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Endocrinology, doi:10.1210/en.2006-0820
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Endocrinology Vol. 147, No. 12 6004-6010
Copyright © 2006 by The Endocrine Society

Brainstem Thyrotropin-Releasing Hormone Regulates Food Intake through Vagal-Dependent Cholinergic Stimulation of Ghrelin Secretion

Yan Ao, Vay Liang W. Go, Natalie Toy, Tei Li, Yu Wang, Moon K. Song, Joseph R. Reeve, Jr., Yanyun Liu and Hong Yang

Center for Ulcer Research and Education: Digestive Diseases Research Center (Y.A., V.L.W.G., N.T., J.R.R., H.Y.), Division of Digestive Diseases, Department of Medicine (Y.A., V.L.W.G., N.T., T.L., Y.W., M.K.S., J.R.R., Y.L., H.Y.), and Brain Research Institute (Y.A., N.T., H.Y.), University of California, Los Angeles, and Department of Veterans Affairs Greater Los Angeles Healthcare System (Y.A., V.L.W.G., N.T., Y.W., M.K.S., J.R.R., Y.L., H.Y.), Los Angeles, California 90073

Address all correspondence and requests for reprints to: Hong Yang, M.D., Ph.D., CURE: Digestive Diseases Research Center, Veterans Affairs Greater Los Angeles Healthcare System, Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: hoyang{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The brainstem is essential for mediating energetic response to starvation. Brain stem TRH is synthesized in caudal raphe nuclei innervating brainstem and spinal vagal and sympathetic motor neurons. Intracisternal injection (ic) of a stable TRH analog RX77368 (7.5–25 ng) dose-dependently stimulated solid food intake by 2.4- to 3-fold in freely fed rats, an effect that lasted for 3 h. By contrast, RX77368 at 25 ng injected into the lateral ventricle induced a delayed and insignificant orexigenic effect only in the first hour. In pentobarbital-anesthetized rats, RX77368 (50 ng) ic induced a significant bipeak increase in serum total ghrelin levels from the basal of 8.7 ± 1.7 ng/ml to 13.4 ± 2.4 ng/ml at 30 min and 14.5 ± 2.0 ng/ml at 90 min, which was prevented by either bilateral vagotomy (–60 min) or atropine pretreatment (2 mg/kg, –30 min) but magnified by bilateral adrenalectomy (–60 min). TRH analog ic-induced food intake in freely fed rats was abolished by either peripheral atropine or ghrelin receptor antagonist (D-Lys-3)-GHRP-6 (10 µmol/kg) or ic Y1 receptor antagonist 122PU91 (10 nmol/5 µl). Brain stem TRH mRNA and TRH receptor 1 mRNA increased by 57–58 and 33–35% in 24- and 48-h fasted rats and returned to the fed levels after a 3-h refeeding. Natural food intake in overnight fasted rats was significantly reduced by ic TRH antibody, ic Y1 antagonist, and peripheral atropine. These data establish a physiological role of brainstem TRH in vagal-ghrelin-mediated stimulation of food intake, which involves interaction with brainstem Y1 receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BRAIN PLAYS a key role in the control of metabolism (1, 2, 3). In particular, neurons in a handful of hypothalamic homeostatic circuits sense multiple hormonal and nutritional signals and control eating behavior, nutrition metabolism, energy expenditure, and insulin sensitivity (1, 2, 3, 4). Increasing evidence shows that the hypothalamus is by no means the only brain area involved in metabolic regulation (1). Lower animals, such as arthropods, do not have a hypothalamus (5), but energy balance, including the regulation of food intake, is maintained. This raises the question whether a more ancient and primary structure of the brain controls food intake. In 1978 Grill and colleagues (6, 7) used a chronically decerebrate rat model and demonstrated that the caudal brainstem appears to be the neural substrate of ingestion because it contains an essential part of the neural mechanism that both detects metabolic need and ameliorates that need by exhibiting autonomic response to the metabolic challenge. Recent studies of this group showed that caudal brainstem is sufficient to mediate many aspects of the energetic response to starvation (8). These findings revealed the importance of the brainstem in maintaining metabolic homeostasis. Indeed, convincing data have evidenced that brainstem neuropeptides, such as melanin-concentrating hormone and neuropeptide Y (NPY), are involved in maintaining energy balance and regulating food intake (9, 10).

Studies in the last 2 decades have well established the physiological role of brainstem TRH, a three-amino acid neuropeptide originally found in the hypothalamus, in autonomic regulation of visceral functions (11, 12). Brain stem TRH is synthesized in the raphe pallidus, raphe obscurus, and parapyramidal regions, nuclei well known to harbor vagal and sympathetic preganglionic motor neurons involved in thermal, cardiovascular, gastrointestinal, and pancreatic regulation (13, 14). TRH-containing projections of the raphe nuclei innervate the dorsal vagal complex (DVC) that contains dense TRH-like immunoreactive nerve terminals and TRH receptor 1 (TRH R1) (15, 16, 17), the sympathoexcitatory region of the rostral ventrolateral medulla (18), and the intermediolateral cell column of the spinal cord containing sympathetic motor neurons (19). When exogenously injected intracisternally (ic) or microinjected into the DVC, TRH or its metabolically stable analog RX77368 induces vagal-cholinergic stimulation of gastric secretion and motility (11, 12), effects that were enhanced by Y1 receptor activation in the DVC and antagonized by peptide YY acting in the DVC through activation of Y2 receptors (20, 21, 22, 23, 24). In humans, TRH-immunoreactive fibers innervating the DVC also constitute the most prominent network, compared with 12 other neuropeptides (25). We recently reported that an unbalanced vagal and sympathetic-adrenal response to brainstem TRH receptor activation in type 2 diabetic Goto-Kakizaki rats contributes to its impaired pancreatic insulin secretion (26, 27), supporting the view that altered autonomic-regulatory function of brainstem TRH participates in the pathophysiology of metabolic disorders.

Previous reports suggest that vagal-cholinergic (muscarinic) activation is involved in stimulating gastric ghrelin gene expression and ghrelin secretion (28, 29, 30). Food deprivation-induced elevation of plasma ghrelin was completely prevented by subdiaphragmatic vagotomy or atropine pretreatment (29). In addition, circulating ghrelin levels in humans can be increased or reduced by cholinergic agonists or antagonists, respectively (30). In this study, we investigated whether brainstem TRH participates in the regulation of food intake through vagal-dependent cholinergic stimulation of gastric ghrelin secretion and the influence of blockage of brainstem Y receptors on the TRH action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley (SD) rats (250–270 g) purchased from Harlan Laboratory (San Diego, CA) were housed in Veterans Affairs/West Los Angeles animal facilities for 1 wk before experiments. The rats were housed under controlled conditions (21–23 C, light on from 0600–1800 h) with free access to standard rat chow (Prolab Lab Diet; PMI Nutrition International, Brentwood, MO) and tap water. Food, but not water, was removed for overnight, 24 h, or 48 h for fasting experiments. Animal protocols were approved by the Veterans Affairs Greater Los Angeles Area Health Care System Animal Research Committee.

Experimental protocol
All experiments were performed between 0930 and 1430 h. Freely fed or fasted rats were treated with ic, intracerebroventricular (icv), ip, iv, and/or sc injection, depending on the experimental design, and then provided with preweighed rat chow and tap water for 3 h. Food intake was measured each hour by subtracting the weight of the remaining rat chow from the previous weight.

In the experiments studying ghrelin secretion, rats were anesthetized with ip pentobarbital (Abbott Laboratories, North Chicago, IL; 50 mg/kg followed by 20 mg/kg each hour). A PE-50 cannula was inserted into the external iliac vein for blood sampling. Bilateral cervical vagotomy, bilateral adrenalectomy, or sham operation was performed immediately after the iv cannulation. Basal blood sampling (0.1 ml) followed by ic injection was performed 60 min after the surgery. In another two groups, atropine (2 mg/kg in saline; Sigma, St. Louis, MO) or saline was sc injected 30 min before ic injection. Blood samples (0.1 ml) were collected at 30, 60, 90, and 120 min after ic injection and 0.1 ml of 0.1% BSA/saline was injected iv after each blood sampling. Serum total ghrelin was measured by RIA kit from Linco Research (St. Charles, MO; catalog no. GHRT-89HK).

In the experiments studying brainstem TRH and TRH R1 gene expression, rats were divided into four groups: freely fed, fasted for 24 h, fasted for 48 h, and fasted for 48 h followed by a 3-h refeeding period. Rats were quickly decapitated and the brainstem removed for total RNA extraction.

Intracisternal injection
Intracisternal injection was performed acutely under short (2–3 min) isoflurane anesthesia (5% vapor concentration in oxygen; Abbott Laboratories). The head of the rat was immobilized with ear bars of a Kopf stereotaxic frame (Kopf Instruments, Tujunga, CA; model 900) and the atlantooccipital membrane was punctured with a 50-µl Hamilton microliter syringe. The successful insertion of the needle into the cisterna magma was verified by aspirating clear cerebrospinal fluid into the syringe before and after the injection of the tested substance. Then the animal was returned to its individual cage where recovery from anesthesia occurred within 1–2 min (31). This procedure did not influence blood glucose levels in SD rats (123.0 ± 2.5 mg/dl in freely fed rats after ic injection).

Chronic lateral ventricle cannulation and icv injection
After 1 wk of habituation to the housing conditions, the rats were anesthetized with pentobarbital (50 mg/kg body weight, ip) and individually mounted on a stereotaxic frame with incisor bar adjusted at –3 mm. A stainless-steel guide cannula (26 gauge; Plastics One, Roanoke, VA) was implanted into the right lateral ventricle, +1.5 mm lateral and –0.8 mm posterior to the bregma and 3.5 mm ventral to the surface of the skull. The guide cannula was fixed to the skull with two screws and dental cement and a dummy cannula was placed into the guide cannula to prevent blockage. After cannulation, the rats were housed in individual cages. Intracerebroventricular injection (10 µl in 1 min) was performed 5 d after the surgery under short (2–3 min) isoflurane anesthesia. Placement of cannula in the lateral ventricle was confirmed after experiment by injecting 10 µl of 0.2% toluidine blue into the cannula before the rats were killed. The dye reached the entire ventricle system within 5 min after the icv injection, including light staining in the fourth ventricle. Only data obtained in rats showing dye in their cerebroventricular system were used.

Tested substances
The metabolically stable TRH analog RX77368 [pGlu-His-(3,3'-dimethyl)-Pro-NH2; Ferring Pharmaceuticals, Feltham, Middlesex, UK] was diluted in sterile saline from aliquots of a stock solution (30 µg/ml in 0.1% BSA, kept at –70 C) before ic injection. The following chemicals in powder form were dissolved in sterile saline immediately before use: GH secretagogue receptor antagonist, (D-Lys-3)-GHRP-6 (Bachem California Inc., Torrance, CA, catalog no. H3108), Y1 receptor antagonist 122PU91 (NeoMPS, San Diego, CA; catalog no. SC126D), and Y2 receptor antagonist D-Tyr (27, 36), S-Tyr32NPY27–36 (Bachem California; catalog no. H-3328). Purified and concentrated anti-TRH IgG (no. 8964, ready for ic injection) and the control IgG, which is obtained from keyhole limpet hemocyanin (a TRH carrier during TRH antibody production) injected rabbit, were provided by the Antibody Core of the Center for Ulcer Research and Education (CURE), UCLA. The TRH antibody has been previously shown to have the in vivo capacity of neutralizing exogenous and endogenous brainstem TRH action on stimulating gastric acid secretion and motility (12, 32, 33).

Northern blot analysis of brainstem TRH mRNA and TRH R1 mRNA
Northern blot was performed as done in our previous studies (34, 35). The primer sequences were designed using Primer Express software (The National Human Genome Research Institute, Bethesda, MD). DNA fragment for probes were synthesized by reverse transcription (RETROscript kit; Ambion Inc. Austin, TX; catalog no. 1710)-PCR (Tag PCR core kit, catalog no. 201225; QIAGEN, Valencia, CA). The primer sequences are as follows: TRH (accession no. NM-013046), 5'-ATTCTTGTGGAAAGACCTCCAGC-3'; TRH R1 (S69160), 5'-AGATGTTTCAACAGCACCGTTTC-3'; and NPY (NM-012614), 5'-GCAGAGGACATGGCCAGATACTAC-3'. DNA fragments were purified from low melting gel by a Wizard PCR Preps DNA purification system (Promega, Madison, WI; catalog no. A7170). Probes were labeled with 32P-dCTP (MP Biomedicals, Irvine, CA; catalog no. 33004X) and purified using nucleotide removal kit (QIAGEN; catalog no. 28304). Hybridization was carried out overnight at 68 C.

Statistical analysis
Data are expressed as mean ± SEM of each experimental group. Statistical comparisons among multiple group-mean values were performed using one-way ANOVA, and between two group-mean values were performed using Student’s t test using SigmaStat program (SPSS, Inc., San Rafael, CA). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intracisternal injection of the stable TRH analog RX77368 stimulated food intake in freely fed SD rats
A bolus ic RX77368 (7.5–25 ng/10 µl) dose-dependently increased food intake (rat chow) in SD rats by 240–300%, compared with controls (100%) receiving ic saline (Fig. 1Go). Eating started immediately after the rats awakened from the short anesthesia and lasted for 3 h. More than half (51–65%) of the total 3-h food intake occurred in the first hour after the ic injection (Fig. 1Go).


Figure 1
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  		FIG. 1. Upper panel, Intracisternal injection of the stable TRH analog RX77368 induced dose-dependent stimulation of food intake in freely fed male SD rats. Lower panel, Effect of icv injection of 25 ng RX77368 on food intake in freely fed male SD rats. The ic or icv injection was performed under a short (2–3 min) isoflurane anesthesia. Each column represents mean ± SEM of number of rats indicated in the bottom of the first hour columns. *, P < 0.05, compared with saline (dose 0)-injected controls; #, P < 0.05, compared with 25 ng RX77368 ic injection group shown in the upper panel.

 
By contrast, icv injection of RX77368 at the highest dose used in the ic injection (25 ng) induced only a insignificant stimulation on food intake in the first hour. The onset of food intake occurred at least 30 min after the icv injection. The 3-h total food intake in the icv RX77368 25-ng group was similar to that in the icv saline group and significantly lower (–46%) than the ic RX77368 25 ng group (Fig. 1Go).

TRH analog RX77368 ic induced a vagal-cholinergic-dependent increase of serum total ghrelin levels in pentobarbital-anesthetized SD rats
In overnight fasted, pentobarbital (50 mg/kg followed by 20 mg/kg/h)-anesthetized male SD rats, RX77368 (50 ng) ic injection increased blood glucose levels from the basal of 86 ± 3 mg% to a peak of 136 ± 12 mg% at 60 min (Fig. 2AGo), which was abolished by acute bilateral adrenalectomy (–60min) (Fig. 2CGo). Serum total ghrelin showed a significant bipeak increase from the basal of 8.7 ± 1.7 (100%) to 13.4 ± 2.4 (154%) at 30 min and 14.5 ± 2.0 ng/ml (167%) at 90 min (Fig. 2EGo), compared with saline ic injection. The increase of ghrelin was completely prevented by either sc atropine pretreatment (2 mg/kg, –30 min) (Fig. 2FGo) or bilateral cervical vagotomy (–60 min) (Fig. 2HGo) but markedly enhanced by bilateral adrenalectomy (–60 min), as shown by a peak of 25.5 ± 2.5 ng/ml at 90 min after ic RX77368 (Fig. 2GGo).


Figure 2
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  		FIG. 2. Effect of ic injection of the stable TRH analog RX77368 on blood glucose (upper panel) and serum total ghrelin levels (lower panel) in pentobarbital-anesthetized SD rats that underwent sham operation and vehicle pretreatment (A and E), atropine pretreatment (B and F), bilateral adrenalectomy (–60 min, C and G), or bilateral cervical vagotomy (–60 min, D and H). Each point represents mean ± SEM of number of rats indicated in the parentheses. *, P < 0.05, compared with ic saline-injected controls; #, P < 0.05, compared with the preinjection levels of the same group.

 
TRH analog RX77368 ic-induced food intake in freely fed SD rats was abolished by simultaneous iv injection of ghrelin receptor antagonist or atropine pretreatment
GH secretagogue receptor antagonist, (D-Lys-3)-GHRP-6 (5 or 10 µmol/kg), or sterile saline was iv injected into the saphenous vein of rat left hindlimb through a small skin cut (sutured immediately after injection) after ic RX77368 (25 ng) under a short isoflurane anesthesia in freely fed SD rats. The ghrelin receptor antagonist significantly reduced ic TRH analog-induced food intake (by 60% in the first hour and 45% for total 3 h) at the dose of 5 µmol/kg and completely abolished it at the dose of 10 µmol/kg (Fig. 3Go, upper panel). Atropine pretreatment (2 mg/kg, sc, –30 min) also prevented ic TRH analog-induced feeding in freely fed SD rats (Fig. 3Go lower panel).


Figure 3
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  		FIG. 3. The TRH analog RX77368 (25 ng) ic injection-induced food intake in freely fed SD rats was dose-dependently prevented by simultaneous iv injection of the ghrelin receptor antagonist (D-Lys-3)-GHRP-6 (5 or 10 µmol/kg) and completely abolished by atropine (2 mg/kg, sc, –30 min) pretreatment. Each column represents mean ± SEM of number of rats indicated in the first hour columns. *, P < 0.05, compared with iv saline-injected controls; #, P < 0.05, compared with iv (D-Lys-3)-GHRP-6 5 µmol/kg group.

 
Brain stem TRH mRNA and TRH R1 mRNA levels elevated during fasting decreased after refeeding
Figure 4Go shows the blood glucose levels and brainstem TRH mRNA and TRH R1 mRNA levels of four groups of SD rats that had undergone either free feeding, 24-h fasting, 48-h fasting, or 48-h fasting followed by a 3-h refeeding period. Blood glucose levels gradually decreased during fasting but remained within the physiological range. Refeeding for 3 h in 48-h fasted rats restored blood glucose to the freely fed levels (Fig. 4Go, left panel). Brain stem TRH mRNA levels were significantly elevated by 57 and 58% in 24- and 48-h fasted rats, compared with the freely fed group, respectively; brainstem TRH R1 mRNA levels increased 33 and 35% at the same time. The elevated TRH mRNA and TRH R1 mRNA levels in 48-h fasted rats returned to the fed levels after a 3-h refeeding period (Fig. 4Go, middle and right panel). Because brainstem TRH R1 mRNA signals were much weaker, compared with TRH mRNA signals, which increases the possibility of error and contributes to a relatively larger individual variation within each group, we added additional rats into some groups for this parameter to reduce the SEM in these groups.


Figure 4
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  		FIG. 4. Blood glucose levels (left panel) and brainstem TRH mRNA (middle panel) and TRH R1 mRNA (right panel) levels in rats undergone free feeding, 24-h fasting, 48-h fasting, or 48-h fasting followed by a 3-h refeeding. Blood glucose levels were measured by One Touch Ultra blood glucose monitoring system (Lifescan, Milpitas, CA) and brainstem mRNAs by Northern blot analysis. Each column represents mean ± SEM of number of rats indicated in the bottom of each column. *, P < 0.05, compared with freely fed rats; #, P < 0.05, compared with 24-h fasted rats; @, P < 0.05, compared with 48-h fasted rats. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

 
Natural food intake in overnight fasted rats was reduced by ic TRH antibody or sc atropine
Purified anti-TRH IgG (210 µg/15 µl) was ic injected under short isoflurane anesthesia into overnight fasted SD rats 10 min before rat chow was provided. Anti-TRH IgG ic injection significantly reduced the first hour food intake by 51%, compared with that in the control antibody-treated group, although the effect was short lasting (Fig. 5Go, upper panel). Atropine pretreatment (2 mg/kg, sc, –30 min) significantly reduced the first hour food intake in overnight fasted rats by 75% and the 3-h total food intake by 57% (Fig. 5Go, lower panel).


Figure 5
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  		FIG. 5. Intracisternal anti-TRH IgG (upper panel) or sc atropine (lower panel) reduced food intake in overnight fasted SD rats. Each column represents mean ± SEM of number of rats indicated in the bottom of the first hour columns.

 
Y1 receptor antagonist completely abolished TRH analog ic-induced food intake in freely fed rats and reduced the natural feeding in overnight fasted rats
Pretreatment ic with the specific Y2 antagonist D-Tyr (27, 36), S-Tyr32NPY27–36 (10 µg/5 µl, –1min) (10) did not influence ic RX77368-induced feeding in freely fed SD rats. By contrast, pretreatment with Y1 antagonist 122PU91 (5 nmol/5 µl) (10) completely abolished ic TRH analog (25 ng/5 µl)-induced food intake in freely fed SD rats (Fig. 6Go, upper panel) and reduced the natural food intake in overnight fasted SD rats (Fig. 6Go, lower panel).


Figure 6
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  		FIG. 6. Intracisternal injection of Y1 receptor antagonist 122PU91 (5 nmol/5 µl) but not Y2 receptor antagonist D-Tyr27,36,S-Tyr32NPY27–36 (10 µg/5 µl) abolished ic TRH analog-induced food intake in freely fed rats (upper panel) and the natural food intake in overnight fasted SD rats (lower panel). Each column represents mean ± SEM of number of rats indicated in the bottom of the first hour columns. *, P < 0.05, compared with ic saline controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results clearly show that vagal efferent activation by brainstem TRH plays a critical role in controlling food intake. The strong orexigenic action of brainstem TRH is mediated by vagal-cholinergic stimulation of gastric ghrelin release. This is based on the facts that ic TRH analog induced a vagal-cholinergic-dependent increase in serum total ghrelin levels and peripheral pretreatment of ghrelin receptor antagonist or atropine completely abolished ic TRH analog-induced food intake. Convincing evidence supports this mechanism. Insulin-induced hypoglycemia, which strongly elevates vagal outflow to the visceral organs, increased gastric ghrelin mRNA (28). Hypo- or hyperthyroidism, in which brainstem TRH gene expression are elevated or reduced, respectively (35, 36), are in accordance with the increase and decrease in vagal efferent function (37), gastric ghrelin mRNA, and blood ghrelin levels (38). In addition, sham feeding, which induces brainstem TRH R1-mediated vagal-dependent cephalic phase of gastric and pancreatic secretion (12), increases ghrelin levels (39). More directly, circulating ghrelin levels in humans can be elevated and reduced by cholinergic (muscarinic) agonists and antagonists, respectively (30). The result of a greater ghrelin response to ic TRH analog in adrenalectomized rats indicates an inhibitory regulation of ghrelin secretion by the activation of sympathetic-adrenal system.

The possibility that ic-injected TRH analog acts on brain structures other than the brainstem cannot be completely excluded; however, it is unlikely to play a major role. First, previous studies established that ic TRH or its analog stimulates vagal efferent activity in nanogram doses by acting on brainstem vagal motor neurons, primarily located in the DVC (11, 12, 40, 41). Second, in the present study, icv injection of RX77368 at the highest dose used for ic injection (25 ng) did not show significant stimulatory effect on food intake, although the experimental condition was identical with that for ic injection. The weaker and the delayed (onset > 30 min) effect of icv RX77368, compared with its ic effect (onset < 5 min), indicate that the insignificant orexigenic effect of icv TRH analog may result from its action in the brainstem, as part of the icv injected chemicals can reach the fourth ventricle within minutes (see Materials and Methods). Our present data are incongruent with some previous reports, which showed that peripheral injection of TRH in doses up to 64 mg/kg or icv injection of TRH (5 µg) or RX77368 (1 µg) decreases, rather than increases, food intake (42, 43). The mechanism and pathways mediating these effects are still to be studied because multiple forebrain sites and pathways can be activated after an icv injection, especially at high doses, and an even more complicated mechanism for post-TRH peripheral administration.

The gene expression of caudal brainstem TRH in the raphe pallidus/raphe obscurus/parapyramidal regions is up-regulated by energy-deficient situations, such as hypothermia (34), a condition with accelerated energy demand and glucose turnover (44), and hypothyroidism, characterized by a reduced metabolic rate (35); both are associated with elevated vagal activity (34, 37). In the present study, fasting rats for 24 or 48 h similarly elevated brainstem TRH mRNA and TRH R1 mRNA levels, and the increases were reversed by refeeding, providing direct evidence that brainstem TRH-vagal regulatory system is responsive to altered metabolism and activated by energy demand. That ic TRH antibody or sc atropine treatment significantly reduced the natural food intake in overnight fasted rats further support the role of brainstem TRH-vagal efferent activation in stimulating food intake. Our data are also compatible with the findings of Williams et al. (29) that the food deprivation-induced elevation of plasma ghrelin was completely prevented by subdiaphragmatic vagotomy and atropine pretreatment. Although brainstem-specific TRH gene knockout mouse is not available at the present time, mice lacking the TRH gene show a decrease in body weight approximately 70% that of controls at the age of 4 wk (45). Consistent with this finding, the body weight of TRH R1-deficient mice is significantly lower than wild-type mice from the time of weaning to the age of 80 d (46). Taken together, these findings suggest that brainstem TRH is a physiological regulator of ghrelin-mediated stimulation of food intake.

An interaction between brainstem TRH and Y receptors in the regulation of vagal efferent functions has been well documented by previous studies using functional, morphological, and electrophysiological approaches (20, 21, 22, 23, 24, 47). The vagal stimulatory action of TRH in the DVC can be potentiated by Y1 agonist in a cumulative manner and antagonized by Y2 agonist (20, 21, 22, 23, 24). The results that ic TRH analog-induced food intake was prevented by ic Y1 antagonist, but not Y2 antagonist, and the abolishment of natural feeding in overnight fasted rats by Y1 antagonist indicate the participation of brainstem NPY in modulating food intake stimulated by brainstem TRH via Y1 receptors. A recent study reported that brainstem Y1 receptors are involved in the stimulation of food intake induced by central injection of ghrelin (10).

To clarify brain areas controlling energy homeostasis and glucose metabolism is an important priority (1). Integrity of endocrine and autonomic regulation is necessary to achieve a physiological or pathophysiological homeostasis. The brainstem performs basic life-maintaining functions, contains glucose sensors (48), and is the target of multiple digestive and nutrition signals including ghrelin, cholecystokinin, and leptin, hormones regulating food intake (49, 50, 51). The present study shows that TRH, a small peptide evolutionarily preserved across the vertebrate phylum (52) and the neurotransmitter that controls vagal and sympathetic-adrenal efferent functions in the brainstem of mammals, is responsive to metabolic state and regulates food intake behavior, in addition to its well-known gastric and pancreatic regulatory functions (11). Thus, brainstem is the primary central structure for controlling metabolism and TRH plays a key role in brainstem regulation of energy homeostasis through integrating endocrine and vagal-sympathetic balance.


   Footnotes
 
This work was supported by a Veterans Affairs Merit Award (to H.Y.) and National Institutes of Health Grant DK-41301 (CURE Center grant Animal Core, Antibody Core, and Peptidomic, Radioimmunoassay, Proteomic Core).

Disclosure statement: the authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: DVC, Dorsal vagal complex; ic, intracisternal; icv, intracerebroventricular; NPY, neuropeptide Y; SD, Sprague Dawley; TRH R1, TRH receptor 1.

Received June 19, 2006.

Accepted for publication August 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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