Maturational Patterns of lodothyronine Phenolic

and Tyrosyl Ring Deiodinase Activities in

Rat Cerebrum, Cerebellum, and Hypothalamus

MICHAEL M. KAPLANand KIMBERLEEA. YASKOSKI, Thyroid Diagnostic Center,Department of Medicine, Brigham and Women's Hospital andHarvard Medical School, Boston, Massachusetts 02115

A B S T RA C T To explore the control of thyroid hor-mone metabolism in brain during maturation, we havemeasured iodothyronine deiodination in homogenatesof rat cerebrum, cerebellum, and hypothalamus from1 d postnatally through adulthood. Homogenates wereincubated with 125I-L-thyroxine (T4) + [13'I]3,5,3'-L-triiodothyronine (T3) + 100 mMdithiothreitol. Non-radioactive T4, T3, and 3,3',5'-triiodothyronine (rT3)were included, as appropriate. The net production rateof [125I]T3 from T4 in 1-d cerebral homogenates wassimilar to the rate in adult cerebral homogenates (9.9+2.5[SEM]% vs. 8.9±1.2% T4 to T3 conversion in 2 h).Production of T3 was not detectable in 1-d cerebellarand hypothalamic homogenates. The net T3 productionrate in adult cerebellar homogenates was twice as greatas, and that in adult hypothalamic homogenates similarto, the rate in cerebral homogenates.

Tyrosyl ring deiodination rates of T4 and T3 weremore than three times as great in cerebral homogenatesfrom 1-d-old rats as in adult cerebral homogenates. Incerebellar homogenates from 1-d-old rats, tyrosyl ringdeiodination rates were much greater than the rates inadult cerebellar homogenates, but less than those in 1-dcerebral homogenates. In 1-d hypothalamic homoge-nates, tyrosyl ring deiodination rates were the highestof all the tissues tested, whereas rates in adult hypo-thalamic homogenates were similar to those in adultcerebral homogenates.

During maturation, T4 5'-deiodination rates in-creased after 7 d and exceeded adult rates between14 and 35 d in cerebral and cerebellar homogenates,and at 28 and 35 d in hypothalamic homogenates. Incerebral homogenates, the peak in reaction rate at 28 dreflected an increase in the maximum enzyme activity

Dr. Kaplan is the recipient of Research Career Develop-ment Award 1-K04-AM 00727-01.

Receivedfor publication 29 August 1980 and in revisedform29 October 1980.

1208

(Vmax) of the reaction. T4 and T3 tyrosyl ring deiodina-tion rates decreased progressively with age down toadult rates, which were attained at 14 d for cerebrumand cerebellum and at 28 d for hypothalamus.

These studies demonstrate quantitative differencesin T4 5'-deiodinase activities in cerebrum, cerebellum,and hypothalamus at all ages, with the overall matura-tional pattern differing from the developmental pat-terns of both the pituitary and hepatic T4 5'-deiodinases.lodothyronine tyrosyl ring deiodinase activities alsovary quantitatively among these same brain regions andexhibit a pattern and a time-course of maturation differ-ent from that of the T4 5'-deiodinase. These enzymescould have important roles in the regulation of intra-cellular T3 concentrations and, hence, on the expres-sion of thyroid hormone effects.

INTRODUCTION

Phenolic ring, or 5'-deiodination of L-thyroxine (T4),lwhich occurs in rat brain in vivo (1-3), supplies muchof the endogenous 3,5,3'-L-triiodothyronine (T3) in thecerebral cortex and cerebellum, including that T3which occupies the nuclear T3 binding sites (2, 3). Afterintravenous injection of 125I-T4, [125I]T3 is also found invarious extranuclear brain fractions, especially thesynaptosomal fraction (4), and more 1251-T3 is found inextracts of whole brain tissue and brain nerve cellbodies in 10-d-old rats than in 30-d-old rats (5). Alsoafter intravenous 125I-T4 injection, [1251]3,3',5'-L-triiodo-thyronine (rT3) and a compound that may be [1251]3,3'-L-diiodothyronine (3,3'-T2) have been found in rat brainextracts (4). The in vivo occurrence of tyrosyl ring de-iodination of T4 and T3 is thus suggested. We have

'Abbreviations used in this paper: DTT, dithiothreitol;3,3'-T2,3,3'-L-diiodothyronine; 3'-T1, 3'-L-iodothyronine; T4,L-thyroxine; T3, 3,5,3'-L-triiodothyronine; rT3, 3,3',5'-L-tri-iodothyronine; Vmax, maximum enzyme activity.

J. Clin. Invest. C The American Society for Clinical Investigation, Inc. * 0021-9738/81/04/1208/07 $1.00Volume 67 April 1981 1208-1214

demonstrated in vitro T4 5'-deiodinase activity inhomogenates of rat cerebral cortex and cerebellum andfound in vitro activity of a separate tyrosyl ring de-iodinase that converts T4 to rT3 and converts T3 se-quentially to 3,3'-T2 and 3'-L-iodothyronine (3'-T1) (6).Both enzymes are largely particulate and require thiolreducing agents for detectable in vitro activity (6).

Thyroid hormone metabolism in fetal and neonatalrat tissues differs from that in adult tissues. T4 5'-deiodination rates in liver homogenates from neonatesare much lower than in adult liver homogenates, butthe differences are abolished by addition of the thiolreducing agent, dithiothreitol (DTT) (7, 8). In contrast,in vitro T4 5'-deiodination is more rapid in neonatalpituitary tissue than in adult pituitary tissue (8, 9). Iforderly changes in patterns of thyroid hormone metabo-lism were also to occur in the brain, then the argumentfor the biological significance of changes in in vitrorates of iodothyronine deiodination reactions in braintissue in altered physiological states would be strength-ened. Further, since T3 is both produced and degradedby brain homogenates, knowledge of relative reactionrates at different ages could facilitate the interpretationof previous (5) and future in vivo studies of brain tissueT3 concentrations. We have therefore investigatedchanges in deiodinative metabolism of thyroid hor-mone in vitro occurring during maturation. Cerebrum,cerebellum, and hypothalamus were examined sepa-rately, since our previous studies showed differencesbetween adult cerebral cortex and cerebellum in ratesof T4 5'-deiodination and of T4 and T3 5-deiodination(6). Determinations of the kinetic parameters for T45'-deiodination in cerebral homogenates were alsomade by modifying the incubation conditions to mini-mize concomitant reactions that interfere with meas-urements of T3 production.

METHODS

Nonradioactive iodothyronines were obtained from the SigmaChemical Co., St. Louis, Mo. and Henning GMBH(Berlin,West Germany). High specific activity tracers were preparedin our laboratory (6). Timed-pregnant Sprague-Dawley ratsand adult male Sprague-Dawley rats were supplied by Zivic-Miller Laboratories, Allison Park, Pa. Male and female pupswere used before age 21 d and only males were used at olderages. Ages of rats weighing .200 g were estimated from theirweights according to growth curves from the supplier. Threeto four pups (ages e43 d) and three to four adult rats (ages>60 d) were used simultaneously, in anticipation of possibleinter-experiment variation. In some experiments two groupsof pups and one group of adults were used. Animals wereanesthetized lightly with ether and decapitated. Brains wereremoved to iced 0.05 M Tris, pH 7.6-0.25 M sucrose (Tris-sucrose) and subsequent operations were performed at 0-4°C.For each group, tissue from the rats was pooled. Brains weredivided into cerebellum, hypothalamus, and cerebral hemi-spheres (with brainstem removed). Whole cerebral hemi-spheres were used since there was no clear demarcation ofthe cerebral cortex in 1- and 7-d-old brains. In some experi-

ments, adult hypothalami were dissected according to Glow-inski and Iversen (10), yielding 90-110 mg tissue per hypo-thalamus. In other experiments, the cephalad cut was madeat the floor of the third ventricle, yielding 30-40 mgtissue perhypothalamus. There was no difference in reaction rates oc-curring in homogenates of the larger or smaller hypothalamicpieces. Hypothalamic weights in the 1-d-old animals were9-13 mg and increased to adult values by 28 d.

Tissue was homogenized in 9 vol Tris-sucrose containing100 mMDTT. Details of the tissue preparation and incubationprocedures have been reported (6). In some experiments, ahigh-speed pellet (material sedimenting between 1,000 and160,000 g) was prepared as described (6), and resuspended tothe original homogenate volume. 90 ILI of the homogenateswere incubated in triplicate at 37°C under nitrogen for 2 h with10 IlI substrate solution, giving concentrations in the incuba-tion mixtures of "=0.2 nM 1251-T4 (40,000 cpm) + =0.1 nM[131I]T3 (20,000 cpm) and either 1 ,uM nonradioactive T3 or1 ,zM nonradioactive rT3. Endogenous T4 and T3 contribute-0.15 nM each to the concentrations in the incubation mix-tures (11). Variations in tracer T4 and T3 concentrations be-tween 0.1 nM and 1 nM do not affect fractional degradationrates. Hypothalamic homogenates from 1-d-old rats were in-cubated only with 1 ,uM T3 because of the small amount oftissue available. Other homogenates were incubated with1 .uM T3 and, separately, with 1 ,uM rT3. T3 inhibits 5-deiodina-tion, but not 5'-deiodination, of T4 and inhibits degradationof newly formed T3 (6). rT3 inhibits T4 5'-deiodination butnot T4 or T3 5-deiodination (6). The data from incubationswith 1 ,uM rT3 thus show maximal T4 5-deiodination rates, notinhibited by T3.

Incubations were terminated by the addition of 200 ,ulethanol and 50 ul of 0.04 N NaOHcontaining nonradioactiveL-T3 + L-T4 + methimazole (6). The ethanolic extracts wereanalyzed by descending paper chromatography in t-amylalcohol:hexane:ammonia, 5:1:6. Further details of the analyti-cal methods and calculations have been published (6). Iden-tifiable products of T4 degradation were T3, rT3, 3,3'-T2, andI-. Identifiable products of T3 degradation were 3,3'-T2, 3'-L-iodothyronine (3'-T,), and I-. Results are expressed as percent1251-T4 or ['31 ]T3 converted to the various products. In all ex-periments reported here, excess 1- production (6) amounted to--5% of 1251-T4 and <2% of [31 I]T3. Experimental protocolsalways included 0 time and 2-h control incubations with notissue in the buffer. Reaction rates in the presence of tissuewere corrected for nonenzymatic reactions observed in thebuffer controls. Boiled tissue controls were not appropriateowing to the occurrence of nonenzymatic deiodination of T4without T3 or rT3 production (6). In the buffer control incuba-tions, <5%of T4 was degraded, <5%of T3 was degraded, andT3 production from T4 was <1.5%. Protein was measured bythe method of Lowry et al. (12), after DTT was removed byperchloric acid precipitation of the protein (6).

In the statistical analyses, each mean of triplicate incuba-tions within an experiment was treated as a single value. Whendata were not normally distributed or variances were nothomogeneous, nonparametric statistics were used. To com-pare reaction rates at different ages, overall significance wastested by Kruskal-Wallis one-way analysis of variance (13). Ifthe x2 was significant, comparisons of rates at different ageswere made by the Mann-Whitney test (13), if three or morevalues were present. All the values for rats 60 d or older weregrouped together, inasmuch as no measurement varied withage within this group. Also, inter-experiment variation of adultresults was found not to differ from intra-experiment adultresults when tissues from individual rats were prepared sepa-rately. Inter-experiment variation, therefore, seems largely toreflect biological variation. Values for 4-d-old and 7-d-old rats

T4 and T3 Metabolism in Maturing Brain 1209

and for 41-48-d-old rats were grouped together to minimizethe data sets with n < 3. This grouping does not materiallyaffect the conclusions.

RESULTS

T4 5'-deiodination in homogenates of cerebrum, cere-bellum, and hypothalamus from 1-d-old and adultrats (Table I). The net production rate of ['25I]T3in 1-d cerebral homogenates was similar to the ratein adult cerebral homogenates. Degradation of [131I]T3and removal of '25I-T4 by conversion to rT3 weremore rapid in the cerebral homogenates from neonatesthan in those from adults (Table I, lines 2 and 5). Netproduction of [1251]3,3'-T2 was 2.50±0.5 (SE)% ofadded T4 in 1-d cerebral homogenates and not detect-able in adult cerebral homogenates. These findingssuggested that the capacity for T4 to T3 conversionmight be greater in neonatal cerebral tissue than inadult cerebral tissue.

In 1-d rats, net production of T3 was not detectablein cerebellar and hypothalamic homogenates. Net pro-duction of [125I]3,3'-T2 from '25I-T4 was also very low orundetectable (<1% conversion) in incubations of cere-bellar and hypothalamic homogenates from 1-d-oldrats; therefore, the possibility can be excluded thatsubstantial quantities of T3 were produced and rapidlydegraded. Even considering the differences betweenthe age groups in homogenate protein content (Table I),and in rates of T4 depletion via 5-deiodination (seebelow), the capacities of neonatal cerebellar and hypo-thalamic homogenates to catalyze T4 5'-deiodinationwere clearly much lower than those of the correspond-

ing adult homogenates. In homogenates from adultrats, the mean T4 5'-deiodination rate was highest incerebellar tissue (Table I), P < 0.001 vs. cerebrum andhypothalamus.

Tyrosyl ring deiodination of T4 and T3 in cerebrum,cerebellum, and hypothalamus from 1-d-old andadult rats (Table I). The mean rates of tyrosyl ringdeiodination of T4 and T3 were more than threetimes as great in cerebral homogenates from 1-d-oldrats than in adult cerebral homogenates in incubationswith 1 ,uM T3. In incubations using 1 uM rT3 instead,61±6% of T4 was converted to rT3 in the 1-d homoge-nates vs. 33 ±3% in the adult homogenates (P < 0.001).In cerebellar homogenates from 1-d-old rats (Table I),tyrosyl ring deiodination rates of T4 and T3 were abouthalf the rates in 1-d cerebral homogenates, but weremuch greater than the rates in adult cerebellar homoge-nates, the latter having very little activity. In cerebellarincubations with 1 ,uM rT3, similar findings were noted:T4 to rT3 conversion was 41.4±3% in the 1-d homoge-nates and 1.1±0.3% in adult homogenates (P < 0.001).In hypothalamic homogenates from 1-d-old rats (TableI), tyrosyl ring deiodination rates of T4 and T3 werethe highest of all the tissues tested, whereas rates inthe adult hypothalamic homogenates were similar tothe rates in adult cerebral homogenates.

To test the effects of differences in protein contenton tyrosyl ring deiodination rates, tyrosyl ring deiodi-nation of 1 ,M T3 was measured in adult tissuehomogenates diluted with 1 vol Tris-sucrose-100 mMDTT. In the three brain regions, rates were reducedin the diluted homogenates to 42-76% of the rates in

TABLE IJodothyronine Deiodination in Cerebral, Cerebellar, and Hypothalamic Homogenates from 1-d-old and Adult Rats

Cerebrum Cerebellum Hypothalamus

Measurement 1 d Adult 1 d Adult 1 d Adult

T4 5'-deiodination(percent T4 converted to T3) 9.9+2.5 8.9+1.2 0.4±0.4* 17.6±1.6 0.2+0.2* 7.6+1.0

T4 5-deiodination(percentT4converted to rT3) 20.8+1.7* 5.6+2.3 10.4±1.7* 0.3±0.5 46.2±7.5* 7.2±1.2

T3 tyrosyl deiodination(percent added T3)

3,3'-T2 27.6±3.2* 9.6±1.3 13.3±2.9* 0.3±0.2 34.3±1.0* 8.2±1.43'-T, 6.8±2.1* 1.0±0.2 2.8±0.5* 0.0±0.1 17.7±3.3* 1.1±0.33,3'-T2 + 3'-T1 34.4±3.8* 10.6±1.4 16.1±2.7* 0.3±0.3 52.0±3.8* 9.3±1.6

Protein content, mglml 5.3±0.5 6.8±0.5 5.9±1.7t 10.0±0.5 5.6±1.0t 8.6±0.5

Results are mean±SEMof individual experimental means for 60-100-d-old adult rats (12 experiments) and 1-d-oldneonatal rats (three experiments). Incubations were performed at 37°C for 2 h with 0.2 nM 125I-T4 + 0.1 nM [131I]T3+ 1 /IM [1271]T3 + 100 mMDTT. Values for neonatal and adult tissues were compared using the t test with n = numberof experiments. No correction was made for degradation of newly formed [1251]T3 or 125I-rT3.*P < 0.001.4 P < 0.01 vs. adult tissue from the same region.

1210 M. M. Kaplan and K. A. Yaskoski

corresponding undiluted homogenates. Correction forthe differences in homogenate protein content wouldtherefore magnify the differences between neonatesand adults in tyrosyl ring deiodination rates seen inTable I, but would not greatly alter the comparisonbetween the different regions.

Changes in T4 5'-deiodination rates during matura-tion. T4 5'-deiodination rates (Figs. 1A, 2A, and 3A)increased after 7 d, and exceeded adult rates between14 and 35 d in cerebral and cerebellar homogenatesand at 28 and 35 d in hypothalamic homogenates. Themean rates at the peak ages were two to three timesthe mean adult rates. T3 production rates in the period14-48 d could be compared directly to adult rates sincethere were no significant differences in T4 depletionand T3 degradation (see below) or homogenate proteincontent.

CEREBRALHEMISPHERES

CEREBELLUM

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FIGURE 2 T4 5'-deiodination and T3 tyrosyl ring deiodinationin homogenates of rat cerebellum. Experimental details are

given in the legend to Fig. 1.

Because of the remaining uncertainty about therelative T4 5'-deiodinase activities in the 1-d and adultcerebral homogenates, and because the peak rates seen

between 14 and 35 d could result from changes ineither the Michaelis constant (Kin) for T4 or maximumenzyme activity (Vmax), or both, we performed T4 dose-response experiments. Cerebral homogenates from 1-,28-d-old and adult rats were incubated for 2 h with2 ,M T3 (instead of 1 ,uM) and with 0.2, 2, 5, 10, and 20nM T4. There was a progressive decrease in all the

HYPOTHALAMUS

o lo 20 30 40 so 60 70 80 90 loo 1,0-1307 24 60 /00 /70 235 300 360 380 402 43042

AGE (DAYS)WE/GHr (GRAMS)

FIGURE 1 T4 5'-deiodination and T3 tyrosyl ring deiodinationin homogenates of rat cerebral hemispheres. Each point isthe mean+SD of triplicate incubations using pooled tissuesfrom three to four rats. Incubations were carried out at 370Cunder N2 for 2 h in the presence of ~=0.2 nM 125I-T4 + "'0.1

nM [31I]T3 + 1 .uM [1271]T3 + 100 mMDTT. Asterisks indicateages at which rates are significantly different (P < 0.05 or

less) from the rates in the adult rats (60 d and older) bythe Mann-Whitney test. Values for weights, in italics belowthe abcissa, are average weights of male rats at the indicatedages. The curves are drawn through the mean rates for ageswith multiple points. (A) Net conversion of 125I-T4 to [125I]_T3 in 2 h. No correction was made for [125I]T3 degradation.(B) Conversion of [31I]T3 to [131I]3,3'-T2 + ['311]3'-T,. Fortechnical reasons, only T3 deiodination could be measuredin the 4 d incubations. More detailed analysis of the 1-d andadult incubations is given in Table I.

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AGE (DAYS)

FIGURE 3 T4 5'-deiodination and T3 tyrosyl ring deiodinationin homogenates of rat hypothalamus. Experimental detailsare given in the legend to Fig. 1.

125

I I I

1-T4 TO 125 T3A

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T4 and T3 Metabolism in Maturing Brain 1211

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homogenates in percent conversion of 125I-T4 to [125I1]T3 as the T4 concentration was increased, but whenrates were translated from percent conversion tofemtomoles T3 produced per minute per milligramprotein, the molar reaction rates were found to increasewith the T4 concentration. In these incubations theextent of [131I]T3 degradation was reduced to 6-15%,the extent of T4 degradation (largely to rT3) was reducedto 16-25% (compare Table I), and the protein concen-trations were similar in the homogenates from the pupsand the adults. The higher T3 concentration thus facili-tated comparisons of T45'-deiodination rates. Eadie-Hofstee plots of (reaction rate . average T4 concentra-tion) vs. molar reaction rate (14) were linear. The kineticparameters calculated from the plots are given in TableII. The apparent Km for T4 was 34% lower in the 1-d-old rats than in adults, whereas it was 45% higher inthe 28-d-old rats than in adults. The Vmax was not sig-nificantly different in the 1-d-old and adult rats, butthe Vmax in the 28 d rats was considerably higher thanin other groups, 2.6 times as high as that of the adult rats.

Changes in T3 tyrosyl ring deiodination rates duringmaturation. Total T3 tyrosyl ring deiodination rates(Figs. 1B, 2B, and 3B) declined progressively with age,attaining adult values at 14 d for cerebrum and cere-bellum and at 28 d for hypothalamus. In parallel in-cubations, using <1 nM T3, fractional T3 tyrosyl ringdeiodination rates were too rapid (>75%) in cerebraland hypothalamic homogenates at all ages to allowdifferences to be detected. However, in cerebellarhomogenates incubated with <1 nM T3, mean frac-tional T3 tyrosyl ring deiodination fell from 70% at 1 dto 6% at 14 d and remained at that level thereafter.These data show that adult cerebellum has real, butlow level, tyrosyl ring deiodinase activity which be-comes saturated at 1 uM T3.

The conversion of 125I-T4 to rT3 in the presence of1 ,M T3 or 1 ,uM rT3 showed the same pattern of de-crease with age as did total tyrosyl ring deiodinationof T3. That is, for each brain region, T4 tyrosyl ringdeiodination rates declined progressively from thehigh neonatal values (Table I) and reached adult levelsat the same age as total T3 tyrosyl ring deiodination.

TABLE IIKinetic Parameters of T4 5'-deiodinase

in Cerebral Homogenates

Age Apparent K. for T4 Vmax

nM fmol T3 per minper mg protein

1 d 9.5 (9.2, 9.9) 2.3 (2.0, 2.9)28 d 21.0 (17.4, 26.4) 3.9 (3.8, 4.1)Adult 14.3 (12.9, 16.1) 1.5 (1.0, 2.4)

Values in parentheses are 95% confidence limits.

DISCUSSION

The present studies extend the information availableabout in vitro thyroid hormone metabolism in brain.From a methodological standpoint, routine inclusion of1-2 ,M L-T3 in the incubations greatly facilitatedquantitation of T4 5'-deiodination, allowing initialestimates of kinetic parameters for T4 5'-deiodinase innormal rat brain. In heterogenous homogenates such asthose employed here, these parameters must be inter-preted cautiously, because nonenzymatic binding ofsubstrate can influence them greatly. They do havesome value in comparing physically similar systems.With that in mind, the apparent Kmfor T4 of T4 5'-de-iodinase in adult rat cerebral homogenates, 14 nM,was found to be very close to the apparent Km for T4of rat anterior pituitary homogenate T4 5'-deiodinase,8.8 nM (15). Both of these values are 35-1,000-foldless than the Kmfor T4 of rat liver and kidney homoge-nate T4 5'-deiodinases (16-23). From this kinetic evi-dence and from the responses of brain and anteriorpituitary T4 5'-deiodinase activity in hyper- and hypo-thyroidism (6, 8, 15), the suggestions may be drawnthat the brain and anterior pituitary T4 5'-deiodinasescould be the same, or very similar, and that both maywell be different from the liver and kidney T4 5'-de-iodinase (17).

The maturational pattern of T4 5'-deiodinase activityin the brain proved to be complex. T4 5'-deiodinaseactivity in homogenates of neonatal tissue was highestin the cerebrum and virtually absent in cerebellumand hypothalamus. In all three regions, the T4 5'-deiodination rates increased over the first 4 wk of life,attaining peaks above adult values, then declining toadult rates. For the cerebrum, this peak reflected anincrease in the total enzyme activity (Vmax); the modestdifference in apparent Km for T4 in the 28-d-old vs.adult rats was in a direction that would tend to lowerrates in the 28-d-old rats at tracer T4 concentrations.The quantitative differences between regions at allages reinforce our previous findings of regional diver-sity of T4 5'-deiodinase activity (6).

Other tissues show different developmental patterns.In rat anterior pituitary tissue, T4 5'-deiodination ratein vitro are elevated in the neonatal period, after 1 d,and decline to adult values by 28-45 d (8, 9). In ratliver homogenates, T4 5'-deiodination rates are muchlower in fetal and neonatal tissue than in adult tissue(7, 8) but the differences between neonatal and adultliver are lessened or abolished by DTT supplementa-tion (7, 8). There is some uncertainty about the timecourse of change of T4 5'-deiodinase activity in liver:Harris et al. (24) found normalization of rates in liverhomogenates at 5 d with (24) or without (7) a peakabove adult rates at 7 d, whereas we observed per-sistently low rates at 9 d and normal adult rates at21 d (8).

1212 M. M. Kaplan and K. A. Yaskoski

For iodothyronine tyrosyl ring deiodination, the pat-tern of changes was completely different. Hypotha-lamic tissue showed the highest rates initially andremained above adult values longest. In all threetissues, there was a progressive decline in iodothyro-nine tyrosyl ring deiodinase activity until a plateauwas reached at the adult values. Tanaka et al. (25) havereported in preliminary form that T4 and T3 tyrosylring deiodination is more rapid in homogenates of fetalrat brain than in homogenates of adult rat brain. Theirobservations are in accord with ours. The parallelchanges of T4 tyrosyl ring deiodination and T3 tyrosylring deiodination in these and previous experiments(6) leave little doubt that a single brain tyrosyl ringdeiodinase accepts T4 and T3 as substrates. In con-trast, the present results support our previous con-clusion (6) that the brain tyrosyl ring deiodinase is notthe same enzyme as the T4 5'-deodinase: neonatalhypothalamic homogenates have abundant tyrosyl ringdeiodinase activity and are devoid of T4 5'-deiodinaseactivity (Table I), whereas cerebellar homogenatesfrom hypothyroid adult rats have abundant T4 5'-deiodinase activity and are devoid of iodothyroninetyrosyl ring deiodinase activity (6). Rat liver andmonkey hepatocarcinoma cells contain iodothyroninetyrosyl ring deiodinases with catalytic propertiesgenerally similar to the rat brain enzyme (26-28),and adult rat liver has higher activity than neonatalrat brain (26, 27). Nonetheless, complete physicalseparation of hepatic phenolic and tyrosyl ring de-iodinase activities has not been achieved (29). Ourfindings in brain homogenates lead us to predict thathepatic phenolic and tyrosyl ring deiodinases willprove to be separate molecules.

By analogy with observations concerning T4 to T3conversion in the anterior pituitary, we have reasonedthat local T3 production in the brain is likely to bephysiologically significant (6). A recent preliminary re-port indicates that after 125I-T4 injection, the fraction ofinjected radioactivity that appears as T3 is greater inbrain extracts from hypothyroid rats than in extractsfrom normal rats (30). This finding is in agreement withour data that in vitro T4 5'-deiodinase activity is muchgreater in cerebral cortex and cerebellum of hypo-thyroid adult rats than in normal tissue (6). The co-herent patterns of change in activities of both de-iodinases in brain tissue during maturation reinforcethe idea that these enzymes may have an importantrole in the expression of thyroid hormone effects. Anobvious possibility is that the balance of intracellularT3 production and T3 degradation is a major deter-minant of steady state T3 tissue concentrations. It isdifficult to relate the in vivo data of Vigoroux et al. (5),to the present results, since they extracted whole brainsand since in vitro T3 production and T3 degradationrates at 10 d are changing rapidly and in opposite direc-

tions. The data in Figs. 1-3 suggest that 1-7, 28 and>60 d would be more suitable ages at which to comparebrain tissue levels of iodothyronines and in vivoiodothyronine metabolism to obtain further directevidence of the physiological significance of theiodothyronine metabolic pathways observed in vitro.

These studies, together with previous reports, sug-gest that there is a complex system of local regulationof thyroid hormone metabolism in target tissues,serving to modulate the expression of thyroid hormoneeffect. Many other factors that may regulate thyroidhormone secretion and actions on target tissuesundergo changes in the first several weeks of life inthe rat. These include the number of T3 nuclearreceptors in different brain regions and in the liver,the sensitivity of thyrotropin secretion to thyrotropin-releasing hormone and to circulating iodothyronineconcentrations, the plasma activity of the proteasesthat degrade thyrotropin-releasing hormone, the sen-sitivity of the thyroid gland to inhibition of secretionby iodide, and the sensitivity of the liver to plasmaT3 concentrations (31-39). Much additional work willbe required to integrate these diverse observations intoa complete picture of the regulation of thyroid hormoneaction in the developmental period.

ACKNOWLEDGMENTSWethank Dr. Ray Gleason for advice in the statistical analyses,Dr. Reed Larsen for helpful discussions and valuable ideas,and Ms. Melissa Jones for expert manuscript preparation.

This work was supported in part by Biomedical ResearchSupport Grant S07 RRO5489 from the Division of ResearchResources, and grant 1-ROl-AM 25340 OlAl, NationalInstitutes of Health.

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1214 M. M. Kaplan and K. A. Yaskoski