Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by ghrelin and 3-TRP-ghrelin

Peptides 36 (2012) 157–167

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Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by ghrelin and 3-TRP-ghrelin A. Eugene Pekary a,c,f,∗ , Albert Sattin a,b,d,e a

Research Service, VA Greater Los Angeles Healthcare System, United States Psychiatry Service, VA Greater Los Angeles Healthcare System, United States c Center for Ulcer Research and Education, VA Greater Los Angeles Healthcare System, United States d Department of Psychiatry & Biobehavioral Sciences, University of California, Los Angeles, CA 90073, United States e Brain Research Institute, University of California, Los Angeles, CA 90024, United States f Department of Medicine, University of California, Los Angeles, CA 90024, United States b

a r t i c l e

i n f o

Article history: Received 15 March 2012 Received in revised form 26 April 2012 Accepted 26 April 2012 Available online 24 May 2012 Keywords: Ghrelin TRH-like peptides Rat Limbic system

a b s t r a c t Ghrelin is not only a modulator of feeding and energy expenditure but also regulates reproductive functions, CNS development and mood. Obesity and major depression are growing public health concerns which may derive, in part, from dysregulation of ghrelin feedback at brain regions regulating feeding and mood. We and others have previously reported that thyrotropin-releasing hormone (TRH, pGluHis-Pro-NH2 ) and TRH-like peptides (pGlu-X-Pro-NH2 , where “X” can be any amino acid residue) have neuroprotective, antidepressant, anti-epileptic, analeptic, anti-ataxic, and anorectic properties. For this reason male Sprague-Dawley rats were injected ip with 0.1 mg/kg rat ghrelin or 0.9 mg/kg 3-Trp-rat ghrelin. Twelve brain regions: cerebellum, medulla oblongata, anterior cingulate, posterior cingulate, frontal cortex, nucleus accumbens, hypothalamus, entorhinal cortex, hippocampus, striatum, amygdala, piriform cortex and 5 peripheral tissues (adrenals, testes, epididymis, pancreas and prostate) were analyzed. Rapid and profound decreases in TRH and TRH-like peptide levels (increased release) occurred throughout brain and peripheral tissues following ip ghrelin. Because ghrelin is rapidly deacylated in vivo we also studied 3-Trp-ghrelin which cannot be deacylated. Significant increases in TRH and TRH-like peptide levels following 3-Trp-ghrelin, relative to those after ghrelin were observed in all brain regions except posterior cingulate and all peripheral tissues except prostate and testis. The rapid stimulation of TRH and TRH-like peptide release by ghrelin in contrast with the inhibition of such release by 3-Trp-TRH is consistent with TRH and TRH-like peptides modulating the downstream effects of both ghrelin and unacylated ghrelin. Published by Elsevier Inc.

1. Introduction Ghrelin is a 28 amino acid residue peptide secreted by gastric tissue originally identified as a gene product regulating food intake, fuel balance and body weight [16,31]. Subsequently, other functions have been identified including antidepressant [37], neurotropic/neuroprotective [1,8,15,33,34,40,41], immunomodulatory [46], insulin release inhibitory [14], gastro-protective [35], beta-cell regenerative [28], anti-epileptic [4], chronobiologic [67], sleep regulatory [42], cardioprotective [62], and fetal developmental [44] properties mediated via the ghrelin receptors in the

∗ Corresponding author at: VA Greater Los Angeles Healthcare System, Bldg. 114, Rm. 229, 11301 Wilshire Blvd., Los Angeles, CA 90073, United States. Tel.: +1 310 268 4430; fax: +1 310 441 1702. E-mail address: [email protected] (A.E. Pekary). 0196-9781/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.peptides.2012.04.021

hypothalamus, hippocampus, frontal cortex, cerebellum, pancreas, adrenals as well as other CNS and peripheral tissues [31,36]. Ghrelin opposes the satiety effects of leptin, an anorexic protein secreted constitutively from adipose tissue in proportion to the triglyceride content of fat cells [13]. Leptin regulates wholebody metabolism by stimulating energy expenditure, inhibiting food intake and restoring euglycemia. Leptin is also neuroprotective, anti-epileptic, and immunomodulatory, like ghrelin [50]. Hyperglucocorticoidemia is a consistent marker for major depression [61]. Recent studies using a TRH receptor 1 mutant mouse revealed a depressive and anxious phenotype [72]. Because TRH (pGlu-His-Pro-NH2 ) and TRH-like peptides (pGlu-X-Pro-NH2 , where “X” can be any amino acid residue) also have antidepressant, neuroprotective [49–57,59,60], anti-epileptic [69], and appetite suppressive properties [32,68], we hypothesized that some of the potential therapeutic effects of ghrelin are mediated by these neuropeptides.

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Table 1 Time-dependent effect of ghrelin or 3-Trp-ghrelin on serum hormone levels (mean ± SD) in male Sprague-Dawley (SD) rats. Ip ghrelin

Leptin (ng/ml)

Control 0.25 h 0.5 h 1.0 h

1.9 1.8 2.2 2.5

Ip 3-Trp-ghrelin

Leptin (ng/ml)

Control 0.25 h 0.5 h 1.0 h

1.5 1.5 1.4 1.7

*

± ± ± ±

± ± ± ±

0.3 0.3 0.7 0.4

0.2 0.1 0.1 0.1

Insulin (ng/ml) 0.20 0.10 0.16 0.64

± ± ± ±

0.20 0.08 0.15 0.38

Insulin (ng/ml) 0.07 0.06 0.06 0.07

± ± ± ±

0.02 0.01 0.01 0.02

Corticosterone (ng/ml) 161 527 519 213

± ± ± ±

89 39* 394* 77

Corticosterone (ng/ml) 428 383 287 203

± ± ± ±

158 133 158 102

Oxytocin (pg/ml) 4 9 14 16

± ± ± ±

5 6 1 12*

Oxytocin (pg/ml) 20 20 24 28

± ± ± ±

1 6 11 8

Testosterone (ng/ml) 115 196 586 213

± ± ± ±

77 100 498* 77

Testosterone (ng/ml) 255 160 118 134

± ± ± ±

236 73 40 56

fT3 (ng/ml) 1.45 1.68 1.28 1.22

± ± ± ±

0.34 0.17 0.28 0.28

TT3 (pg/ml) 51.0 47.2 43.8 45.9

± ± ± ±

3.2 6.8 9.1 3.1

fT4 (ng/ml) 1.39 1.56 1.31 1.29

± ± ± ±

0.16 0.26 0.11 0.19

fT4 (ng/ml) 0.86 0.77 0.76 0.77

± ± ± ±

0.15 0.12 0.18 0.07

Glucose (mg/dl) 167 120 149 188

± ± ± ±

39 34* 29 17

Glucose (mg/dl) 143 148 103 126

± ± ± ±

24 11 21 6

p < 0.05 by one way ANOVA versus the control group.

The present studies focus on the potential role of TRH and TRHlike peptides as mediators of the central and peripheral actions of ghrelin and its metabolites.

2. Materials and methods 2.1. Animals Male Sprague-Dawley (SD) rats (Harlan, Indianapolis, IN) were used for all experiments. These animals were group housed (4 animals per cage), maintained with standard Purina rodent chow #5001 and water ad libitum during a standard one week initial quarantine in a controlled temperature and humidity environment; lights on: 6 am–6 pm. All animals were weighed on the day of receipt and on the morning of each experiment. Initial and final body weights did not differ between experimental groups. Research was approved by the VA Greater Los Angeles Healthcare System Animal Care and Use Committee and conducted in compliance with the Animal Welfare Act and the federal statutes and regulations related to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and use of Laboratory Animals, NRC Publication, 1996 edition. All efforts have been made to minimize the number of animals used and their suffering. All animals were transferred from the Veterinary Medical Unit to the laboratory 12 h before the start of experiments to minimize the stress of a novel environment.

2.2. Time-dependent effects of ghrelin on TRH and TRH-like peptide levels in rat brain and peripheral tissues Rats weighing 299 ± 19 g were either saline injected at 0.5 h (controls, n = 4), or received a single 0.5 ml ip injection of recombinant ghrelin (serine-3 octanoic acid rat/mouse ghrelin, Bachem, Torrance, CA, 62.5 ␮g/ml sterile saline) at 0.25, 0.5 or 1 h before decapitation (n = 4 for each time point). Animals were injected at times that permitted decapitation between 9 and 11 am to minimize the effect of circadian variation in the levels of TRH and TRH-like peptides in rat brain and peripheral tissues [55]. The ghrelin dose was therefore 31.25 ␮g/0.3 kg rat or 104 ␮g/kg. This dose is sufficient for maximum stimulation of 2-h food intake [31].

2.3. Time-dependent effects of 3-Trp-ghrelin on TRH and TRH-like peptide levels in rat brain and peripheral tissues Rats weighing 278 ± 15 g were ip injected with 0.9 mg/kg 3-Trprat ghrelin (Biomatik, Wilmington, DE), which has about 1/20 the potency of ghrelin, and tissues and blood obtained as above.

2.4. Dissection of rat brain, pancreas and reproductive organs Cerebellum, medulla oblongata, anterior cingulate, posterior cingulate, frontal cortex, nucleus accumbens, hypothalamus, entorhinal cortex, hippocampus, striatum, amygdala, piriform cortex, pancreas, prostate, epididymis, and testes were hand dissected, weighed rapidly, and then extracted as previously described [49–57,59,60]. Serum was obtained by collecting 6–8 ml of whole blood in plastic 16 mm × 100 mm test tubes containing 0.5 ml of 25 mg/ml of a proteolytic enzyme inhibitor (4-(2-aminoethyl) benzene sulfonyl fluoride HCl, Sigma, St. Louis, MO) on ice. Tubes were rapidly vortex mixed and maintained at 4 ◦ C during centrifugation [6]. Serum was stored at −20 ◦ C until day of immunoassays for active and total ghrelin. 2.5. Serum hormone assays Serum rat total and active ghrelin, rat leptin, rat insulin, oxytocin, corticosterone, testosterone, free T4 , and free T3 were measured with the following commercial RIA kits: rat total ghrelin, rat active ghrelin, rat leptin and rat insulin (Millipore, St. Charles, MO), oxytocin (Bachem, Torrance, CA), corticosterone (MP Biomedicals, Aurora, OH), testosterone, free T4 and free T3 (DPC Coat-A-Count, Los Angeles, CA). Serum glucose was measured with the OneTouch Ultra Meter (LifeScan, Milpitas, CA). 2.6. HPLC and RIA procedures, HPLC peak identification and quantitation HPLC and RIA procedures, peak identification, and quantitation by co-chromatography with synthetic TRH and TRH-like peptides, relative potency analysis of multiple antibodies to TRH and TRHlike peptides, mass spectrometry, and one way ANOVA have been previously reported in detail [49–57,59,60]. Briefly, after boiling, tissues were dried, re-extracted with methanol, dried and defatted by water-ethyl ether partitioning. Dried samples were dissolved in 0.1% trifluoroacetic acid (TFA), and loaded onto reverse phase C18 Sep-Pak cartridges (Waters, Milford, MA). TRH and TRH-like peptides were eluted with 30% methanol. Dried peptides were again dissolved in TFA, filtered and then fractionated by HPLC using a 4.6 mm × 150 mm Econosphere, 3 ␮m C18 reverse phase column (Alltech Associates, Deerfield, IL) and a 0.2%/min gradient of acetonitrile. The 0.5 ml fractions collected were dried completely, stored at −20 ◦ C, and reconstituted with 0.10 ml of 0.02% NaN3 just before RIA. The antiserum used (8B9) cross-reacts with TRH and eight TRHlike peptides with a relative potency of displacement ranging from 2.31 (Lys-TRH) to 0.288 (Ser-TRH) relative to Tyr-TRH (Table 2: 54). Two of the regularly observed peaks (2a and 2b) consist of a mixture of unidentified TRH-like peptides. Of the eight resolved peptides three have so far been confirmed by mass spectrometry:

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Table 2 Effects of ip ghrelin on HPLC peak areas corresponding to TRH and TRH-like peptide levels in various brain regions of male SD rats involved in regulation of mood, behavior and appetite. Hours

Glu-TRH

Cerebellum 0.29** 0.25 0.50 0.19** 1.00 0.74 Medulla oblongata 2.24* 0.25 0.04* 0.50 1.00 0.18 Anterior cingulate 2.35* 0.25 0.50 3.25* 1.00 0.84 Posterior cingulate 3.20 0.25 1.30 0.50 2.80 1.00 Frontal cortex 1.11 0.25 0.72 0.50 1.00 0.42 Nucleus acumbens 0.47 0.25 0.50 0.32* 1.00 1.27 Hypothalamus 0.34* 0.25 0.50 0.17** 1.00 0.20* Entorhinal cortex 1.05 0.25 0.50 0.25 1.00 0.23 Hippocampus 0.38 0.25 0.50 1.00 1.00 0.99 Striatum 1.24 0.25 0.50 0.14* 1.00 0.25* Amygdala 0.25 0.14 0.50 0.22 1.00 0.66 Piriform cortex 0.02*** 0.25 h 0.5 h 0.18** 1.0 h 0.27*

Peak 2

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

0.12*** 0.07*** 0.49*

0.02** 0.02** 0.59

0.03*** 0.03*** 0.42

0.05** 0.03** 0.45

0.01* 0.006* 0.75

0.04** 0.008** 0.66

0.03* 0.02* 0.46

1.38 0.07** 0.28*

0.27* 0.13* 0.56

0.34* 0.32* 0.76

0.83 0.10* 0.97

0.23 0.04 0.66

0.65 0.03* 0.42

0.89 0.11* 0.70

0.35* 0.09** 0.27*

0.09* 0.16* 0.47

0.07* 0.08* 0.29*

0.21* 0.06** 0.24*

0.22 0.14 0.14

0.99 0.58 0.56

0.22* 0.13* 0.46

1.26 1.58 3.49*

0.44 4.05* 9.36***

0.16 0.56 3.35*

0.28 0.29 2.08*

0.25 0.62 1.83

0.42 0.35 3.58

0.16 0.93 1.27

0.52 0.32 0.40

0.14* 0.08* 0.44

0.25* 0.11* 0.62

0.15*** 0.08*** 0.56

0.27* 0.13* 0.52

0.67 0.42* 0.56

0.53 0.20* 0.58

0.19* 0.13* 0.79

1.95 1.54 7.52**

0.10** 0.08** 0.91

0.16* 0.10* 1.03

0.37 0.21 1.22

1.02 0.46 1.08

0.30 0.49 1.41

0.24* 0.15* 0.17*

0.89 1.17 0.50

0.07** 0.08** 0.25*

0.09** 0.05** 0.22*

0.15** 0.14** 0.18**

0.36* 0.44* 0.20*

0.15** 0.19** 0.21*

0.50* 0.17*** 0.27**

0.03** 0.11* 0.32*

0.02** 0.06** 0.25*

0.04** 0.06** 0.25*

0.07** 0.10** 0.26*

0.22* 0.13* 0.21*

0.08** 0.12** 0.24*

0.15* 0.22* 0.51

0.07* 0.11* 0.30

0.08*** 0.11*** 0.21**

0.06* 0.06* 0.32

0.11* 0.16* 0.24

0.19* 0.21* 0.26*

0.14** 0.14** 0.44*

0.15* 0.04** 0.18*

0.57* 0.03*** 0.16**

0.14* 0.10* 0.44

0.07* 0.01** 0.30*

0.22** 0.04*** 0.16**

0.18*** 0.01*** 0.05***

0.46* 0.04*** 0.22**

0.35* 0.44 0.95

0.18*** 0.18*** 0.70

0.41* 0.76 1.18

0.10* 0.11* 0.51

0.24 0.25 0.70

0.70 0.55 0.90

0.28 0.41 0.93

0.06*** 0.28* 0.91

0.02*** 0.11*** 0.5*

0.15** 0.09*** 0.54

0.18** 0.06*** 0.46*

0.45* 0.14** 0.57

0.91 0.31* 1.11

0.11** 0.14** 0.66

Results are peak area divided by corresponding peak area in saline-injected controls. * p < 0.05 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described [55]. ** p < 0.01 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described [55]. *** p < 0.001 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described [55].

TRH, Glu-TRH and Tyr-TRH [49]. Tissue samples from the four rats within each treatment group were pooled prior to HPLC to provide the minimum amount of immunoreactivity needed for reliable RIA measurements. The mean recovery of TRH and TRH-like peptide immunoreactivity from all tissues studied was 84 ± 15% (mean ± SD). The within-assay and between-assay coefficient of variation for measuring 333 pg/ml TRH was 4.8% and 16.9%, respectively. All HPLC fractions obtained from a given brain region or peripheral tissue were analyzed in the same RIA. The minimum detectable concentration for TRH was 5 pg/ml. The specific binding of [125 I]TRH (Bo /T) was 25%. 2.7. Statistical analysis Statistical comparisons were made with the aid of Statview (Abacus Concepts Inc., Berkeley, CA), a statistical software package

for the Macintosh computer. All multi-group comparisons were carried out by one-way analysis of variance (ANOVA) using post hoc Scheffe contrasts with the control group. The mean within-group coefficient of variation (CV) (SD/mean, CV-within group) for each tissue and TRH/TRH-like peptide combination, across four photoperiod intervals, has been previously reported (circadian rhythm experiment) for untreated SpragueDawley rats [55]. Mean within-group CVs in brain ranged from 6.3% for TRH levels in amygdala to 48% for Leu-TRH in anterior cingulate, and from 3.1% for Val-TRH in testis to 21% for Tyr-TRH in pancreas. These CVs were then used to estimate the level of significance, by one-way ANOVA, of changes in the pooled mean values of TRH and TRH-like peptide levels at each time point following ip injection of ghrelin. The SD measurements of TRH or TRH-like peptide (X-TRH) following ghrelin treatment in a given tissue extract (T), SD (X-TRH, T), was equal to CV (X-TRH, T) [55] multiplied by the mean for the corresponding X-TRH from Tables 2–5.

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Fig. 1. Serum levels of active and total ghrelin versus time following ip injection of 0.1 mg/kg rat ghrelin (left panel) or 0.9 mg/kg rat 3-Trp-ghrelin (right panel). Error bars represent ±1SD.

Fig. 2. Effect of ip ghrelin (open circles) or 3-Trp-ghrelin (closed circles) on levels of TRH and TRH-like peptides versus time in entorhinal cortex. Note the 86 ± 17% (p < 0.01), 89 ± 4% (p < 0.01) and 74 ± 3% (p < 0.01) decline in TRH and TRH-like peptide levels, except Glu-TRH, at 0.25, 0.5, and 1 h, respectively, following ip ghrelin.

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The Students t-test for the difference between the area under the curve (AUC) for TRH and each TRH-like peptide area versus time in response to ip ghrelin (AUGG ) versus the corresponding AUC following ip 3-Trp-ghrelin (AUCTG ) was calculated as follows AUGTG −AUCG [7].tX-TRH,T = √ 1/2 3/2([AUGTG ]2 +[AUCG ]2 )

×CV[X-TRH,T]

161

cingulate (5↑), nucleus accumbens (4↓,1↑) amygdala (2↓), for a total of 106. 3.6. Effect of ghrelin at 0.25 h in peripheral tissues

3. Results

Significant changes were: prostate (7↓), epididymis (5↓), adrenals (5↓), testes (2↑) and pancreas (0) totaling 19, as summarized in Table 3 and Fig. 5.

3.1. Reproducibility of tissue dissection weights

3.7. Effect of ghrelin at 0.5 and 1 h in peripheral tissues

The CVs of brain tissue weights ranged from 10.9% (MED) to 36.2% (FCX). The corresponding CVs for peripheral tissue weights varied from 5.9% (epididymis) to 23.2% (prostate). Prostate weights vary markedly in normal, untreated, young adult male littermates.

Changes in peptide levels for pancreas (15↓), epididymis (12↓), adrenals (10↓) and prostate (6↓) totaled 43 (Table 3 and Fig. 5).

3.2. Serum values of total ghrelin, active ghrelin, leptin, insulin, oxytocin, corticosterone, testosterone, fT3 , fT4 , and glucose (see Table 1 and Fig. 1)

Table 4 summarizes the differences in the area under the curve (AUC) for TRH and TRH like peptide responses to ip 3-Trp-ghrelin versus ghrelin (AUGTG − AUCG ) in brain. The total of the significant differences in each brain region were: striatum (8↑), hypothalamus (7↑), entorhinal cortex (7↑), cerebellum (6↑), piriform cortex (6↑), frontal cortex (4↑), medulla oblongata (3↑), anterior cingulate (3↑, 1↓), hippocampus (2↑), amygdala (2↑), nucleus accumbens (1↑, 1↓), posterior cingulate (1↑, 1↓).

3.3. Overview of HPLC results The acute effect of a single ip injection of ghrelin was, in general, to rapidly decrease TRH and TRH-like peptide levels throughout the brain and peripheral tissues. A significant decrease in peptide level at 1 h or less is consistent with acceleration of release [45,53]. A trend upwards toward control values at 1 h was observed in most tissues except the pancreas where peptide levels continued to decline by 1 h. Peptide levels in the testis were relatively unaffected. TRH and TRH-like peptides are contained within large dense core vesicles (LDCV) that consist of readily releasable LDCVs and reserve LDCVs that require more than 1 h to become activated for release [45]. Replenishment of depleted LDCV pools also requires more than 1 h. Posterior cingulate represents an interesting exception which displayed significant increases in some peptide levels at 1 h consistent with a delayed inhibition of release. The rapid declines in TRH and TRH-like peptide levels were not a nonspecific response to the acute stress of the ip injections administered at 0.25, 0.5 and 1.0 h before decapitation. For example, ip injection of 14.1 mg/kg corticosterone (CORT) resulted in a serum level of 27,806 ng/ml corticosterone at 2 h. Nevertheless, there were no significant changes in TRH or TRH-like peptide levels in brain or peripheral tissues at 2 h compared to uninjected controls [56]. In testis, for example, the levels of TRH-like peptides fell by 86–98% by 4 and 6 h after ip CORT. A follow-up in vitro study demonstrated that this CORT-induced decline in TRH and TRH-like peptide levels was due to peptide release and that this release did not begin until 1.5 h after CORT addition to the decapsulated testis [53]. 3.4. Effect of ghrelin at 0.25 h in brain Since all 8 peptides (TRH and TRH-like) in cerebellum decreased significantly, as seen in Table 2 and Fig. 4, we abbreviate this result as: cerebellum (8↓). Significant changes in TRH and TRHlike peptide levels for other brain regions were: hippocampus (7↓) entorhinal cortex (7↓), hypothalamus (7↓), striatum (7↓), piriform cortex(7↓), frontal cortex (6↓), anterior cingulate (5↓,1↑), amygdala (4↓), medulla oblongata (3↓), nucleus accumbens (3↓), and posterior cingulate (0) for a total of 55. 3.5. Effect of ghrelin at 0.5 and 1 h in brain Significant changes observed in Table 2 and Figs. 2–4 were: striatum (15↓), hypothalamus (14↓), entorhinal cortex (14↓), piriform cortex (11↓), hippocampus (10↓), cerebellum (9↓), anterior cingulate (8↓,1↑), medulla oblongata (8↓), frontal cortex (6↓), posterior

3.8. Effect of 3-Trp-ghrelin relative to ghrelin in brain

3.9. Effect of 3-Trp-ghrelin relative to ghrelin in peripheral tissues Table 5 summarizes the differences in the area under the curve (AUC) for TRH and TRH like peptide responses to ip 3-Trp-ghrelin versus ghrelin (AUGTG − AUCG ) in peripheral tissues. The total significant differences were: pancreas (8↑), epididymis (4↑), adrenals (3↑), testis (0) and prostate (6↓). 4. Discussion This is the first report of the acute effects of ghrelin and 3Trp-ghrelin on the expression of TRH and TRH-like peptides in brain and peripheral tissues of the rat. The autonomic system regulates gastric ghrelin secretion in rats [25,43]. All brain regions examined experienced a rapid and profound decrease in TRH and TRH-like peptide levels (acute peptide release) after ip ghrelin. In hypothalamus, one of the brain regions that coordinates the ghrelin-stimulated effects of hunger [16,31] only the TRH-like peptides fell precipitously. Hypothalamic TRH levels were 20-fold higher, on average, than in other brain regions. TRH levels in the cerebellum, a brain region also involved in the regulation of feeding [63,74], declined 98% at 0.25 h and 0.5 h after ghrelin while hypothalamic TRH levels did not change (Table 2). The amount of immunoreactive TRH released into hypophysial portal blood of female rats is about 2 orders of magnitude greater than gonadotropin releasing hormone and somatostatin [21]. The turnover of TRH, 80% of the total hypothalamic content per hour, is also much greater than that of any other known peptide [21]. The ratio of TRH-Gly (pGlu-His-Pro-Gly), the biosynthetic precursor of TRH, to TRH in the rat hypothalamus is 0.05 [64] while the corresponding ratio for rat prostate is greater than 100 [48]. These results suggest that TRH in the neurosecretory cells of the hypothalamus, which are primarily responsible for the release of pituitary TSH and thyroid hormones, are relatively unresponsive to the acute effects of ghrelin, consistent with the serum fT3 and fT4 results in Table 1. Circulating ghrelin is derived primarily from X/A-like cells lining the fundus of the stomach and epsilon cells of the pancreas and is secreted in anticipation of a meal. This provides a hunger signal to hypothalamic feeding centers mediating energy homeostasis and stimulating gastric acid secretion, gut motility [3], and inhibiting

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Fig. 3. Effect of ip ghrelin (open circles) or 3-Trp-ghrelin (closed circles) on levels of TRH and TRH-like peptides versus time in striatum. Note the marked decline in TRH and TRH-like peptide levels at 0.25 and 0.5 h and return to control values at 1.0 h following 3-Trp-ghrelin but not ghrelin.

pancreatic protein secretions [73]. Ghrelin is considered a counterbalance to the satiety hormone, leptin, which is produced mainly by adipose tissue [8,31,38,50,66,68]. The direct effects of ghrelin are mediated through the growth hormone secretagogue receptor (GHSR) which is an amplifier of GH pulsatility [66]. This receptor also occurs in other brain regions, particularly the cerebellum, hippocampus, frontal cortex, medulla oblongata, amygdala, substantia nigra [31], and peripheral tissues [30,31]. Ghrelin reduces the anxiety and depressive symptoms of chronic stress [37]. Atrophy of certain limbic structures, including the hippocampus, frontal cortex and anterior cingulate characterize major depression [17,18]. Ghrelin’s antidepressant- and anxiolyticlike actions may involve the activation of neurons in the ventral tegmental area or hippocampus, both of which express GHSRs, undergo ghrelin-induced modulation of synapse formation, and are sites of mood regulation [1,15]. Stress increases circulating ghrelin via direct stimulation of ghrelin cells by catecholamines following activation of the sympathetic nervous system [43]. Reduction in the sensitivity of glucocorticoid suppression of CRH and ACTH release within the hippocampus, a characteristic of major depression, can

be reversed by ghrelin [37]. The ghrelin receptor has a high constitutive activity. Development of inverse agonists of GHSR for the treatment of obesity [19] may also have therapeutic potential in the treatment of neuropsychiatric disorders. Reduction in TRH and TRH-like peptide levels, consistent with ghrelin-induced release, was observed in almost all brain regions examined, as seen in Table 2. We have previously reported that these peptides have neuroprotective, antidepressant-like, analeptic, immunomodulatory, and anti-epileptic effects in rats [49–57,59,60]. Interventions that stimulate downstream pathways, rather than ghrelin itself, may represent therapeutic possibilities for all of these pathologies. Disturbance of monoamine neurotransmission is associated with major depression and other psychiatric disorders. Therapeutic agents commonly used for their treatment alter transport, biosynthesis and/or metabolism of this class of neurotransmitters [61]. Mice lacking ghrelin have reduced dopamine release and concentrations of tyrosine hydroxylase, the rate-limiting enzyme for norepinephrine and dopamine synthesis in the nucleus accumbens [2]. These effects were corrected by ghrelin [29]. Consistent with mediation of ghrelin effects in the mesolimbic region by TRH

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Fig. 4. Effect of ip ghrelin (open circles) or 3-Trp-ghrelin (closed circles) on levels of TRH and TRH-like peptides versus time in cerebellum. Note the 93 ± 9% (p < 0.01) and 95 ± 6% (p < 0.01) decline in TRH and TRH-like peptide levels at 0.25 and 0.5 h, respectively, and return to control values at 1.0 h following ip ghrelin.

and TRH-like peptides is the ghrelin-induced release of TRH and TRH-like peptides (acute decrease in levels) throughout the brain (Table 2). Of particular interest is the role of ghrelin in the regulation of reproduction, which is highly dependent on the age and nutritional status of both sexes. Ghrelin increases GnRH release by the hypothalamus of prepubertal and peripubertal rats [20]. A number of neuropeptides and neurotransmitters, including glutamate, are involved in the proestrus surge in GnRH [10]. TRH and TRH-like peptides are colocalized in glutamatergic neurons and may moderate the excitotoxic effects of excessive glutamate release [26,59]. The ghrelin receptor, GHS-R1␣ , immunoreactivity of testis occurs mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes [71]. Serum testosterone levels in men and postmenopausal women are strongly correlated with serum ghrelin levels [24]. Serum testosterone levels were significantly increased 0.5 h after ghrelin injection as seen in Table 1. Acute increases in TRH and TRH-like peptide levels occur throughout the brain and peripheral tissues of rats in response to ip

injection of 100 ␮g of lipopolysaccharide (LPS)/kg body weight [57]. LPS reduces plasma ghrelin levels in fasted rats [70], an effect which is due to inhibition of the circulating ghrelin-acylating enzyme GOAT [65]. The rise in TRH and TRH-like peptides (reduced release) following LPS administration may be, at least in part, a response to a reduction in the circulating levels of the acylated (active) form of ghrelin (AG) [57,65]. TRH is the predominant TRH-like peptide within the adrenal cortex while Trp-TRH is the most abundant TRH-like peptide within the adrenal medulla [51]. These observations are consistent with the release of TRH, an inhibitor of corticosterone, from rat adrenal in vitro [39], playing a role in the ACTH-mediated effects of ghrelin on corticosterone release Table 1 [22]. Ghrelin has a direct stimulating effect on corticosterone output by cultured rat adrenocortical cells [58]. In the rat brain, GHS-R1␣ mRNA has been detected in multiple hypothalamic nuclei, pituitary gland, and dentate gyrus of the hippocampus, CA2 and CA3 regions of the hippocampus, the substantia nigra, ventral tegmental area, and dorsal and median raphe nuclei [11]. The rapid decline in TRH and TRH-like peptide levels after

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Table 3 Effects of ip ghrelin on HPLC peak areas corresponding to TRH and TRH-like peptides in reproductive tissues, pancreas and adrenals of male SD rats.

Epididymis 0.25 h 0.5 h 1.0 h Adrenals 0.25 h 0.5 h 1.0 h Prostate 0.25 h 0.5 h 1.0 h Testis 0.25 h 0.5 h 1.0 h Pancreas 0.25 h 0.5 h 1.0 h * ** ***

Glu-TRH

Peak 2

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

0.28* 0.14** 0.62

0.81 0.07*** 1.06

0.33* 0.01*** 0.44*

0.36* 0.03*** 0.37*

0.97 0.02*** 0.56

0.59 0.06*** 1.00

0.48* 0.02*** 0.30*

0.27* 0.02*** 0.34*

0.34* 0.10** 0.54

0.76 0.09** 0.18**

0.39* 0.37* 0.58

0.26* 0.20** 0.47*

0.35* 0.25** 0.53

1.16 0.22** 0.51

0.51 0.23** 0.58

0.32* 0.25** 0.61

0.06*** 0.02*** 0.78

0.35* 0.67 1.26

0.58 0.21** 0.7

0.19** 0.18** 1.05

0.48* 0.57 1.09

0.29* 0.3* 1.32

0.17** 0.17** 1.29

0.14** 0.27* 1.20

1.21 1.15 1.90

0.93 1.64 1.86

0.89 0.69 0.86

1.63 0.85 1.37

1.58 0.89 0.82

1.85 0.81 0.87

2.54* 0.60 0.86

0.52 0.57 0.12***

0.96 0.25** 0.26*

0.71 0.32* 0.10***

0.75 0.27* 0.14**

1.04 0.20** 0.12***

0.62 0.18* 0.14**

0.80 0.07*** 0.13**

2.30* 10.9 1.28 0.78 0.28* 0.26*

p < 0.05 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described [55]. p < 0.01 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described [55]. p < 0.001 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described [55].

Table 4 Difference in the effects of 3-Trp-ghrelin and ghrelin (single ip injection) on HPLC peak areas in various brain regions of male Sprague-Dawley rats involved in regulation of mood, behavior and appetite.

Cerebellum Medulla oblongata Anterior cingulate Posterior cingulate Frontal cortex Nucleus acumbens Hypothalamus Entorhinal cortex Hippocampus Striatum Amygdala Piriform cortex

Glu-TRH

Peak 2

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

1.22** 1.74 −2.92* −2.02 −0.04 0.96 1.68* 0.42 −0.72 1.42* 2.92 5.27*

1.00** 0.87 2.35* 1.38 1.62 4.66** 2.37* 1.36** 1.15 2.79** 0.63 3.08*

1.93** 1.10* 2.01 −9.94** 1.51 −10.89** 0.11 7.17** 1.35 1.52** 0.66** 1.26*

0.51* 1.97* 1.16 1.85 1.73* 0.72 3.52** 7.80** 3.51*** 2.25* −0.90 2.76**

4.01** 0.32 2.73** 6.13* 4.53** 0.54 2.30** 0.66* 1.00 1.39* 3.96* 3.29*

1.14 3.06* 2.61 5.89 2.51* −0.03 2.06* 3.22** 1.54 3.50** 2.45 4.40*

1.30** 1.78 1.01 6.86 1.23 −0.53 2.82* 3.73* 0.89 3.31*** 1.37 2.02

0.65 1.81 2.67* 5.49 2.61* −0.22 2.87** 2.91** 2.06** 3.79** 2.75 2.45

Results are area under the curve for each TRH and TRH-like peptide versus time after ip 3-Trp-ghrelin minus the corresponding area under the curve following ip ghrelin expressed as a percentage of the corresponding control peak area. * p < 0.05 by one-tailed Student’s t-test. ** p < 0.025 by one-tailed Student’s t-test. *** p < 0.01 by one-tailed Student’s t-test.

ip injection of ghrelin and 3-Trp-ghrelin is consistent with other reports that acylated ghrelin (AG) and unacylated ghrelin (UAG) may have effects via other, yet to be identified receptors [12]. Rapid changes in TRH and TRH-like peptide levels following ip ghrelin also occurred in many tissues which lack GHS-R1␣ including the prostate [23]. Ghrelin can stimulate the release of other hormones, for example, oxytocin (47 and Table 1) that can

mediate some of the biochemical, physiological and behavioral effects of ghrelin [5,9,27,47]. GHS-R1␣ is expressed on pancreatic epsilon-cells where it participates in direct, glucose-dependent, inhibition of insulin secretion by ghrelin [14]. Evidence is accumulating for metabolic effects of unacylated ghrelin (UAG). UAG, for example, improves insulin sensitivity and glucose homeostasis. A UAG receptor,

Table 5 Difference in the effects of Trp-ghrelin and ghrelin (single ip injection) on HPLC peak areas in reproductive tissues, pancreas and adrenals of male SD rats.

Epididymis Adrenals Prostate Testis Pancreas

Glu-TRH

Peak 2

TRH

Val-TRH

Tyr-TRH

Leu-TRH

Phe-TRH

Trp-TRH

2.02* 1.09 0.51 −1.37 3.10*

−0.60 0.98 −0.63 −1.38 4.33*

1.69* – −1.25** 0.66 3.96**

1.29* 0.85 −1.04* −0.60 6.33**

−0.32 11.64** −1.76** −0.16 1.74*

0.63 0.51 −1.23* −0.10 3.97**

2.18* 3.62* −1.44** −1.53 2.39*

1.10 0.86* −1.52** −1.42 2.49*

Results are area under the curve for each TRH and TRH-like peptide versus time after ip 3-Trp-ghrelin minus the corresponding area under the curve following ip ghrelin expressed as a percentage of the corresponding control peak area. * p < 0.05 by one-tailed Student’s t-test. ** p < 0.025 by one-tailed Student’s t-test.

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Fig. 5. Effect of ip ghrelin and 3-Trp-ghrelin on levels of TRH and TRH-like peptides versus time in pancreas. Note the 73 ± 14% (p < 0.05) and 86 ± 6% (p < 0.01) decline in TRH and TRH-like peptide levels at 0.5 h and 1.0 h, respectively, following ip ghrelin which contrasts with the corresponding nonsignificant changes after ip 3-Trp-ghrelin. (Closed circles: 3-Trp-ghrelin; open circles: ghrelin.)

however, has not yet been characterized. Ghrelin prevents streptozotocin induced diabetes [28]. AG and UAG both promote proliferation and inhibit apoptosis of pancreatic ␤-cells [12]. Fig. 1 indicates that measured levels of AG made only a minor contribution to total serum ghrelin levels. Very low levels of measured AG were observed at all times after both ip ghrelin and 3-Trp-ghrelin despite the addition of an esterase inhibitor prior to the processing at 4 ◦ C and storage of the rat serum at −20 ◦ C [6]. AG levels in vivo are, in general only about 10% of the UAG levels because of rapid deacetylation of AG by serum esterases [12]. The profiles of total and active serum ghrelin and 3-Trp-ghrelin in Fig. 1 remained elevated by 1 h after ip injection. This is an interesting observation given that in many tissues TRH and TRH-like peptide levels have returned to control values by 1 h, with the important exception of pancreas after ip ghrelin (Table 3 and Fig. 5).

Growth hormone and AG are diabetogenic: they both stimulate glycogenolysis and gluconeogenesis by the liver and suppress glucose utilization. UAG is antidiabetogenic and restores glucose homeostasis [12]. The decrease in serum glucose seen in Table 1 and the small amount of AG observed in Table 1 and Fig. 1 suggest that the UAG effects predominate when a high dose of AG is administered because of rapid deacetylation by esterases in the circulation. “Peripherally and centrally administered UAG can activate hypthalamic neurons, but the downstream pathway that modulates glucose metabolism remains to be elucidated” [12]. Leptin also stimulates release of TRH and TRH-like peptides throughout the brain and peripheral tissues of rats [50]. Because TRH is anorexogenic, like leptin, we expected that repetition of the present study with a GHS-R1␣ agonist that is not subject to rapid deacylation would inhibit TRH and TRH-like peptide

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