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. 2008 May 8;149(9):4329–4335. doi: 10.1210/en.2008-0411

Differential Effects of Refeeding on Melanocortin-Responsive Neurons in the Hypothalamic Paraventricular Nucleus

Edith Sánchez 1,a, Praful S Singru 1,a, Runa Acharya 1, Monica Bodria 1, Csaba Fekete 1, Ann Marie Zavacki 1, Antonio C Bianco 1, Ronald M Lechan 1
PMCID: PMC2553369  PMID: 18467436

Abstract

To explore the effect of refeeding on recovery of TRH gene expression in the hypothalamic paraventricular nucleus (PVN) and its correlation with the feeding-related neuropeptides in the arcuate nucleus (ARC), c-fos immunoreactivity (IR) in the PVN and ARC 2 h after refeeding and hypothalamic TRH, neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels 4, 12, and 24 h after refeeding were studied in Sprague-Dawley rats subjected to prolonged fasting. Despite rapid reactivation of proopiomelanocortin neurons by refeeding as demonstrated by c-fos IR in ARC α-MSH-IR neurons and ventral parvocellular subdivision PVN neurons, c-fos IR was present in only 9.7 ± 1.1% hypophysiotropic TRH neurons. Serum TSH levels remained suppressed 4 and 12 h after the start of refeeding, returning to fed levels after 24 h. Fasting reduced TRH mRNA compared with fed animals, and similar to TSH, remained suppressed at 4 and 12 h after refeeding, returning toward normal at 24 h. AGRP and NPY gene expression in the ARC were markedly elevated in fasting rats, AGRP mRNA returning to baseline levels 12 h after refeeding and NPY mRNA remaining persistently elevated even at 24 h. These data raise the possibility that refeeding-induced activation of melanocortin signaling exerts differential actions on its target neurons in the PVN, an early action directed at neurons that may be involved in satiety, and a later action on hypophysiotropic TRH neurons involved in energy expenditure, potentially mediated by sustained elevations in AGRP and NPY. This response may be an important homeostatic mechanism to allow replenishment of depleted energy stores associated with fasting.

RECENT EVIDENCE suggests that the endogenous melanocortin system is one of the principal regulators of body weight through effects on both appetite and energy expenditure (1). Genetic alterations affecting proopiomelanocortin (POMC) gene expression or melanocortin receptors in mice result in profound physical, behavioral, and metabolic changes, including an obesity syndrome characterized by hyperphagia, hyperinsulinemia, and reduced energy expenditure (2,3). Similar observations have been made in humans with mutations that interfere with the functions of the melanocortin 4 receptor (MC4-R), POMC gene, or processing enzymes necessary to generate a fully mature α-MSH (4,5,6). Substantial evidence suggests that the hypothalamic paraventricular nucleus (PVN) mediates many of the actions of the melanocortin signaling system on body weight regulation. The PVN receives a particularly high density of axons containing α-MSH (4) and express MC4-R mRNA (5). Furthermore, focal injections of α-MSH or α-MSH agonists directly into the PVN fully replicate the anorexigenic responses observed after intracerebroventricular (icv) administration (6). In addition, reactivation of the MC4-R in the hypothalamic PVN of a MC4-R null mouse transgenic line prevents most of the obesity and hyperphagia associated with deletion of the MC4-R (7). Melanocortin signaling in the PVN may also be involved in the regulation of energy metabolism, at least in part through regulation of the hypothalamic-pituitary-thyroid (HPT) axis (8). TRH neurons in the PVN are heavily innervated by α-MSH-producing neurons of the arcuate nucleus (ARC) (4), and the icv administration of α-MSH to fasting animals rapidly increases cAMP response element binding protein (CREB) phosphorylation in the nucleus of TRH neurons, and increases TRH gene expression (9).

Recent studies in our laboratories have provided evidence that the melanocortin signaling system is activated within 2 h of refeeding after a prolonged fast, and contributes to the development of satiety during the early phase of refeeding, perhaps mediated through direct effects on a discrete population of neurons in the ventral parvocellular subdivision of the PVN (PVNv) (10). This neuronal population shows an increase in c-fos expression shortly after refeeding that can be abolished by pretreatment of the animals with the MC3/4-R antagonist, agouti-related protein (AGRP), before refeeding. These neurons also show evidence of CREB phosphorylation after the administration of α-MSH into the cerebrospinal fluid (9). To determine whether hypophysiotropic TRH neurons are similarly activated in the PVN under these conditions, we studied the effect of refeeding on c-fos activation in TRH neurons in the PVN after 2-h refeeding, and TRH gene expression at various time points after refeeding. On the basis of these data, we propose that hypophysiotropic TRH neurons may be resistant to melanocortin signaling during the early phase of refeeding as part of an important homeostatic mechanism that facilitates the replenishment of energy stores in animals that have undergone a prolonged fast.

Materials and Methods

Animals

Adult, male, Sprague Dawley rats weighing 250–270 g (Taconic Farms, German Town, NY) were used in this study. Animals were acclimatized to standard environmental conditions (light between 0600 and 1800 h; temperature 22 ± 1 C; rat chow and water ad libitum) for 1 wk before the beginning of the experiment. Three groups were studied, including ad libitum fed, fasted for 64 h, and animals given free access to food for 2–24 h after a 64-h fast. During fasting, all animals received water ad libitum. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts Medical Center and Tufts University School of Medicine.

Tissue preparation for double-labeling immunofluorescence

Fed, fasted, and 2-h refed animals (n = 4 for each group) were anesthetized with pentobarbital (50 mg/kg; Ovation Pharmaceuticals, Inc., Deerfield, IL) and perfused transcardially with 20 ml heparinized 0.01 m PBS (pH 7.4), followed subsequently by 100 ml 3% paraformaldehyde containing 1% acrolein and 30 ml 3% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The brains were postfixed in 3% paraformaldehyde overnight and immersed in 25% sucrose in 0.01 m PBS at 4 C. A series of 20-μm thick coronal sections were cut through the rostrocaudal extent of the hypothalamus on a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany) and collected in PBS.

Double-labeling immunofluorescence for c-fos and α-MSH in the ARC

To study the effect of fasting and refeeding on c-fos expression in α-MSH-containing neurons in the ARC, sections through the ARC of fasted and 2-h refed animals were incubated in rabbit c-fos antiserum at 1:10,000 dilution for 2 d at 4 C. After rinsing in PBS, the sections were incubated in biotinylated goat antirabbit IgG (1:400; Vector Laboratories, Burlingame, CA) for 2 h, followed by ABC (Vector Laboratories) at 1:100 dilution for 1 h. Sections were washed in PBS, and the immunoreaction was amplified using the Tyramide Signal Amplification kit for 10 min according to the manufacturer’s instructions (New England Nuclear Life Science Products, Boston, MA). After further washes in PBS, the sections were incubated in Texas Red-Avidin D (1:250; Vector Laboratories) overnight at 4 C and then incubated in sheep α-MSH antiserum for 2 d at 4 C (gift of Dr. Jeffrey Tatro, Tufts Medical Center) at 1:25,000. After additional washes in PBS, the sections were incubated in fluorescein isothiocyanate (FITC)-conjugated donkey antisheep IgG (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 3 h at room temperature, and then mounted on SuperFrost/Plus slides (Fisher Scientific, Pittsburgh, PA) and coverslipped with Vectashield Mounting Medium (Vector Laboratories). Immunofluorescence was observed using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY). The images were captured using a Spot digital camera (Diagnostic Instrument, Sterling Heights, MI), double exposed while switching filter sets for each fluorochrome, and superimposed in Adobe Photoshop CS 8.0 (Adobe Systems, Inc., San Jose, CA) using a Macintosh G4 computer (Apple Computer, Inc., Cupertino, CA) to create a composite image of the same field.

Double-labeling immunofluorescence for c-fos and TRH in the hypothalamic PVN

Sections through the PVN in both fasted and 2-h refed animals were incubated in rabbit c-fos antiserum at 1:10,000 dilution for 2 d at 4 C. The sections were rinsed in PBS and incubated in biotinylated goat antirabbit IgG (1:400) for 2 h, followed by ABC at 1:100 dilution for 1 h. After washing the sections in PBS, the immunoreaction was amplified for 10 min using the Tyramide Signal Amplification kit according to the manufacturer’s instructions and then incubated in Texas Red-Avidin D (1:250) overnight at 4 C. After further washes in PBS, sections were incubated in rabbit pro-TRH (178–199) antiserum diluted 1: 2500 (generous gift from Dr. Éva Rédei, Northwestern University, Chicago, IL) for 2 d at 4 C, and then in FITC-conjugated donkey antirabbit IgG (1:50) for 3 h at room temperature. After rinsing in PBS, the sections were mounted on SuperFrost/Plus slides and coverslipped with Vectashield Mounting Medium. Sections were observed under the Zeiss Axioplan 2 fluorescence microscope and the images captured as described previously.

Tissue preparation for in situ hybridization histochemistry

Animals were divided into five groups, and either given free access to food (fed, n = 4), fasted for 64 h (fasted, n = 4), or fasted for 64 h and then given free access to food for 4 h (n = 4), 12 h (n = 4), or 24 h (n = 4) (refed groups). The timing of fast and reintroduction of food was designed such that animals in all groups were perfused over a similar time frame as those of the fed and fasting groups. Animals were anesthetized with an overdose of pentobarbital (50 mg/kg) and perfused transcardially with 20 ml 0.01 m PBS (BD Diagnostic Systems, Sparks, MD) containing 15,000 U/liter heparin sulfate, followed by 150 ml 4% paraformaldehyde in PBS. The brains were removed from the calvarium and postfixed in the same fixative overnight at 4 C. Hypothalamic tissue blocks were cryoprotected in 25% sucrose solution in PBS at 4 C overnight. A series of 18-μm thick coronal sections through the rostrocaudal extent of the PVN and ARC were cut on a cryostat (Leica CM3050 S). Every fourth section through the PVN was collected and mounted onto the SuperFrost/Plus glass slides to obtain four sets of slides. Sections were desiccated overnight at 42 C and stored at −80 C until processed for in situ hybridization histochemistry.

In situ hybridization histochemistry

Every fourth section of the PVN was hybridized with a 1241-bp single-stranded [35S]uridine 5′-triphosphate-labeled cRNA probe for proTRH as previously described (11,12). In addition, every fourth section through the ARC was hybridized with single-stranded [35S]uridine 5′-triphosphate-labeled cRNA probe for neuropeptide Y (NPY) (12) or AGRP (13), as described previously (11). Hybridization was performed under plastic coverslips in a buffer containing 50% formamide, a 2-fold concentration of standard sodium citrate, 10% dextran sulfate, 0.5% sodium dodecyl sulfate, 250 μg/ml denatured salmon sperm DNA, and 5 × 105 cpm radiolabeled probe for 16 h at 55 C. Slides were dipped into Kodak NTB autoradiography emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 in distilled water, and all the autoradiograms were developed after 2- to 4-d exposure at 4 C, dehydrated in ascending series of ethanol, cleared in Histosol, and coverslipped with DPX histology mounting medium (Fluka, Buchs, Switzerland).

Image analysis

To determine the percentage of α-MSH-containing neurons in the ARC and TRH-containing neurons in the PVN that contain c-fos, three fluorescent-labeled sections through the rostrocaudal extent of the ARC or PVN from each animal were analyzed under a Zeiss Axioplan 2 epifluorescence microscope at ×100 using a dual filter set for Texas Red and FITC (Texas Red excitation 560–585 nm, bandpass 585 nm, emission 600–652 nm; FITC excitation 490–505 nm, bandpass 510 nm, emission 515–545 nm; Chroma Technology Corp., Brattleboro, VT) such that the immunofluorescence for both c-fos and α-MSH, and c-fos and TRH could be visualized simultaneously. The percentage of neurons containing c-fos labeling in fasted and 2-h refed animals was determined and the mean ± sem calculated.

In situ hybridization autoradiograms were analyzed by image analysis under dark-field illumination with a Zeiss Axioplan 2 microscope using a COHU 4910 video camera (COHU, Inc., San Diego, CA). TRH mRNA in the PVN and NPY and AGRP mRNA in the ARC were analyzed with a Macintosh G4 computer using Scion Image software (Scion Corp., Frederick, MD). Background density values were removed by thresholding the image, and integrated density values (density X area) of hybridized neurons in the same region of each side in the PVN or ARC, respectively, were measured in every fourth consecutive rostrocaudal section from each animal.

Hormone measurements

Serum TSH concentration was determined using the rat TSH125 Biotrak Assay System from Amersham Biosciences Inc. (Piscataway, NJ), with little modifications (14,15), with samples all decreasing within the linear range of a curve generated by the serial dilution of hypothyroid mouse serum. Values were calculated using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA) data analysis software.

Statistical analyses

The results are presented as mean ± sem. The data were analyzed using one-way ANOVA, followed by the Newman-Keuls and Mann-Whitney U tests. P < 0.05 was considered statistically significant.

Results

Effect of fasting and refeeding on c-fos expression in α-MSH-containing neurons in the ARC and TRH-containing neurons in the PVN

No c-fos immunofluorescence was apparent in α-MSH neurons in the ARC of fasting animals (Fig. 1A). However, 2 h after refeeding, 90 ± 1.4% α-MSH neurons contained c-fos immunolabeling in their nucleus (Fig. 1B). Similarly, only few, isolated, c-fos-containing cells were observed in the PVN (Fig. 1C) of fasting animals, but 2 h after refeeding, a dramatic increase in c-fos expression was observed primarily in the PVNv (Fig. 1D). By double immunolabeling with TRH antiserum, no c-fos immunolabeling was identified in TRH-immunoreactive (IR) neurons in the PVN in fasting animals (Fig. 1, E and G), and there was only a modest, 9.7 ± 1.1% increase in c-fos-immunopositive TRH neurons in medial and periventricular parvocellular subdivisions of the PVN after refeeding (Fig. 1, F and H).

Figure 1.

Figure 1

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Photomicrographs showing the effect of fasting (A) and 2-h refeeding (B) on c-fos expression (red) in α-MSH-containing neurons (green) in the ARC. Several doubly-labeled neurons are shown by the arrows. In the PVN (C and D), only rare, c-fos-immunoreactive cells are observed in fasting animals (C). D, Two hours after refeeding, numerous, intensely immunoreactive c-fos-containing cells are present in the PVNv, whereas only isolated cells are present in the medial and periventricular parvocellular subdivisions of the PVN (PVNmp and PVNp, respectively). E–H, Double-labeling immunofluorescence photomicrographs showing c-fos-labeled nuclei (red) in TRH-containing neurons (green) in the medial parvocellular subdivision of the hypothalamic PVN in fasting (E and G) and refed (F and H) animals. G and H, High magnification of the area demarcated by the insets in E and F, respectively. Colocalization of c-fos and TRH is absent in the PVN of fasting animals but present in a small number of TRH neurons 2 h after refeeding (F and H, arrows). Scale bar, 25 μm in A and B, and 100 μm in C–H. III, Third ventricle.

Effect of fasting, and refeeding on circulating levels of TSH and TRH mRNA in the PVN

Serum TSH levels were reduced 58.8% in fasting animals, and remained suppressed at both 4 and 12 h after refeeding (Fig. 2). However, 24 h after refeeding, TSH levels returned to levels in fed controls (P > 0.05).

Figure 2.

Figure 2

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Serum TSH levels in fed, fasted, and refed animals. Note a significant decrease in serum TSH level in fasting animals, which returns to normal fed levels only 24 h after refeeding. *, Significantly different (P < 0.05).

TRH mRNA hybridization signal in normal, fed animals was readily observed in the PVN (Fig. 3A), and markedly diminished after fasting (Fig. 3B). Four and 12 h after refeeding, the TRH mRNA signal in the PVN remained suppressed (Fig. 3, C and D), and only appeared similar to fed controls 24 h after refeeding (Fig. 3E). By image analysis, fasting resulted in a significant 2.5-fold reduction in TRH mRNA (Fig. 3F). No significant difference in TRH mRNA levels was found 4 and 12 h after refeeding compared with fasting animals. However, the 24-h refed animals had levels significantly different from fasted animals (P < 0.05), but nonsignificantly different from the fed controls (P > 0.05).

Figure 3.

Figure 3

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Dark-field photomicrographs showing TRH mRNA in the PVN of fed (A), fasted (B), and fasted animals refed for 4 h (C), 12 h (D), or 24 h (E). A marked reduction in the TRH mRNA is seen in the PVN of fasting animals that only returns toward normal in the 24-h refed animals. F, Computerized image analysis of TRH mRNA in the PVN of fed, fast, and refed animals at different time points. *, Significantly different (P < 0.05). Scale bar, 200 μm. III, Third ventricle.

Effect of fasting and refeeding on AGRP and NPY mRNA expression in the ARC

Moderate AGRP (Fig. 4A) and NPY (Fig. 5A) mRNA signal was observed in the ARC by in situ hybridization histochemistry in fed controls, which dramatically increased with fasting (Figs. 4, B and F, and 5, B and F). Compared with fasting animals, a partial reduction in AGRP mRNA was observed 4 h after refeeding, and by 12 h, had returned to control fed levels (Fig. 4, C–F). In contrast, although there was a partial reduction in NPY mRNA levels in the ARC 4 h after refeeding (Fig. 5, C and F), NPY gene expression remained significantly elevated compared with control fed levels even at 24 h after refeeding (Fig. 5, C–F).

Figure 4.

Figure 4

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Dark-field photomicrographs showing AGRP mRNA in the ARC of fed (A), fasted (B), and fasted animals refed for 4 h (C), 12 h (D), or 24 h (E). F, Computerized image analysis of AGRP mRNA in the ARC of fed, fasted, and refed animals at different time points. ***, Significantly different (P < 0.001). **, Significantly different (P < 0.01). *, Significantly different (P < 0.05). Scale bar, 200 μm. III, Third ventricle.

Figure 5.

Figure 5

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Dark-field photomicrographs showing NPY mRNA in the ARC of fed (A), fasted (B), and fasted animals refed for 4 h (C), 12 h (D), or 24 h (E). F, Computerized image analysis of NPY mRNA in the ARC of fed, fasted, and refed animals at different time points. ***, Significantly different (P < 0.001). **, Significantly different (P < 0.01). Scale bar, 200 μm. III, Third ventricle.

Discussion

Previous studies from our laboratory (4,8,9) and by others (16,17) have demonstrated that melanocortin signaling has an important role in the regulation of hypophysiotropic TRH neurons in the hypothalamic PVN. These neurons receive direct monosynaptic projections from α-MSH-producing neurons in the hypothalamic ARC, and when α-MSH is administered intracerebroventricularly to fasting animals, it has a potent effect to restore fasting-induced suppression of TRH mRNA (4) through a phospho-CREB-dependent mechanism (9). Accordingly, we have proposed that inhibition of the HPT axis by fasting may be explained at least in part by inhibition of melanocortin signaling (8). Despite the evidence that the majority of α-MSH-containing neurons in the ARC are activated within 2 h after reintroducing food to fasting animals, presumably as a mechanism to regulate meal size (10), circulating levels of thyroid hormone remain suppressed for up to 24 h after refeeding in animals subjected to a prolonged fast. In studies by Kmiec et al. (18) in rats and Boelen et al. (19) in mice, refeeding did not result in complete recovery of circulating T3 and T4 levels after a prolonged fast, even at 24 h. Similarly in man, recovery of TSH responsiveness to TRH may take several days when refeeding follows a long duration of fasting (20).

To elucidate the effect of refeeding after a prolonged fast on hypophysiotropic TRH neurons in the PVN, we determined whether c-fos is observed in these neurons in concert with c-fos activation in other regions of the PVN shown to be melanocortin responsive, and the time required for recovery of TRH mRNA to fed levels. As previously observed, c-fos IR was absent from α-MSH-containing neurons in the ARC in the fasting animals but present in over 90% immunocytochemically identifiable α-MSH neurons in this region during the first 2 h after refeeding. Presumably early activation of melanocortin neurons in the ARC is an important homeostatic response to limit meal size during the early phase of refeeding as a way to attenuate the initial hyperphagia after a fast (10). c-fos activation was simultaneously observed in the PVNv, which we have shown to be dependent upon melanocortin signaling because these neurons are heavily innervated by axons containing α-MSH, and pretreatment of fasting animals with a melanocortin antagonist just before refeeding prevents their activation. Although refeeding-induced c-fos activation was observed in subdivisions of the PVN where hypophysiotropic TRH neurons reside, only a small minority of TRH neurons in these regions showed evidence of activation as demonstrated by the presence of c-fos in their nucleus. Because both thyroidectomy and cold stress, stimuli that strongly increase TRH gene expression in hypophysiotropic neurons (21,22), activate c-fos in TRH neurons in the PVN (22,23,24), the observations in this study cannot be attributed to the lack of utilization of c-fos in immediate early gene expression in these neurons.

Further evidence for delayed activation of hypophysiotropic TRH neurons in the PVN after refeeding was apparent in the time required for recovery of TRH gene expression in the medial and periventricular parvocellular subdivisions of the PVN. As anticipated, fasting resulted in suppression of TRH mRNA in the PVN, consistent with the concept that central hypothyroidism contributes to the fasting-induced reduction in circulating thyroid hormone levels and TSH (25). However, despite that the animals had resumed eating, TRH mRNA remained suppressed at both 4 and 12 h after refeeding, and was not restored to fed levels until 24 h after the reintroduction of food. A similar delayed recovery in the levels of TSH circulating in the bloodstream was also observed. Therefore, on the basis of these data, we propose that hypophysiotropic TRH neurons in the medial and periventricular parvocellular subdivisions of the PVN may be resistant to the effects of melanocortin signaling during the initial phases of refeeding after a prolonged fast. Because a separate population of melanocortin-responsive neurons in the PVNv retains its sensitivity to melanocortin signaling at this time (10), we presume that there may be an initial dissociation between the effects of melanocortin signaling on populations of neurons involved in satiety from those that promote energy expenditure. This hypothesis is supported by the observation that the refeeding of fasted rats results in a delayed increase in resting oxygen consumption, which peaks only after several days (26). We have hypothesized that neurons in the PVNv mediate the satiety effects of melanocortin signaling on the PVN (10), although further studies are necessary to substantiate this. Nevertheless, substantial evidence suggests that the action of melanocortin signaling on the regulation of appetite and satiety is primarily exerted on the hypothalamic PVN because focal injections of α-MSH or α-MSH agonists directly into the PVN fully replicate the reduced feeding responses observed after icv administration (6), whereas focal injection of the α-MSH antagonist, SHU9119, into the PVN increases feeding (27). In addition, hyperphagia and obesity develop in mice and humans heterozygous for a null allele of the Sim1 transcription factor involved in normal development of the PVN (28). However, most compelling is evidence that reactivation of the MC4-R in the hypothalamic PVN of a MC4-R null mouse transgenic line prevents approximately 60% of the obesity and 100% of the hyperphagia associated with complete deletion of the MC4-R (7). In contrast, by the nature of the fact that hypophysiotropic TRH neurons in the PVN regulate the HPT axis, these neurons contribute to energy expenditure as a result of the effects of thyroid hormone to increase metabolic rate, cellular respiration, and thermogenesis (29,30).

The mechanism(s) by which melanocortin signaling from ARC neurons could affect regions of the PVN involved in satiety differently from regions involved in energy expenditure is unknown. However, it is of interest that, after a prolonged fast, the recovery of NPY neurons in the hypothalamic ARC after refeeding was delayed and remained significantly elevated compared with fed levels even after 24 h. Similar observations have been made in the mouse by Swart (31) and Beck (32) et al., showing persistent elevation of NPY in the PVN after fasting rats had been refed. TRH neurons in the PVN are heavily innervated by NPY-containing axon terminals (33,34) that inhibit TRH gene expression, presumably by reducing cAMP (8). We have also demonstrated that even in the presence of α-MSH, NPY has potent downstream inhibitory effects on TRH gene expression and can reduce the phosphorylation of CREB in the nucleus of these cells (35). Thus, it is conceivable that NPY may be an important factor that allows for the differential affects of melanocortin signaling in the PVN.

In contrast to the effects of refeeding on NPY mRNA, AGRP mRNA showed a more rapid recovery, returning to fed, control levels by 12 h. Nevertheless, AGRP also exerts potent inhibitory effects on TRH gene expression (36) by antagonizing melanocortin receptors and/or by acting as an inverse agonist on TRH neurons (37), and it is contained in axon terminals that heavily inundate all hypophysiotropic TRH neurons (4). Because the effects of AGRP peptide may be sustained well beyond that observed for NPY (38), it is feasible that even after reduction of AGRP mRNA in ARC neurons, there was a persistent inhibitory response on TRH neurons in the PVN. Selective inhibition of hypophysiotropic TRH neurons, but not melanocortin responsive neurons, in the PVN, that are also densely innervated by axons containing AGRP (8) might be secondary to differences in the expression or regulation of proteins that facilitate the action of AGRP such as attractin/mahogany protein and/or syndecans (39,40). Differences in synaptic remodeling in these neuronal groups with fasting and refeeding (41) might also be responsible but would require experimental study. Finally, it should be considered that in addition to the ARC, hypophysiotropic TRH neurons in the PVN might be selectively affected by afferent inputs from other refeeding-sensitive neuronal populations. Potential sources include the dorsomedial nucleus (42), C1–C3 brain stem areas (43,44), and possibly the hippocampus (45).

We conclude that refeeding-induced activation of melanocortin signaling may exert differential actions on its target neurons in the PVN, an early action directed at neurons involved in satiety and a later action on hypophysiotropic TRH neurons, perhaps mediated by sustained elevations in AGRP and NPY. The delayed effect on hypophysiotropic TRH neurons may be an important homeostatic mechanism that allows for replenishment of depleted energy stores associated with a prolonged fast by preventing thyroid hormone-induced energy expenditure.

Footnotes

This work was supported by National Institutes of Health Grant DK-37021.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 8, 2008

Abbreviations: AGRP, Agouti-related protein; ARC, arcuate nucleus; CREB, cAMP response element binding protein; FITC, fluorescein isothiocyanate; HPT, hypothalamic-pituitary-thyroid; icv, intracerebroventricular; IR, immunoreactivity; MC4-R, melanocortin 4 receptor; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; PVNv, ventral parvocellular subdivision of the paraventricular nucleus.

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