THEROLE OF INTERMEDIARY CARBOHYDRATE METABOLISMdm5migu4zj3pb.cloudfront.net/manuscripts/104000/104371/JCI6110… · 3 X10-3 M; KCl, 0.14 M. Usually, homogenates were rehomogenized

THE ROLE OF INTERMEDIARYCARBOHYDRATEMETABOLISMIN REGULATINGORGANICIODINATIONS IN THE

THYROID GLAND*

By GEORGEC. SCHUSSLERAND SIDNEY H. INGBARt WITH THE TECHNICAL ASSISTANCEOF PATRICIA EVELETH

(From the Thorndike Memorial Laboratory, Second and Fourth (Harvard) Medical Services,Boston City Hospital, and the Department of Medicine, Harvard Medical

School, Boston, Mass.)

(Submitted for publication February 16, 1961; accepted April 13, 1961)

As pointed out in recent reviews (1, 2), thesynthesis of hormonally active materials by thethyroid gland proceeds via a sequence of seeminglydiscrete reactions. These may be categorized asfollows: concentration of inorganic iodide from theextracellular fluid, oxidation and organic bindingof iodine to yield iodotyrosines, and coupling ofiodotyrosines to form the hormonally active io-dothyronines. The latter two groups of reactionsoccur within the matrix of the thyroglobulin mole-cule, a large molecular protein for whose syn-thesis and storage the thyroid gland is especiallyadapted.

It seems clear that ultimately the energy forthese synthetic processes, as well as for the main-tenance of glandular architecture, must derive frommetabolic sources. However, little is known ofhow the energy released during intermediary me-tabolism is imparted to those reactions involved inhormonal biosynthesis. Previous studies have in-dicated that the iodide-concentrating function ofthe thyroid is dependent on oxidative processesleading to the formation of high energy phosphatebonds (3, 4). The present studies were under-taken to determine whether a comparable linkto metabolic events could be delineated in the caseof the next stage in hormonal synthesis, the oxi-dation of iodide and formation of monoiodotyro-sine. From the results obtained, a metabolicsequence is postulated whereby the oxidation of

* This investigation was supported in part by Re-search Grant no. A-267 from the National Institute ofArthritis and Metabolic Diseases, Bethesda, Md., andin part by the Medical Research and Development Board,Office of the Surgeon General, Department of the Army,under Contract no. DA-49-007-MD-412. Presented inpart at the 1959 meeting of the Endocrine Society, June2, 1959, Atlantic City, N. J.

t Investigator, Howard Hughes Medical Institute.

iodide is regulated by glucose metabolism via reac-tions coupled to pyridine nucleotide-linked dehy-drogenations. Evidence is presented which sug-gests that the effects of thyrotropin (TSH) onorganic iodinations in the thyroid may be mediatedby an enhancement of glucose metabolism withinthe gland.

METHODS

Experiments were performed with slices or homoge-nates prepared from thyroid glands which had been ob-tained at the abattoir from freshly killed sheep or lambsand brought to the laboratory packed in ice. Through-out the preparative procedure, the tissues were chilled at0 to 40 C.

Preparation of homogenates. Several thyroid glandswere employed in the preparation of a single homogenate.Thyroids were stripped of fat and fascia, minced witha Stadie-Riggs blade and homogenized with a Potter-Elvehjem glass homogenizer in 5 vol of a medium con-taining phosphate buffer (pH 7.4), 6 X 10' M; MgSO4,3 X 10-3 M; KCl, 0.14 M. Usually, homogenates wererehomogenized with a tight-fitting Teflon homogenizerprior to incubation. No further efforts were made toeliminate intact cells. Since it was the purpose of theseexperiments to relate organic iodination to carbohydratemetabolism, the medium chosen was identical in termsof inorganic constituents to that described by Wenner,Dunn and Weinhouse as supporting glycolysis in cell-free systems (5). Aliquots of homogenate (1.7 ml)were pipetted into the main compartment of Warburgvessels. In many experiments, the sodium salt of glu-cose-6-phosphate 1 was added to a final concentration of0.015 M. After gassing of flasks had been completed and

1 The following abbreviations will be used in text,tables and illustrations: glucose-6-phosphate, G-6-P; tri-phosphopyridine nucleotide (oxidized), TPN; triphospho-pyridine nucleotide (reduced), TPNH; diphosphopyri-dine nucleotide (oxidized), DPN; diphosphopyridine nu-cleotide (reduced), DPNH; flavin adenine dinucleotide(oxidized), FAD; flavin mononucleotide (oxidized),FMN; glutathione (oxidized), GSSG; glutathione (re-duced), GSH; methylene blue (oxidized), MB; nor-ethylmaleimide, NEM.

1394

THYROID INTERMEDIARY METABOLISMAND ORGANICIODINATIONS

temperature equilibration achieved, the final volume ofhomogenate was brought to 2 ml by tipping into the maincompartment 300 ,Al of medium containing 23 ,sc of car-rier-free inorganic I131,2 and the substrates, cofactors, en-zymes or inhibitors to be tested. CO. was absorbed by300 ,l of 10 per cent KOHin another side-arm or in thecenter well. Incubation was usually carried out for 1hour in a Warburg apparatus at 380 C, under an atmos-phere of 100 per cent oxygen. A few experiments wereperformed in Erlenmeyer flasks under oxygen in ametabolic shaker.

In certain experiments, an alternative method was em-ployed. When materials to be tested had limited solu-bility, these were dissolved together with the radio-active iodide in 1 ml of medium. A more concentratedhomogenate was then added to achieve a final homoge-nate of the usual volume and concentration. A similarprocedure was followed in homogenate experiments withC14-labeled glucose 3 in order that the exact quantity ofradioactivity added to each vessel could be determined.In the latter experiments, the final concentration of glu-cose was 0.3 to 0.5 mg, containing 1 ,sc of glucose-C", perml.

Preparation of slices. Thyroid slices, rather than ho-mogenates, were employed in experiments designed toassess the effects of TSH on the metabolism of C"-labeled

I'3l organified (per cent) =

glucose. Slices, 50 to 100 mg in weight, were cut with aStadie-Riggs microtome and were incubated in 2 mlof Krebs-Ringer-phosphate medium (KRP) containing1 gc of C"-labeled and 1.5 mg of stable glucose per ml.TSH, 1 U,4 was added to each experimental vessel in100 pul of KRP, and a similar volume of KRP was addedto the controls. Incubation was carried out at 38° Cunder 100 per cent oxygen in a Warburg apparatus.CO. was absorbed by 300 ,l of 10 per cent KOH in aside-arm. Similar experimental conditions were em-ployed to assess the effects of oxidized methylene blue(MB) on glucose metabolism.

Measuremnent of IP1-organification. After incubation,the reaction was stopped by chilling each vessel and add-ing 25 Anl of 0.08 Mmethimazole. The percentile organi-fication of iodide was usually determined by precipitationwith trichloroacetic acid (TCA). Aliquots of homoge-nate (20 Al) were added to 0.7 ml of a solution of hu-man serum albumin (1.4 g per 100 ml) in 13 X 100 mmtest tubes. Two ml of 10 per cent TCA was then added,the resulting precipitate was washed three times with5 per cent TCA, dissolved in 2 N NaOH, and counteddirectly in a well-type scintillation counter. Pooled su-pernatants were alkalinized to prevent loss of iodide, di-luted to standard volume and counted. Percentile organi-fication was calculated according to the following formula:

(I13l in precipitate) X 100(I131 in precipitate) + (I131 in pooled supernatant)

Chromatographic methods were used to characterize thenature of the iodinated products formed by the homoge-nate. Aliquots of homogenate (20 gl) together withcarrier iodide, mono- and diiodotyrosine (MIT and DIT),were applied directly to strips of Whatman no. 1 filterpaper. Following chromatography in butanol: dioxane:2 N ammonia (4: 1: 5) and in butanol: 2 N acetic acid(1: 1) systems,5 radioautographs were prepared. Car-

2 Inorganic I13. was obtained from Abbott Labora-tories, Oak Ridge, Tenn., and was free, by paper chro-matography, of noniodide contaminants described by otherworkers (6).

3 C"-labeled glucose was obtained from the New Eng-land Nuclear Corp., Boston, Mass. Samples wereapparently pure according to radioactive scans of chro-matographic strips. Radioautography of samples chro-matographed in butanol: acetic acid: water (78: 10: 12)occasionally revealed a faint band in the zone occupied bylabeled lactic acid. Direct counting of these zones re-vealed that this area contained no more than 0.1 per centof the total counts on the strip (7). Measurement of thiscontaminant as C"O, could not have accounted for a sig-nificant proportion of the acid volatile counts producedby homogenates under the influence of stimulatory co-factors.

4 Thytropar, Armour.5 Chromatographic solvents were enriched with propyl-

thiouricil in order to prevent oxidation of compoundsduring chromatography.

rier compounds were then localized by spraying with diaz-otized sulfanilic acid for MIT and DIT, and with pal-ladium chloride for iodide. For quantitative analysis, ra-dioactive zones located by radioautography were cutfrom unstained chromatographic strips and were countedin a well-type scintillation counter.

In both chromatographic systems, three major zonesof radioactivity were consistently seen in chromatogramsof unhydrolyzed homogenate: a band at the point ofapplication (origin material), a band coinciding withcarrier inorganic iodide, and a less well defined band seenat the solvent front (front material). The latter com-prised a variable proportion (30 to 60 per cent) of thenon-inorganic I"'. A band between iodide and frontmaterial, similar to the Unknown 2 (U2), first describedby Taurog, Tong and Chaikoff (8), was occasionallyseen but never constituted a significant portion of thetotal I13. After tryptic hydrolysis of the homogenate,the radioactivity at the origin decreased and radioac-tivity appeared in the zone of carrier MIT. This indi-cated that the radioactive material at the origin was pri-marily protein containing MIT in peptide linkage.Unlike the origin material, iodinated material at the sol-vent front was readily eluted from chromatographicstrips by chloroform, methanol, ether and butanol. Fol-lowing rechromatography of the eluted front-runningZ!gmaterial, the radioactivity again appeared at the solventfront and coincided with an area which stained withSudan black. These findings indicated that the front-running material was an iodinated lipid, suggesting that,

1395

GEORGEC. SCHUSSLERAND SIDNEY H. INGBAR

in the homogenate system, oxidation of iodide results inthe iodination of both proteins and lipid.

Chromatography was also performed to validate the use

of TCA-precipitation as a measure of organified iodine.Direct chromatography of the TCA-precipitate showedradioactivity at both the origin and solvent front, butnot in the iodide area. Chromatography of TCA-su-pernatants revealed only inorganic iodide. A chloroform:methanol extract of the TCA-precipitate contained onlyfront-running material; the residual I.3. in the precipitatewas entirely composed of origin material. A saline ex-

tract of the precipitate showed only origin material. Theability to extract the front material prior to chromatog-raphy supported the conclusion that the front materialwas lipid iodinated in the homogenate, rather than a

chromatographic artifact. Thus, both front and originmaterial appeared to be true products of iodination inthe homogenate system. Since both were found in TCA-precipitates and not in supernatants, the precipitationtechnique could be considered as valid as chromatographyfor the assessment of the organification of radioiodide.

C"402 production. The C1402 evolved during incubationof tissues with labeled glucose wvas absorbed in 300 ,Alof KOHin the side-arm of Warburg flasks. At the endof the incubation, tissues and media were removed andreplaced by 2 ml of 5 M H2SO4. Two hundred ,ul of 0.5M Hyamine was placed in the center well and the flaskswere tightly stoppered. The acid and alkali were thenthoroughly mixed and the flasks were gently shaken atroom temperature for 30 minutes. The Hyamine, now

containing CO2 released from the alkali, together withseveral washes of the center well, was transferred to glasscounting vials containing liquid scintillator. Sampleswere cooled overnight and then counted in an automaticliquid scintillation counter. An aliquot of the originalincubation medium was counted as a standard. Internalstandards were used to correct for variations in count-ing efficiency. The quantity of C"402 produced was ex-

pressed as a per cent of added C14-labeled glucose per

gram of tissue.Miscellaneous methods. The reduction of added pyri-

dine nucleotide coenzymes was assessed by measuringthe optical density at 340 ma of supernatants obtainedfrom 1: 3 (vol/vol) mixtures of homogenate and 95 per

cent ethanol (9). When necessary, Celite was added as

a clearing agent. Analyses for stable glucose were per-

formed by an enzymatic method (10).

RESULTS

Duplicate or triplicate vessels were incubated inall experiments. Results, presented represent themean of individual determinations, which, withfew exceptions, agreed closely.

Organic iodinations

Anoxia (Table I). Despite wide variations inpercentile iodinations obtained in control vessels,

TABLE I

The effect of anoxia on organic iodinations in plain andsupplemented homogenates of sheep thyroid glands

Organic 1131E.x )t. Compound

no. added* 02 N2

%total4 0 8.4 0.8

15 0 1.6 0.518 0 3.9 0.5

24 0 3.1 0.6DPN 2.8 0.5

33 0 2.4 0.5DPN 2.3 0.4TPN 3.7 0.4TPNH 3.2 0.4

110 0 0.9 0.3FAD 1.3 0.4

TPN + FAD 6.2 0.6

34 0 11.1 0.4CuCl2 39.8 0.9HgCl12 33.0 1.8

42 MB 5.5 0.3

* Compounds added to a final concentration of 0.0007 M,except in the following instances: CuC12, 0.0125 M; HgC12,0.0005 M; MB) 0.00018 M.

organic iodinations were markedly depressed byanaerobic incubation. Comparable inhibition ofiodination was effected by anoxia, even in thepresence of factors which were highly stimulatoryto iodinations when oxygen was present. At lowerpartial pressures, the system was especially sen-sitive to the partial pressure of oxygen, but maxi-mal rates of iodination were not achieved until theatmosphere was 100 per cent oxygen (Figure 1).

ORGANICI'3'( %total I'3' )

4.0-

3.0

2.0

L1.* I

0 20 40 60

PERCENT02 IN ATMOSPHERE

80 100

FIG. 1. THE EFFECT OF VARYING OXYGENTENSIONS ON

ORGANIC IODINATIONS IN A HOMOGENATEOF SHEEP THY-

ROID GLANDS.

1396


TABLE 11

The effects of dialysis and storage on organic iodinations inhomogenates of sheep thyroid glands *

Organic I131

Control Dialyzed Stored

%total %total %total1 1.3 8.2 2.93 4.9 11.3 7.84 8.4 29.2 9.49 5.4 30.09at 3.3 25.0

16 1.3 13.918 3.9 4.522 1.1 2.122at 0.9 1.726 2.8 13.560 2.7 3.763 3.3 22.268 1.5 20.969 1.9 24.4

* An aliquot of the control homogenate either stored ordialyzed for 16 hours against 25 vol of homogenizationmedium at 4° C prior to incubation with inorganic I'll.

t Glucose-6-phosphate (0.015 M) added to control anddialyzed homogenates prior to incubation.

Dialysis and storage (Table II). Dialysis ofhomogenate for 16 hours at 40 C against 24 volof the homogenization medium consistently in-creased the percentage of added 1131 organified.In 12 experiments, percentile iodinations in dia-lyzed homogenates averaged 650 per cent of thevalues obtained the day before in undialyzed con-trols. Iodinations in homogenates stored at 40 Cfor the same period of time were also consistently,although less markedly, stimulated.

Suilfhydryl inhibitors. The addition of CuCl2markedly stimulated organic iodinations. In eightexperiments, iodinations in the presence of CuCl2(0.0125 M) averaged 692 per cent of control val-ues. In four experiments, performed in the samehomogenates before and after dialysis, the stimu-latory effect of CuCl2 was abolished or greatly di-minished in the dialyzed samples (Table III).

The addition of HgCl2 also produced a pro-nounced stimulation of iodinations which wasabolished by prior dialysis of the homogenate(Table III).

NEM, a specific sulfhydryl inhibitor (11), wasstimulatory at concentrations between 2 x 10-4and 10-3 M; higher concentrations were inhibitory(Figure 2). After dialysis of the homogenate,stimulatory effects of NEMwere diminished orchanged to inhibition (Figure 3).

Glutathione. Iodinations in the homogenate

TABLE III

The effects of heavy metals on organic iodinations in homo-genates of sheep thyroid glands

Organic I131Expt. Concen-

no. tration Control Inhibitor

M % totalCuC12

34 0.0125 11.1 39.866 0.0125 9.0 20.276 0.0125 2.3 24.079 0.0125 2.8 26.6

109 0.0125 3.6 14.74 0.0025 8.4 8.3

0.0040 8.4 14.00.040 8.4 7.2

4-D* 0.0040 29.2 17.026 0.0005 2.8 3.2

0.0025 2.8 8.30.0125 2.8 16.6

26-D 0.0125 13.5 11.368 0.0125 1.5 13.968-D 0.0125 20.9 31.669 0.0125 1.9 18.369-D 0.0125 24.4 31.4

HgC1234 0.0005 11.1 33.026 0.0005 2.8 27.1

0.0025 2.8 4.70.0125 2.8 3.2

26-D 0.0005 13.5 12.6

* D indicates homogenate dialyzed against 25 vol ofhomogenization medium for 16 hours at 40 C prior to addi-tion of inhibitor and incubation with I'3l. Effects ofinhibitor had also been tested in the same homogenateprior to dialysis.

system were inhibited by the addition of glutathi-one. When compared in the same homogenates,0.02 1\1 GSHwas more inhibitory than an equiva-lent concentration of GSSG. In other experi-

ORGANIC(% Cont,

500

400

300

200

100

I

2 5 2 5 2 5x 10-5 XK-4 x10 3

NORETHYLMALEIMIDECONCENTRATION(Molority)

FIG. 2. THE EFFECT OF VARYING CONCENTRATIONSOF

A SULFHYDRYL INHIBITOR, NORETHYLMALEIMIDE, ON OR-

GANIC IODINATIONS IN HOMOGENATESOF SHEEP THYROID

GLANDS.

1397


ORGANIC1i3'( %Control )

400

300

200

100

Undiolyzed Homogenate

Dialyzed Homogenate

, ,---------

2.5x10-4 5x10-4 Ix 10-3 2x10-3NORETHYLMALEIMIDECONCENTRATION(Molority)

FIG. 3. THE EFFECT OF DIALYSIS OF SHEEP THYROID

HOMOGENATESON THE RESPONSEOF ORGANIC IODINATIONSTO THE ADDITION OF NORETHYLMALEIMIDE. Iodinationswere measured prior to and following dialysis of homoge-nate at 4° C against 24 vol of homogenization mediumfor 16 hours. In each instance, the control value is thatobtained in appropriately treated homogenate not sup-

pllemented with norethylmaleimide.

ments, the inhibitory effect of GSSGwas incon-stant. However, when TPN (7 x 10-4 M) was

added, iodinations in the presence of GSSGwere

more often lower than those found in control,unsupplemented vessels (Figure 4). The con-

sistent and pronounced inhibitory effect of GSSG

GSSG TPNH

ORGANIC 3GSH)TPN(% Control) Glutothione Reductose

160

140

120

100--

80

60

40

20 *

GSSG GSH GSSG TPNGSSG

FIG. 4. EFFECT OF OXIDIZED AND REDUCEDGLUTATHIONE

ON ORGANIC IODINATIONS IN SHEEP THYROID HOMOGE-

NATES AND THE EFFECT OF ADDEDTPN ON THE RESPONSE

TO OXIDIZED GLUTATHIONE. Oxidized and reduced glu-

tathione are compared directly in two experiments, as

shown on the left. Enhancement of the response to

oxidized glutathione by TPN is shown on the right.

Each symbol represents results obtained in an individual

experiment. Schema in enclosed box depicts enzymatic

pathway for the reduction of oxidized glutathione.

FIG. 5. MODIFICATION OF THE TPN EFFECT ON ORGANIC

IODINATIONS BY GLUTATHIONE AND BY A LABILE ENDOGE-

NOUSINHIBITOR. Shown on the left are results of ten ex-periments in which the effects of TPN, with and withoutadded oxidized glutathione, were compared. TPN-GSSG/TPN represents ratio of iodinations obtained inthe same homogenate under the two conditions, expressedas a per cent. On the right are shown effects of dialyz-ing or storing homogenates on the response to TPN.Dialysis experiment performed in a single homogenate;storage experiment in another. Values expressed as aper cent of control value obtained in either fresh, dialyzedor stored homogenates, not supplemented with TPN.

in the presence of TPN was best demonstrated,however, when iodinations in vessels containingboth cofactors were compared with those ob-tained with TPN alone (Figure 5).

Glycolytic substrates (Table IV). A numberof substrates was tested. The addition of succinateconsistently stimulated iodination. Other sub-strates did not seem to have a marked or con-sistent effect.

Pyridine nucleotide coenzymes. Both TPN(7 x 10-4 M) and DPN (7 x 10-4 M) usuallystimulated iodinations when addedkto thyroid ho-mogenates (Figure 6). In 45 experiments, or-ganic iodinations in TPN-enriched homogenatesaveraged 182 per cent of control values. The ef-fect of DPNwas less marked and less consistent.In 24 experiments DPNincreased iodinations byan average of only 27 per cent, and in 7 of theseexperiments there was no stimulatory effect. Theimpression that the effect of TPN was more pro-nounced than that of DPNwas confirmed by com-paring effects of the coenzymes in the same ho-mogenate. In each of 20 such experiments, TPNwas the more stimulatory and mean iodination inTPN-supplemented flasks was 153 per cent of

1398


TABLE IV

The effects of glycolytic substrates on organic iodinations inhomogenates of sheep thy'roid glands

Organic I131Expt.

no. Substrate* Control Substrate

%total31 Glucose 3.0 2.732 Glucose 1.7 1.9

4 G-6-P 8.4 5.19 G-6-P 5.4 3.39-D G-6-P 30.0 25.0

22 G-6-P 0.9 1.138 G-6-P 1.8 2.039 G-6-P 2.5 2.335 Fructose 1.1 0.858 Pyruvate 17.6 18.237-D Citrate 58.6 60.158 Citrate 17.6 15.240 Isocitrate 0.6 1.040 a-Ketoglutarate 0.6 0.638 Succinate 1.8 3.139 Succinate 2.5 3.740 Oxaloacetate 0.6 0.858 Oxaloacetate 17.6 21.9

* All substrate added to a final concentration of 0.02 M,except glucose-6-phosphate, which was added to a finalconcentration of 0.015 M.

that obtained in vessels supplemented with DPN.The rate of reduction of added TPN in a homoge-nate containing G-6-P is compared to the rate oforganic iodinations in Figure 7. Following theaddition of TPN, the concentration of TPNHrapidly increased, reaching a virtually constantvalue within 5 minutes. Iodinations, however,continued at an accelerated rate for the remainderof the experiment. Although a second additionof TPN again resulted in a rapid increase in the

ORGANIC Iv'

5001%Control)

400k

300 F

200

100

TPN DPN TPN/DPN.0007M .0007 M

FIG. 6. EFFECTS OF TPN AND DPN ON ORGANIC IO-DINATIONS IN SHEEPTHYROID HOMOGENATES. TPN/DPNrepresents ratio, expressed as per cent, of values foundin 20 experiments in which effects of TPN and DPNwere compared in the same homogenate.

GD. 340A ORGANIGTUI(%@ol

08 - 3h.5 -0~1.24 30 6

1.0-~ ~ ~ ~ '2.5

0.8 -2.0-

0.6 1.5

0.4A 1.0

0.2 J0.5

~~~~~900 90MINUTES MINUTES

FIG. 7. THE RELATION BETWEENORGANIC IODINATIONSANDTHE REDUCTIONOF TPN IN A SHEEPTHYROID HOMOGE-NATE. At the indicated times, TPN tipped into homoge-nates from the side-arm of \Varburg vessels in quanti-ties sufficient to enhance the concentration of TPN by0.0007 M.

concentration of TPNH, there was no furtherstimulation of iodinations. Following the additionof comparable concentrations of DPN, the in-crease in concentration of DPNHwas far lessthan had been the case with TPN.

The effects of TPN and DPN were modifiedby supplementing homogenates with reducirgsystems specifically requiring one of the coenzymes

TABLE V

lModification of the stimulatory effects of pyridine nucleotidecoenzymes on organic iodinations by the addition of their

specific reducing systems

Percentileorganic

iodinations(% control

Compound added Concentration* iodinations)

DPN 0.0007 118

DPN 0.0007 95Ethanol 0.01

DPN 0.0007Ethanol 0 01 87Alcohol dehydrogenaset 0.023

TPN 0.0007 127Isocitrate 0.02 121Isocitric dehydrogenaset 1.0 100

TPN 0.0007Isocitrate 0.02 152Isocitrate dehv-drogenase 1.0

* Molar concentrations of all compounds except alcoholand isocitric dehy'drogenases, for which concentrationsgiven represent mg/ml homogenate.

t Cryst. from yeast, Sigmna Chemical Co., 100 to 150,000U/mg.

$ Type 1, crude, Sigma Chemical Co., 30 U/mg.

-i...- *----.

1399


ORGANICI'V'XX Control)

150 H

125 F

50k

25-

7x 10'- 7x 10-6 7x lo-5 7xO1-4TPN CONCENTRATION(Molority)

FIG. 8. THE EFFECTS OF VARYING CONCENTRATIONSOF TPN ON ORGANIC IODINATIONS IN SHEEP THYROIDHOMOGENATES.

as hydrogen acceptor. The addition of isocitricacid and isocitric dehydrogenase potentiated thestimulatory effect of TPN on iodinations; the ad-dition of ethyl alcohol plus alcohol dehydrogenasechanged the stimulatory effect of DPN to a mod-erate inhibition. Similar results were obtainedin several experiments, one of which is shown inTable V.

When added to the same homogenates in whichthe usual concentration of TPN (7 x 10-4 M) wasstimulatory, lower concentrations of TPN (7 x10-s to 7 x 10-7 M) failed to stimulate iodinationsand, indeed, were moderately inhibitory (Figure8). The effects of DPN (7 x 10-4 M), and ofTPN at 0.1 its usual concentration (i.e., 7 x 10-5M), alone and in combination, are compared in

+ DPN

FIG. 9. SYNERGISTIC EFFECTS OF TPN AND DPN ON

ORGANIC IODINATIONS IN SHEEP THYROID HOMOGENATES.

TPN and DPN, final concentration 0.0007 M.; 0.1 TPN,final concentration 0.00007 M. Each symbol representsresults obtained in a single experiment.

Figure 9. The addition of both coenzymes produceda stimulation greater than that expected from thesum of their individual effects, and iodinationswere then usually equal to or greater than thoseobtained with the higher concentrations of TPNalone.

Prior dialysis of homogenates altered the re-sponse to TPN. Representative results are shownin Figure 5. In a homogenate in which the fullconcentration of TPN (7 x 10-4 M) was notstimulatory prior to dialysis, stimulation was pro-duced by TPN after the homogenate had beendialyzed. The usual inhibitory effect of lowerconcentrations of TPN (7 x 10-5 M) was alsochanged to stimulation by dialysis. In separateexperiments, a similar potentiation of the responseto TPN was achieved by storage.

When TPN (7 x 10-4 M) was added to ho-mogenates containing stimulatory concentrationsof NEM, the resulting increase in iodinationswas considerably greater than the stimulation pro-duced by the addition of TPN alone (Figure 10).Even concentrations of TPN which were inhibi-tory when added alone (7 x 10-5 M) became stim-ulatory when added together with NEM. The ad-dition of GSSGabolished the stimulatory effectof TPN and produced a consistent inhibition ofiodinations, as compared with iodinations obtainedin vessels supplemented with TPN alone (Figure5).

Methylene blue (Table VI). In seven sepa-rate homogenates to which MB (1.8 x 10-4 M)was added, iodinations were increased by anaverage of 94 per cent. The addition of TPN

ORGANICI'5

400[

300F

200

100

. 2.5x10-4 5XIO-4 IxIO-3 2xuI3

NORETHYLMALEIMIDECONCENTRATION(Molority)

FIG. 10. THE EFFECT OF TPN AND NORETHYLMALEI-

MIDE, ALONE AND IN COMBINATION, ON ORGANIC IODINA-

TIONS IN A SHEEP THYROID HOMOGENATE.

- z/v\4+~~~~ NEW

r^P,~~~~E./ 0\__ __ 0"___ ____ _ __ _ __ _

1400

75 h


TABLE VI

The effects of methylene blue and cytochrome C on organiciodinations in homogenates of sheep thyroid glands

Organic 1131

CompoundExpt. no. Control added

%totalMethylene blue (0.00018 M)

42 2.5 5.542a* 5.2 10.145 1.3 3.566 9.0 11.968 1.5 2.268-Dt 20.9 38.669 1.9 4.869-D 24.4 32.276 2.3 4.478 3.5 5.1

Cytochrome C (0.0007 M)57 2.4 1.668 1.5 2.168-D 20.9 26.469 1.9 1.869-D 24.4 24.776 2.3 3.7

* Homogenate supplemented with TPN (0.0007 M).t D indicates homogenates dialyzed against 25 vol of

homogenization medium for 16 hours at 4° C prior to addi-tion of compound and incubation with I'3l. Effects ofcompound also tested in same homogenate prior to dialysis.

with MBproduced a further stimulation of iodina-tions.

Flavin coenzymes. FAD had a weak and in-constant stimulatory effect, producing an averageincrease of 22 per cent in organic iodinations inseven experiments. In the same homogenates,

FAD TPN TPN+ FAD.0007 M .0007 M .0007M .ooO7M

FIG. 11. SYNERGISTIC EFFECTS OF TPN AND FAD ON

ORGANIC IODINATIONS IN SHEEP THYROID HOMOGENATES.

Concentrations shown represent final concentration ofadded compound in homogenate. Each symbol representsthe results of an individual experiment.

TABLE VII

The effect of light on organification of I'll in a solution ofhuman serum albumin and in a homogenate of sheep

thyroid glands

Conditions Organic 1131

Expt. Cofactor ofno. added* experiment No cofactor Cofactor

%totalIncubation in human serum albumin (2.5 g/100 ml)

100 FAD Dark 2.0FMN Dark 2.0FAD Light 2.7FMN Light 2.7

108 TPN Light 0.8FAD Light 0.8TPN+FAD Light 0.8TPN Light-N2t 0.6FAD Light-N2 0.6TPN+FAD Light-N2 0.6

Incubation in thyroid homogenate110 TPN Dark

TPN LightFAD DarkFAD LightTPN+FAD DarkTPN+FAD LightFAD Light-N2TPN+FAD Light-N2

2.12.64.16.9

0.72.82.20.61.00.8

0.9 1.90.7 1.90.9 1.30.7 2.60.9 5.80.7 6.20.3 0.40.3 0.6

* Cofactors added to a final concentration of 0.0007 M.t N2 indicates incubations performed in atmosphere of 100% nitro-

gen. All others performed in 100% oxygen.

TPN alone increased iodinations by an average of80 per cent. However, when the two coenzymes

were added together, iodinations were increasedby 252 per cent (Figure 11). In a single experi-ment, FMN stimulated iodinations moderately.

Cytochrome C (Table VI). Cytochrome C hadonly a slight or inconstant effect on organic iodina-tions, whether added alone or with other cofactors.

Effects of light (Table VII). Iodinations inthe tissue-free system were markedly increasedby the addition of FAD or FMN when flaskswere incubated in room light reinforced by an ul-traviolet lamp. This effect was greatly diminishedby the exclusion of light or oxygen. Even in thepresence of light and oxygen, TPN did not stimu-late iodinations in the tissue-free system, nor didit enhance the stimulatory effect of FAD.

In thyroid homogenates, the presence or absenceof light had no effect upon control iodinations.The increase in iodinations which followed the ad-dition of FAD was partially inhibited in the dark.On the other hand, the increase in organic iodina-tions due to TPN, as well as the marked synergismbetween TPN and FAD, was unaffected by thepresence or absence of light.

Metabolic inhibitors (Table VIII). KCN in-hibited organic iodinations. Antimycin A had a

very slight stimulatory effect when added alone

1401


TABLE VIII

The effect of metabolic inhibitors and antithyroid agents onorganic iodinations in homogenates of sheep thyroid glands

Organic 1131Expt.

no. Compound added Concentration Control Inhibitor

M 0 totalMetabolic inhibitors

4 KCN 0.010 8.4 0.849 KCN 0.010 3.8 0.152 KCN 0.010 4.8 2.130 2,4-Dinitrophenol 0.00010 6.5 6.7

0.00025 6.5 6.50.00050 6.5 6.60.0010 6.5 6.5

38 Malonate 0.010 1.8 3.239 Malonate 0.010 2.5 3.371 Iodoacetate 0.010 1.4 0.2

Norethylmaleimide 0.010 1.4 0.580 Anitmycin A 0.000003 2.4 2.781 Antimycin A 0.000003 1.4 1.5

Antithyroid agents36 Propylthiouracil 0.010 2.2 0.3

Perchlorate 0.001 2.2 2.30.010 2.2 2.2

Thiocyanate 0.050 2.2 0.249 Methimazole 0.003 3.8 0.2

or together with TPN or DPN. Dinitrophenolhad no effect. In two experiments, malonate(0.02 M) stimulated iodinations.

Antithyroid drugs (Table VIII). Methimazole,propylthiouracil and thiocyanate greatly inhibitediodinations; perchlorate had no effect.

Glucose metabolism

The effect on glucose metabolism of variousfactors which stimulate organic iodinations is

TABLE IX

The effect of factors stimulatory to organic iodinations on theproduction of carbon dioxide from glucose of h9mogenates

of sheep thyroid glands

C1402 production (% addedglucose/g tissue)

Control Compound addedExpt. Compound

no. added 1-C1402 6-C1402 1-C1402 6-C1402

96 DPN 0.09 0.01 2.33 0.18DPN+TPN* 0.09 0.01 6.94

97 DPN 0.14 0.02 1.57 0.04TPN 0.14 0.02 1.41 0.12TPN* 0.14 0.02 0.31 0.02DPN+TPN* 0.14 0.02 8.70 0.08

101 DPN 0.23 0.01 1.64 0.02102 TPN 0.44 0.10 0.82 0.16

FAD 0.44 0.10 0.58 0.15TPN+FAD 0.44 0.10 9.10 0.25

103 TPN 0.08 0.01 0.47 0.05TPN+FAD 0.08 0.01 10.08 0.12TPN+FMN 0.08 0.01 10.77 0.17

105 TPN 0.49 0.07 3.86 0.04TPN+FAD 0.49 0.07 3.67 0.07

106 TPN 0.09 0.03 0.40 0.04TPN+FAD 0.09 0.03 0.93 0.02

101 CUC12* 0.23 0.01 0.03 0.02HgCI2* 0.23 0.01 0.02 0.00

* Asterisk indicates TPN, 1 X10-5 M; CuC12, 12.5 X10-3 M; HgCI2,S X10-4 M. All other concentrations, 7 X10-4 M.

shown in Table IX. As compared with slices, un-

supplemented homogenates metabolized glucoseto CO2 at a relatively slow rate. During 1 hourof incubation, the C1402 recovered from glucoselabeled in the 1 and 6 positions (1_Cl402 and6-C1402) averaged 0.23 and 0.04 per cent per g oftissue, respectively, in eight experiments. In seven

experiments with slices incubated for a comparableperiod, the production of 1-C1402 averaged 3.86per cent and 6-C"402 1.53 per cent of added C14-labeled glucose per g of tissue.

Pyridine nucleotide coenzymes. Both DPNand TPN stimulated the production of C1402from labeled glucose. In six experiments, TPNstimulated the production of 1-C1402 by an

average of 756 per cent and the production of6-C1402 by an average of 200 per cent. DPN,in three experiments, stimulated 1-C1402 by an

average of 1,233 per cent and 6-C1402 by 800 per

cent. In two experiments, the effects of full con-

centrations (7 x 10-4 M) of TPN and DPNwere

compared in the same homogenate. In one ofthese, the stimulatory effects of the coenzymes

were essentially the same. In the second experi-ment, in which iodinations were also measured,TPN caused a greater increase in the productionof 1-C140'2 than did DPN. In this experiment,iodinations were slightly stimulated by TPN andwere unaffected by DPN.

The interaction of DPN with TPN at 0.1 itsusual concentration (i.e., 7 x 10-5 M) was alsoevaluated in two experiments. In both, the lowerconcentration of TPN stimulated 1-C1402 pro-

%Ii3Orgonified

C'4 02%MediumGlucose C14

perGm. Tissue

3

2

0

8.8

8.6]

.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2F-i--

I-C"O,H6-C-Oe

nTCONTROL DPN TPN DPN+TPN

.0 007M .00007 M

FIG. 12. THE EFFECTS OF TPN AND DPN, ALONEAND

IN COMBINATION, ON ORGANIC IODINATIONS AND THE

EVOLUTION OF C402 FROM GLUCOSE-1-C"4 AND GLUCOSE-

6-C14 IN A SHEEP THYROID HOMOGENATE.

1402


4

7. 113 3Organiied 2

0 I0CONTROL TPN FAD TPN+FAD

.0007 M .00075

FIG. 13. THE EFFECTS OF TPN AND FAD, ALONEAND

IN COMBINATION, ON ORGANIC IODINATIONS AND THE EVO-

LUTION OF C"O02 FROM GLUCOSE-1-C" AND GLUCOSE-6-C'IN A SHEEP THYROID HOMOGENATE.

duction only slightly as compared with the usualeffect of the tenfold greater concentration of TPN.The generation of 6-C1402 was also increased,but to a relatively slight extent. In one of theseexperiments, iodinations were also measured(Figure 12). These were slightly increased byDPNand markedly increased by the combinationof coenzymes.

Flavin coenzymes. FAD increased the yield of1-C1402, but to a lesser extent than did TPN (Fig-ure 13). The addition of FAD with TPN pro-duced a greater stimulation of both C1402 produc-tion and iodinations than could be accounted for

% I131Orgaified

765432

0.14

C140, 0.10%MediumGlucose C'4

PerGm.Tissue 0.06

002

0J.-oCg

FL LF EL0.00 2x 0-3 Ix10- 5x-4 2-5uxI4NORETHYLMALEIMIDECONCENTRATION(Molarity)

TABLE X

The effect of thyrotropin (TSH) on the production of carbondioxide from differentially labeled glucose by slices of

sheep thyroid gland

C'402 production (% addedglucose/g tissue)

Expt. Control TSHno.

JC14 6-C14 IC14 6-C14

83 7.6 2.3 15.2 3.386a* 1.7 0.1 3.5 0.3

b 2.2 1.0 8.1 1.093 3.9 3.3 4.7 2.794 1.3 0.4 3.1 0.495 2.1 0.2 3.5 1.096 3.5 1.3 5.8 1.0

111 3.3 0.6 9.2 1.0

* Slices from same gland: a, incubated for 45 minutes;b, for 140 minutes.

by their individual effects. Other experimentsconfirmed the ability of FAD to enhance thestimulatory effect of TPN and revealed a similaraction of FMN.

Methylene blue. In two experiments, the addi-tion of MB to thyroid slices increased 1-C1402from an average of 1.68 to 15.46 per cent and6-C1402 from 0.27 to 1.00 per cent of added glu-cose-C'4 per g of tissue.

Sulfhydryl inhibitors. NEM, 2 X 10-3 M,markedly inhibited the production of both 1- and6-C1402; at this concentration iodinations werealso inhibited. With decreasing concentrationsof NEM, the production of 1-C1402 returnedtoward normal. In the case of 6-C1402 a dose-response relationship was not apparent. As theevolution of 1-C1402 approached normal values, astimulation of iodinations became evident (Fig-ure 14). Like NEM, CuCl2 and HgCl2 increasediodinations at concentrations which depressed theproduction of C1402 (Table IX).

TSH (Table X). In seven experiments inwhich thyroid slices were incubated with TSH,yields of 1- and 6-C1402 were increased by anaverage of 107 and 14 per cent, respectively. Inthree experiments in which these were measured,oxygen consumption and utilization of stable glu-cose were increased in TSH-supplemented flasksby an average of 15 and 102 per cent, respectively.

FIG. 14. THE EFFECTS OF NORETHYLMALEIMIDEON OR-GANIC IODINATIONS AND THE EVOLUTION OF C1402 FROM

GLUCOSE-1-C1' AND GLUCOSE-6-C' IN A HOMOGENATEOFSHEEP THYROID GLANDS.

DISCUSSION

The present findings strongly suggest that theorganification of iodide is linked to pathways of

1403

Oi8 .

MME

--------------


carbohydrate metabolism in the thyroid gland.ideally, this relationship would have been charac-terized in the intact animal. However, the diffi-culty of altering the metabolic environment of asingle organ in vivo precluded this approach.Even the surviving slice system was consideredunfavorable for these studies since, in slices, con-trolled changes in the intracellular concentration ofmetabolites could not be produced. Further-more, since intact thyroid slices are capable ofconcentrating inorganic iodide, changes in or-ganically-bound iodine resulting from alterationsin the iodide-concentrating mechanism could notbe differentiated from those due to changes inthe rate of organic binding of intrathyroidal io-dide. These difficulties were overcome by the useof a homogenate system. This abolished the io-dide-concentrating mechanism and also permittedthe direct introduction of the compounds understudy. The whole homogenate, rather than spe-cific subcellular fractions, was chosen for studyin order to preserve, as far as possible, the meta-bolic interrelationships present in the intact cell,and since the primary objective of this study wasan elucidation of the metabolic relationships of theiodinating system, it was considered of great im-portance to preserve glycolytic activity in thepreparation. Because of its reported effectivenessin maintaining glycolysis in liver homogenates(5), a potassium-rich medium was employed.This medium was found to support both glucosemetabolism and iodination in the thyroid homoge-nate.

Several differences between the metabolism ofiodine in the thyroid homogenate and the wholegland, however, raise the question of whether thehomogenate is a suitable medium in which to studyphysiological iodinations. First, as the presentand previous studies have shown (12), iodinationsin the homogenate do not proceed appreciably be-yond the synthesis of MIT; even when the quan-tity of iodine organified was increased by stimula-tory factors or by the addition of inorganic iodide,appreciable quantities of diiodotyrosine were notformed. Second, the protein in which MIT isformed is not thyroglobulin (13). Finally, bothUnknown 2 of Taurog and co-workers and theiodinated front-running material are prominentproducts of iodination in the homogenate, but arenot found in significant proportions in the intact

gland (14). The presence of these unusual iodi-nated products in the homogenate may indicatethe accumulation of iodinated intermediates which,in the intact gland, are too rapidly utilized to beevident. However, if the front material is indeedan iodinated lipid, it would be an unlikely pre-cursor of thyroid hormone but, like MIT, wouldresult from an oxidation of iodide. That the samebasic process determined the rate of formation ofboth MIT and front material is also suggestedby the findings that rates of formation of originand front materials underwent qualitatively simi-lar changes under the influence of stimulatory orinhibitory agents.6 Thus, the evidence suggeststhat, within the homogenate, iodide is oxidized toa more reactive state which, in the face of struc-tural disorganization, randomly iodinates avail-able substrate.

In its inability to carry its iodinations beyondMIT, the thyroid homogenate resembles certainstages in the development of the fetal thyroid (15),certain neoplasms of the gland (16), and glandswhich have been frozen or stored (17). These,too, may show evidence of a preponderant or ex-clusive synthesis of MIT. Thus, the initial oxida-tion of iodide is probably a sturdy function re-sistant to cellular disruption or damage, and thehomogenate system appears well suited to thestudy of this step in the intrathyroidal metabolismof iodine.

A pronounced variation was noted in the per-centile iodinations achieved in unsupplementedsamples of different homogenates. The reasonsfor this variability are not known, although sev-eral factors may have been contributory. Somevariation in the elapsed time between the death ofthe animals and initiation of the experiments in-evitably occurred. Pronounced differences in thesize and vascularity of thyroid glands employedfrom day to day were not infrequent. These dif-ferences in gross appearance may have been re-

6In order to verify increases in iodinations, revealedby TCA-precipitation, chromatographic analysis of ho-mogenates was performed at least once in the case ofevery stimulatory factor. Except for HgCl,, all stimu-latory factors produced proportionate increases in frontand origin materials. Mercury produced a greater in-crease in front than in origin material. Factors stimu-latory to iodinations did not cause the appearance ofsignificant quantities of iodinated products not found incontrol homogenates.

1404


flected in variations in the metabolic activity, inthe rate of endogenous hormonal synthesis or inthe concentration of inorganic iodide in suchglands and in the homogenates to which they con-tributed. Therefore, in order to facilitate com-parison of results obtained in replicate experi-ments, organic iodinations in the presence of modi-fying factors have been calculated not only as afraction of added 1131, but also as a percentage oforganic iodinations obtained in control vessels.

Endogenous inhibition of iodinations. In earlyexperiments, thyroid homogenates were dialyzedin order to determine whether dialyzable factorswere essential for the organification of iodide.On the contrary, percentile iodinations were con-siderably and consistently increased by dialysis.Since percentile iodinations in both fresh anddialyzed homogenates were decreased by the ad-dition of iodide, it seemed likely that the stimula-tory effects of dialysis might have been due atleast in part to a loss of iodide from the homoge-nate. However, an indication that the increase iniodinations observed in dialyzed homogenateswas not due solely to loss of iodide was affordedby the observation that, like dialysis, storage ofhomogenates at 40 C increased subsequent per-centile iodinations, although to a lesser extent.Since the concentration of inorganic iodide, ifanything, increases during storage in the cold(18), the increases in percentile iodinationsachieved in the stored homogenate must have re-flected an increase in chemical iodinations. Thissuggested that the homogenate contained an in-hibitor which was labile to storage and perhapsdialyzable.

These characteristics of the endogenous inhibi-tor suggested that it might be a nonprotein, sulf-hydryl-containing compound, such as glutathione.This tripeptide is present in many tissues (19),including the thyroid (20). It is dialyzable, auto-oxidizes readily, especially in the presence oftraces of copper, and has been shown to inhibitiodinations in the thyroid homogenate system(21). Furthermore, previous workers have re-ported that copper increases organic iodinationsin the homogenate system (21, 22). Althoughthis effect has been ascribed to the participation ofcopper either as a cofactor for a thyroidal iodinase(21) or, nonphysiologically, as a direct oxidant ofiodide (22), the present observations were con-

sistent with the hypothesis that copper increasedorganic iodinations by decreasing the concentra-tion of an endogenous sulfhydryl inhibitor ofiodinations. This could result from either forma-tion of mercaptides or catalysis of sulfhydryl auto-oxidation (11). This conclusion was supportedby the finding that the stimulatory effects of cop-per were shared by both mercury and the evenmore nearly specific sulfhydryl inhibitor, NEM.Furthermore, since dialysis of the homogenate al-tered the response to these agents, rendering stim-ulatory concentrations either less so or franklyinhibitory, it seemed unlikely that the heavy metalscould be acting as direct oxidants of iodide or co-factors for an iodinating enzyme. These obser-vations also indicated that dialysis of homogenatedid more than merely lower the concentration ofinorganic iodide. Rather, dialysis appeared tohave removed the endogenous inhibitor of iodina-tions which the sulfhydryl reagents themselveswere thought to attack. Within this context, theinhibitory effects of high concentrations of sulf-hydryl reagents in the fresh homogenate, as wellas the inhibition of iodinations produced in dia-lyzed homogenates by lower concentrations whichhad previously been stimulatory, could be ex-plained by the sulfhydryl dependency of severalglycolytic enzymes (23) which apparently par-ticipate in the iodination mechanism. With highconcentrations of NEM, this supposition was con-firmed by demonstrating a pronounced decreasein the evolution of CO2 from C14-labeled glucose.

If the endogenous inhibitor were indeed GSH,then its effect on iodinations could be explainedby certain well known reactions of this compound.Thus, GSHmight act as a competitive substratefor an oxidant of iodide, such as H209 (11):H202 + 2 GSHwGSSG+ 2 H20. Alterna-tively, GSHmight reduce an oxidized form of io-dine, as in the following reaction: 2 GSH+ 2 IO>± GSSG+ 2 H+ + 2 I-. Indeed, the facilitywith which the latter reaction proceeds in vitropermits its use in the measurement of GSHandother reducing substances in blood and othertissues (24) .7

7 This technique depends on the re-reduction by GSHand other reducing substances of iodine generated fromiodide by potassium iodate. It appeared to afford a meansof assessing the potential of substances within a thyroidhomogenate to reduce iodine and to behave like the pos-

1405


The greater inhibitory effect of GSH than ofGSSGwas consistent with the foregoing reactions,and indicated some limitation in the ability of theunsupplemented homogenate to reduce the oxi-dized form. This limitation was overcome by theaddition of TPN. This converted the rather vari-able effect of GSSGto a consistent inhibition, aneffect which was even more readily apparent wheniodinations in the presence of TPN and GSSGwere compared with those obtained with TPNalone. Enhancement by TPN of the GSSG-in-duced inhibition of iodinations can be ascribed tothe rapid reduction of TPN within the homoge-nate, since TPNH is a coenzyme for glutathionereductase (25), an enzyme known to be presentin the thyroid gland (26).

If GSH is indeed the endogenous inhibitor ofiodinations,8 the oxygen dependency of iodinationrates may be due in part to the propensity of GSHto undergo oxidation under conditions of increas-ing oxygen tension. However, this alone cannotexplain the absolute oxygen requirement of theiodinating system, since anoxia inhibited iodina-tions in homogenates from which the endogenousinhibitor had been removed by dialysis or by theaddition of sulfhydryl reagents.

The role of carbohydrate metabolism. To de-fine the relation between carbohydrate metabo-lism and organic iodinations in the thyroid homog-enate, several methods were employed to alter theactivity of pathways of glucose catabolism. Ex-tensive studies of the effects of adding substrateswere not performed. However, succinate, andpossibly isocitrate and oxaloacetate, appeared tostimulate iodinations. In view of the proposedmechanism of iodination, to be discussed below,the stimulation produced by succinate is of par-ticular interest, since succinate, unlike other car-tulated endogenous inhibitor of iodinations. lodometrictitrations revealed a virtually complete loss of reducingmaterials following dialysis of the homogenate or addi-tion of stimulatory concentrations of CuCl2. Partialloss of reducing materials occurred upon storage ofhomogenates.

8 The evidence which suggests that GSH is the en-dogenous inhibitor of iodinations would also be consistentwith the possibility that the effect of GSHis mediated inpart or entirely by compounds such as ascorbic acid withwhich glutathione is in oxidation-reduction equilibrium(27). In the present experiments, ascorbic acid provedto be markedly inhibitory to iodinations in the homoge-nate system.

bohydrate intermediates, is directly oxidized bya flavin dehydrogenase (28). The lack of effectof other substrates is difficult to interpret; theirendogenous concentration in the homogenate maynot have imposed any rate-limitation. Sufficientexperiments to determine the effects of addingsubstrates to dialyzed homogenates, in which en-dogenous substrates can be presumed to be de-pleted, were not performed.

It has been suggested that the hexose mono-phosphate (HMP) shunt and the Embden-Meyer-hof glycolytic pathway can be selectively stimu-lated by the addition of pyridine nucleotide coen-zymes specifically associated with the dehydroge-nases of each pathway. Thus, it is thought thatthe addition of TPN increases the activity of theHMPshunt (29), while DPN stimulates glyco-lysis (5, 29). The ability of both TPN and DPNto increase iodinations in the homogenate systemtherefore implicated carbohydrate metabolism inthe regulation of organic iodinations. However,the more consistent and greater stimulatory ef-fect of TPN than of DPNsuggested that the twocoenzymes might stimulate iodinations by dif-ferent mechanisms. It was interesting, therefore,to determine how the effectiveness of each coen-zyme was related to its oxidation-reduction state.The effects of adding the oxidized and reducedforms could not be directly compared because ofthe rapidity with which coenzymes added to tis-sues are brought to oxidation-reduction equi-librium. By adding each coenzyme together withits specific reducing system (isocitrate-isocitratedehydrogenase for TPN; ethyl alcohol-alcohol de-hydrogenase for DPN), it was possible to main-tain within the homogenate higher concentrationsof reduced coenzyme than would have followedthe addition of the coenzyme alone. Under theseconditions, the stimulatory effectiveness of TPNwas enhanced, while DPN became inhibitory.These findings afforded further evidence of a dif-ference in the action of the two coenzymes on or-ganic iodinations, in that TPN stimulated iodina-tions in its reduced form, while DPN was ap-parently effective only in its oxidized form.

As shown in Figure 7, however, there appearedto be a limit beyond which increasing the con-centration of TPNHno longer increased iodina-tions. In this experiment, the stimulation of io-dinations which followed the addition of TPN

1406


continued at a time when the concentration ofTPNHwas no longer increasing. However, thefurther addition of TPN, with the attainment of anew equilibrium at an even higher concentra-tion of TPNH, was attended by no further stimu-lation of iodination. In the presence of excessTPNH, other factors apparently become rate-limiting for iodinations.

An explanation for the seemingly paradoxicalinhibition of iodinations produced by the additionof lower concentrations of TPN was sought inthe interaction between TPN and the endogenousinhibitor. Since exogenous GSSGwas capableof changing the effect of added TPN from stimu-lation to inhibition, it seemed possible that theeffect of TPN could be similarly modified by theendogenous inhibitor. Support for this hypothe-sis was obtained from the observation that eitherdialysis or addition of NEMnot only changed theeffect of the low concentration of TPN (7 x 10-sM) from inhibition to stimulation, but also in-creased the stimulatory effect of full concentra-tions of the coenzyme (7 x 10-4 M). Thus, therelatively low concentration of endogenous inhibi-tor (presumably glutathione) induced inhibitionin conjunction with low concentrations of addedTPN, just as the high concentrations of addedGSSGproduced inhibition with full concentra-tions of added TPN.

Thus, the stimulation of organic iodinationsproduced by TPN is limited by the presence ofan endogenous inhibitor which may be glutathione.This contrasts with the mechanism of action ofthe heavy metals and of NEM; their stimulatoryeffect is evident only in the presence of an en-dogenous inhibitor whose action they prevent.TPN is rapidly reduced in the homogenate, andthe resulting TPNHhas two opposing effects oniodination: a stimulatory action discussed ingreater detail below, and an inhibitory actionwhich is related to the reduction of endogenousGSSG. Increased organic iodinations ensue if,as when large concentrations of TPN are added,the stimulatory effect of TPNHoutstrips the bal-ancing inhibitory action. Under physiologicalcircumstances, such an internally damped systemmight have consideral)le value -in stabilizing therate of organic iodinations during changes inmetabolic activity within the thyroid gland.

Experiments with 1-C14- and 6-C14-labeled glu-

cose were performed in order to gain furtherinsight into the relation between carbohydrate me-tabolism and organic iodinations, to determine thepathways in the thyroid responsible for the reduc-tion of TPN, and to elucidate the mechanism ofaction of other factors stimulatory to iodinations.The complexities of employing differentially la-beled glucose to estimate the relative proportionor absolute amounts of glucose metabolized via theEmbden-Meyerhof pathway vis a vis the HMPshunt have recently been reviewed (30). It isapparent that measurements of C1402 productionfrom glucose-i-C14 and glucose-6-C14 do not alonemake such estimates possible. Nevertheless, apreponderant evolution of C1402 from glucose-1-C14 over that from glucose-6-C"4 provides evi-dence for the operation of the shunt. Further-more, provided that proportionate re-formation ofglucose from products of shunt activity remainsunaltered, an increase in the evolution of C1402,preponderantly from glucose-i-C14, will indicatean increase in the activity of the HMPshunt andconsequently in the rate of generation of TPNH.The present findings with labeled glucose havebeen employed and interpreted in that light.

The greater evolution of C1402 from glucose-1-C14 than from glucose-6-C14 found during thepresent studies in both slices and homogenates ofthyroid tissue indicated that the HMPshunt isoperative in the thyroid gland.9 The data furthersuggested that an acceleration of the HMPshunt,induced by the addition of TPN, was responsi-ble for the reduction of at least a considerableproportion of the added coenzyme and that in thethyroid, as in other tissues, the availability ofTPN is an important rate-limiting factor for thismetabolic pathway.

The stimulatory effect of DPNon organic iodi-nations was more variable and less pronouncedthan that of TPN, suggesting that its action inthe homogenate was more complex. Experimentsin which iodine or glucose metabolism or bothwere measured indicated that this was indeed the

9 Field and his co-workers have also presented evi-deuce for the operation of the HMP shunt in thyroidslices (31). These workers were unable to demonstrategeneration of C14(O from labeled glucose in thyroid ho-mogenates. This discrepancy with the present findingsmay have resulted from the difference in homogenizationmedium employed in the two studies.

1407


case, and that the effect of DPNdepended uponthe concentration of TPN in the homogenate.Thus, in the presence of concentrations of exoge-nous TPN which inhibited iodinations whenadded alone, inhibitory or insignificant effects ofDPNon organic iodinations were changed to stim-ulation and stimulatory effects were increased.

This synergism between the two pyridine nu-cleotides might possibly have been due to the ac-tion of a transhydrogenase (32), oxidizing DPNHand reducing TPNto its stimulatory form, TPNH.However, the stimulatory effect of DPNon iodi-nations was decreased or abolished by the con-comitant addition of a DPN-reducing system.The converse would have been expected had aDPNHto TPN transhydrogenation been respon-sible for the stimulatory effect of DPN. Never-theless, attempts were made to demonstrate sucha transhydrogenase in thyroid homogenates. Bya method analogous to that employed by Colowick,Kaplan, Neufeld and Ciotti (33), the reduction ofTPN was assessed spectrophotometrically fol-lowing addition of a low catalytic concentrationof DPN and a DPN-reducing system (ethyl al-cohol-alcohol dehydrogenase). No transfer ofhydrogen from DPNHto TPN could be dem-onstrated by this method.

Since DPNalso appeared to synergize the ac-tion of TPN on the HMPshunt, other interac-tions between the two pyridine nucleotides seemedpossible. First, in the presence of an activeTPNH to DPN transhydrogenase (33), addedDPN might increase the activity of the HMPshunt by speeding regeneration of the hydrogenacceptor, TPN. However, if such a transhy-drogenase were present, addition of DPNshouldtend to reduce the availability of TPNH andthereby decrease organic iodinations; the conversewas actually the case. Furthermore, direct spec-trophotometric assay by a technique analogous tothat described above failed to reveal a TPNH toDPN transhydrogenase in thyroid homogenates.

A further possible mechanism for the effect ofDPN involves the generation of TPN from DPNvia a DPN-kinase (34). Partial conversion ofDPN to TPN might explain the greater stimula-tion of iodinations achieved by TPN than DPNwhen added to homogenates in equal concentra-tions. The ability of DPN to reverse the inhibi-tion of iodinations produced by 7 x 10-5 MTPN

would also be consistent with this hypothesis.This explanation, however, is not consistent withthe observation that in the presence of 7 X 10-4 MDPN, TPN in a concentration of 7 X 10-5 Mproduced a far greater stimulation of 1-C1402 pro-duction than did 7 X 10-4 MTPN alone.

For these reasons, an alternative explanation,similar to that proposed by others to account forthe synergistic action of TPN and DPN on he-patic synthesis of fatty acids (35) seemed moreattractive. This hypothesis suggests that DPNincreases the activity of the HMPshunt by ac-celerating the metabolism of 3-carbon productsof the shunt which re-enter the Embden-Meyer-hof pathway. Addition of DPN would therebyincrease the generation of TPNH. In the pres-ence of low endogenous concentrations of TPN,DPN might increase the generation of TPNHto the same extent as the addition of low (7 X10-5 M) concentrations of TPN alone. Inhibitionof iodinations would then result from an acceleratedreduction of GSSG. If the endogenous concen-tration of TPN were higher or were increased byexogenous TPN, addition of DPNmight facilitatethe generation of sufficient TPNH so that itsstimulatory effect on iodinations would becomemanifest. Thus, the influence of DPNon organiciodinations would be mediated through its ef-fects on the generation of TPNH.

It seemed unlikely, however, that reduced TPNper se could be responsible for the oxidation ofiodide which is requisite for organification. Rather,TPNHmust serve as an intermediate in the gen-eration of an agent capable of oxidizing iodide.The sensitivity of the iodinating system to inhibi-tion by cyanide and its absolute oxygen depend-ency focused attention on the possibility that fur-ther hydrogen transfers along the cytochromesystem might be required for the formation of thepostulated oxidant. However, in the present ex-periments, in contrast to the results of other stud-ies (36), addition of cytochrome C to the wholehomogenate had little effect on organic iodinations.Antimycin A, which inhibits reduction of the cyto-chromes (37), also failed to inhibit iodinations(38), and in the present experiments was slightlystimulatory. This favored the hypothesis that thecytochromes did not function as part of the io-dinating system. Methylene blue (MB) permitsthe direct transfer of electrons to oxygen, thereby

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bypassing the cytochrome system. MIB was there-fore added to homogenates to compete wvith thecytochrome system as a hydrogen acceptor. Themarked increase in the evolution of 1-C'402 whichfollowed the addition of MBindicated its ability tooxidize TPNH in the intact thyroid slice, as ithas been thought to do in liver slices (39). Fur-thermore, the stimulatory effect of MBon iodina-tions in the homogenate and its synergism withadded TPN in this regard not only seemed to ruleout a necessary participation of the cytochromesin the iodinating system, but also suggested thathydrogen transfers from TPNH to MIB mightconstitute a partial model of the physiologicaliodinating system.

Unlike TPNH, reduced 1\IB is capable of un-dergoing auto-oxidation to yield H.,O1 (40).For some years, it has been thought that thyroidaliodide is oxidized by H202 in a reaction catalyzedby a glandular peroxidase (41, 42). AlthoughH902 has not itself been demonstrated in the thy-roid, a halogen-peroxidase, apparently specificfor iodide has recently been found (43) and anumber of peroxidase inhibitors are known to bepotent antithyroid agents (44). Furthermore,catalase has been shown to inhibit iodinations inboth a soluble tyrosine-iodinating system derivedfrom submaxillary gland (45) and in a thyroidmitochondrial-microsomal preparation (13). Inthe present experiments, catalase was moderatelyinhibitory of organic iodinations in whole thyroidhomogenates. Finally, hydrogen peroxide hasbeen shown to overcome the inhibitory effect ofanoxia on iodinations in the tyrosine-iodinatingsystem (45). It therefore seemed possible thatthe stimulation of iodinations induced by MB inthe present experiments was due to its ability togenerate H202 after reduction by TPNH.

In physiological systems, FAD-linked metallo-proteins are the only known sources of H202.These aerobic dehydrogenases are inhibited bycyanide and are capable of being oxidized by MB.These properties suggested that, in the physio-logical iodinating system, a flavin enzyme mightserve to accept hydrogen from TPNH and thenproduce H202 by auto-oxidation. For these rea-sons, the effects of added flavins in the thyroidhomogenate were assessed. Although FAD aloneinduced only slight stimulation of organic iodina-tions and the evolution of C1402 from labeled glu-

cose, it acted synergistically with added TPN toproduce a marked increase in iodinations and a

profound increase in C1402. Within the contextof the H202 hypothesis, it appeared that FAD po-tentiated the action of TPN on the HMPshuntby maintaining the TPN in the oxidized state,while the resulting reduced FAD underwent auto-oxidation to form the oxidant of iodide, H202.

It was uncertain, however, that the effects ofadded FAD were indeed related to the functionof the physiological iodinating system. Althoughprevious workers had shown that iodinations ina thyroid mitochondrial-microsomal preparationcould be increased by addition of flavins (38),this stimulatory effect was not abolished byanoxia. No explanation is apparent for the dis-crepancy between these and the present findings,in which FAD failed to stimulate iodinations inthe absence of oxygen. More important, however,was the observation that in a tissue-free system,flavins could catalyze a light-dependent oxidationof iodide (46, 47) which could result in the iodi-nation of added serum proteins. For these rea-sons, the effects of light and dark on organic iodi-nations in the present homogenate system, and onthe actions of TPN and flavins therein, were as-sessed. The findings of earlier workers wereconfirmed, in that iodination of human serum al-bumin in a simple buffer medium could be ef-fected by FAD in a reaction inhibited by exclusionof oxygen and light. However, TPN had noeffect on the flavin-stimulated iodinations in thistissue-free system. In the thyroid homogenate,in contrast, exclusion of light had no effect oncontrol iodinations, partially inhibited the increasein iodinations produced by FAD alone, but hadno effect on the pronounced increase in iodinationswhich followed the addition of FAD with TPN.It seemed, therefore, that the added FAD was in-deed participating in the physiological iodinatingsystem of the homogenate, and that the energy forthe flavin-stimulated oxidation of iodide, whichwas derived from incident light in the tissue-freesystem, was, in the thyroid homogenate, suppliedby an endogenous metabolic source.

Avost of these studies were performed withFAD. However, FMNwas similarly effective.This lack of specificity with regard to coenzymeand the fact that the flavin-supplemented systemseemed inefficient in the utilization of metabolic

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energy, causing a proportionately much greaterstimulation of 1-C1402 production than of iodina-tions, makes it unlikely that free flavins are rate-controlling intermediates in the physiological io-dinating system. Rather, it seems likely that inthe thyroid, as elsewhere, the flavins are tightlybound to the enzymes with which they function.

Pronounced differences in the behavior of thy-roid particulate preparations of differing naturehave been noted. Thus, Tong and Chaikoff havereported that cytochrome C stimulated iodinationsin the whole homogenate, but not in a mitochon-drial-microsomal preparation (36), while the con-verse was true of FMN. Earlier, the stimulatoryeffect of flavins in a mitochondrial-microsomalpreparation was reported to persist despite anoxia(38); in the present experiments a moderatestimulatory effect of flavins was obtained in wholehomogenates and was distinctly inhibited byanoxia. Similarly, the present evidence thatTPNH-linked dehydrogenations of the HMPshunt are regulatory to iodinations in the wholehomogenate would presumably not apply to themitochondrial-microsomal preparations. Here,however, mechanisms for the generation of re-duced pyridine nucleotides and flavoproteins viathe Krebs cycle could also lead to the formation ofH202.

Because of these differences in the behavior ofvarious broken cell preparations, experimentswere undertaken to ascertain whether an increasein TPN-linked dehydrogenations could be demon-strated as a characteristic of physiological stimula-tion of the intact thyroid cell. For this purpose,TSH was employed to stimulate organic iodina-tions in thyroid slices. Relative effects on theevolution of C1402 from 1_C14- and 6-C14-labeledglucose were again determined as an index ofchanges in the rate of generation of TPNH.While these studies were in progress, Field, Pas-tan, Johnson and Herring (31) reported the re-sults of similar observations. The present re-sults are entirely in accord with the findings ofthese workers, who observed that TSH increasesthe production of C-402, preponderantly from1-C14- as compared with 6-C14-labeled glucose,and who, like Freinkel (48), reported an increasein total glucose utilization under these conditions.These results indicate that TSH, a physiologicalstimulator of iodinations, is, in the intact thyroid

cell, capable of enhancing those metabolic proc-esses which, in the thyroid homogenate, provideenergy for the oxidation of iodide. For reasonsalready noted, however, the findings do not permit:conclusions as to whether TSH specifically orpreponderantly stimulates the HMP shunt ascompared to the Embden-Meyerhof pathway.Furthermore, they afford no evidence of how theevident increase in the turnover of TPNH is ef-fected. Thus, TSHmight accelerate the reductionof TPN by stimulating the HMPshunt, eitherspecifically or as part of a general increase in glu-cose catabolism. Alternatively, TSHmight stimu-late hydrogen transfers within the physiologicaliodinating mechanism at some point distal toTPNH. The resulting increase in the genera-tion of TPN from TPNHwould increase the ac-tivity of the HMPshunt secondarily. The lattermode of action, analogous in some respects to thatproposed for FAD, would seem more favorableto a stimulation of iodinations, since it would tendto decrease, rather than increase, the availabilityof TPNH for the reduction of GSSG. Finally,TSH might increase the availability of TPN byactivating a DPN-kinase, if such is present inthyroid tissue (34). This explanation would beconsistent with the observation that in the guineapig thyroid, which is relatively inactive, the con-centration of triphosphopyridine nucleotides(TPN + TPNH) is very low and is far exceededby the concentration of diphosphopyridine nucleo-tides (DPN + DPNH) (49).

On the basis of the present findings, it is pro-posed that the energy for the thyroid organifica-tion of iodide is derived from glucose metabolismvia TPN-linked dehydrogenations. TPNH isoxidized by a flavin enzyme which in turn re-duces oxygen to form H202. The latter oxidizesiodide in a reaction catalyzed by a thyroidal perox-idase. DPN has a permissive action on the op-eration of the HMPshunt, but probably does notdirectly contribute hydrogen to the iodinating sys-tem. Dehydrogenations which reduce TPN arealso capable of inhibiting iodinations by accelerat-ing the reduction of GSSG. The latter reactionwould serve as a "governor" on the accelerationof iodinations which would otherwise follow anincrease in glucose catabolism.

Earlier observations have indicated that themaintenance of adequate intrathyroidal stores of

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iodide for hormonal biogenesis depends uponphosphate bond energy derived from glucose ca-tabolism (3, 4). The present observations affordevidence of a relation between glucose catabolismand the next step in hormonal biogenesis, the oxi-dation of iodide. The role of TPN-linked dehy-drogenations in the latter reactions is of particularinterest in view of recent indications that the syn-thesis of protein (50) and fat (35, 51) may beTPNH-dependent. Thus, the energy for in-creased hormonal biogenesis (accelerated syn-thesis of thyroglobin plus increased iodinations)and for its usual concomitant, glandular hyper-plasia, may be derived via the same metabolicpathways. In this way, certain functional andstructural effects of TSH on the thyroid glandcould be mediated by an action upon a singlemetabolic site.

SUMMARY

The role of intermediary carbohydrate metabo-lism in regulating the rate of organic iodinationsin thyroid homogenates has been investigated.Evidence was obtained for the presence of anendogenous inhibitor of iodinations whose ac-tivity was diminished or lost following storage,dialysis, or addition of sulfhydryl inhibitors.

Rates of organic iodination were regulated byTPN-linked dehydrogenations. Reduced TPNparticipated in both inhibitory and stimulatory in-teractions with the physiological iodinating sys-tem. The former appeared to result from an en-hanced generation of the endogenous inhibitor,presumably reduced glutathione. The stimulatoryinteraction was synergized by flavin cofactors andmethylene blue in reactions thought ultimately toyield hydrogen peroxide, an oxidant of iodide.

Dehydrogenations in the hexose monophos-phate shunt appeared to be the principal source ofreduced TPN. DPN and flavin cofactors actedsynergistically with TPN in stimulating shuntactivity and increasing generation of TPNH. Inthe intact thyroid cell, activity of the shunt wasenhanced by TSH, a physiological stimulator ofiodinations.

On the basis of these observations, an internallydamped sequence of hydrogen transfers via TPNand a flavin enzyme is proposed as a means ofutilizing energy from glucose metabolism in theoxidation of iodide.

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THEROLE OF INTERMEDIARY CARBOHYDRATE METABOLISMdm5migu4zj3pb.cloudfront.net/manuscripts/104000/104371/JCI6110… · 3 X10-3 M; KCl, 0.14 M. Usually, homogenates were rehomogenized