Geraldina Dominguez

CART in feeding and obesity.

CART (cocaine- and amphetamine-regulated transcript) peptides are neurotransmitters that have received much attention as mediators of feeding behavior and body-weight regulation in mammals. CART peptides and their mRNAs are found in many brain regions and in peripheral tissues that are involved in feeding, and many animal studies implicate CART as an inhibitor of feeding. Animal studies also demonstrate that CART expression is regulated by both leptin and glucocorticoids, two hormones known to be associated with the regulation of body weight. A recent study also links a mutation in the CART gene to obesity in humans. These peptides might become targets for drug development in the area of obesity.

Characterization of the cocaine- and amphetamine-regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP-dependent signaling pathway in GH3 cells.

Cocaine‐ and amphetamine‐regulated transcript (CART) peptides are regulated neuropeptides that play a role in a variety of physiological processes. CART mRNA is also highly regulated as its levels change in response to psychostimulant drugs and leptin. To understand the mechanisms involved in regulating CART mRNA levels, the mouse CART 5′‐flanking regulatory region was studied. The sequence of 3.4 kb of the mouse CART 5′‐flanking region revealed a proximal promoter that contains a cluster of transcription factor binding sites, including an overlapping STAT/CRE/AP1 site. In addition, the 5′‐most 320 bp of the CART promoter shares 83% nucleotide identity between mouse and human. Three luciferase expressing constructs containing varying amounts of CART 5′ upstream sequence were generated and tested for promoter activity. Transient transfection of GH3 cells with constructs containing 641 and 3451 bp of upstream sequence displayed strong promoter activity, producing 29‐fold and 51‐fold stimulation, respectively, while, a construct containing 102 bp of upstream sequence displayed a 5.4‐fold increase in activity. A␣construct containing the composite STAT/CRE/AP1 site was responsive to cyclic AMP induction by forskolin in GH3 cells. Forskolin treatment also resulted in a 4.5‐fold increase in CART mRNA levels after 6 h and the addition of H89, an inhibitor of protein kinase A, reduced the levels by 50%. These studies indicate that the CART proximal promoter lies within the 5′‐most 641 bp and that in GH3 cells the CART gene is regulated via a cyclic AMP‐dependent pathway.

Cocaine- and Amphetamine-Regulated Transcript Peptides Play a Role in Drug Abuse and Are Potential Therapeutic Targets

Cocaine- and amphetamine-regulated transcript (CART) peptides (55 to 102 and 62 to 102) are neurotransmitters with important roles in a number of physiologic processes. They have a role in drug abuse by virtue of the fact that they are modulators of mesolimbic function. Key findings supporting a role in drug abuse are as follows. First, high densities of CART-containing nerve terminals are localized in mesolimbic areas. Second, CART 55 to 102 blunts some of the behavioral effects of cocaine and dopamine (DA). This functional antagonism suggests that CART peptides be considered as targets for medications development. Third, CREB in the nucleus accumbens has been shown to have an opposing effect on cocaine self-administration. CREB may activate CART expression in that region, and, if so, CART may mediate at least some of the effects of CREB. Fourth, in addition to the effects of CART on DA, DA can influence CART in the accumbens. Thus a complex interacting circuitry likely exists. Fifth, in humans, CART is altered in the ventral tegmental area of cocaine overdose victims, and a mutation in the CART gene associates with alcoholism.Overall, it is clear that there are functional interactions among CART, DA, and cocaine and that plausible cellular mechanisms exist to explain some of these actions. Future studies will clarify and extend these findings.

CART peptides: Modulators of mesolimbic dopamine, feeding, and stress

Abstract: CART peptides have been shown to be peptide neurotransmitters and endocrine factors in a series of cumulative studies over the past eight years or so. This brief review touches on three aspects of CART: CART as a mediator or modulator of mesolimbic dopamine, CARTs regulation by glucocorticoids, and CART as a regulator of feeding, satiety, and body weight. There have been several recent reviews and publications on various aspects of CART peptides. These aspects include the sequence and numbering of the peptides, and their structure, processing, and roles in various physiologic processes.

Genes in drug abuse

Because of the genome projects it will be possible to identify the changes in gene expression that are associated with drug abuse. The ultimate goal will be to determine the role and significance of the gene products. To date about 100 genes have been found with altered expression after administration of drugs. The development of new technologies such as microarrays will greatly facilitate finding such changes in expression. The rate at which levels of some gene products change is compatible with the time course of development of dependence in animals, but we must be able to establish a cause-effect relationship between genes and drug abuse, which this will be difficult. The product of the novel cocaine and amphetamine regulated transcript (CART) gene is a useful example where a gene product has been associated with drug abuse. CART was identified as an mRNA that changed in response to psychostimulant drug administration and injection of CART peptides into the ventral tegmental area produces psychostimulant-like effects. Definitive evidence, if obtainable, that specific genes are responsible for vulnerability to drug abuse could have dramatic effects on public health policy.

CART promoter CRE site binds phosphorylated CREB.

It has been shown previously that: CART (cocaine- and amphetamine-regulated transcript) mRNA is tightly regulated in brain; protein kinase A (PKA) is involved in CART expression in GH3 cells; and a cyclic AMP-responsive element (CRE) site is present in the proximal promoter region of the CART gene. Thus, the goal of this study was to test if CRE binding protein (CREB) can bind to the consensus CRE site and if phosphorylation of CREB occurs in GH3 cells under conditions of enhanced CART gene expression. Electromobility shift assays showed that a 27-bp oligonucleotide containing the CART CRE site was indeed bound by nuclear factors. Western blotting showed that incubation of GH3 cells with forskolin, which enhances CART mRNA expression, caused an increase in phosphorylated CREB (P-CREB) levels. Supershift analyses indicated that the CART CRE oligo/protein complex interacted with a P-CREB antibody. Taken together, these data indicate that P-CREB is a likely regulator of CART expression in GH3 cells.

CART peptide diurnal rhythm in brain and effect of fasting.

We have recently shown that CART peptides exhibit a diurnal rhythm in blood that is affected by food intake and glucocorticoids. In the present study, we extend our observations by demonstrating that CART peptides also exhibit a diurnal rhythm in several brain regions, notably the nucleus accumbens, hypothalamus and amygdala, but not in the midbrain. To examine whether the CART peptide rhythm was dependent on food intake, animals were food-deprived for 24 h. In regular-fed animals, CART peptide levels were lower in the morning compared to evening hours. However, this diurnal variation of CART peptide was not apparent in fasted animals, and CART peptide levels were reduced. The diurnal variation of CART mRNA in the nucleus accumbens paralleled the variation of CART peptide in this region. Similar to the peptide, the mRNA did not change in midbrain. These results show that CART peptide levels and gene expression undergo a diurnal variation in some brain regions, and the variation is altered by fasting. These findings suggest a variety of regulatory mechanisms for CART and additional considerations for CARTs role in brain.