Cocaine-amphetamine-regulated Transcript mRNA Research Articles

Nucleus accumbens cocaine-amphetamine regulated transcript mediates food intake during novelty conflict

Obesity is a persistent and pervasive problem, particularly in industrialized nations. It has come to be appreciated that the metabolic health of an individual can influence brain function and subsequent behavioral patterns. To examine the relationship between metabolic phenotype and central systems that regulate behavior, we tested rats with divergent metabolic phenotypes (Low Capacity Runner: LCR vs. High Capacity Runner: HCR) for behavioral responses to the conflict between hunger and environmental novelty using the novelty suppressed feeding (NSF) paradigm. Additionally, we measured expression of mRNA, for peptides involved in energy management, in response to fasting. Following a 24-h fast, LCR rats showed lower latencies to begin eating in a novel environment compared to HCR rats. A 48-h fast equilibrated the latency to begin eating in the novel environment. A 24-h fast differentially affected expression of cocaine-amphetamine regulated transcript (CART) mRNA in the nucleus accumbens (NAc), where 24-h of fasting reduced CART mRNA in LCR rats. Bilateral microinjections of CART 55-102 peptide into the NAc increased the latency to begin eating in the NSF paradigm following a 24-h fast in LCR rats. These results indicate that metabolic phenotype influences how animals cope with the conflict between hunger and novelty, and that these differences are at least partially mediated by CART signaling in the NAc. For individuals with poor metabolic health who have to navigate food-rich and stressful environments, changes in central systems that mediate conflicting drives may feed into the rates of obesity and exacerbate the difficulty individuals have in maintaining 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.

Regulation of CART mRNA by stress and corticosteroids in the hippocampus and amygdala

CART (Cocaine-Amphetamine-Regulated Transcript) has been shown to be regulated by corticosteroids in the hypothalamus, but its regulation by corticosteroids and stress has not been well examined in the hippocampus or the amygdala. Further, CART has been implicated in the transition to puberty. In this study we examine the effects of acute (30 min) stress on CART mRNA in prepubescent and adult rats. In addition, we examined chronic (21 day × 6 h) restraint stress upon the expression of CART mRNA in the hippocampus and the amygdala and the effects of 7 days of adrenalectomy and corticosteroid replacement upon CART expression in these regions of the adult rat brain. We found an up-regulation of CART mRNA in the central amygdala induced by acute but not chronic stress and an up-regulation in the dentate gyrus induced by chronic but not acute stress. Adrenalectomy reduced CART expression in the dentate gyrus but not the amygdala and this effect was blocked by corticosterone but not RU28,362 or aldosterone replacement, suggesting a synergism of mineralocorticoid and glucocorticoid receptors. Our data establish that CART expression is regulated by stress in a regionally and time specific manner and that CART is regulated by corticosteroid actions in the hippocampus.

Effect of Fasting on Cocaine-Amphetamine-Regulated Transcript, Neuropeptide Y, and Leptin Receptor Expression in the Non-Human Primate Hypothalamus

Leptin is a cytokine produced by white adipose tissue that circulates in direct proportion to adiposity and is an important signal of energy balance. Leptin inhibits food intake in rodents by inhibiting the orexigenic neuropetides neuropeptide Y (NPY) and agouti regulated peptide (AgRP) and stimulating the anorexigenic neuropeptides α-melanocyte-stimulating hormone (α-MSH) and cocaine-amphetamine-regulated transcript (CART). In order to extend our understanding of neuroendocrine regulation of appetite in the primate, we determined the effect of a metabolic challenge on CART, NPY, and leptin receptor (Ob-R) messenger ribonucleic acid (mRNA) in the nonhuman primate (NHP) hypothalamus. Ten adult female rhesus monkeys were either maintained on a regular diet or fasted for two days before euthanasia. CART, NPY, and Ob-R mRNA were measured by in situ hybridization histochemistry (ISHH). A 2-day fast decreased CART expression in the ARC, increased NPY gene expression in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and increased Ob-R expression in the ventromedial nucleus (VMN). This is the first report that fasting inhibits CART expression and stimulates Ob-R expression in monkeys. Increased NPY expression in the SON and PVN, but not the ARC of fasted monkeys also is novel. With some exceptions, our observations are confirmatory of findings in rodent studies. Similarities in the neuroendocrine responses to a metabolic challenge in monkeys and rodents support extending existing hypotheses of neuroendocrine control of energy homeostasis to primates.

Cocaine‐amphetamine‐regulated transcript (CART) gene expression is regulated by intracellular calcium

CART peptides are involved in numerous biochemical processes, including the reinforcement properties of psychostimulants. CART expression appears to be regulated via phosphorylated CREB (pCREB). Because, pCREB and the reinforcing actions of drugs of abuse are affected by Ca2+-mediated signaling, we hypothesized that increased intracellular Ca2+ levels may effect CART expression. GH3 cells, a rat pituitary cell line known to express endogenous CART, were treated with various concentrations of ionomycin, a Ca2+ ionophore. Several subsequent experiments support the involvement of Ca2+ in CART regulation. For instance, electrophoretic mobility shift assays demonstrated increased pCREB binding to the CRE oligo from the CART promoter after ionomycin treatment. Real-time PCR showed a time- and dose- dependent increase in CART mRNA levels. Finally, western blot analysis revealed elevated pCREB following ionomycin treatment. Furthermore, pretreatment with several inhibitors of Ca2+-mediated signalling, including calmidazolium, KN93, and BAPTA, reduced ionomycin-induced CART expression. In-vivo studies demonstrated that intra-accumbal injection of ionomycin increased CART mRNA levels in the rat nucleus accumbens as measured by in situ hybridization. Calmidazolium attenuated ionomycin–induced CART mRNA expression. These results suggest that the CART gene may be regulated by elevated Ca2+ via a calmodulin/pCREB signalling pathway in vitro in GH3 cells and, in vivo in rat nucleus accumbens. Supported by NIH grants RR00165, DA00418, DA15162. 1st and 2nd authors contributed equally to this work

Cocaine-Amphetamine-Regulated Transcript Expression in the Rat Nucleus Accumbens Is Regulated by Adenylyl Cyclase and the Cyclic Adenosine 5′-Monophosphate/Protein Kinase A Second Messenger System

Cocaine-amphetamine-regulated transcript (CART), a neuropeptide involved in the brain's reward/reinforcement circuit, modulates the effects of psychostimulants, including cocaine. The CART gene has been characterized, and binding sites for multiple transcription factors have been identified within the promoter region, including the cAMP-response element, which serves as a binding site for cAMP-response element-binding protein (CREB). CART expression appears to be regulated via cAMP/protein kinase A (PKA)/CREB-mediated signaling in cell culture. Therefore, the goal of these studies was to examine the involvement of cAMP/PKA/CREB-mediated signaling in CART mRNA and peptide expression in vivo in the rat nucleus accumbens. Intra-accumbal injections of forskolin, an adenylyl cyclase activator, stimulated the phosphorylation of CREB and increased both CART mRNA and peptide levels, an effect attenuated by inhibition of PKA with H89 [N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide hydrochloride] and adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In addition, Rp-cAMPS alone decreased CART mRNA compared with saline-injected controls, suggesting that CART expression may be tonically regulated by PKA. Under certain conditions, cocaine increases CART mRNA levels; thus, we examined the effects of cocaine on forskolin-induced CART mRNA expression in the rat nucleus accumbens. Cocaine plus forskolin significantly increased CART mRNA over either of the drugs administered independently, suggesting that under conditions of heightened cAMP signaling, cocaine may impact CART gene expression. These results suggest that CART expression in vivo in the rat nucleus accumbens is regulated by adenylyl cyclase and cAMP/PKA-mediating signaling and, likely, through the activation of CREB.

Effects of cocaine–amphetamine regulated transcript (CART) on hormone release

Objective: Cocaine- and amphetamine-regulated transcript (CART) is a recently described neuropeptide widely expressed in the rat brain. CART is abundant in hypothalamus nuclei controlling anterior pituitary function. In the paraventricular nucleus CART mRNA is colocalized with vasopressin and corticotrophin-releasing factor containing neurons. The data may suggest that CART plays a role in hypothalamic regulation of neuroenocrine functions. Material and methods: Male Wistar–Kyoto rats were investigated. Experiment I: CART was administered intracerebroventricularly (i.c.v.) in a dose of 0.5 μg dissolved in 5 μl vehicle. At 60, 120 min after the infusion of CART or vehicle animals were decapitated and trunk blood was collected until hormonal estimations. Experiment II: CART in a dose of 10 μg was injected intravenously (i.v.). At 60, 120, 240 min the rats were decapitated and the trunk blood was collected. Serum rLH, rFSH, rPRL, rTSH, rGH and plasma leptin, NPY concentrations were measured by RIA methods. Results: CART administered centrally (i.c.v.) simulated significantly GH release after 60 min ( p<0.05) and after 120 min ( p<0.01). CART increased also PRL after 60 min ( p<0.05). A marked increase of corticosterone level was observed at 60 and 120 min ( p<0.01, p<0.01). We did not observe significant changes in LH, FSH and TSH. We found an increase of serum leptin concentrations at 60 min after CART administration ( p<0.01). However, serum NPY levels did not change. After intravenous injection (i.v.) of CART an increase of GH was observed at 120, 240 min ( p<0.01, p<0.01, respectively). A rise in serum PRL was found at 240 min ( p<0.05). Corticosterone concentrations were enhanced at 60, 120, 240 min ( p<0.01, p<0.01, p<0.01, respectively). We did not observe significant changes in LH, FSH and TSH. Conclusions: CART may play a modulating role in the mechanism of pituitary hormone release.

Blunted hypothalamic neuropeptide gene expression in response to fasting, but preservation of feeding responses to AgRP in aging male Brown Norway rats.

Aging mammals lose the ability to maintain energy balance, exhibiting decreased appetite (anorexia) and impaired ability to maintain body weight. To determine the contribution of hypothalamic neuropeptides, two experiments were performed in male Brown Norway rats. To assess the hypothalamic neuropeptide response to food deprivation, young (Y; 4 mo old), middle-aged (M; 13 mo), and old (O; 25 mo) rats were either ad libitum fed or fasted for 72 h (n = 10/group) and killed. Hypothalamic levels of agouti-related peptide (AgRP), proopiomelanocortin (POMC), and cocaine-amphetamine-regulated transcript (CART) mRNA were assessed by in situ hybridization. With aging, arcuate AgRP gene expression decreased and CART mRNA increased, but POMC mRNA did not change. Fasting-induced changes in gene expression of all neuropeptides studied were attenuated with aging. To test the food intake response to appetite-stimulating neuropeptides, Y, M, O, and very old (VO; 33 mo) rats (n = 4-8/group) received one intracerebroventricular injection of each of three treatments: 0.1 nmol AgRP, 2.34 nmol NPY, and saline control. AgRP increased food intake of all groups by 10-20%, compared with saline, and this effect persisted up to 7 days after injection. VO animals were more sensitive to the effects of AgRP than younger animals. In contrast, NPY increased food intake more in Y than in older animals and its effects did not last >24 h. We conclude that the mechanisms by which arcuate nucleus neurons influence appetite are differentially affected by age and speculate that the melanocortin system may be a useful target for treatment of the anorexia of aging.

The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1–/–) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1–/– mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1–/– mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine–regulated transcript (CART), melanin-concentrating hormone (MCH), and prepro-orexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1–/– mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.

The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis.

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1-/-) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1-/- mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1-/- mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and preproorexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1-/- mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.

Systemic 5-hydroxy- l-tryptophan down-regulates the arcuate CART mRNA level in rats

This study was conducted to determine if serotonin (5-hydroxytryptamine; 5-HT) system correlates with the hypothalamic expression of cocaine-amphetamine-regulated transcript (CART) gene. Rats received intraperitoneal 5-hydroxy- l-tryptophan (5-HTP; a single or three daily injections at a dose of 100 mg/kg/10 ml), and CART mRNA level in the hypothalamus was examined by in situ hybridization at different time points. The 5-HT contents of the hypothalamus as well as the brainstem was increased persistently by 5-HTP injections, and food intake and body weight gain reduced. CART mRNA level decreased significantly in the hypothalamic arcuate nucleus by three daily 5-HTP, but not by a single injection. The pair-fed group of the chronic 5-HTP did not show a decrease in the arcuate CART mRNA level. The plasma leptin level markedly decreased in the chronic 5-HTP group, compared to the saline group, however, still higher than the pair-fed group with a statistical significance. These results suggest that 5-HT may suppress CART mRNA expression in the arcuate nucleus, not only by leptin signaling via its anorectic effect on the control of food intake, but also by some non-leptin mediated pathway.

Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids

Cocaine-amphetamine-regulated transcript (CART) is one of the most abundantly expressed mRNAs in the rat hypothalamus. CART mRNA expression in the arcuate nucleus has been shown to be regulated by leptin, and CART peptides have been implicated in feeding behavior and in the regulation of the HPA-axis. To more fully understand the physiological regulation of CART gene expression, we have examined the effects of adrenalectomy and different types of glucocorticoid substitution (corticosterone and dexamethasone) on hypothalamic CART and POMC mRNA levels. In situ hybridization revealed a reduction in CART mRNA levels in both the hypothalamic paraventricular and arcuate nuclei in adrenalectomized rats, which was fully restored upon dexamethasone treatment but not by a subcutaneous 25% corticosterone pellet. Unlike CART mRNA levels hypothalamic POMC expression was unaltered by adrenenalectomy. The present results show that the CART gene is influenced by glucocorticoids, presumably via a GR dependent mechanism.

Testosterone (T)-induced changes in arcuate nucleus cocaine-amphetamine-regulated transcript and NPY mRNA are attenuated in old compared to young male brown Norway rats: contribution of T to age-related changes in cocaine-amphetamine-regulated transcript and NPY gene expression.

The age-related decrease in serum T levels is associated with impairments in food intake and weight regulation and alterations in brain peptides that regulate energy balance. To test the hypothesis that reduced T levels contribute to altered hypothalamic cocaine-amphetamine-regulated transcript (CART) and NPY gene expression, the mRNA content of these neuropeptides was measured by in situ hybridization in sham-operated (intact), castrated, and T-replaced castrated young and old male Brown Norway rats. T levels in T-replaced young and old rats were similar to those in intact young animals. Compared with castrated rats, arcuate nucleus CART mRNA was lower and NPY mRNA was higher in both young and old T-replaced castrated animals, suggesting reciprocal regulation of these peptides by T; these T-induced changes were localized primarily in the rostral arcuate and were markedly attenuated in old animals. Compared with intact animals, paraventricular nucleus CART mRNA was lower in castrated animals and similar in T-replaced young and old rats. We conclude that hypothalamic CART and NPY neurons remain responsive to T regulation in old rats, albeit less so than in young animals, suggesting that the age-related reduction of T contributes in part to altered brain neuropeptide gene expression favoring anorexia and wasting with aging.

Testosterone (T)-Induced Changes in Arcuate Nucleus Cocaine-Amphetamine-Regulated Transcript and NPY mRNA Are Attenuated in Old Compared to Young Male Brown Norway Rats: Contribution of T to Age-Related Changes in Cocaine-Amphetamine-Regulated Transcript and NPY Gene Expression

The age-related decrease in serum T levels is associated with impairments in food intake and weight regulation and alterations in brain peptides that regulate energy balance. To test the hypothesis that reduced T levels contribute to altered hypothalamic cocaine-amphetamine-regulated transcript (CART) and NPY gene expression, the mRNA content of these neuropeptides was measured by in situ hybridization in sham-operated (intact), castrated, and T-replaced castrated young and old male Brown Norway rats. T levels in T-replaced young and old rats were similar to those in intact young animals. Compared with castrated rats, arcuate nucleus CART mRNA was lower and NPY mRNA was higher in both young and old T-replaced castrated animals, suggesting reciprocal regulation of these peptides by T; these T-induced changes were localized primarily in the rostral arcuate and were markedly attenuated in old animals. Compared with intact animals, paraventricular nucleus CART mRNA was lower in castrated animals and similar in T-replaced young and old rats. We conclude that hypothalamic CART and NPY neurons remain responsive to T regulation in old rats, albeit less so than in young animals, suggesting that the age-related reduction of T contributes in part to altered brain neuropeptide gene expression favoring anorexia and wasting with aging.

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.