Measurement of Growth Hormone-releasing Hormone and Somatostatinin Hypothalamic-Portal Plasma of Unanesthetized SheepSpontaneous Secretion and Response to Insulin-induced Hypoglycemia

Lawrence A. Frohman, Thomas R. Downs, lain J. Clarke, and Gregory B. ThomasDivision of Endocrinology and Metabolism, Department of Medicine, University of Cincinnati College of Medicine,Cincinnati, Ohio 45267; and Medical Research Center, Prince Henry Hospital, Melbourne, Australia

Abstract

To elucidate the role of growth hormone (GH)-releasing hor-mone (GRH) and somatostatin (SRIH) in the regulation of thegrowth hormone (GH) secretory pattern, we collected portalblood from five unanesthetized ovariectomized ewes for re-peated measurements of GRHand SRIH simultaneous withthose of peripheral GH. Hormones were measured at 10-minintervals for 5.5 h and their interrelationships analyzed. Meanportal GRHwas 20.4±6.7 (SD) pg/ml and the estimated over-all secretion rate was 13 pg/min. GRHsecretion was pulsatilewith peaks of 25-40 pg/ml and a mean pulse interval of 71min. Mean portal SRIH was 72±33 pg/ml and the estimatedoverall secretion rate was 32 pg/min. SRIH secretion was alsopulsatile with peaks of 65-160 pg/ml and a mean pulse inter-val of 54 min. The GHpulse interval was 62 min. A significantassociation was present between GRHand GHsecretorypeaks though not between GRHand SRIH or SRIH and GH.Insulin hypoglycemia resulted in a rapid and brief stimulationof SRIH secretion followed by a decline in GHlevels. No effectwas observed on GRHsecretion until 90 min, when a slightincrease occurred. The results suggest (a) the presence of anindependent neural rhythmicity of GRHand SRIH secretionwith a primary role of GRHin determining pulsatile GRHsecretion, and (b) that the inhibitory effects of insulin hypogly-cemia on GHin this species are attributable to a combinationof enhanced SRIH secretion and possibly other factors, thoughwithout significant inhibition of GRH. (J. Clin. Invest. 1990.86:17-24.) Key words: growth hormone-releasing hormone-somatostatin- growth hormone - hypothalamic-portal - sheep

Introduction

Growth hormone (GH)' secretion occurs in an episodic man-ner characterized by pulses of relatively short duration inter-spersed with troughs during which time circulating GHlevels

Address correspondence and reprint requests to Lawrence A. Froh-man, M.D., Division of Endocrinology and Metabolism, University ofCincinnati Medical Center, 231 Bethesda Avenue, ML#547, Cincin-nati, OH45267.

Receivedfor publication 5 September 1989 and in revisedform 24January 1990.

1. Abbreviations used in this paper: CV, coefficient of variation; GH,growth hormone; GRH, GH-releasing hormone; hGRH, humanGRH; IR, immunoreactive; oGRH, ovine GRH; SRIH, somatostatin;TFA, trifluoroacetic acid.

approach the sensitivity of detection by current techniques (1).The neuroendocrine mechanism that regulates GHsecretion ismediated by two hypothalamic hormones, one of which isstimulatory (GH-releasing hormone [GRH]) and the other,inhibitory (somatostatin [SRIH]). Although their effects onGHsecretion have been studied extensively, there have beenformidable technical difficulties in obtaining simultaneousmeasurements of the two neurohormones in hypothalamic-hypophysial portal blood and comparing their secretory pat-terns with those of GHin peripheral blood under physiologicconditions. Thus, the pattern of their release from the medianeminence into the hypothalamic-hypophysial portal systemand current knowledge of their precise interaction in regu-lating pulsatile GHsecretion is based almost entirely on indi-rect evidence. Initial results of passive immunization withanti-GRH serum (2) and anti-SRIH serum (3) suggested thatpulsatile GHsecretion was dependent on pulses of GRHandthat the role of SRIH was primarily to set the level of the GHresponses to GRH. However, subsequent studies with infu-sions of anti-SRIH serum (4), comparison of GHresponses toGRHduring trough and pulse periods of GHsecretion (5), andinterruption of intrahypothalamic somatostatinergic neuronalcircuitry (6) suggested the presence of intermittent and possi-bly pulsatile secretion of SRIH as well as GRH.

Attempts to measure GRHand SRIH in peripheral circu-lation have not clarified their hypophysiotropic roles since (a)SRIH immunoreactivity is believed attributable to gastro-pan-creatic rather than hypothalamic origin (7), and (b) GRHim-munoreactivity remains to be critically characterized and mayalso be of nonhypothalamic origin (8). One study has reportedrepeated measurements of immunoreactive GRHand SRIHin portal blood of rats and demonstrated a slight decrease inSRIH levels during a 20-min period before a rise in GRHlevels was observed (9). However, the use of anesthetized ani-mals and the necessity of hypophysectomy in order to collectportal blood limited the overall interpretation of these results.

Recently, a technique has become available for repeatedsampling of hypothalamic-hypophysial portal blood in un-anesthetized sheep and has been used successfully in demon-strating a high degree of concordance between pulsatile secre-tion of gonadotropin-releasing hormone and luteinizing hor-mone (10, 1 1). In the present report, we describe the use of thismodel to examine the interaction of hypothalamic GRHandSRIH secretion in the regulation of GHunder basal conditionsand in response to insulin-induced hypoglycemia.

Methods

Animals and experimental techniquesStudies were performed in five mature Corriedale ewes (38-44 kg) thathad been ovariectomized for at least 3 mobefore portal blood collec-tion. During experiments, the sheep were housed in individual pensunder natural photoperiodic conditions and given a maintenance ra-

GH-releasing Hormone and Somatostatin Levels in Ovine Portal Plasma 17

J. Clin. Invest.C The American Society for Clinical Investigation, Inc.0021-9738/90/07/0017/08 $2.00Volume 86, July 1990, 17-24

tion of lucerne chaff and grain with free access to water. The sheepwere prepared for portal blood collection as previously described (10,11). Briefly, under halothane anesthesia, a tunnel was drilled into thesphenoid bone from the back of the nasal cavity to create an artificialsinus immediately anterior to the pituitary gland. Two 12-G needleswere implanted through the nasal bone so that one was directed at theportal vessels on the face of the gland, and the other opened into thefloor of the artificial sinus. The artificial sinus was then sealed off fromthe nose with dental acrylic and the animal allowed to recover. 24 hlater, a catheter was placed in the external jugular vein for collection ofperipheral blood and the animal was heparinized. A 16-G stilette wasthen passed through the upper needle and used to puncture the portalveins to a depth of 2-3 mmon the anterior surface of the pituitary.Based on previous experience, - 50% of the portal vessels are tran-sected by this procedure. Extreme care is taken when positioning theneedles to avoid cutting too far laterally and damaging the cavernoussinus. The needle position and extent of transection were verified atautopsy in each animal. Portal blood was withdrawn from the artificialsinus by constant suction applied to a catheter in the lower needle.Blood was collected at 10- (or occasionally 5-) min intervals into tubescontaining aprotinin (500 KIU/ml blood) and immediately chilled.The volume of each portal blood sample was - 3.0 ml and was fairlyconstant, varying by only - 20% throughout the experimental period.Bacitracin, used as a peptidase inhibitor in previous experiments (10),was omitted after preliminary studies indicated interference in theGRHRIA. A sample ofjugular blood (5.0 ml) was obtained every 5 or10 min, immediately after the end of each portal collection period. Onthe basis of previous studies ( 12), the contribution of retrograde pitu-itary blood flow to the diverted portal blood is believed to be minimal.Observation of the animals did not indicate any evidence of stressduring the sampling period except that due to handling at the begin-ning of the experiment.

After a 5-h blood sampling period, regular insulin (CommonwealthSerum Laboratories, Melbourne; 5 U/kg) was injected intravenouslyand portal and peripheral blood samples collected for an additional 2h. Previous studies have shown this dose of insulin to reliably lower themean plasma glucose levels within 40 min to - 20 mg/dl (13). Totalblood removal during the entire study was - 320 ml, which repre-sented 8%of blood volume. Hematocrit determinations, performed at2-3-h intervals, revealed no significant change during the study period.Blood samples were centrifuged at 4VC and the plasma stored at-20'C until transported from Melbourne to Cincinnati on dry ice.

Plasma extraction and hormone assaysIn preliminary experiments, ovine plasma was acidified with 0.2 ml 1.0M trifluoroacetic acid (TFA)/ml plasma and extracted on a reversephase C18 Sep-Pak (Waters Associates, Milford, MA) as previouslydescribed (14). Retained substances were eluted with 2 ml 80%acetoni-trile/20% 0.01 MTFA and the eluate was Iyophilized. Initially, peptiderecovery was evaluated by the addition of synthetic '25I-labeled pep-tides to ovine plasma and counting the eluate radioactivity. The recov-ery of added ['25IJSRIH and ['25I]ovine (o)GRH was > 90% and75-80%, respectively (['25"lhuman (h)GRH recovery using identicalconditions was > 90%). The recovery of [125I]oGRH could be slightlyimproved (80-85%) by recycling the acidified plasma sample one ad-ditional time through the Sep-Pak and eluting with 90% acetonitrile/10% 0.01 MTFA. The recovery of unlabeled peptides added to ovineplasma was next evaluated by RIA after resuspension of the Iyophili-sate in assay buffer (0.05 Msodium phosphate, 0.9% NaCl, 0.5% BSA,0.025 MEDTA, 0.02% protamine sulfate, 0.01% Tween-20, pH 7.5).SRIH recovery remained at > 90%k, while hGRHand oGRHrecoverydecreased to 10-20% (hGRH and oGRH recovery from humanplasma using identical conditions remained at > 90% and 80-85%,respectively). The final recovery of oGRHor hGRHfrom the ovineplasma extract was increased to a maximum of 65-70% by resus-pending the Iyophilisate in 5 mMacetic acid, 0.25% BSA, 0.01%Tween-20 (ABT).

A l-ml aliquot of portal and jugular plasma from each animal ateach time point was extracted as a group, together with three l-mlaliquots of an ovine jugular plasma pool with added oGRHand SRIHto monitor recovery. The extracts were 1yophilized, resuspended in 0.3ml ABT, centrifuged (10,000 g, 10 min 4C), and the supernatantsassayed in duplicate for oGRHand SRIH (100- and 25-Ml aliquots,respectively). The recovery of SRIH and oGRHfrom the plasma poolswas 95±2% and 66±2% (mean±SE, n = 5 each), respectively. Resultswere not corrected for recovery.

Plasma oGRHwas measured by a modification of the previouslydescribed RIA for hGRH(14), using anti-hGRH( -40)-OH antiserum(No. 871), [125I]hGRH(l-40)-OH tracer, and an oGRHstandard (Pen-insula Laboratories, Inc., Belmont, CA). The crossreactivity of oGRHwas 80% compared with the hGRH(I-40)-OH standard with paralleldisplacement. All portal and jugular samples from each animal weremeasured in a single assay. The intra- and interassay coefficients ofvariation (CVs) were 12.5 and 10.6%, respectively, and the averagesensitivity of the assay was 2.7 pg/tube (9 pg/ml plasma). Plasma SRIHwas measured by RIA, as previously reported (15, 16), with portal andjugular samples from each animal measured in a single assay. Theaverage sensitivity was 1.5 pg/tube (5 pg/ml plasma), with intra- andinterassay CVs of 7.1 and 11.2%, respectively. The dilution curves ofimmunoreactive (IR) oGRHand SRIH in portal plasma were parallelto those of synthetic oGRHand hGRHand SRIH, respectively.

Ovine GHwas measured by RIA using the reagents provided by theNational Hormone Pituitary Program (Baltimore, MD). All jugularsamples were measured in duplicate with an intraassay CVof 10.4%.The average sensitivity was 25 pg/tube (500 pg/ml plasma) based onthe National Institute of Arthritis, Diabetes, and Digestive and KidneyDiseases oGH-I-4 reference standard. For purposes of statistical analy-sis (see below) the least detectable value for the particular assay wasassigned to all values that were at or beneath that level. Blood glucosewas measured by a glucose oxidase method.

Statistical analysisComparison of spontaneous secretory patterns of GRH, SRIH, andGH. We initially determined whether directional changes in GRH(concordant) and/or SRIH (discordant) occurred immediately before(i.e. in the preceding sample) or simultaneous with changes in GHorthe other hypothalamic hormone. A computer program was developedto tabulate the frequency of such changes between pairs of hormones ineach individual animal during the 5-h period when spontaneous secre-tion was monitored. Multiple criteria for change were examined, rang-ing from 0 to 10 pg/ml (GRH or SRIH) and 0 to 3 ng/ml (GH)(absolute) and 0 to 200% (relative). The resulting frequencies (ex-pressed as a percentage) in the five animals ("experimental") were thencompared with that expected by random association and generated bycomparing directional changes in hormone values from different ani-mals ("random") rather than the same animal. Thus, four separate setsof random frequencies were available for each animal, giving a total of20 random series with which to compare the results in the five individ-ual animals. Differences between the two groups were determined byStudent's t test.

The pulses present in each hormone secretory profile during the 5-hspontaneous secretion period were determined by the program Detect(17) (method 1). The frequency with which pulses of GRHor SRIHoccurred immediately preceding or concurrently with those of GHorof one another in individual animals was compared with the randomfrequency observed using pairs of animals as described above. Statisti-cal differences in frequency were determined by the chi square test. Asa validation of the appropriateness of the Detect algorithm, pulses werealso determined, using as criteria, an increase of at least three times theCV greater than the preceding minimum trough value. The troughminimum was defined as the lowest value in a series of hormonemeasurements beginning with a decreasing value and including allsubsequent values that (a) were no greater than three times the CVofthe lowest included value and (b) did not initiate a progressive rise that

18 L. A. Frohman, T. R. Downs, L J. Clarke, and G. B. Thomas

culminated in a level at least three times the CVof the lowest includedvalue (method 2).

Insulin-induced hypoglycemia. Mean GRH, SRIH, and GHvaluesafter insulin administration were compared by a repeated measuresanalysis of variance. A P < 0.05 was considered significant.

Results

Recovery of immunoreactive GRHfrom ovine portal plasma.The recovery of '251-labeled oGRHfrom either ovine orhuman plasma was always consistently lower (10-15%) thanthat of [1251I]hGRH. This appeared to be primarily the result ofa slightly reduced elution efficiency for oGRHfrom the Sep-Pak cartridge and occurred in the presence or absence ofplasma. The major problem encountered was with the recov-ery of GRHfrom lyophilized ovine plasma extracts. RIA ofovine plasma extracts containing added unlabeled oGRHorhGRH, after resuspension in RIA buffer, revealed that only10-20% of the added peptide was measurable, while the recov-ery of these peptides from human plasma using identical con-ditions was the same as that seen with ['251]oGRH (80-85%)and ['25I]hGRH (> 90%). Resuspension of the lyophilizedovine plasma extract in an acidic solution (pH < 5) or at lowsalt concentration increased the recovery of GRHfrom ovineportal or jugular plasma to a maximail level of 65-70%.Whether this effect is due to the presence of a substance(s) withspecific or nonspecific binding affinity for GRHis unknown.However, the same difficulties have been encountered with therecovery of GRHfrom bovine plasma extracts (data notshown), while GRHrecovery from human, porcine, and mu-rine plasma extracts by our standard protocol has been veryefficient (> 90%).

Jugular venous samples were also assayed for oGRHandSRIH in all five sheep. Of a total of 135 samples, IR-oGRHwas detected in 26 (19%), though in all samples, values were< 30 pg/ml and generally less than double the least detectablevalue for the particular assay. These barely detectable valueswere insufficient for further characterization. SRIH was de-tected in 121 (82%) of 147 jugular plasma samples. Valueswere almost always less than half of those in portal plasma andexhibited an overall mean±SDof 21 ± 13 pg/ml.

Spontaneous secretory pattern of portal GRHand SRIHand of peripheral GH. The secretory patterns of GRHandSRIH in portal plasma and of GHin jugular plasma during the5-h observation period for each of the animals are shown inFig. 1. IR-GRH was detectable in all of the samples from threesheep and in 87 and 58% of samples from the other two ani-mals. Overall mean portal GRHwas 20.4±6.7 pg/ml(mean±SD). On the basis of a 0.3-ml/min blood collection, ahematocrit of 29.0±2.2%, an extraction efficiency of 66%, andassuming a 50% removal of portal blood by the surgical tech-nique (resulting in a corrected portal blood flow of 0.6 ml/min), the mean hypothalamic secretory rate of GRHwas cal-culated to be 13.2 pg/min.

GRHsecretion was pulsatile in each of the animals withpeak values ranging from 25 to 40 pg/ml. The number ofpulses detected during the 5-h period (method 1) ranged from3 to 6 with a mean of 4.2±1.1, representing a mean pulseinterval of 71 min. An identical number of pulses (4.2±1.3)were detected using method 2.

SRIH was detectable in all of the portal samples from eachof the sheep. Overall mean portal SRIH was 72±33 pg/ml and

the mean hypothalamic secretory rate of SRIH was calculated(as above) to be 32.3 pg/min. SRIH secretion was pulsatile ineach of the animals, with peak values ranging from 65 to 160pg/ml. The number of pulses detected during the 5-h period(method 1) ranged from 4 to 9 with a mean of 5.6±2.1, repre-senting a mean pulse interval of 54 min. Using method 2,5.8±1.9 pulses were detected, representing a mean pulse inter-val of 52 min.

Plasma GH levels were detectable in 97% of the jugularsamples. Overall mean jugular plasma GHwas 2.9±2.2 ng/ml.GHsecretion exhibited pulsatility in each of the sheep, withpeak values ranging from 4 to 11 ng/ml. Using method 1, themean number of GHpulses during the 5-h period was 4.6+2.2,representing a mean pulse interval of 65 min, whereas withmethod 2, the values were 5.2±1.6 pulses and 58 min, respec-tively.

Relationships among GRH, SRIH, and GHlevels. Usingthe algorithm described for comparing directional changes inGRH, SRIH, and GHlevels, we found that a substantial per-centage of concordance in directional changes between GRHand GHand discordance in directional changes between SRIHand GHwhen examined at simultaneous time periods or witha one sample lag time for the GHvalue (Fig. 2). However,when these percentages were compared with those that wouldbe expected randomly (by analyzing hormone values from dif-ferent animals), there were no significant differences. Thecurves of the random comparisons were virtually superimpos-able on those derived from the intra-animal comparisons inthe five sheep. Subsequent analyses therefore used GRH,SRIH, and GHpeaks and SRIH troughs as determined bymethods 1 and 2.

Using hormone peaks as defined by method 1, we deter-mined the percentage of GHpeaks occurring coincident withor immediately after (one sample) those of GRHor SRIH andalso the interrelationship of GRHand SRIH peaks. The resultsare shown in Table I. 38% of the GHpeaks occurred coinci-dent with GRHpeaks and 62%occurred either coincident withor immediately (one sample later) after GRHpeaks. Theseassociations were statistically significant when compared withrandom associations from different animals (P < 0.05 and P< 0.02, respectively) or to associations with SRIH peaks (P< 0.02 and P < 0.01, respectively). In contrast, GHpeakscoincident with SRIH peaks were not significantly differentbetween experimental and random pairings. Similarly, the fre-quency of GRHpeaks immediately preceding SRIH peaks orSRIH peaks immediately preceding GRHpeaks did not differsignificantly between experimental and random pairings.

The results of analyses using peaks defined by method 2 areshown in Table II. The association of GRHand GHpeaks wasqualitatively similar to that obtained with method 1 (data notshown). GRHpeaks were found to occur coincident with(28%, P = 0.025) or immediately preceding (48%, P < 0.01)the initiation of the GHpeak. There was no association of GHpeak initiation with SRIH troughs.

Effect of insulin-induced hypoglycemia on portal GRHandSRIH and jugular GHlevels. The results of insulin-inducedhypoglycemia are shown in Fig. 3. All animals developed pro-found hypoglycemia (initial plasma glucose [time 0]: 45±4mg/dl; nadir plasma glucose [time 40 min]: 18±2 mg/dl)which persisted for at least 90 min (18±1 mg/dl). GRHvalueswere unaltered by insulin-induced hypoglycemia until 90 minafter injection, when -a slight increase was observed. SRIH

GH-releasing Hormone and Somatostatin Levels in Ovine Portal Plasma 19

20

9 100

o 11 12 13 14 15 16

Vvv/F V I10 11 12 13 14 15 16

10 11 12 13 14 Is IsTIME of DAY, h

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SHEEP2

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0

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10 11 12 13 14 15 16

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11 12 13 14 1S 16

SHEEP5/vvf AsP I0f

TIME of DAY, h

o 11 12 13 14 1s 16

Sru A0A./~NI Figure 1. GRHand SRIH levels in hypothalamic-pituitary portalI-----------I plasma and GHlevels in jugular plasma of five unanesthetized ovar-010 11 1 iectomized ewes. The interrupted horizontal lines indicate the least

TIME of DAY, h detectable value for each assay.

20 L. A. Frohman, T. R. Downs, I. J. Clarke, and G. B. Thomas

Io

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0 1 2 3 0 1 2CHANGEIn GH, ngaml CHANGEin GH, nglml

values, however, exhibited an increase of 93% (P < 0.01) 20min after insulin injection that remained significantly elevateduntil 50 min after injection, at which time they returned topreinjection levels. GHvalues exhibited a gradual declinewhich was significant from 60 through 110 min.

Discussion

The results of the present studies demonstrate that both GRHand SRIH can be reliably measured in nearly all portal samplesobtained at 10-min intervals from unanesthetized sheep usingthe sampling technique described. Our model therefore per-mits the study of the GRH/SRIH/GH axis in unstressed ani-mals. The sensitivity of the technique is limited only by thedetection limits of each assay and the fraction of hypotha-lamic-pituitary portal blood that is diverted from the pituitary(18). The technique cannot exclude the possibility of admix-ture of portal blood with retrograde flow from the pituitary.Thus, the actual portal GRHlevels may be slightly higher sincemeasurements of GRHin jugular plasma revealed detectablevalues in < 20% of samples.

GRHsecretion, as reflected in portal blood GRHlevels,was observed to be pulsatile in all animals with a mean pulseinterval of 71 min. Two separate statistical methods were usedto determine this value, one of which (Detect [17]) has beenvalidated in studies using peripheral blood measurements andthe other of which was developed specifically for these studies.The comparability of the pulse analyses by the two methodshelps support the conclusion that the results are not techniquedependent. The absence of any recirculation of GRHin theportal vascular system actually simplifies the interpretation of

Figure 2. Frequency ofdiscordance between di-rectional changes inSRIH and GHlevels andof concordance betweendirectional changes inGRHand GHlevels inindividual sheep (n = 5),as compared with the ex-pected frequency of con-cordance and discordancein values using data fromdifferent sheep (n = 20)at the indicated levels ofchange in each parame-ter. See Methods for de-tails. The mean±SE of

, the results in individual: | animals are shown super-

imposed on a stippledarea representing the

3 mean±SEof the resultsin different animals.

the results since hormone metabolic clearance (i.e., half life)need not be considered.

SRIH secretion was also shown to be pulsatile with a pulseinterval of 52-54 min. As with GRH, the two analytic tech-niques used to determine pulse frequency gave comparableresults. Although the SRIH frequency was slightly faster thanthat of GRH, the small number of animals studied, the limitedtime period (5 h) of each study, and the interanimal variabilitydoes not permit a statistical distinction between the pulse fre-quencies of the two hormones.

GHsecretion, as reflected by measurements in jugularplasma, was also pulsatile with a frequency of one pulse every58-65 min, intermediate between that of GRHand SRIH. Aninitial elevation of GHvalues seen in two of the sheep is be-lieved to reflect a stress response before initiation of samplecollection.

The analysis of the interaction between GRHand SRIHalso required a novel analytic approach and we initially at-tempted to compare directional changes in individual hor-mone concentrations. Although considerable concordance(GRH vs. GH) and discordance (SRIH vs. GH) were found,these values were not statistically distinguishable from thoseexpected on the basis of chance observations alone. Whenthesame analysis was applied to the hormone secretory peaksrather than the individual values, however, a highly significantassociation was seen between peaks of GRHsimultaneouswith or immediately preceding both GHpeaks and initiationof the GHpulse. This association could explain the majority(62%) of the GHpeaks observed. In contrast, no such associa-tion was observed between SRIH troughs and GHpeaks orbetween GRHand SRIH peaks in portal plasma.

GH-releasing Hormone and Somatostatin Levels in Ovine Portal Plasma 21

LU0z4ca

0

IL

Table I. Relationships among GRH, SRIH, and GHPeaks(Method 1: Detect)

Comparison No./total (%) x2 P

GHpeak coincident with GRHpeak

ExperimentalRandom

GHpeak coincident or one sampleafter GRHpeak

ExperimentalRandom

GHpeak coincident with SRIHpeak

ExperimentalRandom

GHpeak coincident or one sampleafter SRIH peak

ExperimentalRandom

GRHpeak one sample beforeSRIH peak

ExperimentalRandom

SRIH peak one sample beforeGRHpeak

ExperimentalRandom

GHpeak coincident withGRHpeakSRIH peak

GHpeak coincident or one sampleafter

GRHpeakSRIH peak

GRHpeak one sample beforeSRIH peak

SRIH peak one sample beforeGRHpeak

Experimental data are those from compmals. Random values are those from coone hormone from one sheep with thosefrom the other four sheep.

These findings provide evidencein the pulsatile secretory pattern of 4of an observed association betweepulses suggests that SRIH exerts atiating GHpulses. However, the pctensity of the GHpulse (i.e., pulse 1an extent modified by the level ofGRHpulse. Attempts to demonstithe present study were, however, uiof the small number of pulses in i]interanimal variability in hormoneplete coupling of either GRHor Sraises the possibility of yet anothercontributes to GHpulsatility. Thefactor, however, remains unknown.

8/21 (38)13/84 (15)

13/21 (62)26/84 (31)

2/29 (7)22/116 (19)

6/29 (27)44/116 (38)

4/21 (19)13/84 (15)

6/29 (21)18/116 (16)

8/21 (38)2/27 (7)

13/21 (62)A/oQ 0V71

4.05 0.044

5.63 0.018

1.65 NS

2.34 NS

0.01 NS

Table H. Relationships among GRH, SRIH, and GHPeaks(Method 2:Three CVCriteria)

Comparison No./total (%) x2 P

Initiation of GHpeak coincidentwith GRHpeak

Experimental 8/29 (28)Random 10/106 (9) 5.02 0.025

Initiation of GHpeak coincidentor one sample after GRHpeak

Experimental 14/29 (48)Random 23/106 (22) 6.80 0.009

Initiation of GHpeak coincidentwith SRIH minimum

Experimental 5/29 (17)Random 25/106 (24) 0.23 NS

Initiation of GHpeak coincidentwith or one sample afterSRIH minimum

Experimental 10/29 (34)Random 41/106 (39) 0.04 NS

Experimental and random as defined in Table I.

0.15NS Hypoglycemia in the sheep resulted in a decrease in plasma0.15 NS GHlevels. Similar findings have been previously reported inthe rat (19). Previous studies have suggested a mechanism to

5.59 0.018 explain this response since neuroglycocytopenia induced by5.59 0.018 2-deoxyglucose is a potent stimulus for SRIH release by rathypothalamic fragments in vitro (20). The present resultsclearly demonstrate that insulin-induced hypoglycemia insheep stimulates an immediate release of SRIH from the me-7 1 ) n nnq d st whp l naAla ank/ *1. V.-WO dian eminence into the portal system with peak levels nearlydouble those before injection occurring 20 min after insulin

4/21 (19) injection. GHconcentrations exhibited a corresponding de-

crease, which was significant during the second hour after in-6/29 (27) 0.05 NS jection. In contrast, GRHconcentrations appeared unaltered

by the injection of insulin for 90 min, after which a slightarisons within individual ani- increase was observed. This was well beyond the time whenmparisons of the peaks of SRIH values had returned to preinjection levels. In addition,e of the second hormone GRHpulsatility persisted in three of the five animals during

the first 90 min after insulin administration.There is considerable indirect evidence that suggests an

for a primary role of GRH inhibitory effect of SRIH on GRHsecretion (6, 10, 21). How-GHsecretion. The absence ever, the present results indicate either that (a) the increasedn SRIH troughs and GH release of SRIH into the portal circulation in response to insu-less important role in ini- lin-induced hypoglycemia is unaccompanied by a concomi-)ssibility exists that the in- tant SRIH release at nerve terminals in the region of GRH-height and duration) are to containing cell bodies, or (b) the concentrations of SRIH re-f SRIH at the time of the quired to inhibit GRHrelease are greater than those occurringrate such a relationship in in association with the increased levels detected in the portalnsuccessful in part because circulation. The present results also do not permit a distinctionndividual animals and the to be made between the effects of increased SRIH secretion,levels. The lack of a com- the hypoglycemia induced by insulin, or a separate effect of;RIH with GHpulses also insulin itself on the suppression of GHsecretion. The pro-r hypothalamic factor that longed suppression, well beyond the time that SRIH levels

nature of this postulated returned to baseline, suggests that the effect cannot be ex-plained by SRIH alone. At the dose used in the present study,

22 L. A. Frohman, T. R. Downs, L J. Clarke, and G. B. Thomas

INSULIN, 5U/kg plasma that appear to reflect independent neural rhythmicity.The results indicate a primary role of GRHin determiningpulsatile GHsecretion, with that of SRIH being less impor-tant. Insulin-induced hypoglycemia leads to a rapid and briefincrease in hypothalamic SRIH secretion and a more delayedand prolonged decrease in plasma GHlevels. The persistenceof suppression of GHsecretion, however, also implies the par-ticipation of other inhibitory factors.

I I I I I

0 20 40 60 80 100*

*

*

Wethank Jane Withrow, Heather Francis, Bruce Doughton, and PaulWeston for their excellent technical assistance and the Director of theAnimal Research Institute at Werribee for the use of animal facilities.Materials for the ovine GHassay were provided by the National Insti-tute of Arthritis, Diabetes, and Digestive and Kidney Diseases Hor-mone Distribution Program.

These studies were supported in part by U. S. Public Health Servicegrant DK-30667 and the National Health and Medical ResearchCouncil of Australia.

50

References

0

5

r 2.5I-

CD

~~~~~~~~~~~~~~ll

0 20 40 so so 100

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Figure 3. Mean±SEof portal plasma GRHand SRIH levels and ofjugular plasma GHlevels in five unanesthetized ovariectomized ewesin response to insulin hypoglycemia. Asterisks indicate paired differ-ences between the mean value at individual time points and thoseimmediately before injection of insulin with a P < 0.05.

insulin may have exerted short-term effects through CNSIGF-I receptors to which it binds, though with reduced affinity(22), to stimulate SRIH release (23). In addition, direct effectsof insulin on pituitary IGF-I receptors may have contributedto the more prolonged suppression of GHsecretion, as hasbeen previously reported (23, 24). Moreover, it is possible thatthe hypoglycemia, in addition to its stimulatory effect onSRIH secretion (20), may have affected the release of another,yet unidentified factor that influences GHsecretion.

Finally, our results in the sheep (as in the rat) are in markedcontrast to those in humans where insulin-induced hypoglyce-mia increases circulating GHconcentrations (25) and indirectevidence exists for the suppression of SRIH secretion, based onthe enhancement by insulin of the GHresponse to GRH(26).The explanation for this species difference remains unknown.

In summary, the present studies have described pulsatilepatterns of GRHand SRIH in hypothalamic-pituitary portal

1. Frohman, L. A. 1987. Diseases of the anterior pituitary. InEndocrinology and Metabolism. P. A. Felig, J. Baxter, A. E. Broadus,and L. A. Frohman, editors. 2nd ed. McGraw-Hill Book Company,NewYork. 247-337.

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24 L. A. Frohman, T. R. Downs, I. J. Clarke, and G. B. Thomas