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Endocrinology Vol. 144, No. 3 760-766
Copyright © 2003 by The Endocrine Society

ARTICLE

Proopiomelanocortin-Derived Peptides in Rat Cerebrospinal Fluid and Hypothalamic Extracts: Evidence that Secretion Is Regulated with Respect to Energy Balance

Lynn E. Pritchard, Robert L. Oliver, Julie D. McLoughlin, Susan Birtles, Catherine B. Lawrence, Andrew V. Turnbull and Anne White

School of Biological Sciences & Faculty of Medicine (L.E.P., R.L.O., J.D.M., C.B.L., A.W.), University of Manchester, Manchester M13 9PT, United Kingdom; and AstraZeneca (L.E.P., S.B., A.V.T.), Mereside, Alderley Park, Cheshire SK10 4TG, United Kingdom

Address all correspondence and requests for reprints to: Prof. Anne White, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: awhite{at}man.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of proopiomelanocortin (POMC) is an important means of controlling the central melanocortin system. It has never been established whether the spectrum of POMC-derived peptides synthesized and secreted from the hypothalamus is altered in response to changes in energy homeostasis in vivo. To monitor secretion, we analyzed peptide content of rat cerebrospinal fluid. Strikingly, both the POMC precursor and ACTH were readily detected. Moreover, levels of both were lower in samples from obese Zucker rats (fa/fa) vs. lean Zucker rats (+/+, fa/+) and from fasted vs. fed rats, whereas {alpha}MSH could not be detected. POMC levels were also decreased in hypothalamic extracts from obese and fasted animals. In contrast, despite being the most predominant peptide in extracts, {alpha}MSH levels weren’t significantly changed in any of the rat models. The ratio of precursor to derived peptides in cerebrospinal fluid was significantly higher in obese vs. lean and fed vs. fasted rats, indicating that secretion of POMC-derived peptides is differentially down-regulated during negative energy balance. In contrast to peptide analysis, we found that POMC gene expression was not significantly decreased in fasted rat hypothalami. We conclude that regulation of peptide secretion is an important mechanism by which the POMC system is controlled.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE HYPOTHALAMUS, proopiomelanocortin (POMC)- derived peptides play a central role in the regulation of food intake via the melanocortin-4 receptor (MC4R) (1, 2, 3). {alpha}MSH, the presumed natural ligand of MC4R, is derived from POMC in the brain via a regulated posttranslational cleavage pathway involving the prohormone convertases, PC1 and PC2 (reviewed in Ref. 4). PC1 cleaves POMC to generate ACTH, and this is subsequently cleaved by PC2 to generate {alpha}MSH (Fig. 1Go).



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  		Figure 1. The POMC processing pathway in the hypothalamus. POMC is cleaved by PC1 to generate proACTH and ß-lipotrophin (ß-LPH). ProACTH is further cleaved by PC1 to generate N-POC, joining peptide (JP) and ACTH. PC2 cleaves ßLPH to generate {gamma}-LPH, ß-endorphin (ß-END), and ßMSH. ACTH is further cleaved by PC2 and subsequently processed to mature {alpha}MSH. The POMC IRMA requires binding of two monoclonal antibodies, 1C11 and 1A12, and therefore does not detect ACTH or {alpha}MSH. The ACTH IRMA uses 1A12 and 2A3 and does not detect {alpha}MSH and has minimal cross-reactivity (<0.1%) with POMC.

 
It is thought that the POMC system in the hypothalamus is tightly regulated as a means of controlling activation of the MC4R pathway. Most studies investigating the regulation of POMC have focused on gene expression analysis. Generally, these have detected POMC down-regulation in response to fasting (5, 6, 7) and defective leptin signaling (8, 9). Only a few studies, however, have investigated the effect of changes in energy homeostasis on levels of POMC-derived peptides in the hypothalamus (9, 10). Moreover, these studies do not provide information about peptide secretion, the physiologically relevant parameter. In fact, only one in vivo analysis has ever demonstrated that leptin stimulates {alpha}MSH secretion in the hypothalamus (11). To our knowledge, no studies have addressed whether the relative levels of POMC, ACTH, and {alpha}MSH produced and secreted within the hypothalamus are altered in different states of energy balance.

Analysis of POMC-derived peptide content in cerebrospinal fluid (csf) could provide an indirect measure of peptide secretion from POMC neurons in the brain. POMC peptides have previously been detected in both human (12, 13) and rat csf (14, 15). Furthermore, it has been demonstrated that hypophysectomy has little effect on POMC immunoreactivity in rat csf (16), suggesting that POMC-derived peptides in csf are largely brain derived. POMC is expressed in only two discrete regions of the brain, the arcuate nucleus (ARC) of the hypothalamus and the commissural nucleus of the solitary tract (NTS) of the brain stem (17). There are fewer POMC cells in the NTS than the ARC, and NTS cells produce less peptide than those of the ARC (18). These observations suggest that the majority of POMC-derived peptides detected in csf are likely to be secreted by hypothalamic arcuate neurons. Therefore, comparative analysis of POMC peptide levels in csf from different rat models of altered energy balance may indicate whether POMC processing and peptide secretion is regulated.

The specific aims of this study were 3-fold. First, by analyzing rat csf, we investigated whether POMC, ACTH, and {alpha}MSH levels are altered in lean (+/+, +/fa) vs. obese (fa/fa) Zucker rats and fed vs. fasted Wistar and Sprague Dawley rats. Second, by comparing ratios of POMC to POMC-derived peptides detected in hypothalamic extracts and csf, we investigated the possibility that POMC processing and secretion is altered in the same rat models. Third, we quantified POMC gene expression by Taqman real-time PCR, to investigate whether peptide levels in csf and hypothalamic extracts reflect changes in hypothalamic POMC gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male 10-wk-old obese/lean Zucker rats and Wistar rats were obtained from the animal breeding unit at AstraZeneca Pharmaceuticals (Alderley Park, UK). Sprague Dawley rats were obtained from Charles River Laboratories, Inc. Animals were maintained on a 12-h light, 12-h dark schedule with ad libitum access to standard laboratory diet and water unless otherwise specified. Animal procedures were undertaken by license from the British Home Office in compliance with the Animal Scientific Procedures Act, 1986. Animals were anesthetized by ip injection of Sagatal (60 mg/ml sodium pentobarbitone, Rhone Merieux, Athens, GA). Csf (100–200 µl per animal) was collected from the cisterna magna of anesthetized rats using a 25-gauge butterfly syringe, snap frozen and stored at -80 C until analysis. Csf samples from groups of five individual animals were pooled, to undertake all subsequent peptide measurements in duplicate.

Hypothalamic peptide extraction
Subsequent to csf withdrawal, rats were decapitated and hypothalami were rapidly dissected using consistent landmarks, being bordered by the optic chiasma, mammillary bodies, and hypothalamic sulcus. Hypothalami were snap frozen and stored at -80 C until use. Individual hypothalami were homogenized by sonication in 0.5 ml of 0.1 M hydrochloric acid. Samples were centrifuged (5000 x g, 20 min, 4 C). The supernatant was neutralized to pH 7.5 with sodium hydroxide, then made up to a final volume of 1 ml with 0.1 M Tris (pH 7.5) containing 0.1% wt/vol BSA. Aliqouts were then stored at 80 C until required for assay.

Assays for peptide measurement
POMC and ACTH were measured using two-site immunoradiometric assays as previously described (Fig. 1Go) (19, 20). The POMC assay does not cross-react with ACTH or {alpha}MSH, but cross-reacts 100% with proACTH. Previous chromatographic separation of csf has demonstrated that the assay detects a single peak corresponding to POMC, verifying that POMC is the major precursor peptide in csf, with minimal, if any, proACTH (13). Assay sensitivity during the study was 10 pmol/liter. The ACTH assay does not cross-react with {alpha}MSH, ACTH (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39), or ACTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). It has less than 0.1% cross-reactivity with POMC. Assay sensitivity was 1.1 pmol/liter. {alpha}MSH (Phoenix Pharmaceuticals, Inc., Mountain View, CA) was measured according to manufacturer’s instructions, using RIA. The assays were validated with tissue extracts before use. Cerebral cortex tissues samples were spiked with increasing concentrations of {alpha}MSH, ACTH, and POMC before extraction. Each peptide was quantitatively recovered, with dilutions parallel to the relevant standard curve. Plasma leptin concentrations were determined by ELISA using a commercially available kit (Crystal Chem Inc., Downers Grove, IL), following manufacturer’s instructions.

Taqman RT-PCR analysis
Total RNA was extracted from rat hypothalami, using Tri reagent (Sigma), following manufacturer’s instructions. One microgram of total RNA was used to generate cDNA, using a Taqman RT kit (PE Applied Biosystems, Foster City, CA). For each RNA sample, a corresponding no reverse transcriptase control was prepared to test for genomic DNA contamination of RNA samples. For each gene to be analyzed, probe and primer sequences were designed using Primer Express software (version I, PE Applied Biosystems). Taqman PCR assays for each target gene were performed in quadruplicate in 96-well plates on an ABI Prism 7700 Sequence Detection system (PE Applied Biosystems). For each 25-µl Taqman reaction, 0.01–1 ng cDNA was mixed with final concentrations of 1x Taqman Universal PCR Mastermix (PE Applied Biosystems), 300 nM forward and reverse primers, and 200 nM probe. PCR parameters were 50 C for 2 min, 95 C for 10 min, 40 cycles of 95 C for 15 sec, and 60 C for 1 min. Results were analyzed by the comparative Ct method as previously described in ABI Prism 7700 User Bulletin No. 2 (PE Applied Biosystems). Ct refers to the cycle number at which fluorescence exceeds threshold of detection. Hypoxanthine-guanine-phosphoribosyl-transferase was used as a housekeeping gene to normalize data.

Statistical analysis
Results are presented as mean ± SEM. Comparisons between group mean values of peptide and mRNA levels were performed using an unpaired Student’s t test analysis. A two-tailed probability of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
POMC, ACTH, and {alpha}MSH content in rat csf
Figure 2AGo shows levels of POMC detected in rat csf samples. POMC levels were significantly higher in fed vs. 24-h fasted Wistar rats (112 ± 10.1 pmol/liter vs. 65.2 ± 4.5 pmol/liter; P = 0.0015), fed vs. 48-h fasted Sprague Dawley rats (120.2 ± 8.2 pmol/liter vs. 67 ± 10.4 pmol/liter; P = 0.0035), and lean (+/+, +/fa) vs. obese (fa/fa) Zucker rats (91.5 ± 15.9 pmol/liter vs. 40 ± 9.5 pmol/liter; P = 0.018).



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  		Figure 2. A, POMC concentrations in pooled csf samples from groups of (I) fed/24-h fasted male Wistar rats (n = 5 groups), (II) fed/48-h fasted Sprague Dawley rats (n = 4 groups) and (III) lean (+/+, +/fa)/obese (fa/fa) male Zucker rats (n = 4 groups). Each group consists of pooled csf from five individual animals. B, ACTH concentrations in the same csf samples. C, Ratios of prohormone (POMC) to processed product (ACTH) in each csf sample. *, P < 0.05; **, P < 0.005; ***, P < 0.00005.

 
As with POMC, ACTH levels were also significantly higher in fed vs. 24-h fasted Wistar rats (8.6 ± 1 pmol/liter vs. 4.6 ± 0.7 pmol/liter; P = 0.007), and fed vs. 48-h fasted Sprague Dawley rats (14.1 ± 1.8 pmol/liter vs. 3.1 ± 1.1 pmol/liter; P = 0.001) (Fig. 2BGo). Changes in csf ACTH concentration were particularly striking in lean vs. obese Zucker rats, as ACTH levels were below the detectable limit of the assay in all obese samples analyzed (9.9 ± 0.6 pmol/liter vs. <1.1 pmol/liter; P = 0.000003). {alpha}MSH levels were below the detection limit of the assay (4.5 pmol/liter) in all csf samples analyzed.

To investigate whether the relative quantity of POMC-derived peptides detected in csf changes with respect to energy requirement, we compared the ratio of the prohormone, POMC, to the cleaved product, ACTH (Fig. 2CGo). In obese Zucker rat csf, the POMC:ACTH ratio was significantly higher than in lean controls (>36.4 ± 8.6 vs. 9.3 ± 1.6; P = 0.01). Similarly, in 48-h fasted Sprague Dawley rats, but not 24 h Wistar rats, the ratio of POMC to ACTH was significantly increased compared with ad libitum-fed controls (25.7 ± 5.5 vs. 8.7 ± 0.7; P = 0.01). The difference between 48-h and 24-h fasted animals may be due to strain. However, we found that 48-h fasted animals were essentially leptin deficient, with leptin levels below the detection limit of the assay (0.2 ng/ml), whereas 24-h fasted animals retained significant quantities of circulating leptin (2.8 ± 0.7 ng/ml) compared with fed controls (5.0 ± 0.4 ng/ml). Based on these observations, leptin deficiency and leptin receptor deficiency appear to have a profound influence on the relative levels of POMC-derived peptides secreted from the hypothalamus.

It is possible that POMC immunoreactivity present in csf is a consequence of peptides derived from the pituitary gland crossing the blood brain barrier. To investigate this possibility, we analyzed plasma concentrations in a group of five ad libitum-fed and five 24-h fasted Wistar rats. There were no significant changes in plasma POMC (1.1 ± 0.14 nmol/liter vs. 1.1 ± 0.08 nmol/liter), plasma ACTH (340 ± 105 pmol/liter vs. 332 ± 93 pmol/liter), or plasma {alpha}MSH (122 ± 41 pmol/liter vs. 143 ± 36 pmol/liter). Given that there were no significant changes in plasma peptide concentrations in response to fasting, it would seem unlikely the peptides in csf are derived from the pituitary gland.

POMC, ACTH, and {alpha}MSH content in rat hypothalamic extracts
To determine whether the profile of POMC peptides detected in csf reflect the relative quantities of each peptide produced in the hypothalamus, we analyzed hypothalamic extracts for POMC, ACTH, and {alpha}MSH content. As we were concerned with potential problems of peptide degradation in hypothalamic homogenates, we firstly assessed percentage recovery of known quantities of POMC, ACTH, and {alpha}MSH spiked into test cerebral cortex tissue samples, and identified an extraction procedure that gave maximum recovery for all three peptides. Consistent levels of recovery were obtained for POMC (79 ± 3.8%), ACTH (73 ± 8.5%), and {alpha}MSH (93 ± 1.4%) in three independent experiments.

As in csf, POMC levels were decreased in fasted vs. fed hypothalami (0.85 ± 0.04 pmol/hypothalamus vs. 1.1 ± 0.08 pmol/hypothalamus; P = 0.013) and obese vs. lean hypothalami (0.38 ± 0.03 pmol/hypothalamus vs. 0.56 ± 0.05 pmol/hypothalamus; P = 0.009) (Fig. 3AGo).



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  		Figure 3. Concentrations of A, POMC; B, ACTH; and C, {alpha}MSH in hypothalamic extracts from (I) eight ad libitum-fed and eight 48-h fasted Sprague Dawley rats and (II) five lean and five obese Zucker rats. D, POMC:ACTH ratios; and E, ACTH:{alpha}MSH ratios in each hypothalamic extract. *, P < 0.05; **, P < 0.005.

 
ACTH levels were significantly lower in obese vs. lean Zucker hypothalami (37.2 ± 2.7 fmol/hypothalamus vs. 71.8 ± 4.6 fmol/hypothalamus; P = 0.0001) but were not significantly different between 48-h fasted and ad libitum-fed Sprague Dawley rats (Fig. 3BGo).

{alpha}MSH was readily detected in hypothalamic extracts and was the most predominant of the three peptides quantified (Fig. 3CGo). Surprisingly, in contrast to the other peptides, {alpha}MSH levels were not significantly altered in lean vs. obese, or fed vs. fasted, hypothalamic extracts.

We also compared POMC:ACTH and ACTH:{alpha}MSH ratios in hypothalamic extracts. As in csf, the POMC:ACTH ratio was significantly higher in obese samples vs. lean samples (10.2 ± 0.7 vs. 7.8 ± 0.5; P = 0.02) (Fig. 3DGo). In contrast to csf, there was no significant difference in POMC:ACTH ratio between 48-h fasted and ad libitum-fed Sprague Dawley samples. The ACTH:{alpha}MSH ratio was significantly lower in obese vs. lean samples (0.023 ± 0.002 vs. 0.04 ± 0.003; P = 0.002) (Fig. 3EGo), although there was no significant difference in ACTH:{alpha}MSH ratio between fasted and fed samples. These findings suggest that obesity may lead to a down-regulation of PC1 and an up-regulation of PC2 in the hypothalamus, which is consistent with observations in Cpefat/Cpefat mice (21). Overall, however, the observed changes in the spectrum of peptides in hypothalamic extracts are much less pronounced than those in csf samples from the same animals.

POMC gene expression in rat hypothalamus
We also quantified POMC gene expression by Taqman real-time PCR in lean/obese Zucker rats and fasted/fed rats (Fig. 4Go). Neuropeptide Y (NPY) and agouti-related peptide (AGRP) gene expression were also analyzed as a means of validating the Taqman technology, as both of these genes are regulated in response to changes in energy balance (Ref. 22 and references therein). In obese Zucker rats (n = 8), there was, on average, a 2.5-fold increase in NPY expression compared with lean Zucker rats (P = 0.0002). There was no significant difference in AGRP expression, which is consistent with previous studies (23). POMC expression was significantly decreased in obese vs. lean rats (average 1.75-fold decrease; P = 0.0026). Therefore, in obese Zucker rats, decreases in POMC and ACTH, but not {alpha}MSH, in csf and hypothalamic extracts tightly reflect a concomitant decrease in POMC gene expression.



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  		Figure 4. A, Mean relative hypothalamic expression levels of NPY, AGRP, and POMC in eight lean (clear bars) and eight obese (hatched bars) male Zucker rats, as determined by Taqman analysis (normalized to HPRT expression). B, Expression levels in six ad libitum-fed male Sprague Dawley rats (clear bars) and six male Sprague Dawley rats fasted for 48 h (hatched bars). C, Expression levels in five ad libitum-fed male Wistar rats (clear bars) and five 24-h fasted male Wistar rats (hatched bars). *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

 
In 48-h fasted Sprague Dawley rats (n = 6), we observed significant increases in both NPY expression (average 1.5-fold increase; P = 0.012) and AGRP expression (average 2.8-fold increase; P = 0.00006) vs. ad libitum-fed rats (n = 6) (Fig. 4BGo). However, we did not observe any significant change in POMC expression. This contrasts with peptide measurements in csf. It would appear, therefore, that quantification of secreted peptides is a more sensitive means of assessing changes in regulation of the POMC system than measurement of mRNA levels. This is further demonstrated by Taqman analysis of fed/24-h fasted Wistar rats (Fig. 4CGo), where no significant change in POMC gene expression was detected, despite significant differences in POMC and ACTH levels in csf samples from the same rats.

We undertook a further Taqman experiment to compare the relative levels of POMC expression in the hypothalamus and the brain stem in ad libitum-fed Sprague Dawley rats. We found that the average levels of POMC were 12.5-fold less in the brain stem than the hypothalamus (data not shown). This strongly indicates that the majority of POMC immunoreactivity in the csf is reflective of peptides secreted from POMC neurons within the arcuate nucleus, rather than POMC neurons in the brain stem.

Gene expression analysis of molecules involved in POMC processing
To investigate the potential effect of changes in energy balance on POMC processing, we have quantified the expression of PC1 and PC2 by Taqman in lean/obese Zucker rats and 48-h fasted/fed rats. We have also analyzed proSAAS and 7B2, endogenous inhibitors of PC1 and PC2, respectively (24, 25), as well as PACE4, another prohormone convertase that colocalizes with POMC in the arcuate nucleus (26). We did not detect any significant changes in expression for any of these genes in lean/obese or fed/fasted rats (data not shown). This indicates that changes in gene expression of POMC processing enzymes is unlikely to account for changes in POMC:ACTH ratio in csf.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By using two-site immunoradiometric assays that distinguish 32-kDa POMC precursor and ACTH (19, 20), we have demonstrated that {alpha}MSH precursors are present in rat csf, as is the case in humans (13). In contrast, {alpha}MSH, the presumed natural ligand of the MC4R, could not be detected, which is consistent with a previous study (12). POMC and ACTH were also readily detected in hypothalamic extracts but were less predominant than {alpha}MSH. Clearly, therefore, the relative profile of POMC, ACTH, and {alpha}MSH is different in hypothalamic extracts compared with csf. The extract data indicate that the majority, but not all, of the POMC precursor produced in the hypothalamus is cleaved to completion, to generate {alpha}MSH. This is consistent with previous studies (27, 28). There are a number of potential explanations for the strikingly different relative concentration of {alpha}MSH in csf and hypothalamic extracts. It may be that {alpha}MSH is rapidly and efficiently cleared and/or degraded compared with POMC and ACTH following release from axon terminals, such that relatively little is detected in csf. Alternatively, it is possible that POMC and ACTH are preferentially secreted in comparison to {alpha}MSH in response to changes in energy balance. Despite these uncertainties, this study clearly demonstrates that the {alpha}MSH precursors, ACTH and POMC, are secreted in the hypothalamus.

It is possible that POMC and ACTH have functional significance at melanocortin receptors in the brain. A potential role for ACTH in energy homeostasis is supported by a number of observations. For example, it is known that ACTH has a similar potency to {alpha}MSH at the MC4R in vitro (1, 29). Furthermore, in vivo data have demonstrated that ACTH can inhibit feeding when administered centrally to rats, with an efficacy equipotent to {alpha}MSH (30). The functional role, if any, of POMC in the brain is less clear. However, the observation that circulating glucocorticoid levels are normal in PC1 deficient humans (31) and mice (28), despite a complete lack of POMC processing suggests that the prohormone has the capacity to act as a low potency melanocortin ligand, at least at the MC2R in the adrenal gland. Previous studies have demonstrated that {alpha}MSH biosynthesis (9), and release (11, 32), are up-regulated by leptin. However, our data demonstrate that it is unlikely that {alpha}MSH is the only POMC peptide to produce physiological changes in appetite regulation. Moreover, deficiency of PC2, the enzyme responsible for converting ACTH to {alpha}MSH, is not associated with obesity in mice, suggesting that other POMC-derived peptides can compensate for absence of {alpha}MSH in vivo (33). Which POMC-derived peptides are the most functionally relevant at the MC4R in vivo will require further investigation.

We have demonstrated that, in csf, both POMC and ACTH levels are significantly decreased in obese vs. lean and fed vs. fasted rats. Furthermore, we have found that lowered peptide levels in fasted rat csf return to normal after a 12-h refeeding period (data not shown). Clearly, therefore, analysis of csf with our assays is a very sensitive indicator of hypothalamic regulation of POMC. As csf samples may be taken via a cannula without killing the animal, such analysis could be a convenient methodology to monitor POMC regulation in future in vivo experiments. Moreover, it would be particularly interesting to extend these studies to obese human subjects. Polymorphisms within the POMC locus have been associated with high serum leptin levels and obesity in several human populations (34, 35). A quantification of POMC immunoreactivity in csf of lean and obese subjects in these populations may provide a functional basis for how the POMC locus predisposes to obesity.

This is the first study to assess the relative levels of biosynthesis and secretion of a range of POMC-derived peptides in the hypothalamus. We have observed that the ratio of POMC:ACTH in csf is significantly decreased in obese vs. lean Zucker rats, and in 48-h fasted vs. fed Sprague Dawley rats. This phenomenon may be mediated by leptin, because the ratio was not significantly changed in 24-h fasted rats, which, unlike 48-h fasted animals, retain significant quantities of circulating leptin. We propose two potential explanations for these findings: 1) leptin regulates the extent of POMC processing in secretory granules; and 2) leptin stimulates a preferential release of POMC-derived peptides compared with unprocessed precursor.

With respect to the first possibility, it is interesting to note that convertases involved in POMC processing have been implicated in obesity. It has been observed, for example, that a patient with inactivating mutations in both copies of the PC1 gene displays an extreme obese phenotype, presumably owing to impaired prohormone processing (31). Moreover, preliminary studies have indicated that both PC1 and PC2 are stimulated by leptin in vitro (36). Also, hypothalamic expression/activity of PC1 and PC2 is markedly altered in Nhlh2 and CarboxypeptidaseE deficient mice, which are both models of obesity (21, 37). Furthermore, recent data have implicated defective POMC processing as a molecular mechanism that leads to early-onset obesity in humans (38). We did not find any changes in expression of POMC processing enzymes in lean/obese of fed/fasted hypothalami. However, this observation does not rule out the possibility that they are regulated at the posttranscriptional level, or indeed that they are regulated at the transcriptional level, as measurements in whole hypothalami may not be sensitive enough to detect changes within specific neurons. Future quantitative in situ hybridization experiments and measurements of convertase activity will clarify this issue.

Changes in relative levels of POMC-derived peptides are far more pronounced in csf samples than in hypothalamic extracts. Consequently, we conclude that the major reason POMC:ACTH is significantly increased in csf is that the release of ACTH is differentially decreased in fasted and obese animals. Presumably, therefore, unprocessed POMC and derived peptides must be secreted by different mechanisms. With this in mind, we propose the following model (Fig. 5Go):



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  		Figure 5. A model of hypothalamic POMC secretion in (A) satiated rats and (B) rats in negative energy balance. CLV, Constitutive-like vesicle; ISG, immature secretory granule; TGN, trans-Golgi network.

 
POMC, as a secreted protein, is transported from the endoplasmic reticulum to the trans-Golgi network. From here it is sorted to immature secretory granules, probably via interaction with carboxypeptidase E (39). As the secretory granules mature (MSG), most POMC is cleaved to completion to {alpha}MSH by PC1 and PC2, though some ACTH persists. The activity of the convertases in secretory granules is possibly regulated, via leptin, to control the amounts of endogenous melanocortin receptor ligands produced. MSGs release their contents by exocytosis in response to leptin signaling and possibly other peripheral stimuli, such as insulin and glucocorticoids. Release is regulated to coordinate flux through the melanocortin system with respect to energy requirement. However, not all POMC is secreted in this way. Some material is diverted into constitutive-like vesicles and is released constitutively. This is an integral feature of the regulated pathway and has been implicated as a means by which prohormones, such as pro-insulin and proNPY, are secreted (40, 41, 42). In situations of negative energy balance (Fig. 5BGo), POMC levels in csf and hypothalamic extracts sensitively reflect a decrease in prohormone biosynthesis. Secretion of POMC-derived peptides from MSGs is inhibited and constitutive release of unprocessed POMC is not affected. Consequently, the ratio of POMC:ACTH increases in csf. As both regulated secretion and POMC biosynthesis are inhibited, steady-state {alpha}MSH and ACTH levels in hypothalamic extracts do not necessarily change. Confirmation of this model will require detailed analysis of POMC peptide secretion, both in vivo by push-pull perfusion (11) and ex vivo using hypothalamic explants (32).

In summary, transcriptional control of POMC gene expression is known to be one level at which melanocortin signaling is regulated in energy homeostasis. Overall, this study demonstrates that secretion and possibly processing of POMC in the hypothalamus may be additional key checkpoints that are tightly regulated. An understanding of the underlying mechanisms by which these phenomena are controlled could ultimately identify novel therapeutic strategies for the treatment of obesity.


   Footnotes
 
This work was supported by the Wellcome Trust, AstraZeneca and the Royal Society. We thank Dr. Brian Holloway and Dr. John Brennand of AstraZeneca and the Salford Royal Hospital Trust.

Abbreviations: AGRP, Agouti-related peptide; ARC, arcuate nucleus; csf, cerebrospinal fluid; MC4R, melanocortin-4 receptor; MSG, mature secretory granule; NPY, neuropeptide Y; NTS, nucleus of the solitary tract; POMC, proopiomelanocortin.

Received October 25, 2002.

Accepted for publication November 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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