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Research Report

Rapid modulation of TRH and TRH-like peptide release in ratbrain and peripheral tissues by leptin

A.E. Pekarya,c,f,⁎, Albert Sattina,b,d,e, James Blooda

aResearch Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USAbPsychiatry Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USAcCenter for Ulcer Research and Education, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USAdDepartment of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA 90073, USAeBrain Research Institute, University of California, Los Angeles, CA 90073, USAfDepartment of Medicine, University of California, Los Angeles, CA 90073, USA

A R T I C L E I N F O

⁎ Corresponding author. VA Greater Los AngeFax: +1 310 441 1702.

E-mail address: Eugene.Pekary@va.gov (A

0006-8993/$ – see front matter. Published bydoi:10.1016/j.brainres.2010.05.039

A B S T R A C T

Article history:Accepted 13 May 2010Available online 9 June 2010

Leptin is not only a feedbackmodulator of feeding and energy expenditure but also regulatesreproductive functions, CNS development and mood. Obesity and major depression aregrowing public health concerns which may derive, in part, from disregulation of leptinfeedback at the level of the hypothalamic feeding centers and mood regulators within thelimbic system. Identifying downstream mediators of leptin action may provide therapeuticopportunities. We and others have previously reported that thyrotropin-releasing hormone(TRH, pGlu-His-Pro-NH2) and TRH-like peptides (pGlu-X-Pro-NH2, where “X” can be anyamino acid residue) have neuroprotective, antidepressant, anti-epileptic, analeptic, anti-ataxic, and anorectic properties. For this reason, young, adult male Sprague–Dawley ratswere injected ip with 1 mg/kg rat leptin and peptide and protein levels were measured inbrain and peripheral tissues at 0, 0.5, 1 and 2 h later. Eleven brain regions: pyriform cortex(PYR), entorhinal cortex (ENT), cerebellum (CBL), nucleus accumbens (NA), frontal cortex(FCX), amygdala (AY), posterior cingulate (PCNG), striatum (STR), hippocampus (HC),medulla oblongata (MED) and anterior cingulate (ACNG) and five peripheral tissues(adrenals, testes, epididymis, pancreas and prostate) were analyzed. TRH and six TRH-likepeptide levels in STR fell by 0.5 h consistent with leptin-induced release of these peptides:STR (7↓). Significant changes in TRH and TRH-like peptide levels for other brain regionswere: CBL (5↓), ENT (5↓), HC (4↓), AY (4↓), FCX (3↓), and ACNG (1↓). The rapid modulation ofTRH and TRH-like peptide release combined with their similarity in behavioral,neuroendocrine, immunomodulatory, metabolic and steroidogenic effects to that of leptinis consistent with these peptides participating in downstream signaling.

Published by Elsevier B.V.

Keywords:LeptinTRH-like peptideRatLimbic system

les Healthcare System, Bldg. 114, Rm. 229, 11301 Wilshire Blvd., Los Angeles, CA 90073, USA.

.E. Pekary).

Elsevier B.V.

Fig. 1 – Serum levels of leptin versus time following ipinjection of 1 mg/kg rat leptin. Error bars represent ±1 SD.

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1. Introduction

Leptin is a protein secreted by adipose tissue originally iden-tified as a gene product regulating food intake, fuel balance andbody weight (Bjorbaek and Kahn, 2004; Meyers, 2006). Subse-quently, other functions have been identified including antide-pressant, neurotropic, immunomodulatory, anti-epileptic, andanti-ataxia properties mediated via the leptin receptors in thehippocampus, frontal cortex, cerebellum, adrenals as well asother CNS and peripheral tissues (Andreasson et al., 2007; Lagoet al., 2008; Lu et al., 2006; Lu, 2007; Shibusawa et al., 2008; Xuet al., 2008). Leptin has a potential therapeutic advantage com-pared to other neuropeptides in that it crosses the blood–brainbarrier by a saturable transport system when administeredperipherally (Kastin and Pan, 2004) and by a direct route whenapplied intranasally (Xu et al., 2008).

Hyperglucocorticoidemia is a consistent marker for majordepression (Lu, 2007). Chronic leptin infusion in mice withmutations in the leptin gene (ob/ob) or leptin receptor (db/db)can reverse their elevated serum corticosterone (CORT) levelsbefore significant weight loss suggesting an inhibitory rolefor leptin in HPA regulation independent of alterations inenergy homeostasis (Lu, 2007). This inhibitory effect ap-pears to operate on multiple levels including suppressionof hypothalamic CRH release, enhancement of CORT nega-tive feedback, and direct inhibition of CORT release by theadrenals (Lu, 2007).

Leptin modulates the hypothalamic–pituitary–thyroid(HPT) axis in humans and rodents. Starvation suppressesserum T3 and T4 markedly reducing basal metabolism. Thisdecrease is accompanied by a fall in hypothalamic TRHmRNAand serum TSH levels that can be reversed by leptin. Leptineffects on the HPT axis results from both direct action on TRHneurons of the hypothalamus and indirect effectsmediated byother neurons that project to the hypothalamus. Leptin canstimulate TRH gene expression via a STAT3 response elementon the TRH promoter in cells in vitro (Bjorbaek and Kahn, 2004)and in vivo (Guo et al., 2004).

Recent studies using a TRH receptor 1 mutant mouserevealed a depressive and anxious phenotype (Zeng et al.,2007). Because TRH (pGlu-His-Pro-NH2) and TRH-like pep-tides (pGlu-X-Pro-NH2, where “X” can be any amino acidresidue) also has antidepressant, neuroprotective (Pekaryet al., 2005, 2006a,b,c,d), anti-epileptic (Veronesi et al., 2007),and appetite suppressive properties (Lechan and Fekete,2006; Valassi et al., 2008), we hypothesized that some of thepotential therapeutic effects of leptin are mediated by theseneuropeptides.

Leptin suppresses epileptiform-like bursting and eleva-tions in intracellular calcium in hippocampal (HC) cells byopening large-conductance calcium-activated neuronal po-tassium (BK) channels (Harvey, 2007; Xu et al., 2008). Theanticonvulsive effects of TRH result, at least in part, fromenhanced release of GABA from inhibitory HC interneuronsafter blockade of k2P channels in these cells (Yarbrough et al.,2007).

The present studies focus on the potential role of TRH andTRH-like peptides as mediators of the central and peripheralactions of leptin.

2. Results

2.1. Reproducibility of tissue dissection weights

The CVs of brain tissue weights ranged from 8.4% (PYR)to 30.9% (FCX). The corresponding CVs for peripheral tissueweights varied from 6.1% (testis) to 23.8% (prostate). Prostateweights varymarkedly in normal, untreated, young adultmalelittermates.

2.2. Serum values of leptin, insulin, corticosterone,testosterone, T3, fT4, and glucose

Serum values of leptin were (ng/ml, mean±SD): 1.2±0.6 at 0 h,915±233 at 0.5 h (p<0.01 vs. 0 h), 202±102 at 1 h (n.s.), and 197±59 at 2 h (p<0.05 versus 0 h; Fig. 1). Serum insulin (ng/ml,mean±SD): 0.28±0.06 at 0 h, 0.17±0.06 at 0.5 h, 0.16±0.06at 1 h, and 0.44±0.15 at 2 h (all n.s. versus 0 h). Testosterone(ng/ml, mean±SD): 368±106 at 0 h, 685±162 at 0.5 h, 648±436at 1 h and 543±306 at 2 h (all n.s. versus 0 h). Serum total T3

(ng/ml, mean±SD): 64.1±9.8 at 0 h, 64.3±3.8 at 0.5 h, 71.3±3.6at 1 h, and 64.8±9.7 at 2 h (all n.s. versus 0 h). Serum free T4

(ng/ml, mean±SD): 2.02±0.45 at 0 h; 1.94±0.31 at 0.5 h; 2.09±0.37 at 1 h, and 2.40±0.20 at 2 h (all n.s. versus 0 h). Serumcorticosterone (ng/ml, mean±SD): 104±119 at 0 h, 103±79 at0.5 h, 154±120 at 1 h, and 116±85 at 2 h (all n.s. versus 0 h).Glucose (mg/dl, mean±SD): 181±14 at 0 h, 179±19 at 0.5 h, 173±15 at 1 h, and 205±10 at 2 h (all n.s. versus 0 h).

2.3. Overview of HPLC results

The acute effect of a single ip injection of leptin on levelsof TRH and TRH-like peptides in brain and peripheral tissueswas, in general, heterogeneous (Figs. 2, 3, and 4). Dependingon the particular peptide and tissue or brain region, responsesin peptide levels ranged from (a) no effect, (b) a rapid andsustained rise, (c) a rapid rise followed by a gradual decline

Fig. 2 – Effect of ip leptin on levels of TRH and TRH-like peptides versus time in hippocampus.

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towards the control value, (d) a rapid and sustained decline, ora rapid decline followed by a recovery to the control value. Asignificant decrease in peptide level at 2 h is consistent withacceleration of release (Dahm et al., 2001). TRH and TRH-likepeptides are contained within large dense core vesicles (LDCV)that consist of readily releasable LDCVs and reserve LDCVs thatrequire more than 2 h to become activated for release (Dahmetal., 2001). Replenishmentof depletedLDCVpools also requiresmore than 2 h. Conversely, a significant rise in peptide level at2 h suggests inhibition of release. Changes after 2 h may resultfrom a combination of altered LDCV release and regeneration,making mechanistic interpretations more difficult.

2.4. Effect of leptin at 0.5 h in brain

TRH and six TRH-like peptide levels in STR fell by 0.5 h con-sistent with leptin-induced release of these peptides, as seenin Table 1. We abbreviate this result as: STR (7↓). Significantchanges in TRH and TRH-like peptide levels for other brain

regions were: ENT (5↓), CBL (5↓), HC (4↓), AY (4↓), FCX (3↓), andACNG (1↓) for a total of 29.

2.5. Effect of leptin at 1 and 2 h in brain

Significant changes from Table 1 were: CBL (1↑, 9↓), HC (7↓), AY(6↓), STR (6↓), FCX (1↑, 5↓), MED (5↑), ENT (4↓), PCNG (4↓), ACNG(3↓), NA (1↑, 2↓), and PYR (1↑) for a total of 59.

2.6. Effect of leptin at 0.5 h in peripheral tissues

Significant changes in epididymis (6↓), prostate (4↑), adrenals(4↑), pancreas (1↑), and testes (1↑) totaled 16 as summarizedin Table 2.

2.7. Effect of leptin at 1 and 2 h in peripheral tissues

Changes in epididymis (12↓), prostate (8↑), pancreas (4↑), testes(2↓), and adrenals (2↑) totaled 28 (Table 2).

Fig. 3 – Effect of ip leptin on levels of TRH and TRH-like peptides versus time in striatum.

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3. Discussion

This is the first report of the acute effects of leptin on theexpression of TRH and TRH-like peptides in extrahypothalamicbrain and peripheral tissues of the rat. Circulating leptin isderivedprimarily fromadipose tissueandsecretedconstitutivelyin proportion to the triglyceride content of fat cells. Thisprovidesa satiety signal to hypothalamic feeding centers mediatingenergy homeostasis. The direct effects of leptin require thelong formof the leptin receptor,Ob-Rb,which ishighlyexpressedinhypothalamicnuclei involved in the regulationof appetite andmetabolism. This receptor also occurs in other brain regions,particularly the CBL, HC, FCX, MED, AY and substantia nigra(Harvey, 2007; Lu, 2007), and peripheral tissues.

A role in depression is a novel function of leptin (Lu, 2007).Atrophy of certain limbic structures, including the HC, FCXand ACNG characterize major depression. These changes are

reversible with leptin treatment (Duman andMonteggia, 2006;Matochik et al., 2005). Leptin has antidepressant effects inanimal models of depression such as the forced swim, tailsuspension and chronic unpredictable stress tests (Lu, 2007;Lu et al., 2006). Reduction in the sensitivity of glucocorticoidsuppression of CRH and ACTH release within the HC, acharacteristic of major depression, can be reversed by leptin(Lu, 2007). The high incidence of depression in obesity hasbeen attributed to leptin resistance (Lu, 2007). Reduction inTRH and TRH-like peptide levels, consistent with leptin-induced release, was observed in the CBL, STR, HC, AY, ENT,FCX and ACNG, as seen in Table 1. We have previouslyreported that these peptides have neuroprotective, antide-pressant-like, analeptic, immunomodulatory, and antiepilep-tic effects in rats (Pekary et al., 2005, 2006a,b,c,d, 2007).Interventions that stimulate downstream pathways, ratherthan leptin itself, may represent therapeutic possibilities forboth of these pathologies.

Fig. 4 – Effect of ip leptin on levels of TRH and TRH-like peptides versus time in cerebellum.

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Disturbance of monoamine neurotransmission is associ-ated with major depression and other psychiatric disorders.Therapeutic agents commonly used for their treatment altertransport, biosynthesis and/or metabolism of this class ofneurotransmitters. Mice lacking leptin have reduced dopa-mine release and reduced concentrations of tyrosine hydrox-ylase, the rate-limiting enzyme for dopamine synthesis in theNA. These effects were corrected by leptin (Fulton et al., 2006).Consistent with mediation of leptin effects in the mesolimbicregion by TRH and TRH-like peptides is the leptin-inducedreduction of seven of the eight peptides (acute decrease inlevels) in STR (Table 1).

TRH and TRH-like peptide levels in the CBL were the mostresponsive to the acute effects of leptin of all the brain regionsstudied. OB-Rb isoform is widely expressed in the humanbrain with highest levels in the CBL (Burguera et al., 2000). Inrat brain, 125I-leptin binding sites are located in the thalamus,PYR, and granular and molecular cell layer of the CBL (Dal

Farra et al., 2000). On the other hand, leptin receptors arebarely detectable in the mouse brain except in the choroidplexus and leptomeninges (Dal Farra et al., 2000). TRH pro-duction is regulated directly at the transcriptional level byleptin (Bjorbaek and Kahn, 2004; Guo et al., 2004). Leptin-deficient rodents have reduced mobility unrelated to obesity.Leptin facilitates NR2B NMDA receptor-mediated Ca2+ influxin cerebellar granule cells via a MAP kinase-dependent path-way which is essential for normal mobility (Irving et al.,2006). The improvement of ataxic gait is one of the importantpharmacological properties of TRH. In the cerebellum, cyclicGMP mediates this effect of TRH (Shibusawa et al., 2008). TheCBL is also involved in feeding control. Hunger, satiation andthirst activate the CBL (Zhu and Wang, 2008).

Intraventricular (ICV) injection of both TRH and leptin intothe fourth ventricle produces a dramatic increase in brownadipose tissue (BAT) temperature while injection of leptinalone has no effect (Hermann et al., 2006, 2008). According to

Table 1 – Effects of leptin (single ip injection) on HPLC peak areas corresponding to TRH and TRH-like peptide levels invarious brain regions of male Sprague–Dawley rats involved in regulation of mood and behavior.

Glu-TRH Peak 2 TRH Val-TRH Tyr-TRH Leu-TRH Phe-TRH Trp-TRH

Nucleus acumbens0.5 H 0.937 0.804 0.507 1.075 1.476 0.780 1.135 0.4171.0 H 1.345 0.772 0.772 1.399 1.400 0.857 3.671** 0.6612.0 H 0.532 0.636 0.327* 1.171 1.648 0.351 1.351 0.298*

Hippocampus0.5 H 1.273 0.337 0.144* 0.303* 2.391 0.552 0.408* 0.163*1.0 H 0.992 0.456 0.115* 0.371* 4.045 0.433 0.400* 0.215*2.0 H 0.717 0.676 0.561 0.537* 1.157 0.380 0.327* 0.508*

Amygdala0.5 H 0.687 0.265* 0.583* 0.520* 0.833 0.927 0.249* 0.2801.0 H 1.105 0.309* 0.669* 0.560* 1.853 0.556 0.464* 0.2722.0 H 0.675 0.519* 0.789* 0.833 0.643 0.960 0.496 0.705

Pyriform cortex0.5 H 0.703 0.469 0.557 2.239 1.020 0.731 1.113 0.4851.0 H 0.551 0.503 0.477 2.172 0.960 1.467 1.028 0.5032.0 H 0.603 1.357 1.348 4.915* 0.524 1.528 1.949 1.571

Anterior cingulate0.5 H 0.821 0.589 0.279 0.291* 0.693 0.801 2.159 0.5611.0 H 0.669 0.537 0.459 0.343* 1.400 0.621 3.485 0.4892.0 H 0.387* 0.623 0.549 0.368 0.879 0.519 0.791 0.481

Posterior cingulate0.5 H 2.207 2.903 2.817 1.129 2.052 4.383 1.685 3.8201.0 H 1.888 1.489 1.459 1.768 1.877 4.840 2.443 2.6202.0 H 1.445 4.669* 8.523* 3.723* 2.483 4.825 2.085 8.549**

Entorhinal cortex0.5 H 0.744 0.269* 0.139* 0.125* 0.500 0.388* 0.692 0.223*1.0 H 0.591 0.480* 0.333* 1.503 2.131 0.392* 0.368 0.155*2.0 H 0.837 1.013 1.011 0.513 1.219 0.808 0.584 0.717

Striatum0.5 H 0.640 0.325* 0.17* 0.280* 0.312* 0.185* 0.640* 0.188*1.0 H 0.971 0.237* 0.195* 0.292* 0.295* 0.243* 0.849 0.193*2.0 H 0.763 0.813 1.219 1.096 0.273* 0.795 1.083 0.924

Frontal cortex0.5 H 0.375* 0.459 0.152* 0.808 1.000 0.672 0.613 0.145*1.0 H 0.792 0.513 0.341 1.127 2.777* 0.451 0.640 0.224*2.0 H 0.869 0.876 0.565 0.783 1.100 0.980 1.537 0.381*

Cerebellum0.5 H 0.381* 0.785 0.220* 0.356* 1.773 0.885 0.359* 0.144*1.0 H 0.701 0.503* 0.461* 0.509* 3.276 1.339 0.425* 0.104*2.0 H 0.545* 0.983 0.483* 0.420* 5.021* 1.587 0.471* 0.373

Medulla oblongata0.5 H 0.768 0.432 0.351 0.591 0.716 0.857 0.943 0.2161.0 H 1.123 0.697 0.496 0.591 1.235 1.231 3.027 0.4162.0 H 1.529 3.411* 1.885* 4.151* 2.385 4.489 6.004* 3.089*

Results are peak area divided by corresponding peak area in saline-injected controls. *p<0.05, **p<0.01 by one-way ANOVA using post hocScheffe contrast versus the control group as previously described (Pekary et al., 2006a).

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Table 1, leptin has an inhibitory effect on TRH and TRH-likepeptide release from the MED at 2 h which is very similar tothe effect seen 6 h after ingestion of Glu-TRH (Pekary andSattin, 2009; and unpublished data). While TRH has a ther-mogenic effect which can be amplified by leptin, its release

may be under feedback regulation by leptin. Leptin receptorsand Type I TRH receptors (TRHR1) are widely distributed with-in the MED and coregulate thermogenesis (Hermann et al.,2006, 2008). Deletion of the tumor necrosis factor-alphareceptor 1 (TNFR1) in rats fed a high-fat diet resulted in a

Table 2 – Effects of leptin (single ip injection) on HPLC peak areas corresponding to TRH and TRH-like peptides inreproductive tissues, pancreas and adrenals of male SD rats.

Glu-TRH Peak 2 TRH Val-TRH Tyr-TRH Leu-TRH Phe-TRH Trp-TRH

Adrenal0.5 H 2.060* 1.163 1.396 2.313* 2.576* 1.699 2.696* 0.7971.0 H 1.248 0.977 1.085 1.557 1.944 1.261 2.2589* 0.6312.0 H 0.945 0.919 0.883 0.828 1.567 1.019 2.688* 1.108

Epididymis0.5 H 0.367* 0.447* 0.445* 0.277* 0.948 0.563 0.144** 0.305*1.0 H 0.180** 0.297* 0.409* 0.267* 1.156 0.3623* 0.183** 0.113**2.0 H 0.136** 0.437* 0.401* 0.820 0.592 0.708 0.152** 0.229**

Prostate0.5 H 1.432 2.056* 1.523 2.109* 1.933 2.685* 3.117* 1.1161.0 H 3.096* 5.189** 2.452* 3.193* 2.469* 4.793** 7.123** 4.273**2.0 H 1.823 1.565 1.305 0.549 0.781 1.848 1.576 1.435

Pancreas0.5 H 2.329* 0.812 1.072 1.548 0.771 0.640 0.915 1.2191.0 H 2.344* 1.376 2.357* 0.860 1.089 0.819 2.069* 0.6592.0 H 2.887* 0.673 0.715 0.459 0.948 0.599 1.499 0.679

Testis0.5 H 1.207 0.935 1.075 1.141 1.700 2.155* 1.724 0.7641.0 H 0.839 1.231 1.689 1.632 0.768 0.129** 1.481 0.8092.0 H 0.616 0.660 0.784 0.419* 0.728 0.787 0.712 0.593

*p<0.05; **p<0.01 by one-way ANOVA using post hoc Scheffe contrast versus the control group as previously described (Pekary et al., 2006a).

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significantly increased expression of TRH and protection fromobesity (Romanatto et al., 2009).

Of particular interest is the role of leptin in the regulation ofreproduction, which is highly dependent on the age andnutritional status of both sexes. Leptin increases GnRH releaseby the HY of prepubertal and peripubertal rats. In peripubertalrats this increase was accompanied by a significant decreasein HY release of GABA and enhanced release of glutamate andNMDA receptor activation (Carbone et al., 2005). TRH and TRH-like peptides are colocalized in glutamatergic neurons andmoderate the excitoxic effects of excessive glutamate release(Sattin, 1999).

In the testes of mice, leptin expression is mainly restrictedto gonocytes at 5 days, and spermatogonia at 10 days. Fromday 20 and beyond, leptin expression predominated in sper-matocytes at stages VII–XIII of the seminiferous epithelium.Receptor isoforms Ob-Ra, Ob-Rb and Ob-Re were all expressedin testes from 5- to 60-day-old mice. The mRNA for Ob-Raand Ob-Re, but not Ob-Rb or leptin was identified in bothimmature (14-day old) and adult (60-day old) isolated Leydigcells. These results suggest that leptin has direct effects inthe proliferation, differentiation of germ cells and the mod-ulation of testicular steroidogenesis, using both autocrine andparacrine mechanisms (Herrid et al., 2008). TRH and TRH-likepeptides, and TRH and TRH receptor mRNA are all localizedwithin the Leydig cells (Pekary et al., 2006d) where theymodulate testosterone biosynthesis (Gerendai et al., 2000)and potentially act as mediators for the steroidogenic effectsof leptin.

Leptin injection into the white adipose tissue (WAT) ofthe epididymis evokes afferent activity to the central nervous

system. A reflex activation of sympathetic outflow to WATmay accelerate lipolysis (Niijima, 1998). A role for TRH andTRH-like peptides in this process is suggested by the depletionof these peptides from the epididymis by leptin observed inTable 2. Leptin receptors are observed in all epithelial cells ofboth immature and mature epididymides (Rago et al., 2009).Obesity results in clear morphological changes in the headsof the epididymides and sperm (Vigueras-Villasenor et al.,2010).

The leptin receptor is expressed on pancreatic beta-cellswhere it participates in direct, glucose-dependent, suppressionof insulin secretion by leptin (Anubhuti andArora, 2008; Haddadet al., 2006). Acute leptin, and Glu-TRH ingestion (Pekary andSattin, 2009 and unpublished data), also suppressed TRH andTRH-like peptide release (increased TRH and TRH-like peptidelevels, Table 2) consistentwith the colocalizationof insulin, TRH,and TRH-like peptides in the large dense core vesicles of thepancreatic beta cell (Pekary et al., 2006d). Leptin restores normalserum glucose in Type I diabetes (Wang et al., 2010).

Leptin inhibits steroid hormone secretion from the adrenal,in part by a direct interaction with leptin receptors in theadrenal cortex (Glasow et al., 1998; Malendowicz et al., 2007;Tena-Sempere et al., 2000). On the other hand, leptin enhancescatecholamine release from the adrenal medulla (Malendo-dowicz et al., 2007). Prazosin, a brain-active, α1-adrenoceptorblocker, stimulates CORT, TRH and TRH-like peptide release(Pekary et al., 2008) while leptin inhibits CORT, TRH and TRH-like peptide release from the adrenals of SD rats (Resultssection and Table 2). TRH is the predominant peptide withinthe adrenal cortex while Trp-TRH is the most abundant TRH-like peptide within the adrenal medulla (Pekary et al., 2008).

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These results are consistent with TRH, an inhibitor ofcoticosterone release from rat adrenal in vitro (Mitsuma etal., 1996, and likely TRH-like peptides which can increase TRHlevels and action), playing a mediating role in direct effects ofleptin and prazosin on corticosterone release.

In summary, a single ip injection of 1 mg/kg leptin hasa profound effect on the rate of TRH and TRH-like peptiderelease throughout the brain and peripheral tissues of young,adult male SD rats. The rapid modulation of TRH and TRH-like peptide release, both stimulatory and inhibitory, and theoverlap in their known behavioral, neuroendocrine, immuno-modulatory, metabolic and steroidogenic effects with that ofleptin suggests that these peptides are downstreammediatorsof leptin. We conclude that pathophysiological conditionswhich have been attributed to leptin resistance may thereforebe treatable with TRH-like peptides or analogs which havegreater in vivo stability and bioavailability than TRH itself(Pekary et al., 1999).

4. Experimental procedures

4.1. Animals

Male Sprague–Dawley (SD) rats (Harlan, Indianapolis, IN) wereused for all experiments. These animals were group housed(four animals per cage), maintained with standard Purinarodent chow #5001 and water ad libitum during a standard 1-week initial quarantine in a controlled temperature andhumidity environment; lights on: 6 am to 6 pm. All animalswereweighed on the day of receipt and on themorning of eachexperiment. Initial and final body weights did not differbetween experimental groups. Research was approved bythe VA Greater Los Angeles Healthcare System Animal Careand Use Committee and conducted in compliance with theAnimal Welfare Act and the federal statutes and regulationsrelated to animals and experiments involving animals andadheres to principles stated in the Guide for the Care and useof Laboratory Animals, NRC Publication, 1996 edition. Allefforts have been made to minimize the number of animalsused and their suffering. All animals were transferred fromthe Veterinary Medical Unit to the laboratory 12 h before thestart of experiments to minimize the stress of a novelenvironment.

4.2. Time-dependent effects of leptin on TRH and TRH-likepeptide levels in rat brain and peripheral tissues

Rats weighing 236±7 g were either uninjected (controls,n=4), or received a single 0.5 ml injection of recombinantrat leptin (Imgenex, San Diego, CA, 0.5 mg/ml sterile distilledwater) at 0.5, 1 or 2 h (n=4 for each time point) beforedecapitation.

4.3. Dissection of rat brain, pancreas and reproductiveorgans

Rats were decapitated. Nucleus accumbens (NA), amygdala(AY), frontal cortex (FCX), cerebellum (CBL), medulla oblongata(MED), anterior cingulate (ACNG), posterior cingulate (PCNG),

striatum (STR), pyriform cortex (PYR), hippocampus (HC),entorhinal cortex (ENT), pancreas, prostate, epididymis, andtestes were hand dissected, weighed rapidly, and thenextracted as previously described (Pekary et al., 2006a,b,c,d,2005, 2004, 2007, 2008).

4.4. Serum hormone assays

Serum rat leptin, rat insulin, corticosterone, testosterone,free T4, total T3 and glucose were measured with thefollowing commercial RIA kits: rat leptin and rat insulin(Linco Research, Inc., St. Charles, MO), corticosterone (MPBiomedicals, Aurora, OH), testosterone, free T4 and total T3

(DPC Coat-A-Count, Los Angeles, CA). Serum glucose wasmeasured with the OneTouch Ultra Meter (LifeScan, Milpitas,CA).

4.5. HPLC and RIA procedures, HPLC peak identificationand quantitation

HPLC and RIA procedures, peak identification, and quantita-tion by co-chromatography with synthetic TRH and TRH-likepeptides, relative potency analysis of multiple antibodies toTRH and TRH-like peptides, mass spectrometry and resolutionof overlapping peaks by least squares fitting of a 2-Gaussianstatistical model have been previously reported in detail(Pekary et al., 2007, 2005).

Briefly, after boiling, tissues were dried, re-extracted withmethanol, dried and defatted by water–ethyl ether partition-ing. Dried samples were dissolved in 0.1% trifluroacetic acid(TFA), and loaded onto reverse phase C18 Sep-Pak cartridges(Water, Milford, MA). TRH and TRH-like peptides were elutedwith 30% methanol. Dried peptides were again dissolved inTFA, filtered and then fractionated by HPLC using a4.6×150 mm Econosphere, 3 µm C18 reverse phase column(Alltech Associates, Deerfield, IL) and a 0.2%/min gradient ofacetonitrile. The 0.5 ml fractions collected were dried com-pletely and reconstituted with 0.15 ml of 0.02% NaN3 justbefore RIA.

The antiserum used (8B9) cross-reacts with TRH andeight TRH-like peptides with a relative potency of displace-ment ranging from 2.31 (Lys-TRH) to 0.288 (Ser-TRH) relativeto Tyr-TRH (Table 2, Pekary et al., 2004). Two of the regularlyobserved peaks (2a and 2b) consist of a mixtureof unidentified TRH-like peptides. Of the seven observedpeptides three have so far been confirmed by massspectrometry: TRH, Glu-TRH and Tyr-TRH (Pekary et al.,2005) and mass spectrometry of the remaining four isplanned. Tissue samples from the four rats within eachtreatment group were pooled prior to HPLC to provide theminimum amount of immunoreactivity needed for reliableRIA measurements.

The mean recovery of TRH and TRH-like peptide immuno-reactivity from all tissues studied was 84±15% (mean±SD).The within-assay and between-assay coefficient of variationfor measuring 333 pg/ml TRH was 4.8% and 16.9%, respective-ly. All HPLC fractions obtained from a given brain region orperipheral tissue were analyzed in the same RIA. Theminimum detectable dose for TRH was 5 pg/ml. The specificbinding of [1215I]TRH (Bo/T) was 25%.

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4.6. Statistical analysis

Statistical comparisons were made with the aid of Statview(Abacus Concepts, Inc., Berkeley, CA), a statistical softwarepackage for the Macintosh computer. All multi-group compar-isons were carried out by one-way analysis of variance usingpost 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-likepeptide combination, across four photoperiod intervals, hasbeen previously reported (circadian rhythm experiment) foruntreated Sprague–Dawley rats (Pekary et al., 2006c). Meanwithin-groupCVs inbrain ranged from6.3% for TRH levels inAYto 48% for Leu-TRH inACNG, and from3.1% forVal-TRH in testisto 21% for Tyr-TRH in pancreas. These CVs were then used toestimate the level of significance, by one-way ANOVA, ofchanges in thepooledmean values of TRHandTRH-like peptidelevels at each time point following ip injection of leptin. The SDformeasurements of TRHorTRH-like peptide (X-TRH) followingleptin treatment in a given tissue extract (T), SD (X-TRH,T), wasequal to CV (X-TRH,T; Pekary et al., 2006c) multiplied by themean for the corresponding X-TRH from Tables 1 and 2.

Acknowledgments

This work was supported by the Department of VeteransAffairs Medical Research funds (AEP and AS) and the PekaryFamily Trust.

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