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Endocrinology, doi:10.1210/en.2003-1648
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Endocrinology Vol. 145, No. 9 4119-4124
Copyright © 2004 by The Endocrine Society

Cocaine- and Amphetamine-Regulated Transcript Peptide Levels in Blood Exhibit a Diurnal Rhythm: Regulation by Glucocorticoids

A. Vicentic, G. Dominguez, R. G. Hunter, K. Philpot, M. Wilson and M. J. Kuhar

Neuroscience Division, Yerkes National Primate Research Center of Emory University, Atlanta, Georgia 30329

Address all correspondence and requests for reprints to: Aleksandra Vicentic, Ph.D., Yerkes National Primate Research Center of Emory University, 954 Gatewood Road NE, Atlanta, Georgia 30329. E-mail: avicen2{at}emory.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cocaine- and amphetamine-regulated transcript (CART) peptides are novel neurotransmitters that are implicated in several physiological functions such as control of feeding behavior, drug reward, sensory processing, stress, and development. Although a majority of studies have examined the role of CART in the brain, less is known about its function in the periphery. Therefore, the goals of this study were to examine the levels and species of CART peptides in blood, to determine whether they undergo diurnal rhythms, and to elucidate their sources and regulatory factors. RIA showed that CART peptides are present in the blood of rats and monkeys and that they exhibit a diurnal variation. Western blotting confirmed the pattern of diurnal variation in rats and, additionally, showed that CART immunoreactivity was due to a single predominant fragment with an apparent molecular weight in the range of the active CART 55–102 peptide. Adrenalectomy caused a 70% reduction in CART peptide levels in rat blood, and this was reversed by corticosterone replacement. CART levels paralleled glucocorticoid levels in rat and monkey blood. Control of CART levels by corticosterone suggests the possibility that CART peptides in blood may be influenced by hypothalamic-pituitary-adrenal interactions and that they may play a role in glucocorticoid-related processes such as stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COCAINE- AND AMPHETAMINE-regulated transcript (CART) was originally isolated as a transcript that was up-regulated by the psychostimulants cocaine and amphetamine in the rat striatum (1). CART peptides are neurotransmitters that became a focus of research investigations partially because of their potent anorexigenic properties (2, 3). Their role in feeding has been documented by the evidence that CART is a major determinant of body weight in humans because mutations in the CART gene have been associated with increased body weight (4, 5). CART peptides act also as neurotransmitters implicated in drug reward and reinforcement based on the demonstration of CART-dependent modulation of mesolimbic dopamine (6) and CART mRNA up-regulation in human cocaine abusers (7). Furthermore, CART peptides are involved in endocrine control and stress (8, 9).

CART peptides and mRNA are widely expressed in the central nervous system, and CART mRNA is among the most abundant mRNAs in the rat hypothalamus (10, 11). Although the role of CART in the central nervous system has been studied, the effects of CART peptides in the periphery have received less attention. CART peptides are found in the peripheral nervous system (12), the adrenal medulla, throughout the gut (11, 13, 14), and in islet somatostatin cells (15); they have been identified in some of these tissues by immunoprecipitation, sequencing, and Western blotting (16, 17, 18). These studies have established a strong presence of CART peptide in the periphery and suggested several functional roles for peripheral CART peptide. For example, its expression in various cell types in the gut and the ability to attenuate NO-induced relaxations suggests a role as a gut hormone or neuromodulator (14, 19). CART peptides are also found in sympathetic ganglion neurons and adrenal gland (12, 20), where they may act as signaling molecules in the sympatho-adrenal axis. Furthermore, a study by Jensen et al. (15) showed that CART peptide is expressed in the normal islet of Langerhans as well as in different islet tumors, whereas Cowles et al. (21) demonstrated a CART-induced increase in amylase secretion from the pancreas in rats after femoral infusion. Thus, CART peptide may potentially have endocrine and/or paracrine functions. CART immunoreactivity is also prominent in islets of the developing pancreas, where it may play a role in islet development (22). The significance of peripheral CART was also corroborated through a report that iv administration of CART stimulates prolactin and GH secretion and TSH release (23). This collection of data has established CART as a regulatory peptide with interesting physiological roles in the periphery.

In addition to being expressed in peripheral nerves and in neuroendocrine cells, CART peptide is found in blood (15, 24) from where it can rapidly reach the brain (25). However, the source, fate, and regulatory mechanisms of circulating CART peptide are poorly understood. Therefore, based on the previous studies that demonstrated the physiological effects of CART in the periphery and the suggestion that CART peptides can cross the blood-brain barrier, the goals of this study were to examine the levels and species of CART peptide in blood and to attempt to elucidate their sources and controlling factors. Based on reports by Balkan et al. (26, 27) and Vrang et al. (28) that hypothalamic CART peptide is regulated by glucocorticoids, we also aimed to test whether glucocorticoids are involved in regulation of circulating CART levels. Because a number of hormones, such as corticosterone, and physiological processes, such as feeding, exhibit a diurnal rhythm (29, 30), another goal of our study was to determine whether CART peptides in blood undergo diurnal variations; demonstration of a rhythm for CART could link the peptide to these processes or hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) weighing 375 g and female rhesus macaques weighing approximately 3–4 kg were housed in groups of two in separate facilities. Animals were maintained on a 12-h normal light/dark cycle (lights on at 0700 h) with a temperature range of 22–25 C. Water was available ad libitum. Food was also available ad libitum except for the group of rats that were fasted 24 h before blood collection. Animals were handled according to the NIH Guide for Care and Use of Laboratory Animals. The experimental protocol was approved by the Emory Institutional Animal Care and Use Committee.

Surgical procedures and blood sampling
Adrenalectomized and sham adrenalectomized rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and maintained 7 d in our care before the experiment. All animals received NaCl (0.9%) and sucrose (1%) in their drinking water. In adrenalectomized animals, corticosterone (30 µg/ml; Sigma Chemical Co., St. Louis, MO) was replaced in drinking water for 7 d (27). Animals were killed by decapitation, and trunk blood was collected in centrifuge tubes kept on ice. Samples were collected at 0500, 1100, 1200, 1700, and 2300 h. Lights were turned briefly on when decapitation was performed at 2300 h. Blood tubes were centrifuged at 3500 rpm for 15 min, and serum aliquots were stored at –80 C until CART peptide levels were determined via RIA and Western blotting and corticosterone levels were determined via RIA. Rat pituitary glands were collected by dissection immediately after decapitation and frozen in liquid nitrogen. The samples were stored at –80 C until RIA.

Female rhesus monkeys were habituated to the blood sampling regimen so that samples could be obtained without anesthesia (31). In habituated animals, these procedures have no adverse effect on behavior, reproduction, or growth and development (32, 33). Samples were collected at 1000 and 2200 h. Room lights were turned on before the collection of the 2200-h samples. For sample collection, a monkey was transferred from her home cage, by entering an aluminum transfer box, to a single cage located in the same room. The cage was modified for conscious venipuncture allowing the female to voluntarily extend her leg through a hole in the bottom front of the cage. Four milliliters of blood were collected from the saphenous vein by venipuncture. Tubes with blood were centrifuged at 3500 rpm for 15 min, and serum was harvested and stored at –80 C until used for RIA of CART and cortisol. After collection of the sample, the monkey was returned immediately to her home cage.

RIA
The concentration of immunoreactive CART peptide was determined in blood and pituitary gland by RIA. The procedure was carried out generally as previously described (13) but with a commercially available 125I-RIA kit (Phoenix Pharmaceuticals, Belmont, MA). The RIA kit was validated before use with tissue extracts and dose-response curves, and increasing concentrations of authentic rat CART 55–102 standard (Peptide International, Louisville, KY) added to tissue extracts were parallel to the standard curve. The assay sensitivity was 10 pg/tube, and intraassay variability was 5%. To determine the efficiency of the procedure, internal standards (80 pg/tube) of authentic CART 55–102 were added and were recovered to a level of 57%, and the values deviated less than 5%. All values are corrected for the loss of CART peptide using the recovery factor. Samples were purified by Sep Pak C18-E columns (Phoenix Pharmaceuticals). The eluates were freeze-dried overnight using a lyophilizer and later were dissolved in RIA buffer.

Extraction of CART peptide from tissues was carried out as previously described by Murphy and co-workers (13). Briefly, 0.1 M acetic acid was added to each tissue sample, which was then transferred toa boiling water bath for 15 min. After cooling on ice, samples were homogenized and centrifuged at 13,000 x g for 15 min. The supernatant was collected and the pellet reextracted with acetic acid. The supernatant was divided into two tubes. One tube was used for RIA analysis of CART peptides and the other for quantification of soluble protein. The supernatants were further purified on Sep Pak C18-E columns. After extraction, the eluates were freeze-dried overnight using a lyophilizer, and samples were dissolved in RIA buffer for assay.

Corticosterone levels were measured in rat serum samples (assay volume = 50 µl) using commercially available 125I RIA kits (Coat-a-Count, Diagnostic Products, Los Angeles, CA) in the Yerkes Endocrine Core Laboratory (26). The assay sensitivity was 5 ng/ml, and the intraassay and interassay coefficients of variation were 1.31% and 5.8%, respectively. Cortisol concentration in rhesus macaque serum was determined using the commercially available 125I RIA kit (Diagnostic Products) in the Yerkes Endocrine Core Laboratory (34). The sensitivity of the assay (assay volume = 10 µl) was 0.4 µg/dl, and the intraassay and interassay coefficients of variation were 10% and 3%, respectively.

Western blots
Western blot analysis was carried out as previously described by Kuhar and Yoho (17). Tissue samples were placed in lysis buffer with protease inhibitors, homogenized, and placed in a boiling water bath for 10 min. Samples were centrifuged at 14,000 rpm for 10 min. Blood samples were extracted and purified on Sep Pak C18-E column, and the eluates were freeze-dried in a lyophilizer overnight and dissolved in PBS buffer. Samples were loaded in the equivalent of 50 µg of protein on SDS-PAGE 16% trycine gels. The authentic rat CART 55–102 peptide (Peptide International) of known molecular weight was used as a marker. Gels were transferred overnight at 50 V and 40 C. Membranes were blocked for 2 h in nonfat milk diluent (Invitrogen, Carlsbad, CA) and incubated with polyclonal antiserum to CART 55–102 peptide overnight at 4 C. After washing and rinsing three times with Tris-buffered saline with Tween 20, membranes were then incubated for 1 h with horseradish peroxidase-coupled antirabbit antiserum (Rockland Immunochemicals, Gilbertsville, PA), reconstituted in 1 ml, and then diluted 1:50,000 in milk diluent. The specificity of stained CART immunoreactive bands was confirmed by the absence of an approximate 6-kDa CART band when the blot was preadsorbed with CART 55–102 but not an irrelevant peptide, CART 55–78. Membranes were washed three times for 10 min in Tris-buffered saline with Tween 20, and the immunoreactivity was detected with enhanced chemiluminescent substrate after varying exposures.

Nomenclature
The CART peptide numbering used in this paper is based on the rat long form of proCART, which contains 102 amino acids (1, 35).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CART peptide levels were determined in rat serum at various times of day by RIA as described in Materials and Methods. There was a clear variation in levels from approximately 50 pg/ml at early morning hours to approximately 150 pg/ml in early evening hours (Fig. 1Go). As shown by one-way ANOVA followed by a Newman Keul’s post hoc test, levels of CART peptide at 1700 and 2300 h were significantly different (P < 0.001) from those obtained 0500, 1100, and 1200 h; (F4,23 = 36.7; P < 0.0001).



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  		FIG. 1. The diurnal profile of CART peptide in rat serum. The data represent the mean ± SEM obtained from eight to 10 rats per time. Data were analyzed by one-way ANOVA (time, F4,23 = 36.7; P < 0.0001) followed by a Newman Keul’s post hoc test. *, Levels of CART peptide at 1700 and 2300 h that were significantly different (P < 0.001) from those obtained at 0500, 1100, and 1200 h; #, levels of CART that were significantly different (P < 0.001) from those obtained at 0500 h.

 
To provide additional confirmation that CART peptide levels in blood display a diurnal rhythm and to determine the molecular weights of the CART immunoreactive species in blood, Western blot analysis was carried out (Fig. 2Go). A previous Western blotting analysis of various tissues indicated that there are a number of immunoreactive CART peptides of differing molecular weights due to processing (17, 36). The antibody used was directed against the conserved carboxyl region of CART 55–102 (11) and identifies all precursors and processed CART peptide species, including the two active peptides, CART 55–102 and CART 62–102. The results in Fig. 2AGo demonstrate that one major immunoreactive species with approximately the same molecular mass as authentic CART 55–102 or 62–102 (~6 kDa) was abundant in blood (at 0500 and 1700 h) and that it varied from lower levels in the morning (0500 h) to higher levels in the evening (1700 h), which is in agreement with the RIA analyses. Western blots of extracts of hypothalamus, adrenals, and pituitary were included to show the similarity between the 6-kDa band in tissues and blood. In Fig. 2BGo, the antibody was preincubated with authentic CART 55–102, which blocked the staining of the band at approximately 6 kDa; this confirms that the band at 6 kDa is specific CART peptide immunoreactivity.



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  		FIG. 2. Western blot analysis of the diurnal profile of CART peptide in rat serum. A, CART peptide levels obtained at 0500 and 1700 h. Rabbit polyclonal serum (1:5000) against CART 79–102 identified the most prominent molecular mass band of approximately 6 kDa that corresponded to CART 55–102 or CART 62–102. Levels were lower at 0500 h compared with 1700 h. B, To determine the specificity of the immunostaining, identical samples were run in parallel, and preadsorption with the authentic CART 55–102 peptide (100 ng/ml) blocked the staining of the lower band of approximately 6 kDa, demonstrating that it is specific for CART peptide. Molecular mass (kilodalton) markers are shown on the left. See text for details.

 
Because adrenal glucocorticoids have been shown to influence levels of other peptides (32, 33), we determined whether the adrenal gland could influence the levels of CART peptides in blood. CART levels in blood were assessed in animals that were sham-operated, that had undergone adrenalectomy (ADX), and that had ADX plus corticosterone replacement. A two-way ANOVA followed by a Newman Keul’s post hoc test indicated that a significant reduction of approximately 70% was found in adrenalectomized animals, but this was reversed by corticosterone (Fig. 3Go). The same statistical test demonstrated that corticosterone replacement did not reinstate the diurnal rhythm (for time, F1,13 = 15 and P < 0.001; treatment, F1,13 = 40 and P < 0.0001; time x treatment, F1,13 = 5.6 and P < 0.05).



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  		FIG. 3. Effects of ADX, sham operation, and corticosterone (Cort) replacement on the diurnal rhythm of CART peptide levels in rat serum measured at 1200 and 1700 h. The data represent the mean ± SEM obtained from six rats. The difference between sham and adrenalectomized animals was determined by two-way ANOVA followed by a Newman Keul’s post hoc test (time, F1,13 = 15 and P < 0.001; treatment, F1,13 = 40 and P < 0.0001; time x treatment, F1,13 = 5.6 and P < 0.05). *, Significantly different (P < 0.001) from CART levels obtained in sham-operated animals at 1700 h; #, significantly different (P < 0.001) compared with CART levels obtained in adrenalectomized animals at the same time point.

 
Because the experiment involving ADX suggested a role for glucocorticoids in regulation of CART in blood, additional experiments were carried out. Glucocorticoids are known to exhibit a reversed diurnal pattern in monkey blood compared with rat, with the higher values measured in the morning and lower in the evening (37). We confirmed this in our samples as shown in Table 1Go. We therefore examined whether CART peptides also displayed a reversed diurnal rhythm in the blood of female rhesus macaques. As demonstrated in Fig. 4Go, CART indeed exhibited a diurnal variation opposite to that in rat; namely, higher values were observed in the morning and the lower values in the evening as determined by Student’s t test. This parallel between the diurnal rhythm of cortisol and the rhythm of CART in rhesus macaque (37) further supports a role for glucocorticoids in regulating CART in blood.


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  		TABLE 1. Diurnal levels of corticosterone in rat and cortisol in rhesus macaque serum

 


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  		FIG. 4. The diurnal profile of CART peptide levels in rhesus macaque serum. The data represent the mean ± SEM obtained from nine female monkeys. The difference between 1000 h and 2200 h was determined by Student’s t test. *, Significantly different (P < 0.001) compared with CART levels obtained at 2200 h.

 
Because fasting is known to elevate glucocorticoids in blood (38, 39, 40), we tested whether fasting influenced CART peptide levels. Levels of CART peptides were assayed by RIA at 0500 and 1700 h in both fasted (24 h) and regularly fed animals. Figure 5Go shows that the expected diurnal variation was found in regularly fed animals but was abolished in fasted animals. As indicated by two-way ANOVA followed by Newman Keul’s post hoc test, levels at both the early and later times were similarly high in fasted animals and similar to the high levels found in regularly fed animals at 1700 h, (time, F1,21 = 27.23, P < 0.0001; treatment, F(1, 21 = 6.24, P < 0.05; time x treatment, F1,21 = 10.07, P < 0.0001). Because high levels were found in fasted animals at both time points, the change cannot be due simply to a shift in the rhythm. This finding further suggests that glucocorticoids control CART levels in blood because fasting increases corticosterone levels in blood (38, 40, 41).



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  		FIG. 5. Effect of fasting on diurnal rhythm of CART peptide levels in rats. The data represent the mean ± SEM obtained from six to eight rats. Data were analyzed by a two-way ANOVA followed by a Newman Keul’s post hoc test (time, F1,21 = 27.23 and P < 0.0001; treatment, F1,21 = 6.24 and P < 0.05; time x treatment, F1,21 = 10.07 and P < 0.0001). *, Significantly different (P < 0.001) compared with CART levels obtained at 1700 h in regularly fed animals; #, significantly different (P < 0.001) compared with CART levels obtained at 0500 h in fasted animals.

 
Because the pituitary both contains CART (11) and can be regulated by glucocorticoids (42, 43), it is a possible source of CART peptides in blood. Accordingly, CART levels were examined in the pituitary at 0500 and 1700 h (Fig. 6Go). A diurnal variation was found, and the direction of the variation was the same as that found for blood; namely, the morning values were lower than those in the evening, as demonstrated by a Student’s t test. These results suggest that the pituitary is a source of CART in blood.



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  		FIG. 6. CART peptide levels in the rat pituitary gland. The tissue samples were collected in parallel with blood samples from the same animals killed at 0500 and 1700 h. The data represent the mean ± SEM obtained from eight to 10 rats per group. The difference between 0500 h and 1700 h was determined by Student’s t test. *, Significantly different (P < 0.001) compared with CART levels obtained at 0500 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results of this study demonstrate for the first time that CART levels in the blood of rats and monkeys undergo a diurnal rhythm that depends at least partly on glucocorticoids. CART levels measured at a midday time point in rats appeared to be in the low range (~10 pM) of those found by Jensen and co-workers (15). Diurnal variations in levels of other peptides have been previously found in peripheral blood as well (38, 44, 45, 46).

CART peptides in rat blood were also examined by Western blotting. This approach not only provides semiquantitative levels of immunoreactive peptides but also provides a measure of the molecular mass. Western blots indicated the presence of a single major CART peptide with a molecular mass of approximately 6 kDa, which is similar to that for the active peptides CART 55–102 and CART 62–102 (17). This new finding suggests that the major CART peptide that circulates in the blood is one shown to be active (8).

Because adrenal glucocorticoids are known to influence the levels of many peptides in blood (38, 47, 48), we examined the effect of ADX on blood CART. Indeed, ADX caused a 70% reduction in CART peptide levels, and this was reversed by corticosterone replacement. These findings suggest that corticosterone at least partially controls CART levels in blood. A reasonable hypothesis is that corticosterone from the adrenals influences CART levels in blood, perhaps through the hypothalamus and pituitary where CART is abundant (10, 11). This is supported by our observation that CART levels in the pituitary undergo a diurnal variation that parallels the variation in blood. Also, CART mRNA and peptide levels in the paraventricular and arcuate nuclei of the hypothalamus are reduced by ADX, and the reduction is at least partially reversed by corticosterone replacement (26, 27, 28, 30). Because 30% of CART remained in blood after ADX, at least some blood CART could come from non-adrenal-influenced tissues. For example, CART is abundant in the gut in a variety of cell types (14, 19) that could contribute to CART blood levels.

Additional findings in this study also support the hypothesis that the CART in blood is under corticosterone control. Glucocorticoids exhibit a diurnal rhythm in rat blood (44) that parallels the CART rhythm demonstrated here. In monkey blood, however, the rhythm of glucocorticoids is shifted compared with the rat (37), and indeed, the CART rhythm was also found to be shifted in monkey blood. Furthermore, glucocorticoids in rat blood are increased by fasting at early hours (39, 40), and we find that CART levels are also increased at early times in fasted animals.

The discovery that a diurnal variation in CART peptide levels is influenced by corticosteroids has potential physiological implications. A role for glucocorticoids in regulation of food intake and the development of obesity has been widely established. Likewise, the majority of evidence (3, 18, 49, 50), including human genetic and CART knockout mice studies (51), suggest that centrally administered CART peptide influences body weight. Because the diurnal rhythm of CART parallels that of corticosteroids, and the peak and nadir of glucocorticoid secretion coincide with the initiation and termination of feeding, it is possible that the diurnal rhythm of CART regulates the diurnal pattern of feeding. Indeed, several research studies suggested that the diurnal rhythm of feeding behavior is evoked by appetite-regulating peptides (48, 52, 53). The demonstration that fasting changed the CART diurnal variation and that blood levels of CART peptides are increased in anorexics (54) further corroborates the hypothesis that blood CART plays a role in feeding.

In addition, the immunohistochemical localization of CART to several peripheral organs (14, 15, 22, 55) and the presence of its diurnal rhythmicity in blood suggest that CART may modulate the function of some of these organs. Indeed, administration of CART peptide has been shown to stimulate amylase secretion from the rat pancreas (21). Amylase secretion exhibits a distinct rhythm characterized by a rise in enzyme and fluid secretion in the dark period and accompanied by a decrease in the light period (56, 57). Therefore, diurnal rhythmicity of CART peptide could coordinate the diurnal pattern of amylase secretion. It is worth mentioning that the vagus nerve, which is a central regulator of pancreatic secretion, also expresses CART peptide and that interruption of the vagus nerve completely abolishes CART-induced increases in pancreatic amylase secretion (21). Because circulating CART can cross the blood-brain barrier and possibly activate the vagus nerve, increase in blood CART observed in the evening hours may underlie a rise in amylase secretion at the same time period.

In summary, our studies clearly indicate that CART peptide levels are found in blood, that the peptide has the molecular weight of an active CART fragment, and that the levels undergo a diurnal variation and are influenced by corticosterone. Future studies will be needed to more fully elucidate the mechanisms, sources, and the functional significance of CART peptide and its diurnal variation in blood.


   Footnotes
 
This work was supported by National Institutes of Health Grants RR00165, DA00418, DA10732, HD37583, and DA15513.

Abbreviations: ADX, Adrenalectomy; CART, cocaine- and amphetamine-regulated transcript.

Received December 3, 2003.

Accepted for publication May 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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