Asiatic Herpetological Research

April 1990

Vol.3, pp. 46-51

Relationships Between Serum T 4 T 3 Cortisol and the Metabolism ofChemical Energy Sources in the Cobra During Pre-hibernation, Hibernation
and Post-hibernation,,RUIMIN WU AND JlE HUANG
11^Department of Biology, Fujian Medical College, Fujian, China

Abstract. -This study analyzed the variation of the serum thyroxine (T4 ), triiodothyronine (T 3 ), andCortisol in the cobra (Naja naja Linnaeus) in pre-hibernation, hibernation, and post-hibernation. Thevariations were compared with the changes of indexes of cobra metabolism in those three periods, such as

oxygen consumption, serum glucose, serum triglyceride, glycogen content of liver, and triglyceride content
of fat bodies. From pre-hibernation to hibernation, the change of metabolic indexes of the cobra indicatedthat the metabolic rate tends to decline because of the very low levels of the three serum hormones in prehibernation and falling temperature. Low temperature during hibernation limited the activities of the threeserum hormones which were rising at higher levels during hibernation. From hibernation to post
hibernation, the rising temperature allowed the three serum hormones to increase markedly, stimulating
metabolism gradually. Therefore the metabolic rate of the cobra during post-hibernation tended to riseagain. The metabolic indexes of the cobra in post-hibernation showed a significant increase in metabolic
The indexes ofrate, which had close relation with the high levels of the three serum hormones.
metabolism of the cobra indicate that it is hepatic glycogen and not fat that hibernating cobras use as theirmain energy source.

Key Words:

Reptilia, Serpentes, Elapidae, Naja naja, cobra, thyroxine, triiodothyronine, Cortisol,metabolism, energy metabolites, chemical energy sources.

Introduction

and guidance for maintaining snakes.

Hibernation is an adaptive strategy ofectotherms for keeping out of the cold
during winter. The metabolic rate of theisapparently different

hibernating reptiles
from that of active ones, as a result ofphysiological adaptation. The endocrine
system and its close relation to metabolism
may play an important role in physiologicaladaptation. Maher (1965) pointed out thatthe maximum metabolic response in lizards
to thyroxine

occurred

at3°C; Wilhoft(1966) found that injections of thyroxine inlizards increased their metabolism.

Musucchia (1984) suggested

that

theinjection of Cortisol into hamsters duringhibernation or hypothermia could achieve
the same result as the injection of glucose,which might prolong the survival of
hamsters under the same conditions.

We

consider that the metabolism of energy
metabolites in hibernating snakes must havea close relation to the control of serum
thyroxine (T4 ), triiodothyronine (T 3 ), andThis study may provide a way tofind the mechanism of snake hibernation

Cortisol.

1990 by Asiatic Herpetological ResearchMethods
study was carried out from
September 1987 to May 1988. Cobras
Naja naja (Linnaeus) used as experimental
animals were captured at Changlou, FujianThis

We studied serum T4 T 3and Cortisol, and related indexes ofmetabolism in cobras during preProvince, China.

hibernation,hibernation,,and,post-hibernation.

Cobra groups: 12 adult cobras (6 males, 6females) were captured in May 1987,weighing 176-273 g. These cobras had
been fed in our college snake garden for 4the experiment.
On
September 9, 1987, 6 cobras were takenout of the snake garden and tested as a prehibernation group (Pre G). On October 2,1987, the remaining 6 cobras weretransferred from the snake garden into adark cement pool with a perforated metalmonths before

window above. The temperature in thepool was similar to the atmosphere. On

Asiatic Herpetological Research

April 1990

Vol. 3, p. 47

February 10, 1988, 3 cobras were takenout from the pool and tested as ahibernation group (H G), and on May 7,
the last three cobras were tested as a post-Triglycerides of fat bodies extracted in nheptane were analyzed by clinical chemical

hibernation group (Post G).

Triglycerides of fat body(g/Kg(BW))
content in fat body/body weight (BW)methods.

Determination of oxygen consumption:
The oxygen consumption of each group
was determined using the method of Dong

Analysis of three hormones: Blood wasdrawn from the posterior vena cava.
Serum T 4 T3 and Cortisol of each group,were analyzed by radioimmunoassay bymeans of kits produced by the Institute ofShanghai Biologicals. Each sample wasanalyzed twice.

Analysis of serum energy metabolites:
Serum glucose and serum triglycerides ofeach group of snakes were analyzed byclinical chemistry methods, and eachsample was analyzed twice.
Analysis of hepatic glycogen: Fresh snake
liver of each group was weighed and cut
into pieces. After having been in boilingwater for 5 minutes, the liver pieces werehomogenized.

The homogenate was inboiling water for another twenty minutes

andfilteredestimated by the following calculation:
Hepatic glycogen (g/Kg(BW)) =totalglycogencontent in liver/body weight (BW)Analysis of fat body triglycerides: Freshsnake fat bodies of each group wasweighed and a small weighed part washomogenized in a fixed volume of n-extracted(C 7 H 16 ). Thein

thiswayfattissue

threewastimes.

HG and Post G

The levels of serum T4 T 3 and Cortisolof Pre G were the lowest in the three cobragroups. The level of serum Cortisol of
was markedly lower than that of post G.
were notThe levels of T4 and T 3 offromthoseofPost G
different
distincdy,,

HG

HG(Table 1).
2. Variations of the oxygen consumption

and energy metabolites during Pre G, HGand Post G

The oxygen consumption of the HG
group was significantly the lowest of theThe oxygen
three cobra groups.
consumption of Pre G was not significantly
different from that of Post G.

The contents of serum glucose of both

TABLE 1.

Experimental results of the threeserum hormones and t tests.

The filtered

immediately.

homogenate was mixed with 95% alcohol A. R. (mean analysis reagent) to twice thevolume and kept at room temperature forten minutes before it was centrifuged forThetwenty minutes at 3000 rpm.
supernate was drawn out, the sediment(glycogen) soluble in hot water was

heptaneVariations of the three serum hormones

during Pre G,Heart beats and respiratory state: Heart rate
and respiratory state of each group were
measured by routine methods.

,Results1.

et al. (1986) corrected by the authors.

= triglycerideGroupsVol. 3, p. 48

Asiatic Herpetological Research

TABLE 2. Experimental results of oxygen consumption by energy substances, andGroups

April 1990ttests.

Asiatic Herpetological Research

April 1990

Compared with that of pre G, theoxygen consumption of post G wasVol. 3, p. 49

The secreted T 4 is converted into T 3 in theblood or in the tissues and organs. The

relatively large, but the serum glucose andfunction of stimulating metabolism by T 3 is
at least three times stronger than that by T 4

relatively low. The reasons may be: 1) thatthe pre G cobras were not fasting so that
their contents of serum glucose andThe activity of the cobra thyroid gland was
at a low level in pre-hibernation. The
synthesized and secreted T4 was very low
and a portion of T4 was converted into T 3thus the concentration of serum T4 was at alow level and the level of T 3 was relatively

serum triglyceride content of post G weretriglycerides were relatively high, 2) thatcobras had so little hepatic
the postasto be unable to raise theirglycogenserum glucose and triglyceride contents as

GGgroup, and 3)
high as those of the Pre
that the rising temperature and the highserum hormone

level after hibernation

accelerated the organs and tissues to take inglucose and triglycerides from the blood.
Among the three reasons the latter was themost important because of the apparent riseThis amountin oxygen consumption.
indicated that the metabolism in the organs
and tissues had been enhanced markedly.
2. Variation of the three serum hormones

Serum T 3 of HG was 56.6% higher thanG, and serum T 3 of Post G
51.7% higher than that of HG. Similar toserum T 3 serum T4 of Pre G was too low,

bebuttested,

T3ofHG

roseGT

continued tosignificantly, and 3 of Postrise 43.2% more than that of HG. As aresult,high. After the cobra entered hibernation,
the secretive function of the thyroid gland
began to become active and produce more

and more T4 therefore serum T4 dominatedat a higher level though both T 4 and T 3
increased in the blood. The variation ofserum T4 and T 3 in post G was the same as,HG. The very high levels of serum T4and T3 in post G were of great advantage tothe cobra enhancing its metabolic rate forarising from hibernation. Besides these,
the results in Table 1 and Table 2 also
indicate that the levels of serum T4 and T 3of HG were higher than that of Pre G, but
the amount of oxygen consumption of HGwas less than that of Pre G. This seemed
to be self-contradictory because both T 4
and T 3 were able to stimulate metabolism.
These phenomena resulted in the falling
temperature, inhibiting the function ofhormones. Wilhoft (1966) pointed out thatthe function of T4 stimulating metabolism
appeared to be inactive in many reptiles at
low body temperature, and to be active only
Stimulation ofat higher temperature.
metabolism was carried out in such a waythat T4 and T 3 were capable of inducing thesynthesis of aerobic metabolic enzymes.
The low levels of serum T4 and T 3 in prein,that of Preto.

serum T 3 and T4 were increasing

steadily during hibernation. This partem ofserum T4 was similar to the results reported

by Nauleau, et al. (1987).

A comparison between the variations ofserum T 3 and serum T4 can provide some
important information about the secretive
state of the thyroid gland.
BeforeTwasandserum
low,hibernation,
4very
serum T3 was higher, being in a dominantposition, but in hibernation, serum T4 was
twice as great as serum T3 though both of,them had increased. These variations werecaused by the increased activity of the
thyroid gland during hibernation.

Turnerand Bagnara (1976) suggested

that the,,,hibernation indicated that the metabolic rateof cobras in this period tends to fall.

The metabolic enzymes which had beensynthesized were still in an active statebecause of the higher temperature, thus thepre G cobras had a high oxygenconsumption. The metabolic rate of HG,
though serum T4 and T 3 levels had risen,hibernation, reaching a peak during posthibernation.
The thyroid gland mainly

to the lowwhichhibernation
temperature duringtheTandof
inhibited the function
3 (T 4 )activity of metabolic enzymes synthesized
had a small
before. The cobras ofsecretes T4

oxygen consumption. The very high levels

activity of the thyroid gland of ectotherms
was low both in summer and during pre-hibernation.

,It

was enhanced during

which is the precursor of T 3.

wasatalow level due

HGAsiatic Herpetological ResearchVol. 3, p. 50of serum T4 and T3 gradually performed thefunction of stimulating metabolism in thephase at the end of hibernation as thetemperature was rising.

SerumCortisol

wasincreasing

continuously during hibernation. Serum
Cortisol ofwas 2.8 times higher thanthat of pre G, and that of post G which
continued to rise by 3.6 times more thanthat of HG. Musacchia (1984) reported
that the survival of animals duringhibernation or hypothermia had a close
relation to the level of serum Cortisol intheir bodies. An injection of Cortisol into
hamsters during hibernation or hypothermia
could yield the same result as an injectionof glucose, and might increase the survivalof hamsters under the same conditions. HeHG

April 1990

under the control of the enzymes which hadbeen synthesized before. The metabolic
rate of cobras tended to decrease from prehibernation to hibernation as thetemperature was falling and as the amountof enzyme reduced (synthesizing less anddegrading). During hibernation, when thetemperature was low, cobras had a low
metabolism under the control of the lowtemperature isozymes, though the levels ofserum hormones were at relatively higher.

The low serum glucose and triglyceride
contents implied that the decreased
metabolism of cobras in hibernation, andthe reduction of glycogen in the liverindicated the metabolic rate of cobras at a
In post-hibernation, hightemperature provided a favorable factor forcertain level.

hormones to perform their functions, andalso reported that glucocorticosteroids

T (T 4 induced synthesis of enzymes andplayed an important role in animals arisingfrom hibernation or hypothermia. Someclues perhaps could be found for reptiles

Cortisol accelerated the gluconeogenesis andfrom the experimental results on hamsters,
although they were very different in other)3

fatcatabolism.

Serum glucose wasincreasing markedly though the hepaticglycogen was low, and quite a lot of

wasglucose

producedrespects.

The results in Table 2 indicated that thehepatic glycogen content of HG was very
low. It was difficult for cobras to continue
hibernating while only using so little an
amount of hepatic glycogen as a energy
source.
They must receive energy
inmetabolites
otherways.

Gluconeogenesis may be an important
process in which Cortisol has a close

The surprarenal cortex of cobras
hibernation
became active graduallyduringand the level of serum Cortisol became
higher and higher, which was related torelation.

accelerating gluconeogenesis and to thecobra's arising from hibernation.
Thevariation of Cortisol showed that fat
metabolism became active after cobras werearoused out of hibernation.

important role in accelerating somatic cells
to take in energy metabolites and in raisingmetabolism.
In short, during hibernation the cobra

nearly used up all the hepatic glycogen but

consumed little fat body triglyceride. Body
weight tended to be lost significantly during
the period after the snake was aroused outof hibernation. Perhaps fat bodies wereused up because there was little hepaticThe high level of Cortisol
glycogen.
indicated the tendency for fat catabolism.
4. One putative pattern

From the above results, we consider thatperhaps the cobra has formed a seasonal
regulation of endocrine and that hibernationis controlled by this mechanism.

3. Relationship between the three serum

hormones and energy metabolites
In pre-hibernation, the function of T4 andTfromgluconeogenesis. Serum T4 and T3 play anin inducing synthesis of aerobic
metabolic enzymes was weak because of
the low levels of serum T4 and T3The
metabolism of cobras in this period was3.

When theseason for hibernation comes, the contentsof serum T4 T 3 and Cortisol decrease sothat the metabolic system of all organs andtissues in cobras is controlled effectively.
The metabolic rate of cobras tends to go,,

With temperature falling and theserum hormones reducing, theremaining metabolic enzymes in cobras
down.
three

Asiatic Herpetological Research

April 1990

Literature Cited

become inactive gradually (content less andactivity low) and cobras go into hibernation

Vol. 3, p. 51

keeping their metabolic rate at a fairly low
level under the control of the low

DONG, Y. J. ZHAO, J. YANG, Y. CUI, G. LI, AND1986. Studies on physiological
B. CHEN.

temperature isozymes, and after physicalregulation. This suggests that the external
factor for cobra hibernation is the fall oftemperature, while the internal factor is thedrop of the three serum hormone levels. In
hibernation, the contents of serum T4 T3

ecology of Rana spinosa during hybernation
and activation. Acta Herpetologica Sinica 1986,,,and Cortisol increases gradually, but thelevels of the three serum hormones are nothigh enough to accelerate metabolism of theorgans and tissues, and the low temperature
inhibits the activities of the three serum
hormones. When the levels of serum T4
T 3 and Cortisol are high enough, themetabolism of the organs and tissues inWith thecobras tends to go up.
serum
three
thetemperature rising,hormones induce the synthesis of aerobic
metabolic enzymes in the organs andtissues and accelerate the metabolism of5(4):24 1-245.

1965. The role of the thyroid
gland in the oxygen consumption of lizards.
General Comparative Endocrinology 5(3):320-MAHER, M. T.
325.

MUSACCIA, X. J.

1984.
Comparative
biochemicalandaspects ofphysiologicalhypothermia as a model for hibernation.

Cryobiology 21(6):583-592.

,,energy metabolites. The metabolic rate
rises and the cobra recovers from

NAULLEAU, G., F. FLEURY, AND J. BOISSIN.
1987. Annual cycles in plasma testosterone andthyroxine in male Aspic Vipera aspis L.
(Reptilia, Viperidae), in relation to the sexual

cycle and hibernation. General Comparative
Endocrinology 65(2):254-263.

TURNER, G. AND T. J. BAGNARA.
endocrinology,

sixthed.

1976. GeneralW.R.

Co.

Philadelphia.

hibernation. This suggests that the external
factor for the cobra to come out of

WILHOFT, D. C. 1966. The metabolic response tohibernation is the rise of temperature, while
the internal factor is the rise of the threethyroxine of lizards maintained in a thermal
gradient. General Comparative Endocrinology

serum hormone levels.

7(3):445-451.

5. Conclusion

Serum T4 T 3 and Cortisol at low levels
were involved in the regulation of,,depressing the metabolism of cobras before
hibernation. The low temperature duringhibernation inhibited activity of the threeserum hormones, and the metabolism wasWith the rising temperature,
the levels of serum T 4 T 3 and Cortisol at

at a low level.

,,

very high levels accelerated the metabolic

which resulted in arousal
from hibernation. Cobras in hibernation
used hepatic glycogen as their main energy

rate of cobras

source instead of fat body triglycerides.