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Departments of Surgery (J.-Y.L., Y.-K.Y., Q.Z., S.-Y.Q., I.G.) and Pediatrics (S.F., C.D.), University of Michigan, Ann Arbor, Michigan 48109-0682; and Howard Hughes Medical Institute and Departments of Pediatrics and Genetics (G.B.), Stanford University, Stanford, California 94305-5428
Address all correspondence and requests for reprints to: Ira Gantz, M.D., 6504 MSRB I, 1150 W. Medical Center Drive, Ann Arbor, Michigan 48109-0682. E-mail: IGantz{at}UMich.edu
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-MSH, orexin B,
melanin-concentrating hormone, or serotonin. HPLC analysis of AGRP-LI
in rat serum identified a single peak that eluted in close proximity to
synthetic AGRP (87–132) and mouse [Leu127Pro]AGRP and that was
identical to the peak seen in hypothalamic and adrenal tissue extracts.
The serum concentration of AGRP-LI in rats fed ad
libitum was 0.865 ± 0.323 nmol/liter (mean ±
SE). Food deprivation resulted in a slow, but statistically
significant rise in serum immunoreactivity at 48 h [1.174 ±
0.118 nmol/liter (mean ± SE)]. Bilateral
adrenalectomy did not change serum levels of AGRP-LI. These studies
demonstrate that in the rat there are different levels of basal
hypothalamic AGRP-LI release in fed and fasted states and that in the
fasted rat this release can be profoundly suppressed by leptin. These
studies also suggest that AGRP is present in the systemic circulation
of rats. |
Introduction |
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-MSH, a potent satiety-inducing agent produced by
POMC-producing neurons located more laterally in the arcuate nucleus
(2, 3, 4, 6, 7). Pharmacological studies using the cloned melanocortin
receptors (MCRs), have demonstrated that AGRP is a very potent
antagonist of -MSH action at the MC3R and MC4R and that it also has
some potency at the MC5R (6). Both the MC3R and MC4R are expressed within hypothalamic feeding centers. Therefore, both of these MCR subtypes must be considered potential targets for AGRP action in central processes regulating food intake. However, while genetic and pharmacological models have firmly established the role of the MC4R in feeding behavior, the participation of the MC3R in these events can only been inferred from the distribution of its messenger RNA (mRNA) (5, 8, 9, 10, 11).
Release of AGRP in the hypothalamus has not previously been examined due to the lack of a reliable RIA. Therefore, factors affecting the acute release of AGRP have only been surmised from studies that have examined changes in the level of its mRNA under various conditions. Factors presently known to alter AGRP mRNA levels in the arcuate nucleus include fasting and leptin. AGRP mRNA levels are increased in normal mice that are fasted and genetically obese mice that lack leptin (ob/ob) or its receptor (db/db) (1, 3, 7). Administration of exogenous leptin to normal mice and ob/ob mice has also been shown to reduce AGRP mRNA levels (12).
Because AGRP and NPY are produced by the same neuron (4), agents known to affect NPY synthesis and release might conceivably also influence the synthesis and release of AGRP. Leptin has been shown to decrease NPY mRNA levels (13, 14). However, the acute effects of leptin on NPY release have been conflicting. Leptin was reported to decrease NPY release from perfused hypothalami of lean Zucker rats (15). In contrast, in vivo studies with Long-Evans rats failed to demonstrate an acute effect of leptin on NPY release from the paraventricular nucleus (PVN), the hypothalamic region believed to be the major site of release of NPY produced in the arcuate nucleus (16). In other in vivo studies, the serotonin antagonist methysergide has been shown to increase NPY secretion and the 5-HT1B/2C receptor agonist m-CPP has been shown to reduce NPY protein levels in the PVN of the rat (17, 18). Therefore, leptin and serotonin might be expected to have an acute effect on hypothalamic AGRP release.
In both the rat and human arcuate nucleus AGRP-NPY- and
-MSH-containing neurons send projections to the lateral hypothalamus
(19, 20). This hypothalamic region contains neurons that produce the
orexigenic agents melanin-concentrating hormone (MCH) and orexin A and
B. This anatomical arrangement is consistent with the widely held
concept that the arcuate nucleus provides important neurochemical input
to the lateral hypothalamus. However, fibers from the lateral
hypothalamic area have also been reported to project to the medial
hypothalamus (21, 22). This raises the possibility that the orexigenic
agents produced in the lateral hypothalamus could reciprocally
influence the release of NPY and AGRP from AGRP-NPY-containing
neurons.
To date the majority of investigations have focused on AGRP’s orexigenic role in the hypothalamus. Nonetheless, it is worthwhile to note that expression of AGRP mRNA has been reported in a number of other sites. In humans and rodents, the adrenal glands (cortex and medulla), appear to be the site of the highest level of AGRP mRNA expression after the hypothalamus (1, 3). AGRP mRNA has also been documented in the subthalamic nucleus, lung, and testis of humans and in rodents it has been reported in lung, kidney, testis, ovary, and muscle tissues (1, 3). The function of AGRP in those extra-hypothalamic sites is presently unknown. Although the distribution of MCRs in the peripheral tissues of humans and rodents has not been extensively studied, expression of several MCR subtypes sensitive to the action of AGRP has been reported in peripheral tissues. Available data indicates that in humans and rodents MC3R and MC5R mRNA is expressed in the gastrointestinal tract and/or pancreas (23, 24). In rodents expression of MC5R mRNA has been reported in the adrenal glands, skeletal muscle, and lacrimal glands (23, 24, 25).
Within the context of this information we sought to establish a RIA that could be used to further explore the central and peripheral actions of AGRP. The present studies used this RIA to characterize basal AGRP-LI release from perfused rat hypothalamic slices and identify factors that influence that release. In addition the RIA was used to explore whether or not rodent serum contains AGRP-LI and attempted to identify the tissue source of the immunoreactivity.
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Materials and Methods |
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-MSH, ACTH, and
[Nle4, DPhe7] -MSH
(NDP-MSH), were purchased from Peninsula Laboratories, Inc. Leptin was purchased from Calbiochem (San
Diego, CA). Orexin A and B and MCH were purchased from Phoenix Pharmaceuticals, Inc. (Mountain View, CA). NPY was purchased
from American Peptide Co. (Sunnyvale, CA). Serotonin and
1-(m-chlorophenyl)-piperazine (m-CPP) were obtained from
Sigma (St. Louis, MO). A rabbit polyclonal anti-AGRP antibody raised against partially purified recombinant human AGRP was diluted to a titer of 1:200,000. This antibody has been previously characterized in immunohistochemical studies where it was found to be specific for AGRP (7). 125I-AGRP (87–132) was prepared by simple oxidative methods using chloramine-T and Na125I (Amersham Pharmacia Biotech) as previously described (6). Briefly, 10 µl of a 2.4 mg/ml solution of chloramine-T (Sigma) in 50 mM sodium phosphate (pH 7.4) was added for 15 sec and the reaction stopped with 50 µl of a 4.8 mg/ml solution of sodium metabisulfite (Sigma). The reaction mixture was then diluted in 800 µl of 50 mM ammonium acetate (pH 5.8) and purified by reverse phase chromatography using a C18 column. 100 µl of a 2% solution of BSA was immediately added to all fractions containing radioactivity.
RIAs were performed in a 0.05 M sodium phosphate buffer (pH
7.0) containing 2 mM EDTA, 200 mg/liter sodium azide, and 1
g/liter BSA. When antibody was diluted 1:200,000 and using 5,000 cpm of
125I-AGRP (87–132) the dose of half-maximal
inhibition of specific binding (ID50) was 10
fmol/ml. Under these conditions, no cross-reactivity was observed with
recombinant agouti protein, -MSH, ACTH, NDP-MSH, leptin, orexin B,
or neuropeptide Y. However, 0.0038% cross-reactivity was noted for
orexin A.
Measurement of AGRP-like immunoreactivity in tissue
For characterization of AGRP-LI in tissue, freshly dissected rat
tissues were homogenized in 3% acetic acid (10 ml/g tissue) and
centrifuged at 3,000 rpm at 4 C for 10 min. Supernatants were titrated
to pH 7.0 and stored at -20 C until used. For chromatographic analysis
of AGRP-LI molecular forms, samples were diluted 1 to 10 in buffer A
(50 mM ammonium acetate, pH 5.3). The material to be
assayed was then loaded on a FPLC HR 5/5 pepRPC (C18) column
(Amersham Pharmacia Biotech, Piscataway, NJ) previously
equilibrated with buffer A. After the absorbance at 280 nm
(A280) had returned to baseline following sample
application, the column was eluted with buffer B (buffer A + 75%
acetonitrile) in a linear gradient at a flow rate of 1 ml/min and 1 ml
fractions were collected. The recovery of synthetic AGRP (87–132) from
the C18 column was 80%.
Measurement of AGRP-like immunoreactivity in serum
Serum could not be directly assayed for AGRP-LI because of
interfering proteins in blood. Therefore, serum AGRP-LI was
characterized by HPLC as described above. In addition, because the
measurement of serum AGRP-LI in experiments using groups of rats
required the assay of a large number of samples of limited volume, a
serum AGRP-LI assay was developed using a C18 cartridge. For these
experiments serum was diluted 10-fold in buffer A and loaded onto a C18
environmental cartridge (Waters Corp., Milford, MA).
Samples were eluted with 60% acetonitrile before lyophilization.
Lyophilized samples were reconstituted in distilled water for assay.
Recovery of synthetic AGRP (87–132) was 30%. Because of the
relatively poor recovery, we sought to compare serum measurement using
the C18 cartridge with standard HPLC. The two methods of measurement
resulted in nearly identical determinations of serum AGRP
concentration. Therefore, the two methods only differed in the
percentage of AGRP-LI recovered from serum.
AGRP-LI in tissues and serum was also characterized by ion-exchange chromatography. Tissue extracts or sera were diluted 10-fold in 50 mM MES (pH 5.5) and applied to a FPLC HR 5/5 Mono S cation exchange column previously equilibrated in the diluting buffer. After the A280 had returned to baseline following sample application, the column was eluted with a NaCl gradient at a flow rate of 1 ml/min and 1 ml fractions collected.
Hypothalamic tissue perfusion
Male Sprague Dawley rats weighing 280–300 g were housed under
conditions of controlled temperature (20 ± 2 C) and lighting
(0700–1900 h). Animals were housed in the Unit of Laboratory Animal
Medicine facilities at the University of Michigan and all animal
experiments were approved by the University Committee on Use and Care
of Animals. Rats were killed by cervical dislocation then decapitated.
Hypothalami were harvested between 1000 h and 1100 h to avoid
circadian variation. Hypothalami were rapidly removed by dissecting the
brain at the optic chiasm rostrally, the hypothalamic fissures
laterally, the mammillary bodies caudally, and the ventral surface of
the thalamus dorsally. Hypothalami were then separated into five
coronal slices using a razor blade. Eight hypothalami were used in each
experiment. The perfusion system consisted of a fixed stem jacketed
tissue vessel (Harvard Apparatus, Cambridge, MA), a constant
temperature circulating water bath (HAAKE, Karlsruhe, Germany)
and peristaltic pump (Harvard Apparatus). The perfusion chamber was
kept at 37 C and continuously perfused with modified
Krebs-Ringer-bicarbonate buffer (composition in mM: NaCl
120, KCl 5, CaCl2 2.6,
MgSO4 0.7,
KH2PO4 1.2,
NaHCO3 27.5, pH 7.4) supplemented with 2 g/liter
glucose and 0.1% BSA and aerated with 95%
O2/5% CO2 at a flow rate
of 0.2 ml/min. Samples were collected on ice at 10 min intervals and
stored at -70 C. Before RIA determinations the samples (2 ml) were
lyophilized and reconstituted in 0.65 ml of distilled water. NPY was
measured using a commercially available NPY RIA kit (Peninsula Laboratories, Inc. Belmont, CA). Each experiment was repeated at
least four times. The effect of each concentration of leptin on
hypothalamic AGRP-LI release was determined in separate experiments. 50
mM potassium chloride (KCl) depolarization was used to test
tissue viability at the end of all experiments. In perfusion studies
AGRP-LI was expressed as the fmol release per mg of hypothalamic
protein to minimize for small differences that might result from the
use of slightly differing amounts of tissue. Total protein was
determined by colorometric assay (Bio-Rad Laboratories, Inc., Hercules, CA).
Bilateral adrenalectomy
Three groups of rats were used in this experiment. Group 1
consisted of 8 animals that had bilateral adrenalectomies, Group 2
consisted of 8 animals that had bilateral adrenalectomies +
corticosterone replacement, and Group 3 consisted of 8 animals that had
a sham operation. Bilateral adrenalectomies were performed through a
flank incision using xylazine (3 mg/kg) and ketamine (120 mg/kg)
anesthesia. Sham operations were performed by manipulating the
animal in an identical manner, but without removal of the adrenal
glands. Completeness of bilateral adrenalectomy was confirmed by
measurement of serum corticosterone using a RIA (Diagnostic Products Corp., Los Angeles, CA). Adrenalectomized animals
received 0.9% NaCl in their drinking water. Group 2 received
corticosterone replacement by daily injection of 1.5 mg/kg sc between
18.00 and 18.30 as previously described (26). 1.5 ml of blood was
collected under ether anesthesia for assay of AGRP-LI at 1 week, 2
weeks, and 3 weeks.
Northern blot analysis of AGRP mRNA
Individual hypothalami of fed or fasted rats were
dissected and stored in liquid nitrogen. Total RNA was extracted using
Trizol Reagent (Life Technologies, Inc., Grand Island,
NY). Twenty-five micrograms of total RNA was loaded per well and
electrophoresed on a denaturing gel (2.2 M formaldehyde,
1 x MOPS). RNA was then transferred to a nylon membrane and
hybridized with a random primed 32P labeled
partial length rat AGRP complementary DNA (cDNA) probe. A random primed
32P labeled glyceraldehyde-3-phosphate
dehydrogenase (GAPD) was used to check equivalency of well loading.
Blots were hybridized overnight at 42 C in buffer containing 50%
formamide, 1 x Denhardt’s, 6 x SSPE, 0.5% SDS and salmon
sperm DNA (100 µg/ml). Final wash conditions were 55 C in 0.1% SSC,
0.2% SDS. Blots were exposed for 12 h using Kodak
XAR film (Eastman Kodak Co., Rochester, NY). RNA was
quantified using a Hewlett-Packard Co. ScanJet 4C/T
scanner and the NIH Image 1.55 program.
Statistical analysis
Physiological data are expressed as a mean ±
SE. Two way ANOVA with repeated measures was used to
compare the basal release of AGRP-LI and NPY from the hypothalami of
fed and 48 h fasted rats (see Figs. 2
and 6) and for the
comparison of serum levels of AGRP-LI in adrenalectomy experiments (see
Fig. 9). One way ANOVA followed by Tukey-Kramer posthoc test was used
to analyze leptin inhibition of AGRP-LI and NPY release from
hypothalami of 48 h fasted rats (see Figs. 3 and 7). One-way ANOVA
followed by Dunnett’s method for multiple comparison was used to
analyze the dose-dependency of leptin inhibition of AGRP-LI release
from hypothalami of 48 h fasted rats (see Fig. 4) and the rise in
serum AGRP-LI in 24 and 48 h fasted rats (see Fig. 8). Student’s
t test was used to determine the statistical significance of
the change in hypothalamic AGRP mRNA in 48 h fasted rats (see Fig. 5).
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). The greatest concentrations of
AGRP-LI were present in hypothalamus and adrenal gland. Very low levels
of AGRP-LI were noted in brain striatum and thalamus. No AGRP-LI was
detectable in brain cortex or liver tissue. When characterized by
reverse phase C18 HPLC, synthetic AGRP (87–132) eluted as a single
peak earlier than the peak of full-length [Leu127Pro]AGRP.
Hypothalamic tissue AGRP-LI eluted as a single peak close to the
elution position of AGRP (87–132) (Fig. 1A). Although not depicted in Fig. 1, the
peak of adrenal tissue AGRP-LI was superimposable with the peak
observed from hypothalamic tissue except that the amount of
immunoreactivity was less. AGRP-LI eluted from the C18 environmental
cartridge was found to coelute with serum applied directly to the C18
HPLC column. AGRP-LI present in rat serum eluted near the
immunoreactivity found in hypothalamus (Fig. 1A). Further
characterization of rat tissue and serum AGRP-LI by cation-exchange
chromatography revealed that AGRP-LI eluted as single peak but somewhat
later than synthetic AGRP (87–132), suggesting that it was more
positively charged than the synthetic peptide (Fig. 1B). While the
chromatography data suggest that there is posttranslational processing
of tissue and serum AGRP further analysis will be required.
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, a continuous low
level of AGRP-LI was released from the hypothalami of rats fed ad
libitum before they were killed (<0.5 fmol release/mg
hypothalamic protein). In contrast, a significantly greater amount of
AGRP-LI (1.0 to 1.2 fmol release/mg hypothalamic protein) was released
from the hypothalami of 48 h fasted rats. Comparison of the 13
time points before administration of 50 mM KCl
revealed that the difference in AGRP-LI release from hypothalami of fed
and 48 h fasted animals was statistically significant
(P < 0.001). On average AGRP-LI release was 3-fold
greater in 48 h fasted rats. Analysis of the last two time periods
in Fig. 2 revealed that there was also a statistically significant
greater amount of KCl-induced AGRP-LI released from the hypothalami of
48 h fasted vs. fed rats (P < 0.001).
In time period 15, the perfusion of hypothalami of 48 h fasted
rats with 50 mM KCl resulted in a 262% increase
of AGRP-LI over mean basal release. The response to KCl administration
toward the end of all perfusion studies demonstrates not only tissue
viability, but also the presence of substantial additional AGRP-LI
stores. Time did not appear to have any effect on basal AGRP-LI release
(or basal NPY release, see Fig. 7) during the time course of these
studies.
As shown in Fig. 3, addition of leptin to
the perfusate caused inhibition of AGRP-LI release from the hypothalami
of 48 h fasted rats. Comparison of the 40 min before the
administration of 10-7 M leptin
(baseline period) to the 40 min during which leptin was infused
revealed that this inhibition was statistically significant
(P < 0.001). During the time period of leptin infusion
inhibition reached statistical significance at 30 min
(P < 0.03). A persistent leptin effect remained for at
least 30 min after cessation of the leptin infusion with maximal
inhibition of AGRP-LI occurring 70 min after initiation of leptin.
Compared with AGRP-LI released during the 40 min baseline time period
maximal inhibition was 90%. Figure 4
demonstrates the dose-dependency of leptin inhibition of AGRP-LI. In
comparison to 10-7 M
leptin, 10-6 M leptin also
caused maximal inhibition at 70 min at which time 95% inhibition was
observed. No increase in the basal release of AGRP-LI was observed in
fed animals in response to -MSH (10-8
M, 10-7
M, or 10-6
M), orexin B (10-6
M), MCH (10-7
M or 10-6
M), serotonin (10-6
M), or m-CPP
(10-6 M). In 48 h
fasted rats neither orexin B (10-6
M) nor serotonin (10-6
M) altered baseline release of AGRP-LI.
Cross-reactivity of the antibody made it impossible to accurately
determine the effect of orexin A on hypothalamic AGRP-LI release at
high doses of orexin A, but no effect was seen at
10-7 M or
10-8 M orexin A. We also
measured AGRP mRNA levels and detected a statistically significant
2.8-fold increase in fasted vs. fed animals
(P < 0.01) (Fig. 5).
NPY release from perfused hypothalamic tissue slices
NPY release was determined from the same samples that were used to
determine AGRP-LI release. As shown in Fig. 6, the amount of NPY release was greater
from the hypothalami of 48 h fasted rats than from the hypothalami
of rats fed ad libitum before they were killed
(P < 0.001). On average fasted animals released 80%
more NPY. As shown in Fig. 7, leptin
(10-6 M) inhibited NPY
release from the hypothalami of 48 h fasted rats. Comparison of
the 40-min time period before the administration of leptin (baseline
time period) to the 40-min time period during which leptin was infused
reveals that this inhibition was statistically significant
(P < 0.01). Statistically significant inhibition was
reached at 40 min (P < 0.03). As was the case with
AGRP-LI, a residual effect of leptin on NPY release persisted for at
least 30 min after cessation of the NPY infusion. Compared with the
average release of NPY during the 40 min baseline time period the
maximal inhibition of NPY occurred 1 h after beginning the leptin
infusion and was 33%. We did not observe any increase in NPY release
in fed animals with the administration of orexin B
(10-6 M) or MCH
(10-6 M). Although we
could not determine the effects of high dose orexin A on AGRP-LI
because of the cross-reactivity previously mentioned, we did not
observe any increase in NPY release in fed animals in response to high
dose orexin A (10-6
M).
Effect of fasting and bilateral adrenalectomy on serum AGRP-like
immunoreactivity
A slow rise in serum AGRP-LI occurred during the 48 h fast
(Fig. 8). At 24 h, the increase in
serum AGRP-LI was not statistically significant. However, the increase
in serum AGRP-LI did reach statistical significance at 48 h.
Bilateral adrenalectomy with or without corticosterone replacement had
no significant effect on serum AGRP-like immunoreactivity in fed
animals (Fig. 9). Serum corticosterone
levels were found to be negligible in rats that had undergone bilateral
adrenalectomy. Bilateral adrenalectomy also did not lead to any changes
in the level of hypothalamic AGRP mRNA (data not shown).
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Specificity of the AGRP RIA
To ensure the veracity of our findings we critically examined the
specificity of our AGRP RIA. Several lines of evidence support our
contention that the RIA is specific. First, in preliminary experiments
we found a low ID50 [10 fmol/ml for AGRP
(87–132)] and no cross-reactivity to a number of other peptides
including -MSH, NPY, orexin B, leptin, and agouti protein. The
latter is most notable in that agouti protein shares with AGRP a very
similar cysteine-rich C-terminal motif. Moreover, the antibody
recognizes AGRP’s C-terminus [AGRP (87–132)], which is a portion of
the molecule that is critical to bioactivity in pharmacological studies
(6).
Second, the concentrations of AGRP-LI observed in the tissue extracts
listed in Table 1 correlate well with the levels of mRNA known to be
present in those tissues. For example, AGRP mRNA is expressed at its
highest levels in the hypothalamus with the next highest level of
expression in the adrenal glands (1, 3). Consistent with that data, we
observed that AGRP-LI was greatest in the hypothalamus followed by the
adrenal gland. Similarly, no AGRP mRNA has been reported in liver by
Northern blot or PCR (1, 3) and consistent with those data no AGRP-LI
was detected in extracted hepatic tissue. In addition, the increase in
basal release of hypothalamic AGRP-LI observed in fasted rats
correlated with the increase in mRNA observed in similarly fasted
rats.
Third, the results of our chromatographic analysis strongly support the specificity of the RIA. Both tissue and serum AGRP-LI eluted in a single peak close to the elution position of synthetic AGRP (87–132). An important control in serum data were the observation that AGRP-LI eluted from the C18 environmental cartridge coeluted with serum applied directly to the C18 HPLC column in similar amounts. This observation indicates that the only significant difference between the two assay methods (C18 cartridge vs. C18 HPLC) is that the C18 cartridge results in significantly lower recovery of AGRP-LI. Nonetheless, the cartridge method facilitates the analysis of multiple samples.
Release of AGRP-LI from hypothalamic slices
In these studies, we were able to confirm that release of AGRP-LI
correlates with the changes in AGRP mRNA previously observed in fed and
fasted states or with leptin administration (7, 12). In our studies, we
noted a 3-fold increase in hypothalamic AGRP-LI released from the
hypothalami of 48 h fasted rats. This change correlates nicely
with the nearly 3-fold increase in AGRP mRNA observed in our studies
(Fig. 5). However, the increase in AGRP mRNA observed in these rat
studies are much less than the 15-fold increase in AGRP mRNA noted in
the mouse (1, 7) and may reflect interspecies differences. Recently,
Harrold et al. described a 91% reduction in AGRP
concentrations in 10 day food-restricted rats and hypothesized that the
marked reduction in AGRP (in a physiological state that might be
expected to be associated with increased AGRP concentrations) was due
to increased peptide release (27). Although we did not measure static
concentrations of AGRP-LI in 48 fasted rats, our results are consistent
with their hypothesis of increased AGRP release in physiological states
associated with hunger.
In our experiments, we observed that the basal release of AGRP-LI from the hypothalamus varies with the nutritional state of the animal (fed vs. fasted). We also observed that the fat hormone leptin has a profound influence on this release. This effect was most obvious in the fasted animals because their AGRP-LI levels are elevated. The observed suppression of AGRP-LI by leptin in fasted rats is what one would expect from a hormone that provides feedback to the hypothalamus regarding the adequacy of nutritional intake. We also observed a significant decrease of NPY release with leptin administration, which has previously been noted by Lee et al. (15). These data indicate that leptin concomitantly affects AGRP and NPY release from AGRP-NPY-containing arcuate nucleus neuron.
While the present studies are unable to distinguish whether the observed AGRP-LI release is governed by the constitutive or regulated pathways (or released through both pathways), the fact that KCl depolarization caused a large release of AGRP-LI suggests that at least some AGRP is stored and released with the regulated pathway. This is consistent with the notion that AGRP is an important regulatory neuropeptide and not merely a protein released constitutively from cells.
Several pieces of data indicate that the observed leptin suppression of AGRP-LI release from the hypothalamus of fasted animals was a specific effect. Importantly, the response was dose dependent and was not induced by any of the other peptide agents used in these studies. In addition, a simultaneous inhibition of NPY release was observed which makes the likelihood of a nonspecific AGRP response less likely. Thus, we hypothesize that the effect of leptin on AGRP-LI release is a result of the action of leptin on leptin receptors known to be present on the AGRP-NPY-containing neuron (7, 20). However, in our in vitro system the perfusate bathed the entire hypothalamus and the leptin-containing perfusate could have influenced the release of chemical messengers from other hypothalamic neurons expressing leptin receptors that also affect AGRP release. It is therefore conceivable that the leptin effect observed in these studies represents a summation response of the AGRP-NPY-containing neuron. Nonetheless, our data are consistent with the hypothesis that leptin is a predominant regulator of AGRP release.
Contrary to our expectations we found no increase in the release of AGRP-LI with the administration of orexin B from hypothalami of fed animals. This was surprising in view of the work of van den Pol et al. who found that arcuate nucleus neurons have nanomolar sensitivity to orexin and are capable of releasing GABA and glutamate from axons terminals adjacent to the AGRP-NPY-containing cell in response to orexin (28). Electron microscopy studies performed by Horvath et al. have also identified an abundance of presynaptic orexin-immunoreactive boutons abutting NPY-containing neurons (21). This latter observation would strongly suggest the presence of orexin receptors on the AGRP-NPY-containing neuron.
We also had anticipated that MCH might have an effect on hypothalamic AGRP-LI release because MCH projections from the lateral hypothalamus to the arcuate nucleus have previously been observed (22). However, it should be noted that the actual presence of MCH receptors on the AGRP-NPY-containing neuron has not, to our knowledge, been documented.
That m-CPP did not cause a change in hypothalamic AGRP-LI release in fasted rats was also somewhat unexpected. Because the hypophagic effect of serotonin is thought to be mediated by the serotonin 5HT1B/2C receptor, the observation that serotonin did not cause a change in AGRP-LI might be interpreted as consistent with the conflicting positive and negative effects of this biogenic amine on NPY release reported in the literature (17, 18). However, the lack of suppression of basal AGRP-LI release with the administration of the selective 5HT1B/2C agonist m-CPP could be construed as inconsistent with the data of Dryden et al. who observed a decrease in NPY levels in the PVN (which was presumed to reflect an effect of m-CPP on the AGRP-NPY-containing neuron in the arcuate nucleus) with sc or ip administration of that agent (18). Nonetheless, the in vivo model used in the latter study differs significantly from our perfusion model and, therefore, no firm conclusions can be drawn regarding this potential discrepancy.
Several possible explanations exist for the lack of change in AGRP-LI in response to orexin B, MCH and m-CPP. First we must acknowledge that the hypothalamic perfusion system itself might not be sensitive enough as a system to detect small changes in AGRP-LI release produced by these agents. However, if this is the case, an important corollary might be that orexin B, MCH, and m-CPP are relatively weak regulators of AGRP. We do not feel that the sensitivity of the RIA was an issue because the half-dose of AGRP (87–132) suggests that the assay should have been sensitive enough to detect reasonably small changes in AGRP-LI.
A second possibility is that when orexin, MCH, and m-CPP are applied to hypothalamic slices they stimulate neurons bearing their receptors in other hypothalamic nuclei. The summation response might not result in an observable change in AGRP-LI release.
A third possible explanation is that orexin B, MCH, and m-CPP do not directly affect the acute release of AGRP or NPY. Rather, it is possible that these agents act by modulating the membrane potential of the AGRP-NPY-containing neuron and by this mechanism determine the subsequent response of the AGRP-NPY-containing neuron to agents such as leptin. In this scenario, the effect of orexin B, MCH, and m-CPP on AGRP release would only be indirect and in our experimental model we would not observe a change in AGRP-LI release in response to of any of these three agents. A similar lack of observable effect would occur if orexin, MCH, and serotonin acted by regulating the transcription of AGRP mRNA and subsequently influencing the pool of AGRP available to be released under physiological conditions such as decreasing serum leptin levels.
Comparison of NPY and AGRP-LI release from hypothalamic slices
Because AGRP and NPY are produced and released from the same
arcuate nucleus neurons, we simultaneously examined AGRP-LI and NPY
release from the perfused hypothalami of fed and fasted rats. Similar
to AGRP-LI release, a continuous basal NPY release was observed in both
the fed and fasted state (Figs. 2 and 6). NPY release was 80% greater
from the hypothalami of 48 h fasted vs. fed animal
(Figs. 2 and 6). An increase of NPY release from PVN of food deprived
rats has previously been reported (29, 30). These latter studies
suggest that the PVN is the predominant source of the NPY released
from the perfused hypothalami in the present studies. Comparing the
percent increase in NPY and AGRP-LI release that occurred in fasted
animals reveals that NPY release increased to a lesser degree than
AGRP-LI (80% vs. 216%). Similar to AGRP-LI release, NPY
release from perfused hypothalami was inhibited by leptin and the time
course of leptin-induced NPY suppression and recovery paralleled that
of AGRP-LI (Figs. 3 and 7). However, leptin inhibition reached
statistical significance sooner in the case of AGRP-LI release
vs. NPY release (30 min vs. 40 min). In addition,
comparing the maximal inhibition of AGRP-LI and NPY release by
10-6 M leptin reveals that
NPY release was inhibited to a lesser extent than AGRP-LI release (33%
vs. 95%). Although purely speculative, these data may
indicate that leptin has a greater role in regulating AGRP release than
NPY release and/or that leptin mediates its effects more through its
regulation of AGRP release than through its regulation of NPY release.
The last two statements should be viewed with caution because the
physiological consequences of the relative percent increases or
decreases in release of the two agents cannot be determined from the
present studies.
Presence of AGRP-LI in adrenal tissue and serum
Our studies indicate that the level of AGRP mRNA expression in
adrenal tissue correlates well with the substantial stores of AGRP
protein that we detected in that organ. On the basis of wet tissue
weight the adrenal has approximately 16% of the AGRP protein observed
in the hypothalamus of the fed rat.
Our chromatographic characterization of AGRP-LI suggests that AGRP is present in the systemic circulation at slightly less than nanomolar concentrations in the fed animal and increases to slightly more than one nanomolar in the 48 h fasted rat. However, the origin of serum AGRP-LI detected in our studies remains a crucial and unresolved question. Our adrenalectomy experiments indicate that adrenal AGRP is unlikely to be the source of serum AGRP-LI. The results of those experiments would also suggest that adrenal AGRP is likely to have an as yet unidentified paracrine role in adrenal physiology.
One potential source for serum AGRP-LI could be the hypothalamus. In the rat, AGRP-containing fibers have recently been noted to project from the arcuate nucleus through the median eminence to the posterior pituitary, where it conceivably could be released into the systemic circulation (7). In this regard, it is important to note that no compensatory increase in hypothalamic AGRP mRNA was seen in adrenalectomized animals. This is certainly consistent with the lack of change in the serum concentration of AGRP-LI observed in those animals. However, more direct studies would be required to conclude that hypothalamic AGRP actually reaches the systemic circulation.
Despite the fact that the origin of serum AGRP-LI is unclear, the levels of AGRP-LI observed in rat serum are potentially in a physiological range. Although purely speculative, AGRP in the bloodstream would likely function as a modulator of peripheral MCR activity in tissues such as the pancreas or gastrointestinal tract that contain receptors sensitive to its actions. The concentration of AGRP found in rat serum is close to the IC50 of AGRP (87–132) observed in our pharmacological studies of the cloned human MC3R (approximately 1 nanomolar) (6). However, AGRP (87–132) has a much lower affinity for the human MC5R, another peripherally expressed MCR that could be a potential target of serum AGRP. In this regard, we have not examined the binding affinity of AGRP at the rat MC3R and MC5R. Species differences in ligand binding affinity are not uncommon and the binding affinity of AGRP could be quite different at the rat receptors. The recent identification of the mahogany protein, which is postulated to be a low affinity receptor for agouti protein, may also have relevance for this discussion (31, 32). Whether mahogany protein or a similar molecule exists that functions to increase local concentrations of AGRP in peripheral tissues is presently unknown.
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Acknowledgments |
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Footnotes |
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2 Associate Investigator of the Howard Hughes Medical Institute.
Received August 24, 1999.
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References |
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-MSH or POMC concentrations in
dietary-obese and food-restricted rats. Biochem Biophys Res Commun 258:574–577[CrossRef][Medline]
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