Cocaine- And Amphetamine-regulated Transcript Expression Research Articles

Cocaine and amphetamine regulated transcript (CART): a newly characterized neuropeptide in human prostate

ABSTRACT Cocaine and amphetamine regulated transcript (CART) is a somatostatin-like polypeptide. CART has been localized in the CNS, hypothalamo-pituitary-adrenocortical (HPA) axis, pancreatic islets and enteric nervous system. We investigated the cellular localization of CART in normal human prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia and acinar adenocarcinoma. CART was assessed using immunohistochemistry (IHC) and in situ hybridization (ISH), and its gene expression was identified by RTqPCR. We found cellular expression of CART in both normal prostatic luminal secretory epithelial cells neuroendocrine cells (NEC) of both ducts and acini. The cellular appearance indicated a cycle of neuropeptide synthesis and secretion as validated by ISH/IHC concordance. RTqPCR analysis also validated the immunohistochemical data and gene expression, which both indicated low to moderate expression in prostatic tissues. CART expression also was increased in both neuroendocrine and glandular epithelial cell populations from samples of benign prostatic hyperplasia as validated by IHC, ISH and RTqPCR. CART expression was markedly diminished and, in some cases, entirely absent in tissues of prostatic intraepithelial neoplasia and adenocarcinoma. Owing to loss of CART expression in adenocarcinoma and its increase in benign prostatic hyperplasia, CART may prove to be an important prostate marker.

Cocaine- and Amphetamine-Regulated Transcript (CART) Peptide Is Co-Expressed with Parvalbumin, Neuropeptide Y and Somatostatin in the Claustrum of the Chinchilla.

Although for many years, researchers have been working on understanding the function of the cocaine- and amphetamine-regulated transcript (CART) peptide at the central- and peripheral-nervous-system level, data describing the presence of CART in the claustrum are still missing. Therefore, the aim of the present study was to immunohistochemically investigate the CART expression in the claustrum neurons in chinchillas as well as the CART co-localization with somatostatin (SOM), parvalbumin (PV), and neuropeptide Y (NPY) using double-immunohistochemical staining. The claustrum is divided into two main parts: the dorsal segment (CL), which is located above the rhinal fissure, and the ventral segment (EN), located below the rhinal fissure. The presence of HU C/D-IR CART-IR-positive neurons was detected in both the insular claustrum (CL) and the endopiriform nucleus (EN). The vast majority of CART-IR neurons were predominantly small and medium in size and were evenly scattered throughout the claustrum. CART co-localization with selected neurotransmitters/neuromodulators (SOM, NPY, and PV) showed the presence of a CART-IR reaction only in the neurons, while the nerve fibers were, in all cases, devoid of the CART-IR response. Our research supplements missing knowledge about the distribution and co-localization pattern of CART with SOM, NPY, and PV in the chinchilla claustrum, and also provides a better understanding of the similarities and differences compared to other species of rodents and other mammals.

Cocaine and Amphetamine Regulated Transcript (CART) Expression Changes in the Stomach Wall Affected by Experimentally Induced Gastric Ulcerations.

Cocaine- and amphetamine-regulated transcript (CART) is a peptide suggested to play a role in gastrointestinal tract tissue reaction to pathology. Gastric ulceration is a common disorder affecting huge number of people, and additionally, it contributes to the loss of pig livestock production. Importantly, ulceration as a focal disruption affecting deeper layers of the stomach wall differs from other gastrointestinal pathologies and should be studied individually. The pig’s gastrointestinal tract, due to its many similarities to the human counterpart, provides a valuable experimental model for studying digestive system pathologies. To date, the role of CART in gastric ulceration and the expression of the gene encoding CART in porcine gastrointestinal tube are completely unknown. Therefore, we aimed to verify the changes in the CART expression by Q-PCR (gene encoding CART in the tissue) and double immunofluorescence staining combined with confocal microscopy (CART immunofluorescence in enteric nervous system) in the porcine stomach tissues adjacent to gastric ulcerations. Surprisingly, we found that gastric ulcer caused a significant decrease in the expression of CART-encoding gene and huge reduction in the percentage of CART-immunofluorescent myenteric perikarya and neuronal fibers located within the circular muscle layer. Our results indicate a unique CART-dependent gastric response to ulcer disease.

Regulation of Pituitary Cocaine- and Amphetamine-Regulated Transcript Expression and Secretion by Hypothalamic Gonadotropin-Releasing Hormone in Chickens.

There is increasing evidence that cocaine- and amphetamine-regulated transcript (CART) peptide is abundantly expressed in the anterior pituitary of birds and mammals, suggesting that CART peptide may be a novel pituitary hormone and its expression and secretion is likely controlled by the hypothalamic factor(s). To substantiate this hypothesis, using chicken as an animal model, we examined the effects of gonadotropin-releasing hormone (GnRH) on pituitary CART secretion and expression and investigated whether GnRH could modulate plasma CART levels. The results showed that: (1) chicken GnRH (GnRH1 and GnRH2) could potently stimulate CART peptide secretion in intact pituitaries incubated in vitro, as detected by Western blot; (2) GnRH could also stimulate CART mRNA expression in cultured pituitary cells, as revealed by quantitative real-time polymerase chain reaction (qPCR) assay; (3) GnRH actions on pituitary CART expression and secretion are likely mediated by GnRH receptor coupled to the intracellular Ca2+, MEK/ERK, and cAMP/PKA signaling pathways; and (4) plasma CART levels are high in chickens at various developmental stages (1.2–3.5 ng/ml) and show an increasing trend towards sexual maturity, as detected by enzyme-linked immunosorbent assay (ELISA). Moreover, plasma CART levels could be significantly induced by intraperitoneal administration of GnRH in chicks. Taken together, our data provide the first collective evidence that CART peptide is a novel pituitary hormone and its expression and secretion are tightly controlled by hypothalamic GnRH, thus likely being an active player in the hypothalamic-pituitary-gonadal (HPG) axis.

Experimental Pediatric Obesity Impairs Central Control of Food Behavior

According to WHO in 2016, 41 million children in their frist5 years of age are either overweight or obese. Overweight and obese children have a higher risk to stay obese into adulthood and present more incidences to develop chronic diseases. The control of the food and energy intake is essentially controlled and integrated by hypothalamic neuropeptide with anorexigenic and orexigenic functions such as ‘cocaine‐and‐amphetamine‐regulated transcript (CART) co‐expressing neurons, which suppress feeding behavior or activation of agouti related‐peptide (AgRP) co‐expressing neurons that induces an increase in appetite response. Additionally to the peptides, specific receptors also play a key role in the control of the food behavior, such as 5‐HT1b and 5‐HT2c, acting regulating meal size and eating rate in association to the neuropeptides. Until the moment the effect of pediatric obesity and the expression of these neuropeptides and receptors remains to be more explored. Three days after delivery, the puppies were randomly assigned into two groups: Controls (nine pups per mother, n=6) or Overfed (three pups per mother, n=6). At 21 days, all male pups began to receive water and commercial chow ad libitum until 60 days of age. We evaluate body weight‐BW, 24 hours food intake‐FI, blood glucose and triglycerides levels, CART, AgRP, 5‐HT1b, and 5‐HT2c gene expression. Our data demonstrate that reduction in litter size induce the increase in BW since the seventh day of life and maintains until the sacrifice (7, 22, 60th, p<0.001). Associated with BW, we observed an increase in FI in dark cycle (p<0.01) and in light cycle (p<0.05). Evaluating serum patterns, we verify an increase in glucose (p<0.05) and in triglycerides (p<0.001). At the molecular levels, we observe an impressive decrease in CART expression (p<0.001) in the overfed group. Taken our data together, we suggest that neonatal overfed induce energetic disturb increasing the risk of metabolic syndrome in a possible mechanism mediated to alteration on neuropeptide signaling in the central control of food behavior.Support or Funding InformationFACEPE APQ grant #0164‐4.05/15; This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance code 001This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P5-05-07: Expression of the cocaine- and amphetamine-regulated transcript (CART) recruits SWI/SNF chromatin remodelling complexes to the estrogen receptor

Abstract Introduction Cocaine- and amphetamine-regulated transcript (CART) peptides are neuropeptides involved in regulating physiological processes, such as feeding and drug reward. Recent studies have associated high CART expression with worse overall survival in patients with small-bowel carcinoid tumours and estrogen receptor-positive (ER+), lymph node-negative breast cancer. CART was also shown to be associated with poor patient response to tamoxifen, suggesting CART may play a role in conferring tamoxifen resistance. Materials and methods We have previously demonstrated that CART can impact the transcriptional activity of ERα through the use of western blotting and qPCR for specific ERα gene targets. RNA sequencing was carried out using a stable CART-inducible cell line model to identify genes which are upregulated/downregulated in cells expressing CART. Further, using our stable CART-inducible cell line model, we preformed ERα-Immunoprecipitation followed by in-solution mass spectrometry to identify differentially recruited protein complexes +/- CART expression. Results and discussion RNA sequencing revealed 156 significantly downregulated, and 100 significantly upregulated, genes in cells expressing CART (p<0.05). Through mining of publicly available ERα ChIP-seq data sets, both upregulated and downregulated gene sets were found to contain genes which have previously been shown to contain ERα binding events within their promotor regions. Mass spectrometry analysis revealed that the majority of proteins recruited to ERα in the presence of CART were members of the SWI/SNF (BAF) chromatin remodelling complex. The identification of SMARCD1 within this complex was of particular interest to this study, as this protein has previously been reported to be a critical mediator of nuclear receptor function. Further in silico analysis demonstrated high expression of SMARCD1 correlates with poor overall survival (OS) (p<0.00001) and distant metastasis free survival (DMFS) (p=0.00708) in a cohort of ER+ breast cancer patients. Intriguingly, SMARCD1 expression did not correlate with poor OS or DMFS in a cohort of ER- breast cancer patients, suggesting that this negative impact on survival is dependent on ER status. Conclusion In conclusion, we suggest that CART expression results in the recruitment of chromatin remodelling complexes to ERα in order to facilitate the regulation of receptor function and this impacts on patient outcome. Citation Format: O'Connor DP, Mooney B, Das S, Klinger R, Moran B, Ni Chonghaile T, Cagney G, Bracken A, Gallagher WM. Expression of the cocaine- and amphetamine-regulated transcript (CART) recruits SWI/SNF chromatin remodelling complexes to the estrogen receptor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-07.

Chronic Stress Combined with a Fructose Diet Reduces Hypothalamic Insulin Signaling and Antioxidative Defense in Female Rats

Background: Increased fructose consumption and chronic exposure to stress have been associated with the development of obesity and insulin resistance. In the hypothalamus, a crossroad of stress responses and energy balance, insulin and glucocorticoids regulate the expression of orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), and anorexigenic neuropeptides, proopio­melanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Objectives: We investigated whether chronic stress and fructose diet disrupt these hormonal signaling pathways and appetite control in the hypothalamus, contributing to the development of insulin resistance and obesity. Potential roles of hypothalamic inflammation and oxidative stress in the development of insulin resistance were also analyzed. Methods: Insulin, glucocorticoid, and leptin signaling, expression of orexigenic and anorexigenic neuropeptides, and antioxidative and inflammatory statuses in the whole hypothalamus of fructose-fed female rats exposed to unpredictable stress for 9 weeks were analyzed using quantitative PCR and Western blotting. Results: Chronic stress combined with a fructose-enriched diet reduced protein content and stimulatory phosphorylation of Akt kinase, and elevated 11β-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression, while alterations in appetite regulation (NPY, AgRP, POMC, CART, leptin receptor, and SOCS3 expression) were not observed. The expression of antioxidative defense enzymes (mitochondrial manganese superoxide dismutase 2, glutathione reductase, and catalase) and proinflammatory cytokines (IL-1β, IL-6, and TNFα) was reduced. Conclusions: Our results underline the combination of long-term stress exposure and fructose overconsumption as more detrimental for hypothalamic function than for either of the factors separately, as it enhanced glucocorticoid and impaired insulin signaling, antioxidative ­defense, and inflammatory responses of this homeostasis- regulating center.

Cocaine‑ and amphetamine‑regulated transcript (CART) is associated with dopamine and is protective against ischemic stroke.

Cocaine and amphetamine-regulated transcript (CART) is a neuropeptide that can protect brains against ischemic injury. The aim of the present study was to investigate the effects of the CART within ischemic stroke and it possible mechanism. The expression levels of dopamine (DA) and CART in ischemic brain tissues of mice were measured following middle cerebral artery occlusion (MCAO). After receiving the treatment of DA and CART, the infarct volume of brain was measured in mice with MCAO. In addition, the function and potential mechanism of CART in ischemic stroke were further investigated. DA and CART expression was significantly decreased in mice with MCAO compared with normal control mice. Treatment of mice with MCAO with exogenous CART significantly decreased the extent of brain injury compared with untreated mice with MCAO. Treatment with exogenous CART promoted the survival of ex vivo neurons following oxygen-glucose deprivation (OGD), while exogenous DA induced CART mRNA expression in a dose-dependent manner, which suggested an association between CART and DA. Apoptosis of ex vivo neurons was significantly increased following OGD, however treatment with exogenous CART significantly inhibited this effect. The potential mechanism of CART was determined to be associated with inflammatory cytokines and related apoptotic genes. CART therefore appears to be associated with DA in its effect on ischemic stroke and is protective against brain injury following ischemic stroke by reducing inflammation activation; it may provide a promising means to treat ischemic stroke patients.

Impaired hypothalamic cocaine- and amphetamine-regulated transcript expression in lateral hypothalamic area and paraventricular nuclei of dehydration-induced anorexic rats.

Negative energy balance promotes physiological adaptations that ensure the survival of animals. The hypothalamic-pituitary-thyroid axis regulates basal energy expenditure and its down-regulating adaptation to negative energy balance is well described: in fasting, the serum content of thyrotrophin (TSH) and thyroid hormones (TH) decreases, enhancing the survival odds of individuals. By contrast, dehydration-induced anorexic (DIA) rats present an impaired hypothalamic-pituitary-thyroid (HPT) axis adaptation despite their negative energy balance: increased circulating TSH levels. The implication of cocaine- and amphetamine-regulated transcript (CART), an anorexic peptide, in HPT axis function impairment and food-avoidance behaviour displayed by DIA animals is unknown. Because CART is co-expressed with the peptide that regulates the HPT axis in hypophysiotrophic paraventricular nucleus (PVN) neurones (TSH-releasing hormone), we analysed CART expression and possible implications with respect to high TSH levels of DIA animals. We examined whether changes in CART expression from the lateral hypothalamic area (LHA) and arcuate nucleus (ARC) could participate in food-avoidance of DIA rats. DIA and forced-food restricted (FFR) animals reduced their body weight and food intake. FFR rats had a down-regulation of their HPT axis (reduced serum TH and TSH content), whereas DIA animals had reduced TH but increased TSH levels. CART mRNA expression in the ARC decreased similarly between experimental groups and diminished in anterior, medial PVN and in LHA of FFR animals, whereas DIA animals showed unchanged levels. This impaired CART mRNA expression in the anterior PVN and LHA could be related to the aberrant feeding behaviour of DIA rats but not to their deregulated HPT axis function.

Short-Term High-Fat Diet Increases Leptin Activation of CART Neurons and Advances Puberty in Female Mice.

Leptin is a permissive factor for puberty initiation, participating as a metabolic cue in the activation of the kisspeptin (Kiss1)-gonadotropin-releasing hormone neuronal circuitry; however, it has no direct effect on Kiss1 neurons. Leptin acts on hypothalamic cocaine- and amphetamine-regulated transcript (CART) neurons, participating in the regulation of energy homeostasis. We investigated the influence of a short-term high-fat diet (HFD) on the effect of leptin on puberty timing. Kiss1-hrGFP female mice received a HFD or regular diet (RD) after weaning at postnatal day (PN)21 and were studied at PN28 and PN32. The HFD increased body weight and plasma leptin concentrations and decreased the age at vaginal opening (HFD, 32 ± 0.53 days; RD, 38 ± 0.67 days). Similar colocalization of neurokinin B and dynorphin in Kiss1-hrGFP neurons of the arcuate nucleus (ARC) was observed between the HFD and RD groups. The HFD increased CART expression in the ARC and Kiss1 messenger RNA expression in the anteroventral periventricular (AVPV)/anterior periventricular (Pe). The HFD also increased the number of ARC CART neurons expressing leptin-induced phosphorylated STAT3 (signal transducer and activator of transcription 3) at PN32. Close apposition of CART fibers to Kiss1-hrGFP neurons was observed in the ARC of both RD- and HFD-fed mice. In conclusion, these data reinforce the notion that a HFD increases kisspeptin expression in the AVPV/Pe and advances puberty initiation. Furthermore, we have demonstrated that the HFD-induced earlier puberty is associated with an increase in CART expression in the ARC. Therefore, these data indicate that CART neurons in the ARC can mediate the effect of leptin on Kiss1 neurons in early puberty induced by a HFD.

Roles of hypothalamic neuropeptide gene expression in body mass regulation in Eothenomys miletus (Mammalia: Rodentia: Cricetidae)

To investigate the roles of hypothalamic neuropeptide expression in body mass regulation in Eothenomys miletus, body mass, food intake, serum leptin levels and hypothalamic neuropeptide expression were measured in seasonal-acclimatized and lab-acclimated animals. The results showed that body mass, body fat mass and food intake showed seasonal variations. Serum leptin levels also appeared similar seasonal variation, showing a similar trend to body fat mass. Expression of hypothalamic neuropeptide Y (NPY) and agouti-related protein (AgRP) levels showed significant seasonal differences, while pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) expression showed no significant differences among seasons. Leptin had negative correlation with NPY and AgRP expression, positive correlation with POMC and CART expression among seasons. Cold and food restriction reduced body mass, body fat mass and serum leptin levels, and increased NPY and AgRP expression, but decreased POMC and CART expression. Photoperiods had no significant effect on these characters. Leptin was positively correlated with body fat mass, CART and POMC, and negatively correlated with NPY and AgRP under different temperatures, photoperiods and food quantity acclimation. All of these results showed that E. miletus reduced body mass and body fat mass, and increased food intake to survive in winter under seasonal changes. Temperature and food resources were the key factors influencing the body mass and energy metabolism in E. miletus. Leptin may play an important role in regulating body mass and energy metabolism by acting on hypothalamic neuropeptide expression in E. miletus.

Mediation of oxidative stress in hypothalamic ghrelin-associated appetite control in rats treated with phenylpropanolamine

Phenylpropanolamine (PPA)-induced appetite control is associated with oxidative stress in the hypothalamus. This study explored whether hypothalamic antioxidants participated in hypothalamic ghrelin system-associated appetite control in PPA-treated rats. Rats were given PPA daily for 4 days, and changes in food intake and the expression of neuropeptide Y (NPY), the cocaine- and amphetamine-regulated transcript (CART), superoxide dismutase, catalase, ghrelin, acyl ghrelin (AG), ghrelin O-acyltransferase (GOAT) and the ghrelin receptor (GHSR1a) were examined and compared. Results showed that both food intake and the expression of NPY and ghrelin/AG/GOAT/GHSR1a decreased in response to PPA treatment with maximum decrease on Day 2 of the treatment. In contrast, the expression of antioxidants and CART increased, with the maximum increase on Day 2, with the expression opposite to that of NPY and ghrelin. A cerebral infusion of either a GHSR1a antagonist or reactive oxygen species scavenger modulated feeding behavior and NPY, CART, antioxidants and ghrelin system expression, showing the involvement of ghrelin signaling and oxidative stress in regulating PPA-mediated appetite control. We suggest that hypothalamic ghrelin signaling system, with the help of antioxidants, may participate in NPY/CART-mediated appetite control in PPA-treated rats.

Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland

Cocaine-and Amphetamine Regulated Transcript (CART) peptide is expressed in the brain, endocrine and neuroendocrine systems and secreted into the serum. It is thought to play a role in regulation of hypothalamic pituitary functions. Here we report a spatial and temporal analysis of Cart expression in the pituitaries of adult and developing normal and mutant mice with hypopituitarism. We found that Prop1 is not necessary for initiation of Cart expression in the fetal pituitary at e14.5, but it is required indirectly for maintenance of Cart expression in the postnatal anterior pituitary gland. Pou1f1 deficiency has no effect on Cart expression before or after birth. There is no 1:1 correspondence between CART and any particular cell type. In neonates, CART is detected primarily in non-proliferating, POU1F1-positive cells. CART is also found in some cells that express TSH and GH suggesting a correspondence with committed progenitors of the POU1F1 lineage. In summary, we have characterized the normal temporal and cell specific expression of CART in mouse development and demonstrate that postnatal CART expression in the pituitary gland requires PROP1.

Abstract 2771: Epigenetic regulation of the cocaine and amphetamine regulated transcript (CART) in cancer

Abstract The cocaine and amphetamine regulated transcript (CART) is well documented in the central nervous system for its role in appetite regulation and reward pathways. In recent studies CART has been proven to be a poor prognostic marker in lymph node-negative, estrogen receptor (ER)-positive breast cancer patients, and has also been shown to be associated with worse survival in small bowel carcinoid tumours. The regulation of CART in these different cancer types however remains largely unknown. The presence of a large CpG island in the putative promoter region and the lack of expression of CART in many cancer cell lines suggest a possible epigenetic mechanism of suppression. A panel of various cancer cell lines (breast, colorectal, endometrial and ovarian) were assessed for endogenous CART expression via western blot and RT-PCR. Cells were then treated with forskolin and leptin; agents which have been previously shown to induce CART expression in the CNS. Cells were also treated with CART conditioned media, which has been previously shown to be the only factor capable of inducing CART expression. Treatment was carried out with varying doses of two DNA methyltransferase inhibitors, 5-Aza-2′-deoxycytidine and RG108, followed by examination of CART expression post treatment. Bisulfite sequencing of the promoter region of CART was performed in all cell lines and a small selection of formalin fixed paraffin embedded (FFPE) tissue samples. Whole methylome sequencing was carried out to confirm this promoter region of CART and study the global effects of treatment with epigenetic modifiers and CART peptide. Assessment of a collection of cell lines via western blot has shown CART expression to be negative across the panel. Moreover, treatment with forskolin and leptin did not result in re-expression of CART. Treatment with 5-Aza-2′-deoxycytidine has however resulted in increased CART mRNA levels, and subsequent bisulfite sequencing of the promoter region of CART has revealed dense methylation at the CpG island. Treatment with CART conditioned media may also induce promoter demethylation and subsequent re-expression of CART. Collectively, this data suggests the potential involvement of DNA methylation in the regulation of CART. Citation Format: Kate Connor, Sudipto Das, Bruce Moran, Laoighse Mulrane, William M. Gallagher, Darran O’Connor. Epigenetic regulation of the cocaine and amphetamine regulated transcript (CART) in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2771.

Abstract 4561: Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer

Abstract The cocaine- and amphetamine-regulated transcript (CART) was first discovered as a peptide up-regulated by the administration of cocaine and amphetamine to rats. CART peptides are involved in regulating physiological processes, such as feeding, reward and neuro-protection. However, recent studies have associated high CART expression with worse overall survival in patients with small-bowel carcinoid tumours [1] and estrogen receptor-positive (ER+), lymph node-negative breast cancer [2]. Interestingly, in ER+ breast cancer, CART was also shown to be associated with poor patient response to tamoxifen, suggesting CART may play a role in conferring tamoxifen resistance [2]. The aim of this study was to elucidate the mechanism(s) by which CART signals in ER+ breast cancer. In order to test whether CART could mediate the ligand-independent activation of ER-alpha, MAP-Kinase pathway activation and levels of downstream gene-targets of ER-alpha were assessed post CART stimulation. Additionally, the ability of CART to activate ER-alpha using three LXD motifs (nuclear receptor co-activator recognition motifs) present within the CART sequence was also assessed. This was achieved through selectively mutating these motifs and testing whether CART still possessed the ability to activate ER-alpha using an ERE-Dual luciferase reporter assay. Treatment of cells with CART demonstrated an increase in MAP-Kinase activity through the detection of increasing phosphorylated ERK levels. An increase in the phosphorylation of ER-alpha at serine 118 (a phosphorylation site thought to be involved in tamoxifen resistance) was also evident following CART stimulation. CART stimulation also resulted in an increase in levels of the progesterone receptor, a known ER-alpha gene target. Mutagenesis of each LXD motif within CART resulted in significant decreases in ER-alpha activity, suggesting a potential structure-function relationship between CART and ER-alpha. In conclusion, we suggest that CART can activate ER-alpha in a ligand-independent manner through the MAP-Kinase pathway, and also potentially through specific LXD motifs within its sequence. Further investigation into the relationship between CART and ER-alpha will help us gain a better understanding of, not only the potential structure-function relationship between CART and ER-alpha, but also the role CART plays with regards to tamoxifen resistance in ER+ breast cancer. [1] Landerholm K et al., Expression of cocaine- and amphetamine-regulated transcript is associated with worse survival in small bowel carcinoid tumors. Clin Cancer Res. 2012 Jul 1;18(13):3668-76 [2] DJ Brennan, DP O’Connor et al., The cocaine- and amphetamine-regulated transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer. Oncogene 2011 Dec 5. Doi: 10.1038/onc.2011.519 Citation Format: Brian Mooney, Sudipto Das, Rut Klinger, William Gallagher, Darran O’Connor. Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4561.

Nicotine regulates cocaine-amphetamine-Regulated Transcript (Cart) in the mesocorticolimbic system.

Cocaine-and-Amphetamine Regulated Transcript (CART) mRNA and peptides are intensely expressed in the brain regions comprising mesocorticolimbic system. Studies suggest that CART peptides may have a role in the regulation of reward circuitry. The present study aimed to examine the effect of nicotine on CART expression in the mesocorticolimbic system. Three different doses of nicotine (0.2, 0.4, 0.6 mg/kg free base) were injected subcutaneously for 5 days, and on day 6, rats were decapitated following a challenge dose. CART mRNA and peptide levels in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum (DST), amygdala (AMG), lateral hypothalamic area (LHA), and ventral tegmental area (VTA) were measured by quantitative real-time PCR (qPCR) and Western Blot analysis, respectively. In the mPFC, 0.4 and 0.6 mg/kg nicotine, decreased CART peptide levels whereas there was no effect on CART mRNA levels. In the VTA, a down-regulation of CART peptide expression was observed with 0.2 and 0.6 mg/kg nicotine. Conversely, 0.4 and 0.6 mg/kg nicotine increased CART mRNA levels in the AMG without affecting the CART peptide expression. Nicotine did not regulate CART mRNA or CART peptide expression in the NAc, DST, and LHA. We conclude that nicotine regulates CART expression in the mesocorticolimbic system and this regulation may play an important role in nicotine reward. Synapse 70:283-292, 2016. © 2016 Wiley Periodicals, Inc.

CART peptide and opioid addiction: Expression changes in male rat brain

Previous studies have shown the prominence of cocaine- and amphetamine-regulated transcript (CART) peptide in rewarding and reinforcing effects of drugs of abuse specially psychostimulants. The data regarding the effects of different stages of opioid addiction on CART expression and the interconnection between CART and opioids are not much available. Here we have studied the changes in the expression level of CART mRNA and protein in various parts of the brain reward pathway in different stages of opioid addiction. Groups of male rats received acute low-dose (10mg/kg), acute high-dose (80mg/kg) and chronic escalating doses of morphine. In addition, withdrawal and abstinence states were evaluated after injection of naloxone (1mg/kg) and long-term maintenance of addicted animals, respectively. Expression of CART mRNA in the brain was measured by real-time PCR method. Western blotting was used to quantify the protein level. CART mRNA and protein were both up-regulated in high-dose morphine-administered animals and also in the withdrawal group in the nucleus accumbens (NAc), striatum and prefrontal cortex (PFC). In the addicted group, CART mRNA and protein were both down-regulated in NAc and striatum. In the abstinent group, CART mRNA was down-regulated in NAc. In the hippocampus, the only observed change was the up-regulation of CART mRNA in the withdrawal group. We suggest that the modulatory role of CART peptide in rewarding and reinforcing effects of opioids weakens when opioids are used for a long time and is stimulated when acute stress such as naloxone-induced withdrawal syndrome or acute high-dose administration of morphine occurs to the animal.

Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells.

Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine-regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts.

Involvement of oxidative stress in the regulation of NPY/CART-mediated appetite control in amphetamine-treated rats

Amphetamine (AMPH) treatment can suppress appetite and increase oxidative stress in the brain. AMPH-induced appetite suppression is associated with the regulation of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. The present study explored whether antioxidants, including glutathione S-transferase (GST) and glutathione peroxidase (GP), were involved in this NPY/CART-mediated appetite control. Rats were treated daily with AMPH for four days. Changes in food intake and expression levels of hypothalamic NPY, CART, GST, and GP were examined and compared. Results showed that, in AMPH-treated rats, (1) food intake and NPY expression decreased, while CART, GST, and GP expression increased; (2) NPY knockdown in the brain enhanced the decrease in NPY and the increases in CART, GST, and GP expression; and (3) central inhibition of reactive oxygen species production decreased GST and GP and modulated AMPH anorexia and the expression levels of NPY and CART. The present results suggest that oxidative stress in the brain participates in regulating NPY/CART-mediated appetite control in AMPH-treated rats. These results may advance the knowledge regarding the molecular mechanism of AMPH-evoked or NPY/CART-mediated appetite suppression.

Analysis of sequence variability in the pig CART gene and association of polymorphism with fatness traits in a F2 population.

CART (cocaine- and amphetamine-regulated transcript) peptides are neuromodulators that are involved in appetite control and energy homeostasis. It can inhibit food intake and reduce body weight that have received much attention, but a direct and comprehensive relationship with pigs differing in fatness which could be applied to breeding well has not been established. This study aims to search for polymorphism within the porcine CART gene and evaluate the effect of specific genotypes with regards to an association with fatness traits in a F2 population consisting of 230 individuals. Screening of 2264 bp DNA fragment covering the entire CART gene revealed 29 mutations and four indels (insertion or a deletion), in which four unlinked SNPs (single-nucleotide polymorphisms) could be digested by enzymes and subsequently genotyped in two purebreds and a F2 population. Landrace (lean-type), one of purebreds, presented significantly higher CART expression level than Lantang (obese-type) in most tissues studied. Association analysis revealed that three SNPs (T415C, C640T and C847T) displayed significantly association (p < 0.05) with fatness traits. Additionally, they are in almost complete linkage disequilibrium. Western blotting experiments on these three SNPs loci revealed difference in CART expression among individuals with different genotypes, and the individuals with lower average live backfat thickness (BFAW) expressed CART protein at a bit higher level than others. Our study screened and mapped the genetic variations in the porcine CART gene, and confirmed three functional SNPs which are promising molecular markers for pig production traits.