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Evidence for the Role of Thyroxine as a Hormone of a Lizard

in the Physiology

Department of zoology, Garhwal University, Srinagar, Garhwal, U.P., India 246174 Accepted June 8, 1989 In vivo effects of thyroxine (T4) and triiodothyronine (T,) were studied in male lizards. T., or T, (0.5 to 2 nmol) was administered per day over 10 days to surgically thyroidectomized male lizards (35 + 2.0 g) and the responses were measured in terms of scale shedding, whole body oxygen consumption (OC), and testicular weight. T3 was more effective in stimulating OC as compared to T,. T4 accelerated scale shedding to a greater extent as compared to T3. T4 was more effective than T, in restoring the decline in the gonadal weight of thyroidectomized animals. Effect of inhibition of peripheral conversion of T4 + T, by iopanoic acid (IOP) was studied on the above response parameters. IOP at both dose levels inhibited extrathyroidal conversion of T4 + T3. The T&imulated increase in OC of IOP-treated animals was suppressed, clearly indicating that the T., effect could be attributed to its conversion to T,. But in these same animals, IOP failed to inhibit T.,-stimulated scale shedding and gonadal weight. As the proportion of T4 converted to T, decreased as a result of IOP treatment, the effectiveness of T, increased in terms of scale shedding and restoration of gonadal weight. From these studies it appears that all effects of T4 in lizards need not necessarily be mediated via conversion to T,. o IWOAcademic press, IK.

The thyroid gland has long been known to be involved in the regulation of a number of physiological phenomena in lizards viz scale shedding, oxygen consumption (OC), and gonadal function (see reviews Lynn, 1970; Thapliyal, 1980; Chiu, 1982). Thyroxine (T4) and triiodothyronine (T,), as in other vertebrates, are the major circulating thyroid hormones in reptiles, and extrathyroidaI conversion of T4 to T3 has also been demonstrated in this phylum (Chiu, 1982; Bona-Gallo et al., 1980; Sellers et al., 1982; Kar and Chandola-Saklani, 1985a, b). It is believed that T4 is a prohormone; T3, its monodeiodinated form being the principal biologically active compound (reviews, Chopra et al., 1973; Ingbar and Braverman, 1975; Larsen et al., 1980; Escobar et al., 1981). Our recent studies with nonmammalian vertebrates on T4 and T3 profiles in re’ Present address: Devi Ahilya Vishwavidyalaya dore, India.

In-

lation to seasonal physiological events and effects of thyroid hormone administrations suggested that T4 may be more effective in eliciting certain physiological responses and that T3 may not always mimic the action of T4 (Chandola and Bhatt, 1982; Chandola et al., 1982; Kar and ChandolaSaklani, 1985a, b; Pathak and Chandola, 1981). In the present paper we investigated the relative in vivo activities of T4 and Ts in a lizard and present evidence that all effects of T, need not necessarily be mediated through its conversion to T,. MATERIALS

AND METHODS

Animals The garden lizard, Calotes versicolor, is a commonly available reptile in the Indian subcontinent. It is a summer breeder and undergoes hibernation in December-January. Animals were procured from supphers and were acclimatized to laboratory conditions for at least a week before use. Experiments 1 and 2a were conducted under ambient conditions of temperature

173 001~6480/90 $1.50 Copyright 0 1!3!30by Academic Press, Inc. All rights of repmduction in my form resetved.

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and photoperiod (minimum-maximum 18-28”; 12 hr-30’ light:11 hr-30’ dark). Experiment 2b was conducted at constant temperature (26 2 2”) and photoperiod (13 hrL: 11 hr D). Automatic time switches were used to switch lights on and off. Only male lizards weighing 35 + 2.0 g and measuring 10 f 1.5 cm, snout to vent, were used. Lizards were fed live maggots and water nd lib. and remained healthy and active during the course of the study.

Experiment

1

Effects of T., and T3 on scale shedding, oxygen consumption, and testicular weight in rhyroidecromized (TX) lizards. About 60 lizards were surgically thyroidectomized under ether anesthesia. The TX lizards were given 30 ~1~1 ‘13’1 ip, as carrier-free NaI to ensure complete destruction of thyroid tissue. Animals were used 3 weeks later when the TX lizards were exsanguinated under light ether anaesthesia. B!ood was centrifuged and aliquots of plasma were stored at - u)” for subsequent analysis of T4 and T, by radioimmunoassay following the methods of Brown et al. (1970) modified for reptile serum (for assay characteristics see Kar and Chandola-Saklani, 1985a). It may be mentioned that, as in other nonmammalian vertebrates, e.g., fowl, small birds, reptiles, frogs, and fish (Higgs and Eales, 1977; Kuhn and Nouwen, 1978; Klandorfer al., 1978; Bona-Gall0 et al., 1980; Pathak and Chandola, 1981; Suzuki and Suzuki, 1981) the circulating levels of T4 (0.25-5 rig/ml) are rather low in this lizard and that T, (O-l .76 rig/ml) is comparable to that in man with a T,:T, ratio varying between 0.1 and 1.54 (Kar and Chandola-SakIani, 1985a). Earlier experiments revealed that exogenous administration of 0.5 to 1 pg T,/day can maintain the normal summer concentration in TX lizards (Kar and Chandola-Saklani, 198513). These doses were therefore presumed to be physiological and were used in the present experiment. Seven groups of 6 to 10 TX lizards each were established. Group 1,2, and 3 received im 0.5, 1.O, and 2.0 kg (or 0.56, 1.12, and 2.24 nmol) of T4 (sodium salt, Sigma) per day/lizard, respectively, and groups 4, 5, TABLE EFFECT

OF IOP-INDUCED INHIBITION RESPIRATION IN MALE

KAR

and 6 received equimolar doses of T, (sodium salt, Sigma) per lizard/day, respectively, in 0.1 ml of 0.9% alkaline NaCl over a period of 10 days. Group 7 (TX) and group 8 (sham operated intact) received 0.1 ml vehicle/day and served as controls. During the course of experimentation, molting was studied by the method described earlier (Kar and Chandola-Saklani, 1985a) and expressed as the total number of scales molted over the period. Twenty-four hours after the last injection, whole body respiration was measured manometrically following the technique described by Zarrow et al. (1964) and was expressed as milliliters of O2 consumed per hour/per gram of body weight. After recording the above observations, lizards were sacrificed by decapitation and testes were quickly removed, cleaned, and weighed.

Experiment

2

Effect of inhibition of peripheral conversion of T4 + T3 on oxygen consumption, scale shedding and testis weight (Table I). Thirty TX lizards were prepared as in experiment 1 and used 3 weeks later. Three groups of 8-10 lizards each were established. Group I was injected with 1 .O n&f T, im and group 2 with 1.O nM T, + 2.6 FM iopanoic acid (IOP, Winthrop) ip per animal/day over 10 days. Group 3 received the vehicle only im and ip and served as control. Scale shedding was checked every day and whole body oxygen consumption was studied 24 hr after the last injection. Four groups of 12 TX lizards each were established (prepared as in experiment 1). Group 1 was injected im with 0.5 nM of T,/day per lizard. Groups 2 and 3, in addition to 0.5 n&f T,, received 1.3 and 2.6 JLM IOP, respectively, per lizard per day. Group 4 received vehicle im and ip and served as control. Twenty-four hours after the first injection, four lizards from each group were exsanguinated, blood was centrifuged, and plasma was stored at - 20” for subsequent RIA of T, and T,. Injections were continued in the remaining lizards up to a period of 10 days. Scale shedding and oxygen consumption were recorded 24 hr after the last 1

OF T4 TO T, CONVERHON ON MOLTING LIZARD, Calores versicolor (EXPERIMENT

Treatment

Total number of scales molted (F f SD)

TX vehicle TxT,(l.OnM) TX + T4 (1.0 nM) + IOP (2.6 @M)

0.0 2.33 k 0.33 8.00 2 1.73**

AND WHOLE 2~)

BODY

Whole body Otionsumption WWW (X ” SE) 0.48 + 0.11 0.81 2 0.08 0.50 * 0.01*

Note. TX, thyroidectomized; IOP, iopanoic acid. * P < 0.05 and **P < 0.01, compared to the corresponding values of TX lizards treated with T4 only.

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injection when testes were removed and weighed following decapitation. Significance of differences in group means was assessed by Students t test and ANOVA. Dose-response regression analysis was carried out by the method of least squares.

RESULTS Experiment 1

T, and T3 were undetectable in the plasma of TX lizards (assay sensitivity: T3, 0.07 rig/ml; Tq, 0.15 rig/ml). Oxygen consumption. OC in TX lizards (0.91 + 0.09 ml per g&n) was significantly less (P < 0.001) than that in intact controls (2.85 + 0.35 ml per g/hr). Compared to the controls, T, significantly stimulated OC at two higher dose levels (1 nMlday, P c 0.01, 2 nh4/day, P C 0.05). T4 significantly stimulated OC at one dose level only (2 nM/day, P < 0.05) (Fig. la). Scale shedding. The number of scales shed in TX lizards (2.75 rt 1.49 per lizard) was significantly reduced (P < 0.001) compared to that in intact controls (12.25 f 1.45 per lizard). Administration of T, significantly stimulated scale shedding at all levels (0.5 nMlday P < 0.05; 1.0 nMlday P < 0.05; 2.0 nM/day P < 0.01; TX controls vs treated), but T, was only effective at the highest dose level (2 nM/day, P C 0.05, TX control vs treated). Regression analysis revealed a positive correlation (T4, r = 0.89, T,, r = 0.5), which was highly significant in the case of TX (P < 0.005) (Figs. lb and 2). Gonads. Gonadal weight in TX lizards (16.66 + 4.4. mg/lOO g body weight) was significantly low (P < 0.01) as compared to that in intact controls (40.50 f 3.79 mg/lOO g body wt). T4 stimulated gonadal development at all doses but the effect was only significant at the highest dose level (2 nM/ day, P < 0.05 treated vs TX control). T3 stimulated gonadal development in TX lizard at the two higher dose levels, but the effect was not significant (Fig. lc). Experiment 2a

OC and scale shedding were significantly

of6 0

0.5 Hormone

2.0

1.0

administered

(n Moles/lizard

)

FIG. 1. Effect of 10 daily injections of T, or T, (0.5, 1.0, and 2.0 niMliiday) on (a) oxygen consumption, (b) number of scales molted, and (c) testicular weight in thyroidectomized male lizards. Vertical bars indicate means + SE of six to eight animals (Experiment 1).

higher in the TX lizards receiving 1.0 I%/ day of T, as compared to TX controls (P < 0.05). OC significantly declined and scale shedding significantly increased in the T4 + IOP-treated group as compared to that in the T, alone-treated group (P < 0.01 for both) (Table 1). Experiment 2b

Scale shedding, OC, and testicular weight registered significant increases in TX lizards treated with 0.5 nM T4 as compared

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0

a5

1.0

2.0

Hormone administered (nMoles/iizard)

FIG. 2. Correlation between dose of hormone administered (T., or T,) to thyroidectomized male lizards and parameters as indicated. Data correspond to Experiment 1.

to that in TX controls (P C 0.01). OC declined and testicular weight increased in both groups receiving T4 + IOP, the effect being significant in the group receiving higher doses of IOP (2.6 @, P < 0.05, compared to TX lizards receiving T4 alone). Scale shedding was significantly increased in TX lizards receiving both doses of IOP in addition to 0.5 nA4 T4 (both doses P < 0.001, compared to TX lizards receiving 0.5 nA4, T4 alone) (Fig. 3). IOP at both dose levels (1.3 and 2.6 pA4) significantly inhibited extrathyroidal conversion of T4 --) T3 (Fig. 4). DISCUSSION

The results clearly indicate that T4 and T, may have varied effects in the same species, depending on the responding system. Thus T, and Tj both stimulated O2 consumption and T4 had a significantly greater

AND KAR

effect (at all dose levels employed) on scale shedding and gonadal growth (at two dose levels out of the three used) (Fig. 1). How far the alteration in food intake due to T4 or T,, if any, may have influenced the responses is not known. When the extrathyroidal conversion of T4 to T, was inhibited by IOP in thyroidectomized lizards (Table 1, Fig. 3), the T,-stimulated increase in O2 consumption was suppressed, clearly indicating that the T4 action may be attributed to its conversion to T,. However, in these same animals, scale shedding was not reduced. In fact, as the proportion of T4 converted to T, decreased as a result of IOP treatment, the effectiveness of T4 increased in terms of scale shedding. IOP alone had no effect whatsoever in thyroidectomized lizards. Further, inhibition of conversion of T, + T, did not inhibit the stimulatory effect of T, on gonadal function in thyroidectomized lizards. Similar results have been obtained in birds with feather regeneration, respiration, and gonadal growth as response parameters (see Chandola et al., 1982; Kar and Chandola, 1985). It is clear that all of the T4 effects need not necessarily be mediated via T, conversion to T,. Morphological and biochemical studies on induction of metamorphosis in amphibia also indicated that, apart from T,, T4 is also active, at least in the early phase of metamorphosis (Cohen et al., 1978; Galton et al., 1982). Further, T4 and T, have been shown to be equipotent in stimulating body weight, comb growth, liver glycogen, and oxygen consumption in thyroidectomized birds (Newcomer, 1957)and that T, is more effective than T, in preventing goiter in thiouracil-treated chickens (Mellen and Wentworth, 1959). Interestingly, when the circulating T4 and T, profiles are examined in relation to the seasonal physiological events (molting, reproduction, migratory fattening, etc.), scale shedding in lizards (Kar and ChandolaSaklani, 1985a)was found to coincide with

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TX Tx+TL(05nM

TX TX< ,$FmM)

TX [email protected]

FIG. 3. Oxygen consumption, scale shedding, and testicular weight following 10 days of treatment of thyroidectomized (TX) lizards with 0.5 IN T&y alone or in combination with 2.6 or 1.30 pA4 IOP/lizard/day (Experiment 2b).

increased levels of circulating T4 and not T,. Similarly, the pro- or antigonadal relationship of thyroid (as evidenced by thyroid extirpation and replacement therapy) is reflected with T, and T, (Kar and Chandola, 1985). On the other hand, premigratory fat deposition in buntings is preceded by a significant increase in plasma T, and not T,

(Pathak and Chandola, 1981). A number of clinical and experimental situations have also been recognized in which serum T, concentrations correlate better than T, with clinical data of physiological status, including serum TSH regulation and erythropoiesis (Chopra et al., 1978; Andreoli, 1981). However, most of these have been ex0.2L-

ZL-

016.

16. 0

.m

I t m 0.6 l-

8.

OTX Tx.T&%i

01 -& TX TX.-)

TX Tx+TL(O.SnM)

FIG. 4. Plasma T, and T, (&ml) and Tn., ratio in thyroidectomized (TX) male lizards treated with vehicle, 0.5 IN T, alone, or in combination with 1.3 or 2.6 $M IOPkard.

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plained on the basis of an intracellular conversion of T, + T, which varies from tissue to tissue (see Larsen et al., 1980). The apparent relationship between circulating T4 concentration and the physiological manifestations of thyroid hormone excess or deficiency observed in our studies above cannot be argued on this basis since we used IOP which is a potent inhibitor of intracelluar T, + T3 conversion. Nor can they be explained on the basis of quicker T, turnover in blood since T4 and T, in birds both bind to prealbumin or albumin and have similar half-lives (Wentworth and Ringer, 1986). Our results, although obtained from in viva studies alone, suggest two possibilities; (a) an independent action of T4 in certain situations or (b) the local production of T4 metabolites other than T, which are biologically active. Apparently, further studies on subcellular distribution and metabolism are required to test these ideas. It seems that the peripheral conversion of T, to T3 which has been demonstrated in all phyla ranging from fish to man may be a strategy used by animals for adjusting seasonal events like molting, reproduction, migration, and hibernation in accordance with the annual energy budget to the demands of the seasonally changing environment. Its role in exercising a subtle organ-specific, action-specific, metabolic control aside, the fact that peripheral thyroid hormone metabolism may be influenced by environmental and physiological alterations, including season, diet, and malnutrition (Ingbar and Galton, 1975; Higgs and Eales, 1977; Chandola-Saklani et al., 1989), imparts to it a great selective value. ACKNOWLEDGMENTS The senior author gratefully acknowledges the gift of iopanoic acid (Winthrop) from Professor G. Morreale de Escobar, Department0 de Endocrinologia Experimental, Instituto G Maraiion, Madrid, and Dey’s Medical Stores, Calcutta. The authors thank UGC, DOEn & DAE, India, for financial assistance and Mr.

AND

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P. C. Lakhera Department of Zoology, Garhwal University, for help in conducting some experiments and in the preparation of the manuscript. Some of the experiments described were carried out at Banaras Hindu University, Varanasi.

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