Effect of thyroid deficiency on oxidative phosphorylation in rat liver, kidney, and brain mitochondria

ARCHIVES

OF

BIOCHEMISTRY

Effect of Thyroid S. S. KATYARE, Biochemistry

AND

BIOPHYSICS

182, 155-163 (1977)

Deficiency on Oxidative Phosphorylation Kidney, and Brain Mitochondria M. V. JOSHI,

and Food Technology

Division,

P. FATTERPAKER, Bhabha

Received

Atomic

February

Research

AND

in Rat Liver,

A. SREENIVASAN’

Centre, Bombay

400 085, India

4, 1977

The effect of thyroidectomy on oxidative metabolism of rat liver, kidney, and brain mitochondria has been examined. The respiration in liver, kidney, and brain mitochondria was affected differentially after thyroidectomy, the common effect in all the tissues being the impairment in state 3 as well as state 4 rates of succinate oxidation. Thyroidectomy did not have any effect on ADP/O ratios; however, compared to normal, respiratory control indexes were, in general, somewhat higher. Thyroidectomy also did not alter total ATPase activity of liver, kidney, and brain mitochondria, although the basal ATPase activity had decreased significantly under these conditions. The cytochrome content of the mitochondria also showed tissue-specific changes after thyroidectomy; however, no significant changes in the absorption characteristics of the cytochromes were seen. The succinate and glutamate dehydrogenase activities of mitochondria from liver, kidney, and brain were not affected by thyroidectomy, thereby ruling out the possibility that the decrease in substrate oxidation’may be due to alterations in the primary dehydrogenase levels. It is concluded that thyroid hormone(s) may have a tissue-specific role in regulating the metabolic functions of mitochondria.

Influence of thyroid hormone(s) on mitochondrial metabolism has been noted by several workers (l-8). Hypothyroidism is characterized by decreased metabolism with a concomitant lowering of BMR*; treatment of hypothyroid animals with thyroid hormones results in elevation of these activities in terms of increased rate of respiration (l-8) which has been attributed to a selective increase in the respiratory components in mitochondria (1, 2, 6). Protein synthesis, both cellular as well as mitochondrial, is similarly affected under these conditions. Studies carried out in our laboratory have indicated that thyroid hormones have a tissue-specific role in regulating turnover of mitochondrial membrane proteins and DNA (9, 10). In particular, the synchrony of turnover of protein components in liver and kidney mitochondria

was lost after thyroidectomy (9). It was therefore of interest to see if this tissue specificity of the hormone effect is also reflected in terms of mitochondrial ftinction. The present investigation deals with a study of oxidative phosphorylation in mitochondria from liver, kidney, and brain of thyroidectomized rats using substrates involving three, two, and one site(s) of phosphorylation. Attempts are made to correlate the observed changes in the rates of substrate oxidation with cytochrome contents and the levels of primary dehydrogenases under the experimental condition. Observations on mitochondrial ATPase are also included. MATERIALS

AND

METHODS

All chemicals used were of analytical reagent grade. Sodium succinate, sodium salt of ascorbic acid, ATP, ADP, rotenone, antimycin A, Triton X100, and sodium deoxycholate were obtained from Sigma Chemical Co., St. Louis, Missouri. L-Glutamic acid was from E. Merck, Darmstadt A.-G., Germany, and N,N,N’N’-tetramethyl-p-phenylene diamine (TMPD) was from Eastman Organic Co., Rochester, New York. NAD was purchased either

1 Present address: 72 Pali Hill, Bombay 400 050. * Abbreviations used: BMR, basal metabolic rates; RCI, respiratory control index; DNP, 2,4-dinitrophenol; TMPD, N,N,N’,N’-tetramethyl-p-phenylene diamine; DCIP, dichloroindophenol. 155 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved

ISSN

0003-9861

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KATYARE

from Sigma or V.P. Chest Institute, New Delhi, India. Animals. Thyroidectomy was performed on weanling male rats of Wistar strain (20-22 days old, 30-35 g body weight) by surgery as described previously (7-9). The animals were allowed to grow for 8-10 weeks; a periodic record of their body weights and basal metabolic rates (BMR) was kept. Only those animals which showed a considerable decrease in body weight (30-35% decrease) and basal metabolic rate (3540% decrease) compared to their littermate unoperated controls were used for further studies. Stock laboratory diet and tap water were given ad libitum. Isolation

Isolation of liver, kidof mitochondriu. ney and brain mitochondria was carried out as described previously (9). Mitochondria were washed once and suspended in their respective isolation media (0.25 M sucrose containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA for liver and kidney mitochondria; 0.3 M mannitol containing 0.1 mM EDTA, pH 7.4, for brain mitochondria) at a final concentration of 30-40 mg of protein/ml. The rates of oxidation of different substrates were followed in a Gilson oxygraph, Model KM (Gilson Medical Electronics, Middleton, Wisconsin), as described previously (11). The reaction medium used for liver and kidney mitochondria contained 16 mM potassium phosphate buffer, pH 7.4,38 mM NaCl, 40 mM KCl, 12 mM KF, 6 mM MgCl*, 10 mM substrate, and 4-6 mg of mitochondrial proteins in a total volume of 2.0 ml (11). For studies with brain mitochondria, the reaction medium containing 0.3 M mannitol, 10 mM KCl, 10 mM Tris-HCl buffer, pH 7.4, 5 mM potassium phosphate buffer, pH 7.4, and 0.02 mM EDTA was employed (12). The oxidation of substrates was monitored at 25°C. In experiments with succinate as substrate, rotenone dissolved in absolute ethanol was added in small aliquots of 20 ~1 to give a final concentration of 1 PM. With ascorbate as substrate, 0.338 mM TMPD was used as a mediator of electrons; in addition, 10 pg of antimycin A was also included (11). Additions of small aliquots of ADP (20 ~1; 0.2-0.4 pmol) were made from time to time and rates of respiration in the presence of added ADP (state 3) and after its depletion (state 4) were determined. ADP/O ratios and respiratory control index (RCI) were calculated as described by Chance and Williams (13). Determination of cytochrome content of mitochondriu. To about 1.0 ml of mitochondrial suspension,

1.0 ml of 1.0 M potassium phosphate buffer, pH 7.4, was added and mitochondria were solubilized by addition of a suitable aliquot of 10% (v/v) Trimn X100 (6). For brain mitochondria, deoxycholate was also used for solubilization. The volume was made up to 10.0 ml with the respective isolation media and

ET AL.

thoroughly mixed. Samples in.the reference cuvette were completely oxidized by the addition of a few crystals of potassium ferricyanide and those in the experimental cuvette were reduced by the addition of a few milligrams of sodium dithionite. The difference spectra of cytochromes were recorded using a Perkin-Elmer double-beam spectrophotometer Model 126. The spectrophotometer signal was amplitied 10 times so that the full scale on a Perkin-Elmer Model 165 recorder represented 0.1 A unit. The samples were scanned from 650 to 500 nm with a slit of 0.5 or 2.0 mm. Cytochrome contents were calculated by using the wavelength pairs and extinction coefticients as given by Estabrook and Holowinsky (14). Enzyme assays. Succinate dehydrogenase (15) and glutamate dehydrogenase (16) activities in mitochondria were determined according to published procedures. Solubilization of brain mitochondria for determination of glutamate dehydrogenase activity was achieved with Triton X-100 as described by Neidle et al. (17). ATPase activity of liver mitochondria was determined as described previously (7, 11). Conditions for assay of ATPase activity of kidney mitochondria were standardized in separate experiments. It was observed that the activity could be stimulated either by 4 mM Mg2+ or 0.1 mM DNP. The reaction mediuin used for assay of kidney mitochondrial ATPase was essentially the same as the one described for liver mitochondria (7, 11) except for MgZ+, which was 4 mM wherever indicated. The ATPase activity of brain mitochondria was determined by the procedure of Ozawa et al. (12). Estimation of inorganic phosphate liberated in the supematant was according to the procedure of Fiske and SubbaRow (18). Protein was estimated according to Lowry et al. (19) using crystalline bovine serum albumin as a standard. RESULTS

The results on the effects of thyroidectomy on oxidative phosphorylation in liver, kidney, and brain mitochondria are summarized in Tables I-III. Three different substrates, i.e., glutamate, succinate, and ascorbate-TMPD, which involve three, two, and one site(s) of phosphorylaCon, were used for these studies. It was observed that thyroidectomy had differential effects on state 3 and state 4 respiratory rates in the mitochondria of all of the tissues under study. Thus, in liver mitochondria (Table I), oxidation with glutamate as substrate decreased significantly with and without added ADP (30 and 45% decrease, respec-

THYROID

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TABLE

157

PHOSPHORYLATION

I

EFFECT OF THYROIDECTOMY ON OXIDATIVE PHOSPHORYLATION IN LIVER MITOCHONDRIA” ADP:O ratio RCI Rate of oxidation (nmol of OJminlmg Substrate of protein)

Animals

-ADP

+ADP Control

Glutamate Succinate Ascorbate

Thyroidectomized

Glutamate Succinate Ascorbate

+ TMPD

17.34 +- 1.06 44.31 k 1.18 59.16 -c 4.24

5.81 + 0.26 12.31 + 0.14 45.16 k 2.34

2.98 3.56 1.31

3.04 + 0.104 1.92 r+ 0.010 0.78 + 0.048

+ TMPD

12.53 r?: 1.47* 34.70 + 1.20** 66.22 2 2.36 (NS)

3.23 f 0.33** 9.83 k 0.76*** 51.73 2 1.53 (NW

3.88 3.53 1.28

2.99 2 0.037 1.93 2 0.042 0.66 z!z0.028

u Oxygen consumption rates in the presence and absence of ADP were determined as described in the text and the ADP/O ratios and respiratory control index (RCI) were calculated according to Chance and Williams (13). The results are given as mean 4 SEM of five independent experiments. * P < 0.05 compared with values for controls. ** P < 0.001 compared with values for controls. *** P < 0.01 compared with values for controls. TABLE

II

EFFECT OF THYROIDECTOMY ON OXIDATIVE PHWPHORYLATION IN KIDNEY MITOCHONDRIA~ ADPO Rate of oxidation (nmol of O.Jmin/mg RCI Substrate of protein)

Animals

+ADP Control

Glutamate Succinate Ascorbate

Thyroidectomized D The given as *P ** P *** P

Glutamate Succinate Ascorbate

ratio

-ADP

+ TMPD

12.47 k 0.75 55.58 k 2.30 126.08 + 5.37

7.97 k 0.03 25.62 k 1.28 86.00 2 0.30

1.56 2.17 1.47

2.45 f 0.12 1.55 2 0.061 0.83 f 0.021

+ TMPD

8.54 t 0.60* 48.34 t 1.54** 76.39 Z!Y3.29***

7.39 k 0.63 (NS) 16.72 f 0.63*“* 53.71 2 4.44***

1.16 2.89 1.42

2.47 f 0.13 1.86 ” 0.034 0.75 A 0.032

ADP/O ratios and RCI were determined as described mean k SEM of five independent experiments. < 0.01 compared with values for controls. < 0.05 compared with values for controls. < 0.001 compared with values for controls.

TABLE

in the text and in Table I. The values

are

III

EFFECT OF THYROIDECTOMY ON OXIDATIVE PHOSPHORYLATION IN BRAIN MITOCHONDRIA~ Animals

Substrate

Rate of oxidation (nmol of OZ/min/mg of protein) +ADP

Control

Thyroidectomized

Glutamate Succinate Ascorbate Glutamate Succinate Ascorbate

+ TMPD

+ TMPD

10.59 2 0.84 54.38 f 4.64 106.81 k 9.65 10.76 f 0.22 (NS) 29.20 ‘- 1.52* 91.89 t 4.30 (NS)

RCI

ADP:O ratio

5.34 2 0.62 19.84 rt 0.89 81.13 ? 9.70

1.98 2.74 1.32

3.31 + 0.110 2.25 k 0.068 1.02 f 0.065

4.22 k 0.21 (NS) 11.04 2 0.34* 72.98 k 4.32 (NS)

2.54 2.64 1.26

3.79 f 0.012 2.41 k 0.121 0.93 + 0.037

-ADP

a The incubation medium given by Ozawa et al. (12) and as described in the text was used to determine oxygen consumption in the presence and absence of added ADP. The results are given as mean f SEM of five independent experiments. * P < 0.001 compared with values for controls.

158

KATYARE

tively), similar to the case with succinate as substrate, where approximately a 20% decrease in state 3 and state 4 respiration was seen. With ascorbate-TMPD, both state 3 and 4 respiration rates remained practically unaltered. Similar observations have been reported for glutamate and succinate oxidation in liver mitochondria from hypothyroid rats by Tata et al. (4) and Hoch (20). For kidney mitochondria (Table II), state 3 respiration with all substrates was inhibited significantly from 15 to 40%, similar to the case for state 4 respiration except in the case of glutamate, where practically no change was seen. In the case of brain mitochondria also (Table III), state 3 as well as state 4 respiration with succinate decreased signiflcantly (46 and 44% decrease, respectively). State 4 respiration using glutamate as substrate decreased by about 20% but was not statistically significant. Respiration with ascorbate-TMPD changed only marginally. The respiratory control indexes (RCI) were comparable for normal as well as thyroidectomized rats (Tables I-III), the mitochondria from hypothyroid rats showing slightly higher RCI at times, indicating that these mitochondria may be supercoupled. Such an observation has also been reported by several other workers (1, 2, 21, 22). The ADP/O ratios showed values close to the theoretical ones in the case of liver and kidney mitochondria from both normal as well as thyroidectomized animals (Tables I and II). These values were somewhat higher in the case of brain mitochondria (Table III); however, no differences were seen between the two groups, normal and thyroidectomized. The reason for high ADP/O ratios is unclear as yet, but it may be due to incomplete utilization of ADP, thus deceptively giving higher values for ADP/O ratios. It is likely that part of the ADP may also be used for some other reactions. Similar to these observations, Chance (23) has reported a high ADP/O ratio for pigeon breast muscle mitochondria with glutamate as substrate. We next examined the effect of thyroid-

ET AL.

ectomy on ATPase activity, yet another parameter pertaining to the energy metabolism of mitochondria. It was observed that the basal ATPase activity (-Mg2+DNP) was significantly low in all three mitochondrial fractions from thyroidectomized rats compared with the corresponding values for their euthyroid controls. Addition of Mg2+ and DNP stimulated the ATPase activity in liver, kidney, and brain mitochondria maximally, although to varying extents (Table IV). The total enzyme activities, however, were not affected after thyroidectomy. Mitochondria from liver, kidney, and brain of normal as well as thyroidectomized rats showed typical difference spectra (Figs. 1 and 2), although a slight variation in the absorption maxima is noted. Thus, cytochrome a in all of the tissues shows an absorption maximum at 605 nm. Cytochrome b shows an absorption maximum at 556.5 nm in liver and kidney but has an absorption maximum of 560 nm in brain. Cytochromes c + c, show an absorption maximum of 551.5 in liver, in accordance with values reported in the literature, while this respiratory component in kidney and brain shows an absorption maximum at a slightly lower wavelength, 550.5 nm (Fig. 1). Thyroidectomy appeared to have no effect on absorption maxima (Fig. 2). However, it may be pointed out that in the case of kidney mitochondria, several individual scans of cytochrome spectra have consistently indicated a shift of 2 nm in the lower wavelength region for cytochrome b. This may perhaps relate to altered association of cytochrome b with membrane proteins. Such alterations in absorption characteristics have also been observed by other workers (24). The specific cytochrome contents of liver, kidney, and brain mitochondria are shown in Table V. It is evident that even in the normal euthyroid animals the mitochondria from the three tissues differ with respect to their cytochrome contents and composition. This is also reflected in terms of the ratios of cytochrome a:b:c + c,. Thus, in the normal euthyroid rat, kidney and brain mitochondria have relatively low amounts of cytochrome b compared

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TABLE IV EFFECT OF THYROIDECTOMY ON ATPase ACTIVITY OF LIVER, KIDNEY, AND BRAIN MITOCHONDRIA”

Tissue

Additions

Micromoles of P, liberated per hour per milligram of protein Normal

Thyroidectomized

Liver

None +Mg2+ +DNP +Mg2+ + DNP

0.670 t 0.032 2.122 2 0.357 6.958 -+ 0.346 7,868 2 0.376

0.338 -I- 0.068* 1.276 k 0.395 (NS) 6.088 f 0.726 (NS) 6.092 ” 0.740 (NS)

Kidney

None +Mg*+ +DNP +Mg*+ + DNP

2.217 2 0.337 7.878 ?- 0.214 9.776 2 1.000 12.560 2 0.548

1.370 2 0.135** 7.960 e 0.483 (NS) 9.120 r 0.982 (NS) 11.810 2 1.507 (NS)

Brain

None +Mg2+ +DNP +Mg*+ + DNP

1.949 ‘2.980 ‘4.450 t5.730 2

0.377 0.252 0.622 0.388

0.852 2 2.780 f 3.840 + 5.110 zi

0.113*** 0.533 (NS) 0.343 (NS) 0.656 (NS)

a ATPase activities of liver, kidney, and brain mitochondria were determined by estimating the amount of ATP hydrolyzed in terms of inorganic phosphate (Pi) liberated in the supernatant (18). Concentrations of Mg*+ and DNP and other experimental details are as indicated in the text. The results are given as mean f SEM of six independent experiments. * P < 0.001 compared with values for normal animals. ** P < 0.05 compared with values for normal animals. *** P < 0.02 compared with values for normal animals.

with liver. In addition, the cytochrome c + c, content of brain mitochondria also seems to be relatively low. From comparison of absolute contents of cytochromes in the three mitochondria, it is evident that the kidney mitochondria are, relatively. speaking, the richest in cytochrome content. This may perhaps relate to the high energy requirement of the kidney (25). The hypothyroid state of the animals affected the specific contents of the cytochromes (Table V). Thus, in liver mitochondria, the contents of cytochromes a and b fell by about 20%, with a marginal decrease in cytochromes c + c, content. In kidney mitochondria, cytochromes a and c + c, showed about a 20% decrease, a significantly large decrease of 65% being observed in cytochrome b content. In the case of brain mitochondria, while cytochromes a and c + c, showed a decrease of 25 and 16%, respectively, cytochrome b content remained unaltered. Succinate and glutamate dehydrogenase activities in the three mitochondrial fractions are shown in Tables VI and VII, respectively. It is apparent that both en-

zyme activities were not affected by thyroidectomy in any of the tissues. Under these conditions, mitochondrial protein contents also did not show any significant changes (9). DISCUSSION

The results of the present studies have indicated that the effects of thyroidectomy on the metabolic activities of liver, kidney, and brain mitochondria with respect to their respiration (Tables I-III) are different. The effect of thyroidectomy on cytochrome contents is also evident (Table V). These results can be briefly summarized as follows. In liver mitochondria, the oxidation of glutamate and succinate decreased, in kidney the effect was seen on the rate of oxidation of all three substrates used, while in brain the oxidation of succinate. was affected most significantly with only a marginal decrease in the oxidation of ascorbate-TMPD. The common point of action in all tissues, therefore, seems to be succinate oxidation. It has been reported by some workers that a decrease in Qo, of liver, kidney,

160

KATYAKE 04

ET AL. O-1

NORMAL

n

THVRO~DECTOM~ZED

KIONE Y

BRAIN

500

550

600 WAVELENGTH.

650 n,,,

FIG. 1. Typical difference spectra of cytochromes of liver, kidney, and brain mitochondria from normal euthyroid rata. Mitochondria were solubilized using Triton X-100; deoxylate in addition was used for brain mitochondria. Samples in the reference cuvette were oxidized with potassium ferricyanide and those in the experimental cuvette were reduced with sodium dithionite. The difference spectra of reduced minus oxidized cytochromes were recorded for a wavelength span of 650 to 500 nm. Final concentrations of liver, kidney, and brain mitochondria ’ were 8.2, 4.2, and 4.9 mglml, respectively. Other details are as described in the text.

and brain parallels the fall in BMR as a function of thyroidectomy and that the succinoxidase activity in these tissues is enhanced in hyperthyroidism (cf. Ref. 1). However, the data presented by several others with respect to brain seem to be more controversial and need some comment. There is disagreement in the literature as to whether the brain in myxedema does (26) or does not (27) consume oxygen at a rate slower than that in the normal brain. It may, however, be pointed out that, in these studies (271, hype- or hyperthyroid patients themselves served as controls after treatment. The data would have been more meaningful if they were compared with normal euthyroid individuals. Despite this fact, the difference in the rate of cerebral oxygen consumption between the two group of human subjects, hypoand hyperthyroid, was significantly large, thus emphasizing dependence of rate of

500

550

600 WAVELENGTH.

650 nm

FIG. 2. Typical difference spectra of cytochromes of liver, kidney, and brain mitochondria from thyroidectomized rats. Final concentrations of mitochondrial proteins were 6.8, 5.7, and 8.0 mg/ml, respectively. Other details are as in Fig. 1 and the text.

cerebral oxygen consumption on the thyroid status. Fazekas et al. (28) and Barker (29) also did not find depression in the rate of oxygen consumption in the hypothyroid rats. These results, however, cannot be strictly compared with our present fmdings because of the differences in the methodology; in their studies, glucose was used as a respiratory substrate (28) and brain tissue was homogenized in distilled water (29). Besides, a critical appraisal of the above data (26-29) becomes difficult in view of the fact that the hormone effects may be species specific, being related to the stage of development as well as to the dose of the hormone (1, 2). Observation on humans and rats may not be, therefore, strictly correlated. Attempts to correlate the foregoing observations with the cytochrome contents in the three types of mitochondria indicated that, in this respect also, the effect of thyroidectomy was differential. The decrease in tissue respiration could partly be correlated with their cytochrome content, although no strict parallelism could be drawn. Thus, in liver, respiration with glutamate and succinate decreased along

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TABLE V EFFECT OF THYROIDECTOMY ON CYTOCHROME CONTENT OF LIVER, KIDNEY, AND BRAIN MITOCHONDRIA~ Tissue

Liver

Cytochrome

a b

c +c, Kidney

a b

c + c, Brain

a b

c + c, e The X-100 + chromes *P ** P ***P **** P

Nanomoles of cytochrome per milligram of mitochondrial protein

Ratio of a:b:c + c, Thyroidectomized

Normal

Thyroidectomized

Normal

0.145 2 0.0072 0.196 * 0.0071 0.256 f 0.0087

0.115 2 0.0021* 0.160 ? 0.0092** 0.227 f 0.0088***

1:1.35:1.76

1:1.39:1.97

0.246 ? 0.0097 0.240 f 0.0130 0.401 + 0.0083

0.201 f 0.0097* 0.083 f 0.0038**** 0.321 k 0.0153****

1:0.98:1.63

1:0.41:1.62

0.170 2 0.0094 0.082 k 0.0030 0.223 k 0.0072

0.128 ?.0.0061* 0.083 2 0.0074 (NS) 0.187 f 0.0062*

1:0.48:1.31

1:0.65:1.46

cytochromes were determined in Triton X-100~solubilized liver and kidney mitochondria and Triton deoxycholate-solubilized brain mitochondria by difference spectra of reduced and oxidized cytoas described in the text. The results are given as mean -e SEM of six independent experiments. < 0.01 compared with values for normal animals. < 0.02 compared with values for normal animals. < 0.05 compared with values for normal animals. < 0.001 compared with values for normal animals. TABLE VI

TABLE VII

EFFECT OF THYROIDECTOMY ON SUCCINATE DEHYDROGENASE ACTIVITY OF LIVER, KIDNEY, AND BRAIN MITOCHONDRIA”

EFFECT OF THYROIDECTOMY ON GLUTAMATE DEHYDROGENASE ACTIVITY OF LIVER, KIDNEY, AND BRAIN MITOCHONDRIA”

Tissue

Nanomoles of DCIP reduced per minute per milligram of protein Normal

Liver Kidney Brain

26.14 f 3.05 72.67 2 6.76 28.86 2 3.95

Tissue

Thyroidectomized 24.33 + 0.76 (NS) 74.95 + 0.81(NS)31.14 f 1.10 (NS)

n Succinate dehydrogenase activity in mitochondria was determined as described by Caplan and Greenwalt (15) using DCIP as an artificial electron acceptor. Other details are as described in the text. The results are given as mean 2 SEM of six independent experiments. NS, not significant compared with values for normal animals.

with a decrease in the amounts of cytochromes b and a; in kidney, the respiration with all three substrates decreased with decreased contents of all of the cytochromes, while in brain the effect was seen on succinate oxidation and cytochromes a and c + c, contents. Similar to our observations, a decreased cytochrome content in mitochondria in the hypothyroid state has been noted by several workers. Bronk (30) has reported a decrease in cytochromes a and c + c, in liver mitochondria of thyroidectomized

Nanomoles of NADH formed per minute per milligram of protein Normal

Liver Kidney Brain

87.87 2 8.60 46.00 f 1.87 28.61 ? 1.67

Thyroidectomized 90.92 -r- 6.15 (NS) 52.88 2 4.35 (NS) 28.48 -r- 1.40 (NS)

a Glutamate dehydrogenase activity in Triton X100~solubilized mitochondria (17) was determined according to Leighton et al. (16). Other details are as in the text. The results are given as mean f SEM of five independent experiments. NS, not significant compared with values for normal animals.

rats. Kadenbach (31) observed a decrease in cytochromes a and c in liver, cytochromes b and c in kidney, but no changes in brain mitochondrial cytochromes. Volfin et al. (32) found that the content of cytochromes a and u3 decreased in liver mitochondria of thyroidectomized rats. The results reported here generally agree with the above observations. However, it should be pointed out that, in Kadenbach’s (31) experiments, hypothyroidism was achieved by feeding rats with propylthiouracil. This antithyroid drug is known to have marked extrathyroidal effects on the

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utilization of the hormones (33) as well as on liver-protein metabolism (34). Examination of the effect of thyroidectomy on the levels of succinate dehydrogenase activity revealed that this enzyme activity was not affected by thyroidectomy (Table VI) and it did not quite parallel the rates of succinate oxidation (Tables I-III). This, however, may be explained on the basis of the fact that succinate dehydrogenase activity was determined using artificial electron acceptors and perhaps this may be the best estimate of the enzyme activity we could get under the experimental conditions. Modulation of succinate dehydrogenase activity by several metabolic effecters has been discussed recently (35, 36); there are many reports which indicate that this enzyme activity was not altered after thyroidectomy and under a variety of physiological and other stress conditions (37-39). It is likely that the latter observation may also hold true for glutamate dehydrogenase (Table VII). In fact, it has been reported that, during metamorphosis of Xenopus larvae, while several enzymes related to energy metabolism showed a decrease in activity to the extent of 50-70%, glutamate dehydrogenase remained unchanged (40). It would therefore seem that levels of primary dehydrogenases may not be the rate-limiting step during metabolism of substrates such as succinate and glutamate. Impairment in substrate transport across the mitochondrial membranes could perhaps provide an alternate explanation for the decreased rate of substrate oxidation. We have, however, not explored this possibility in our present studies. It is interesting to note in this context that submitochondrial particles obtained from liver mitochondria of hypothyroid rats do not show an altered rate of succinate oxidation, presumably because the permeability barrier for substrate transport does not exist in these vesicles (41). Recently, it has also been reported that treatment with Tq resulted in the increased transport of succinate in rat liver slices (42). It is likely that the cytochrome b-c segment of the respiratory chain may also be affected by this physiological condition. Indeed, this seems to be the case from the

ET AL.

studies of Hassinen et al. (431, who observed that the kinetics of cytochrome b reduction were altered in thyroidectomized rat liver. Bronk (30) has similarly observed that cytochromes b and c may be the points of thyroid action during succinate oxidation in rat liver. Similar observations have also been reported by Hoch (20). The possibility that redistribution of available energy in hypothyroidism favors transhydrogenation energy-dependent over phosphorylation possibly because of altered fatty acid composition (44) seems interesting and also has to be considered in this context. On the whole, the results of the present studies indicate that the effect of thyroid hormone(s) is tissue specific with respect to the mitochondrial respiration, which could be traced partly to the cytochrome contents in these tissues, the common point of action in all tissues being oxidation of succinate. The thyroid hormone(s) deficiency thus could lead to altered metabolic function. Although ADP/O ratios remained unaltered (Tables I-III), a decreased rate of substrate oxidation would result in impairment of energy production. Instances of such an impairment of energy metabolism in terms of decreased respiration rate are becoming evident in senescent rats (45) and in genetic deseases like achondroplasia (461, where, apparently, the first site of phosphorylation is inoperative. Results of our present studies, however, indicate that ATPase may not be rate limiting in the energy metabolism of mitochondria from thyroidectomized rats (Table IV). From the studies presented here as well as from our earlier observations (9), it is apparent that a simple and single hypothesis for thyroid effects cannot be adopted even for mitochondria. These results thus perhaps emphasize the fact that the mechanism underlying the regulation of tissue metabolism by thyroid hormone(s) may be much more complex than is anticipated. REFERENCES

1. TATA, J. R. (1964) in Action of Hormones on Molecular Processes (Litwack, G., and Kritchevsky, D. eds.), pp. 66-131, Wiley, New York.

THYROID

DEFICIENCY

AND OXIDATIVE

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