REDUCED AEROBIC CAPACITY AND LOCOMOTORY ENDURANCE … · Animals were fed a mixed diet of lettuce and blossoms of common butter-weed (Senecio sp.) thoughout the experimental period,

J. exp. Biol. 109, 175-189 (1984) \75Printed in Great Britain © The Company of Biologists Limited 1984

REDUCED AEROBIC CAPACITY AND LOCOMOTORYENDURANCE IN THYROID-DEFICIENT LIZARDS

BY HENRY B. JOHN-ALDER

Department of Developmental and Cell Biology, University of California,Irvine, California, 92717 U.SA.

Accepted 26 August 1983

SUMMARY

This study was undertaken to examine the effects of thyroid hormonaldeficiency on (1) standard (SMR) and maximal (Vozmax) rates of O2 con-sumption, (2) tissue glycolytic and oxidative capacities and (3) submaximallocomotory endurance in a lizard (Dipsosaurus dorsalis). Surgicalthyroidectomy induced hypothyroidism in all animals as determined bylevels of plasma thyroxine. Hypothyroid lizards had lower levels of SMR(—48 %), Vo2max (—16 %) and citrate synthase activity in liver, heart andskeletal muscle compared to controls. There was a correlated decrease inlocomotory endurance in thyroid-deficient animals. Pyruvate kinase activity(an index of glycolytic capacity) in all tissues, and myofibrillar ATPaseactivity (an index of contractile velocity) in white iliofibularis muscle,showed no significant changes in thyroid-deficient animals. Thyroid hor-mones appear to be important in ultimately establishing an animal's capacityfor locomotory endurance. These findings suggest a new selective contextfor understanding the evolution of thyroid function.

INTRODUCTION

Thyroid hormones are present in all classes of vertebrates and are associated witha diverse array of metabolic processes (see Shambaugh, 1978). One of the mostcharacteristic effects of thyroid hormones is a stimulatory effect on standard metabolicrate (SMR), and this calorigenic effect has been interpreted as evidence for a thermo-regulatory role of the thyroid gland in endothermic vertebrates. A more recent viewis that thyroid hormones may not play a major role in cold acclimation or otherphysiological adjustments to environmental temperature (Galton, 1978), but ratherthat primary effects of thyroid hormones may be alterations in the activities ofenzymes in energetic pathways (Gorbman, 1978). Functional benefits accrued by ananimal through these enzymatic effects are still unclear. Interpreting the stimulatoryeffect of thyroid hormones on SMR in ectothermic vertebrates is even moreproblematical, because metabolic heat production makes a negligible contribution tothe control of body temperature in these animals.

Lizards present a good model for studying non-thermoregulatory actions of thyroidhormones because SMR is influenced by thyroid status in these animals (Maher &

|Key words: Thyroid, lizard, aerobic capacity.

176 H. B. JOHN-ALDER

Levedahl, 1959; Maher, 1965; Wilhoft, 1966) but there is no obligatory change ilbody temperature or thermal conductance. Recently, John-Alder (1983) reported astimulatory effect of thyroxine (T4) not only on SMR but also on maximal rates ofO2 consumption (Vc^max) and muscle oxidative capacity in the desert iguana, Dipso-saurus dorsalis. An enhancement of Vozmax suggests an improvement in submaximalendurance (John-Alder & Bennett, 1981), and improved endurance could translateinto improved performance during natural activities. If this were true, then selectionfor the stimulatory effects of T4 on metabolic capacities could have operated indirect-ly through selection for improved endurance.

The present experiments were designed to extend our understanding of metaboliceffects of thyroid hormones and the resultant effects on activity capacity in lizards.Previous experimental T4-injection protocols have resulted in non-physiological plas-ma T4 concentrations (John-Alder, 1983). Such data are difficult to interpret sincephysiological responses to progressively higher levels of thyroid hormone levels aregenerally biphasic, being anabolic within some lower range of hormone concentra-tions and catabolic at higher concentrations (Shambaugh, 1978). It is difficult tointerpret the resuts of earlier studies within the natural biological context of lizards.I have found that surgical thyroidectomy can be performed easily to inducehypothyroidism in Dipsosaurus, and the range of experimental plasma T4 concentra-tions induced by thyroidectomy is very similar to that of Dipsosaurus retrieved fromwinter hibernation (H. B. John-Alder, in preparation). Thus, similar physiologicalresponses may occur in experimentally thyroidectomized lizards and in field-activelizards during the seasons marked by low thyroid activity.

The study was made upon Dipsosaurus dorsalis, for which there is considerableinformation on ecology (Norris, 1953; Muth, 1980), thermal biology (DeWitt,1967), and physiology (Moberly, 1963; Bennett & Dawson, 1972; Gleeson, Putnam& Bennett, 1980; John-Alder & Bennett, 1981). This species is the subject of acontinuing series of experimental and field studies designed to identify thephysiological importance of thyroid hormones in lizards (John-Alder, 1983, and inpreparation).

MATERIALS AND METHODS

Adult male desert iguanas (mean body mass ±S.E . = 65-2 ± l-9g) were capturedin May 1982 in Riverside County, CA (CA Scientific Collecting Permit No. 2005) andwere transferred to the laboratory at the University of California, Irvine. Animalswere maintained on a 13:11 h L: D photoperiod, the onset of light being at 06.00PDT. The daily temperature cycle was as follows: 24 °C from 00.00 to 07.30; increas-ing temperature from 07.30 to 09.00; 40°C from 09.00 to 17.00; decreasing tem-perature from 17.00 to 18.30; 24°C from 18.30 to 24.00. These temperatures wereselected because the average activity temperature of Dipsosaurus is 42-1 °C (Norris,1953) and the substrate temperature at a depth of 20 cm was 24 °C at the time ofcapture. Animals were fed a mixed diet of lettuce and blossoms of common butter-weed (Senecio sp.) thoughout the experimental period, with the exception of 36-hfasts prior to measurements of SMR. Water was available ad libitum.

Thyroid deficiency in lizards 111

Experimental design

Initial measurements of SMR, Vozmax during locomotory activity, and sub-maximal endurance were made within 4 days of capture before animals had beensegregated into experimental groups. All measurements were made at 40 °C. At thecompletion of these initial measurements of metabolic and performance capacities,animals were arbitrarily assigned to one of two groups for experimental treatment.Blood samples were collected via the infraorbital sinus for determinations of initialT4 concentrations. Subsequently, one group underwent surgical thyroidectomy, theother a sham operation. Lizards were maintained as described for 35 days until thefinal measurements were made. Metabolic and performance capacities were measuredagain during the sixth week after surgery. After the final metabolic and performancemeasurements were completed, lizards were killed by decapitation and were exsan-guinated via the neck wound. Blood was collected in heparinized tubes, and plasmawas immediately separated from formed elements. Plasma was frozen at — 70 °C andstored for determination of T4 concentrations by radioimmunoassay. The heart, liverand hindlimb muscles were quickly dissected free of the carcass and were frozen andstored at — 70 °C for subsequent determinations of enzyme activities.

Thyroidectomy and sham operations

Lizards were initially anaesthetized by having them rebreathe air inside a chambersaturated with vaporous Halothane. Subsequent administrations of Halothane weredone as required via the barrel of a 20-ml syringe placed over the lizards head. Allsurgical equipment and the lizard's neck were bathed with 95 % ethanol prior tosurgery. A ventral midline incision was made through the skin anteriorly from thecaudal plane of the pectoral girdle to a point above the posterior third of the hyo-glossum. Muscles of the throat and the underlying fat body were separated by bluntdissection. The thyroid gland in Dipsosaurus is a bi-lobed, encapsulated structurelying on either side of the trachea and connected by a narrow isthmus on the ventralsurface of the trachea. The entire thyroid was removed. An electrically-heated loopof steel wire was used to cauterize superficial vessels and the thyroid arteries and veinsas they were encountered. Blood loss was usually negligible by this procedure. Thethroat cavity was then bathed with sterile saline, and the incision was closed with threeor four disconnected ties of 3-0 silk. Sham operations involved anaesthesia and ex-posure of the thyroid gland. Lizards were given several hours in isolation for recoveryand then were returned to the common housing facilities. Thyroidectomized andsham-operated animals were not segregated for housing.

Metabolic measurements

Standard metabolic rates were measured on lizards placed individually into open-circuit metabolic chambers constructed from 22-cm sections of transparent Plexiglascylinders (i.d. 8 cm). Five metabolic chambers were arranged inside an environmen-tal chamber regulated at 40 ± 0-1 °C. Air flowed sequentially through a column con-taining Drierite and Ascarite for the removal of H2O vapour and CO2, respectively,gfcrough an upstream rotameter for measuring incurrent flow rate, through the


metabolism chamber, through a downstream column of Drierite and Ascarite, ancLfinally into a sampling manifold connected to a switching device. Excurrent air w ^vented either directly into the room or into a sampling port, as determined by theswitching device. The switching device sampled the excurrent air from each chamberonce each hour for 5-8 min. Three samples of room air were interspersed with thechamber air samples. Flow rates through the metabolism chambers were120-150 ml min"1. Samples of excurrent air were pumped through an AppliedElectrochemistry model S-3A O2 Analyzer at a rate of 20 ml min"1. Animals werefasted for 36 h prior to entry into the chambers. Body masses were recorded before andafter metabolic measurements, and the average of the two masses was used insubsequent calculations. Animals were placed inside the chambers at about 17.00 h.The lowest single record of O2 consumption was used to calculate SMR.

Maximal V02 values were measured on animals forced to run through a series ofgraded speeds on a treadmill as described previously (John-Alder, 1983). The initialspeed was 0-9 km h"1, and increments of about 0-2 km h"1 were adjusted atapproximately 2-min intervals. Air was drawn through the mask covering the lizard'shead at a rate of 470mlmin"1 (STPD; about 500mlmin"1 ambient). Continuousrecords of O2 and CO2 concentrations in the excurrent air were made on a recorder,and the record of the highest O2 consumption was used to calculate Vc^max. Record-ings not less than 1 min in duration were used for calculations. Rates of CO2 produc-tion were calculated only insofar as they are required for calculating Vo2max whenCO2 is not removed from the air. Equation 3b of Withers (1977) was used to calculateV02 , and equation 2 of Gleeson (1979) was used to calculate Vco2 •

Performance measurementsEndurance was measured as walking time to exhaustion at 1* 1 kmh"1 on a motor-

driven treadmill. This speed was selected because it was the lowest speed that was stilladequate to discriminate among animals with high and low endurance capacities.Endurance trials were terminated by exhaustion of the animal, as indicated by thesecond failure to sustain the tread speed, or at 30 min by the investigator.

Tissue preparations and enzyme assaysCitrate synthase and pyruvate kinase activities were assayed on the same tissue

homogenates. Tissues were homogenized in 100 mM potassium phosphate buffercontaining 5 mM-EDTA (pH 7-4 at 0-4°C). All assays were done at 40 °C. Dilutionfactors for the final homogenates of each tissue were as follows: liver, 50X; heart500X; red iliofibularis muscle, 100X; white iliofibularis muscle, 25X;gastrocnemium muscle, 50 X. Citrate synthase was assayed by the method of Srere(1969) using the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid). Assays were per-formed in a Beckman model 25 thermostatted spectrophotometer in 1 ml reactionvolumes, and the change in absorbance at 412 nm was recorded. Pyruvate kinaseactivity was assayed in all tissues as described by Somero & Childress (1980). Assayswere performed in 2 ml reaction volumes in thermostatted cuvettes, and the changein absorbance at 366 nm was recorded. All enzyme activities for these two assays areexpressed as Ug"1 at 40 °C. Protein concentrations in homogenates were measuredusing Biuret reagent and BSA standards.

Thyroid deficiency in lizards 179Myofibrillar ATPase activity was assayed using the white iliofibularis from the

ptmaining hindlimb. Myofibrils were prepared at 0-4 °C as described by Marsh &Wickler (1982). The ATPase assays were conducted in stirred 1-dram vials at 40 °Cwith a reaction volume of 15 ml containing 100mM-KCl, 20mM-Tris, 2mM-MgCl2,0-25 mM-CaCh, 2 mM-MgATP at pH 7-4 (adjusted at 40 °C). The assay was initiatedby the addition of ATP and was terminated by the addition of 0-1 ml of 50% tri-chloroacetic acid (TCA) after 30 s. Zero time controls were done by adding 50%TCA first. Vials were placed on ice and subsequently analysed for free phosphate bythe method of LeBel, Poirier & Beaudoin (1978). Protein was measured by the Biuretmethod with BSA standards. Activities were expressed as Umg"1 myofibrillarprotein.

Thyroxine radioimmunoassayPlasma T4 concentrations were determined by a radioimmunoassay (RIA)

modified from MacKenzie, Licht & Papkoff (1979). Antiserum T4-15 was obtainedfrom Endocrine Sciences (Tarzana, CA) and 125I-labelled T4 (NEX-11IX) from NewEngland Nuclear. Standards were prepared from crystalline L-thyroxine, Na-salt(Sigma T-2501) in 0-11 M-Na-barbital buffer (Barbital Sodium C-IV, Fisher B-22),pH8-6, containing lgl"1 bovine gamma-globulins (Sigma G-3500) and 0-01 gl"1

Thimerosal (Sigma T-5125). Assays were conducted in polystyrene gamma-countervials. To begin the assay, 25 \A of sample or standard were pipetted into the vial, and150/il of 125I-T4, diluted 2300X in barbital buffer containing 2-Omgml"1 8-anilino-1-naphthalene sulphonic acid (sodium salt; Pfaltz & Bauer A32940) in addition to thegamma globulins and Thimerosal, were added. Vials were vortexed and pre-incubated at room temperature for 1 h. Antiserum was diluted 1300X in barbitalbuffer, and 150 ̂ 1 of diluted antiserum was pipetted into each vial. Vials were coveredand incubated in the dark for 12—15 h. Vials were immersed in an ice-water slurry for20min, and 500^1 of polyethylene glycol (25%w/v; Carbowax PEG 8000; FisherP-156) was pipetted into each vial. Each vial was vortexed for 5 s and returned to theice-water slurry for 20 min. Vials were centrifuged at maximum speed in a Beckmanmodel TJ-6 refrigerated bench top centrifuge for 20 min. Supernatants were decantedand discarded. Vials were inverted over absorbent paper for 2-3 h prior to counting.Vials were counted for 1 min in a Beckman model 4000 gamma counter. Results wereexpressed as the quoteint of counts in the sample or standard divided by counts in thevial containing no unlabelled T4 (Bo). Quotients for standards were plotted on log-logit plots, and sample T4 levels were determined directly from the resultant linearplot. The limit of detection of the assay was 0-55 ngml"1. Sample T4 concentrationswere linear through a series of four serial dilutions. Preparation of standards inDipsosaurus plasma stripped of thyroid hormones had no effect on the standard curve.

Statistical analysesAll measured variables were analysed by analysis of covariance (Kleinbaum &

Kupper, 1978) to adjust for differences in body mass among individuals. Initialmeasurements of SMR and Vc^max were pooled for analysis. In cases where body masswas not a significant covariate, subsequent comparisons were done by f-tests. En-

durance measurements were analysed by the Wilcoxon signed ranks test. All statistical


analyses were done on an Apple II Plus computer uing STATPRO (BlueComputing, Madison, WI).

RESULTS

Surgical thyroidectomy resulted in a significant decrement in plasma T4 concentra-tions pre- and post-surgically (mean±s.E. 13-0 ±5-1 vs 0-83 ± 0-28 ngml"1;P = 0-044). There was also a significant decrease in plasma T4 of sham-controls(9-5 ±2-2 vs 5-8± l ^ n g m r 1 ; P = 0-015). Nevertheless, the final mean T4 con-centration of thyroidectomized lizards was significantly less than that of sham-controls (0-83 ±0-28 vs S^ l l ^ng im 1 " 1 ; P = 0007) (Table 1). By the directcriterion of plasma T4 concentration, hypothyroidism was successfully induced in allthyroidectomized lizards relative to sham-controls. Individual final T4 concentra-tions in thyroidectomized animals ranged from below the limit of detection of the T4assay to l-Sngml"1, a level below the range of sham-controls. The most likely ex-planation for the highest T4 levels in thyroidectomized animals is that some extra-thyroidal follicles had partially restored normal thyroid secretory activity by the endof the experiment.

The average body mass of sham-operated controls showed a small decline duringthe experiment (60-9 ± 2-8g vs 56-6 ± 3-3 g; P < 0 0 2 ) , whereas there was no sig-nificant change in body mass of thyroidectomized animals (66-1 +3-0 g vs63-5±3-0g; 0-4<P<0-5). The differences between final mean body masses ofsham-operated controls and thyroidectomized animals were not significant.

Organismal metabolic rates and locomotory endurance

By analysis of covariance, SMR was lower in thyroidectomized animals than insham-controls (P<0001) and lower in sham-controls than in the initial group(P< 0-005). These differences are illustrated in Fig. 1, in which log SMR is plottedas a function of log mass. This presentation allows comparisons of SMRs at all bodymasses, and differences among groups are seen as adjustments in intercepts. The 95 %confidence interval around the slope of log SMR on log mass is large (±0-48), and theslope should be interpreted with appropriate caution.

The final Vozmax of thyroidectomized animals was significantly less than that ofboth sham-controls and the initial measurements (P < 0-005; Fig. 2). Sham-operated

Table 1. Plasma T4 concentrations (ngml~') before and after experimentaltreatments

Group (N)Sham-control (8) Thyroidectomized (6) Probability (between)

Initial T4 9-5 ± 2-2 13-0 ±5-1 0-509Final T4 5-8 ±1-6 0-83 ± 0-28 0-007

Probability (within) 0015 0044

Values are means + 1 S.E.Probability levels were calculated from paired t statistics (within groups) and from Student's t statistics

(between groups).


fcpntrols showed no significant change in Vozmax during the experiment and have^een pooled with the initial measurements for this analysis.

Endurance is presented as walking time to exhaustion in Fig. 3. Some walking trialswere terminated at 30 min by the investigator, and this protocol necessitated non-parametric data analysis. Endurance of thyroidectomized lizards declined significant-ly between the initial and final measurements (one-tailed P = 0-0078; Wilcoxonsigned ranks test). There was no significant change in endurance of controls

Relative organ masses and tissue protein contents

Liver, heart and three skeletal muscles were analysed for tissue and cellular res-ponses to thyroid deficiency. The relative heart mass of thyroidectomized lizards, i.e.the ratio of heart mass to body mass, (gg"1), was significantly lower than in sham-controls (mean±s.E. 0-0019 ± 0-0008 vs 0-00214 ± 0-0006; P = 0-015: Student's ttest on arcsin-transformed values) (Fig. 4). The relative masses of liver and skeletalmuscles were not different between the two experimental groups.

Protein contents of heart, red iliofibularis muscle and gastrocnemius muscle weresignificantly lower in thyroidectomized lizards than in sham-controls (Table 2).

1-5 1-7Log body mass (g)

1-8 1-9

Fig. 1. Log SMR (standard metabolic rate) as a function of log body mass in (A) pre-experimental( • ) , (B) sham-control ( • ) , and (C) thyroid-deficient ( • ) Dipsosaurus. The following equationsdescribe SMR as a function of body mass for each of the groups: A, log SMR = - 0-894 + 1-084 (logmass); B, log SMR = - 1025 + 1084 (log mass); C, log SMR = - 1-247 + 1084 (log mass). Byanalysis of covariance, all differences among groups are significant at P< 0-005.


+ 12

+8

I +41 023

-a

i -4

U -8

- 1 2

- 1 6

Sham

1-6 1-7Log body mass (g)

Fig. 2

1-9

Thyroidectomy

Each bar represents one animal

Fig. 3


Sham Thyroid-deficient

Fig. 4. Relative heart masses (g/g X 1000) of sham-control and thyroid-deficient lizards. The linessuperimposed over the points indicate means ± s.E. The difference between means is significant(P = 0-015; i-test on arcsin-transformed values).

There were no significant differences between the two groups in white, non-oxidativeiliofibularis muscle nor, surprisingly, in liver.

Enzyme activities

The activities of citrate synthase (Table 3) and pyruvate kinase (Table 4) werecalculated both in mass-specific and in protein-specific units. On a mass-specific basis,citrate synthase activity was significantly lower in all tissues of thyroidectomizedlizards compared to controls. These results indicate that the oxidative capacity per

Fig. 2. Log Vo2max as a function of log body mass in (A) pre-experimental ( • ) , (B) sham-control(D), and (C) thyroid-deficient ( • ) , Dipsosaurus. The following equations describe Vo2max as a func-tion of body mass for each of the groups: A and B, log V02max = 0-348 + 0-945 (log mass); C, logV^max = 0-271 + 0-945 (log mass). By analysis of covariance, V02max of thyroid-deficient lizards isless than that of both other groups (P< 0-005).

Fig. 3. Change in endurance at 1-1 km h~' between initial and final measurements for each in-dividual. There was a significant decrease in endurance of thyroidectomized lizards pre- and post-surgically (one-tailed P= 0-0078).


Table 2. Tissue protein contents (mgg ') in liver, heart and three skeletal muscles ojsham-control and thyroidectomized (Thy-X) Dipsosaurus

Liver Heart Red IF White IF Gastroc.

Sham-control (8)Thy-X (6)

Difference (%)P

194 ± 9-4190±ll-4

-1-90-798

144 ±4-8136 + 2-4

-5-60021

149 ±5-4114 + 4-4-23-4

0-001

199 ±3-9190 ±9-5-4-3

0-622

144 ±5-212713-3-11-8

0-025

Values are means ± 1 s.E.Probability levels were calculated from Student's t statistics.Iliofibularis muscle = IF; gastrocnemius muscle = Gastroc.

Table 3. Citrate synthase activity of liver, heart and three skeletal muscles of sham-control and thyroidectomized (Thy-X) Dipsosaurus

Citratesynthaseactivity

ug-

Ug""1 protein

Group (N)


Difference (%)Probability



Liver

40-113-217-713-7

-5600001

20811894122

-5510001

Heart

162-517-2132-216-9

— 18-60012

1133127977171

-13-70-289

Tissue

Red IF

45-313-230-613-2

-32-50008

306 ± 27271135

-11-50-604

White IF

8-710-95-610-2

-35-50012

44153012

-31-80-022

Gastroc.

14010-810-810-4

-2310008

98158615

-12-70-094

Enzyme activity is expressed both on a mass-specific basis (U g ') and on a protein specific basis (U g ' protein).Values are means 1 1 s.E.Probability levels were calculated from Student's t statistics. Iliofibularis muscle = IF; gastrocnemius muscle

= Gastroc.

gram of all tissues was lowered by thyroid deficiency. On a protein-specific basis,however, there were significant decreases in citrate synthase activity only in liver andwhite iliofibularis muscle. The percentage decreases in citrate synthase activity ofheart, red iliofibularis and gastrocnemius were, however, greater than the percentagedecreases in protein content of these tissues.

Pyruvate kinase activity was not significantly changed by thyroid deficiency (Table4). This result is unchanged whether pyruvate kinase activity is expressed on a mass-specific or on a protein-specific basis. It should be noted, however, that all tissues ofthyroidectomized animals had somewhat higher protein-specific pyruvate kinaseactivity than did tissues of controls, whereas the same tissues had lower protein-specific citrate synthase activity than did controls.

Myofibrillar ATPase activity of white iliofibularis muscle is reported per mg ofmyofibrillar protein (Table 5) due to the nature of the assay. There was no significad


fable 4. Pyruvate kinase activity of liver, heart and three skeletal muscles from sham-control and thyroidectomized (Thy-X) Dipsosaurus

Pyruvatekinaseactivity Group (N) Liver Heart

Tissue

Red IF White IF Gastroc.





4-6 ±0-34-6 + 0-7

00-982

24-3 ±3-824-1 ±1-9

+ 1-00-949

68-5 ±2-270-1 ±4-7

+2-30-733

517 ± 4 0477 ± 14

+8-40-267

397 ± 67306 ± 32

-23-40-285

2673 ± 2752642 ± 423

+ 1-20-951

407 ± 24427+ 12

+4-80-530

2258+ 832043 + 104

+ 10-50-124

405 ± 29418 + 21

+3-20-740

3315 ±2002855 ± 258

+ 1610-509

Ug"'protein

Enzyme activity is expressed both on a mass-specific (Ug ') and on a protein-specific (Ug ' protein) basis.Probability levels were calculated from Student's statistics,lliofibularis muscle = IF; gastrocnemius muscle = Gastroc.

Table 5. Myofibrillar ATPase activity of white iliofibularis, a fast-glycolytic muscle

Group (TV) Myofibrillar ATPase activity

Sham-control (8)Thyroidectomy (6)P

Values are means ± 1 S.E.Units are Umg~' myofibrillar protein.The probability level was calculated from Student's statistics.

0-81 ±0-070-96 ±006

0137

difference between thyroidectomized and sham-control animals (mean ± S.E. 0-957 ±0-063 vs 0-809 ± 0-070; P = 0-137).

DISCUSSION

Capacities for aerobic energy metabolism in Dipsosaurus are dependent on thyroidstatus. Both standard (—40%) and maximal (—16%) rates of O2 consumption ofthyroid-deficient lizards were below those of controls. In an earlier study, John-Alder(1983) reported a 60 % increase in SMR and a 15 % increase in Vc^max of T4-injectedlizards. However, since T4-injection protocols used previously in lizards (Maher,1965; Wilhoft, 1966; John-Alder, 1983) resulted in plasma T4 concentrations thatwere transiently two orders of magnitude above normal, the conclusions of suchstudies can be criticized for the non-physiological experimental T4 levels. In thepresent study, plasma T4 concentrations of thyroidectomized Dipsosaurus were in thenormal range observed in these lizards retrieved from hibernation (mean ± S.E.•^#62 ± 0-183ngml~1; H. B. John-Alder, in preparation). Physiological responses to


thyroid deficiency can thus be interpreted in the context of a naturally realistic thyroidstatus.

Standard metabolic rates of sham-operated controls decreased significantly duringthe experiment (Fig. 1). It is intriguing that there is a similar decrease in SMR of field-active Dipsosaurus at a comparable time of year (H.B. John-Alder, in preparation).One interpretation of this observation is that natural seasonal variation in physiologi-cal functions was not interrupted by the experimental animal-housing conditions.Two other results of the present experiments support this interpretation. First, therewas a significant decrease in plasma T4 concentrations of sham-controls (Table 1),similar to the decrease in plasma T4 of field-active Dipsosaurus at comparable dates(H. B. John-Alder, in preparation). Secondly, citrate synthase activities reportedhere for sham-controls (Table 3) are comparable to those of field-active Dipsosaurusin mid-June and higher than those of field-active animals at any other time of year (H.B. John-Alder, in preparation).

The functional significance of reduced Vc^max in thyroid-deficient lizards can beseen in the concomitant reduction in locomotory endurance. On the basis of demon-strated correlations between Vc^max and endurance in lizards (John-Alder & Ben-nett, 1981; John-Alder, Lowe & Bennett, 1983) and mammals (Davies, Packer &Brooks, 1981), it was argued that T4-supplemented Dipsosaurus would probablyhave had improved locomotory endurance. Lowered endurance has been reported inthyroid-deficient rats (Baldwin, Hooker, Herrick & Schrader, 1980), and improvedendurance has been reported in T4-supplemented rats (Naito & Griffith, 1977).Thus, it would seem clear that thyroid hormonal effects on aerobic capacity aredirectly reflected in submaximal locomotory performance. However, reduced en-durance has been reported not only in thyroidectomized dogs (Kaciuba-Uscilko,Brzezinska & Kobryn, 1979) but also in T4- and T3- supplemented dogs (Brzezinska& Kaciuba-Uscilko, 1979a,b). In the latter two reports, reduced endurance wasassociated with lowered pre-exercise levels of liver and muscle glycogen. Reducedliver and muscle glycogen have also been reported in T4-supplemented frogs(McNabb, 1969; Packard & Randall, 1975). Given the potential for a reduction inenergy stores in hyperthyroid animals, it is important that any prediction of an im-provement in endurance of T4-supplemented animals be verified experimentally.

Thyroid hormones affect both the potential for cardiovascular O2 delivery capacityand the oxidative capacity of skeletal muscle. The reduced relative heart mass ofthyroid-deficient lizards suggests a reduced cardiovascular capacity for muscle per-fusion during activity and consequently a reduced maximum work capacity (seeDavies et al. 1981). Mass-specific citrate synthase activity, a frequently-used indexof mitochondrial oxidative capacity, was lower in all tissues of thyroidectomizedDipsosaurus than in controls. The reduction in muscle oxidative capacity is directlyassociated with impaired submaximal locomotory endurance. High oxidative capacityfavours oxidation of fatty acids and sparing of glycogen. Glycogen sparing is impor-tant in the context of endurance because glycogen depletion is likely to be the ultimatefactor responsible for exhaustion during submaximal activity (Holloszy et al. 1977).Baldwin et al. (1980) and Kaciuba-Uscilko et al. (1979) presented evidence of im-paired lipid mobilization in thyroid-deficient rats and dogs, respectively, and Paul(1971) reported that glucose provides a greater contribution to substrate utilization

Thyroid deficiency in lizards 187during exercise in thyroidectomized than in control dogs. Thus, thyroidectomizeddipsosaurus would be expected to have reduced submaximal endurance even if pre-activity glycogen levels were unaffected by thyroid deficiency. Protein contents ofliver and white iliofibularis muscle were not affected by thyroidectomy (Table 2). Thedecrements in citrate synthase activity of these tissues (Table 4) thus appear to bespecific effects of thyroid deficiency. In heart, red iliofibularis and gastrocnemiusmuscles, however, decreases in protein contents can account for the decreases incitrate synthase activity.

It is of interest that the mass-specific citrate synthase activity of all skeletal muscleswas lower in thyroidectomized lizards compared to controls, whereas T4-supplementation of intact animals failed to induce an increase in citrate synthaseactivity of white, non-oxidative iliofibularis muscle (John-Alder, 1983). Similar find-ings have been reported in mammals. Baldwin et al. (1980) and Janssen, van Harde-veld & Kassenar (1978) have reported decrements in the oxidative capacities of red,oxidative and white, non-oxidative mammalian skeletal muscles, whereas Janssen etal. (1978) and Winder & Holloszy (1977) have reported white mammalian skeletalmuscle to be much less responsive to thyroid hormones than red muscle. Thesedifferences among muscles may be explained on the basis of different concentrationsof thyroid hormone receptors in different muscles.

The effects of thyroid hormones on energetic pathways are apparently restricted toenzymes of aerobic metabolism. Pyruvate kinase activity, an index of glycolytic capac-ity (Zammit, Beis & Newsholme, 1978), was unchanged in all tissues that wereexamined. Furthermore, myofibrillar ATPase, the dominant ATPase of skeletalmuscle was not affected by thyroidectomy. It appears, then, that there is a dichotomybetween responses in those energetic pathways used in sustained activities and res-ponses in those pathways of rapid ATP synthesis and hydrolysis used during intenseactivity. It would be expected that the capacity of Dipsosaurus to engage in briefperiods of strenuous activity requiring high power output would be unimpaired bythyroid deficiency.

This new experimental information on physiological responses to thyroid manipu-lation in lizards (see also John-Alder, 1983) establishes a new selective context forunderstanding the evolution of thyroid function. Previous field studies on seasonalityof thyroid glandular activity in field-active lizards were not successful in identifyingphysiological correlates of thyroid glandular activity, and laboratory studies havefailed to establish the functional significance of changes in SMR in thyroid-manipulated lizards (see Lynn, 1970). However, an intriguing, recurrent observationhas been that peak thyroid activity in field-active lizards occurs at the time of highestphysical activity (Lynn, 1970). Recently, John-Alder (1982) reported preliminaryfindings showing that seasonal changes in thyroid function parallel seasonal changesin aerobic capacity and endurance in Dipsosaurus (H. B. John-Alder, in preparation).Selection for metabolic responses to thyroid hormones in lizards may have operatedon the advantages associated with improved endurance in addition to some otheraspects of increased metabolic rate per se. Increases in SMR associated with thyroidactivity would thus be seen as metabolic costs associated with the maintenance of highenergetic capacities, and these costs would be outweighed by the advantages of im-Jiroved endurance.


Dr Kenneth W. Baldwin, Dr Albert, F. Bennett, Dr Paul Licht and Dr RichariL. Marsh made contributions to the development of this manuscript. Ms Kathleen Jj[John-Alder skillfully prepared the figures and helped in the preparation of themanuscript. Mr Theodore Garland provided advice on statistical analyses. Mr BrettA. Adams read a preliminary draft of the manuscript. This study is part of my doctoraldissertation. I am grateful to Dr Albert P. Bennett for chairing my dissertationcommittee and for opening his laboratory to me. Supported by NSF Grant DEB 81-19797 to HBJ-A and AFB and by an award from the Patent Fund of the Regents ofthe University of California to HBJ-A.

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