CARBOHYDRATE UTILIZATION BY BEEF THYROID TISSUE*

BY BENJAMIN WEISS

WITH TIIE TECHNICAL ASSISTANCE OF CAROL CASTRICUM

(From the Research Division, Harper Hospital, Detroit, Michigan)

(Received for publication, July 9, 1951)

Preliminary to a study of various pathological thyroid states, it was considered necessary to investigate first the carbohydrate metabolism of the normal gland. Also, it was thought that the information gained re- garding the function and energetics of the normal gland might eventually provide an insight into the mechanism of formation of the hormone, thyroxine.

Although numerous studies related to thyroid function have appeared in the literature, little is known of the pattern of carbohydrate utilization in this gland. Values for oxygen consumption (1) and the influence of goitrogenic and related compounds on the cytochrome oxidase activity of surviving thyroid slices (2) have been reported. That several enzyme systems involved in carbohydrate metabolism are present in the thyroid gland has been demonstrated by means of chemocytological procedures (3).

Because of the important Ale of the thyroid gland in regulating the rate of cellular metabolism, considerable effort in the past has been spent in studying the effects of thyroxine separately and in conjunction with the thyrotropic hormone of the pituitary gland in biological oxidations. Within the large volume of data that have accumulated, varying results have been reported as to the capacity of thyroxine (4-9) and thyrotropic hormone (10-12) to exert a stimulative effect when added to isolated surviving tissues.

It is the purpose of this investigation to study the carbohydrate metab- olism of the normal thyroid gland and to reexamine the hormonal effect on respiration.

EXPERIMENTAL

Preparation-Mature steers’ were killed by exsanguination. In a few experiments the animals were first stunned by a blow on the head before being bled. Both lobes of the thyroid gland were rapidly excised, placed in a wide mouthed bottle containing the suspending medium to be used

* This work was aided in part by a grant from the Kresge Foundation. 1 We wish to express our gratitude to Mr. Emil Hofman of the Hygrade Food

Products Corporation, Detroit, Michigan, for making available a generous supply of material.

509

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510 CARBOHYDRATE UTILIZATION BY THYROID

in the experiment, and conveyed from the abattoir to the laboratory in a Dewar flask packed with cracked ice. The gland was trimmed of ad- herent fat and connective tissue, and then slices were prepared with a Stadie-Riggs microtome. In the homogenate studies, the trimmed gland was blended for 1 minute in a Waring blendor with 4 parts of 0.05 M phos- phate buffer, pH 7.4, and promptly used after being filtered through a double layer of cheese-cloth. Approximately 1 hour elapsed from the time the gland was removed from the animal until the tissue was ready for incubation.

Substrates and other factors were added to the tissue from the side arm after a 10 minute equilibration period. Acidic components were brought to pH 7.4 with either 0.3 N NaOH or 0.1 M KzC03. In the slice experi- ments, all substrates were employed in a final concentration of 0.033 M;

the suspending medium was Ringer-phosphate solution unless otherwise indicated. At the end of the experimental period the slices were removed, pressed free of salt solution, and dried to constant weight for 24 hours in an oven at 110”. Nitrogen analyses were performed on the dried tissue. Generally, oxygen consumption was measured at 30 minute intervals for 1 hour at 38” by means of a Summerson differential manometer.

Methods-Adenosinetriphosphate (ATP) and oxalacetic acid were ob- tained as outlined in Umbreit et al. (13). The purity of the ATP was 91 per cent as assayed by the acid-labile phosphorus released in 7 minutes at 100”. Crystalline sodium pyruvate was prepared from the corresponding commercial acid by the procedure of Robertson (14). The sodium salts of fructose-6-phosphoric and fructose-l ,6-diphosphoric acids were pre- pared by dissolving the commercial products in 0.1 N HCI, decolorizing with norit, and centrifuging after the addition of saturated NazSOI in slight excess. The supernatant liquid was used after neutralization with 1 N NaOH. In a few experiments, the fructose-l ,6-diphosphate was further purified (15). Acetoacetic acid was derived from the corresponding ester (16). Cytochrome c was prepared from fresh horse heart (17), and stand- ardized spectrophotometrically. In the final step of dialysis of the cytochrome c against distilled water, the time was considerably shortened by using a mechanical dialyzer with a Cramer synchronous motor made by the American Instrument Company (18). Diphosphopyridine nu- cleotide (DPN) was a commercial product (Schwarz), assaying 54 per cent in the Beckman spectrophotometer. DPN which had been prepared from bakers’ yeast (19) and which assayed 41 per cent was also used. The cortisone, obtained as a saline suspension, was employed in alcoholic solu- tion. The hormone concentration was adjusted by the addition of the required amount of 50 per cent ethanol. The thyrotropic hormone was prepared as an aqueous solution and used directly.

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B. WEISS 511

The hormone preparations were obtained as follows: m-thyroxine from the Delta Chemical Works, thyrotropic hormone from Parke, Davis and Company, and cortisone from Merck and Company. All other materials employed were of reagent grade.

Glucose was determined by the Nelson (20) method, and fructose and its esters by the procedure of Roe (21). The method of Barker and Sum- merson (22) was used to estimate lactic acid. Analyses for pyruvate, cu-ketoglutarate, oxalacetate, and total keto acids were made according to Friedemann and Haugen (23). Citric acid was measured according to the method of Perlman, Lardy, and Johnson (24) as modified by Speck, Moulder, and Evans (25). Amino acids were determined according to Frame, Russell, and Wilhelmi (26). Tyrosine analyses were performed on metaphosphoric acid filtrates (27) by the method of Bernhart (28). Adenosinetriphosphatase (ATPase) activity was measured by the pro- cedure of DuBois and Potter (29) ; the liberated inorganic phosphate was determined by the method of Lowry and Lopez (30).

Results

Oxygen Quotient-It was occasionally observed in these experiments that a considerable variation in the Qo, attended the incubation of slices prepared from the same gland. This was attributed to trauma, presum- ably arising from the unequal handling and cutting of the tissue during its preparation into slices, or to the fact that not all parts of the gland function to an equal extent at the same time, which has been demon- strated with the Geiger-Miiller counter on intact humans or dogs and by means of autoradiography (31). In Table I are presented QO, values for thyroid tissue obtained from normal animals. It is seen that the Q,,, of steer thyroid lies in the region of 3.0. In order to ascertain how the Qo, is affected, the conditions of the experiment were varied. Starting with the suspending medium, it was found that incubation in Ringer-bicar- bonate or phosphate buffer (pH 7.4) solution produced no effect on the rate of respiration. A change in the gas phase from air to pure oxygen gave similar results. The addition of various substrates from the glyco- lytic and tricarboxylic acid cycles to the medium also failed to show any significant change in the oxygen consumption.

In consideration of the effect of castration upon the rate of oxygen utilization, a comparison with other members of the species disclosed that little difference appeared in the QoZ values of thyroid from bull, cow, heifer, or calf. In the same manner, no significant change is observed with slices, containing blood elements, from glands of animals which were first stunned before being sacrificed.

Since the thyroid is infiltrated to a large extent with such non-respira-

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512 CARBOHYDRATE UTILIZATION BY THYROID

tory elements as follicular colloid and connective tissue, the expression of the Qo, as a function of the dry tissue weight was checked by employing another tissue referent. That the Qo,, when measured on a dry weight basis, is representative of the active components of the tissue is essen- tially confirmed by agreement with similar calculations based on the total nitrogen content.

The respiratory quotient was determined by the “direct” and “indirect” methods of Warburg and by the differential method of Summerson; the values, corrected for bound CO2 and representing the average of triplicate determinations, are 1.07, 1.16, and Oy97 respectively. The suspending

TABLE I

Oxygen Consumption of Beef Thyroid Slices

Animal QCl,* Qo, (NH

Steer....................................... “ . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I‘ (stunned; see text) . . Bull . . . . . cow. . Heifer......................................

Calf. . Steer 11.

3.1$

“ ,

2.89

3.4 3.5 3.8 2.7

2.8 2.8 3.0

“ 3.3

24.4 25.4

28.9

* Microliters of oxygen taken up per mg. of dry tissue per hour. t Microliters of oxygen taken up per mg. of tissue nitrogen per hour. $ Average obtained from thirty-four experiments; range, 1.9 to 5.0.

0 In an atmosphere of pure oxygen, 2.7 ~1. of oxygen were taken up. 11 Slices prepared from separate glands.

medium was Ringer’s solution. It was observed that slices were able to respire at a sustained rate in the absence of added substrate. After more than 10 hours, the oxygen consumption gradually begins to decline. It may be that the ability of the slice to respire for such long periods is re- lated to the slow rate of utilization of its metabolites.

Aerobic and Anaerobic Glycolysis-The production of lactate was studied with slices incubated in an aerobic and an anaerobic environment, and it was found that no lactate was formed in excess of the controls under either set of conditions. Warren and Ebaugh (32) studied the influence of various ions on the anaerobic glycolysis of rat liver slices. In view of their findings, a medium of the same ionic composition that was employed in their experiments was tried. The results were essentially the same as those previously obtained, even in the presence of added glucose, fructose,

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B. WEISS 513

or a-glycerophosphate. The small amount of lactate which consistently appeared in the suspending medium (5 to 10 y per mg. of dry tissue) is probably due to “leakage” from injured or broken cells incurred during the preparation of the slice.

Effect of Added Substrates-It was mentioned earlier that the addition of a number of carbohydrate intermediates failed to influence the respira- tory rate. The following compounds were tested and found to be without effect: glucose, glucose-l-phosphate, fructose, fructoseS-phosphate (F6P), fructose-l ,6-diphosphate (HDP), CY- and P-glycerophosphates, lactate, pyruvate, citrate, cY-ketoglutarate, succinate, fumarate, malate, oxalace- tate, acetate, acetoacetate, butyrate, glycine, alanine, glutamate, tyrosine, and 3,5-diiodotyrosine. Pyruvate (33) and citrate were used in a cal- cium-free medium. The above compounds in various combinations were also ineffective. Chemical analyses for the substrates added to the medium were entirely in agreement with the manometric observations. Although it is known that certain hexose phosphates and Cd acids are inert to a variety of cells and tissues (34), this explanation for the results observed seemed unlikely on the basis of the importance and number of the compounds examined. It appeared more reasonable to suppose that the tissue is saturated with its own metabolites and therefore, as a con- sequence of the greater access, preferentially utilizes them. It was thought that, if the tissue were allowed to respire long enough to exhaust its foodstuffs, then the addition of substrate might result in its utilization, with an increase in the oxygen uptake, provided that the integrity of the tissue remained intact throughout the given period. Such an attempt, however, proved unsuccessful, since, in a series of experiments in which the substrates were added to the tissue when the oxygen uptake was less than 20 per cent (after 11 to 13 hours for a 15 mg. slice, dry weight) of the first hour reading, in no case was an increase in the respiratory rate observed. Instead, the respiration diminished at the same rate as in the absence of substrate.

E$ect of Various Compounds on Respiration-Several compounds, whose effect on respiration is well known, were tested; however, their use on a heterogeneous system such as a tissue slice yields information primarily of a general nature. Of the compounds examined (Table II), an appreciable inhibition is exerted by iodoacetate and cyanide, whereas with cysteine and sulfide there is a marked stimulation. The presence of sulfhydryl and iron-containing enzymes is in accord with these results. Thiocyanate and thiouracil were examined because of their goitrogenic action in the intact organism. Despite the comparable oxidation-reduction potential of cys- teine and sulfide, their effect on respiration is of unequal magnitude. Moreover, the pronounced increase in the oxygen consumption in the

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514 CARBOHYDRATE UTILIZATION BY THYROID

presence of sulfide is of interest in view of its inhibitory effect on cytochrome oxidase.

Carbohydrate Balance-Since the methods employed thus far revealed little as to the nature of the compounds being utilized within the thyroid cell, the following experiment was conducted; the procedure of Lyman and Barron (35) was adopted with a few modifications. Approximately 400 mg. of slices (wet weight) were added to 25 ml. Erlenmeyer flasks con- taining 5 ml. of Ringer-phosphate solution. In the early experiments, the flasks were charged with 95 per cent 02-5 per cent COZ; however, this step was found unnecessary since similar results were obtained in the

TABLE II Effect of Various Compounds on Respiration of Steer Thyroid Slices

Final concentration of compounds 0.033 M unless otherwise indicated.

Compound Q0** Per cent inhibition or stimulation

None...................................... 3.0 Arsenate. . . . . . . . . . . . . . . . . 3.4 Cyanide? (1.7 X lo-’ M). 2.0 -33

“ (1.7 x 10-s ‘0. 1.0 -66 Cysteine 6.0 t100 Fluoride................................... 3.3 Iodoacetate (3.3 X 10-s M). 1.6 -47

‘I (3.3 x 10-r “). 0.7 -77 Malonate.................................. 3.1 Sulfide. . . 10.7 t-257 Thiocyanate............................... 3.5 Thiouracil (8 X lo-+ M). . 2.9

- * Each value is an average of duplicate determinations. t Alkali-cyanide mixture for center well, as in Umbreit et al. (13).

presence of air. The flasks, open to the atmosphere, were then placed in a metal rack and shaken for 4 hours in a constant temperature bath at 38”. After the incubation period, the reaction was stopped by the addi- tion of 1 ml. of 5 N HzSOc and the contents of each flask digested for 3 hours on a steam bath. During the early phase of digestion, the slices were macerated with a stirring rod to aid solution of the tissue. The digested contents were quantitatively transferred to 25 ml. volumetric flasks, and, after dilution to the mark, poured into 250 ml. beakers con- taining 4 gm. of BaC03. The contents of each beaker were swirled, and, if the solution were still acidic when the evolution of CO2 ceased, more BaC03 was added. The BaSOa was removed by centrifugation and anal- yses were performed on aliquots of the neutral supernatant fluid.

Although no detailed fractionation of the mixture was undertaken, it

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B. WEISS 515

was believed that a general trend would be indicated in so far as the com- pounds determined probably represent the bulk of the carbohydrate con- stituents present in the tissue. The results of a typical carbohydrate balance experiment are shown in Table III. It is evident that lactate is the only metabolite of those determined which undergoes disappearance.

Hormonab E$ect on Respkation-The influence of thyroxine, thyrotropic

TABLE III

Carbohydrate Balance of Steer Thyroid Slices

Non-incubated* Incubated Metabolite determined

Sample 1 Sample 2 Sample 1 Sample 2 ~~~____

Y 7 Y Y

“Apparent” glucose. . . . 465 405 435 450 Lactate................................... 180 181 90 95 Total hydrazones. . . 85 87 80 90

All values based on 500 mg. of wet tissue; dry weight, 10 per cent of wet weight. For “apparent fructose” and citrate the concentration was too low for measurement.

* Samples inactivated at zero time.

TABLE IV

Hormonal Effect on Respiration of Steer Thyroid Slices Final concentration of hormone indicated in parentheses.

None............................ 2.8 2.9 2.9 2.3 2.7 Thyroxine (3.3 X 1O-3 M). 2.5 2.7 2.6 2.7 2.5 Thyrotropict (0.066%) 2.9 2.9 3.0 2.9 2.7 Cortisone (0.033%). 3.1 3.0 3.1 2.9 2.9

1st 2nd. 3rd 4th 5th ---

* Hormones added to tissue at the beginning of the hour. t Assay, 6 to 8 units per mg.

hormone, and cortisone on the rate of respiration was examined. In the experiments which follow, the same slice was employed in both a control and test capacity. This eliminated the conventional use of two groups of slices; i.e., a control and test group from which average results are cal- oulated and compared. In this manner, the variations inherent among individual slices are avoided and the results are reported as a function of a single slice. Obviously, this approach was made possible by the char- acteristic behavior of the slice in carrying on respiration at a sustained and linear rate. In Table IV Qoz values are given for 5 successive hours. The 1st hour is in the absence of hormone and thus serves as a control

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516 CARBOHYDRATE UTILIZATION BY THYROID

reading. In the hours which follow with hormone present, it is seen that the Qo, readings remain practically unchanged. It has been assumed that, if an elapse of time or “induction period” is necessary before the activation effect can occur, it would be detected during the observed ex- perimental period.

Homogenate Requirements-By employing homogenates supplemented with the necessary cofactors, it was believed that most of the difficulties encountered previously with the slice in obtaining the utilization of added substrate would be obviated. Preliminary tests with a number of sub- strates involved in the sequence of oxidations in the carbohydrate cycle revealed that oxygen uptake was obtained with homogenates fortified

TABLE V Optimum Conditions for Utilization of Fructose-1,6-diphosphate

The data for each component added were obtained from separate experiments. The concentration of each component was varied and all the other components were held constant and at their optimum concentration.

01 uptake per flask DPN

Oa uptake per flask

On uptake Cytochrome c ATP

02 uptake HDP

per hr. per flask per hr. per hr.

per flask per hr.

-- II x 10-r cl. per cent d. al d. M Pl.

0 18 0 50 0 74 0 42 2.3 160 0.25 166 0.0088 194 0.05 121 3.8 208 0.50 178 0.0176 202 0.10 163 7.6 202 1.0 202 0.0352 209 0.20 201 3.8* 201 2.0 198 0.30 204

* 0.1 ml. of 0.2 M nicotinamide present in addition to the other components of the reaction mixture.

with cytochrome c, DPN, and ATP. Since, of the compounds examined, the greatest activity was obtained with HDP, it was used as the substrate in establishing the optimum conditions of the system. Data are pre- sented in Table V showing the effect of concentration of the various co- factors on the oxygen uptake. It is evident that with the omission of either cytochrome c, DPN, ATP, or HDP the oxygen consumption is con- siderably reduced. The addition of nicotinamide in varying concentra- tions to the complete system produced no noticeable effect on the rate of respiration. The optimum concentration of the components of the final reaction mixture is as follows: 1.5 ml. of 20 per cent homogenate, 0.5 ml. of 3.8 X lOA M cytochrome c, 0.1 ml. of 1 per cent DPN (54 per cent purity), 0.1 ml. of 0.0196 M ATP, 0.2 ml. of 0.2 M HDP, and Hz0 to a final volume of 3.0 ml. The center well contained 0.2 ml. of 20 per cent KOH. This system was used in all of the subsequent experiments in regard to changing the substrate or adding other factors. In either case,

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the concentration of substrate and the final volume of the reaction mix- ture were maintained the same. The oxygen uptake of the system is linear with the optimum concentration of cofactors during the first 45 minutes, after which the rate of uptake begins to decline.

Effect of Di$erent Media and Storage-The effect on the activity of homogenates prepared in various suspending media was examined with HDP as substrate. In previous studies, Hz0 homogenates have been employed in the determination of the malic dehydrogenase (36) and glyco- lytic (37) systems, whereas isotonic homogenates were demonstrated to be more suitable in the oxidation of oxalacetate (38) and octanoate (39). Potter et al. (38) state that the cytoplasmic particles of isotonic homo- genates appear to be functionally different from the particles present in water homogenates. Thyroid homogenates of 20 per cent concentration were prepared in 0.05 M phosphate buffer, (pH 7.4), distilled water, and isotonic KCl; the microliters of oxygen taken up per mg. of tissue nitrogen per hour were 26.6, 20.4, and 15.8 respectively. Each value represents an average of duplicate determinations. It is seen that the greatest oxy- gen uptake occurs with hypotonic phosphate buffer. The greater degree of cytolysis that would be expected to occur with the use of Hz0 in the preparation of the homogenate probably accounts for its greater activity as compared to the homogenate prepared with isotonic KCl. A compari- son of the water and hypotonic phosphate homogenates immediately re- flects the importance of inorganic phosphate.

Storage of the homogenate for 24 hours at 5” results in a progressive loss in oxygen consumption with the substrates, F6P, HDP, lactate, suc- cinate, fumarate, and malate. The loss of activity that occurs when the homogenate is dialyzed at room temperature for 4 hours against phos- phate buffer is apparently due to standing, since an undialyzed portion of the same homogenate held for an equivalent length of time as that taken for the dialysis exhibited a similar loss. of activity. It was found that, although respiration diminishes with storage, the glycolytic activity with either F6P or HDP as substrate is only slightly affected. In a typi- cal experiment the oxygen uptake per flask per hour for the fresh homo- genate with F6P and HDP as substrates was 168 and 197 ~1. respectively; with storage the uptake correspondingly decreased to 104 and 124 ~1. In the same manner, the lactate formed per flask per hour was 1194 and 1211 y for the fresh homogenate and 1086 and 1188 y for the stored prep- aration. In an attempt to ascertain whether or not the decreased oxygen uptake toward F6P and HDP with storage might be associated with the inactivation of sulfhydryl groups, the activity of the fresh and stored homogenate was determined in the presence of cysteine. In each case, the same relative loss in activity occurred.

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518 CARBOHYDRATE UTILIZATION BY THYROID

Substrates Utilized-The compounds oxidized by the present system are shown in Table VI; the system contained the optimum concentration of cofactors found for the utilization of HDP. Lactate formation and oxy- gen uptake are greatest when either F6P or HDP acts as substrate. In the presence of magnesium ions, a considerable increase in the formation of lactate from F6P occurs, along with a depression in the oxygen uptake. However, if fluoride is added in place of magnesium ions, the reverse effect takes place, in which the accumulation of lactate is decreased and

TABLE VI Oxygen Consumption in Presence of Various Substrates

0.1 ml. of 0.1 M of either K glutamate, MgC&, or Na fluoride was added as indi- cated; otherwise, the components of the reaction mixture are as described in the text.

Substrate

None. ..............................

Fructose-6-phosphate ................ “ + Mg. ........ I‘ + fluoride.

Fructose-l ,6-diphosphate ............ a-Glycerophosphate ................. Lactate ............................. Pyruvate ........................... Succinate ........................... Fumarate ........................... Malate. ............................. Gxalacetate ......................... Lactate + glutamate ................ Fumarate + glutamate .............. Malate + glutamate. ...............

-

C

--

-

Y--

h uptake per flask per hr.

d. 13

212 178 267 224 73

+;;* 111 60 56

+22 110 102 109

.actate formed er flask per hr

7

9 860

1470 538 958 202

129 76

112 113

-

.-

Pyruvate forked per flask per hr.

Y

1 180 123 15

196 19

131

10 26 28

* The depression in oxygen uptake below that of the control is designated by +.

the oxygen uptake increased. A net increase, although slight, in both lactate and pyruvate occurs when succinate, fumarate, or malate serves as substrate. The effect of pyruvate on the oxygen consumption was consistently inhibitory. In the presence of oxalacetate, the oxygen up- take varied, an inhibition being observed in most cases. When lactate or pyruvate was incubated with any of the member acids of the cycle with the present tissue preparations, the oxygen consumption in the presence of both compounds together never exceeded the sum of the values ob- tained with each compound separately. The increase in oxygen uptake when glutamate is incubated with lactate, fumarate, or malate (Table VI) is probably due to transamination. The oxygen consumption of the con-

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trol was due to the oxidation of lactate, which was initially present in the homogenate.

The compounds that were examined but which gave no oxygen uptake above the control were as follows: glucose, glucose-l-phosphate, fructose, &glycerophosphate, citrate, cu-ketoglutarate, acetate, butyrate, acetoace- tate, glycine, alanine, glutamate, tyrosine, and diiodotyrosine. The fatty acids were tested under a variety of conditions including the addition of magnesium ions and a cooxidant such as malate (39), but with negative results. Similarly, the conditions for glucose and fructose utiliiation were also varied. Incubation in an anaerobic atmosphere with the addition of either fluoride or increased amounts of ATP was without effect. In order to ascertain the extent to which the dephosphorylation of ATP was re- sponsible for the observed results with glucose and fructose, the ATPase activity of the homogenate was determined. It can be seen from Table

TABLE VII Adenosinetriphosphatase Activity of Steer Thyroid Tissue

167 7 of ATPase phosphorus was present in the incubation mixture. A 10 per cent, water homogenate was used as the enzyme source.

Homogenate used Phosphorus liberated in 15 min.

ml. per cm;

0.1 35.4 0.2 66.4

VII that with 0.2 ml. of a 10 per cent homogenate approximately 66 per cent of the ATPase phosphorus is cleaved in 15 minutes. Therefore, it is

likely that the ATP of the reaction mixture exists intact for only a short time. However, in the presence of fluoride a significant inhibition of ATPase activity is obtained. Meyerhof and Wilson (40) showed that in brain preparations most of the dephosphorylating activity toward ATP was associated with the structurally bound elements and could be removed by moderate centrifugation. Extracts prepared in this manner were ca- pable of rapidly phosphorylating glucose or fructose. Thyroid homo- genates treated similarly by centrifuging at 3000 r.p.m. failed to show any evidence of glucose or fructose disappearance as well as any formation of lactate.

It seems to appear that the high ATPase activity of the homogenate is advantageous for the utilization of HDP, for the faster ATP is dephos- phorylated the more rapidly can it accommodate the discharge of phosphopyruvic acid. In the presence of F6P as substrate, its conversion to HDP aids further the role of phosphate acceptor by the adenylic system through the formation of adenosinediphosphate. Since magnesium ions

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520 CARBOHYDRATE UTILIZATION BY THYROID

catalyze the conversion of F6P to HDP, and since fluoride inhibits the breakdown of ATP, the results above regarding the formation of lactate from F6P are readily explained.

An examination for the possible occurrence of D- or L-amino acid oxidase was made on the basis of the presence of flavin mononucleotide and flavin- adenine dinucleotide in thyroid tissue (41). When the procedure of Krebs (42) was employed, no activity was found toward nn-alanine with either whole homogenates or extracts.

E$ect of Compounds on Glycolysis and Respiration-A number of com-

TABLE VIII Effect of Various Compounds on Glycolysis and Respiration with

Fructose-l ,6-&phosphate As Substrate

All of the compounds were prepared in 0.2 M concentration except cyanide and thiouracil, which were 6.7 X lo+ M and 3.4 X lOma M respectively. 0.2 ml. of each compound was added to the flasks containing the reaction mixture as described in the text.

Compound added

None. .......................................... Arsenate ........................................ Cyanide ....................................... Cysteine ........................................ Fluoride ........................................ Iodoacetate ..................................... Malonate. ...................................... Sulfide .......................................... Thiocyanate .................................... Thiouracil.. ....................................

1 uptake per flash per hr.

d. -Y

180 912 183 828 24 975

416 948 236 389 98 292

180 835 274 930 194 980 198 830

Lactate formed per 5ask per hr.

pounds were examined for their effect on glycolysis and respiration with HDP as substrate. Although cyanide and iodoacetate have a marked inhibitory effect on the oxygen uptake (Table VIII), the accumulation of lactate remains relatively unaffected by the former. A decrease in the formation of lactate is obtained with fluoride. In the presence of cysteine or sulfide, the pronounced stimulation in respiration is unaccompanied in either case by a change in the formation of lactate. That glycolysis is dissociated from respiration is evident from the diametrical action of these substances.

Since the addition of either cysteine or sulfide to the reaction mixture results in an augmented oxygen uptake, the possibility was considered that perhaps this effect was due to the fact that the sensitive SH groups of the enzymes were air-oxidized during the blending of the tissue in the

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preparation of the homogenate. Homogenates prepared by grinding the tissue with sand in a glass mortar or by blending the tissue in a Waring blendor under a nitrogen atmosphere gave results that were not dissimilar to those previously obtained.

It was thought of interest to compare aerobic and anaerobic glycolysis with HDP as substrate. In a particular experiment, the aerobic and anaerobic formation of lactate per flask per hour was 1026 and 811 y respectively. At first sight, these results might indicate the absence of a Pasteur effect; however, this finding can be adequately explained by the rapid disappearance of ATP (Table VII).

DISCUSSION

It was stated earlier that the indifference of steer thyroid slices to all of the test substrates may be due to the fact that the tissue is saturated with its own metabolites, thus rendering any additional source superfluous. In the long term respiration experiments for the purpose of examining this hypothesis, attempts to show a utilization of added substrate failed. This failure, however, to take up substrate added during the declining respiration of the tissue slice may have been the result of a deterioration of the enzymatic mechanisms, in which event the original aim of depleting the tissue’s endogenous food supply would have been unfulfilled, The results obtained by employing homogenates amply bear out this view. In the carbohydrate summation studies, the immobilization of glucose is apparent. This lack of glucose utilization might be attributed to an in- activation of hexokinase or to a depletion of ATP, either by diffusion or dephosphorylation. Similarly, this might apply to the inability of homo- genates to remove glucose. Further support of this explanation is evi- denced by the repeated failure to observe any formation of lactate with slices in the experiments on glycolysis, and by the high glycolyzing ac- tivity of the homogenate when suitable substrates are added. In addition, the conclusion must be drawn from the balance experiments that other substrates beside lactate are being oxidized, since the disappearance of lactate alone cannot account entirely for the oxygen consumed if a Qo, of 3.0 is assumed.

The evidence in the literature is conflicting as to the effect of thyroxine and thyrotropic hormone on the respiration of thyroid tissue in vitro. Since in most instances the same laboratory animal was employed, species differences cannot be advanced as a cause for the variable results. It may be that the frequent use of serum as a suspending medium and the purity of the hormone preparations are in part responsible for the different re- ports. Since no hormonal effect on the respiration of thyroid slices was observed in these studies, the problem was further pursued with the cell-

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522 CARBOHYDRATE UTILIZATION BY THYROID

free system. The formation of lactate and pyruvate, along with measure- ments of the oxygen consumption, was taken as the possible areas in which any effect of the hormone might be immediately observed. When a variety of substrates was employed, the effect of thyrotropic hormone and cortisone in all cases was negative. However, in the presence of thyroxine the results obtained were irregular; in some instances the oxy- gen uptake and the lactate and pyruvate formed were depressed, while in others no effect at all was exerted.

SUMMARY

1. The oxygen consumption of steer thyroid slices was determined under various conditions. A respiratory quotient of 1.0 suggested the oxidation of carbohydrate. In the absence of added substrate, slices were able to respire for many hours at a sustained linear rate.

2. Slices were unable to utilize a number of added components of the Meyerhof-Embden and tricarboxylic acid cycles. Since homogenates sup- plemented with cytochrome c, DPN, and ATP were able to oxidize many of these same compounds, it was concluded that the tissue was saturated with its own reservoir of intermediates and thus preferentially used them. In the carbohydrate balance studies, lactate was the only metabolite found to undergo disappearance. However, it followed that other substrates in addition to lactate were being oxidized, since the disappearance of lactate alone could not fully account for the oxygen consumed.

3. Of the compounds tested for their effect on respiration in the case of both the slice and the homogenate a marked inhibition was obtained with iodoacetate and cyanide, whereas with cysteine and sulfide an intense stimulation was observed. However, the presence of iodoacetate de- creased the aerobic glycolysis of the homogenate, while the remaining compounds exerted no effect.

4. The oxygen uptake of the slice and the homogenate remained un- changed in the presence of thyroxine, thyrotropic hormone, and cortisone.

BIBLIOGRAPHY

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B. WEISS 523

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