Cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide that has garnered significant attention for its diverse physiological roles, particularly in energy homeostasis. Since its discovery in 1995, research has revealed its involvement in stress, feeding behavior, immune function, autonomic regulation, fluid balance, metabolic processes, sexual function, and endocrine control. This article explores the localization and function of CART peptide (CARTp) with a focus on energy homeostasis, its potential as an anorexigenic signal, and its role in the pathophysiology of obesity.
Discovery and Structure of CART
In 1995, Douglass and colleagues identified CART as a transcript in the rat ventral striatum that was differentially displayed in response to cocaine and amphetamine. Further studies confirmed that the transcript was translated into protein, with CARTp found to be highly conserved between species. In rats, mRNA splicing and polyadenylation of CART mRNA (Cartpt) give rise to two different pro-peptides of 116 or 129 amino acids, which are post-translationally processed into two stable, biologically active forms: CART (55-102) and CART (62-102). The amino acid sequence of both the short-form CART (49-89) and the long-form CART (40-89) in humans shares 95% amino acid sequence homology with the respective rodent forms.
Distribution of CART in the Brain and Periphery
Early studies on Cartpt expression patterns provided insight into its potential function. Immunohistochemical and in situ hybridization studies have revealed the presence of CARTp and Cartpt mRNA throughout the brain, including the frontal part, midbrain, hypothalamus, and hindbrain. CART is present in both the central nervous system (CNS) and the periphery. In the periphery, CART expression has been identified in the islet endocrine cells, ganglionic cells, as well as the sensory and autonomic nerve fibers of the pancreas.
Frontal Brain Regions
In the frontal part of the mouse brain, Cartpt expressing neurons are distributed across different nuclei including the inner plexiform layer of the olfactory bulb and olfactory tubercle and the prefrontal cortex (PFC). Furthermore, Cartpt expression in the somatosensory, piriform, and insular cortices was also reported in rats and humans. These regions are actively involved in olfaction, integration of sensory information, as well as decision making.
Midbrain Regions
Cartpt transcript was originally identified in the striatum of rats. It has since been reported to be expressed in a number of other midbrain nuclei important in the control of feeding behavior. In the amygdala, changes in CARTp or Cartpt expression patterns in both CeA and BMA have recently been suggested to modulate stress response, hedonic feeding and energy homeostasis. The original finding that Cartpt is upregulated in response to drugs of abuse highlights a potential role for CARTp in reward and reinforcement. Cartpt expression in NAc has been associated with food consumption. In the hippocampus, the exact functions of CARTp remain unclear.
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Hypothalamus
Notably, high levels of CART expression have been identified to localize in brain regions that include the arcuate nucleus (Arc), the lateral hypothalamus area (LHA), the paraventricular nucleus (PVN), and the nucleus accumbens (Acb), suggesting an important role for CART in the regulation of food intake and energy homeostasis. The arcuate nucleus is often described as having two separate populations of neurons that express either Cartpt and pro-opiomelanocortin (CART/POMC) or neuropeptide Y and agouti-related peptide (NPY/AgRP), with these two neuronal populations having opposing metabolic functions.
Hindbrain
In situ hybridization indicates that there are significant Cartpt expressing neurons in the nucleus tractus solitarius (NTS) and area postrema (AP), with few soma in the Parabrachial nucleus (PBN). At the protein level, high to moderate density of CARTp stained fibers were also observed in the NTS, AP, PBN, locus coeruleus, and raphe nucleus.
CART and Energy Homeostasis
CART plays a crucial role in energy homeostasis, primarily through its anorexigenic effects. It is involved in the circuits that control the overall regulation of energy balance. The major site of action is the hypothalamic arcuate nucleus located at the base of the hypothalamus in an area where the blood-brain barrier is semi-permeable, hence uniquely assessable to circulating humoral and metabolic mediators.
Anorexigenic Effects
As a leptin-regulated neurotransmitter with potent appetite-suppressing activity, CART expression in the CNS is highly localized to distinct brain areas critically involved in the control of energy homeostasis, limbic and sensory functions, as well as throughout the HPA axis. Injections of CART peptide into the nucleus accumbens have shown an inhibition of feeding in rodents. In the Arc, CART is co-localized with α-melanocyte stimulating hormone (α-MSH), which is produced from the proopiomelanocortin (POMC) precursor and is a major inhibitor of appetite and food intake. In the Arc, CART mRNA levels are regulated by circulating leptin and are increased by peripheral leptin administration, again indicative of a critical role in energy balance regulation.
Regulation by Hormones
In the Arc, Cartpt expression is regulated by peripheral hormones. Leptin, which is released from adipose tissue, activates Arc CART/POMC neurons and upregulates Cartpt expression but inhibits NPY/AgRP neurons expression and activity. Cartpt expression is almost absent from the Arc of genetically obese Zucker (fa/fa) rats (have dysfunctional leptin receptor) and ob/ob mice (have disrupted leptin gene) and downregulated in DMH of ob/ob mice. Treatment of ob/ob mice with leptin injected intraperitoneally restored Cartpt levels in the Arc and DMH. In contrast, ghrelin activates Arc NPY/AgRP neurons and inhibit POMC/CART neurons.
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Interaction with Other Neuropeptides
Interestingly, CARTp is reported to co-express within hypothalamic neurons known to have opposing effects on feeding anorexigenic POMC neurons in Arc and orexigenic NPY neurons in DMH. CARTp has been demonstrated to stimulate the release of NPY and AgRP but not α-MSH from hypothalamic explants. Furthermore, while Cartpt is expressed in POMC neurons in rodents, it is also colocalized with other orexigenic neuropeptides, including melanin-concentrating hormone neurons in the LHA. Therefore, it is likely that hypothalamic CARTp not only has anorectic properties, rather plays a multifaceted role in controlling energy homeostasis.
CART and Obesity
Increasing evidence supports a role for CARTp in the pathophysiology of obesity. This is supported by polymorphisms in human populations that show an association between CARTp and obesity. Most studies reported only a few specific single nucleotide polymorphisms (SNPs) in Cartpt gene, for instance, higher allele frequency of the 1475A>G SNP in the individuals with obesity. While there are various challenges on the translation of observations from animal studies to human system, mutations in Cartpt have been documented to promote weight gain and thus endorsing the anorectic role of the CARTp.
Expression Changes in Obesity
In diet-induced obese mice, Cartpt increased after 10 weeks of fed high fat diet (40-60% kcal fat) in the DMH, but not the Arc. Cartpt expression in LHA was also reported to be increased along with decreased in Arc and PVN of obesity-prone mice compared to obesity-resistant mice fed high-fat diets. Furthermore, hypothalamic Cartpt expression and protein levels were also reported to be downregulated in other models of obesity such as gold thioglucose-treated mice, VMH-lesioned rats and some but not all studies using streptozotocin-diabetic rats.
CART and Energy Expenditure
Injection of CART I (55-102) into the PVN or Arc of rats markedly enhanced the mRNA expression for the uncoupling protein-1 (UCP-1) in brown adipose tissue, relating CART peptides not only to the control of feeding but also the modulation of energy expenditure. There is also evidence that cold exposure, which promotes weight loss mainly due to increase in thermogenesis, was associated with an increase in Cartpt mRNA abundance in the Arc of cold-acclimatized, compared to warm-maintained rats.
CART Receptor and Signaling
While a specific CART receptor(s) has not been identified to date, there is strong evidence that CART signaling can be blocked by pertussis toxin (PTX), indicative of the involvement of an inhibitory G-protein-coupling receptor that couples to Gi/o proteins. For example, CART I (55-102) has been described to inhibit voltage-gated L-type Ca2+ channels in hippocampal neurons, an effect that was blocked by treatment with PTX. Furthermore, central administration of CART I (55-102) stimulated the phosphorylation of cyclic AMP-response-element-binding protein (CREB) in CRH neurons in the PVN of fasted and fed rats, which again is classified as a PTX-sensitive mechanism.
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