Effects of Inorganic Iodide on the Intermediary Carbohydrate Metabolism of Surviving Sheep Thyroid Slices

EFFECTS OF INORGANIC IODIDE ON THE INTERMEDIARY CARBOHYDRATE METABOLISM OF SURVIVING SHEEP THYROID SLICES

William L. Green, Sidney H. Ingbar

J Clin Invest. 1963;42(11):1802-1815. https://doi.org/10.1172/JCI104865.

Research Article

Find the latest version: https://jci.me/104865/pdf Journal of Clinical Investigation Vol. 42, No. 11, 1963 EFFECTS OF INORGANIC IODIDE ON THE INTERMEDIARY CARBOHYDRATE METABOLISM OF SURVIVING SHEEP THYROID SLICES *

By WILLIAM L. GREEN t AND SIDNEY H. INGBAR t (From the Thorndike Memorial Laboratory and Second and Fourth [Harvard] Medical Services, Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston, Mass.; and the Department of Medicine, Seton Hall College of Medicine, Jersey City, N. J.) (Submitted for publication April 2, 1963; accepted July 31, 1963)

The rate of hormone formation by the thyroid changes in the rate of hormone formation in- in vivo is greatly influenced by acute alterations duced by varying the availability of iodide to thy- in the concentration of inorganic iodide in the roid slices in vitro are associated with, or can be extracellular fluid. In the rat (1-3) and in man ascribed to, alterations of intermediary metabolism (4, 5), increasing concentrations of iodide in the within the thyroid. A preliminary report of these plasma are associated with increased thyroidal studies has appeared (17). uptake of iodine until a critical concentration of iodide is reached. Beyond this range, the forma- METHODS tion of hormone declines. A similar relationship Preparation of slices. Thyroid glands were obtained between extracellular iodide concentration and from freshly killed sheep and were brought from the organic iodinations occurs in vitro in slices of abattoir to the laboratory packed in ice. A single gland sheep thyroid (6), whole lobes of rat thyroid (7), was used in each experiment. Slices were cut with a of rat This Stadie-Riggs microtome, washed twice in ice-cold physio- and homogenates salivary gland (8). logical saline solution, weighed on a torsion balance, and relative inhibition of thyroid hormone formation placed in Warburg vessels containing 2 or 3 ml of ice- induced by high concentrations of iodide is often cold medium. Tissue weights were quite uniform termed the Wolff-Chaikoff phenomenon, and has within individual experiments, but varied between ap- never been fully explained. In normal animals, proximately 50 and 300 mg per flask in different such inhibition is transient (9). Rarely, however, experiments. Preparation of media. The standard medium em- prolonged treatment with iodine does cause goiter ployed in control vessels was Krebs-Ringer-phosphate and myxedema in man (10), possibly because the buffer (KRP), pH 7.4, with one-third the recommended inhibitory effect of iodide persists. concentration of CaClJ1 (18). Each milliliter of medium Recently, evidence has accumulated that the contained 1.5 or 2.0 mg of stable glucose, labeled with 0.5 formation of hormone and oxidative metabolism in to 1.5 /uc of C14.2 Vessels were incubated at 380 C under 100% oxygen in a Warburg apparatus for from 60 the thyroid are closely linked. First, the iodide- to 200 minutes. CO2 was absorbed by 0.2 ml of 10% concentrating mechanism is dependent on phos- KOH in a sidearm. phate bond energy (11, 12), and second, organic Media containing desired concentrations of iodide binding of thyroidal iodide appears to de- were prepared by isosmotic substitution of NaI for pend on electron transfers leading to generation NaCl in KRP. Several factors, however, might cause of the ultimate concentration of iodide in the medium to hydrogen peroxide (13-16). The present deviate from that produced by the addition of NaI. studies were designed to determine whether First, as judged by iodine analyses performed in 14 different preparations,8 the concentration of iodide in con- *This investigation was supported in part by U. S. trol contamination Public Health Service research grants AM-00267-10 and media, representing of the reagents AM-06650-01 from the National Institute of Arthritis 1 Modified Krebs-Ringer-phosphate buffer: NaCl, 0.132 and Metabolic Diseases, Bethesda, Md. M; KCI, 0.005 M; CaCl,, 0.0009 M; MgSO4, 0.0013 M; tA portion of this investigation was carried out dur- and phosphate buffer, 0.009 M. ing the tenure of a U. S. Public Health Service post- 2 C14-labeled glucose was obtained from the New doctoral fellowship from the National Institute of England Nuclear Corporation, Boston, Mass. Arthritis and Metabolic Diseases. 3 Performed at Boston Medical Laboratory, Boston. t: Investigator, Howard Hughes Medical Institute. Mass. 1802 EFFECTS OF IODIDE ON THYROIDAL INTERMEDIARY METABOLISM 1803 used, was considered negligible (average, 5.2 ug per incubated without tissue was not determined in the ma- 100 ml, or 4 X 10- M). Second, the quantity of iodide jority of experiments. C14-labeled lactate in suspending that might be leached f rom thyroid tissue during the media was estimated by ascending paper chromatography incubation period was considered. Speciments of 9 sepa- in butanol: acetic acid: water (100: 12: 88). That por- rate thyroid glands were boiled in a test tube containing a tion of the filter paper containing lactate was located by small volume of distilled water. The entire contents of radioautography, excised, and placed in a vial of liquid the tube were then placed in a dialysis sac and dialyzed scintillator for counting. Radioactivity in the lactate overnight against distilled water at 40 C. The calcu- zone was compared with that in a sample of the initial lated content of dialyzable iodine in thyroids, based medium applied to filter paper and counted under similar upon iodine analyses in the dialyzing solution, averaged geometric conditions. Values for lactate production re- 2.1 ,g per 100 mg of tissue. In view of the subse- ported are the means of duplicate vessels; in no case quent demonstration that thyroid tissue contains sub- did the value for an individual vessel differ from the stantial quantities of iodinated peptides that are slowly reported mean by more than 7%. Concentrations of dialyzable (19-21), this value was undoubtedly an over- stable glucose were measured in duplicate by the estimation of the true concentration of inorganic iodide Somogyi-Nelson method (25); control studies indicated in these specimens. Even if this were not the case, that iodide concentrations in the medium as high as 1 X flasks containing 100 mg of tissue and added iodide in 10' M did not influence the glucose measurement. Total a concentration of 1 X 10' M (1.27 jg per ml) would CO2 production was determined by BaCO3 precipitation, contain at least twice as much iodide as flasks contain- with care taken to prevent contamination with atmos- ing tissue without added iodide. Furthermore, the glandu- pheric C02- lar contribution of iodide would be insignificant in flasks Statistical treatment. With but few exceptions, all re- in which media had been enriched to a concentration of sults presented are the average of closely agreeing values 1 X 10' M or more. A third consideration was the obtained in duplicate or triplicate vessels. With some of ability of thyroid slices to perform iodinations and thereby the results, analysis of variance and tests of significance to decrease the concentration of iodide in the medium. were performed as described by Snedecor (26). For Here, too, this effect would almost always be negligible comparison of results obtained in many thyroid glands, at iodide concentrations greater than 1 X 10i M, where however, of markedly different metabolic activity, from percentile organic binding of iodide seldom exceeds animals whose age and history of iodide ingestion were 0.5% (6, 22).4 Finally, it is likely that any metabolic unknown, it seemed preferable to use nonparametric effects of iodide would be more closely related to con- methods, which do not assume a normal distribution of centration in the slice than to that in the medium, and results. Therefore, the "sign test" was also employed that the activity of the iodide-concentrating mechanism (27, 28). The null hypothesis tested was that the num- would therefore influence the results obtained. Since ber of experiments showing an increase in a given func- slice/medium (S/M) iodide concentration gradients tion (e.g., Q02) at a given concentration of iodide would greater than one may exist at concentrations of iodide equal the number showing a decrease. The probability in the medium ranging up to 1 X 10' M (11), this fac- that the observed number of positive and negative dif- tor was operative in most of the present studies. In ferences occurred by chance was then taken from a stand- them, no systematic attempt was made to correct for ard table (28). these factors. The iodide concentrations hereafter re- ferred to therefore represent merely the concentration of RESULTS iodide added to the medium, and flasks with no iodide Oxygen consumption. In 41 experiments, oxy- will be termed "controls." Measurements. Oxygen consumption was measured by gen consumption in control vessels averaged standard manometric methods (18). C402 derived from 28.6 ± 8.7 Mul per hour per 100 mg (mean + SD). labeled glucose was measured by methods previously The changes in Q02 induced by varying concen- described (15). In brief, these consisted of redistilling trations of iodide in the medium are depicted in CO2 from KOH into 0.5 M Hyamine, which was then Figure 1, and their statistical significance is sum- transferred to vials containing liquid scintillator. Count- ing was carried out in a liquid scintillation counter, and marized in Table I. Low concentrations of iodide counts in C'402 were compared with counts in a sample in the incubation medium (5 x 10-6 to 1 X 10-5 of the initial medium, after correction for quenching. M) did not affect Q02 with significant consist- The recovery of counts from labeled glucose in vessels ency. At iodide concentrations between 5 x 105 4That this effect might also be unimportant at lower and 5 x 10(4 M, however, there was almost in- concentrations of iodide is suggested by the work of variably an increase of Q02 above control values Kondo (23). He found that, during active uptake of (average, + 17%), and this change was highly I"31 by pig thyroid slices, iodide concentration in the significant (p < 0.001) by the sign test (Table I). medium remained constant or increased. Release of stable iodide from human and canine thyroid slices has Significant stimulation of Q02 above control been shown by Molnar, Albert, Keating, and Orvis (24). values also occurred at iodide concentrations of 1804 WILLIAM L. GREEN AND SIDNEY H. INGBAR

160- 0 150- 140

130- 0 9S 120- Qo, 3 00 S0 % CONTROL I o in, 0@ 0 S looIrtet lI Ia le9I lI

90- S S . 80i

I 70- 5X1O-6to XIX0'1 5x 105to 5x104 Ixl03to WVl0- 5 x 10-2 MOLAR CONCENRATON OFADDED IODIDE FIG. 1. EFFECTS OF INORGANIC IODIDE ON OXYGEN CONSUMPTION IN SHEEP THYROID SLICES. Within each concentration range, each point represents closely agreeing duplicate, triplicate, or quadruplicate results from a single thyroid gland.

1 X 10-3 to 5 x 10-3 M (p < 0.001), but was of second hour averaged 7 % less than during the smaller magnitude (average, + 11%) and, when first hour. In six experiments lasting 3 hours, directly compared, was usually less than the stimu- QO2 s during the third hour averaged 9%0o less lation occurring at 5 x 10-5 to 5 x 10-4 M. At than during the second hour. The proportionate the higher iodide concentration of 5 X 10-2 M, decline in Q02 with time was uninfluenced by the QO,'s did not differ consistently from control val- addition of iodide. ues, but were consistently less than QO2's at the Experiments with uniformly-labeled glucose. lower, stimulatory concentration ranges of iodide In eight experiments, tissue was incubated in a (5 x 10-5 to 5 x 10-4 M and 1 X 10-3 to 5 x 10-3 standard concentration of stable glucose (2 mg M). per ml) labeled with glucose-U-C14, and two In prolonged experiments, the rate of oxygen standard concentrations of iodide (1 x 10-4 M consumption declined slightly. In nine experi- and 5 X 10-2 M). Tissue weights in these ex- ments 2 hours long or longer, QO,'s during the periments averaged 196 mg, and incubations were

TABLE I Effect of added iodide on oxygen consumption by sheep thyroid slices seen in comparisons by the "sign test"

Iodide concentrations compared A B n* 4 Pt 0 5 X 10-6 to 1 X 1-5 10 4 >0.2 0 5 X 10-5 to 5 X 10-4 28 26 <0.001 0 1 X 10-3 to 5 X 10-3 18 16 <0.001 0 5 X 10-1 18 8 >0.2 5 X 10-6 to 1 X 10-5 5 X 10-5 to 5 X 10-4 9 9 0.002 1X10-3to5X10-3 5X10-'to5X10-4 17 12 0.072 5 X 10-2 5 X 10-5 to 5 X 10-4 15 15 <0.001 5 X 10-2 1 X 10-3 to 5 X 10-3 10 9 0.011

* n = number of experiments in which comparisons of QO2's were made at the indicated concentrations of added iodide. t x = number of experiments in which Q02 was greater at iodide concentration B than at iodide concentration A. t p = probability of difference between observed value of x and x = n/2. EFFECTS OF IODIDE ON THYROIDAL INTERMEDIARY METABOLISM 1805

TABLE II Effect of inorganic iodide on C1402 production from glucose-i-C14 and -6-C14 in sheep thyroid slices*

Glucose-i-C14 Glucose-6-C'4 Proportion Proportion C1402 production of exp'ts C1402 production of exp'ts No. of with p No. of w-with p exp'ts At B -At B > A§ (sign test) exp'ts At B - A\ B > A§ (sign test)

% of initial radioactivity/ % of initial radioactivity/ 100 mg/hour 100 mg/hour Control (A) vs 5 X 10-6 to 1 X 10-5 M iodide (B) 10 0.185 +0.122 8/10 0.055 511 0.058 +0.016 4/4 0.062 ±0.111 ±0.036 +0.019 ±0.005 Control (A) vs 1 X 10-4 to 1 X 10-3 MI iodide (B) 19 0.161 +0.228 19/19 0.001 11 0.074 +0.072 11/11 0.001 40.074 ±0.049 ±0.041 ±0.026 Control (A) vs 5 X 10-2 M iodide (B) 9 0.163 +0.131 8/9 0.002 911 0.073 +0.031 7/8 0.004 ±0.040 ±0.052 ±0.045 ±0.016 1 X 10-4 to 1 X 10-3 M iodide (A) vs 5 X 10-2 M iodide (B) 6 0.414 -0.106 0/6 0.016 6 0.202 -0.077 0/6 0.016 ±0.318 ±0.042 ±0.130 ±0.020 * Standard conditions for these experiments were 3 mg glucose in 2 ml medium in each flask, and incubation time (;f 60 minutes. t C1402 production at iodide concentration A (mean ±4SD). T Difference between C1402 production at iodide concentration B and that at concentration A (mean difference ± SE of mean difference). § Figures represent no. of experiments where C1402 production at concentration B exceeded that at concentration A/ no. of experiments where B # A. In the sign test, experiments where B = A are not included (27, 28). 11 In one experiment in this group, B = A.

allowed to proceed for an average of 165 minutes. there was in each experiment a greater percentile Results obtained in this group of experiments are stimulation of C140. production than of QO., summarized in Table II; to facilitate comparisons (mean increase, 71 against 19%). At 5 X 10-2 among the various functions measured, data are .M iodide, some glands (experiments 37 and 67) expressed in micromoles. displayed distinct stimulation of C1402 produc- In each of these eight experiments, changes in tion when compared with controls, while others oxygen consumption were as noted above: stimu- showed essentially no change. lation at 10-4 MKI iodide and loss of stimulation at From a comparison of moles of oxygen con- 5 x 10-2 M iodide. Changes in the generation sumed with the estimate of moles of glucose car- of C1402 roughly paralleled those in oxygen bon converted to CO2, the proportion of oxygen consumption.5 At 1 x 10-4 M iodide, however, consumption attributable to oxidation of exoge- Hoskin has demonstrated that preparations of C'4- nous glucose was calculated. This proportion was labeled glucose contain volatile radioactivity that is col- relatively small, averaging 12% in control flasks, lected into alkali, even in the absence of tissue (29). 18% at 1 x 10-4 M iodide, and 14% at 5 X 10-2 Tissue-free blanks were measured in three of these ex- .M iodide. of periments, and blank radioactivity was less than 0.008% However, the increase in oxygen of initially added radioactivity. In 24 subsequent ex- consumption induced by 1 x 10-4 1\1 iodide (mean periments with glucose-U-C'4 from the same source, increment, 0.25 /Amoles per hour per 100 mg tis- not reported here, the amount of radioactivity recovered suie), 487 could be ascribed to increased oxidation in KOH in vessels free of tissue (thermobarometers) of averaged 0.009 ± 0.002% of medium radioactivity, and exogenous glucose. was uninfluenced by the addition of iodide. In the eight Assimilation of glucose (disappearance from experiments reported here, radioactivity collected in al- the medium) was measured in six experiments. kali in vessels containing tissue and no added iodide averaged 0.46% of medium radioactivity (range, 0.21 to production differed materially from those presented in 0.88). Thus, it is unlikely that the true values for C'402 Table II. 1806 WILLIAM L. GREEN AND SIDNEY H. INGBAR

No consistent change induced by iodide could be r 0 0% Ht \0 m detected. Errors in this estimation are relatively C 0% x c0 0-i0 -q large, however, and the data do not exclude the t) ._ 0 possibility that small changes did occur.6 From 1. the data in Table II, the proportion of assimilated C 0_ C') 0% glucose converted to CO2 x n.4 o _4 averaged 2.9% in con- 4i trol flasks, 5.9% in those containing 1 x 1i4 M 00 Mc 0 iodide, and 3.7% in the presence of x . 5 10-2 M . iodide. - 0 Respiratory quotients were determined in four U a.. experiments. Control values averaged 1.0, and ba -q'ICIO%-0%ONC %O0 0%C no consistent e oo666666 6 changes were seen at either stand- 0I 0 0 ard concentration of added z) A iodide. Data with re- ._ x gard to the effects of iodide on the production of M\OerI't00 00e 6ao 20 "do -- .,40O NcC,004 0 C14-labeled lactate yielded no evidence that the 0a, 0x 66666666 6 t, ot Ca, v 0 production of lactate was stimulated, and in two U) V at-C% ON)c1%0o % r0 0 experiments (no. 37 and 67), iodide appeared to . 0. *. - 0 *4 6 0 to 66666666 cause a decline in lactate production greater than Ci 0 the error of the method. 04 -!tU)%0 0\00 C' Analysis of variance of the data in Table II 00 00- \0 t-C'm revealed that iodide produced significant changes x ._. IOen only in oxygen consumption in ._ and the production C) AD of C1402. With the criteria suggested by Snede- a) 0 C1400 00 1C0 0 Cd 0 W cor (26), values for both functions in flasks con- x 0 ba C#)U) 54 \~'0 V4 taining 1 X 10-4 M iodide were significantly 0 ¢ U) .0 ca, on- o-- - Cd higher than values in control flasks, or in those Ca) containing 5 X 10-2 M iodide. Differences be- U)T tween control flasks and those containing 5 x .0 10-2 M iodide were not significant. U) -N - -t CK 0o la Experiments with glucose-l-C"4 and -6-C14. ._ Tissue-free blanks were not routinely incubated 4) in experiments with either glucose-i-C14 or -6-C14; x 100 LJi 0 in two experiments, a blank with glucose-6-C14 x la 0I0I V .0 was incubated, and 0.01%o of medium radioac- x -It 00 tivity was found in KOH. In these studies with 0 CCd 0 -. 0._C.)C specifically labeled glucose, smaller amounts of 045 x +j CS tissue and shorter incubation times were em- no than uP C) ployed was the case in experiments with a,) U) '0 glucose-U-C"4. Therefore, failure to correct for S ._C nonmetabolically generated radioactivity may have Cd 8 Ce 1o 0 t4 .0 6The actual changes in medium glucose concentration C6 0I -m- -4m C- N c t-- O O. during incubation in these experiments averaged about U)j0 It wUl) X-0Ul) U') 0 -4.4.--0vci - '4-- Cd 25 mg per 100 ml, a difference easily measurable by the VN method used. As noted above, however, the mean incre- r m CL) 00 C~-4 E \CE)4 ment in oxygen consumption induced by 1 X 10' M iodide . . . 4 .4 .' -* - - was 0.25 /Amoles per hour per 100 mg tissue. If this in- .0 crement had been expended solely in the formation of zn0 * CO. from an increase in assimilated glucose, the differ- 6 -

u ence in final glucose concentration between control flasks 1- U) 00 Ce o e - '0 e et It U) U) 'C \0 -- X x .CSZC S and those at 1 10' M would have been only about cS'a 2.5 mg per 100 ml. EFFECTS OF IODIDE ON THYROIDAL INTERMEDIARY METABOLISM 1807

WIAR CO t'EN7RAT/ONV OFOAE IODIDE 5xIOlo Ix10-5 I x 1O4to I x10-3 5 X 10' (5) , (7) (4)

1 1-4

-,-. i. . 1. P.qbk 1q-.R-5

C1.3 "I-. Aj-;;z

.; 't" -

I*.- xll. I.a

(f Glucose-I-C'4 G cose-6-C"o FIG. 2. COMPARATIVE EFFECTS OF IODIDE ON C1402 PRODUCTION FROM GLU- COSE-1-C14 AND -6-C14. Only experiments in which glucose-i-C14 and -6-C1 were used concurrently are depicted. In each experiment, each flask contained 3 mg glucose in 2 ml medium. C402 production in control flasks is indicated downward from the zero line (mean + SE); increases induced by the given concentrations of iodide are indicated upward from the zero line (mean difference + SE) ; number of experiments is in parentheses. Open columns represent results with glucose-i-C14; hatched columns represent results with glucose-6-C14. introduced significant errors in the determination concentrations of iodide were greater than those of C1402 production. Blank values may be as- of the highest concentration.7 sumed to be constant, however, among all the In a number of instances, the effects of iodide flasks in a single experiment; therefore, the on the generation of C1402 from C-1- and C-6- amount of change in the generation of C1402 in- labeled glucose were compared in the same ex- duced by iodide in the medium can be accurately periment. Results obtained in these experiments determined. are shown in Figure 2. In all cases, the increase Results obtained in experiments with glucose- in formation of C1402 from glucose-i-C'4 was 1-C14 and -6-C14 are shown in Table III. At the far greater than from glucose-6-C14. lowest range of iodide concentrations employed Effect of thionamide derivatives. The effects (5 X 10-6 to 1 X 10-5 M), generation of C140O of thiouracil and methimazole in concentrations from C-1- and C-6-labeled glucose was increased, known to inhibit organic iodinations (11) were and the changes were of borderline statistical sig- assessed in four experiments (Figure 3). In ves- nificance by the sign test. At intermediate con- sels free of added iodide, these agents slightly, centrations of iodide (1 x 10-4 to 1 x 10-3 M), 7 It is not immediately apparent why C1402 produc- and at the highest concentration of iodide em- tion was not consistently stimulated at an iodide concen- ployed (5 x 10-2 M), generation of C140' from tration of 5 X 10 M in experiments using glucose-U-C14, both and was although such stimulation occurred rather consistently in C-1- C-6-labeled glucose significantly experiments with glucose-i-C1' and -6-C14. Variation increased. In experiments in which they were between the individual thyroid glands studied may be the directly compared, effects of the intermediate best explanation. 1808 WILLIAM L. GREEN AND SIDNEY H. INGBAR but consistently, inhibited the generation of C1402 200- = I Br= from glucose-i-C'4, but had no appreciable effect on QO,. Both thiouracil and methimazole, how- 150- 0 ever, completely or almost completely inhibited the stimulatory response of these functions to 100- added iodide, and a methimazole concentration of 5 x 10-3 M did not appear to be more effective 50- than one of 1 x 10-4 M. It may be presumed that both the stimulation of 0 11leLaDLUllSlll1az~tshaicrn%UD)LJO1VeUlkcArxrlraA Zdot arnnAArntf111OUeI arc rannttWs11CCRFLIttUM5 0of iodide and the loss of stimulation at higher cloncentrations of iodide were more closely re- 200 ated to the concentration of iodide in the thyroid tlhan to its concentration in the medium. In this r egard, it has been clearly established that agents, -010Q- luch as methimazole, that inhibit organic binding i vivo concomitantly increase the concentration e 50- 0 f iodide in the thyroid relative to that in the

t: 0 - 10 10 5x10 5x10 150 0 without T/uniomide, 2 with Thionomide Mo/or Concentration of Hal/ogen Medium

FIG. 4. THE EFFECT OF BROMIDE ON OXYGEN CONSUMP- 100 TION AND GLUCOSE OXIDATION IN SHEEP THYROID SLICES. Each symbol represents results obtained with duplicate flasks in a In 50 single experiment. experiments 46 (A), 54 (0), and 56 (0), glucose-U-C14 was used; in experiment 59 (A), glucose-i-C14 was used. Diagonally hatched columns represent flasks supplemented with iodide; cross- 300- hatched columns represent flasks supplemented with 2 bromide. a 250- a 0 A serum (30, 31).8 Thus, methimazole might have t 200- abolished the stimulatory effect of iodide by in- 0 q)t 150- creasing the intrathyroidal concentration of iodide beyond the stimulatory range. The results of ex- 100- periment 34, however, suggested that this was not the case. Here, in the absence of methima- 50- zole, stimulatory effects of iodide were evident at concentrations ranging between 1 x 10-5 M and O- 0 10-5 lo-4 10-3 1 X 10-3 M (Figure 3). In the presence of Mo/or Concentration of Iodide in Medium methimazole, no stimulation of metabolism was evident at an iodide concentration of 1 x 10-5 M, FIG. 3. THE EFFECT OF ANTITHYROIDAL AGENTS ON IODIDE-INDUCED STIMULATION OF OXYGEN CONSUMPTION 8 To test whether the same phenomenon occurs in vitro, AND C140 PRODUCTION FROM GLUCOSE-1-C14 IN SHEEP THY- slices were incubated in media containing 1 X 10' M ROID SLICES. Hatched columns represent flasks con- iodide, with and without 1 X 10' M methimazole, and taining the antithyroidal agent. Each symbol represents with tracer amounts of P131. After an hour of incubation, results obtained with duplicate flasks in a single ex- F" in slice and medium was partitioned into organic and periment. The agents used, and their concentrations, inorganic moieties by precipitation with trichloroacetic were: in experiment no. 14 (v), 2 X 10- M thiouracil; acid. Without methimazole, 58% of the IP"' was organi- in no. 32 (A), 5 X 10- M methimazole; in no. 34 (0), cally bound, and the S/M iodide gradient was 20. With 1 X 10' M methimazole; and in no. 35 (A), 1 X 104 M methimazole, less than 1% of P"31 was organically bound, methimazole. and the S/M iodide gradient was 46. EFFECTS OF IODIDE ON THYROIDAL INTERMEDIARY METABOLISM 1809

TABLE IV lFfect of iodide on the metabolism of sheep liver and kidney slices

Q02 C1402 p)roduction from glucose-CI4 5 X 10-2 Labeled 5 X 10-2 Exp't no. Tissue Control 10-4 M I-* M I- carbon Control 10-4 M I- M I- Mt/hour/ % C'4/hour/ 100 mg (c control % control 100 mg C% control C- control 12a Kidnev 133 93 C-1 1.0 73 C-6 0.67 84 20a Kidney 102 99 C-1 0.70 94 C-6 0.70 68 73a Kidney 166 96 93 C-U 0.42 90 83 76a Kidney 132 105 C-U 0.56 98 12b Liver C-1 0.058 71 C-6 0.017 88 20b Liver 12.3 92 C-1 0.072 96 C-6 0.022 77 73b Liver 23.4 81 55 C-u 0.030 103 93 74 Liver 32.3 110 85 C-U 0.013 96 91 75 Liver 32.8 92 96 C-U 0.100 88 114 76b Liver 23.8 96 C-U 0.026 108 * Conceiltration of iodide added to incubation medium. and by direct measurement, S/M gradients for generation of C1402 f rom glucose- i-C14, -6-C14, 1131 were 30. Thus, methimazole, in vessels con- and -U-C14.9 Tissue-free blanks were incubated taining 1 x 10-5 M iodine, could not have in- in all the experiments with glucose-U-C14, and creased intrathyroidal iodide to a concentration correction was made for blank radioactivity. Such higher than that present in flasks containing 1 x blanks were not performed in the experiments 10-3 M iodide and no methimazole. with glucose-i-C14 and -6-C14. Effect of bromide. Four experiments were performed to test the effects of supplementing incu- DISCUSSION bation media with bromide, rather than iodide The data presented here are generally in ac- (Figure 4). Since preparations of bromide may cord with earlier observations concerning the be contaminated with iodide, the iodide concen- rate of oxygen consumption and glucose oxidation trations of media enriched with bromide were de- by sheep thyroid slices. They confirm previous termined. These, however, did not differ sig- studies indicating that the oxidation of exoge::ous nificantly from those of simultaneously prepared glucose accounts for a relatively minor proportion KRP (KRP, 3.6 ltg I per 100 ml; KRP and 5 x (in the present studies, approximately 12%) of M 4.0 I 100 Bromide 10-2 bromide, ,tg per ml). 9 If the intracellular concentration of iodide is the de- had no consistent effects either on Q02, or on the terminant of its metabolic effects, direct comparisons be- production of C1402 from glucose-U-C14 (3 ex- tween the effects of standard extracellular concentra- periments) or glucose-1-C'4 (1 experiment), even tions of iodide on the thyroid and on other tissues that at the intermediate concentrations at which iodide do not concentrate iodide are not possible. In liver and kidney, iodide is not actively concentrated (32, 33). was distinctly stimulatory. Therefore, intracellular iodide concentrations in liver or Experiments Znith sheep liver and kidney slices. kidney are probably lower than in thyroid when tissue The concentration of iodide, 1 x 10-4 A, that slices from these respective organs are placed in media most consistently stimulated oxidations in thyroid enriched with iodide at 1 X 10V M; at this concentration, slices had no similar effect on slices of sheep liver thyroid slices maintain S/M gradients greater than unity (11). At a concentration of 5 X 10' M iodide, however, and kidney (Table IV). At the higher concentra- thyroidal S/M gradients decline below unity (11), and tion of 5 x 10-2 M, however, iodide had a con- intracellular iodide concentrations in thyroid, liver, and sistent inhibitory effect on Q09 and decreased the kidney are probably quite similar. 1810 WILLIAM L. GREEN AND SIDNEY H. INGBAR total oxygen consumption (34). This is con- Evidence can be adduced concerning the path- sornant with the observation that the QO2 of ways over which stimulatory concentrations of io- thyroid slices is well-maintained although no sub- dide produced their second major effect, increased strate is added to the medium (35-37). The en- production of CO2 from exogenous glucose. It is dogenous substrate whose oxidation accounts for clear that the quantity of exogenous glucose oxi- most of the oxygen consumed is, however, un- dized in the hexose monophosphate (HMP) known. It has been reported that the concentra- shunt increased, in view of the greater increase tion of glycogen in thyroid tissue is very low that iodide induced in C-1 than in C-6 oxidation. (38) and is insufficient to support observed rates It cannot be concluded, however, that iodide in- of oxygen consumption (39). On the other hand, creased the proportion of glucose metabolized via in agreement with the findings of Weiss (35), the HMP shunt.'0 Any factor that generally respiratory quotients in the present studies were stimulates glucose metabolism will be manifested near unity, suggesting that a major portion of the by a prompter increase in C-1 than in C-6 oxida- endogenous substrate is derived from carbohy- tion, since C-1 is immediately available for oxida- drate. No data are presently available on the tion by the shunt, whereas C-6 must traverse a catabolism by thyroid slices of either endogenous much longer series of reactions and be diluted lipid or the large stores of protein and pro- in a greater number of intermediate pools before tein-bound hexoses that the thyroid contains (40). appearing as CO2. This is reflected in Dumont's The fate of the major proportion of assimilated finding that the ratio between C1402 production glucose is unknown, since a very small proportion from glucose-i-C'4 and from glucose-6-C14 (the (3 to 6%o) is converted to CO2, and only about "C-1/C-6 ratio") declines progressively with 40%o is converted to lactate. time (44). This decline also occurs at stimulatory The major finding of this study is that pro- concentrations of iodide (22). nounced effects on certain aspects of thyroidal Several considerations indicate that the HMP intermediatry metabolism can be acutely induced shunt was not the sole metabolic pathway stimu- by variations in extracellular iodide concentra- lated by iodide. These follow from the increase tion. These effects are biphasic: oxygen con- in the generation of C1402 from glucose-6-C14 sumption and conversion of exogenous glucose to that iodide induced. This could have occurred CO2 are stimulated by concentrations of iodide merely as a result of increased formation of triose- ranging up to approximately 1 x 104 M, a find- C14, without any alteration in its proportionate ing which has also been reported by Dumont conversion to CO2. Had this been the case, then (41), whereas at higher concentrations of iodide, the percentile increase in formation of lactate- these oxidative processes return toward normal. C14 should have equalled the percentile increase Tentative conclusions can be drawn concerning in formation of C140, from glucose-6-C14. A the nature of the stimulatory metabolic effects of change of this magnitude (an increase of over iodide observed in the present study. First, iodide 50% at 1 x 10-4 M iodide) should readily have caused increased oxygen consumption. In the 10 Katz and Wood have recently described a method absence of added iodide, oxidation of endogenous for calculating the proportion of glucose metabolized via substrate in the thyroid appeared to account for the HMP shunt by use of the fractional yields of C140. much of the oxygen consumed. Although stimu- from assimilated C-1- and C-6-labeled glucose (42). latory concentrations of iodide increased the pro- Although yields of C1402 from C-1- and C-6-labeled glu- portion of the oxygen consumed that was uti- cose were not precisely determined in the present studies, owing to failure to correct values for volatile contami- lized for the metabolism of exogenous glucose, nants in the glucose, this method was used to provide endogenous compounds of unknown nature re- a first-order approximation of the percentile contri- mained the principal oxidative substrate, and their bution of the HMP shunt to exogenous glucose me- metabolism must have been increased. Further- tabolism in sheep thyroid slices. Calculations revealed more, absence of significant change in respira- this to be no more than a few per cent in control slices. Such low values are consonant with recent observations tory quotients in association with increased Q02 that the yields of C,4-labeled lactate from glucose-U-C4 suggests that iodide changed the amount, rather or -1-C4 do not differ appreciably from the yields of la- than the nature, of the substrates concerned. beled lactate obtained with glucose-6-C'4 (43). EFFECTS OF IODIDE ON THYROIDAL INTERMEDIARY METABOLISM 1811 been detectable by the method employed for lactate i.e., iodide, can increase the rate of oxidation of measurement, and no such change occurred. other substrates, can be explained in several ways. Thus, the increased forniation of C1402 from First, studies in thyroid homogenates and sub- glucose-6-C14 appears to have resulted from an in- cellular fractions have demonstrated a peroxidase crease in the proportionate conversion of triose to capable of oxidizing iodide in the presence of CO,. This increase could occur for several rea- H120 (13, 14). Evidence has recently been pre- sons. First, acetate-C14 derived from exogenous sented suggesting that the same enzyme may cata- glucose may, in the presence of iodide, have been lyze both the formation of H202 and subsequent diluted to a lesser extent by acetate derived from peroxidations (49). The reactions involved in unlabeled endogenous sources. This explanation the generation of H202 probably occur according seems inconsistent with the large increase in the to the following sequence (15, 16): oxidation of endogenous substrate that iodide in- TPNH (or DPNH) + H+ + flavoprotein > duced. Second, a proportion of C14 reaching the TPN+ (or DPN+) + flavoprotein * H2 [ 1 ] 3-carbon pool may have been diverted from vari- flavoprotein H2 + O2-> H202 + flavoprotein [2] ous synthetic reactions (e.g., formation of lipids and amino acids) to the formation of CO2. The H2o2 + 21- + 2H+ 21 + 2H20. [3] present data do not exclude this possibility. Fi- Over-all: nally, iodide may have directly stimulated oxida- TPNH + 3H+ + 21- ±+ 2->TPN+ + 2H20 tive disposition of Krebs-cycle intermediates. + 21. [4] Such an increase in Krebs-cycle oxidations could If this is the correct mechanism, then it would have resulted f rom an uncoupling of oxidative follow from the present results that the rate of phosphorylation, such as iodide induces in rat the over-all reaction depends on the concentra- liver mitochondria (45). Alternatively, it might tion of iodide. In this way, oxidation of iodide reflect an increase in the availability of cof actors, could directly increase oxygen consumption and such as oxidized pyridine nucleotides and flavins. could indirectly stimulate other substrate oxi- This explanation seems especially attractive in dations by increasing the availability of oxidized \view of the evidence of increased activity of both pyridine nucleotides. Implicit in this argument, the HMIP shunt and the Krebs cycle, as discussed however, is the suggestion that the rate of perox- above. ide formation is controlled by the rate of peroxide Several findings indicate that the stimulatory utilization, a situation not clearly shown to oc- effects of moderate concentrations of iodide on cur in biological systems.1' thyroidal metabolism, whatever the pathways me- One argument against the foregoing mechanism diating them, are related to the effects of iodide derives from stoichiometric considerations. In on the rate of hormone synthesis. First, like the postulated sequence of reactions, one mole of the presently demonstrated increases in Q02 and oxygen should be consumed for every two moles C1402 production, total iodinations in sheep thy- of iodide oxidized. An iodide concentration of roid slices increase progressively as the iodide 1 X 10-4 M in the medium, however, increased concentration in the medium is increased to ap- oxygen consumption by as much as 1.5 tumoles per proximately 1 X 10-4 M (6). Second, when or- flask, although the flask contained only 0.2 umole ganic iodinations are inhibited by methimazole of iodide, of which only a small proportion would or thiouracil, stimulation of metabolism by io- be expected to have undergone organic binding dide is abolished. Furthermore, no metabolic (6). This objection can be overcome, however, stimulation is induced by bromide, a halogen that 11 As an alternative to this mechanism, it may be that undergoes negligible organic binding (46-48). reactions 1 and 2 proceed at a constant rate and that Finally, iodide induces no metabolic stimulation only reaction 3 is stimulated by iodide. If this were the in liver and kidney, tissues that do not organically case, and if, when the supply of iodide was limited, bind iodide. These findings suggest that the oxi- H20. were attacked by catalase, iodide would still stimulate net oxygen consumption, since the catalatic process dation of iodide is linked to and somehow influ- returns one mole of oxygen for each two moles of H202 ences the rate of other thyroidal oxidations. The degraded. This mechanism per se, however, would not apparent paradox, that oxidation of one substrate, provide for increased substrate oxidations. 1812 WILLIAM L. GREEN AND SIDNEY H. INGBAR by postulation of a cyclic oxidation and reduction count for the stimulatory effects of moderate of iodine. This could come about in either of two concentrations of iodide on thyroidal oxidative ways. First, the thyroid appears to contain in- metabolism, there remains to be considered the hibitors of organic iodinations, such as ascorbic cause of the decline in or loss of stimulation oc- acid or glutathione (GSH) (15, 49, 50), that curring when concentrations of iodide are further could reduce oxidized iodine before it is organi- increased. This biphasic metabolic response re- cally bound. If the inhibitor were GSH, the sembles the biphasic response of total organic reaction (no. 5) would be: 2GSH + 210 > GSSG iodinations to increasing concentrations of extra- + 2H+ + 21. Since glutathione reductase is pres- cellular iodide, and thus provides a further sug- ent in thyroid tissue (15, 51), the following reac- gestion that the oxidative metabolism of the thy- tion (no. 6) would also be expected: GSSG + roid gland and its rate of hormone formation are

TPNH + H+ -* 2GSH + TPN+. closely related. Both in vitro and in vivo, the A second, less direct mechanism for the reduc- thyroid responds to acute increases in extracellu- tion of iodine involves the deiodination of iodo- lar iodide concentration with increasing organic tyrosines by the thyroidal iodotyrosine deiodinase iodinations. Beyond a critical concentration (52). This enzyme is TPNH-dependent (53, range, however, which in sheep slices is approxi- 54), and its operation presumably results in the mately 1 x 10-4 M (6), organic iodinations de- generation of TPN from TPNH. In view of the cline, although not necessarily to control levels. recent demonstration that the thyroid contains Two types of relationship between this so-called free iodotyrosines that are rapidly turning over Wolff-Chaikoff effect and the biphasic effects of (55), a substantial recycling of iodide through extracellular iodide on thyroidal oxidative metab- iodotyrosines may occur. olism can be envisioned. First, if the rate of Both mechanisms cited above for the reduction iodide oxidation in some measure regulates oxi- to iodide of an oxidized form of iodine provide dative intermediary metabolism in the thyroid, as for the generation of TPN from TPNH, and suggested above, then declining oxidation of io- both might therefore be expected to enhance glu- dide during the Wolff-Chaikoff effect may ex- cose oxidation. Indeed, addition of iodotyrosines plain the lessening of metabolic stimulation. It to thyroid tissue has been shown to have this effect has been suggested that the Wolff-Chaikoff ef- (38, 41, 56). If reactions 1 to 4 are also relevant, fect is due to complexing of oxidized iodine with each mole of iodide cycled through an oxidation iodide, in the presence of high concentrations of and reduction would consume j mole of oxygen the latter, to form the triiodide ion I31, which is and would generate one mole of TPN. To the incapable of carrying out iodination (8). This extent that extensive recycling of iodine occurs, theory, still unproven, does not predicate a de- therefore, especially when intrathyroidal iodide creased initial oxidation of iodide. It does imply, is moderately enhanced, a cascade of electron however, that the TPNH-oxidizing reactions sec- transfers would occur and would be associated ondary to the recycling of iodide, which appear with increases in both Q02 and substrate oxida- to be critical to the production of an appreciable tions. Quantitatively, this would be poorly re- metabolic effect, would be diminished or abolished. flected in net organic iodinations. An alternate relationship between the Wolff- Finally, a third mechanism by which iodide Chaikoff effect and the decline in metabolic stimu- may stimulate substrate oxidations has been sug- lation occurring at high concentrations of iodide gested by de Groot and Davis, who demonstrated also seems plausible. In vitro, high concentrations that an enzyme solubilized from a sheep-thyroid of iodide have been shown to inhibit two en- particulate fraction can peroxidize reduced pyri- zymes involved in oxidative metabolism: glucose- dine nucleotides (57). The activity of this en- 6-phosphate dehydrogenase (58) and fumarase zyme, and hence the generation of oxidized pyri- (59). Such inhibition could account for the dine nucleotides, is directly related to iodide con- decrease in Q02 and C1402 production that high centration, since iodide apparently protects the concentrations of iodide induced in slices of liver enzyme from degradation. and kidney. Other related enzymes, such as Although the foregoing mechanisms may ac- aldolase (60) and lactic dehydrogenase (61), are EFFECTS OF IODIDE ON THYROIDAL INTERMEDIARY METABOLISM 1813 inhibited by molecular iodine, possibly through REFERENCES oxidation of sulfhydryl groups (62). If physio- 1. Wolff, J., and I. L. Chaikoff. 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