CART is overexpressed in human type 2 diabetic islets and inhibits glucagon secretion and increases insulin secretion.

AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.

Collegial ethics: supporting our colleagues.

The goal of collegial ethics is to actively support our colleagues and to develop the skills needed to do so. While collegial interactions are key for our careers, there is little or no training in this. Many of our actions and reactions with our colleagues are instinctive. Human nature has evolved to be self-protective, but many evolved and automatic responses to others are not always in the best interests of our society or of us. Developing courage and a style of supportive language, avoiding destructive acts, and adhering to the golden rule will improve our relationships and provide a more positive environment for all.

CART peptide inhibits locomotor activity induced by simultaneous stimulation of D1 and D2 receptors, but not by stimulation of individual dopamine receptors.

CART (Cocaine- and amphetamine-regulated transcript) peptide has been implicated in playing a modulatory role in reward and reinforcement. Previously, our laboratory demonstrated that injections of CART peptide (CART 55-102) into the nucleus accumbens (NAc) attenuated both cocaine- and dopamine-induced increases in locomotor activity (LMA), and attenuated cocaine reward as well. In this study, the effects of CART peptide on LMA induced by dopamine receptor agonists were evaluated after intraaccumbal injections in male, Sprague-Dawley rats. Effects of the D1 receptor agonist SKF-81,297, saline, CART 55-102, or CART 55-102 and SKF-81,297 together were compared. The SKF-81,297-induced increase in LMA was potentiated by coadministration of CART, while injection of CART alone had no significant effect. Injection of the D2 agonist 7-OH-DPAT had no effect on LMA, and the combination of both 7-OH-DPAT and CART peptide also had no effect. Quinelorane, a D3 receptor agonist, did not alter LMA, nor did the combination of CART peptide and quinelorane. The next experiment examined the effects of CART peptide on LMA induced by coinjection of both the D1 agonist SKF-81,297 and the D2 agonist 7-OH-DPAT. The combination of SKF-81,297 and 7-OH-DPAT induced greater LMA than SKF-81,297 alone. Coadministration of CART peptide along with the D1 and D2 agonists reduced LMA. These results strongly suggest that CART peptide reduces the effects of psychostimulants by modulating the simultaneous activation of both D1 and D2 dopamine receptors rather than by affecting the action of any individual dopamine receptor.

CART peptides as modulators of dopamine and psychostimulants and interactions with the mesolimbic dopaminergic system.

Cocaine- and amphetamine-regulated transcript (CART) peptides (CART 55-102 and CART 62-102) are peptidergic neurotransmitters that are widely but specifically distributed throughout the brain, gut and other parts of the body. They are found in many brain regions associated with drug addiction including the nucleus accumbens, ventral tegmental area and ventral pallidum. Injections of CART 55-102 into the nucleus accumbens have no effect on basal locomotor activity. However, an injection of CART just before an i.p. injection of cocaine reduces the locomotor activating effects of cocaine. These and other data suggest that CART in the accumbens blunts the effects of cocaine. A hypothesis is that CART is homeostatic in the accumbens and tends to oppose large increases in dopamine signaling. These actions would therefore be able to regulate the effects of some abused drugs such as the psychostimulants.

High dose CART peptide induces abnormal EEG activity and behavioral seizures.

Cocaine- and amphetamine-regulated transcript (CART) peptides are neurotransmitters found throughout the nervous system and in the periphery. CART has an important role in the regulation of food intake, anxiety, endocrine function, and in mesolimbic-mediated reward and reinforcement. This short report casts light upon previous descriptions of presumed behavioral seizure and tremor activity following administration of CART into the central nervous system. By employing electroencephalographic (EEG) recordings, we document the state of cerebrocortical activity. We find that intracerebroventricular (icv) administration of 5 microg of CART 55-102 readily produces an abnormal EEG characterized initially by high amplitude hypersynchronous alpha in the 8-10 Hz range during behavioral wakefulness as manifest in both cortical and hippocampal theta EEG channels. This reliably progressed in three of three animals tested to unequivocal epileptiform activity accompanied by tremors and assumption of a rigid, tonic body posture. The neural substrates underlying this finding are unclear. This novel description of the epileptogenic quality of CART should lend caution to interpretations of the behaviors attributed to CART in other experimental paradigms.

Injection of CART (cocaine- and amphetamine-regulated transcript) peptide into the nucleus accumbens reduces cocaine self-administration in rats.

Cocaine- and amphetamine-regulated transcript (CART) peptides appear to modulate various effects of psychostimulant drugs. Injections of CART peptide into the nucleus accumbens (NAcc) inhibit locomotion produced by systemic injections of the psychostimulants cocaine and amphetamine. Intra-NAcc injections of CART peptide also inhibit locomotion produced by microinfusions of dopamine into the NAcc, suggesting that the effects of CART peptides may be due to an interaction with the dopaminergic system in the NAcc. We sought to determine if this inhibitory effect of CART peptide generalizes to other measures of dopaminergic function such as reward/reinforcement by testing the effect of bilateral intra-NAcc CART infusions (0, 0.25, 1.0 and 2.5 microg per side) on cocaine and food self-administration. One group of rats self-administered cocaine (0.75 mg/kg per 140 microl IV infusion) on a progressive ratio schedule. A separate group received 45 mg food pellets on the same progressive ratio schedule. Bilateral intra-NAcc injections of CART peptide dose-dependently decreased the number of cocaine infusions, the breakpoint of cocaine self-administration, and the total number of bar presses on the cocaine-associated lever. There were no effects of CART injections on the breakpoint for food reward. Thus, we conclude that injections of CART into the NAcc appear to functionally antagonize a major site of action for cocaine self-administration in rats.

CART regulates islet hormone secretion and is expressed in the beta-cells of type 2 diabetic rats.

Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic peptide widely expressed in the central, peripheral, and enteric nervous systems. CART is also expressed in endocrine cells, including beta-cells during rat development and delta-cells of adult rats. We examined the effect of CART 55-102 on islet hormone secretion, using INS-1(832/13) cells and isolated rat islets. In addition, islet CART expression was examined in two rat models of type 2 diabetes: Goto-Kakizaki (GK) rats and dexamethasone (DEX)-treated rats. At high glucose, CART potentiated cAMP-enhanced insulin secretion via the cAMP/protein kinase A-dependent pathway. In the absence of cAMP-elevating agents, CART was without effect on INS-1 cells but modestly inhibited secretion of insulin, glucagon, and somatostatin from isolated islets. CART was markedly upregulated in the beta-cells of both diabetes models. Thus, in DEX-treated rats, islet CART mRNA expression, and the number of CART-immunoreactive beta-cells were 10-fold higher than in control rats. In GK rats, the relative number of CART-expressing beta-cells was 30-fold higher than in control rats. We conclude that CART is a regulator of islet hormone secretion and that CART is upregulated in the beta-cells of type 2 diabetic rats.

Synthesis, monoamine transporter binding, properties, and functional monoamine uptake activity of 3beta-[4-methylphenyl and 4-chlorophenyl]-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes.

Synthetic methods were developed for the synthesis of the 3beta-(4-substituted phenyl)-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes (4a-s). The compounds were evaluated for their monoamine transporter binding and monoamine uptake inhibition properties using both rat brain tissue and cloned transporter assays. In general, the compounds showed higher dopamine transporter (DAT) affinity relative to the serotonin and norepinephrine transporters (SERT and NET, respectively) and greater [3H]dopamine uptake inhibition potency relative to [3H]serotonin and [3H]norepinephrine uptake inhibition. Several compounds were DAT selective relative to the SERT and NET in the monoamine transporter binding assays. The most potent and selective analog in the functional monoamine uptake inhibition test was 3beta-(4-methylphenyl-2 beta-[5-(3-nitrophenyl)thiazol-2-yl]tropane (4p).

Intra-VTA CART 55-102 reduces the locomotor effect of systemic cocaine in rats: an isobolographic analysis.

CART (cocaine- and amphetamine-regulated transcript) peptides appear to be mediators or modulators of psychostimulant drugs. An interesting result in the nucleus accumbens has been that injection of CART peptide has no effect by itself on locomotor activity, but it reduces the locomotor activity induced by cocaine or amphetamine. However, in the ventral tegmental area (VTA), injections of CART peptide have been shown to increase locomotor activity, although to a lesser degree [Kimmel, H.L., Gong, W., Vechia, S.D., Hunter, R.G., Kuhar, M.J., 2000. Intra-ventral tegmental area injection of rat cocaine and amphetamine-regulated transcript peptide 55-102 induces locomotor activity and promotes conditioned place preference. J. Pharmacol. Exp. Ther. 294, 784-792]. This study was carried out to clarify the interaction of intra-VTA CART 55-102 and systemic cocaine on locomotor activity. The CART-cocaine interaction has been examined using the rigorous isobolographic approach. This type of analysis permits an assessment of additivity, subadditivity, or synergism of two substances. By measuring locomotor activity and using a range of doses of CART peptide and cocaine, both alone and together, with different dosing strategies, clear evidence of subadditivity was found. CART reduced the locomotor activating effects of systemic cocaine, especially at higher doses of CART. These results imply that intra-VTA CART is not simply acting in the same manner as cocaine, and is likely to oppose the action of cocaine. This has implications for the physiological significance of CART-DA (dopamine) interactions and for medications development.

CART peptide in the nucleus accumbens regulates psychostimulants: Correlations between psychostimulant and CART peptide effects.

In this study, we reexamined the effect of Cocaine-and-Amphetamine-Regulated-Transcript (CART) peptide on psychostimulant (PS)-induced locomotor activity (LMA) in individual rats. The Methods utilized were as previously published. The PS-induced LMA was defined as the distance traveled after PS administration (intraperitoneal), and the CART peptide effect was defined as the change in the PS-induced activity after bilateral intra-NAc administration of CART peptide. The experiments included both male and female Sprague-Dawley rats, and varying the CART peptide dose and the PS dose. While the average effect of CART peptide was to inhibit PS-induced LMA, the effect of CART peptide on individual PS-treated animals was not always inhibitory and sometimes even produced an increase or no change in PS-induced LMA. Upon further analysis, we observed a linear correlation, reported for the first time, between the magnitude of PS-induced LMA and the CART peptide effect. Because CART peptide inhibits PS-induced LMA when it is large, and increases PS-induced LMA when it is small, the peptide can be considered a homeostatic regulator of dopamine-induced LMA, which supports our earlier homeostatic hypothesis.

The Leu34Phe ProCART mutation leads to cocaine- and amphetamine-regulated transcript (CART) deficiency: a possible cause for obesity in humans.

Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic neuropeptide synthesized in the hypothalamus. A Leu34Phe missense mutation in proCART has been found in an obese family in humans. Here we show that humans bearing the Leu34Phe mutation in proCART have severely diminished levels of bioactive CART, but elevated amounts of partially processed proCART in their serum. Expression of wild-type proCART in AtT-20 cells showed that it was sorted to the regulated secretory pathway, a necessity for proper processing to bioactive CART. However, expressed Leu34Phe proCART was missorted, poorly processed, and secreted constitutively. The defective intracellular sorting of Leu34Phe proCART would account for the reduced levels of bioactive CART in affected humans. These results suggest that the obesity observed in humans bearing the Leu34Phe mutation could be due to a putative deficiency in hypothalamic bioactive CART.

Regulation of CART mRNA in the rat nucleus accumbens via D3 dopamine receptors.

A variety of studies indicate that CART in the nucleus accumbens (NAcc) is involved in the action of psychostimulants. In order to understand in more detail if and how dopamine is involved in the regulation of CART mRNA in the NAcc, the present studies of individual receptors were performed. The D1 agonist, dihydrexidine, and the D1 antagonist, SCH23,390, were administered separately and in combination to adult male rats; however, no changes were found in CART mRNA as measured by in situ hybridization. The D2/3 agonist, quinpirole, was administered either separately or in combination with the D2 selective antagonist, L741,626, or the D3 selective antagonist, GR103,691. Quinpirole produced a decrease in CART mRNA of up to 43%. This effect was blocked by pretreatment with the D3 antagonist GR103, 691, but not by the D2 antagonist, L741,626. CART peptide levels showed a similar decrement after acute quinpirole. CART mRNA levels in the NAcc of D3 mutant mice were found to be higher than that in wild-type animals, but treating the mutants with quinpirole failed to produce a decrease in CART expression like that observed in wild-type rodents. These findings demonstrate that CART is regulated by dopamine in the NAcc, at least partly by D3 dopamine receptors.

Intrathecal CART (55-102) attenuates hyperlagesia and allodynia in a mouse model of neuropathic but not inflammatory pain.

CART peptides are found in brain and spinal cord areas involved in pain transmission. In the present study, we investigated the role of rat CART (55-102) in the modulation of chronic pain using models of chronic neuropathic (nerve injury model) and inflammatory (carrageenan test) pain models in the mouse after intrathecal administration. The results show that CART (55-102) was highly effective in reversing the hyperalgesia and allodynia signs of chronic neuropathic pain in a dose-related manner at doses (0.05-2 microg/mouse) that did not affect motor coordination of the animals. These effects lasted for at least 3 h after injection and were not blocked by naloxone, an opiate antagonist. Although CART (55-102) attenuated carrageenan-induced hyperalgesia, it failed to reduce the inflammation associated with this model. These results suggest the involvement of the CART peptides in the development of hyperalgesia and allodynia associated with neuropathic pain.

Cocaine and amphetamine regulated transcript (CART) and the stress response.

CART is expressed abundantly in the hypothalamic paraventricular nucleus and locus coeruleus, major corticotropin releasing factor (CRF) and noradrenaline sources, respectively. There is a bidirectional relation between CART and hypothalamo-pituitary-adrenal axis activity. CART stimulates CRF, adrenocorticotropic hormone and glucocorticoid secretion, whereas CRF and glucocorticoids increase the transcriptional activity of the CART gene; adrenalectomy declines CART expression in the hypothalamus. Stress exposure modulates CART expression in hypothalamus and amygdala in rat brain in a region and sex specific manner. CART may be a mediator peptide in the interaction between stress, drug abuse, and feeding. The review discusses the established role of CART as it relates to the stress response.

Colocalization of CART peptide with prodynorphin and dopamine D1 receptors in the rat nucleus accumbens.

CART peptide is a peptidergic neurotransmitter that is expressed in brain regions involved in critical biological processes such as feeding and stress, and in areas associated with drug reward and abuse including the dopamine-rich nucleus accumbens (NAcc), which can be considered part of the basal ganglia. Because CART has been shown to colocalize with substance P, a marker of the basal ganglia direct pathway, we now test for colocalization with other markers of the direct pathway to determine if CART colocalizes with dynorphin and dopamine D1 receptors. In the NAcc, CART peptide immunoreactivity (IR) was colocalized with prodynorphin-IR in neurons. Approximately 80.1% of CART-IR cells colocalized with prodynorphin-IR, while only 27.6% of prodynorphin-IR neurons contained CART-IR, suggesting that CART cells are a subset of dynorphin cells. In contrast, only about 25% of CART-IR cell bodies demonstrated dopamine D1 receptor-IR. Because dynorphin and D1 receptors are markers for the basal ganglia direct pathway, from the NAcc to the basal ganglia output nuclei, and because CART significantly colocalizes with these markers, some CART neurons are part of the direct pathway or some comparable pathway in the accumbens. The presence of CART in NAcc neurons and the fact that NAcc projection neurons have extensive local collaterals suggest that CART may have effects in both terminal and cell body regions of the accumbens and may therefore affect information processing in the NAcc by modulating accumbal neurons.

CART expression in limbic regions of rat brain following forced swim stress: sex differences.

Our previous studies showed the modulation of cocaine and amphetamine regulated transcript (CART) positive neurons and CART mRNA by adrenalectomy and corticosterone replacement in hypothalamic nuclei of male rat brain. More recently, we have shown by CART immunohistochemistry that restraint and forced swim (FS) stress have sexually dimorphic and regionally specific effects on CART expression in the hypothalamic nuclei of male and female Sprague-Dawley rats. This study aimed to evaluate the effects of FS stress on CART peptide expression in hypothalamus, amygdala and hippocampus of male and female (in or near estrus) Sprague-Dawley rats. Initially basal CART levels in regions of interest were determined in male and female rats; no sex differences were observed. In FS test, rats were forced to swim on two consecutive days, in a Plexiglas cylinder for 15 and 6 min, respectively. Rats were decapitated on the second day, 10 min after the stress procedure. Hypothalami, amygdalae and hippocampi were dissected and homogenized. CART peptide expression in these regions was measured by Western blotting. In males, FS increased CART expression in hypothalamus and amygdala. On the other hand, in females, FS lowered CART expression in amygdala. CART expression in hippocampus was not affected by the stress procedure in either sex. Our results suggest sexually dimorphic modulation of CART expression in hypothalamus and amygdala by FS procedure. Although modulation of the CART peptide by glucocorticoids and gonadal hormones appears likely, future studies are needed to elucidate the underlying mechanisms in the involvement of CART peptide in stress response.

Effects of dopamine transporter selective 3-phenyltropane analogs on locomotor activity, drug discrimination, and cocaine self-administration after oral administration.

Several studies suggest that a dopamine transporter uptake inhibitor that has a slower onset and longer duration of action than cocaine in animal behavioral measures and decreases cocaine self-administration would be useful as an indirect dopamine agonist pharmacotherapy to treat cocaine addiction. In the present study, we compared five 3-phenyltropane analogs administered orally in locomotor activity in mice and drug discrimination in rats to gain information concerning relative potency, onset, and duration of action. The compounds were also evaluated for reduction of cocaine self-administration in rats after oral administration. In general, the compounds had a slower onset of action than cocaine and reduced cocaine self-administration. 3beta-(4-Chlorophenyl)-2beta-(3-(4'-methylphenyl)-isoxazol-5-yl)tropane (RTI-336) was the most potent in locomotor activity and drug discrimination; it was less stimulatory than cocaine in the first hour and had the slowest onset and longest duration of action. It also reduced self-administration of two infusion doses of cocaine in the rat.

CART Peptides and Drugs of Abuse: A Review of Recent Progress.

Earlier studies suggesting an involvement of cocaine and amphetamine regulated transcript peptide (CARTp) in the actions of drugs of abuse are confirmed in the most recent publications. This seems especially true for the psychostimulants where CARTp in the nucleus accumbens inhibits or regulates the actions of these drugs; the regulation is lost after repeated drug use which may be an important mechanism in addiction. The other drugs, including nicotine, alcohol, opiates, and perhaps caffeine can affect CARTp or CART mRNA levels. While the exact mechanism is not always clear, the hope is that these findings may provide some insight for the development of medications. While binding studies indicate the existence of specific G-protein coupled receptors (GPCR) receptors for CARTp, major work to be done is the cloning of these receptors.

Synthesis and monoamine transporter binding properties of 3beta-(3',4'-disubstituted phenyl)tropane-2beta-carboxylic acid methyl esters.

3beta-(3'-Methyl-4'-chlorophenyl)tropane-2-carboxylic acid methyl ester (3b, RTI-112) is a 3-phenyltropane analogue that has high affinity for both the dopamine and serotonin transporters (DAT and 5-HTT, respectively). Compound 3b shows significant reduction of cocaine self-administration in rhesus monkeys, yet fails to maintain robust drug self-administration. PET studies revealed that unlike more DAT selective analogues such as GBR 12 909 and 3-(4-chlorophenyl)tropane-2-carboxylic acid phenyl ester (RTI-113), 3b shows no detectible DAT occupancy when dosed at its ED(50) for reduction of cocaine self-administration. In contrast, it highly occupies the 5-HTT at this dose. In this study, we report the synthesis and monoamine transporter binding potency of several new 3-(3',4'-disubstituted phenyl)tropane-2-carboxylic acid methyl esters (3c-k), which have binding properties very similar to 3b. With the exception of the 3',4'-dimethyl analogue 3k, all of the compounds possess subnanomolar IC(50) and K(i) values at the DAT and 5-HTT, respectively. The 3'-chloro-4'-bromo analogue 3e (IC(50) = 0.12 nM) and the 3'-bromo-4'-iodo analogue 3i (K(i) = 0.14 nM) are the most potent analogues at the DAT and 5-HTT, respectively. These compounds will be useful to further characterize the highly interesting behavioral profile of 3b.