CART peptides: regulators of body weight, reward and other functions

Abstract

Over the past decade or so, CART (cocaine- and amphetamine-regulated transcript) peptides have emerged as major neurotransmitters and hormones. CART peptides are widely distributed in the CNS and are involved in regulating many processes, including food intake and the maintenance of body weight, reward and endocrine functions. Recent studies have produced a wealth of information about the location, regulation, processing and functions of CART peptides, but additional studies aimed at elucidating the physiological effects of the peptides and at characterizing the CART receptor(s) are needed to take advantage of possible therapeutic applications.

Figure 1. Amino-acid sequences of rat proCART and CART peptides

a ∣ In the rat, CART (cocaine- and amphetamine-regulated transcript) mRNA has two splice variants (not shown): one that encodes a long form of proCART and one that encodes a short form. The mRNA that encodes the long form is translated into a 102-amino-acid sequence (top). In the other variant, the section that encodes amino acids 27–39 of the long form (shown in blue) is spliced out, and the resulting short-form CART mRNA is therefore translated into an 89-amino-acid sequence (bottom). The fragments of the long form of proCART that have been reliably shown to be active are amino acids 55–102 and 62–102. In the short form of proCART the numbers of the active amino acids are 42–89 and 49–89, but these amino acids are identical to those of the long form in a given species; this has led to some confusion in the literature because different numbers refer to the same amino-acid sequences. Only the 89-amino-acid form (the short form) of proCART has been found in humans. The amino-acid sequence of this human peptide is slightly different from the amino-acid sequence of the rat peptide. Amino-acid 42, which lies in the active fragment, is isoleucine in the rat peptide but is valine in the human peptide. Pairs of basic amino acids shown in bold are the sites of processing by prohormone convertases. b ∣ The structure of CART 55–102, with the disulphide bridges that are required for activity. The other major active peptide is CART 62–102, which has the same general structure. Part a modified, with permission, from REF. © (2008) Elsevier Science. Part b reproduced, with permission, from REF. © Elsevier Science B. V.

Figure 2. Proposed CART receptor signalling

Several studies on CART (cocaine- and amphetamine-regulated transcript)-peptide-induced cell signalling have demonstrated that CART peptides activate at least three signalling mechanisms. First, CART 55–102 inhibited voltage-gated L-type Ca²⁺ channels through a pertussis toxin (PTX; an inhibitor of inhibitory-G-protein (G_i/G_o)-dependent signalling)-sensitive mechanism in hippocampal neurons. Second, CART 55–102 increased the phosphorylation of cyclic AMP-response-element-binding protein (CREB) in the nuclei of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus in fasted and fed rats. Third, CART 55–102 increased extracellular signal-regulated kinase (ERK) phosphorylation in AtT20 and GH3 cells, an effect that was blocked by U0126, an inhibitor of MEK kinases, and by PTX. The dashed arrow indicates that it is not yet known whether this effect of CART 55–102 is mediated by inhibitory G proteins.

Figure 3. Effects of a missense mutation resulting in Leu34Phe in proCART

Western-blot analysis of CART (cocaine- and amphetamine-regulated transcript)-peptide production, processing and release in transfected AtT20 cells reveals the effects of a Leu34Phe mutation in the CARTPT gene. a ∣ Non-transfected AtT20 cells (C) showed no detectable CART peptide after either low or high exposure. Transfection of cells with a gene encoding wild-type (WT) proCART resulted in WT proCART largely being processed into intermediate CART peptides (~8 kDa) and active CART 42–89 (5.2 kDa) after high exposure. Transfection with a gene encoding mutated Leu34Phe proCART (M) resulted in M proCART being partially processed to intermediate CART peptides but only minimally processed to bioactive CART 42–89 after high exposure. b,c ∣ CART peptides secreted into the medium from cells transfected with either a gene encoding WT proCART (b) or a gene encoding M proCART (c) were analysed. Cells were incubated in basal medium (DMEM) for two 30-minute periods (B1 and B2) and then were incubated in stimulation medium for 30 minutes (S). Western blots of cell media show that in cells expressing WT CART peptides, basal secretion of an active form of CART peptide (5.2 kDa) is very small, but secretion is increased with stimulation. Cells expressing a gene encoding M proCART showed higher basal secretion of M proCART (10 kDa) and intermediate proCART (~8 kDa), but no increase was observed with stimulation. It is therefore thought that the Leu34Phe mutation results in a deficiency of bioactive CART peptides that are released by Ca²⁺-dependent mechanisms. The mutation seems to promote constitutive release of CART peptides, whereas WT CART peptides are mostly released by stimulation. The sera from the carriers of the mutation produced consistent results (not shown). Figure reproduced, with permission, from REF. © (2006) Endocrine Society.

Figure 4. CART peptides as potential regulators of dopamine activity in the nucleus accumbens

a ∣ The effect of bilateral CART (cocaine- and amphetamine-regulated transcript)-peptide infusions (0.0, 1.0 or 2.5 μg per side) into the nucleus accumbens on cocaine self-administration. Bilateral CART peptide infusions reduced the break point of cocaine self-administration in a dose-dependent manner. The break point is a reflection of how hard the animals are willing to work for a cocaine injection, and can be considered to be a measure of reward. b ∣ Cocaine administration elevates dopamine levels and therefore increases the locomotor activity that is associated with elevated dopamine levels. According to one hypothesis, CART peptides in the nucleus accumbens act to blunt this increased activity — perhaps through normal regulatory responses in this region, although the precise mechanism of the blunting is not yet clear. aCSF, artificial cerebrospinal fluid; SEM, standard error of the mean. Data in part a from REF. .

Figure 5. The involvement of the CART system in the stress response

CART (cocaine- and amphetamine-regulated transcript) peptides are, strikingly, present in many parts of the stress axis, and CART-peptide-containing neurons respond to stress presumably by releasing CART peptides. a ∣ Staining cells in the arcuate nucleus for the presence of CART peptide. The top image shows normal (untreated) tissue. The middle image shows tissue from an adrenalectomized animal: the number of CART-peptide-containing cells was reduced by ~40%. The bottom image shows tissue from an adrenalectomized animal that was given hormone replacement. The number of CART-peptide-containing cells in the replacement condition was not significantly different from that in the control condition shown in the top image. b ∣ CART peptides are strikingly found in most of the key regions and tissues that are involved in the stress response^-. ChAT, choline acetyltransferase; POMC, pro-opiomelanocortin; TH, tyrosine hydroxylase; TRH, thyrotropin-releasing hormone; VSP, vasopressin. Part a reproduced, with permission, from REF. © (2003) Elsevier/North-Holland Biomedical Press. Part b modified, with permission, from REF. © (2006) Elsevier Science Inc.