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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Physiol Behav. 2021 Mar 8;235:113380. doi: 10.1016/j.physbeh.2021.113380

Past, Present and Future of Cocaine- and Amphetamine-Regulated Transcript Peptide

Gina LC Yosten 1,2, Christopher J Haddock 1, Caron M Harada 1,2, Gislaine Almeida-Pereira 1, Grant R Kolar 2,3, Lauren M Stein 4, Matthew R Hayes 4, Daniela Salvemini 1,2, Willis K Samson 1,2
PMCID: PMC8058305  NIHMSID: NIHMS1685137  PMID: 33705816

Abstract

The existence of the peptide encoded by the cocaine- and amphetamine-regulated transcript (Cartpt) has been recognized since 1981, but it was not until 1995, that the gene encoding CART peptide (CART) was identified. With the availability of the predicted protein sequence of CART investigators were able to identify sites of peptide localization, which then led to numerous approaches attempting to clarify CART’s multiple pharmacologic effects and even provide evidence of potential physiologic relevance. Although not without controversy, a picture emerged of the importance of CART in ingestive behaviors, reward behaviors and even pain sensation. Despite the wealth of data hinting at the significance of CART, in the absence of an identified receptor, the full potential for this peptide or its analogs to be developed into therapeutic agents remained unrealized. There was evidence favoring the action of CART via a G protein-coupled receptor (GPCR), but despite multiple attempts the identity of that receptor eluded investigators until recently. Now with the identification of the previously orphaned GPCR, GPR160, as a receptor for CART, focus on this pluripotent neuropeptide will in all likelihood experience a renaissance and the potential for the development of pharmcotherapies targeting GPR160 seems within reach.

The Past

In 1981, Spiess and colleagues (1) reported that during the purification of the somatostatin prohormone a somatostatin-like peptide was identified in ovine hypothalamus; however, they did not publish additional studies on that peptide sequence, focusing their attention instead on the posttranslational processing of the somatostatin prohormone, while simultaneously identifing the amino acid sequence of corticotropin releasing factor (CRF) (2). It wasn’t until fourteen years later in 1995, that investigators studying the effects of psychomotor stimulants on gene transcription using PCR differential display approaches identified a transcript that was up-regulated by both cocaine and amphetamine in several brain nuclei (3). A homologous transcript would later be demonstrated in human brain that encoded a peptide with only minor amino acid differences from that produced in the rat (4) and from that identified originally in ovine hypothalamus (1). A similar brain distribution of the human mRNA was observed to that reported earlier in the rat (3,4). The distribution of CART-encoding mRNA in rat brain matched that reported one year later as identified by polyclonal antibodies raised against peptide fragments (5,6) predicted from the gene sequencing work by Douglass and colleagues (3). Notably peptide and mRNA were demonstrated in areas of brain relevant to ingestive behaviors and reward, as well as the anterior and posterior pituitary, and the adrenal medulla (5). This same group reported an extensive update on CART-like immunoreactivity across multiple fore- and mid-brain areas and the dorsal horn of the spinal cord (6). Soon thereafter, tissue specific processing of the CARTpreprohormone was recognized (7) and the importance of prohormone convertases PC2 and PC1/3 identified (8).

A major advance occurred in 1998 when the first recombinant peptide was produced and was demonstrated to decrease food intake in rats (9). Both lateral and third cerebroventricular administrations were effective (9,10), and in 2001 Moran’s group provided evidence that a hindbrain site of action was essential (10). Earlier that same year, Berthoud’s group using synthetic CART (55–102) demonstrated fourth ventricular (4V) administration to exert a more potent effect than previously seen in more rostral sites, leading to the hypothesis that CART acted within brainstem structures to exert at least its anorexigenic effect (11). In 2006, Moran’s group would go on to demonstrate that the anorexigenic effect of CART was blocked by pretreatment with a glucagon-like peptide −1 (GLP-1) antagonist des-His1-Glu9-exendin4 (12), while others would similarly show that the hypothermic effects of brainstem CART also were blocked by GLP-1 antagonism (13). Interestingly, a hindbrain action of CART to reduce gastric emptying was reported to require activation of the CRF receptor (14). Thus to date, at least two unique neural circuits were known to communicate CART’s diverse activities.

Attempts to identify the site(s) of action of CART initially employed screens for early gene activation (c-fos) and revealed potential sites in the hypothalamus including the arcuate (ARC), paraventricular (PVN) and supraoptic (SON) nuclei, as well as nuclei in the brainstem including the nucleus tractus solitarius (NTS), lateral parabrachial nuclei (LPBN), inferior olive and raphe nuclei (12, 15). Once those sites were identified, investigators performed intraparenchymal injections in an attempt to tease out the multiple actions of CART based upon localization of action (16–18). Surprisingly the effects of these CART microinjections revealed contrasting results. While intracerebroventricular administration of CART reduced food intake (9–11), site-specific injection of the peptide into the ARC, ventromedial and dorsomedial hypothalamic areas, PVN and lateral hypothalamic area (LHA) resulted in increased feeding responses (16–19). Attempts then were made to untangle these opposing, pharmacological findings using molecular approaches. Overexpression of the gene encoding CART (Cartpt) in the PVN increased food intake and weight gain (19), while reintroduction of ARC Cartpt expression in Cartpt knockout mice decreased energy expenditure and physical activity, without significant effects on food intake (20). Reintroduction in the LHA resulted in increased fat mass due to decreased energy expenditure and not increased food intake (20). Clearly, more was going on than simply effects on food intake. This was highlighted by findings in microinjection studies targeting the NTS. Injections targeting this region resulted in decreased body temperature, as well as decreased food intake and body weight (13, 21).

Not only is CART produced in hypothalamic neurons, but also in brainstem feeding centers such as the NTS. In the arcuate nucleus, CART is co-produced with other important regulators of appetite, particularly proopiomelanocortin (POMC). Although the majority of POMC neurons in the rodent hypothalamus co-localize CART, most CART neurons do not contain POMC (15, 20, 22,23) suggesting that CART interacts with, but is not dependent upon melanocortin circuits to exert its effects on appetite. Like other centrally expressed anorexigenic peptides, CART exhibits functional changes in mRNA expression depending on metabolic status. In particular, CART mRNA levels are reduced in the fasted state, but return to baseline levels upon refeeding and like POMC-expressing neurons in the arcuate, those producing CART are controlled by ambient levels of leptin (21,24,25). In hindbrain, CART activates an anorexigenic signaling cascade (13). Downstream signaling of this vagally delivered and the resident hindbrain CART-producing neurons couple via the parabrachial nucleus to eventual inactivation of feeding circuits in the hypothalamus. It appears that the anorexigenic effect of CART in those circuits requires the recruitment of locally produced glucagon-like peptide 1 (GLP-1) (13) although the potential role of oxytocin (OXY) should not be ignored as well (26). A broad distribution of one or more unique CART receptors was suggested by the reports of multiple sites of action of the peptide. While it was clear that site specific actions, some orexigenic and others anorexigenic, could be demonstrated under pharmacologic conditions, the primary action of endogenously produced peptide, at least that delivered by the vagus, appeared to be anorexigenic. However, those pharmacologic studies required more physiologic approaches to come to any firm conclusions.

Passive immunoneutralization approaches supported a role for endogenous CART in the physiologic regulation of food intake. Two groups reported in 1998 that ventricular administration of anti-CART antibodies resulted in increased food intake (21,27). More recent studies, published in 2020, demonstrated that immunoneutralization of endogenous CART in NTS resulted in increased feeding in fed but not fasted animals (28). Thus, as summarized below, when considered along with earlier work from two groups, a CNS site of action mediating the anorexigenic and body weight suppressive effect of CART action appear to be in the brainstem, particularly in the NTS (10–13,28).

It was hoped that genetic approaches might sort out inconsistencies in the reported effects of CART on food intake. Studies employing embryonic gene compromise (knockout) revealed that global Cartpt depletion resulted in increased food intake and body weight, due to fat mass accumulation, under high-fat feeding conditions (29,30), an effect that was delayed in onset in animals fed a standard chow diet. But were these results due to the loss of an anorexigenic action of CART or changes in reward behaviors (31) or physical activity (31,32)?

The identification of polymorphisms in the human CART (CARTPT) gene led to some important clues that might offer more information with regard to the role of endogenous CART signaling in the control of energy balance. A leucine 34 to phenylalanine (Leu34Phe) mutation resulted in reduced energy expenditure and hyperphagia leading to early onset obesity in an Italian family (33,34). This mutation resulted in decreased circulating levels of bioactive CART and increased amounts of the inactive form, CART preprohormone, in serum (35). When a Leu34Phe encoding plasmid was transfected into AtT20 cells, compared to the wild type CART gene (Cartpt), the mutation hindered splicing and trafficking of CART preprohormone leading to a deficit in fully processed and bioactive peptide (36,37). Another study in a French and Swiss Caucasian population concluded that the −368T>C mutation also resulted in obesity (38). A sequencing study of a Japanese population identified several polymorphisms in the 5′ upstream promoter region of the Cartpt. In particular, a polymorphism ALA-156GLY predisposed individuals to obesity (36). In summary, polymorphisms in the human population support the hypothesis that CART pre-propeptide and the final posttranslational product, CART, are important contributors to the homeostatic mechanisms controlling energy balance. In addition, these polymorphisms may have led to even more questions (39), including the potential role of CART in human anxiety and depression. Indeed, a missense mutation in the gene encoding CART was observed in a group of adolescents experiencing higher than expected levels of anxiety and depression (34). However, the situation appears to be the opposite in experimental animals. Increased anxiety-like behaviors following CART administration have been identified in rodent studies employing the elevated plus maze (40–41) and stress-like behaviors have been described in social interaction protocols (41). As with the effects of CART when delivered to the brain of rodents, the sign of the behavioral effects (orexigenic versus anorexigenic) of the peptide may depend upon the exact site of administration. Likewise, global Cartpt compromise or site-specific, conditional knockdown of GPR160 levels, may be required to answer this apparent between-species behavioral disparity.

The Present

Taking advantage of the wealth of information on the pharmacologic actions of CART and its sites of production and action, more recent studies have focused not only on CNS-derived CART, but also CART produced outside of the central neural axis. Localization studies detailed a broad distribution of CART-positive cells within the brain (5,6,24,43,44). In addition, CART of peripheral origin clearly accesses brain sites important in energy regulation via vagal afferents (45–46). Some of the controversy over orexigenic versus anorexigenic actions of CART can be explained on the basis of site of action, or even perhaps pharmacologic versus physiologic effects. It is safe to say that the overwhelming weight of evidence favors a physiologically relevant action for vagus nerve-derived CART in the control of food intake. Nodose ganglion (NG) cells express not only Cartpt but also receptors for leptin and cholecystokinin (CCK). Of notable interest is the finding that knockdown of Cartpt expression in NG eliminated the short-term satiety response to leptin (46). More recently, a comprehensive examination of the role of vagally delivered CART in the hindbrain control of feeding was published by Lee and colleagues (28). In an imaginative series of experiments these authors demonstrated that pharmacologic administration of CART to the site of vagal afferent termination in the NTS inhibited food intake, while compromise of endogenous CART released from the vagal afferents into the NTS increased food intake. These authors also employed shRNA-mediated compromise of vagal afferent nerve CART levels to support their hypothesis that the vagus nerve, through delivery of CART to the NTS, is a physiologically relevant regulator of food intake through effects on meal size and meal frequency. These data also suggest that the reduced sensitivity of vagal neurons to satiating hormones (47), distension (48) and nutrients (49) might be therapeutically reversed by CART administration. The problem with harnessing CART as a potential therapeutic for the treatment of metabolic disease lies in the labile nature of small peptides, including CART, and the necessity to ensure that it or any biologically active analog can access the NTS. Traditionally, for the development of such ligands, the identity of the cognate receptor for the endogenous peptide must first be identified. For nearly forty years following the first description of an endogenous neuropeptide that turned out to be CART, and the extensive work on the pharmacology of this potent inhibitor of food intake, efforts to identify the CART receptor had failed.

Early reports identified the possibility that the CART receptor was a GPCR. In addition to its actions in vivo to elicit c-FOS in select brain sites (10,12,21), in vitro studies revealed CART activation of extracellular signal-regulated kinase (ERK) phosphorylation as a signaling mechanism. The effect to increase ERK phosphorylation was pertussis-toxin sensitive suggesting signaling through a Gαi/o linked GPCR both in transformed pituitary cells and differentiated PC-12 cells (50,51). GTP-gamma-S membrane binding also was stimulated by CART in PC-12 cells, further suggesting the receptor to be a GPCR. The observation that hippocampal neurons responded to CART also indicated that, similar to transformed cells, primary neurons must express a cognate receptor (52). We made the assumption that CART signaled through one of the approximately 140 orphan GPCRs (oGPCRs), receptors for which a ligand had yet to be identified. We developed a novel, deductive reasoning strategy for “de-orphanizing” members of this class of GPCRs (53–56). With this approach we already had identified the cognate receptors for two novel neuropeptides we had discovered, neuronostatin (57) and phoenixin (58), to be GPR107 (53) and GPR173 (54), respectively, and had de-orphanized the receptors for adropin (55), and pro-insulin connecting peptide (56).

We had been interested in CART for some time because of its multiple pharmacologic actions and the fact that a cognate receptor had not been identified. We started by examining several cell lines for measurable responses to the peptide and focused our attention on differentiated PC-12 cells (ERK phosphorylation) and the gastric tumor cell line, KATO III cells (cFos) (59). We were aware of the data indicating a possible involvement of CART in the transmission of neuropathic pain (60) and the localization of CART-like immunoreactivity in dorsal horn of the lumbar spinal cord (6,60) and employed a receptomic and unbiased transcriptomic approach to identify oGPCR expression changes in the spinal cord during the onset of neuropathic pain (59). We identified 61 transcripts as potential GPCR candidates. Phylogenetic analysis segregated these candidates into four major classes: opioid, purinergic, serotonergic/cannabinoid-like, and arachidonic acid-like receptors. Thirty-one of the candidates turned out to be oGPCRs. We then turned to a model of neuropathic pain, sciatic nerve injury, and by PCR and RNAseq analysis identified 10 candidates, one of which, Gpr160, was significantly elevated in the dorsal horn of the injured animals compared to controls, only on the side ipsilateral to the injury. These findings were verified by immunohistochemistry. Furthermore, using RNAscope technology we identified increased Gpr160 expression on neurons, as well as astrocytes and microglia on the injured side of the spinal cord (59).

Was GPR160 responsible for CART activation of cfos and ERK phosphorylation we observed KATO III and PC-12 cells (59) and was it necessary for the transmission of nerve injury-induced pain? In cultured cells, knockdown of Gpr160 mRNA and protein levels with siRNA constructs prevented the stimulatory effect of the peptide on early gene expression and ERK phosphorylation. We provided evidence that CART and GPR160 can be co-immunoprecipitated, suggesting but not establishing physical interaction. However, using the Proximity Ligation Assay approach we were able to establish the close proximity of CART bound to KATO III cells and GPR160. CART signaling in spinal cord in vivo was similarly dependent upon the presence of GPR160. Further support for the role of GPR160 in pain sensation and the requirement of GPR160 signaling came from in vivo studies. Intrathecal CART administration evoked GPR-160-dependent activation of early gene expression and phosphorylation of ERK in dorsal horn, while knockdown of spinal cord GPR160 attenuated the development of neuropathic pain and even reversed established CCI-induced mechano-allodynia (59).

In summary, our data (59) support the notion that CART signals via GPR160 and provides the foundation for the development of CART analogs or small molecule mimetics for the treatment of chronic pain. But what about the other actions of CART? Is GPR160 required for the anorexigenic actions of the peptide? We recently reported evidence that antibodies targeting the extracellular domain of GPR160 administered into the 4V interrupted the pharmacologic action of CART to inhibit both food and water intakes in ad libitum fed and watered animals (61). In additional experiments, following two injections of anti-GPR160 antiserum into the 4V at 15-minute intervals just prior to lights out (1800 h) drinking, but not food intake was increased compared to that following vehicle injected control rats over the next two hours of ad libitum eating and drinking. However, by the following morning (lights on at 0600 h) antiserum treated animals had consumed significantly more water and food than controls. Those results suggested that activation of GPR160 initially attenuated the diurnal drinking response that accompanies the onset of the dark cycle and that only after multiple hours was the inhibition of food intake apparent. Could it be that the anorexigenic action was secondary to an initial, antidipsogenic action? Alternatively, the effect of endogenous CARTpt during the lights out period might reflect a longer-term action on satiety and not be related to any alteration in drinking behavior. While we cannot answer these questions yet, we are in the process of trying to establish whether the two behaviors are interdependent or unique. We have presented initial evidences suggesting that they may be independent (61).

The Future

To be sure, the research discoveries being made with regard to CART and GPR160 have created a foundation for many new directions of research and for possible therapeutic development. In recent studies we validated a commercially available antibody and began mapping GPR160-like immunoreactivity localization in the rat central nervous system (59,61). While by no means extensive, these studies identified GPR160 in many places predicted by the pharmacologic actions of CART including, but not limited to, laminae I-III of the dorsal horn of the lumbar spinal cord, the dorsal vagal complex, nucleus accumbens shell, hippocampus and multiple hypothalamic nuclei. Notably the densest staining was observed in the amygdala suggesting to us that potential, physiologically relevant actions of CART may be revealed by peptide microinjections into that region. Importantly, those localization studies provide a map enabling site-specific compromise of function studies to begin. We currently are examining the effect of GPR160 compromise in selective brain sites using AAVshRNA approaches on several CART driven behaviors. In addition, we have now developed a transgenic rat line harboring a floxed, Gpr160 gene (Gpr160flx/flx) and are examining the consequences of site-specific receptor knockout in those behavioral paradigms. Ongoing work in our group also examines the observations made in the initial publications (59,61) that GPR160 appears to be present on glia as well as neurons. Could it be that activation of glia is essential for the pharmacologic and physiologic actions of the peptide? It will be important to validate the immunohistochemical distribution results using in situ hybridization techniques. In addition, our preliminary use of RNAscope approaches is being expanded by recently initiated, cellular colocalization studies using snRNAseq.

Clearly for any progress to be made in the development of CART-based therapeutics the identification of a cognate receptor was a major advance and we are currently screening libraries and synthetic analogs for selectivity. One problem that will have to overcome is the broad pharmacologic profile of CART itself. While we can envision topical application of a GPR160 antagonist for the treatment of peripheral neuropathy, perhaps in patch or gel formulation, a systemic administration of an antagonist for the treatment of pain might bring with it an untoward effect on food intake, metabolism or thirst. Similarly, any formulation envisioned for appetite regulation (e.g. GPR160 agonists) might precipitate pain responses. These are potential roadblocks we hope to bypass in the future with computational biology (biased ligand development) and site-specific administration approaches.

Highlights.

  • Cocaine- and amphetamine-regulated transcript peptide (CART) acts pharmacologically to alter numerous behaviors, including food and water ingestion, pain sensation and motivation/depression.

  • The physiological relevance of those pharmacologic effects has been demonstrated for pain sensation and ingestive behaviors.

  • A G protein-coupled receptor, GPR160, has been identified that appears necessary for the actions of CART, opening the door to the development of therapeutic analogs of CART for the treatment of a wide variety of clinical conditions including obesity, chronic pain, and anxiety/depression.

Acknowledgments

Our work on CART and GPR160 was supported by Start-up Funds provided by the Saint Louis University School of Medicine (GLCY and DS) and NIH Grants: DK118340 (GLCY), NS113257 (DS and GLCY), HL121456 (WS), F32 DK18818 (LMS) and DK 115762 (MRH).

Abbreviations:

CART

Cocaine- and amphetamine regulated transcript peptide

Cartpt

rat CART gene

CARTPT

human CART gene

GPCR

G protein-coupled receptor

GPR160

G protein-coupled receptor 160 protein

Gpr160

G protein-coupled receptor 160 gene

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Disclosures: GLCY, DS and WKS are holders of Patent No. 10,85,378 (Methods for treating pain using anti-GPR160 Antibodies)

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