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Department of Neuroscience, Neurocrine Biosciences, Inc., San Diego, California 92121
Address all correspondence and requests for reprints to: Mary Ann Pelleymounter Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, California 92121. E-mail: MPelleymounter{at}neurocrine.com
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whether or not the effects of central CRF on energy balance can be separated from its effects on glucocorticoid release is unclear. Two receptor populations mediate the functional effects of CRF with quite distinct distributions in brain. For example, CRF1 receptors are localized in the cerebral cortex and the anterior lobe of the pituitary gland, whereas CRF2 receptors are localized in the ventromedial and paraventricular hypothalamus (19). Interestingly, CRF2 but not CRF1 antisense administration attenuates the effect of CRF on appetite (20). Finally, the mammalian CRF-related peptide, urocortin (UCN), which has a 20- to 40-fold higher affinity for the CRF2 receptor than CRF itself (21, 22), reduces food intake when infused centrally (20, 23, 30). Whether or not centrally administered UCN also activates the sympathetic nervous system and promotes glucocorticoid release is not known. If the role of the brain CRF2 receptor could be dissociated from pituitary activation, it is possible that a receptor agonist specific to the CRF2 receptor would promote appetite suppression without the deleterious side effects that accompany chronic glucocorticoid release. Alternatively, a receptor antagonist specific to the CRF2 receptor could possibly facilitate eating in cachexic disorders without affecting pituitary function. Therefore, it would be important to know whether the effects of CRF on energy balance were receptor subtype specific and whether the proposed CRF2-selective agonist, UCN, would effect energy balance without elevating corticosterone. We have addressed these questions in two ways: 1) by comparing the effects of centrally infused CRF and UCN on energy balance, and 2) by assessing the effects of a recently described peptide CRF2-selective antagonist (antisauvigine-30) (24) on the negative energy balance induced by central CRF or UCN infusion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides
Antisauvagine-30 (ASV-30) was synthesized by solid phase
methodology on a Beckman Coulter, Inc. (Fullerton,
CA) 990 peptide synthesizer using t-Boc-protected amino acids and the
assembled peptide was deprotected with hydrogen fluoride. The crude
peptide product was purified by preparative HPLC. The purity and
identity of the final product was ascertained by analytical HPLC and
mass spectrometric analysis using an ion-spray ion source. CRF and
urocortin (UCN) were purchased from American Peptide Co.
(Sunnyvale, CA). All peptides were dissolved in a 0.1% BSA
vehicle.
CRF and UCN were placed into osmotic mini-pumps at concentrations
varying from 2.1–210 µg/ml. In the case where CRF and
antisauvigine-30 were coinfused, the concentration of CRF was 0.21
mg/ml and antisauvigine-30 was at 3.02 mg/ml. Antisauvigine-30 is
approximately 100-fold more potent at CRF2
receptors, with a Ki of 153.6 nM at
CRF1 receptors, and 1.4 nM at
CRF2
receptors (24).
Design
In the first study, where the objective was to compare the
dose-response functions of CRF and UCN on body weight, rats were given
either CRF (0.01–1.0 nmol/day), deamidated CRF (1 nmol/day), or UCN
(0.01–1.0 nmol/day) by continuous infusion into the lateral ventricle.
Deamidated CRF (CRF-OH) does not bind to the CRF receptor and was used
to assess the overall receptor specificity of CRF’s effects on body
weight. Body weight was monitored for the entire infusion period, which
was 13 days. On the last day of infusion, rats were killed, and brains
were examined to ascertain accuracy of cannula placement. A second
study assessed the importance of food intake on the effects of CRF or
UCN on body weight. In this study, the food intake of vehicle-treated
rats was matched to rats given a high dose of either CRF or UCN (1
nmol/day). Change in body mass and tissue weights were the dependent
variables in this second study. In the third and final study, the
objective was to assess the importance of the
CRF2 receptor in the effects of CRF on body
weight and to examine in more detail the importance of this receptor in
the effects of CRF on energy balance. Therefore, rats were treated with
either CRF (1 nmol/day), CRF (1 nmol/day) plus ASV-30 (30 nmol/day),
UCN (1 nmol/day), UCN (1 nmol/day) plus ASV-30 (30 nmol/day), ASV-30
(30 nmol/day) alone or vehicle alone for a 13-day period. Body weight
and food intake were monitored as in the first study, and on day 13,
these animals were killed, with tissue weights and blood chemistry
assessed as dependent variables.
Procedures
Surgery. All rats were implanted with a 22 g cannula
(Plastics One, Inc.) into the lateral ventricle (AP = -0.8 mm,
ML = +1.2 mm and DV = -4.5 mm). An osmotic mini-pump
(Alza Corp., Model 2001) was attached to the cannula with
silicone tubing, and implanted into the intrascapular space. Pumps
delivered peptides or vehicle at 1.0 µl/h for a 7-day period and were
replaced once under isoflurane anesthesia. At the time the rats were
killed, the pump was disconnected, and cresyl violet (1 mg/ml) was
injected into the cannula tubing to verify placement and flow. Animals
with evidence of misplaced cannulae or impaired flow of dye were not
included in the study.
Food intake and body weight (Experiments 1–3). Food intake and body weight were always measured at the same time each day, which was between 1600 and 1700 h. In the pair-feeding studies, vehicle-treated rats were given the amount of food eaten each day by an assigned CRF or UCN-treated counterpart. This assigned amount was divided between two feedings: one in the morning (0900 h) and one in the evening (1700 h).
Tissue weights (Experiments 2 and 3). The rats were anesthetized with isoflurane for a maximum of 2 min to minimize anesthesia effects on corticosterone or glucose levels. During this 2-min period of anesthesia, blood was drawn by cardiac puncture, and brains were taken for verification of cannula placement. Thymus, adrenals, heart, and spleen were then dissected from the remaining carcass and weighed. In addition, brown adipose tissue from the intrascapular area, along with retroperitoneal and inguinal white adipose fat pads were dissected and weighed.
Blood chemistry (Exp 3 only). Blood was collected between 0900 and 1100 h on the day the rats were killed. Rats were not food deprived the night before to assure low baseline corticosterone levels. Corticosterone and insulin were measured in nonextracted serum using RIA (ICN Pharmaceuticals, Inc., Orangeburg, NY; and Linco Research, Inc., St. Charles, MO, respectively). Glucose, serum nonesterified FFA (NEFA), cholesterol, and triglycerides were analyzed using a Hitachi 717 blood chemistry analyzer (Anilytics, Inc., Gaithersburg, MD).
Statistics
All data collected over time was analyzed by repeated measures
ANOVA, with treatment group as the between groups variable and time as
the within-groups variable. Significant group effects or interactions
were then analyzed by Fisher’s PLSD (Statview). Single measure
variables were analyzed by one-way ANOVA, with Fisher’s PLSD as the
posthoc test for group differences. Body weight was analyzed as change
from baseline weight, and tissue weights were normalized to final body
mass before ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). UCN also produced a significant,
dose-dependent, attenuation of weight gain [F(3,192) = 3.2;
P < 0.05] (Fig. 1B). Both peptides produced
significant attenuation of weight gain only at the 1 nmol/day dose
(ps < 0.004–0.01). CRF produced significantly more attenuation
of weight gain than UCN [F(5,588) = 8.2; P < 0.0001]
(posthoc P < 0.0001). The effects of CRF on body mass
at the highest dose were obvious within the first 48 h in all
three studies (ps < 0.02–0.0002 vs. vehicle) and
sustained throughout the study period (ps < 0.007–0.0001
vs. vehicle, day 13). The pattern and magnitude of effect
for UCN varied from study to study, with sustained weight gain
attenuation only statistically significant in the first experiment
(P < 0.003 vs. vehicle on day 13).
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. In contrast, weight gain of rats that
were pair-fed to UCN-treated counterparts was not different from that
of UCN-treated animals, as shown in Fig. 2B. However, in this study,
weight gain in the UCN-treated rats themselves was parallel to that of
vehicle controls after an initial attenuation of 25–32%.
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). UCN produced a
statistically signficant reduction in food intake that was time and
dose dependent, as suggested by a significant interaction between time
and dose [F(36,180) = 1.54; P < 0.03], as shown in
Fig. 4B. When the food intake of rats treated with the highest dose of
CRF and UCN was compared over time in Exp 1, CRF and UCN produced a
similar degree of overall appetite suppression (13.2–13.1%). In the
third study, however, CRF reduced overall food intake more than UCN
(-21% vs. -6%), a difference that was statistically
significant [F(5,1188) = 8.88; P < 0.0001] (posthoc
P < 0.0001). ASV-30 completely reversed the effects of
both CRF and UCN on food intake [F(5,1188) = 8.88; P
< 0.0001)] (posthoc ps < 0.0002–0.0001 vs. UCN or
CRF, respectively). Infusion of ASV-30 alone also produced a slight
increase (5%) in overall food intake (P < 0.006).
These effects are illustrated in Fig. 5.
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and 2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, rats that were pair-fed to CRF-treated counterparts did not lose as much weight as those actually treated with CRF, consistent with the hypothesis that CRF-induced weight loss could have a metabolic component in addition to its effects on appetite. If CRF-induced weight loss does involve a metabolic component, it is probably related to the sympathetic nervous system (SNS)-activating properties of CRF, as previously suggested (1, 5, 8). Consistent with this idea, brown adipose fat pad weights and adrenal tissue weights were both increased in CRF-treated animals, suggesting prolonged SNS stimulation. These changes in BAT weight were probably a function of direct CRF stimulation of the SNS, rather than an indirect result of the prolonged appetite suppression induced by CRF because these changes were not observed in pair-fed counterparts. Further, chronic central administration of CRF resulted in elevated corticosterone, cholesterol, triglycerides and FFA, all of which suggest SNS-induced lipolysis. These elevations in serum lipids, however, were not accompanied by significant reductions in fat pad weights. Perhaps fat pad weights were too insensitive a measure, because Rivest (7) have shown that carcass fat was reduced with chronic central CRF infusion.
As previously reported, chronic central administration of CRF resulted in elevated plasma corticosterone and adrenal weights, along with a reduction in thymus and spleen weight that was independent of weight loss itself (5, 6, 17, 25), and presumably a function of the chronic increase in serum corticosterone (17, 25). Again, these effects of CRF were probably not an indirect function of sustained appetite suppression because pair-fed controls did not show increased adrenal weights or reduced thymus and spleen weights.
Our findings that centrally administered UCN failed to alter any of these HPA-related variables agrees with earlier work showing that anti-UCN serum did not inhibit the release of ACTH induced by stress (26) or adrenalectomy (27) and that adrenalectomy did not alter UCN IR in the hypothalamus (28). However, these data were somewhat unexpected in view of the fact that like CRF, UCN binds with very high affinity to CRF1 receptors (21, 22). Further, previous work has shown that peripherally administered UCN increased plasma ACTH and corticosterone (22, 29), which is consistent with the idea that UCN can have direct effects on CRF1 receptors in the pituitary in vivo (22).
In addition, Grill et al. (30) recently reported that a single injection (0.6 nmol) of UCN into either the lateral or fourth ventricle at the onset of the dark cycle produced a 2-fold increase in plasma corticosterone. Perhaps the slow (1 µl/h) infusion of UCN used in our methods did not result in the same parenchymal concentration as the bolus injection approach used in the Grill (30) study. It is also possible that CRF has better parenchymal penetration than UCN.
Another unexpected finding with chronic UCN administration was that it did not produce more appetite suppression than CRF. This was unexpected in view of the fact that UCN binds with higher affinity to the CRF2 receptor than CRF itself (21, 22), which has been used as evidence that UCN is a CRF2 agonist. It must be noted, however, that CRF also binds with high affinity to the CRF2 receptor (13–17 nM) (21, 22). Whether or not a 20-fold difference at such high affinities would actually translate into an in vivo distinction between the two ligands is not clear, although there is a report showing that UCN is a more potent appetite suppressant than CRF when administered as a single icv bolus (23). As suggested above, perhaps the protein-binding properties of the peptide, combined with the slow infusion rate used in chronic intraventricular delivery, resulted in more binding of UCN than CRF to blood vessels lining the ventricles, which also contain CRF2 receptors (19). Thus, it is difficult to make a generalization about the role of the CRF2 receptor and energy balance based upon the effects of UCN alone.
ASV-30, however, clearly attenuated the effects of both UCN and CRF on food intake. Because ASV-30 has been portrayed as a selective antagonist of the CRF2 receptor, these results are consistent with previous data showing that antisense to the CRF2 receptor attenuated the effects of CRF on food intake (20) and that mice lacking the CRF2 receptor show a blunted response to the effects of UCN on food intake (31). Interestingly, ASV-30 alone also increased food intake, providing the first line of evidence that antagonism of endogenous CRF in an adult can result in increased feeding.
Despite the fact that ASV-30 completely reversed the effects of CRF on food intake, it did not alter the effects of CRF on glucose levels. ASV-30, however, reversed the CRF-induced reduction in insulin levels, actually overshooting the levels observed in vehicle controls. The insulin overcompensation produced by ASV-30 is probably the reason that glucose levels did not normalize along with food intake in animals treated with the CRF/ASV-30 combination. Because it has been suggested that the effects of CRF on insulin are a component of its general effects on the SNS (5, 18, 32), it is possible that ASV-30 does influence a subset of the SNS-activating aspects of CRF. Exactly why ASV-30 would influence only one component of CRF-induced sympathetic activation is not clear.
ASV-30 did not attenuate the increase in serum lipids that are typically associated with increased sympathetic nervous system activity, however. Further, ASV-30 did not have any effect on the BAT hyperplasia observed in CRF-treated animals, which also indicated that ASV-30 did not attenuate the SNS activation induced by CRF. In addition, ASV-30 had no effect on the CRF-induced changes in adrenal, thymus, and spleen weights, or on the CRF-induced elevation in corticosterone. Taken together, these data suggest that ASV-30 did not effect the SNS or HPA variables that were altered by chronic CRF infusion.
Therefore, our data indicate that when administered centrally, ASV-30 has effects on a very specific subset of variables associated with energy balance; food intake and insulin levels. If the fairly specific functional profile of this peptide as well as that of UCN reflects receptor selectivity, then our data suggest that the role of the CRF2 receptor in energy balance may be primarily related to food intake. Earlier work from this laboratory is consistent with this hypothesis, in that the reduction in food intake induced by a single icv administration of CRF was only attenuated by ASV-30 or the mixed CRF antagonist, astressin, but not by a specific CRF1 antagonist (33).
In summary, our data show that chronic, central infusion of CRF induces a state of negative energy balance that is partially a function of its effects on food intake, and partially a function of its activating effects on the sympathetic nervous system. In contrast, UCN appears to induce a less dramatic state of negative energy balance that is primarily dependent upon changes in food intake and does not appear to involve sympathetic nervous system activation. Further, chronic central administration of the peptide CRF2 antagonist, ASV-30, completely reversed the effects of CRF and UCN on food intake, and increased food intake when given alone. ASV-30 also completely reversed the effects of CRF on insulin levels and dramatically attenuated the effects of CRF and UCN on body weight. ASV-30 did not, however, alter the effects of CRF on HPA-related variables such as increased corticosterone levels, increased adrenal weight and reduced thymus and spleen weight. Similarly, ASV-30 did not change the effects of CRF on SNS-related variables such as elevated serum lipids and brown adipose fat pad weight. These data suggest that the central CRF2 receptor may primarily mediate the appetite suppressing effects of CRF rather than the metabolic effects of this peptide.
Received May 17, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-selective antisauvigine-30. Proc Natl
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