the levels of CART peptide and mRNA in the brain of M. ornata suggests its conserved role in seasonal control of appetite and reproduction.

Involvement of neuropeptide CART in the central effects of insulin on feeding and body weight

While insulin secreted from pancreas plays a pivotal role in the control of glucose homeostasis, it also interacts with hypothalamic sites and negatively influences the energy balance. The present study was undertaken to reveal the functional interaction between cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, and insulin within the framework of hypothalamus in the regulation of feeding behavior and body weight. Insulin was administered daily by intracerebroventricular (icv) route, alone or in combination with CART (icv) for a period of seven days. Immediately thereafter, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, brains of insulin-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with insulin (6 mU, icv) for a period of 7 days caused a significant decrease in food intake and body weight as compared to control. Concomitant administration of CART (0.5 μg, icv) potentiated insulin-induced anorexia and weight loss. Insulin administration resulted in a significant increase in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that increased CART contents in the hypothalamus may be causally linked with anorexia and weight loss induced by insulin.

A role of neuropeptide CART in hyperphagia and weight gain induced by olanzapine treatment in rats

Although olanzapine is highly efficacious and most widely used second generation antipsychotic drug, the success of treatment has been hampered by its propensity to induce weight gain. While the underlying neuronal mechanisms are unclear, their elucidation may help to target alternative pathways regulating energy balance. The present study was undertaken to define the role of cocaine- and amphetamine-regulated transcript (CART), a well-known anorexic peptide, in olanzapine-induced hyperphagia and body weight gain in female rats. Olanzapine was administered daily by intraperitoneal route, alone or in combination with CART (intracerebroventricular) for a period of two weeks. Immediately after drug administrations, preweighed food was offered to the animals at the commencement of the dark phase. The food intake and body weight were measured daily just prior to next injection. Furthermore, the brains of olanzapine-treated rats were processed for the immunohistochemical analysis of CART-containing elements in the hypothalamus. Treatment with olanzapine (0.5 mg/kg) for the duration of 14 days produced a significant increase in food intake and body weight as compared to control. However, concomitant administration of CART (0.5 µg) attenuated the olanzapine-induced hyperphagia and weight gain. Olanzapine administration resulted in a significant reduction in CART immunoreactivity in the hypothalamic arcuate, paraventricular, dorsomedial and ventromedial nuclei. We suggest that decreased CART contents in the hypothalamus may be causally linked with the hyperphagia and weight gain induced by olanzapine.

Changes in the Distribution of Cocaine- and Amphetamine-Regulated Transcript-Containing Neural Structures in the Human Colon Affected by the Neoplastic Process.

The present study analysed changes in the distribution pattern of cocaine- and amphetamine-regulated transcript (CART) in the enteric nervous system (ENS) of the human colon challenged by adenocarcinoma invasion, using the double-labelling immunofluorescence technique. In control specimens, CART immunoreactivity was found in neurons of all studied plexuses, representing 30.1 ± 4.1%, 12.9 ± 5.2%, and 4.1 ± 1.3% of all neurons forming the myenteric plexus (MP), outer submucous plexus (OSP), and inner submucous plexus (ISP), respectively. Tumour growth into the colon wall caused an increase in the relative frequency of CART-like immunoreactive (CART-LI) neurons in enteric plexuses located in the vicinity of the infiltrating neoplasm (to 36.1 ± 6.7%, 32.7 ± 7.3% and 12.1 ± 3.8% of all neurons in MP, OSP and ISP, respectively). The density of CART-LI nerves within particular layers of the intestinal wall did not differ between control and adenocarcinoma-affected areas of the human colon. This is the first detailed description of the CART distribution pattern within the ENS during the adenocarcinoma invasion of the human colon wall. The obtained results suggest that CART probably acts as a neuroprotective factor and may be involved in neuronal plasticity evoked by the progression of a neoplastic process.

Levels of Cocaine- and Amphetamine-Regulated Transcript in Vagal Afferents in the Mouse Are Unaltered in Response to Metabolic Challenges.

Cocaine- and amphetamine-regulated transcript (CART) is one of the most abundant neuropeptides in vagal afferents, including those involved in regulating feeding. Recent observations indicate that metabolic challenges dramatically alter the neuropeptidergic profile of CART-producing vagal afferents. Here, using confocal microscopy, we reassessed the distribution and regulation of CART(55-102) immunoreactivity in vagal afferents of the male mouse in response to metabolic challenges, including fasting and high-fat-diet feeding. Importantly, the perikarya and axons of vagal C-fibers were labeled using mice expressing channelrodhopsin-2 (ChR2-YFP) in Nav1.8-Cre-expressing neurons. In these mice, approximately 82% of the nodose ganglion neurons were labeled with ChR2-YFP. Furthermore, ChR2-YFP-labeled axons could easily be identified in the dorsovagal complex. CART(55-102) immunoreactivity was observed in 55% of the ChR2-YFP-labeled neurons in the nodose ganglion and 22% of the ChR2-YFP-labeled varicosities within the area postrema of fed, fasted, and obese mice. The distribution of positive profiles was also identical across the full range of CART staining in fed, fasted, and obese mice. In contrast to previous studies, fasting did not induce melanin-concentrating hormone (MCH) immunoreactivity in vagal afferents. Moreover, prepro-MCH mRNA was undetectable in the nodose ganglion of fasted mice. In summary, this study showed that the perikarya and central terminals of vagal afferents are invariably enriched in CART and devoid of MCH.

Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig

In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed.

CART treatment improves memory and synaptic structure in APP/PS1 mice.

Major characteristics of Alzheimer’s disease (AD) include deposits of β-amyloid (Aβ) peptide in the brain, loss of synapses, and cognitive dysfunction. Cocaine- and amphetamine-regulated transcript (CART) has recently been reported to attenuate Aβ-induced toxicity. In this study, CART localization in APP/PS1 mice was characterized and the protective effects of exogenous CART treatment were examined. Compared to age-matched wild type mice, 8-month-old APP/PS1 mice had significantly greater CART immunoreactivity in the hippocampus and cortex. A strikingly similar pattern of Aβ plaque-associated CART immunoreactivity was observed in the cortex of AD cases. Treatment of APP/PS1 mice with exogenous CART ameliorated memory deficits; this effect was associated with improvements in synaptic ultrastructure and long-term potentiation, but not a reduction of the Aβ plaques. Exogenous CART treatment in APP/PS1 mice prevented depolarization of the mitochondrial membrane and stimulated mitochondrial complex I and II activities, resulting in an increase in ATP levels. CART treatment of APP/PS1 mice also reduced reactive oxygen species and 4-hydroxynonenal, and mitigated oxidative DNA damage. In summary, CART treatment reduced multiple neuropathological measures and improved memory in APP/PS1 mice, and may therefore be a promising and novel therapy for AD.

Increase in cocaine- and amphetamine-regulated transcript (CART) in specific areas of the mouse brain by acute caffeine administration

Caffeine produces a variety of behavioral effects including increased alertness, reduced food intake, anxiogenic effects, and dependence upon repeated exposure. Although many of the effects of caffeine are mediated by its ability to block adenosine receptors, it is possible that other neural substrates, such as cocaine- and amphetamine-regulated transcript (CART), may be involved in the effects of caffeine. Indeed, a recent study demonstrated that repeated caffeine administration increases CART in the mouse striatum. However, it is not clear whether acute caffeine administration alters CART in other areas of the brain. To explore this possibility, we investigated the dose- and time-dependent changes in CART immunoreactivity (CART-IR) after a single dose of caffeine in mice. We found that a high dose of caffeine (100 mg/kg) significantly increased CART-IR 2 h after administration in the nucleus accumbens shell (AcbSh), dorsal bed nucleus of the stria terminalis (dBNST), central nucleus of the amygdala (CeA), paraventricular hypothalamic nucleus (PVN), arcuate hypothalamic nucleus (Arc), and locus coeruleus (LC), and returned to control levels after 8 h. But this increase was not observed in other brain areas. In addition, caffeine administration at doses of 25 and 50 mg/kg appears to produce dose-dependent increases in CART-IR in these brain areas; however, the magnitude of increase in CART-IR observed at a dose of 50 mg/kg was similar or greater than that observed at a dose of 100 mg/kg. This result suggests that CART-IR in AcbSh, dBNST, CeA, PVN, Arc, and LC is selectively affected by caffeine administration.

Temperature dependent changes in cocaine- and amphetamine regulated transcript (CART) peptide in the brain of tadpole, Sylvirana temporalis

Cocaine- and amphetamine-regulated transcript peptide (CARTp) has emerged as a novel neurotransmitter in the brain. Although the physiological role of the peptide has been intensely investigated in mammals, its role in amphibians has not been investigated. In the present study, an attempt has been undertaken to study the expression of CART in the tadpole brain of frog Sylvirana temporalis, subjected to thermal stress. Cells with strong CART-immunoreactivity were observed in the nucleus preoptic area (NPO) of tadpoles exposed to high temperature (37±2°C) as compared to those in the tadpoles exposed to low (12±2°C) and normal (24±2°C) temperatures. In the ventromedial thalamic nucleus (VM) and nucleus posterocentralis thalami (NPC), moderate CART-ir cells were observed in the control groups while number of cells and intensity of immunoreactivity was increased in tadpoles at low and high temperatures. In the nucleus infundibularis ventralis (NIV) and raphe nucleus (RA), CART immunoreactivity increased in the low as well as high temperature treated groups. Intensely stained CART cells were observed in the pituitary of tadpoles exposed to high temperature as compared to low temperature and control groups. We suggest that CART system in the brain and pituitary of tadpole may play a very important role in mediating responses to temperature variations in the environment.

The cocaine- and amphetamine-regulated transcript, calbindin, calretinin and parvalbumin immunoreactivity in the medial geniculate body of the guinea pig

The purpose of this study was to describe the distribution and colocalization of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins (calbindin, calretinin and parvalbumin) in each main division of the medial geniculate body (MGB) in the guinea pig. From low to moderate CART immunoreactivity was observed in all divisions of the MGB, although in most of its length only fibers and neuropil were labeled. A small number of CART immunoreactive somata were observed in the caudal segment of the MGB. The central parts of all divisions contained a distinctly smaller number of CART immunoreactive fibers relative to their outer borders, where CART fibers formed patchy clusters. As a whole, the intense CART immunoreactive borders formed a shell around the weakly CART labeled core. Double-labeling immunofluorescence showed that CART did not colocalize with either calbindin, calretinin or parvalbumin, whose immunoreactivity was predominantly restricted to perikarya. The distribution pattern of calretinin was more similar to that of calbindin than to that of parvalbumin. Calretinin and calbindin exhibited higher immunoreactivity in the medial and dorsal divisions of the MGB, where parvalbumin staining was low. In general, although parvalbumin exhibited the weakest immunoreactivity of all studied Ca2+ binding proteins, it was most highly expressed in the ventral division of the MGB. Our results indicate that CART could be involved in hearing, although its immunoreactivity in the medial geniculate complex was not as intense as in other sensory brain regions. In the guinea pig the heterogeneous and complementary pattern of calbindin, calretinin and parvalbumin is evident, however, the overlap in staining appears to be more extensive than that seen in other rodents.

Effect of desipramine and citalopram treatment on forced swimming test-induced changes in cocaine- and amphetamine-regulated transcript (CART) immunoreactivity in mice.

Recent study demonstrates antidepressant-like effect of cocaine- and amphetamine-regulated transcript (CART) in the forced swimming test (FST), but less is known about whether antidepressant treatments alter levels of CART immunoreactivity (CART-IR) in the FST. To explore this possibility, we assessed the treatment effects of desipramine and citalopram, which inhibit the reuptake of norepinephrine and serotonin into the presynaptic terminals, respectively, on changes in levels of CART-IR before and after the test swim in mouse brain. Levels of CART-IR in the nucleus accumbens shell (AcbSh), dorsal bed nucleus of the stria terminalis (dBNST), and hypothalamic paraventricular nucleus (PVN) were significantly increased before the test swim by desipramine and citalopram treatments. This increase in CART-IR in the AcbSh, dBNST, and PVN before the test swim remained elevated by desipramine treatment after the test swim, but this increase in these brain areas returned to near control levels after test swim by citalopram treatment. Citalopram, but not desipramine, treatment increased levels of CART-IR in the central nucleus of the amygdala (CeA) and the locus ceruleus (LC) before the test swim, and this increase was returned to control levels after the test swim in the CeA, but not in the LC. These results suggest common and distinct regulation of CART by desipramine and citalopram treatments in the FST and raise the possibility that CART in the AcbSh, dBNST, and CeA may be involved in antidepressant-like effect in the FST.

Involvement of cocaine- and amphetamine-regulated transcript peptide in the hyperphagic and body weight promoting effects of allopregnanolone in rats

Allopregnanolone (ALLO), a gamma-aminobutyric acid (GABA) type A receptor active neurosteroid, elicits hyperphagic response in rodents. Since GABA-A receptors are present on the peptidergic neurons in the hypothalamus, we were interested in finding out if ALLO and neuropeptide cocaine- and amphetamine-regulated transcript (CART) interact and influence feeding behavior. While subcutaneous ALLO treatment, for a period of 7 days, produced a significant increase in food intake and body weight, pretreatment with subthreshold dose of CART (intracerebroventricular) attenuated both the effects. On the other hand, subcutaneous administration of dehydroepiandrosterone sulfate (DHEAS; GABA-A inhibitor neurosteroid) for a period of 7 days resulted in a significant reduction in food intake and body weight. These effects of DHEAS were potentiated by intracerebroventricular pretreatment with subeffective dose of CART. The brains of ALLO-treated rats were processed for the immunohistochemical analysis of CART immunoreactive elements. ALLO treatment resulted in a significant reduction in CART immunoreactivity in the hypothalamic arcuate, paraventricular and lateral nuclei, and nucleus accumbens shell. The results of the present study suggest that ALLO and CART might interact in the brain, and influence food intake and body weight. However, further investigations are needed to clarify the precise mechanisms by which ALLO modulate feeding behavior.

Ontogeny of cocaine- and amphetamine-regulated transcript peptide in brain of frog, Microhyla ornata

The cocaine- and amphetamine-regulated transcript (CART) peptide is widely distributed in the brains of adult vertebrates including amphibians. Several physiological roles of CART have been intensely investigated in mammals. Despite these studies, the expression of CART during development of brain has not been studied in amphibians. In the present study, distribution of CART was investigated during development in the post hatched stage 23 to premetamorphic stage 30 of frog Microhyla ornata. CART is expressed as early as in stage 23 in ventral thalamus and rhombencephalon. As development progressed, CART immunoreactivity was observed in the olfactory bulb, telencephalon, rhombencephalon and spinal cord in stage 24. At stage 25, the CART immunoreactivity was observed in the ventromedial thalamic nucleus, posterocentral thalamic nucleus, torus nucleus, central gray and inferior reticular nucleus. In stage 26, CART reactivity was seen in the medial septum, preoptic area, nucleus entopeduncularis, magnocellular nucleus, median eminence, optic tectum, hypophysis and cerebellum. Additionally, CART immunoreactivity was observed in the medial pallium, anterior commissure, nucleus infundibularis dorsalis, ventralis and raphe nucleus at stage 30. The occurrences of CART immunoreactivity at early stage of development suggest that the peptide may have a functional significance during development. The wider appearance of CART in the brain of tadpoles, M. ornata suggests that the peptide may act as a neurohormone during the ontogeny.

Distribution of the Cocaine and Amphetamine - Regulated Transcript (Cart) and Calcium Binding Proteins Immunoreactivity in the Preoptic Area of the Ram

Abstract The aim of the study was to describe the distribution of cocaine and amphetamine-regulated transcript (CART) and calcium binding proteins (CaBPs) of EF-hand family, namely calbindin, calretinin, and parvalbumin in the preoptic area (POA) of the ram. Frozen sections were processed for a routine immunofluorescence labelling. CART, calbindin, and calretinin immunoreactivity was present in neurons and fibers of the preoptic area, whereas parvalbumin showed immunoreactivity only in the POA fibers. CART displayed from a moderate to low immunoreactivity in cells and a high immunoreactivity in fibers. The highest immunoreactivity of all studied CaBPs exhibited calbindin, whereas the lowest parvalbumin. The results of the present study suggest that among the studied CaBPs, calbindin is the most likely to be involved in the participation of the important regulatory functions in the ram’s POA and the rich CART innervation seems to be strictly related to its control of the reproduction.

Reproductive phase‐related variations in cocaine‐ and amphetamine‐regulated transcript (CART) in the olfactory system, forebrain, and pituitary of the female catfish, Clarias batrachus (Linn.)

The annual sexual cycle of Clarias batrachus is divisible into resting (December-January), preparatory (February-April), prespawning (May-June), spawning (July- August), and postspawning (September-November) phases. The gonosomatic indices rose steadily through the preparatory and prespawning phases, peaked in the spawning phase, and were greatly reduced during the postspawning and resting phases. A clear pattern of change was also identified in the immunocytochemical profile of the luteinizing hormone (LH) cells in the pituitary. These changes were correlated with the cocaine- and amphetamine-regulated transcript (CART)-immunoreactive system in the forebrain and pituitary. In the olfactory bulb, CART immunoreactivity in the terminal fields of the mitral cell layer, granule cells, and medial olfactory tracts gradually decreased during the resting through prespawning phases. However, it was considerably augmented during spawning (P < 0.001) and showed highest activity in the postspawning phase (P < 0.001). A different pattern was noticed in the fibers and/or neurons of the lateral part of ventral telencephalic area, the entopeduncular nucleus, and the dorsal part of the nucleus preopticus periventricularis. In these areas, intense immunoreactivity seen in preparatory phase, declined during prespawning (P < 0.01) then through spawning, and was partially augmented during the postspawning and resting phases (P < 0.05). A similar pattern was also seen in the nucleus preglomerulosus lateralis and medialis, nucleus dorsalis posterioris of thalamus, lobobulbar nucleus, and the nucleus of posterior recess. CART was transiently expressed in LH cells in the pituitary during the preparatory period. We suggest that the CART system may play a role in triggering the brain-pituitary-ovary axis at the onset of the preparatory phase.

Cocaine- and amphetamine-regulated transcript peptide immunoreactivity in the brain of the CCK-1 receptor deficient obese OLETF rat

Cocaine- and amphetamine-regulated transcript (CART) peptide is expressed in brain areas involved in homeostatic regulation and reward. CART has been shown to reduce food intake, but the underlying mechanisms and the relevance of this effect on obesity yet remain unknown. Therefore, we used immunohistochemistry to investigate the expression of CART peptide in various brain regions of the obese Otsuka Long Evans Tokushima Fatty (OLETF) rats lacking the CCK-1 receptor. Analysis revealed that whereas the distribution of CART-peptide immunoreactive neurons and axonal networks was identical in OLETF rats and lean controls, the intensity of CART immunoreactivity was significantly reduced in the rostral part of the nucleus accumbens (p < 0.01), the basolateral complex of the amygdala (p < 0.05) and the rostro-medial nucleus of the solitary tract (p < 0.001) of the OLETF rats. These areas are involved in reward and integration of taste and viscerosensory information and have been previously associated with altered functions in this strain. The findings suggest that in addition to previously described deficits in peripheral satiety signals and augmented orexigenic regulation, the anorectic effect of CART peptide may also be diminished in OLETF rats.

Elevated Cocaine- and Amphetamine-Regulated Transcript Immunoreactivity in the Circulation of Patients with Neuroendocrine Malignancy

Cocaine- and amphetamine-regulated transcript (CART) codes for a peptide widely distributed in nervous and endocrine tissues. CART immunoreactivity (CART-LI) has been detected in human insulinomas. The objective of the study was to investigate the measurement of plasma CART-LI as a tumor marker of neuroendocrine malignancy. Plasma CART-LI levels were measured in 401 patients with a range of diagnoses: neuroendocrine malignancy (n = 131), after removal of neuroendocrine malignancy (n = 27), without any form of tumor or renal impairment (n = 192), with renal impairment (n = 17) and with nonneuroendocrine tumors (n = 34). Chromatography methods were used to investigate CART-LI circulating in human plasma. The upper limit of normal calculated for CART-LI was 150 pmol/liter. Mean circulating plasma CART-LI among neuroendocrine tumor patients was 440 pmol/liter, 56% of subjects having levels greater than 150 pmol/liter. Measuring CART-LI in addition to chromogranin (Cg)-A improved the sensitivity for neuroendocrine malignancy from 85 to 91%, whereas combined use of CgA and CgB had a joint sensitivity of 89%. Of 38 patients with pancreatic neuroendocrine tumors, 71% had plasma CART-LI levels greater than 150 pmol/liter, increasing to 95% in those classified with progressive disease (n = 20, mean CART-LI 625 pmol/liter), compared with 80% for CgA. Chromatographic analysis suggests that circulating CART-LI is present as one major form, which may correspond to CART (62-102) or another unknown form. We demonstrate CART-LI as a specific tumor marker in patients with a range of neuroendocrine tumors. Used in combination with CgA, CART-LI measurement has the potential to improve sensitivity in diagnosis and follow-up of neuroendocrine tumors, in particular progressive pancreatic neuroendocrine tumors.

Cocaine- and Amphetamine-Regulated Transcript: Stimulation of Expression in Rat Vagal Afferent Neurons by Cholecystokinin and Suppression by Ghrelin

The neuropeptide transmitter cocaine- and amphetamine-regulated transcript (CART) inhibits food intake and is expressed by both vagal afferent and hypothalamic neurons. Here we report that cholecystokinin (CCK) regulates CART expression in rat vagal afferent neurons. Thus, CART was virtually undetectable after energy restriction for 24 h, but administration of CCK to fasted rats increased CART immunoreactivity, and refeeding of fasted animals promptly increased CART by a mechanism sensitive to a CCK-1 receptor antagonist. In vagal afferent neurons incubated in serum-free medium, CART was virtually undetectable, whereas the orexigenic peptide melanin-concentrating hormone (MCH) was readily detected. The addition of CCK rapidly induced CART expression and downregulated MCH. Using a CART promoter-luciferase reporter vector transfected into cultured vagal afferent neurons, we showed that CCK stimulation of CART transcription was mediated by activation of protein kinase C and cAMP response element-binding protein (CREB). The action of CCK on CART expression was inhibited by the orexigenic peptide ghrelin, through a mechanism that involved exclusion of phosphorylated CREB from the nucleus. Thus, CCK reciprocally regulates expression of CART and MCH within the same vagal afferent neuron; ghrelin inhibits the effect of CCK at least in part through control of the nuclear localization of phosphoCREB, revealing previously unsuspected modulation of gut-brain signals implicated in control of food intake.

Characterization of Leptin-Responsive Neurons in the Caudal Brainstem

The central melanocortin system plays a key role in the regulation of energy homeostasis. Neurons containing the peptide precursor proopiomelanocortin (POMC) are found at two sites in the brain, the arcuate nucleus of the hypothalamus (ARC) and the caudal region of the nucleus of the solitary tract (NTS). ARC POMC neurons, which also express cocaine- and amphetamine-regulated transcript (CART), are known to mediate part of the response to factors regulating energy homeostasis, such as leptin and ghrelin. In contrast, the physiological role(s) of the POMC neurons in the caudal brainstem are not well characterized. However, development of a transgenic mouse expressing green fluorescent protein under the control of the POMC promoter [POMC-enhanced green fluorescent protein (EGFP) mouse] has aided the study of these neurons. Indeed, recent studies have shown significant activation of NTS POMC-EGFP cells by the gut released satiety factor cholecystokinin (CCK). Here we show that peripheral leptin administration induces the expression of phospho-signal transducer and activator of transcription 3 immunoreactivity (pSTAT3-IR), a marker of leptin receptor signaling, in more than 50% of NTS POMC-EGFP neurons. Furthermore, these POMC-EGFP neurons comprise 30% of all pSTAT3-IR cells in the NTS. Additionally, we also show that in contrast to the ARC population, NTS POMC-EGFP neurons do not coexpress CART immunoreactivity. These data suggest that NTS POMC neurons may participate with ARC POMC cells in mediating some of the effects of leptin and thus comprise a novel cell group regulated by both long-term adipostatic signals and satiety factors such as CCK.