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Neurobiology of Nutrition Laboratory, Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808
Address all correspondence and requests for reprints to: Hans-Rudolf Berthoud, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808. E-mail: berthohr{at}pbrc.edu.
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Abstract |
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ERKCREB cascade may thus constitute a molecular integrator for converging satiety signals from the gut and adiposity signals from the hypothalamus in the control of meal size and food intake. |
Introduction |
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Numerous studies show that normal ingestion of food (4, 5), delivery of food to the stomach and small intestine (5, 6, 7), or distending the stomach (7, 8, 9) induce c-Fos in solitary nucleus (NTS) neurons, an event indicating adaptive neuronal responses involving gene transcription (10). We have recently demonstrated that peripheral administration of cholecystokinin (CCK), the classical satiety hormone, stimulates ERK phosphorylation in NTS neurons and that activation of this intracellular signaling cascade is necessary for CCK’s food intake suppression (11). Given the modulatory character of descending inputs from the hypothalamus and other forebrain sites, this signaling pathway may therefore represent the molecular substrate by which direct and indirect controls of meal size are integrated.
Descending inputs from the hypothalamus include melanocortinergic projections originating in the arcuate nucleus (12), oxytocin projections from the paraventricular nucleus (13), and orexin and melanin-concentrating hormone projections from the lateral hypothalamus (14, 15). The central melanocortin system has been strongly implicated in the control of food intake and energy balance (16). Attention to the caudal brainstem as a site of action for the central melanocortin system to modulate food intake was drawn when melanocortin receptor ligands were injected into the fourth ventricle or the dorsal vagal complex (17, 18, 19). Melanotan II (MTII) administered to the fourth ventricle dose-dependently reduced 30 min glucose intake in rats without disruption of lick motor performance, suggesting that activation of brainstem MC4-melanocortin receptor (MC4R) reduces intake by amplifying satiation mechanisms (19). Strong expression of the MC4R in the dorsal vagal complex has also been reported (20, 21). Furthermore, prior fourth ventricular administration of the MC4R antagonist SHU9119 completely blocked exogenous CCK-induced feeding suppression, suggesting a direct interaction between melanocortin and peripheral satiety signals in the caudal brainstem (22).
Here we test the hypotheses that the ERK signaling pathway in NTS neurons is modulated by MC4R ligands administered to the fourth ventricle and might act as an integrator of melanocortin- and CCK-mediated signals controlling satiation and meal size.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides and antibodies
MTII and SHU9119 were obtained from Phoenix Pharmaceuticals (no. 043–23, no. 043–24; Belmont, CA). Cholecystokinin (sulfated octapeptide, CCK 26–33) was purchased from Bachem-Peninsula (no. 7183; San Carlos, CA). MAPK kinase (MEK) inhibitor U0126 (no. V1121) was obtained from Promega (Madison, WI). The cAMP receptor protein-Rp isomer (cAMP-Rp) triethylammonium salt was supplied by Tocris Cookson (no. 1337; Ellisville, MO). Cell Signaling Technology (Beverly, MA) supplied primary antibodies for phospho-p44/42 MAPK (Thr 202/Tyr 204 phospho-ERK1/2; no. 9101), Ser 133 phospho-CREB (no. 9191), p44/42 MAPK (ERK 1/2; no. 9102), and cAMP response element-binding protein (CREB; no. 9192). With immunoblot analysis, all antibodies gave clear signals at the predicted molecular sizes of the investigated proteins.
Fourth ventricular cannulations
Animals were anesthetized with ketamine-xylazin-acepromazine (80–4-1.6 mg/kg sc) and given atropine (1 mg/kg ip). A 24-Ga stainless steel guide cannula was aimed at the fourth ventricle (2.5 mm anterior to the posterior occipital suture, on the midline, 5.0 mm below the dura). A 30-Ga beveled injector was designed to protrude for 1.0 mm from the guide cannula. Rats were given 10 d to recover, after which cannula placement and patency was verified using the 5-thio-glucose test (23), consisting of injecting 5-thio-glucose (210 µg in 3 µl sterile saline) and measuring plasma glucose concentration after 30 min. Only animals responding with an increase of plasma glucose concentration of at least 80 mg/ml were used for experiments.
Experimental protocols
Nine rats were implanted with fourth ventricular cannulas for the MTII dose-response experiment. Animals were adapted to the test procedure during the 7-d recovery period. On test days, between 0900 and 1000 h (2–3 h after lights on), one group of overnight-fasted rats was infused with either 0.05 or 2.0 nmol of MTII (in 3 µl sterile saline) or saline alone into the fourth ventricle over 2 min. Thirty minutes after injections, rats were killed by guillotine in a separate room and brains were rapidly extracted, blocked, and snap frozen in isopentane cooled in dry ice. The tissue was held at –80 C until ready for protein extraction.
To study the combined effect of MTII and CCK, 24 rats were cannulated and adapted as above. Six rats were assigned to each treatment group. On test days between 0900 and 1000 h, overnight food-deprived rats were infused with either MTII (0.05 nmol in 3 µl sterile saline) or saline alone into the fourth ventricle. Thirty minutes later, rats were injected ip with either CCK (2 µg/kg in sterile saline) or saline alone and killed 15 min later. The injections were separated by 30 min to guarantee sufficient diffusion of MTII before the relatively fast action of ip CCK. Brains were harvested and frozen as mentioned above.
To determine whether U0126 blocked MTII-induced increases in phospho-ERK, 16 naïve rats with fourth ventricular cannulas divided into four weight-matched groups were used for Western blotting. Overnight (16 h) food-deprived rats were first infused with either U0126 (2 µg in 3 µl 50% dimethylsulfoxide/sterile saline) or vehicle alone and 1 h later with either MTII (0.05 nmol in 3 µl sterile saline) or saline alone into the fourth ventricle. Without allowing access to food, rats were killed 30 min after the second injection and brains were rapidly removed and frozen as described above.
Similarly, to test the role of cAMP signaling in MTII-induced ERK phosphorylation, 12 naïve rats with fourth ventricular cannulas, divided into four weight-matched groups were used. On test days, overnight-fasted rats were first infused with the cAMP-inhibitor cAMPS-Rp (100 nmol in 3 µl sterile saline) or saline alone and 30 min later with MTII (0.05 nmol in 3 µl sterile saline) or saline alone into the fourth ventricle. They were killed 30 min later, without access to food. Brains were harvested and frozen as described above.
To investigate the ability of the MEK inhibitor U0126 to reverse MTII-induced reduction of food intake, 10 naïve rats with fourth ventricular cannulas were used. In a counterbalanced crossover design, 16-h food-deprived rats were first injected with either U0126 (5 µg in 50% dimethylsulfoxide/sterile saline) or vehicle alone and 1 h later with either MTII (0.05 nmol in sterile saline) or saline alone during the light phase of the light-dark cycle. All infusions were given over a period of 2 min in a volume of 3 µl. After the second infusion, rats were returned to their home cage and a preweighed amount of chow was made available. Food intake was measured at 2 and 4 h by weighing the food and taking spillage into consideration. The four test days were separated by at least 3 d to avoid possible carryover effects.
To determine the effects of SHU9119 on CCK-induced ERK cascade phosphorylation, 12 cannulated rats were used. On test days, overnight-fasted rats were first given fourth ventricular infusions of either SHU9119 (1 nmol in 3 µl sterile saline) or saline alone and 2 h later ip injections of either CCK (10 µg/kg in sterile saline) or saline alone. Rats were killed 15 min after the second injection without access to food. Brains were harvested and frozen as described previously.
To determine the effects of SHU9119 on meal structure, six rats with fourth ventricular cannulas were habituated to an automated feeding system consisting of wire-mesh cages with an access hole to a food cup containing powdered chow and resting on a balance. The weight of the food cup was continuously monitored by a computer and meal size, meal duration, intermeal interval, meal frequency, and satiety ratio (time in minutes of intermeal interval per gram eaten) was calculated using custom-made software. A meal was defined as ingestive activity not interrupted by more than 10 min and amounting to at least 0.2 g eaten. On test days, rats were injected with SHU9119 (0.5 nmol in 3 µl sterile saline) or saline into the fourth ventricle 1 h before dark onset and returned to their cage. Feeding activity throughout the dark period for a total of 16 h was monitored and analyzed. Each rat received the drug and control injection in a counterbalanced fashion, with at least 2 d between injections.
Tissue collection
Animals were decapitated with a guillotine and the brain rapidly removed, blocked, and frozen in isopentane cooled in dry ice before being stored at –80 C until use. Two hundred-micrometer-thick brainstem sections throughout the NTS (–14.08 to –12.72 mm from Bregma) were cut in a cryostat. Slices were placed on a chilled slide under a dissecting microscope. The NTS was removed with a micropunch tool and placed into 2% sodium dodecyl sulfate (SDS) (24). Tissue was homogenized by trituration and centrifuged at 150,000 x g for 15 min at 4 C to remove any particulate matter. An aliquot of solubilized tissue was used for total protein determination by the bicinchoninic acid assay (Pierce, Rockford, IL).
Western blotting
An aliquot of the frozen supernatant was diluted with an equal volume of 2x electrophoresis sample buffer [final concentration, 50 mM Tris-HCl (pH 6.7), 4% (wt/vol) glycerol, 4% SDS, 1% 2-mercaptoethanol, and 0.02 mg/ml bromphenol blue] and boiled for 10 min. Twenty micrograms were separated by size on a 10% SDS-polyacrylamide gel using the buffer system of Laemmli (25) and transferred to Immobilon-P polyvinylidene fluoride membranes in Towbin-transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, and 0.01% SDS). After transfer, the blot was washed with PBS containing 0.05% Tween 20 (PBST). The membranes were blocked in PBST containing 5% nonfat dry milk and 1% BSA for 1 h at room temperature with agitation. The membranes were then incubated with an antibody specific for phospho-ERK 1/2 or total ERK, phospho-CREB, or total CREB, diluted 1:1000 in PBST containing 0.5% nonfat dry milk and 0.1% BSA for 1 h at room temperature. The membranes were washed twice in PBST for 5 min, once for 15 min, and then twice for 5 min in PBST. This was followed by a 1-h incubation with agitation at room temperature with horseradish peroxidase-conjugated goat antirabbit immunoglobulin diluted 1:100 in PBST containing 0.5% nonfat dry milk and 0.1% BSA.
After this incubation, the membranes were washed as above and the antigen-antibody-peroxidase complex was detected by enhanced chemiluminescence according to the manufacturer’s instructions and visualized by exposure to Hyperfilm enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were then stripped by incubation in stripping buffer [100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCL (pH 6.7)] for 30 min at 50 C with gentle agitation. Membranes were blocked as above and reprobed with anti-ERK 1/2 or CREB total antibody as described above. Film autoradiograms were analyzed and quantified by computer-assisted densitometry (HP Scanjet 5200 and Quantity One 4.4.1 software; Bio-Rad Laboratories, Hercules, CA). Only ERK 2 bands were quantified for densitometry.
Data analysis
Densitometry scores were analyzed by two-way ANOVA followed by Bonferroni-adjusted least significant difference post hoc comparisons. Food intake in the U0126 experiment was analyzed using repeated measures ANOVA with specific a priori hypotheses tested with linear contrasts. Meal parameters were analyzed using paired t tests. All statistics were computed using the appropriate procedures of SYSTAT 10.0 (SPSS 1998; SYSTAT for Windows; SPSS Inc., Chicago, IL).
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Results |
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CREB cascade and food intake
, phosphorylated ERK1/2 but not total ERK1/2 was dose-dependently increased 30 min after administration of MTII. The lower dose of 0.05 nmol of MTII increased pERK2 about 2-fold (P < 0.05) and the higher dose of 2 nmol more than 3-fold (P < 0.01).
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and 3, the effects of CCK and MTII given in combination were additive for both ERK and CREB phosphorylation. A low dose of 2 µg/kg ip CCK significantly increased pERK2 (P < 0.05) and pCREB (P < 0.05), and, if given 15 min after the lower dose of fourth ventricular MTII, there was a significant further increase of pERK2 (P < 0.01) and pCREB (P < 0.05). Total ERK and CREB were not significantly changed by any treatment.
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). As shown above, MTII (0.05 nmol) significantly increased ERK2 phosphorylation (P < 0.01). Pretreatment with U0126 (2 µg) significantly (P < 0.05) attenuated MTII-induced ERK phosphorylation by about 50%, whereas U0126 alone did not change it.
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). As expected, MTII suppressed 2-h food intake by about 50% (vehicle/vehicle 7.41 ± 0.26 g, vehicle/MTII 3.78 ± 0.12 g, P < 0.01). Prior treatment with this dose of U0126 almost completely reversed the effect of MTII (U0126/MTII 7.11 ± 0.25 g, P < 0.01) and by itself did not affect chow intake (vehicle/U0126 7.29 ± 0.29 g, ns). To directly compare the effect of MTII in the absence and presence of prior U0126 treatment, we used a paired t test on the individual difference scores. In the presence of U0126, the average MTII-induced decrease of 2-h food intake (–0.29 g) was significantly less than in the absence of U0126 (–3.79 g, P < 0.01). Similarly, 4-h food intake was also significantly suppressed by MTII, and this effect was completely reversed by prior U0126 treatment (data not shown). No adverse behavioral effects were noted after fourth ventricular U0126 injections.
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).
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CREB cascade) and pCREB (Fig. 8). Injection of SHU9119 in the absence of CCK did not significantly change the already low levels of phosphorylated proteins. Because Fan et al. (22) recently demonstrated that fourth ventricular injection of SHU9119 prevented the food-suppressing effects of exogenous CCK, we did not carry out the behavioral counterpart to this experiment.
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, SHU9119 (0.5 nmol) significantly increased 16-h powdered chow intake [(F [1, 5]) = 39.5, P < 0.01] by selectively increasing meal size (F = 11.1, P < 0.05) but not meal frequency (F = 0.16, ns). SHU9119 treatment also significantly decreased the satiety ratio (F = 15.8, P = 0.011), a measure of the capacity of a given amount of food to generate satiety. The increase in SHU9119-induced meal duration was marginally significant (F = 6.8, P = 0.47).
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Discussion |
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Here we show that feeding suppression induced by both local hindbrain MC4R-agonism and exogenous CCK administration depend on activation of the ERK1/2 signaling pathway in the NTS. We have demonstrated earlier that this is also the case for feeding suppression induced by exogenous CCK (11). Fourth ventricular administration of the MC4R agonist MTII dose-dependently stimulates phosphorylation of ERK1/2 and CREB in the NTS, just as reported earlier for exogenous CCK (11), and, when given in combination, the effects are additive. In contrast, administration of the MC4R antagonist SHU9119, completely blocks the CCK-induced phosphorylation of ERK and CREB in the NTS. Partial blockade of MTII-induced ERK-phosphorylation by the cAMP antagonist cAMPS-Rp further implicates cAMP signaling in MC4R stimulation-induced increases in ERK phosphorylation. To demonstrate that these changes in protein phosphorylation are necessary for the behavioral effects, we further show that blockade of the ERK signaling pathway by fourth ventricular pretreatment with the MEK inhibitor U0126 abolishes the food intake-suppressing potency of MTII in food-deprived rats. We further demonstrate that fourth ventricular MTII selectively decreases and SHU9119 increases meal size and satiety ratio, but not meal frequency, in the freely feeding rat. These findings suggest that the ERK signaling pathway in the rat NTS is involved in the integration of direct and indirect controls of meal termination.
Mechanisms of MC4 receptor-mediated changes in ERK-phosphorylation
The MC4, but not the MC3 receptor, is highly expressed in the dorsal motor nucleus and NTS (21, 32). MC4R stimulation increases the amount of intracellular cAMP through the G protein Gs, and increases in cAMP are known to activate PKA (33, 34, 35). Whereas studies in multiple cell types as well as whole brain extracts (36, 37, 38) have shown that the cAMP-PKA cascade inhibits ERK1/2 through phosphorylation of Raf-1, studies in PC12 cells using a PKA-activated isoform of Raf (Raf-B), demonstrated that cAMP/PKA can stimulate ERK phosphorylation through the Ras homologue Rap-1 (39). The present findings in the NTS and studies in the hypothalamus (34) demonstrate that the ERK cascade is coupled to the MC4R in the brain. The ERK cascade is classically activated through a linear series of kinases starting at the cell surface with p21RAS, which activates Raf, which in turn activates MAP/ERK kinase MEK (40). The involvement of PKA is suggested by our finding that MTII-induced ERK-phosphorylation was significantly reduced by more than 50% after treatment with cAMPS-Rp that is known to block cAMP-induced activation of PKA. In addition, a recently characterized cAMP-responsive guanidine nucleotide exchange factor was discovered that can activate ERK1/2 through a PKA-independent mechanism (41) (Fig. 10).
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Mechanisms of CCK-induced ERK activation
The small intestinal hormone CCK fulfills all the criteria for a satiety signal (45, 46), and exogenous administration has been widely used as a model satiety-inducing manipulation in rodents and humans. Although there is strong evidence that exogenous CCK’s food intake-suppressing effect is mediated by subdiaphragmatic vagal afferents (47, 48), the circulating hormone can also act directly on the brain (49). However, without clear evidence of direct effects of CCK on NTS neurons, we assume that its effects on ERK phosphorylation in our studies are mediated by activation of vagal afferents releasing glutamate and stimulating ionotropic glutamate receptors resulting in increased intracellular calcium concentration (for review see Ref. 1). Activation of ERK1/2 can then occur through PKA-dependent or -independent pathways (Fig. 10
). The calcium-calmodulin complex can activate RAS and Raf through either calmodulin-kinase or guanine nucleotide exchange factors. However, because phosphorylation of calcium calmodulin-dependent protein kinase II was not increased after peripheral CCK (11), its involvement is unlikely. Activation of PKA can occur through calcium-dependent activation of adenylyl cyclase isoforms 1 and 8, or through binding calcium/calmodulin (50), and the subsequent activation of ERK1/2 would proceed as described above.
We predict that this proposed mechanism of activation occurs in MC4R-expressing NTS integrator neurons that also receive input from vagal afferent fibers. Expression of signaling molecules such as Rap-1, Raf-B, and cAMP/guanidine nucleotide exchange factor or the calcium-dependent isoforms of adenylyl cyclase in NTS neurons remains to be demonstrated.
Integration of melanocortin and CCK-induced signals
Melanocortin signaling in the NTS could interact with vagal afferent signal processing through either a pre- or postsynaptic mechanism or both (Fig. 10). Although it has been demonstrated that the MC4R is located on vagal cholinergic preganglionic neurons in the dorsal motor nucleus (21, 32), it is not known whether the MC4R is located on NTS neurons or presynaptically on vagal afferent terminals innervating NTS neurons. The present results could be explained with either arrangement. Activation (with MTII) or inhibition (with SHU9119) of presynaptic MC4-Rs could simply gate CCK-induced release of glutamate from vagal afferent terminals, leading to more or less glutamate receptor activation and ultimately ERK and CREB phosphorylation (see above). Alternatively, activation and inhibition of postsynaptic MC4Rs could interact with CCK-induced vagal afferent input through convergence of intracellular signaling pathways within special "integrator" neurons. For both scenarios, it would be necessary to stipulate a constitutively active MC4R to account for the inhibition of CCK-induced ERK activation produced by the MC4R antagonist SHU9119. This inverse agonism has been demonstrated for the endogenous antagonist agouti-related protein (see Ref. 51). It is also possible that MC4R and vagal afferent-mediated effects are generated in different populations of NTS neurons whose outputs converge on a third set of NTS neurons to affect the ERK pathway. Using immunohistochemistry we have shown that CCK-induced pERK is not expressed in all NTS neurons (11) but more specific phenotypical identification of activated neurons has not yet been carried out.
Another open question is the endogenous source of MC4R ligands acting on the dorsal vagal complex. 
MSH could originate from proopiomelanocortin (POMC) neurons within the caudal NTS (12, 22, 52), or alternatively, from POMC neurons in the hypothalamus (12). Using a transgenic mouse model with green fluorescent protein under the control of the POMC promoter, it was recently demonstrated that exogenous CCK-induced c-Fos expression in about 30% of POMC neurons of the NTS (22). Thus, NTS POMC neurons could be directly involved in mediating CCK-induced suppression of food intake. It is also known that a few POMC neurons in the arcuate nucleus project to the NTS (12, 27). Selective ablation or silencing of medullary or forebrain POMC neurons will be necessary to demonstrate their respective contribution.
Relevance of ERK pathway for satiation and meal termination
Suppression of food intake in food-deprived rats and mice by both ip CCK (22, 49) and fourth ventricular MTII (18) and stimulation of food intake by SHU9119 (17, 22) has been clearly demonstrated. Here we show that these effects on food intake are paralleled by reciprocal effects on ERK and CREB phosphorylation in the NTS. More importantly, experiments using the ERK pathway inhibitor U0126 show that MTII-induced (present results) and CCK-induced (11) suppression of food intake are at least partially mediated by this signaling cascade. Because brainstem administration of SHU9119 (present study) and MTII (27) change food intake in the freely feeding rat by modulating meal size but not meal frequency, the most parsimonious explanation is that the ERK pathway is involved in the natural processes of satiation and meal termination induced by endogenous CCK and melanocortinergic signaling. Therefore, it could have been expected that fourth ventricular injection of U0126 alone would block the action of endogenous CCK and result in higher food intake (meal size) than under control conditions. However, this was not the case, and we believe that the high level of food intake under deprivation conditions in the present experiment prevented even higher intake because of a ceiling effect. Further substantiation of our hypothesis will thus require additional experiments in freely feeding animals.
The current view is that meal size is determined by direct and indirect controls (3). The direct controls are provided by signals generated by ingested foods interacting with the alimentary canal, such as CCK, and operate exclusively at the level of the caudal brainstem (28). The indirect controls are represented by metabolic, cognitive, and environmental factors acting on the hypothalamus and other forebrain areas and are thought to exert their effect on food intake by changing the potency of the direct controls to determine meal size (3). However, beyond the fact that gastrointestinal satiety signals can activate NTS neurons as measured by electrophysiological recording (e.g. Ref. 29) or c-Fos expression (e.g. Ref. 30), nothing is known about the mechanisms translating activity of NTS neurons into terminating an ongoing meal.
As indicated by studies in decerebrate rats, cumulative and integrated activity of NTS neurons ultimately inhibits the premotor and motor neurons in the brainstem controlling ingestion. Given that the average duration of a chow meal is about 15–20 min (27, 53) (Fig. 8), it is unlikely that meal termination is controlled by transcriptional events because they typically require more time. It is also unlikely that the rapid electrical events induced by ionotropic glutamate receptor activation alone controls meal termination. We suggest that an event with an intermediate time course in the neighborhood of minutes seems to be responsible for meal termination. The ERK-signaling pathway with its nontranscriptional downstream effector mechanisms fits into this category because we have shown that ERK phosphorylation is first significantly increased at 8 min and is maximally stimulated around 15 min after peripheral CCK administration (11).
But how does ERK activation lead to meal termination? We have previously reported that CCK-induced ERK activation results in increased phosphorylation of the A-type potassium channel Kv4.2 in the NTS (11). Phosphorylation of Kv4.2 at ERK-dependent sites has been shown to inhibit channel conductance, leading to increased neuronal depolarization and neuronal excitability in hippocampal neurons (54, 55), and may also play a role in NTS neurons. MTII-induced PKA activation may also play a role in neuronal excitability. Biochemical studies have demonstrated that PKA can phosphorylate the N-methyl-D-aspartate glutamate receptor on the C-terminus of the NR1 subunit, resulting in increased receptor function (56, 57). The combined effects of both of these stimuli on potassium channels and N-methyl-D-aspartate receptor function in NTS neurons may produce a synergistic effect shutting off ingestion.
Clearly, the transcriptional effects of CREB are among the major downstream effectors of ERK (Fig. 10). That CCK would activate this system has long been assumed from the robust expression of c-Fos in NTS neurons. It is also interesting that combined administration of leptin and CCK in rats results in additive effects on c-Fos expression in the NTS (30). Here we show that CCK and MTII produce a similarly additive effect on CREB phosphorylation and that SHU9119 completely blocks the CCK-induced effect. Because both ERK and PKA can phosphorylate CREB at Ser133, CCK and MTII are likely to produce their effects through combined activation of these pathways.
Because transcription typically takes more than 20 min, it is unlikely that it is directly involved in meal termination. However, CREB-mediated transcriptional effects would be in an ideal position to integrate the various relevant signals into longer-term plasticity of the system that modulates future ingestive behavior through the control of intermeal intervals and meal size.
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Footnotes |
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First Published Online June 16, 2005
Abbreviations: CCK, Cholecystokinin; CREB, cAMP response element-binding protein; MCR, melanocortin-receptor; MEK, MAPK kinase; MTII, melanotan II; NTS, solitary nucleus; PBST, PBS containing Tween 20; PKA, protein kinase A; POMC, proopiomelanocortin; SDS, sodium dodecyl sulfate.
Received May 10, 2005.
Accepted for publication June 8, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH) in brainstem baroreceptor areas of the rat. Brain Res 436:323–338[CrossRef][Medline]
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