Thyroid hormone modulation of the hypothalamic growth hormone (GH) releasing factor pituitary GH axis in the rat

Thyroid hormone modulation of the

hypothalamic growth hormone (GH)-releasing

factor-pituitary GH axis in the rat.

N Miki, … , T Aoki, H Demura

J Clin Invest. 1992;90(1):113-120. https://doi.org/10.1172/JCI115823.

Both thyroid hormone and hypothalamic growth hormone (GH)-releasing factor (GRF) facilitate pituitary somatotroph function. However, the pathophysiological role of thyroid hormone in GRF secretion is less well understood. Thyrotoxicosis, induced by

administration of thyroxine (T4) in rats, inhibited both pituitary GH levels and

immunoreactive GRF secretion from incubated hypothalamus. At the highest dose of T4 given for 12 d, GRF secretion and pituitary GH decreased by 50 and 39%, respectively. Hypothyroidism induced by thyroidectomy (Tx) enhanced GRF secretion approximately twofold while depleting pituitary GH by greater than 99%. Both of these hypothalamic and pituitary effects were reversed by replacement of T4 but not human GH for 7 or 14 d. Human GH was as potent as T4 in restoring decreased body weight gains or serum insulin-like growth factor-1 levels in Tx rats. These results indicate that at both physiological and pathological concentrations in serum, thyroid hormone acts as an inhibitory modulator of GRF secretion, probably not involving a feedback mechanism through GH. A biphasic effect of thyroid hormone on pituitary GH levels appears to derive from the difference in primary target tissues of hyper- and hypothyroidism, the hypothalamus and the pituitary,

respectively. Research Article

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Thyroid Hormone

Modulation

of the Hypothalamic Growth

Hormone

(GH)-releasing

Factor-Pituitary GH

Axis in the Rat

Nobuhiro Miki,MasamiOno,NaomiHizuka, Tomoko Aoki, and HiroshiDemura

DepartmentofMedicine, InstituteofClinical Endocrinology, Tokyo Women'sMedicalCollege, Tokyo 162,Japan

Abstract

Both thyroid hormone and hypothalamic growth hormone (GH)-releasingfactor (GRF) facilitate pituitary somatotroph function. However, the pathophysiologicalrole ofthyroid hor-mone inGRFsecretion isless wellunderstood. Thyrotoxicosis, induced byadministrationof thyroxine (T4) in rats,inhibited both pituitary GH levelsand immunoreactive GRF secretion from incubated hypothalamus.At the highest dose ofT4given for 12 d,GRF secretion and pituitary GH decreasedby50and 39%, respectively. Hypothyroidism induced bythyroidectomy

(T.)

enhanced GRF secretion approximately twofold while de-pleting pituitary GH by >99%. Both, ofthese hypothalamic andpituitary effectswerereversed by replacement of T4but not human GH for 7or 14 d.HumanGHwas as potent as T4in restoring decreased body weight gains or serum insulin-like growth

factor-i

levels in

_T.

rats.These results indicate thatat both physiological and pathological concentrationsin serum, thyroidhormone acts as aninhibitory modulator of GRF secre-tion, probably not involving a feedback mechanism through GH. A biphasic effect of thyroid hormone on pituitary GH levels appearstoderive from the difference in primarytarget tissues of hyper- and hypothyroidism, the hypothalamus and thepituitary,respectively. (J.Clin.Invest.1992.90:113-120.) Key words: hyperthyroidism-hypothyroidism*

somatotropin-somatotropin-releasingfactor *insulin-like growth factor-I

Introduction

Secretionofpituitarygrowth hormone

(GH)'

ispositively regu-latedby thehypothalamic peptide, GH-releasing factor(GRF) ( 1-3). GRF generates spontaneous burstsofGHsecretion (4-6)and also increases GHgenetranscription andGHmRNA levels(7, 8).Somatostatin (SRIF)is the hypothalamicpeptide

Portionsofthis work were presented in abstract form at the1st Interna-tionalCongressofNeuroendocrinology,San Francisco, CA, 9-1 1 July 1986, andatthe 8th International Congress of Endocrinology, Kyoto, Japan, 17-23 July 1988.

Addressreprintrequests to N. Miki, Department ofMedicine,

Insti-tuteofClinicalEndocrinology,Tokyo Women's Medical College, 8-1 Kawada-cho,Shinjuku-ku, Tokyo 162, Japan.

Receivedfor publication 23 September 1991 and in finalform 31 January1992.

1.Abbreviations used in this paper: BW, body weight; GH, growth

hormone; GRF, GH-releasing factor; hGH, human GH; HKRB,

Hepes-bufferedKrebs-Ringer bicarbonate solution;IGF-I,insulin-like growthfactor-I;SRIF, somatostatin;T3,triiodothyronine; T4,

thyrox-ine;TX,thyroidectomyorthyroidectomized.

thatinhibitsGHsecretion(9, 10) andmaybe also involved in theregulation ofGRFsecretion withinthehypothalamus (11, 12). Although GRF and SRIF playaprimaryrole in GH secre-tion, thereareotherimportanthormones that modulate pitu-itaryGHsecretion, probably througheffectsonthe GH-regula-torypeptides,GRFand/orSRIF. These includethyroid hor-mone, adrenal glucocorticoid, and gonadal steroid of peripheral endocrine organs (13). The most important for growth and development of mammals is thyroid hormone. Thyroid hormone stimulates synthesis andsecretion ofGH (14-16) and also increases GH gene transcription and GH mRNAlevels (17-19). HypothyroidismdepletespituitaryGH ( 14-16), inhibits GH secretion (20-22), and causes severe growthretardation(23).

Thus, boththyroidhormone and GRF directly stimulate pituitary somatotroph function,andtheirhypophysealactions aresimilarinmany aspects.However, the

pathophysiological

roleofthyroidhormone in

hypothalamic

GRFsecretionis less well understood. It appears more complex because GRF

re-ceivesanegative feedback signal fromGH

(24-27),

so

thyroid

hormone might influence GRF secretion indirectly through theeffectonpituitaryGH. Furthermore,

thyroid

hormone has a biphasic effect on growth rate. In immature animals and youngpatients, growth is retarded by

hypothyroidism,

is

re-storedbyreplacementdosesof

thyroxine

(T4),and is inhibited bythyrotoxic dosesof T4 (28).Weand others

previously

re-ported thatthyroidectomy

(T,)

enhanced invitro secretion of hypothalamic GRF in rats (26, 29). Whether this effect is caused by thyroid hormone deficiency per se or mediated through associatedGHdeficiency remainsunknown. Also

un-known is theeffect of hyperthyroidism onGRFsecretion.

Inthepresentstudiesinrats, wehavecompared effects of thyroidhormone excess and deficiency in vivo on

hypotha-lamicGRFsecretioninvitro. Toexaminethemechanism of action of

hypothyroidism,

we have

investigated

whether the

TX-induced

rise in GRFsecretion is reversibleby replacement of T4or human GH(hGH). We have also studied effectof hGH in combination with T4, because GHgivenwithT4 pro-motesthegrowthof

thyroid-deficient

animalsmorethan GH alone(23, 30).Inexperiments with hGH,serumlevelsof insu-lin-like growth factor-I

(IGF-I)

aswell asbodyweight

gains

weremeasured as anindex ofabiologicaleffectof hGH. Fi-nally,wehave characterizedGRF secreted in the mediumto

examine whetheranabnormalthyroidstatusaltersamolecular form ofGRF.

Methods

Chemicals

SodiumL-thyroxine (T4)wasobtained fromSigmaChemical Co.(St.

Louis, MO). Recombinant hGHwasprovidedby Sumitomo Pharma-ceutical Co. Ltd. (Osaka, Japan). Acetonitrile (CH3CN)and

2-pro-panolwereobtained from KantoChemical Co. Ltd.(Tokyo, Japan) ThyroidHormoneand GrowthHormone-releasingFactor 113

J. Clin. Invest.

©TheAmericanSociety for Clinical Investigation, Inc. 0021-9738/92/07/0113/08 $2.00

and trifluoroacetic acid(TFA)from Wako Pure ChemicalIndustries

Ltd. (Osaka,Japan).SyntheticratGRF waspurchasedfrom Peninsula

Laboratories,Inc. (Belmont, CA).

Animals and experimental protocols

MaleSprague-Dawleyrats wereobtainedfromJapan SLCInc.

(Hama-matsu City, Shizouka, Japan)at5 wkofage.Theywerehoused in a room undercontrolledtemperature(22±10C), humidity (50-55%)

andlight/darkconditions(lightson0800-2000h). Foodand water

were availablead libitum.

Hyperthyroidismwasinduced at 10-1 1 wk of ageby subcutane-ously injecting T4oncedailyfor6 or 12 d. Animalswereweighed daily

at1700-1730 h, and T4 wasgivenon adailyweight basisin dosesof25,

50,or100_,g/100 gbodyweight (BW).Anintermediate50,ug/100 g BW doseofT4 was the dose thatinhibitedBWgainbutdidnot reduce

pretreatment BW,accordingtotheresultsofapreliminaryexperiment.

T4 wasdissolvedin50%propyleneglycolanddiluted four timeswith 0.0033 N NaOH beforesubcutaneousadministration.Controlrats

re-ceived vehicle injection. The animalswerekilled by decapitation20 h

afterthe lastinjection. Hypothalamic tissueswereobtained for subse-quentincubation experimentsand anteriorpituitaries forGH

measure-ment.The trunk blood was alsocollectedinexperimentsinwhichT4 or

vehiclewasadministered for 12 d.

Hypothyroidismwas producedbysurgicalthyroidectomy

_(T.)

at6 wkofage. BecauseparathyroidectomyattenuatespulsatileGH secre-tion in rats(31),atleastthreeparathyroid glandswereleft,and

cal-ciumwas notsupplementedindrinkingwater.From 4 wkafter

opera-tion,

T,

rats werereplacedoncedaily witha"physiological"doseofT4

(1 or 2,g/100gBW)ortreatedtwicedailywith hGH (100ig/rat)for 7 d. hGH wasdissolved in physiological saline. Inthe next

experi-ments,

_T.

ratsreceived thesame treatmentsfor14d.Finally,we inves-tigatedwhether hGH could affectGRF secretionsynergisticallywith

thyroidhormone.TXrats weresupplementedwithT4 alone (2,g/100 gBW, oncedaily)orin combination withhGH(100ag/rat,twice

daily) for 7 d.T4wasinjectedsubcutaneouslyat 1730-1800 h and hGH at0830-0900hand 1730-1800 h.ControlTXandsham-operated

rats were treatedwith vehiclesfor T4, hGH,orboth. Theanimalswere

decapitated 18 hafterthe lastinjection;and thehypothalamus, ante-riorpituitary,and trunk blood were collected.

Hypothalamic

incubation

Themethodof hypothalamic incubationhas beendescribed elsewhere (24).Inbrief,thebrainwasobtainedafter cuttingthepituitarystalks

carefully,and thehypothalamiwere cutsagittally throughthemedian eminence withthe ventralsurfaceup. The areabetweentheposterior

borderof theopticchiasma, theanteriorborder of themammillary body,the lateralhypothalamic sulcus,and 2 mmdeep fromthe ventral

surfacewasdissected intotwofragments.Thefragmentswereplacedin

polyethylene vials containing0.6 ml of 11 mMHepes-buffered

Krebs-Ringerbicarbonate solution (HKRB),pH7.40,which waspregassed with95%02-5%CO2and supplementedwith5.5 mM glucose,0.1% BSA,and500 mg/mlbacitracin.Fourhypothalamicfragments from

two rats wereincubatedin one vial. Allincubationswere 30 min at

37°Cunder an atmosphere of 95%02-5%CO2in a shaking water bath (60cycles/min).Afterpreincubation,the tissueswere incubatedin HKRB (basal secretion) and then in 50 mMK+-containingHKRB

(K+-stimulated secretion). The medium was collected every 30min

and replaced with fresh buffer. The collected medium wascentrifuged

at 3,000 rpm for 5 min at4°C, and thesupernatants were quickly frozen on dry ice and kept at-20°Cuntilassayed.Eachhypothalamic

incubation used 18-24 animals, and asecretionexperiment consisted of two to three separateincubations.

Tissue

extraction

Immediately afterthehypothalamic incubations,fourhypothalamic fragmentswerepooled andGRF in the tissueswasextractedas de-scribed_(24).Thefragmentswereboiled for7min andhomogenized by

polytroninIN acetic acid and 0.02 N HCIcontaining10 mMEDTA,

1 gg/mlpepstatin,and 0.01%0-mercaptoethanol.Thehomogenates werecentrifugedat 10,000g for 30 minat4VC,and thesupernatants

were frozen in adryice-methanol bath, lyophilized, and stored at

-20'Cuntilassayed.The anteriorpituitarieswerehomogenized by

polytronin 0.05 MNaHCO3-Na2C03buffer, pH 9.96. The

homoge-natesweresupplementedwithanequalvolume of0.01 M PBS

contain-ing 1% BSA, 0.1% TritonX-100, and 25 mM EDTA and storedat

-20'CuntilassayedforratGH.

Reverse-phase

HPLC

A total of 10-12hypothalamiofhyper-orhypothyroidratsand their controlswereincubated inadepolarizing (50mMK+)condition for

twosuccessive30-min periods.The medium(6.0-7.2 ml)waspooled,

acidifiedbythe addition of 0.3 M TFAto afinal concentration of 0.06

M,and extractedondisposableSep-Pak C18 cartridges (Waters

Chro-matographic Division, Millipore Corp.,Bedford, MA)asdescribedby

Frohman and Downs(32).The cartridgeswerepreactivated by

succes-sive4-mlwashes of 0.01 M TFA, 80% (vol/vol) CH3CN/20%(vol/ vol)0.01 MTFA,and0.01MTFA. After the samples were applied, the

cartridgeswerewashed with 4 ml of 0.01 MTFA,and GRFwaseluted with2 ml of 80%(vol/vol)CH3CN/20% (vol/vol) 0.01 M TFA. The eluantwasfreeze-dried and reconstituted inatwentieth theoriginal

medium volume of0.01 M TFA for HPLC analysis. The HPLC system (WatersChromatographic Division,MilliporeCorp.) consisted of600 E multisolvent delivery system, U6K universal liquidchromatography injector,and programmable multiwavelength detector.Reverse-phase

HPLC columnwasVydac218TP54(C 18,0.46X25 cm, 5-,Amparticle size) (The Separationgroup,Hesperia, CA). Solvent A was 0.1%(vol/

vol) TFA and solvent B was 80% (vol/vol) CH3CN/20%(vol/vol)

2-propanol/0. 1% (vol/vol) TFA. The samples were eluted with a

lin-eargradient of 25-45% (vol/vol) solvent B in 32 min and then 45-80% (vol/vol) solvent B in 28 min. Flow rate was 1 ml/mmin. -ml fractions

werecollected, lyophilized, and assayed for GRF. Blankinjections

werecarriedoutbefore each sample injection, and fractions collected didnotcontain any detectable GRF-like immunoreactivity. The final recovery ofsyntheticGRFwas- 64%.

RIAsforratGRF, GH, T4, and IGF-I

GRF in the medium and tissue samples was determined by RIAas

described elsewhere (24). The minimum detectable dose(BO-2SD)

was 2pg/tube, with half-maximaldisplacementof22-25 pg/tube. The nonspecific binding was < 2%. The samples were measured directly

without extraction or concentration. In assays ofthe medium samples, HKRB buffer was added to all standard curves in a volume of 100-200

tL equivalent to that ofthe unknown. The total incubation volumewas

500 ,u.PituitaryGHcontents weremeasured induplicate byaspecific ratGH RIA (5, 12,24) at two dilutions using materials provided by the Rat Pituitary Hormone DistributionProgram, National Institute of Diabetes and Digestive andKidneyDiseases(NIDDK)National Insti-tutesof Health. Results wereexpressedintermsof NIDDKratGH RP-2reference standard. Serum T4 levels were determined byadouble antibody method with a RIA kit purchased from Eiken ICL(Tokyo,

Japan). The sensitivity of the T4 assay was 1Ag/dl.Serum IGF-I levels

weremeasuredbyRIAasdescribed(33),using biosynthetichuman IGF-Iasstandards andanantiserum notcross-reactive with IGF-II. Serum samples were extracted by the aceton-formic acid method of Bowsher et al. (34). The least detectable level of IGF-I assaywas 10 pg/tube.

Data analysis

Resultswereexpressedasmeans±SE.Analysisofvariancewasused for statisticalanalysis. RelationshipbetweenserumT4and GRF secretion inhyperthyroidrats wasanalysedusinglinearregression analysis. Sub-sequent toanalysis ofvariance,thesignificanceof differences between experimental groups was determined by theStudent-Newman-Keuls

?

50

40

~

0

30

dered

6

a

3

₁

0

50

100

0

25

50

100

14

Dose

(pg/b10g BW/Day)

Figure1. GRFsecretionfrom hypothalamic fragmentsofrats

ren-deredhyperthyroid byadministration of 14 or6(left panel)or12 d

(rightpanel). Closed and open columns indicate basal and 50 mM

K+-stimulatedGRF release,respectively.Twohypothalamiwere in-cubated in onevial,and the secretion of GRF wasexpressedas

pico-grams (means + SE) perhypothalamus/30 min. The numbers of

ob-servationswereshownonthe top of closed columns. *P<0.05, **P

<0.01,***P<0.001vs. control indicated by a zero doseofT4.

Results

Effects ofhyperthyroidism

on

hypothalamic

GRF, pituitary

GH, and

serum T4

Fig. 1 shows the results of secretion experiments in hyperthy-roidrats. Asshown intheleft panel, administration ofT4for6 d hadnoeffecton basalGRFsecretion (closed columns)but

significantly inhibited K+-evoked GRF secretion (open col-umns). Theextent of GRF suppressionwas notdifferent be-tween two doses ofT4: -28.9% (P < 0.05) by 50

_qg

and

-31.3% (P<0.01) by 100

jig

T4/ 100g perd.Administration ofT4 for 12dinhibitedGRFsecretion (right panel) and in-creasedserum T4 levels(Table I).Botheffectsweredose

re-lated, and theinhibitory effectonGRF secretionwasgreater than that observed after 6 d of T4 administration. Basal GRF secretion was inhibited below the assay detection limit (P

<0.01)by both of the 50 and 100 ug/ 100gdoses.K+-evoked GRF secretion was suppressed by 10.3% (NS), 27.3% (P <0.0l),and5o.1%(P<0.00l)bythe25,50,and

l0,g/100

gdosesofT4,respectively. Linear regression analysisshoweda

significant negative relationship(r= -0.77,P< 0.0001) be-tween serumT4 levels and K+-evoked GRFsecretion (datanot

illustrated).

The T4-induced suppression ofGRFsecretion after 12 d wasassociatedwith aparallel dose-related decline in pituitary GH(TableI).Thedeclinein GHwassignificantat an interme-diate doseofT4(- 18.5%,P<0.01), and the minimum GH levelatthe highest dosewas60.7% (P<0.001) ofthecontrol

value. T4 at thehighest dose also reducedpituitaryGHafter6 d, butto alesserextent(-10.6%, P< 0.05)than after 12 d (TableI). HypothalamicGRFcontents werediminishedonly afteradministration ofthe highestdosefor 12 d (-16.4%, P

<0.05)(Table I).

AtthebeginningofT4 treatmentfor 12 d,mean(±SE)BW of allanimalswas378±5g(n=90), and those aftertreatment were412±4 (n = 26), 399±6(n = _{14), 366±7 (n} = _26),or 329±6g(n =24) (P<0.001 vs.initial weight)inratsgiven0 (vehicle), 25, 50,or

100,ug

T4/100gperd,respectively. After administration ofT4 for 6 d, theinitial BW wassignificantly decreased alsoatthehighestdoseonly(-7.6%,P<0.01).

Effects

of hypothyroidism and

T4 or

hGH replacement

on

hypothalamic

GRF secretion in vitro

As shown in Fig. 2,

Tx

significantly enhanced both basal (closed columns)andK+-stimulatedGRFsecretion(open col-umns)at5 wkafteroperation. ReplacementofT4for7dat2 ,ug/100 g per d partially reversed the

_T,-induced

rise in

K+-stimulatedGRFsecretion(-53%,P<0.01), whereas the enhanced basal GRF secretionwas reversed (P < 0.01) to a

level statistically indistinguishable from that of the

sham-Tx

rats(leftpanel). However, hGHtreatmentfor7dat200

jig/

rat perd hadnoinhibitory effectontheenhanced GRFsecretion either basallyorunderK+ stimulation in

Tx

rats(rightpanel).

Tx

rats

weighed

30% less than sham

_Tx

controlsat4wk after

TableI. Serum T4, PituitaryGH,and HypothalamicGRFLevelsinHyperthyroidRats

Number

Treatment Dose Length of rats SerumT4 Pituitary GH Hypothalamic GRF

Alg days sg/dl ug/gland pg/tissue

Vehicle 6 12 ND 459±15 858±53

T4 50 6 12 ND 459±23 817±47

T4 100 6 12 ND 410±22* 902±23

Vehicle 12 26 5.4±0.1 453±22 719±23

T4 25 12 14 12.7±0.5§ 408±20 775±41

T4 50 12 26 15.4±0.8§ 369±16t 781±32

T4 100 12 24 18.2±0.6§ 275±12§ 601±27*

Results are means + SE. ND, notdetermined. DosesofT4 are expressed asqg/100g bodyweight/day. Tissue GRF levels were measured by

poolingtwohypothalamiandareexpressedaspicogramsper onehypothalamus. *P<0.05, *_{P <0.01,} §P<0.001 vs. vehicle.

1001

80

E

~60

-c

40

C 0

1120

6

6

U

66

Sham

Tx

Tx

Sham

Tx

Tx

Tx

T4

Tx

GH

Figure 2. Effects ofthyroidectomy (Tx)and replacement of T4(left panel)orhGH(rightpanel)for7d onGRFsecretionfromincubated hypothalamicfragments.Tx rats were treatedoncedailywith T4 ( 2

yg/100g)ortwicedailywith hGH(100jig/rat)beginningat 4 wk

afterTx.Closed and open columnsindicatebasal and 50 mM K+-evokedsecretion,respectively. The results were expressed as

pico-grams(means +SE)ofGRF secretedfromonehypothalamusper30 min.The numbersof observationswereshown on the topofclosed columns. *P<0.05,** P<0.001vs.sham

_T,.

tP<0.01 vs. Tx.

operation. hGH increased theirBWsat a rate comparable to thatofT4. During7dof treatments, the mean (±SE) weight gainof sham

Tx,

T.,

or

_Tx

+T4 groups was

29.2+2.3,

4.7+2.5,

or 22.5±1.4 g/rat, and that ofsham

Tx,

Tx, or

Tx

+ hGH groups was 33.5±3.1, 4.2±1.5, or 25.2±2.7 g/rat (n = 12 each), respectively.

Tx

rats were thenreplaced with T4 or hGHfor14d at the same dailydose (Fig. 3). T4 reversed the

Tx-induced

risein K+-stimulated GRFsecretion by 76% (P<0.01). Although

v _Sham

Tx Tx

Figure 3. Effects of

replace-mentof T4orhGH for14 donGRFsecretion inTX

rats.T4wasgivenonce

dailyat2,ug/100 g and hGHtwicedailyat100 ,ug/ ** ratbeginningat4wkafter

TX.ShamTXand controlTX

rats weretreated with

vehi-*+ clesfor both T4 and hGH.

Closedand open columns indicate basal and 50 mM K+-evoked GRFsecretion,

6 respectively. The secretion of GRF was expressed as

Tx - ** picograms(means

+ _SE)

Tx -

_per

hypothalamus/30

min.

Tx Tx _ *P<0.01, **P<0.001vs. T4 GH shamTx.tP<0.01vs.Tx.

notcomplete, this reversibility was greater than that observed after 7 d (Fig. 2). T4 suppressed the enhanced basal GRF secre-tion completely (P < 0.01 ), as it did after 7 d. At a half daily dose of 1

jtg/

100 g, T4 replaced for 14 dwas also effective, though to a lesser extent, in inhibiting the enhanced K+-evoked GRF secretion (data not shown). In contrast, hGH did not significantly reduce either basal or K+-stimulated GRF secretion in

TX

rats. However, hGH restored their diminished weight gains to normal. During 14 d of treatments, themean (±SE) weight gain of sham

Tx,

T1,

Tx

+ T4, or

Tx

+ hGH groups was48.8±5.0, 5.3±1.2, 49.4±2.8, or 51.1±2.8 g/rat (n = 12-16), respectively.

The thirdexperiment with hGH was carried out to study the effect ofcombined hGH and T4 replacement in

Tx

rats (Fig. 4).The doses of T4 and hGH were the same with those shown inFigs. 2 and 3, and the length of treatments was 7 d. T4 alone partially reversed the enhancedK+-evokedGRF secretion (P <0.01)whilenormalizingtheincreased basal secretion. hGH incombination withT4similarlyreversed theenhanced K+-evoked GRF secretion significantly (P<0.01), but the level reversedby the combined hGH and T4 therapy was statistically not different from that attained by T4 alone. hGH and T4 re-placedtogether increased the mean (±SE) weight gainof

Tx

ratsfrom 3.0±2.1 to 33.4±0.9 g/rat during 7 d of treatment, which was significantly greater than that of sham

_Tx

rats (26.7±3. 1,P<0.05)

orT4-treatedTx

rats(21.6±3.5,P<0.01) (n = 12 each).

Serum T4, serum

IGF-I,

pituitary

GH,

and

hypothalamic

GRF levels in

hypothyroid

rats

and

effects

of

T4 or

hGH

replacement

Asshown in Table II,

Tx

reduced serum T4to undetectable levels(< 1 ,g/dl) (P<0.001),serumIGF-Iby 36.5-38.6% (P <0.001),andhypothalamicGRF contentsby25.7-34.3% (P <0.05-0.01). Replacement ofT4for7and 14d restored the decreased serum T4 and hypothalamicGRF to levels higher than those in sham

Tx

rats, whereas hGH treatment was with-outeffectoneither parameter. The decreasedserumIGF-I lev-elsweresignificantlyrestoredbyT4 (P<0.01 vs.

_Tj)

to74.3 and 85.6% of the value of sham

Tx

animals after7and 14d, respectively. hGH treatmentfor7dhad noeffectonthe

Tx-in-100 Figure 4. Effects of

replace-.' mentofhGH in

combina-E _**

tion with T4 for 7

d'on

GRFsecretioninTXrats.

8

The dosesof T4and hGH E

werethesamewith those

° 60 **+ shown in Figs. 2 and 3: T4,

QT1 *t 2

_12g/

100g perd ina_single T1 dose and hGH, 200 ,g/rat

I--,

perd intwodivided doses.

.0 ShamTXandcontrolTX

H animals received vehicles

°)

20 ii 6 ~ for bothT4and hGH.

1 * 6 6 Treatments started at 4 wk

0ix

*L-

6 - + after T.. Closed and open

*_-columnsindicate basal and

Sham Tx Tx Tx 50 mMK+-evoked GRF

TX T4 T4 +GH secretion, respectively.

Thesecretion of GRFwas

expressedaspicograms (means+SE) per hypothalamus/30 min. *P<0.01, **P<0.001 vs.shamTx.tp<0.01 vs.Tx.

116 N.Miki,M.Ono,N.Hizuka, T.Aoki,and H.Demura

- 80

._C

E

1-U)

E 60

s

-C

z40

g 20

0

u) lL

vx

**

Table II. Serum T4, SerumIGF-I,andHypothalamicGRF Levels in Thyroidectomized(Tx)Rats Treated with T4 orhGH

Number

Group Length ofrats Serum T4 Serum IGF-I Hypothalamic GRF

days ug/dl ng/ml pg/tissue

ShamTX 7 12 5.9±0.2 441±6 803±35

TX 7 12 <1.0 280±8§ 596±58*

TX+T4 7 12 8.6±0.7*1 328±7*1 1068±481

ShamTX 7 12 5.6±0.1 391±14 726±65

TX 7 12 <1.0 243±7§ 501±24*

TX+hGH 7 12 <1.0§ 261±8§ 543±52*

ShamTX 14 16 6.1±0.1 396±14 1023±68

TX 14 12 <1.0§ 243±9§

672±740

TX+T4 14 12 8.9±1.211 339±15*' 1359±37*1

TX+hGH 14 12

_<1.0§

329±8$' 734±85*

Resultsarethemeans± SE.Tissue GRF levelsweremeasuredby poolingtwohypothalami andareexpressedaspicograms perone hypothala-mus. *P<0.05, tP<0.01, §P<0.00Ivs.shamTX. IP<0.01, 'P<0.001 vs.TX.

duceddecrease inserumIGF-Ilevels. However,hGH given for 14dincreasedit significantly (P < 0.001 vs.

TX)

to 83.0% ofthe euthyroid value, whichwas comparable to that in hypothyroid rats treatedwithT4 forthe same duration.

TX

depletedpituitary GHcontents to < 1% ofthat of euthy-roidrats(P<0.001)at 5-6 wkafter operation,and the reduced pituitaryGH wassignificantly(P <0.001)reversed by T4 (Ta-ble III). Thisreversibilitywas greater byreplacement of T4 at a higher dose (2vs. 1 ,ug/100 g per d) when the length of treat-mentwas same, or by replacementfor a longer duration ( 14 vs. 7d) when the doseofT4 was same.Withthereplacement of T4 at 2,ug/100g perd for 14 d, the decreasedpituitary GH was restored tothemaximumlevelof 82% of that of sham

TX

rats, although this level remained significantly lower (P < 0.001) than theeuthyroidvalue.

Characterization

ofhypothalamic

GRF in

the medium

Fig.5showsreverse-phase HPLCelution profiles ofimmunore-activeGRFinhyper- and hypothyroid animals and their

con-TableIII. Effectsof Thyroidectomy

(TxJ

and Replacementof T4 orhGHonPituitary GH Contents

Number

Group Treatment Dose Length of rats Pituitary GH

Ag days Mg/gland

ShamTX Vehicle 7, 14 80 393±5 (100)

TX Vehicle 7, 14 64 2.1±0.1(0.5)

Tx T4 1 7 14 142±16*₍₃₆₎

Tx T4 2 7 12 183±11*(46)

Tx T4 1 14 14 243±12*(61)

Tx T4 2 14 12 324±10*₍₈₂₎

Tx hGH 200 7 12 1.7±0.4(0.4)

Tx hGH 200 14 12 2.8±0.6(0.7)

Results are means±SE; values in parentheses arepercentages.

Pitu-itaryGHcontentsin shamTxorTxrats treatedwith vehicle for 7 or

14dayswerecombined. *P <0.001 vs. shamTx+vehicle orTx+

vehicle.

trols, which wassecreted in vitro inresponse to 50 mMK+. Rats weregivenvehicleorT4at100,ug/ 100g perdfor 6 d(left panels),orreceived sham

TX

or

_T.

5wkago(right panels). In all ofthese groups, a major proportion of immunoreactive GRF was eluted ata position identical to that of synthetic GRE. This indicates thatbyHPLCcriteria, ratGRF secreted into the mediumwasindistinguishable fromsynthetic peptide.

A smallportion ofGRF-like immunoreactivity also was de-tectedatlaterfraction(s)than that of authenticratGRF,but the natureofthese smallimmunoreactive peaks is unknown. Anelutionprofile of GRF of

TX

ratstreated with T4(2

,gg/

100 g perd)orhGH(200,ug/ratperd)for7dwassimilartothat of sham

TX

or

TX

rats(datanotillustrated).

Discussion

Boththyroidhormone andGRFarethephysiologicalfactors thatactdirectlyatthepituitary somatotrophand facilitate its functionalactivity.Their actionsweresimilar inmany aspects, which include stimulation ofGH genetranscription, mRNA levels,biosynthesis,andsecretion (4-8, 14-19).Theresults of thepresentstudiesdemonstrate thatthyroidhormone also af-fectsthesecretion of hypothalamic GRF, theGH-regulatory peptide.Theeffects of thyroidhormoneexcessanddeficiency

onGRF secretionwereopposite,and therewasaninverse

rela-tionshipbetweenserumT4 levels and GRF secretion. This find-ing clearly indicatesaninhibitory role ofthyroidhormonein GRF secretion. Characterization ofGRF in the medium by reverse-phase HPLC revealed that the molecular form of GRF peptidewasnotaltered byanabnormalthyroidstatus.

Theinhibition of GRF secretion by thyroidhormone oc-curs atphysiological concentrationsin serum.Thisisindicated bythefindingsthatthyroidgland ablation enhancedGRF

se-cretionand thatthis effectwashighlyreversible by replacement of T4atphysiologicaldoses.Also,atthyrotoxic doses,T4 sup-pressed GRF secretioninnormalrats.Theseeffects ofthyroid hormone status onGRF secretionareprobablynotsimplythe result ofweightchanges or altered food intake. First, hyper-andhypothyroidism similarlydiminished BWs, while exhibit-inganopposite effectonGRFsecretion. Second,theinhibition ofGRF secretionbythyrotoxicosiswas alreadysignificantat

c

0

U _

m

L-

ck:-1 10 20 30 40 60 1 1 0 20 30 40 60 Fraction (number)

theintermediatedoseofT4,whichdidnotreduceinitialBWs

ofanimals.Third, hyperthyroidismpromotesand hypothyroid-ismdepressesfood intake(35), whereasfood deprivation de-creasesGRF mRNAlevels (36) and GRFsecretionin therat (unpublished observation). Therefore, if thyroid hormone actedbyaltering food intake,it would have been the hypothy-roid but not hyperthyroid rats who showed the diminished GRFsecretion.

Consistent with a modulatory role in GH secretion, the effectthatthyroidhormone exertedonGRFsecretionwasofa delayed type at both physiological and thyrotoxic doses. Al-though the decreased GRFcontentsinhypothyroid rats were

fullyrecoveredafter7dofT4replacement, the enhanced GRF secretionwas notcompletely reversedafter either7 or 14dof replacement. T4 was withouteffect on GRFsecretion when replacedfor4d in

TX

rats(26). Thesuppression ofGRF

secre-tioninhyperthyroidrats wasgreaterafteradministration ofthe highestdoseofT4 for 12than 6 d, and the dose-response rela-tionship becameapparentafter12d. These resultssuggestthat theeffectiveness ofthyroidhormoneonGRFsecretion is de-termined by the duration of itstreatment aswellasthe dose administered.

Itis well establishedthathypothyroidism causes a severe

depletion ofpituitary GH ( 14-16).Assoon as6 hafter replace-ment, T4 can start to increase the diminished GH (16), whereaslittle isknown on aprecise timecourseofits recovery toeuthyroid level. Thepresentstudies givesomeinformation on thisissuebyreplacingT4 atphysiological dosesof1 or 2

,g/ 100g,eachfor7and14d. However, replacement ofT4 for

14dwas notsufficientto recover thediminishedGH content completely. This is consistent witha morphological studyof DeFesi et al. that 17-24 d oftriiodothyronine (T3) replace-ment wasrequiredtonormalize decreasedsomatotroph num-ber in

TX

rats(37).In contrast,diminishedGH secretion inTX

rats was normalizedwithin 4 dafter initiation of T4 replace-ment(22).Thesefindings,togetherwiththe present results on

GRF, indicate that it takes > 14 d for replacement of T4 to

Figure 5. Reverse-phase HPLC of a immunoreactivehypothalamicGRF

0 secreted invitro fromhypothalami * of hyper- and hypothyroid rats and

E their controls.Leftpanels, elution

0

a} profiles of GRF ofratsadministered

< with vehicle

(upper

panel)orT4

(100,ug/100 g per d)(lowerpanel) for 6 d.Rightpanels, elution profiles of GRFofsham Tx (upper panel) orT1 (lowerpanel) rats5wkafter

operation.Arrowsindicate elution positions of syntheticratGRF. Re-sults wereexpressed as

picograms/hypothalamus per 30 min perfraction. Brokenlines indi-catethedetectionlimit.

normalize the whole ofthe altered hypothalamic GRF-pitu-itaryGHaxisinhypothyroidrats.

In additionto depleting pituitary GHcontents, hypothy-roidism also abolishes spontaneous GH secretion (20-22),

suppressesGHresponse toGRF(38-40), andcausesgrowth retardation (23). Qualitatively similar effects can be repro-ducedby GRF antibodies(4-6,41)oritsantagonists (42, 43). Thesefindings initiallyledustothehypothesisthat hypothy-roidism might cause somatotroph failure, at least in part, throughinhibiting hypothalamicGRF secretion.By observing

adecrease in GRFcontentafter

_TX

anditsrapidrecoveryby replacementofT4, Katakami et al.(22)also have suggested thatacriticalroleofthyroidhormone in GHsecretion is re-latedtoits effectonhypothalamicGRF. Later, weandothers demonstrated that GRF secretion in vitrowasrather enhanced inhypothyroid, GH-deficientrats(26, 29).We conclude, there-fore,that thepituitary somatotroph failureinhypothyroidism is not causedby GRF deficiencyand so most probably isa

directconsequenceofthyroidhormonedeficiency.Thesame

conclusionwasobtained also inaninvivostudy withneonatal hypothyroidrats(44).

Theeffectofhyperthyroidism on the hypothalamic-pitu-itaryGH axis has been less clear. Thereissome evidenceto suggestthat

thyrotoxicosis

hasaninhibitoryinfluenceonGH. Hyperthyroidism causes amoderatedeclineinbothpituitary GH andBWsin the neonatal rat(45,46).Physiological GH secretionwasdiminished in humansubjectswith

thyrotoxico-sis andwasnormalized aftertheybecameeuthyroid (47).The present studies have demonstrated that hyperthyroidism in-hibits GRFsecretion, concomitantly decreasingbothBWs and pituitary GH in the adultrat. Thisfinding, together with the report of Clark and Robinson (48) that GRF accelerates growth and increases GH content in rats, indicates that the decreased invitrorelease of GRF in

hyperthyroid

ratsisnot

dueto anincreasedturnover rateof GRF in vivo.The

parallel

decline in GRFsecretion,GHcontent, and

possibly

GH

tissue ofhyperthyroidism in GH secretion. Thisview is sup-ported bythe finding that pituitary GH responsivenesstoGRF invivo was notaffected or only minimally decreased in hyper-thyroid rats (49, 50). Thus,atpathological conditions, thyroid hormone appears to negatively influence pituitary somato-troph functionby two pathways: oneinhyperthyroidism via the hypothalamus and the other in hypothyroidism leading directly tothepituitary. Thisdifference in the primarytarget tissue could explain biphasic effects of thyroid hormone on growthrate(28) andpituitaryGHcontents.

At a supraphysiological concentration of 1.55 X 10-6 M, theaddition of T3 stimulateshypothalamic SRIFsecretion in vitro (51 ). This effect is inhibitory for pituitary GH secretion, as wastheeffect thatthyroidhormoneexcessexertedonGRF inthe presentstudies. Hyperthyroidism may depresspituitary GH secretion through adualeffectonGRF and SRIF in the hypothalamus. ModulationofSRIF bythyroidhormone defi-ciencyisconflicting. Hypothalamic SRIF secretion in vitro is reported to be decreased (51), unaltered (21), orenhanced (29) inhypothyroidrats.Thismay be due in parttothe

differ-ence in theinduction time orlength ofhypothyroidism orin the methodofoperation, thyroidectomyvs. thyroparathyroid-ectomy. Anyway, thesediscrepantstudiesseemtoagreeonthat hypothyroidrats cancontinueto secrete aconsiderable propor-tion of SRIF, if itscapacityisevendecreased.However, it re-mains unknown whether SRIF inhibition of GHsecretionisof anysignificanceinseverehypothyroidism,inwhich GH is vir-tually absent in the bloodstream.

With replacement ofhGH, we could not reversethe en-hancedGRFsecretion associatedwith GHdeficiencyin hypo-thyroidrats.Atthe dose anddurationused, hGHwasaspotent asT4 inrestoringdecreasedbodyweight gainsof

_T.

rats.hGH also restored thediminishedserumlevelsof IGF-I,amediator ofanabolic effects of GH(52). Theeffect ofhGHon IGF-I induction wascomparableto thatofT4after 14dof replace-ment, although not significant after 7 d. This finding agrees wellwith the earlier reports on hypothyroid adult rats treated withbovineGHfor7d(53)orhGHfor 14d (54). Further-more, unlike the effect on growth (23, 30, 55), a combined hGH and T4 therapywas not moreeffectivethan T4 alonein inhibiting GRF secretion in hypothyroid rats. These results suggest that the increased GRF secretion inhypothyroidism is

notmediated through the feedback mechanism involving ei-ther GHorIGF-Ideficiency.

Thereis biochemical evidence to support that thyroid hor-mone actsdirectlyatthe levelof the hypothalamus. The con-version of T4toitsactive metabolite T3 is an important step in mediatingtheaction of thyroid hormone on its nuclear recep-torand has beenconsideredthemainsourceofT3in thebrain (56,57). Type II 5 ' -monodeionase isanenzyme that deiodin-atesT4toT3 in the brain (56), and the activity of this enzyme is highest in the arcuate nucleus and median eminence in brains of hypothyroidrats (58). Because thesehypothalamic regionspossessbothcellbodiesand nerveterminals ofthe

hy-pophysiotrophic GRFneurons(59, 60), T3 generated locally in thehypothalamus could directly influence the functional activity of GRF neurons. This viewmay be supported by the

significantcorrelationbetween serumT4levelsand GRF secre-tionthat weobserved inhyperthyroidrats. Alternatively, thy-roid hormone may act indirectly through altering metabolic and/orotherfactors ofthe centralorperipheral origin.

Other laboratories have

recently

reported

that

hypotha-lamicGRFmRNAlevels inratswereincreased in

hypothyroid-ism

(26, 27,

61

)

anddecreased in

hyperthyroidism (61 ).

This

negative relationship

betweenGRFmRNAand

_thyroid

func-tionissimilartothat whichwefoundbetween

GRF

secretion

andserum

T4.

Intheir studieson

hypothyroid

rats,

however,

GH showed an

_inhibitory

effect on GRF mRNA

levels,

whereasweobserved that GHwaswithouteffecton

GRF

se-cretion.Theincreased GRFmRNA levelswere

completely

re-versed

by

5 dofratGH

replacement

in adult

_hypothyroid

rats, andratGHwas morepotentthan

T4

(26).

Itwasalso reversed

by

hGH in

prepubertal

hypothyroid

rats,

although

14d of

re-placement

was

required

for

complete

reversel

(27). Thus,

thy-roid hormone modulates GRFmRNA levels and

_peptide

se-cretioninasimilar

inhibitory

mode.

However,

the

_underlying

mechanismsare

probably

different,

involving

the GH feedback for GRFmRNAbut notfor GRF

peptide

secretion.

Alterna-tively,

GRF secretion

might

be lesssensitivethan

GRF

mRNA

to the GH feedback

signal

orcould

respond

toGH adminis-tered ina

physiological

pulsatile

pattern

only.

Acknowledgments

Wethank the NationalInstituteof Diabetes and

_Digestive

and

_Kidney

Diseasesforsupplyingmaterials forratGH RIA andSumitomo

Phar-maceuticalCo. Ltd. for the generous

gift

ofrecombinanthGH. This work was supported in _partby a _{grant from} the Growth ScienceFoundation.

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