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The regulation and metabolism of glucose in the green anole (Anolis carolinensis)

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Title:
The regulation and metabolism of glucose in the green anole (Anolis carolinensis)
Creator:
McCoy, Jerry, 1958-
Publication Date:
Language:
English
Physical Description:
vii, 113 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Blood ( jstor )
Enzymes ( jstor )
Glycogen ( jstor )
Insulin ( jstor )
Lactates ( jstor )
Liver ( jstor )
Lizards ( jstor )
Muscles ( jstor )
Plasmas ( jstor )
Reptiles ( jstor )
Dissertations, Academic -- Zoology -- UF
Glucose ( lcsh )
Green anole ( lcsh )
Zoology thesis Ph.D
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jerry McCoy.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Full Text


THE REGULATION AND
GREEN ANOLE
METABOLISM OF GLUCOSE
(AM.Q.LJ.S C.A.RQLINENSIS)
IN THE
By
JERRY MCCOY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERTSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1 9U7


ACKNOWLEDGEMENTS
The author wihes to thank the members of his advisory
committee, Dr. Harvey Lillywhite (chairman),
Dr* John Anderson, Dr* Brian McNab, Dr. Michel Collopy,
and Dr. F. Wayne King, for helpful suggestions during the
course of the research and preparation of the manuscript.
I would also like to tnanx Dr. Donald Allison of the
Biochemistry Department for helpful suggestions with the
enzyme assays.


TABLE OF CONTENTS
Lsjlzl
ACKNOWLEDGEMENTS.. ...... ii
ABSTRACT v
CHAPTERS
I BACKGROUND AND PERPESPECTIVE................ 1
The Problem....,.,...,.... 1
Approach....... 7
IISEASONAL VARIATION IN BLOOD GLUCOSE AND
GLYCOGEN STORES........ 9
Introduction................................. 9
Methods.............. 9
Results...................................... 11
Discussion................. ....... ,17
IIIACTIVITIES OF REGULATORY METABOLIC ENZYMES.,. 22
Introduction..*.............................. 22
Methods................ 27
Results...................................... 30
Discussion 33
IVTHE EFFECTS OF ACTIVITY AND FASTING ON TISSUE
GLYCOGEN AND PLASMA GLUCOSE LEVELS 42
Introduction........ 42
Methods.............. 43
Results........... 47
Discussion........*.. 69
VGLUCOSE TOLERANCES AND hORMONAL INFLUENCES IN
GLUCOSE REGULATION 76
Introduction*......... 76
Methods... 79
Results.*............... ............ 60
Discussion................................... 94
VI SUMMARY AND CONCLUSIONS 98
iii


I
£.a&g.
LITERATURE CITED 102
APPENDIX
CALCULATIONS OF ENERGY EXPENDITURES FOR THE
OXIDATION OF LACTATE AND GLUCONEOGENESIS 111
BIOGRAPHICAL SKETCH.. . 113
IV


Abstract of Dissertation Presented to tne
Graduate School of the University of Florida in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
THE REGULATION AND METABOLISM OF GLUCOSE IN THE
GREEN ANOLE (AtiflLIS CARQLINENSIS)
By
Jerry McCoy
December 1987
Chairman: Dr. Harvey Lillywhite
Major Department: Zoology
The ways in which glucose is regulated and metabolized
in the green anole, Ano 1 is carollnensis. were
examined and compared with mammalian systems.
Parameters examined were seasonal variation in stored
glucose, regulatory enzyme activities associated with
glucose metabolism and the effects of exercise,
fasting, insulin, glucagon ana carbohyarate loading on
glucose.
Plasma glucose ana tissue glycogen show seasonal
variation. Plasma glucose levels are highest in the
spring and lowest in late summer and early fall. Muscle
and liver glycogen levels peak during winter and are
lowest auring the summer. The low levels of tissue
glycogen and plasma glucose during the summer coincide
with the reproauctive season of the green anole.
v


The decrease in glucose during the breeding season may
be due to the influence of reproductive hormones on the
behavior of the lizards, and the increased catabolism
of glucose.
Anaerobic enzyme activities with the exception of
hexokinase are higher in Anolis carolinensis
than in mammals, hitochonarial enzyme activities in
Anolis are lower than those in mammalian
mitochondrial enzymes. The lower mitochondrial
activities in the lizard are probably due to lower
mitochondrial volume densities and mitochondrial
membrane surface areas. These factors are correlated
with differences in the metabolic rates of the two
groups and the reliance of Anolis on anaerobiosis to
support burst activity.
The most probable fate of lactate produced during
exercise in Anolis is gluconeogenesis. Recovery
from lactate acidosis occurs at a faster rate than
tissue glycogen repletion, and excess glucose is removed
preferentially by the liver after exercise. Fasting
depresses tissue glycogen levels,but does not affect
the manner in which glucose is used.
vi


Carbohydrate loading in resting animals produced long
lasting hyperglycemia. Relatively high insulin
concentrations are required to elicit hypoglycemia,
which is of short duration and is followed by a
compensatory hyperglycemia. Hyperglycemia is
probably due to glucagon secretion.


CHAPTER 1
BACKGROUND AND PERSPECTIVE
Uia Lna^lera
Vertebrate ectothermic poikiIotherms rely principally
on anerobic metabolism to meet the energy demands of
intense activity Reliance on anaerobic pathways reduce
the ability to maintain long episodes of intense activity
because of the production of large amounts of lactate
(Bennett 1978, Bennett et al* 1981, Gleeson 1980a,
1980b, 1982, Gaesser and Brooks 1984, Gratz and Hutchison
1977, Hutchison et al* 1977, Hutchison and Turney 1975,
Loumbourdis and Hailey 1985, Putnam 1979a, 1979b,
harnock et al, 1965). The increase in blood and tissue
lactate concentrations may be as high as 10 times resting
levels after prolonged activity and may cause major
pertubations in blood and muscle pH (Bennett 1973,
Bennett and Licht 1972, 1973). A decrease in the oxygen
transporting ability of blood as well as rates of enzyme
catalysis may occur as a result of lactate production.
Metabolic products other than lactate formed during
anaerobiosis presumably are not important in the
synthesis of high energy phosphate compounds in vertebrate
ectotherms. Removal of excess blood lactate produced
during activity may take several hours (Gleeson 1980a, 1980b)
1


2
Repayment of oxygen debt is usually much shorter than the
time for lactate reduction (Brooks et al 1973, Gaesser
and Brooks 1984, Gleeson 1980, Gratz and Hutchison 1977)*
In mammals, lactate is rapidly oxidized to carbon dioxide
and water (Brooks et a 1* 1973, Drury and Wick 1956)* The
fate of anaerobically produced lactate is not known in
reptiles* Although muscle and liver glycogen levels are
reestablished, the location of lactate catabolism is not
known* The skeletal muscle of amphibians have shown
some ability to oxidize lactate (Bendall and Taylor 1970,
Gourley and Suh 1967) One potential site of lactate
catabolism is the liver* Relatively high specific
activities of lactate dehydrogenase have been found in
this tissue in reptiles (Baldwin and Seymour 1977)*
Due to reliance on glycolysis, the regulation of
glucose levels must be of physiological importance in
ectotherms. Most studies concerning anaerobiosis in
terrestrial ectothermic vertebrates focus on either lactate
production or muscle glycogen utilization (Bennett 1978,
Bennett and Gorman 1981, Bennett and Ruben 1975, Callard
et al. 1975, Coulson and Hernandez 1953, Gleeson 1980a,
1980b, Loumbourdis and Hailey 1985, Pough and Andrews 1985,
Putnam 1979a, 1979b), Few studies examine both lactate
production and glucose use simultaneously (Gleeson 1982,
1986 )


I
3
The role of liver glycogen during and after activity
is not known. Liver glycogen stores of some ectothermic
vertebrates may increase or decrease during bouts of
exercise (Dean and Goodnight 1964, Gleeson 1982, Gratz
and Hutchison 1977)* According to one study liver
glycogen is not an important source of energy during and
after activity (Gratz and Hutchison 1977)*
In mammals regulation of blood glucose levels is
important to maintain brain function Approximately one-half
of all the glucose formed by g1uconeogenesis during
interfeeding periods is used for brain metabolism (Guyton
1971)* Most reptiles and birds possess blood glucose
levels significantly higher than mammals; however, among
the reptiles there is considerable variation in levels of
blood glucose (Epple and Lewis 1973 Dessauer 1970,
Penhos and Ramey 1973 Umminger 1977). Lizards appear to
possess the highest levels, with concentrations over 200
mg % being measured (Callard and Chan 1972,
Callard et al 1975, Haggag et al, 1966, Umminger 1977,
Zain-ul-abedin and Qazi 1965) Lower levels are found in
other reptilian groups (Algauhari 1967, Coulson and
Hernandez 1953, Dessauer 1970, Epple and Lewis 1973,
Gratz and Hutchison 1977, Houssay and Penhos 1960, Hutton
1958, Penhos and Ramey 1973, Skoczylas and Sidorkiewicz
1974, Umminger 1977, Zain-ul-abedin and Qazi 1965).
During exercise blood glucose may increase or decrease
(Bennett 1978). In one case where glucose increased


4
during and after exercise, it remained high for several
hours during recovery while liver levels remained
unchanged (Gratz and Hutchison 1977)* The source of this
increase in blood glucose is not known. One possibility
is gluconeogenesis from lactate via the Cori cycle.
Several factors may contribute to the maintenance of
high blood glucose in reptiles. First, reptiles, as with
mammals, may maintain high levels of glucose for brain
function. Second, reptiles may maintain relatively high
levels of blood glucose because of their heavy reliance
on anaerobiosis. Free glucose in the blood can serve as
additional storage. In this form glucose is available for
sudden bursts of activity but is not critical for resting
metabolism. Third, high plasma glucose may be due to an
inherently low insulin concentration in the blood or
low production by the pancreas. The latter has been shown
in birds (Hazelwood 1973)*
The most obvious effect of insulin given to a mammal
is a rapid decrease in the blood glucose level. This
decrease is a consequence of the enhanced transport of
glucose across cellular membranes. In addition to causing
a drop in blood glucose levels, insulin has several
secondary effects including the indirect activation of
glycogen synthetase. In terrestrial ectothermic
vertebrates the effect of insulin is not as clear. In the
snake Psammophls sibilans. insulin has no effect except
when injected with very high dosages (Algauhari 1966).


5
Even with these high dosages a lowering of blood sugar is
only seen 10 hours after the injection. In Rana pipjens,
insulin stimulates the uptake of glucose into cells and
the synthesis of glycogen (Gourley and Suh 1967, 1969),
It also accelerates glycolysis through an enhancement of
the activity of phosphofructokinase (Ozand and Narahara
1964), Rana catesbeiana. however, has a very slow
response to exogenous insulin (Wright 1959)
High levels of glycolytic enzymes and regulation of
those that are rate limiting are adaptations allowing for
high rates of glycolytic pathways in vertebrates (Baldwin
and Seymour 1977) Hexokinase and phosphofructokinase
catalyze irreversible steps in glycolysis, and
phosphofructokinase is the rate limiting enzyme.
Phosphorylase is important in the sequence through which
glycogen enters glycolysis, Because glycogen is the main
substrate for anaerobically active muscle tissue, this
enzyme plays a vital role in the production of adenosine
triphosphate (ATP), The activities of these enzymes
provide a measure of the maximum rate of glucose breakdown
anaerobically (Baldwin et al. 1977, Bennett 1974, Crabtree
and Newsholme 1972, Ozand and Narahara 1964), Low levels
of hexokinase have been measured in those reptiles
examined, which may indicate they are unable to utilize
glucose very rapidly as an energy source (Baldwin and
Seymour 1977)


6
The nutritional state of an animal should, in part, be
responsible for the level of energy reserves found in the
body Many ectotherms become inactive during part of their
annual cycle. The animal must rely on its energy reserves
during this temporal inactivity* Certain ectotherms show
resting body glycogen levels that vary 100% or more
seasonally (Gleeson 1962, Moore 1967, Patterson 1978,
Smith 1954). In other reptiles little seasonal variation
occurs (Skoczylas and Siedorkiewicz 1974). In some
reptiles fasting has no effect on blood glucose levels
for up to 20 weeks (Algauhari 1967). Carbohydrate
digestion leads to increased glycogenesis in some lizards
(Gleeson 1982). Glucose present after exercise is polymerized
mainly in the liver where the amount of glycogen stored
exceeds the pre-exercise levels ( o v ercompensation).
Overcompensation occurs in muscle only if a second
glucose feeding is given during recovery (Gleeson 1982),
which is similar to the response seen in mammals
(Bergstrom et al 1967, Gaesser and Brooks I960, Pernow
and Sa1 tin 1971).
Because anaerobiosis has such an important role in the
metabolism of activity in ectothermic vertebrate the
following questions arise, khat is the specific role of
glucose in anaerobiosis, and how is it utilized in these
organisms? Is blood glucose and/or tissue glycogen
regulated as in higher vertebrates? If so what is the


regulating mechanism(s)? Is body glucose a limiting
factor in the metabolism of activity in reptiles? If so,
how?
7
This study examines ways in which glucose may be
regulated, metabolized and stored in a terrestrial
vertebrate ectotherm, the green anole, Ano 1 is carollnensls.
This species will serve as a model system for glucose
utilization in a reptile and may then be used for comparison
with other vertebrates, ectothermic and endothermic.
The study animal is a representative of one of the
largest lizard families in the world, It is typical in
many regards to other members of its family and species
found in other families. The effects of activity are a
major portion of this study, A lizard species was
therefore chosen as the study animal oecause of all
reptiles, lacertilians tend to lead more active
lifestyles. If glucose Is a limiting factor in the
metabolism of activity, a lizard species is a prime
candidate for this study.
This study is organized into six chapters.
Chapter II deals with seasonal variation in blood glucose,
skeletal muscle and liver glycogen levels, Chapter III
presents the activities of various anaerobic and aerobic


8
regulatory enzymes. These activities are compared to those
measured in mammals, Chapter IV examines the effect of
exercise on tissue glycogen and blood glucose levels*
Chapter IV also details the effect of carbohydrate
loading and the relationship between it and glycogen
The production and removal of lactate is also examinea
in Chapter IV* Chapter V looks at the effect of insulin
and glucagon on blood glucose and tissue glycogen.
Chapter VI is a summary of all findings and presents a
model system of glucose regulation and utilization based
on all the interrelationships presented in the previous
chapters.


CHAPTER II
SEASONAL VARIATION IN BLOOD GLUCOSE AND GLYCOGEN STORES
Introduction
An analysis of any physiological function that is
affected by a number of exogenous factors requires
information on the seasonal variability inherent in that
process. Data on seasonal variation in body glucose
levels in reptiles is sparse (Dessauer 1953, Moore 1967).
Here I characterize seasonal variability in plasma
glucose and/or tissue glycogen. These data shall form the
basis for all further experimentation in the later
chapters.
Methods
Green anoles of both sexes were collected monthly
(n = 7), around Gainesville, Florida, from December 19to4
to April 1987 They were returned to the laboratory after
capture. The lizards were kept in a constant temperature
cabinet set at the amoient temperature at which the
animals were captured for 24 hours. After the 24-hour
acclimation period, blood and tissue samples were taken
and analyzed. All sampling was done between 10:00 a.ra.
9


10
and 12:00 p*ra* E*S*T*, which corresponds to the times of
capture on the previous day* Others were obtained by
purchased from an animal dealer in Louisiana when the
numbers of lizards captured locally were small*
Blood samples were collected in heparinized capillary
tubes after decapitation* The samples were then centrifuged
on a raicropipette centrifuge for 3 minutes* Plasma glucose
concentration was measured colorimetrical ly using Glucostat
test kits (Worthington Biochemical Corporation) and a model
6/20 Coleman Junior 2 spectrophotometer set at 500 nm*
Plasma glucose concentrations are expressed as mg/al or mg%*
Different animals from those used for plasma glucose were
used for liver and muscle glycogen analyses to avoid any
problem associated with struggling* To quickly immobilize
the animals, they were plunged into liquid nitrogen
and stored at -5C until assayed*
To assay for tissue glycogen, the liver was dissected
free along with the musculature of the fore and hind limbs
excluding the manus and tne pes. The skeletal muscle samples
from each animal were combined (fore and hind limb) and
analyzed as a unit* All tissues were ground using a chilled
mortar and pestle* The tissues were weighed to the nearest
0*001 gram Glycogen was extracted in hot KOh and hydrolyzed
to glucose by the Good et al* method (1933) The glucose


11
was then measured using the pheno1-su 1furic acid method
(Montgomery 1957)* The tissue glycogen levels are expressed
as milligrams glycogen per gram tissue (mg/g). All means
are expressed with their standard errors*
&§.§. Ltg.
Data on seasonal variation in plasma glucose and
tissue glycogen are summarized in Figures I1-1 and
II-2 Plasma glucose levels are highest during the months
of April (223 6*9 mg/dl) and May (230 7*5 mg/dl). They
are lowest during August (125 3*1 mg/dl)* Muscle glycogen
concentrations peak during November (53 +. 0.06 mg/g) and
are lowest during July (3.9 0.03 mg/g)* Liver glycogen
concentrations are highest auring November
(38.6 1.06 mg/g) and lowest during August (4.9 . 0.53
mg/g)* In all three cases the differences between the
highest and lowest concentrations are significant at the
0.05 level (small sample two tailed T-test).
There are no significant correlations between plasma
glucose levels and muscle glycogen levels, or plasma
glucose levels and liver glycogen levels (Table II-1)* A
significant positive correlation occurs between muscle
and liver glycogen concentrations (Table II-1),


Figure II-1. Seasonal variation in plasma glucose. Abcissa is
labeled with the first letter of each month. December 1985
was omitted for lack of data. The vertical bars are one
standard error.


ho 5
CONCENTRATION (mg/dl)
cn
I
r>o
cn
1
cn
_L
2 2 5H


Figure 11-2. Seasonal variation in liver and limb musculature
glycogen levels. Squares = muscle glycogen, circles = liver
glycogen. The vertical bars are one standard error*


CONCENTRATION (mg/g)
St


16
Table II-1* Statistical correlations of seasonal data.
Accepted level of confidence equals 0*05
£.gxr.sJLa-v..g.a£ n L y.&Jl.as.
plasma glucose
vs
muscle glycogen -0.20 > 0,05
plasma glucose
vs
liver glycogen 0.049 0.40
liver glycogen
vs
muscle glycogen 0.567 0.001


17
Discussion
These data show that season affects both plasma
glucose ana tissue glycogen concentrations, The most
marked effect is seen in the liver glycogen levels with
an almost 8-fold difference existing between the highest
and lowest levels Plasma glucose and muscle glycogen
levels also follow a seasonal trend, although with less
variation than liver glycogen.
Liver and muscle glycogen levels appear somewhat in
synchrony (Figure II-2), These seasonal data follow the same
pattern as seen in other lizards including Ano 1is
carolinensis (Dessauer 1953, Gleeson 19b2, Patterson
et al, 197b). During late fall both muscle and liver
glycogen levels begin to increase. This is probably in
preparation for overwintering similar to that seen in
temperate zone mammals. However, the genus Ano 1 is is
mainly tropical in its distribution* The species
caro 1inensis has the most northern range of the genus,
extending up to North Carolina, Tennessee, Arkansas and
southeast Oklahoma (Conant 1975), These animals are known
to emerge on warm days during the winter over part of their
range. Therefore, anoles probably are not true hibernators
as are other sympatric lizard species, which may reflect
tropical affinities


18
Low environmental temperatures may cause a reduced rate
of plasma glucose utilization due to the concomitant
reduction of metabolic activity (Coulson and Hernandez 1953,
Dimaggio and Dessauer 1963)* Although plasma glucose
levels increase during late autumn through winter, liver
glycogen levels start to decline during winter. This
suggests that hepatic glycogenolysis is occurring and the
free glucose is entering the blood stream. Rapatz and
Musacchia (1957) found slight changes in blood glucose
but extensive hepatic glycogenolysis in turtles held at
low ambient temperatures for 2 months* Blood glucose levels
have been reported to increase in amphibians and lizards
maintained at low temperatures (Miller 1961, hoore 1967).
Increases in blood glucose during winter may be due to a
release of glucose by the liver and the body not
catabolizing it due to a decreased metabolic rate. Peak
levels of hepatic glycogen are almost 8 times higher than
the lowest values. Even though a large increase in
carbohydrate storage occurs during the late fall, this
represents only a small fraction of the total reserve
energy. Using standard caloric values, Dessauer (1953)
calculated that the stored energy contributed by
carbohydrate reserves amounts to only about 3 of the
total storage. Similar results were reported for Lacerta
vivpara (Patterson et al. 1973). In these two species


19
the amount of stored glucose is too small to be a
significant source of energy during cold torpor; however,
tissue glycogen may act as an important source of
glucosyl residues (Patterson et al. 1978)* If so, tissue
glycogen would tena to reduce the catabolism of amino
acids which are the last resort to supply glucosyl residues.
Blood glucose continues to rise in tne spring, which
may be due to carbohydrate intake through feeding. In
amphibians no correlation exists between f'eeaing and blood
glucose concentration (Wright 1959)* However, in Eumeces
obsoletus blood glucose concentrations increase with an
increase in feeding (Miller and Wurster 1958). In
Ano 11s during the summer, when temperatures are highest
and presumably the animals are operating at their
eccritic temperatures, blooa glucose levels are
declining. One would expect that feeding during this time
would be as high or higher as during the spring, which
may be the case with female lizards, but not in males.
Ano 1 is is typical of other small iguania species
in its reproductive behavior. Males are territorial and
actively defend territories against other males (Greenberg
and Nobel 1944). Active defense in many cases may preclude
other activities, such as feeaing, and can be
metabo 1ica11y taxing (Pough and Andrews 1985). From late
winter through early autumn liver glycogen reserves are


20
low (Figure II-2). This period overlaps the reproductive
season of Ano 1is carolinensis (Hamlett 1952). The
increase in male-male aggression and territoriality
during the breeding season probably reflect the increase
in testoterone secretion by the gonads (Crews 1979)
Androgen injections are known to produce typical sexual
behavior in castrated male anoles (Adkins and Schlesinger
1979, Mason and Adkins 1976).
Females are not territorial and any increase in
activity during the reproductive season may simply
reflect the higher ambient temperatures during this time of
the year. Because no differences were found between males
and females in their blood glucose and tissue glycogen
levels, something obviously reduces female liver
glycogen and blood glucose during the reproductive season.
The answer may also be attributable to hormones.
Ano 1is carolinensis has a very peculiar form of
ovulation, termed monoa11ochronic ovulation (Smith et al*
1973). Females ovulate a single egg alternately from each
ovary approximately every 14 days during the breeding
season (Hamlett 1952, Jones et al, 1953, Nobel and
Greenberg 1941). Each egg is oviposited 18 or 19 days
after ovulation (Hamlett 1952), Therefore a period of
4 or 5 days exists when one oviduct contains a mature
shelled egg while the other oviduct contains a recently


21
ovulated unshelled egg. During the reproductive
season plasma concentration of estradiol-17 beta (E-2) and
progesterone are high (Jones et al, 1983)* Marschall and Gist
(1973) reported reductions in both plasma glucose and liver
glycogen levels following estradiol and follicle stimulating
hormone (FSH) administration. They suggested that
mobilization of hepatic glycogen and blood glucose
utilization following hormonal treatments is due to the
metabolism of carbohydrates contributing part of the energy
required for the synthesis of yolk proteins.
Reducing blood glucose may also have secondary effects
on other forms of stored energy, such as lipids. In
mammals lipid degradation can result from hormone sensitive
lipases or may depend on the availability of glucose.
Glucose is required for the reesterification of free
fatty acids through the generation of glycerol-3-
phosphate. Therefore, if glucose availability to the
adipose cells is reduced, it results in the release of
free fatty acids (Marschall and Gist 1973). Estrogen
treatment results in fatty acid release in Uta
stans curiana (Hahn and Tinkle 1 965 ). A mechanism for
triacylglycerol degradation involving plasma glucose
reduction in JL*. caro 1 inensis may occur. Such a
mechanism was suggested by Marschall and Gist (1973)
because even when presented with a glucose load, the
incorporation of labeled glucose carbon into fat bodies
was reduced during estrogen treatment.


CHAPTER III
ACTIVITIES OF REGULATORY METABOLIC ENZYMES
Introduction
Carbohydrates usually make up a large proportion of the
diet of most animals. Some of the carbohydrate intake
may be converted to, and metabolized as, fat However
the major function of carbohydrates is a source of energy
through cellular oxidation. The major form of carbohydrate
for oxidation is the hexose glucose. Other principal
monosaccharides which may be utilized include fructose, the
major form found in fruits, and galactose, which is found in
dairy products Fructose is only important if the diet
includes large amounts of sucrose, a disaccharide composed
of glucose and fructose Both fructose and galactose can
be converted into glucose through hepatic mechanisms.
Carbohydrate metabolism can occur through several
pathways (Figure III-I), The major pathway during
activity in reptiles is the oxidation of glucose or
glycogen to lactate by theEmbden-Meyerhof pathway
(Figure III-2) High levels of glycolytic enzymes and
regulation of those that are rate limiting are adaptations
22


23
GLUCOSE
.^OX ALO ACETATE
PHOSPHOENOLPYRUVATE
1
PYRUVATE
> LACTATE
ACETYL-Co A
FUMERATE
SUCCINYL-Co A<-
CITRATE
V
OC-KETOGLUTERATE
Figure III-1, Schematic of
catabolism. Double headed
utilizing the same enzyme,
pathways.
pathways of glucose and glycogen
arrows indicate reversible reactions
Multiple arrows indicate multiple


24
PHOSPHORYLATED HEXOSES
TRIOSE PHOSPhATE RUVATE
TRIOSE PhOSPHATE
V
LACTATE
Figure III-2. Simplified schematic of the Embden-Meyerhof
pathway.


25
allowing for high rates of sudstrate passage through
glycolytic pathways. If anaerobiosis represents the major
pathway for energy production during activity, one would
expect to find high activities of anaerobic enzymes.
Enzymes examinee were hexokinase, phosphorylase,
lactate dehydrogenase and phosphofructokinase, key
regulatory catalysts in glycolysis. These four enzymes are
used to estimate the maximum rates of glycolysis in
muscle (Crabtree and Newsholme 1972). Phosphorylase is
not an enzyme of the Embden-Meyerhof pathway proper
(Figure III-3). Phosphorylase is involved in the
degradation of glycogen through the mobilization of
glucose-1-phosphate, hexokinase controls the entry of free
glucose into the glycolytic pathway through the
phospnorylation of glucose. Under physiological conditions
this reaction can be considered to be irreversible
because of the considerable loss of free energy as heat.
Although lactate dehydrogenase is not a rate-1imiting
enzyme, high levels are usually associated with nigh
anaerobic scopes. These four enzymes are all found in the
soluble fraction (cytosol) of the cell.
The mitochondrial enzymes, nicotinamide adenine
dinucleotioe (NAD)-linked isocitrate dehydrogenase ano
succinic dehydrogenase are both involved in aerobic
metabolism and the generation of electron transporting


26
PHOSPHORYLASE B
(active R form)

AMP
GLUCOSE b-PHOSPHATE
ATP
PHOSPHORYLASE B
(inactive T form)
(inactive T form)
PhOSPHORYLASE A
(active R form)
Figure II1-3* Control of the activity of phosphorylase in
hepatic and skeletal muscle tissue* The enzyme can change from
an inactive T (tense) form, to active R (relaxed) form. The
proportion of the active conformation is determined mainly by
the rates of phosphorylation and dephosphorylation. Adapted
from Stryer 1961*


27
molecules* Thus, both are linked to the proauction of ATP
via the Kreb's cycle and they are also key regulatory steps
in the Kreb's cycle.
Methods
Green anoles were obtained from a dealer in Louisiana
and held in captivity for 7 days in a thermal gradient
(22 50 C) on a 12:12 light and dark cycle. The lizards
were fed crickets and mealworms daily. Five animals
were used to measure each enzyme activity. Animals were
fasted for 48 hours prior to Deing assayea. Lizards were
killed by freezing in liquid nitrogen. After freezing the
lizards were assayed immediately or storea frozen until
assayed at a later date. To assay, the entire liver was
removed along with the musculature of both fore ana hind
limbs. All the dissected tissue was weighed and placed in
an ice bath. All preparations were performed in an ice oath.
Muscle and liver samples were homogenized separately
in ice cola bufferea Ringer's solution (1 gram tissue
in 4 ml solution, Guillette 1 982) with a mortar and
pestle. Triton X-1G0 was added to give a final
concentration of 1$. Triton X-100 increases the soluble
hexokinase activity (Burleigh and Schimke 19b9). After
the Triton X-100 addition, the homogenate was incubated on
ice for 30 minutes followed by centrifugation at 17000
rpms at 4 C.


28
Enzyme activities were measured according to the methods
outlined for each enzyme* Concentrations are expressed in
the total volume of the final reaction mixture.
All measurements were made with a model 6/20 Coleman Junior
2 spectrophotometer. Samples were kept at 33 0 C
(eccritic temperature) in a water bath and the optical
density measured every 60 seconds. Mean specific activities
are expressed in n-moles of product formed per minute per
milligram protein nitrogen* Protein was estimated by the
biuret method according to Gornall et al. (1949)*
Purified bovine serum aloumin solutions were used as
standards (Sigma Chemical Company)* All means are given
with their standard errors.
fle.SQMn.a.sg. 1
The reaction mixture contained 0.76 m M N A D P, 3 ni H ATP,
7*5 mM magnesium chloride, 0*2 mM glucose, 15mM potassium
chloride, 2 I g1ucose-6-phosphate dehydrogenase,
5 mM mercaptoethano1, and 50 mM sodium phosphate buffer
pH 7.4 (Baldwin ano Seymour 1977). The reaction was
started with the addition of homogenate* Enzyme activity
was measured by following the reduction of NADP at 340 nm.
Lactate Dehvdroaenase ( E C* 1 1 .1*27.)
The reaction mixture contained 33 mM phosphate buffer
pH 7*4, 0*067 mM NADh, 0*33 mM sodium pyruvate and
homogenate. The reaction was started Dy the addition of


29
pyruvate. The specific activity was measured from the
decrease in optical density at 340 nm.
£.j}Q.s_B.iiojf.u.St.P.k.1ng,g.e 1 )
The reaction mixture contained 5 mM magnesium
chloride, 200 mM potassium chloride, 50 mM Tris hCl pH 7.4,
0*1 mM N A D H 1 mM ATP, 2 mM AMP, 0*3 mM sodium cyanide,
2 mM glucose-6-phosphate, 2 IU alpha-glycerophosphate
dehydrogenase and homogenate (Baldwin and Seymour 1977).
The reaction was started by the addition of glucose-6-
phosphate. The decrease in optical density at 340 nm was
used to calculate specific activity.
tiAP-Mnfrad. Xg.os.-v.fa.tg. p.g.hY.dr.pggnaag (. c. i. i i .41)
The reaction mixture contained 20 mM phosphate
buffer pH 6.5, 1*7 mM magnesium chloride, 0*17 mM
manganese chloride, 0.50 mM buffered potassium cyanide,
pH 7*5, 0.030 mM dich1oropheno1 indophenol, 0.17 mM NAD,
6.7 mM sodium DL-isocitrate and homogenate (Bennett 1972).
The reaction was started by adding the isocitrate, and the
decrease in optical density at 600 nm was used to
calculate specific activity.
Succinic Dehydrogenase (,£.x£.i.i^3...9.a.i.-U.
The reaction mixture contained 40 mM phosphate
buffer, pH 7.5, 3*o mM Duffered potassium cyanide, pH 7.5,
0.040 mM dichlorophenol indophenol, 0.13 mM phenazine


30
methosu1phate, 26 mh buffered sodium succinate, pH 7,5
and homogenate (Bennett 1972). Succinate was added to
start the reaction. Activity was determinea from the
decrease in optical density at 600 nm.
Results
The specific activities of hexokinase, phosphorylase,
lactate dehydrogenase, phosphofructokinase, NAD-linked
isocitrate dehydrogenase and succinic dehydrogenase
compared with those of mammalian values in Tables III-1
and III-2 (Bennett 1972, Lamb et al. 1969)
Of the four skeletal muscle glycolytic enzyme
comparisons, only hexokinase showed greater activities in
the mammals* Lactate aehydrogenase, phosphory1 ase and
phosphofructokinase were greater in the reptile, However,
in comparing the activities of mitochondrial enzymes,
mammalian activities were greater. Reptilian hexokinase
and lactate dehydrogenase activities were greater in the
hepatic comparisons. Phosphofructokinase activities of the
reptile were lower than those of the mammals, botn liver
mitochondrial enzyme activities of the mammals were
higher than the reptilian values.


31
Table III-1. Comparisons of anaerobic ana aerobic enzyme
activities between skeletal muscle of mammals and green
anoles, Values taken from Craotree and hewsholme were
converted to nmoles/mg protein min assuming a ratio of 29:1
muscle tissue to muscle protein nitrogen and 10t:1 liver
tissue to liver protein nitrogen. Assay temperature and
pH equals 33 C and 7.4 respectively.
MlSC-LE
Hexokinase
Lactate Dehydronase
Phosphofructokinase
Phosphorylase
Isocitrate Dehydrogenase
Succinic Dehydrogenase
LIZARD
65,5*
0.013
473G**
74200
238**
1051
4 1 3 b *
2522
25.6**
5.6
114**
22.4
* From Crabtree and Newsholm 1972.
** From Bennett 1972,
*** From Prichard and Schofield 1968.


32
Table III-2, Comparisons of anaerobic and aerobic enzyme
activities in liver tissue of mammals and green anoles.
Values taken from Crabtree ana Newsholme were converted to
nmoles/mg protein min assuming a 29:1 ratio of muscle tissue
to muscle protein nitrogen and a 108:1 ratio of liver tissue
to liver protein nitrogen, Assay temperature and pH
equals 33 0 C and 7.4 respectively.
um
MMhALS.
LIZARDS
Hexokinase
1.2***
3.92
Lactate Dehydrogenase
18800**
27600
Phosphofructokinase
93**
56.6
Phosphorylase
1058.4
Isocitrate Dehydrogenase
87.8**
24.4
Succinic Dehydrogenase
261 **
73.6
* From Crabtree and Newsholme 1972.
* From Bennett 1972.
*** From Prichard and Schofield 1968


33
Discussion
The comparatively higher activities of glycolytic
enzymes in the lizard suggest a greater capacity for
anaerobic metabolism than in mammals* Likewise, the higher
activities of aerobic enzymes in the mammal reflect a
greater aerobic capacity. Utilizing the less efficient
glycolytic pathway for energy production has the
advantage of the rapid production of ATP* Other
ectothermic species examined reveal the same pattern
(Baldwin 1975, Baldwin and Seymour 1977, Baldwin et al.
1977, Bennett 1972, 1974, Crabtree and Newsholm 1972).
All glycolytic enzymes excluding hexokinase exhibit
greater activities in skeletal muscle than in the liver.
The greater activity is expected because of rapid depletion
of available oxygen in skeletal muscle during activity
and subsequent anaerobiosis*
Phosphorylase activity in skeletal muscle is
approximately 2.5 times higher than its activity in liver,
Phosphory1 ase in its active form, phosphory1ase-a (Figure
III-3), catalyzes the breakdown of glycogen at a very high
rate to glucose-1-phosphate. G1ucose-1-phosphate can then
be shuttled through glycolysis after isomerization into
glucose 6-phosphate. This mobilization of glucose-1 -
phosphate from glycogen is very important in the liver
and skeletal muscle. Under most physiological conditions


34
phosphorylase-b is inactive because of the inhibitory
effects of ATP and g1ucose-6-phosphate (Stryer 1961), The
greater activity of phosphory1 ase in skeletal muscle may
be due to the speed at which the end product is used.
Phosphorylase is the control point in an amplification
cascade in hepatic and extrahepatic tissues (Figure III-
4). In liver tissue the end result of this cascade is the
release of free glucose into the bloodstream* Skeletal
muscle lacks glucose-6-phosphatase ana cannot produce free
glucose. Thus all glycogen stored in skeletal muscle is
only used by muscle: skeletal muscle does not act as a
glycogen reserve for other tissues,
Several minutes may be required for glycogen
phosphory1 ase to reach peak activity in the liver
(Lehninger 19d2). Because of the inherent amplification
involved with each reaction step, hepatic phosphory1 ase may
possess lower activity because of the tremendous production
from each subsequent step in the cascade. The amplification
is estimated to be some 25 million fold, The cascade in
skeletal muscle is lower and, therefore, the amplification
is reauced In addition, under certain conditions,
the energy required by the muscles must be instantaneous.
In situations where rapid escape behavior or prey ambush
is required, any delay in glucose utilization may be
deleterious


35
STIMULUS >ADRENAL MEDULLA
I
EPINEPHRINE
V
ADENYLATE CYCLASE
CELL MEMBRANE
ATP-
PROTEIN KINASE
(inactive)
-> CAMP + PPi
C a + +
PROTEIN KINASE + CAMP
(active)
ATP + DEPHOSPHO-PHOSPHORYLASE
KINASE Ca++
(inactive)
>PHOSPHO-PHOSPHORYLASE + ADP
KINASE
(active)
ATP + PHOSPHORYLASE B
(inactive)
-> PHOSPhORYLASE A + ADP
(active)
GLYCOGEN + Pi
-^GLUCOSE 1-PHOSPHATE
I
GLUCOSE 6-PHOSPHATE
I
GLUCOSE + Pi
CELL MEMBRANE
BLOOD GLUCOSE
Figure III-4. Epinephrine produced amplification cascade in
hepatic cells. Epinephrine arrives at the receptor sites on
the cell membrane at a concentration of approximately one nM.
The free glucose released into the blood Increases its
concentration by 5 mM The multiplier effect produced is
about 3 milllion times.


36
Low hexokinase activities in skeletal muscle may
reflect the inability of this tissue to use free glucose
from the blood as a readily available energy source. Low
hexokinase activities have been reported for other
poikilotherms (Baldwin and Seymour 1977, Baldwin et al.
1977, Bennett 1972, 1974, Crabtree and Newsholm 1972)
Enzymes with low specific activities that form part of
the chain of a metabolic pathway usually catalyze
non-equilibrium reactions (hewsholme and Start 1973).
This is indicative of fewer active sites in the
enzyme molecule than is required to reach equilibrium.
The greater catalytic capacities of preceeding and
subsequent enzymes form substrates and remove products
faster than the non-equilibrium enzyme can inter-convert
them* Thus, enzymes that control non-equilibrium reactions
form a bottleneck in metabolic pathways, Hexokinase,
pnosphofructokinase and phosphory1 ase are prime
candidates for catalyzing non-equilibrium reactions,
By comparing mass-action ratios (t) with apparent
equilibrium constants (K') a reaction can be evaluated as
being no n-e q u i 1 i b r i um or not. If the value of t is much
smaller than tC', the reaction is displaced far from
equilibrium. Comparisons between t and K' in mammalian
tissues show hexokinase, phosphofructokinase and
phosphory1 ase all catalyze non-equilibrium reactions
(Newsholm and Start 1973)* Similar results were also
found in spiders (Prestwich 1962),


37
Mass action ratios were not measured here However,
the similar enzyme activities found here and in studies
of other ectotherms, indicate that very few if any
differences in the glycolytic pathway exists (Baldwin and
Seymour 1977, Baldwin et a 1. 1977, Bennett 1972, hacleod
et al, 1963) If no differences exist in the descriptive
aspects of the pathway, the same bottlenecks will occur at
the same locations. However, the higher activities of
certain key steps in poiki1otherms, enhance the output of
this pathway.
In emergency situations or during periods of fasting the
liver, mobilizing glycogen stores, releases free glucose
into the blood. Glucose molecules enter cells by means of
a symport (Figure III-5). Once inside the cell glucose is
phospnorylated by hexokinase at the expense of one ATP.
By phosphory1ating all the glucose that enters the cell,
a large glucose concentration gradient is maintained
between the blood and the intracellular compartment. Low
hexokinase activity reduces this potential gradient
througn the reduction of glucose-6-phosphate formation.
One potential solution to low hexokinase activity is
maintaining high blood glucose levels* High blood glucose
levels may serve to continously flood the intracellular
environment with glucose. Maintaining high levels of
glucose intracellularly may allow the low levels of
hexokinase to phosphory1 ate large numbers of glucose
molecules. This is discussed further in Chapter IV.


Figure III-5* The glucose sodium symport* The sodium graaient
produced powers the transport of glucose* Adapted from Stryer
1961*


Cell membrane
ATP
v
riJ k +
V
Na+
^ADP+ P
>Na+
>Glucose
Symport
v
^Glucose


40
Seasonal variation may be involved in enzyme activities,
as may the rate of metabolism* All enzyme assays were
performed on tissue samples from resting animals during late
summer when plasma glucose levels are declining and tissue
glycogen levels are low. During this time enzymes are
expected to be most effective. If true, muscle hexokinase
activities would be even more reduced during times when
glucose and glycogen levels are high* Ozand and Narahara
(1964) reported increases in the phosphofructokinase
reaction rate in contracting muscle. They suggested that
under conditions in which the phosphofructokinase of the
muscle is saturated with fructose-6-phosphate, an
increase in Vmax of the reaction may involved. If we
assume that regulatory enzyme function follows a
conservative pattern, hexokinase activity may also show
an increase with an increase in metabolism.
The nigher activities of the mitochondrial enzymes in
the mammal may be due to either or both of two reasons,
First it may be simply due to a more rapid conversion of
substrate with no difference in the concentration of
enzyme between the anole and the rat. However the amount
of enzyme present in the mammalian tissue may be greater
due to either a greater proportion of mitochondria,
greater relative membrane surface area, or both. In
comparing the activity of cytochrome oxidase in four


41
different tissues of a reptile ana a mammal, Else and
Hulbert (1981) reported higher activities in the latter in
all four tissues. They attributed this to greater
mitochondrial volume densities and mitichondrial membrane
surface areas in the mammal, A greater mitochondrial
volume density in mammals is probably related to the use
of fatty acids as a metabolic substrate (Bennett 1972,
Drummond 1971) Because of the preferential use of glucose
as an energy substrate for activity in reptiles, fatty
acid metabolism is only utilized during specific times
such as fasting and reproduction (Dessauer 1955,
Smith 1968)


CHAPTER IV
THE EFFECTS OF ACTIVITY AND FASTING ON
TISSUE GLYCOGEN AND PLASMA GLUCOSE LEVELS
Introduction
Poik1othermic ectotherms are generally regarded as low
energy systems This is especially true when they are
comparea metabo 1ica1ly to endothermic homeotherms
(Bennett 1972) Reptiles generally are not as active as
are mammals or birds. The basis for this less active
lifestyle is the preferential use of anaerooic,
glycolytic pathways instead of aerobiosis during intense
activity More than 50% of the total energy produced from
activity is derived from anaerooiosis (Bennett and Licht
1972, Gleeson 1982, Gratz and Hutchison 1977, Pough and
Andrews 1983)* Although glycolysis is less efficient than
aerobiosis (ie., Kreo's cycle), the former produces ATP's
at a much faster rate.
Reliance on glycolysis for energy production suggests
that glucose is a limiting factor in metabolism during
activity in poikilotherms. Muscle glycogen depletion
occurs in most vertebrates after intense activity (Dean
and Goodnight 1964, Gaesser and brooks 1980, Gleeson
42


43
1582, Gratz and Hutchison 1977, Johnston and Goldspink
19731 MacDougall et al* 1977, Piehl 1974), During the
recovery period following depletion of muscle glycogen,
small reptiles are particularly susceptible to predation.
Their susceptibility increases with the time course of
glycogen replenishment.
This chapter examines the effects of burst activity on
tissue glycogen, blood glucose, lactate production and the
effects of fasting on carbohydrate utilization before,
during and after exercise. Additionally the
relationship between carbohydrate loading ana glycogen
replacement is also evaluated.
Glucose and glycogen analyses were performed during
July and August 1986, as described in Chapter II, Animals
were exercised in a water-driven treadmill at their
eccritic temperature (33 0 C), until exhausted.
Exhaustion is defined as loss of the righting response.
Immediately upon exhaustion the animals were either
dropped into liquid nitrogen and assayed for tissue
glycogen and lactate or decapitated and the olood assayed
for glucose or lactate. Glucose and glycogen
concentrations were measured at rest, 3U seconds after
the onset of exercise, at the point of exhaustion and at


44
various times up to 720 minutes during recovery* Recovery
here and in all subsequent assays is defined as the time
from the end of exercise to when pre-exercise (resting)
levels of the parameter in question are reached. Plasma
glucose is expressed as mg/dl. Tissue glycogen is
expressed as mg glycogen/g of tissue. All animals were
maintained in a photothermal gradient (22 C to 50 C,
12:12 light:dark cycle). All means are expressed with
their standard errors.
Lactate ft.n.a.ly.si.g.
Ten groups of five animals each were analyzed at rest,
after exhaustion, or 10, 60 or 120 minutes during the
recovery period. One half of the groups were usea to analyze
whole body lactate concentrations. Lizards were
decapitated and dropped into liquid nitrogen. After
freezing, the bodies were homogenized in volumes of cold
0.6 h perchloric acid equivalent to 12 times body mass.
To study compartmenti1ization of lactate, samples of
blood, liver and leg muscle from the animals of the other
five groups were weighed, pooled in cold perchloric acid
(5 times the sample weight) and homogenized* All
homogenates were centrifuged at 3000 rpms at 4 C for 10
minutes. The supernatant was filtered and recentrifugea
at 10000 rpms at 4 C for 10 minutes. The resulting
supernatant was assayed with an enzymatic test kit (Sigma


45
Chemical Company)* Samples were read on a model 6/20
Coleman Junior 2 spectrophotometer at 340 nm. Lactate
concentrations are expressed as mg lactate/g tissue
weight*
Q.xy.ge.n G.Q.nsvupp.t J-pn
Oxygen consumption (Vq2) was measured manometrically
using a Gilson Differential Respirometer during July and
August 1 986 Lizards (n = 5) were placed in 125 ml
respirometer flasks. Each flask contained approximately
12 grams of soda lime to absorb CO^. All determinations
were made at 33 C (i.e., eccritic temperature). Resting
rates were measured at 10:00 pm E*S.T*
After several hours of acclimation in the flasks Vq2's
were measured, To measure oxygen consumption after
exhaustion and during recovery, animals were exercised at
9:00 p.m. in the respirometry flasks by the motorized
shaking of the flasks. To increase the activity of the
lizards in the flasks, small glass spheres were added,
The movement of the spheres agitated the lizards thereby
causing activity. During the recovery period rates were
measured every 2 minutes. Rates are expressed as
ml G 2 /g h.


46
Carbohydrate Loading
The experiments were performed in August, the time when
body sugar levels are lowest The role of these experiments
was to explore the relationship between glucose
supplementation and glycogen resynthesis Lizards were fasted
for 48 hours (n = 120). After exercising to exhaustion,
half the lizards were fed a glucose solution, 20 ul/g body
weight at a concentration of 1 mg/4 ul by stomach tube
and allowed to recover for 12 hours. This volume and
concentration is equivalent to 5 mg glucose per g body
weight. The other lizards were given an equivalent volume
of distilled water via a stomach tube. After 12 hours one
half of the glucose supplemented lizards were assayed for
tissue glycogen as previously described. One half of the
control group were assayed for tissue glycogen. The other
glucose supplemented lizards were given a second glucose
feeding and were assayed 12 and 24 hours later. The rest
of the control lizards were also assayed at these times.
These experiments examined the effect of fasting on
glucose concentrations at rest, during exercise and
during recovery. Lizards fasted for 2 weeks during August.
After fasting the animals were exercised as previously
described until exhaustion. Glucose and glycogen levels
were measured at the same intervals as previously
described


47
Results
Oxygen consumption and whole body lactate at rest,
during exercise and recovery are summarized in Figure 1V-1.
9
Maximum Vq2 occurred during exercise
(1.65 0*201 ml/g h). With a resting VQ2 of 0.262
0.036 ml/g h, the aerobic scope equals 6.3. Oxygen
consumption declined steadily throughout the recovery
period. At 75 minutes of recovery no statistically
significant difference existed in the recovery and
resting rates of oxygen consumption (P > 0*05, T-test
for paired data). TaDle IV-1 summarizes the net recovery
oxygen consumption and the change in lactate
concentration during recovery.
Whole body lactate increased 6.4 times from resting
values, 0.29 . 0.01 mg/g, to exercise values
2*43 0.21 mg/g. Over the first 10 minutes of
recovery lactate declined very little (2.36 + 0.26 mg/g).
Sixty minutes into recovery, lactate had dropped
drastically (0.68 +. 0.14 mg/g). By 120 minutes the
lactate levels were not statistically different from those
of resting values (P > 0.05, paired T-test).
Figure IV-2 summarizes the data for lactate
compartmenta1ization. All compartments show the same trend.
Maximal lactate concentrations occur during exercise. Muscle
and blood lactate levels after 2 hours of recovery were


Figure 1V-1* Lactate concentration and oxygen comsumption
before, during and after exercise. Circles = oxygen
consumption Triangles = lactate. Each symbol represents
the mean of 5 lizards.


TIME (minutes)
LACTATE CONCENTRATION (mg/g)
6t/


50
Table IV-1. Resting ana post exercise lactate and glycogen
levels Units are mg lactate/glycogen per g tissue
a.QA£A&XIfaL& E.AS.XIM.
Muscle
REST.
g.Q.aXEm£ISE
EE3.X
£aaXE£EEC.X&E
Lactate
0.62
2.53


Glycogen
4.1
1.6
3.9
1 .9
Liver
Lactate
0.15
1 .44


Glycogen
7.1
3.9
5.4
2.3


Figure IV-2. The compartraenta1ization of lactate befor
during and after exercise. Squares = plasma lactate.
Triangles = skeletal muscle lactate. Circles = liver
lactate. Each symbol represents the mean of 5 lizards.


TIME (minutes)
CONCENTRATION (mg/g)
29


53
statistically indistinguishable from resting values
(P > 0*05, T-test). Liver lactate levels during recovery
were the same as resting levels by 2*5 hours. Resting
muscle lactate levels were significantly greater than
those of the liver and blood (P > 0.05, T-test). Resting
liver and blood lactate levels are very similar
(0.15 mg/g and 0.12 mg/g respectively).
Muscle glycogen and lactate levels show the expected
trends (Figure IV-3). As exercise proceeds glycogen levels
drop and lactate concentrations increase. As activity
ceases lactate begins to decrease with a concomitant
increase in muscle glycogen. At 120 minutes into recovery
lactate levels are not statistically different from resting
levels (0.59 0.07 mg/g and 0.62 0.05 mg/g,
respectively, P > 0.05, T-test). Muscle glycogen returns
to resting levels 6 h after the cessation of activity
(4.3 +. 0.17 mg/g ana 4.1 0.33 mg/g, respectively,
P > 0.05, T-test).
Liver glycogen and lactate concentrations also show
opposite changes (Figure IV-4). Liver glycogen levels do
not drop significantly until the end of exercise, ho
significant change in liver glycogen occurs after one
minute into exercise (7.1 0.83 mg/g and 7.0 0.67 mg/g
respectively, P > 0.05, T-test). At the end of activity,
the glycogen level drops to 3.9 +. 0.44 mg/g. Liver lactate
increased from a resting level of 0.15 . 0.02 mg/g to


Figure IV-3 Muscle glycogen and lactate before, during and
after exercise. Circles = glycogen. Triangles = lactate.
Each symbol represents the mean of 5 lizards. The vertical
bars are one standard error.


LACTATE CONCENTRATION (mg/g)
h5
TIME (minutes)
GLYCOGEN CONCENTRATION (mg/g)


Figure 1V-4 Liver lactate and glycogen before, during and
after exercise. Triangles = lactate. Circles = glycogen.
Each symbol represents the mean of 5 lizards.


LACTATE CONCENTRATION (mg/g
-8.0
IME (minutes)
T
cn
GLYCOGEN CONCENTRATION (mg/g)


58
1.44 +. 0.11 mg/g at the end of exercise. Lactate levels
continue to rise after exercise reaching a peak value of
1.47 +. 0*14 mg/g after 10 minutes of recovery. Lactate
levels then declined to 0*78 +. 0.03 mg/g after 60 minutes
of recovery. After 2.5 hours of recovery lactate levels
returned to resting levels (0.13 +. 0*008 mg/g and
0*15 +. 0.02 mg/g respectively, P > 0*05, T-test).
Resting and peak lactate and glycogen concentrations in
muscle and liver with time to recovery are summarized in
Table IV-2.
Plasma glucose concentrations before, during and after
exercise are summarized in Figure IV-5. Plasma glucose in
the post-exercise period reached higher levels than
pre-exercise levels. During exercise plasma glucose
values fell from 179*6 14.3 mg/dl to 149.6 12.3
mg/dl The plasma glucose level reaches its maximum
concentration, 239*4 +. 12.1 mg/dl two hours into recovery.
This level remains fairly constant for the next three
hours. After 12 hours, plasma glucose levels decline to
134*1 6.7 mg/dl,
Carbohydrate loading during recovery produced different
results for muscle and liver glycogen replenishment. The
initial glucose supplement had no effect on muscle
glycogen replenishment (Figure IV-6). After 12 hours control
and experimental values were not different (4.5 +. 0.07 mg/g
and 4.6 + 0.04 mg/g, P > 0.05, T-test). Compensation


59
Table IV-2* Oxygen consumption requirements for lactate
oxidation and gluconeogenesis from lactate* Numbers are based
on data tasen from Figure IV-2.
Q.U.bbk .E.kkltxft.P, lifl-U
TIME
(MIN )
LACTATE NET
(mm) OXYGEw
RECOVERY
CONSUMPTION (ml)
LACTATE TO
GLUCOSE b-P
LACTATE
OXIDATION
0-4
0.072
0*098
7.6
X
10 "7
4.838
4-1 5
0.070
0.074
7.4
X
10 -7
4.704
U1
1
-tr
U1
0.020
0.029
r\)

o
X
10 "7
1.344
45-75
0.019
0.010
2.0
X
10 -7
1 .277
75-120
0.009
0.002
9.5
X
10 "9
0.605


Figure IV-5. Plasma glucose concentration before, during and
after exercise. Triangles = fasting animals. Squares =
nonfasting animals* Each symbol represents the mean of 5
lizards. The vertical bars are one standard error.


250
200
150
100
50
i ai r~
R Ex 60
r
120
r-
300
TIME (minutes)
720
cr>


Figure IV-6. Effects of post exercise carbohydrate loading on
muscle glyocgen. Squares = controls Triangles = animals
receiving glucose load. N = 120. The vertical bars are
one standard error. Glucose is given at the end of
exercise.


TIME (hours)
MUSCLE GLYCOGEN (mg/g)
£9


64
occurred only in muscle after the second glucose feeding*
After the second feeding muscle glycogen levels increased
almost 2 times (4*6 0*04 mg/g to 6*4 +. 0*23 mg/g*
Overcompensation occurred after both glucose feedings in
liver tissue (Figure IV-7)% Liver glycogen had increased
10 times over resting levels after the second feeding
(7.0 +. 0.66 mg/g to 70.1 +. 4.08 mg/g).
Plasma glucose is affected differently during exercise
in fasting and nonfasting animals (Figure IV-5). Plasma
glucose of fasting lizards increased from
149.6 131 mg/dl to 177.1 16.1 mg/dl. Glucose levels
declined rapidly, and after 60 minutes of recovery, plasma
levels were equal to resting levels (151*2 10*3 mg/dl
and 14 9*6 +. 13-1 mg/dl respectively, P > 0.05, T-test).
Plasma levels then dropped and remained slightly below
resting levels*
Resting tissue glycogen concentrations are lower in
fasting animals than nonfasting animals (Figure IV-8),
The same patterns during and after activity are
produced among fasted lizards as seen among nonfasted
individuals (Figures IV-3, IV-4). however glycogen levels
are generally depressed among fasted animals. Peak
resting and recovered muscle and liver glycogen values
are summarized in Table IV-1,


Figure IV 7 Effects of post exercise carbohydrate loading on
liver glycogen. Squares = controls* Triangles = animals
receiving glucose load* N = 120. The vertical bars are
one standard error* Glucose is given at the end of
exercise.


80
70
60
50
40
30
20
10
12
TIME (hours)
TEST
R Ex
T~
24
36
os
OS


Figure IV-b. Tissue glycogen before, during ana after
exercise in fasted lizards. Squares = muscle glycogen.
Triangles = liver glycogen. Each symbol represents the
mean of 5 lizards. The vertical bars are one standard
error


TIME (minutes)
TISSUE GLYCOGEN (mg/g)
ro
i
OJ
L_
L_
TO -
CT>
O
ro
o
OJ
o
o
CT
O -|
O
89


69
Discussion
A characteristic prolonged elevation of oxygen
consumption occurs after exercise in vertebrates.
Hill et al* (1924) originally termed this the oxygen
debt. This elevated oxygen consumption was interpreted as
the oxidation of lactic acid produced as the result of
tissue glycogen catabolism, The original hypothesis
stated that only a fraction of the lactate, approximately
20%, was actually oxidized. The lactate oxidized provided
the energy required to resynthesize glycogen from the
remaining lactate. Results from several studies have
questioned the validity of this hypothesis (Bennett and
Licht 1973, Brooks et al, 1971, 1973, Gleeson 1980a, 19&0b,
Gratz and Hutchison 1977, Hutchison et al* 1977)* Results
of this study also appear to refute the classic oxygen
debt hypothesis*
Seventy-five minutes into the post-exercise recovery
period oxygen consumption has returned to resting values
(Figure IV-1). Post exercise lactate levels require two
hours to return to resting levels* These data show that
net lactate removal is still occurring after oxygen
consumption has returned to normal, however, muscle and
liver glycogen levels return to pre-exercise levels
without any carbohydrate intake* The time course for this
tissue glycogen replenishment is several hours (Figures
IV-3 IV -4 )


70
Lactate removal appears, in Figure IV 1, not to be the
result of elevated post-exercise oxygen consumption
(EPOC), however, the temporal uncoupling between lactate
removal and EPOC is misleading. The metabolic pathway(s)
used for lactate removal cannot be determined without
using radio-1 a be 1ed compounds. The fate of lactate can be
suggested by calculating the amount of oxygen required to
resynthesize glucose from lactate (i.e, g1uconeogenesis),
or to completely oxidize lactate to carbon dioxide and
water (Appendix), and by comparing these estimates with
those actually measured. Table IV-2 is such a summary.
Obviously the increase in oxygen consumption during
recovery cannot supply the energy required to completely
oxidize lactate (Table IV-2). The net recovery oxygen
consumption only accounts for approximately 03 of
the energy required for complete lactate oxidation.
However, the net recovery oxygen consumptionis more than
adequate for the resynthesis of glucose from lactate.
These comparisons suggest that most if not all of the
lactate produced during forced activity is reconverted
into glycogen via g1uconeogenesis. This differs from the
situation in mammals, in which the primary fate of
lactate is oxidation (Brooks et al* 1973, Drury and Vvick
1956, Gaesser and Brooks 1980). The production of
glycogen from lactate appears to occur in other reptiles
(Gleeson 1985, Moberly 1968). Gleeson (1985), using


71
Table IV-2. Oxygen consumption requirements for lactate
oxidation and gluconeogenesis from lactate. Numbers are based
on data taken from Figure IV-2.
Sim REQUIRED (ml)
TIME
(MIN )
LACTATE NET
(mM) OXYGEN
RECOVERY
CONSUMPTION (ml)
LACTATE TO
GLUCOSE 6-P
LACTATE
OXIDATION
0-4
0.072
0.098
7.6
X
10 "7
4.838
4-15
0.070
0.074
7.4
X
10 7
4.704
15-45
0.020
0.029
2.0
X
10 "7
1.344
45-75
0.019
0.010
2.0
X
10 _7
1.277
75-120
0.009
0.002
9.5
X
10 "9
0.605


72
inferential data reported seasonal variation in the
ability of red muscle to produce glycogen from lactate,
He suggested that muscle gluconeogenesis may account for
approximately 20% of the lactate removed during recovery
from exhaustive activity, because many reptiles rely on
burst activity in many of their behaviors, utilizing
the gluconeogenic pathway for lactate removal allows them
to resynthesize the much needed substrate for its reuse.
The compartmentalization of lactate also suggests the
possibility of a gluconeogenic fate for lactate
(Figure IV-2). Liver lactate levels during recovery
remain elevated after blood and muscle lactate levels
have returned to pre-activity levels, which suggests that
the liver may be responsible for the reduction of lactate
levels. The liver has been shown to possess relatively
high specific activities of lactate dehydrogenase
(Chapter III), an enzyme that is required to convert
lactate back into pyruvate before it is metabolized. This
conversion is necessary to regenerate NAD+ so the
glycolytic pathway remains active as long as possible. As
such the metabolic burden of energy production does not
fall on the skeletal muscle alone. Part is shifted from
muscle to the liver (Stryer 1961).
The potential for the liver as the primary site of
gluconeogenesis is strengthened when one examines the
plasma glucose levels during recovery (Figure IV-5).


73
Plasma glucose levels remain elevated for at least 5
hours following forced activity, which may be due to the
reconversion of lactate to glucose by the liver, After
the conversion of lactate to glucose by the liver, free
glucose enters the bloodstream from which it is stored by
various tissues as glycogen. Glycogen then serves as the
primary substrate for catabolic energy production.
Plasma glucose levels are probably not rapidly reduced
during activity due to the low hexokinase activity
(Chapter III), A high concentration would then be
required to establish a suitable concentration
gradient between the plasma and intracellular
compartments. Such a gradient permits the passage of
glucose into the cell. Once glucose moves inside, the
gradient prevents the leakage of glucose to the outside
until it can be phosphory1ated by hexokinase and acted
upon by various other enzymes for storage as glycogen.
The liver glycogen level requires more time to return
to resting levels after exercise than is required for
repletion of muscle glycogen (Figures IV-3, IV-4),
suggesting that the primary course of glycogen
replenishment is first in muscle and then in the liver,
this sequence is logical because muscle glycogen is utilized
first during episodes of intense activity. The glycogen
stored in the muscles is for use by muscles only,


74
whereas the liver acts as a storage site for glycogen to
be shuttled to extrahepatic tissues at the onset of
activity*
During intense activity the liver apparently releases
free glucose into the bloodstream. However, the total
amount of glycogen stored in muscles limits the amount of
anaerobically produced work that occurs during intense
activity. If an anaerobically dependent animal depletes
part of its glycogen store through activity or fasting,
it then reduces the amount of work that it can perform*
The same pattern of tissue glycogen utilization occurs
during activity and recovery in fed and fasted Ano 1 is
(Figure IV-8). Fasted lizards, however, exhausted much
faster than those that were fed. During activity, plasma
glucose levels increase and then decline (Figure IV-5), a
pattern during periods of glycogen depletion that
produces a concentration gradient for glucose uptake by
skeletal muscles. Muscle glycogen stores are not replaced
more quickly in fasting versus nonfasting lizards (Figure
IV b ) However the establishment of a plasma
intracellular glucose gradient may allow for anaerobiosis
to occur after a shorter recovery period (i*e, the free
glucose entering the cell is used immediately for
glycolysis ) .


75
Otner studies have shown that repetitive bouts of
activity result in decreased times to exhaustion
(Putnam 1979a, 1979b)* Decreased glycogen stores are part
of the reason for this reduction in performance. Lactate
buildup would play a major role in performance reduction
if lactate levels were still elevated. Ano 1 is possesses
the ability to quickly replace reduced muscle glycogen
stores compared to mammals (Conlee et al, 1978). This
ability may involve a mechanism that minimizes any
potential reduction of activity resulting from fatigue
or fasting. Muscle glycogen repletion appears to occur
without the need for dietary carbohydrate intake.
A large part (approximately 6 0 %) of an oral load of
glucose is taken up preferentially by the liver in resting
humans (Maehlum et al, 1977). Less than 15% ends up as
muscle glycogen. However during post exercise recovery,
a greater percentage of splanchnic glucose ends up as
muscle glycogen. Muscle glycogen repletion has a higher
priority over hepatic retention during the recovery period.
The picture is different during recovery in Ano 1 is.
Carbohydrate loading appears to increase glycogenesis. The
ingestion of glucose after exercise causes an increase in
hepatic glycogen. An oral glucose load has no initial
effect on muscle glycogen. Skeletal muscle only shows an
increase in storage after a second glucose feeding. This
preferential overcompensation effect in liver tissue


76
suggests that muscle cells are storing glycogen at or near
their maximum rate and further strengthens the
conclusion that these animals rely heavily on
stored glycogen in muscle for activity metabolism.
Muscle tissue increases its storage of glycogen
apparently only after saturating the hepatic glycogen
storage rate. Whether this increase in storage is temporary
is not known. If so, the only way the glycogen
concentration can be reduced is through the catabolic
activities of the muscle. Muscle cells do not possess
glucose-6-phosphatase. Therefore, muscle glycogen cannot
be utilized as a precursor to fat storage and can only be
used by the muscle.
The storage of glycogen increases the time a lizard
may be engaged in vigorous activity, but it will occur
only if the lactate produced is not the maximum amount
that can be tolerated. If not, the upper limit for lactate
tolerance would prevent any increase in the time a lizard
may engage in intense activity, regardless of the amount
of glycogen stored.
The cost of using anaerobiosis coupled to
gluconeogenesis is unfavorable. Consider that three high
energy phosphate bonds are produced per glucose molecule
utilized and derived from tissue glycogen. Six high energy
phosphate bonds are used in the reconversion of glucose
from pyruvate* Consequently, a net loss of three high energy


77
phosphate bonds results from coupling glycolysis with
gluconeogensis. The loss in bond energy is equivalent to
21,9 kcal/mole of phosphate bond hydrolyzed
The evolutionary benefits derived from an anaerobic
system outweigh its costs* The metabolic machinery for
aerobic metabolism is present although its performance is
somewhat reduced (Chapter III) By utilizing glycolysis
for the production of energy during activity Ano 1 is is
limited in the length of time it may engage in burst
activity. Because of this limitation a large amount of
stored energy is not required to engage in short term
bouts of intense activity.


CHAPTER V
GLUCOSE TOLERANCE AND HORMONAL INFLUENCES
IN GLUCOSE REGULATION
Introduction
The effects of insulin and glucagon have been examined
in every order and suborder of reptiles except for
rhyncocephalians (Penhos and Ramey 1973)* The physiological
effects are variaDle. Insulin is least effective in the
lizards examined, and shows the most markea effect in
alligators. Resting levels of plasma glucagon have not
been determined in ectothermic vertebrates, however,
injected glucagon raises the blood sugar level in all
reptile species examined.
Glucose tolerance tests allow the effects of an added
glucose load on body sugar levels to be evaluated. The
usual procedure involves the intraperitoneal injection of
a glucose solution and subsequent monitoring of blood
glucose and/or tissue glycogen levels over time.
Alligators show the greatest deviations from glucose
homeostasis. This study is an attempt to characterize
glucose homeostatis in green anoles and compare them to
other species previously examined. Glucose tolerance,
insulin and glucagon responses were used as criteria
for this characterization,
78


79
Methods
G-i.g.P.gg, 1 ole r.S.nge
Gastric glucose tolerance was examined during August,
Large adult lizards (S-V length 600 mm), were
purchased from an animal dealer. The lizards fasted for
two days prior to testing (n = 70), Glucose was
administered by gastric intubation at a concentration of
2 and 5 mg/g body weight. Plasma glucose and tissue
glycogen were analyzed as described in Chapter II. Sugar
concentrations were measured hourly for 10 hours, then
24, 4b and 72 hours after intubation. Plasma glucose is
expressed as mg/dl +. 1 S.E,M, Skeletal muscle and liver
glycogen are expressed as mg glycogen/g tissue 1 SEM,
Insulin
Mammalian insulin was obtained as a zinc salt from
ICN Biomedical Inc. The insulin was injected
intraperitoneally at concentrations of 1000 units/kg and
2000 units/kg. Animals were assayed for plasma glucose,
muscle and liver glycogen at 10, 20, 30, 60, 90, 120, 300
minutes and 24 hours after the injection (n = 108),
The results are expressed as previously described.


80
Glucagon
Mammalian glucagon was obtained from ICN Biomedical Inc.
The hormone was injected intraperitoneally at
concentrations of 10 ug/kg and 100 ug/kg (n = "108).
Animals were assayed at the same intervals as in the
insulin experiment, Results are expressed as previously
described.
hesults
Glucose Tolerance
Results are summarized in Figures \-1 ana V-2. Plasma
glucose levels peaked between 2 and 3 hours after
ingestion (4S2 mg/dl). The plasma glucose levels
were back to resting levels after 48 hours
(135 +. 4.7 mg/dl). Muscle glycogen showed little effect
of added glucose. Liver glycogen levels peaked between 10
and 24 hours (52.3 3.33 mg/g).
lijsy.IiD.
Insulin responses are summarized in Figures V-3 and V-4.
In both cases there is a rapid onset of hypoglycemia. Plasma
glucose levels dropped from 132 +. 2*1 mg/dl to
99 1*4 mg/dl and 134 +. 1.7 mg/dl for the low and high
insulin concentrations respectively. Within 60 minutes
after the onset of hypoglycemia plasma glucose levels
returned to normal values. At 90 minutes after
administering insulin, the lizards showed a plasma


Figure V-1. Glucose tolerance for plasma glucose. Each
symbol represents the mean of five animals.


PLASMA GLUCOSE (mg/dl)
1iiiiIi i i i r
0 1 5 10
TIME (hours)
24 48 72
00
ro


Figure V-2. Glucose tolerance for muscle and liver glycogen.
Triangles = muscle glycogen. Circles = liver glycogen.
Each symbol represents the mean of five animals.


TIME (hours)
GLYCOGEN CONCENTRATION (mg/g)
t/8


Figure V-3. The effect of insulin on plasma glucose, muscle,
and liver glycogen Insulin concentration = 1000 units/kg,
Squares = plasma glucose Triangles = muscle glycogen
Circles = liver glycogen Insulin was injected at time
zero*


GLYCOGEN CONCENTRATION (mg/g)
0 10 20 60 90 120 300 24
TIME (minutes//hours)
00
cr>
PLASMA GLUCOSE (mg/dl)


Figure V -4. The effects of insulin on plasma glucose,
and liver glycogen Insulin = 2000 units/kg# Squares
glucose. Triangles = muscle glycogen. Circles = liver
glycogen. Insulin was injected at time zero.
muscle,
p1 asma


TIME (minutes //hours)
CO
00
PLASMA GLUCOSE (mg/dl)


89
compensatory hyperglycemia which ended at 120 minutes.
Muscle glycogen levels showed a rapid increase after the
insulin injection. Within 10-20 minutes glycogen
reached peak levels. Liver glycogen concentrations show
the same trend, a rapid increase in stored glycogen
followed by the maintenance of a relatively constant level,
both dosage groups produced the same results.
G.ims.agpp.
Glucagon responses are summarized in figures V-5 ana
V-6. Animals injected with the higher concentration
(100 ug/kg) showed the greatest effect. Plasma glucose
concentration increased rapidly in both groups In the
lower concentration group (10 ug/kg), this
hyperglycemia continued for 6 hours. After 24 hours
plasma glucose levels returned to pre-injection levels.
In the 100 ug/kg group plasma glucose levels remained
elevated for 46 hours. After 72 hours plasma glucose levels
returned to normal. No effect of glucagon on muscle
glycogen levels appeared in either group. In the liver
stored glycogen levels decreased. In the 10 ug/kg group
this reduction continued for 5 hours* Liver glycogen
returned to resting levels after 24 hours (Figure V-5).
In the higher concentration group, the reduction in liver
glycogen continued for 24 hours. After 72 hours the
glycogen levels returnea to normal (Figure V-6).


Figure V-5. The effects of glucagon on plasma glucose, and
muscle and liver glycogen. Glucagon concentration = 10 ug/kg.
Squares = plasma glucose. Triangles = muscle glycogen.
Circles = liver glycogen. Glucagon was injected at time
zero


GLYCOGEN CONCENTRATION (mg/g)
9
1irn 1 1 i 1 t
O 10 30 60 90 120 300 24
TIME (minutes//hours)
PLASMA GLUCOSE (mg/g)


Figure V-6. The effects of glucagon on plasma glucose and
muscle and liver glycogen Glucagon concentration = 100
ug/kg. Squares = plasma glucose Triangles = muscle glycogen.
Circles = liver glycogen Glucagon was injected at time
zero


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9 b



THE REGULATION AND
GREEN ANOLE
METABOLISM OF GLUCOSE
(MQ.LJ.S C.A.RQLINENSIS)
IN THE
By
JERRY MCCOY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERTSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1 9U7

ACKNOWLEDGEMENTS
The author wihes to thank the members of his advisory
committee, Dr. Harvey Lillywhite (chairman),
Dr* John Anderson, Dr* Brian McNab, Dr. Micheál Collopy,
and Dr. F. Wayne King, for helpful suggestions during the
course of the research and preparation of the manuscript.
I would also like to tnanx Dr. Donald Allison of the
Biochemistry Department for helpful suggestions with the
enzyme assays.

TABLE OF CONTENTS
£iL&£
ACKNOWLEDGEMENTS.. ...... ............... ii
ABSTRACT v
CHAPTERS
I BACKGROUND AND PERPESPECTIVE................ . 1
The Problem....,.,...,..». 1
Approach....... 7
IISEASONAL VARIATION IN BLOOD GLUCOSE AND
GLYCOGEN STORES...... 9
Introduction................................. 9
Methods............ 9
Results...................................... 11
Discussion............ 17
IIIACTIVITIES OF REGULATORY METABOLIC ENZYMES.,. 22
Introduction............ 22
Methods...... .......... ............ .......... 27
Results...................................... 30
Discussion. 33
IVTHE EFFECTS OF ACTIVITY AND FASTING ON TISSUE
GLYCOGEN AND PLASMA GLUCOSE LEVELS 42
Introduction. 42
Methods...•••••.••,•».•••••••••••............ 43
Results......... 47
Discussion........••••.•,••••••••••••••••»... 69
VGLUCOSE TOLERANCES AND hORMONAL INFLUENCES IN
GLUCOSE REGULATION 7b
Introduction.,.*».»». 7 8
Methods.,.................................... 79
Results.*................ 80
Discussion................................... 94
VI SUMMARY AND CONCLUSIONS 98
iii

I
E.S&g.
LITERATURE CITED • 102
APPENDIX
CALCULATIONS OF ENERGY EXPENDITURES FOR THE
OXIDATION OF LACTATE AND GLUCONEOGENESIS 111
BIOGRAPHICAL SKETCH,» ...* 113
IV

Abstract of Dissertation Presented to tne
Graduate School of the University of Florida in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
THE REGULATION AND METABOLISM OF GLUCOSE IN THE
GREEN ANOLE (AtiflLIS CARQLINENSIS)
By
Jerry McCoy
December 1987
Chairman: Dr. Harvey Lillywhite
Major Department: Zoology
The ways in which glucose is regulated and metabolized
in the green anole, Ano 1 is carollnensis. were
examined and compared with mammalian systems.
Parameters examined were seasonal variation in stored
glucose, regulatory enzyme activities associated with
glucose metabolism and the effects of exercise,
fasting, insulin, glucagon ana carbohydrate loading on
glucose.
Plasma glucose ana tissue glycogen show seasonal
variation. Plasma glucose levels are highest in the
spring and lowest in late summer and early fall. Muscle
and liver glycogen levels peak during winter and are
lowest during the summer. The low levels of tissue
glycogen and plasma glucose during the summer coincide
with the reproductive season of the green anole.
v

The decrease in glucose during the breeding season may
be due to the influence of reproductive hormones on the
behavior of the lizards, and the increased catabolism
of glucose.
Anaerobic enzyme activities with the exception of
hexokinase are higher in Anolis carolinensis
than in mammals. Mitochondrial enzyme activities in
Anolis are lower than those in mammalian
mitochondrial enzymes. The lower mitochondrial
activities in the lizard are probably due to lower
mitochondrial volume densities and mitochondrial
membrane surface areas. These factors are correlated
with differences in the metabolic rates of the two
groups and the reliance of Anolis on anaerobiosis to
support burst activity.
The most probable fate of lactate produced during
exercise in Anolis is gluconeogenesis. Recovery
from lactate acidosis occurs at a faster rate than
tissue glycogen repletion, and excess glucose is removed
preferentially by the liver after exercise. Fasting
depresses tissue glycogen levels,but does not affect
the manner in which glucose is used.
vi

Carbohydrate loading in resting animals produced long
lasting hyperglycemia. Relatively high insulin
concentrations are required to elicit hypoglycemia,
which is of short duration and is followed by a
compensatory hyperglycemia. Hyperglycemia is
probably due to glucagon secretion.

CHAPTER 1
BACKGROUND AND PERSPECTIVE
Uia Lnafelera
Vertebrate ectothermic poikiIotherms rely principally
on anerobic metabolism to meet the energy demands of
intense activity» Reliance on anaerobic pathways reduce
the ability to maintain long episodes of intense activity
because of the production of large amounts of lactate
(Bennett 1978, Bennett et al* 1981, Gleeson 1980a,
1980b, 1982, Gaesser and Brooks 1984, Gratz and Hutchison
1977, Hutchison et al* 1977, Hutchison and Turney 1975,
Loumbourdis and Hailey 1985, Putnam 1979a, 1979b,
Vvarnock et al, 1965). The increase in blood and tissue
lactate concentrations may be as high as 10 times resting
levels after prolonged activity and may cause major
pertubations in blood and muscle pH (Bennett 1973,
Bennett and Licht 1972, 1973). A decrease in the oxygen
transporting ability of blood as well as rates of enzyme
catalysis may occur as a result of lactate production.
Metabolic products other than lactate formed during
anaerobiosis presumably are not important in the
synthesis of high energy phosphate compounds in vertebrate
ectotherms. Removal of excess blood lactate produced
during activity may take several hours (Gleeson 1980a, 1980b)»
1

2
Repayment of oxygen debt is usually much shorter than the
time for lactate reduction (Brooks et al» 1973, Gaesser
and Brooks 1984, Gleeson 1980, Gratz and Hutchison 1977)*
In mammals, lactate is rapidly oxidized to carbon dioxide
and water (Brooks et a 1* 1973, Drury and Wick 1956)* The
fate of anaerobically produced lactate is not known in
reptiles* Although muscle and liver glycogen levels are
reestablished, the location of lactate catabolism is not
known* The skeletal muscle of amphibians have shown
some ability to oxidize lactate (Bendall and Taylor 1970,
Gourley and Suh 1967)» One potential site of lactate
catabolism is the liver* Relatively high specific
activities of lactate dehydrogenase have been found in
this tissue in reptiles (Baldwin and Seymour 1977)*
Due to reliance on glycolysis, the regulation of
glucose levels must be of physiological importance in
ectotherms. Most studies concerning anaerobiosis in
terrestrial ectothermic vertebrates focus on either lactate
production or muscle glycogen utilization (Bennett 1978,
Bennett and Gorman 1981, Bennett and Ruben 1975, Callard
et al. 1975, Coulson and Hernandez 1953, Gleeson 1960a,
1980b, Loumbourdis and Hailey 1985, Pough and Andrews 1985,
Putnam 1979a, 1979b), Few studies examine both lactate
production and glucose use simultaneously (Gleeson 1982,
1986 )

I
3
The role of liver glycogen during and after activity
is not known. Liver glycogen stores of some ectothermic
vertebrates may increase or decrease during bouts of
exercise (Dean and Goodnight 1964, Gleeson 1982, Gratz
and Hutchison 1977)* According to one study liver
glycogen is not an important source of energy during and
after activity (Gratz and Hutchison 1977)*
In mammals regulation of blood glucose levels is
important to maintain brain function» Approximately one-half
of all the glucose formed by g1uconeogenesis during
interfeeding periods is used for brain metabolism (Guyton
1971)* Most reptiles and birds possess blood glucose
levels significantly higher than mammals; however, among
the reptiles there is considerable variation in levels of
blood glucose (Epple and Lewis 1973» Dessauer 1970,
Penhos and Ramey 1973» Umminger 1977). Lizards appear to
possess the highest levels, with concentrations over 200
mg % being measured (Callard and Chan 1972,
Callard et al» 1975, Haggag et al* 1966, Umminger 1977,
Zain-ul-abedin and Qazi 1965)» Lower levels are found in
other reptilian groups (Algauhari 1967, Coulson and
Hernandez 1953, Dessauer 1970, Epple and Lewis 1973,
Gratz and Hutchison 1977, Houssay and Penhos 1960, Hutton
1958, Penhos and Ramey 1973, Skoczylas and Sidorkiewicz
1974, Umminger 1977, Zain-ul-abedin and Qazi 1965).
During exercise blood glucose may increase or decrease
(Bennett 1978). In one case where glucose increased

4
during and after exercise, it remained high for several
hours during recovery while liver levels remained
unchanged (Gratz and Hutchison 1977)* The source of this
increase in blood glucose is not known. One possibility
is gluconeogenesis from lactate via the Cori cycle.
Several factors may contribute to the maintenance of
high blood glucose in reptiles. First, reptiles, as with
mammals, may maintain high levels of glucose for brain
function. Second, reptiles may maintain relatively high
levels of blood glucose because of their heavy reliance
on anaerobiosis. Free glucose in the blood can serve as
additional storage. In this form glucose is available for
sudden bursts of activity but is not critical for resting
metabolism. Third, high plasma glucose may be due to an
inherently low insulin concentration in the blood or
low production by the pancreas. The latter has been shown
in birds (Hazelwood 1973)*
The most obvious effect of insulin given to a mammal
is a rapid decrease in the blood glucose level. This
decrease is a consequence of the enhanced transport of
glucose across cellular membranes. In addition to causing
a drop in blood glucose levels, insulin has several
secondary effects including the indirect activation of
glycogen synthetase. In terrestrial ectothermic
vertebrates the effect of insulin is not as clear. In the
snake Psammophls sibilans. insulin has no effect except
when injected with very high dosages (Algauhari 1966).

5
Even with these high dosages a lowering of blood sugar is
only seen 10 hours after the injection. In Rana pipjens,
insulin stimulates the uptake of glucose into cells and
the synthesis of glycogen (Gourley and Suh 1967, 1969),
It also accelerates glycolysis through an enhancement of
the activity of phosphofructokinase (Ozand and Narahara
1964), Rana catesbeiana. however, has a very slow
response to exogenous insulin (Wright 1959)»
High levels of glycolytic enzymes and regulation of
those that are rate limiting are adaptations allowing for
high rates of glycolytic pathways in vertebrates (Baldwin
and Seymour 1977)» Hexokinase and phosphofructokinase
catalyze irreversible steps in glycolysis, and
phosphofructokinase is the rate limiting enzyme.
Phosphorylase is important in the sequence through which
glycogen enters glycolysis, Because glycogen is the main
substrate for anaerobically active muscle tissue, this
enzyme plays a vital role in the production of adenosine
triphosphate (ATP), The activities of these enzymes
provide a measure of the maximum rate of glucose breakdown
anaerobically (Baldwin et al. 1977, Bennett 1974, Crabtree
and Newsholme 1972, Ozand and Narahara 1964), Low levels
of hexokinase have been measured in those reptiles
examined, which may indicate they are unable to utilize
glucose very rapidly as an energy source (Baldwin and
Seymour 1977)»

6
The nutritional state of an animal should, in part, be
responsible for the level of energy reserves found in the
body» Many ectotherms become inactive during part of their
annual cycle. The animal must rely on its energy reserves
during this temporal inactivity* Certain ectotherms show
resting body glycogen levels that vary 100% or more
seasonally (Gleeson 1962, Moore 1967, Patterson 1978,
Smith 1954). In other reptiles little seasonal variation
occurs (Skoczylas and Siedorkiewicz 1974). In some
reptiles fasting has no effect on blood glucose levels
for up to 20 weeks (Algauhari 1967). Carbohydrate
digestion leads to increased glycogenesis in some lizards
(Gleeson 1982). Glucose present after exercise is polymerized
mainly in the liver where the amount of glycogen stored
exceeds the pre-exercise levels ( o v ercompensation).
Overcompensation occurs in muscle only if a second
glucose feeding is given during recovery (Gleeson 1982),
which is similar to the response seen in mammals
(Bergstrom et al» 1967, Gaesser and Brooks 1980, Pernow
and Sa1 tin 1971).
Because anaerobiosis has such an important role in the
metabolism of activity in ectothermic vertebrate the
following questions arise, khat is the specific role of
glucose in anaerobiosis, and how is it utilized in these
organisms? Is blood glucose and/or tissue glycogen
regulated as in higher vertebrates? If so what is the

regulating mechanism(s)? Is body glucose a limiting
factor in the metabolism of activity in reptiles? If so,
how?
7
This study examines ways in which glucose may be
regulated, metabolized and stored in a terrestrial
vertebrate ectotherm, the green anole, Ano 1 is carollnensls.
This species will serve as a model system for glucose
utilization in a reptile and may then be used for comparison
with other vertebrates, ectothermic and endothermic.
The study animal is a representative of one of the
largest lizard families in the world, It is typical in
many regards to other members of its family and species
found in other families. The effects of activity are a
major portion of this study, A lizard species was
therefore chosen as the study animal oecause of all
reptiles, lacertilians tend to lead more active
lifestyles. If glucose Is a limiting factor in the
metabolism of activity, a lizard species is a prime
candidate for this study.
This study is organized into six chapters.
Chapter II deals with seasonal variation in blood glucose,
skeletal muscle and liver glycogen levels, Chapter III
presents the activities of various anaerobic and aerobic

8
regulatory enzymes. These activities are compared to those
measured in mammals, Chapter IV examines the effect of
exercise on tissue glycogen and blood glucose levels*
Chapter IV also details the effect of carbohydrate
loading and the relationship between it and glycogen
The production and removal of lactate is also examinea
in Chapter IV* Chapter V looks at the effect of insulin
and glucagon on blood glucose and tissue glycogen.
Chapter VI is a summary of all findings and presents a
model system of glucose regulation and utilization based
on all the interrelationships presented in the previous
chapters.

CHAPTER II
SEASONAL VARIATION IN BLOOD GLUCOSE AND GLYCOGEN STORES
Introduction
An analysis of any physiological function that is
affected by a number of exogenous factors requires
information on the seasonal variability inherent in that
process. Data on seasonal variation in body glucose
levels in reptiles is sparse (Dessauer 1953, Moore 1967).
Here I characterize seasonal variability in plasma
glucose and/or tissue glycogen. These data shall form the
basis for all further experimentation in the later
chapters.
Methods
Green anoles of both sexes were collected monthly
(n = 7), around Gainesville, Florida, from December 19to4
to April 1987» They were returned to the laboratory after
capture. The lizards were kept in a constant temperature
cabinet set at the amoient temperature at which the
animals were captured for 24 hours. After the 24-hour
acclimation period, blood and tissue samples were taken
and analyzed. All sampling was done between 10:00 a.ra.
9

10
and 12:00 p*nu E*S*T*, which corresponds to the times of
capture on the previous day* Others were obtained by
purchased from an animal dealer in Louisiana when the
numbers of lizards captured locally were small*
Blood samples were collected in heparinized capillary
tubes after decapitation* The samples were then centrifuged
on a raicropipette centrifuge for 3 minutes* Plasma glucose
concentration was measured colorimetrical ly using Glucostat
test kits (Worthington Biochemical Corporation) and a model
6/20 Coleman Junior 2 spectrophotometer set at 500 nm*
Plasma glucose concentrations are expressed as mg/al or mg%*
Different animals from those used for plasma glucose were
used for liver and muscle glycogen analyses to avoid any
problem associated with struggling* To quickly immobilize
the animals, they were plunged into liquid nitrogen
and stored at -5°C until assayed*
To assay for tissue glycogen, the liver was dissected
free along with the musculature of the fore and hind limbs
excluding the manus and tne pes. The skeletal muscle samples
from each animal were combined (fore and hind limb) and
analyzed as a unit* All tissues were ground using a chilled
mortar and pestle* The tissues were weighed to the nearest
0*001 gram» Glycogen was extracted in hot KOh and hydrolyzed
to glucose by the Good et al* method (1933)» The glucose

11
was then measured using the pheno1-su 1furic acid method
(Montgomery 1957)* The tissue glycogen levels are expressed
as milligrams glycogen per gram tissue (mg/g). All means
are expressed with their standard errors*
&§.§„». Lt.g.
Data on seasonal variation in plasma glucose and
tissue glycogen are summarized in Figures I1-1 and
II-2» Plasma glucose levels are highest during the months
of April (223 ± 6*9 mg/dl) and May (230 ± 7*5 mg/dl). They
are lowest during August (125 ± 3*1 mg/dl)* Muscle glycogen
concentrations peak during November (5*3 +. 0.06 mg/g) and
are lowest during July (3.9 ± 0.03 mg/g)* Liver glycogen
concentrations are highest during November
(38.6 ± 1.06 mg/g) and lowest during August (4.9 ±. 0.53
mg/g)« In all three cases the differences between the
highest and lowest concentrations are significant at the
0.05 level (small sample two tailed T-test).
There are no significant correlations between plasma
glucose levels and muscle glycogen levels, or plasma
glucose levels and liver glycogen levels (Table II-1), A
significant positive correlation occurs between muscle
and liver glycogen concentrations (Table II-1),

Figure II-1, Seasonal variation in plasma glucose. Abcissa is
labeled with the first letter of each month, December 1985
was omitted for lack of data. The vertical bars are one
standard error.

5 OH
CONCENTRATION (mg/dl)
en
_L
r>o
en
1
en
_L
2 2 5H

Figure 11-2. Seasonal variation in liver and limb musculature
glycogen levels. Squares = muscle glycogen, circles = liver
glycogen. The vertical bars are one standard error*

CONCENTRATION (mg/g)
St

16
Table II-1* Statistical correlations of seasonal data.
Accepted level of confidence equals 0*05»
£.pxr.sJLa-v.¿.g.a£ n ¿ y.&Jl.ws.
plasma glucose
vs
muscle glycogen -0.20 > 0,05
plasma glucose
vs
liver glycogen 0.049 0.4G
liver glycogen
vs
muscle glycogen 0.567 0.001

17
Discussion
These data show that season affects both plasma
glucose ana tissue glycogen concentrations* The most
marked effect is seen in the liver glycogen levels with
an almost 8-fold difference existing between the highest
and lowest levels* Plasma glucose and muscle glycogen
levels also follow a seasonal trend, although with less
variation than liver glycogen*
Liver and muscle glycogen levels appear somewhat in
synchrony (Figure II-2)» These seasonal data follow the same
pattern as seen in other lizards including Ano 1is
carolinensis (Dessauer 1953* Gleeson 19b2, Patterson
et al. 197b). During late fall both muscle and liver
glycogen levels begin to increase. This is probably in
preparation for overwintering similar to that seen in
temperate zone mammals. However, the genus Ano 1 is is
mainly tropical in its distribution* The species
caro 1inensis has the most northern range of the genus,
extending up to Worth Carolina, Tennessee, Arkansas and
southeast Oklahoma (Conant 1975)* These animals are known
to emerge on warm days during the winter over part of their
range. Therefore, anoles probably are not true hibernators
as are other sympatric lizard species, which may reflect
tropical affinities

18
Low environmental temperatures may cause a reduced rate
of plasma glucose utilization due to the concomitant
reduction of metabolic activity (Coulson and Hernandez 1953,
Dimaggio and Dessauer 1963)* Although plasma glucose
levels increase during late autumn through winter, liver
glycogen levels start to decline during winter. This
suggests that hepatic glycogenolysis is occurring and the
free glucose is entering the blood stream. Rapatz and
Musacchia (1957) found slight changes in blood glucose
but extensive hepatic glycogenolysis in turtles held at
low ambient temperatures for 2 months* Blood glucose levels
have been reported to increase in amphibians and lizards
maintained at low temperatures (Miller 1961, hoore 1967).
Increases in blood glucose during winter may be due to a
release of glucose by the liver and the body not
catabolizing it due to a decreased metabolic rate. Peak
levels of hepatic glycogen are almost 8 times higher than
the lowest values. Even though a large increase in
carbohydrate storage occurs during the late fall, this
represents only a small fraction of the total reserve
energy. Using standard caloric values, Dessauer (1953)
calculated that the stored energy contributed by
carbohydrate reserves amounts to only about 3% of the
total storage. Similar results were reported for Lacerta
vivípara (Patterson et al. 1973). In these two species

19
the amount of stored glucose is too small to be a
significant source of energy during cold torpor; however,
tissue glycogen may act as an important source of
glucosyl residues (Patterson et al. 1978)* If so, tissue
glycogen would tena to reduce the catabolism of amino
acids which are the last resort to supply glucosyl residues.
Blood glucose continues to rise in tne spring, which
may be due to carbohydrate intake through feeding. In
amphibians no correlation exists between f'eeaing and blood
glucose concentration (Wright 1959)* However, in Eumeces
obsoletus blood glucose concentrations increase with an
increase in feeding (Miller and Wurster 1958). In
Ano 11s during the summer, when temperatures are highest
and presumably the animals are operating at their
eccritic temperatures, blooa glucose levels are
declining. One would expect that feeding during this time
would be as high or higher as during the spring, which
may be the case with female lizards, but not in males.
Ano 1 is is typical of other small iguania species
in its reproductive behavior. Males are territorial and
actively defend territories against other males (Greenberg
and Nobel 1944). Active defense in many cases may preclude
other activities, such as feeaing, and can be
metabo 1ica11y taxing (Pough and Andrews 1985). From late
winter through early autumn liver glycogen reserves are

20
low (Figure II-2). This period overlaps the reproductive
season of Ano 1is carolinensis (Hamlett 1952). The
increase in male-male aggression and territoriality
during the breeding season probably reflect the increase
in testoterone secretion by the gonads (Crews 1979)»
Androgen injections are known to produce typical sexual
behavior in castrated male anoles (Adkins and Schlesinger
1979, Mason and Adkins 1976).
Females are not territorial and any increase in
activity during the reproductive season may simply
reflect the higher ambient temperatures during this time of
the year. Because no differences were found between males
and females in their blood glucose and tissue glycogen
levels, something obviously reduces female liver
glycogen and blood glucose during the reproductive season.
The answer may also be attributable to hormones.
Ano 1is carolinensis has a very peculiar form of
ovulation, termed monoa11ochronic ovulation (Smith et al*
1973). Females ovulate a single egg alternately from each
ovary approximately every 14 days during the breeding
season (Hamlett 1952, Jones et al, 1963, Nobel and
Greenberg 1941). Each egg is oviposited 18 or 19 days
after ovulation (Hamlett 1952), Therefore a period of
4 or 5 days exists when one oviduct contains a mature
shelled egg while the other oviduct contains a recently

21
ovulated unshelled egg. During the reproductive
season plasma concentration of estradiol-17 beta (E-2) and
progesterone are high (Jones et al, 1983)* Marschall and Gist
(1973) reported reductions in both plasma glucose and liver
glycogen levels following estradiol and follicle stimulating
hormone (FSH) administration. They suggested that
mobilization of hepatic glycogen and blood glucose
utilization following hormonal treatments is due to the
metabolism of carbohydrates contributing part of the energy
required for the synthesis of yolk proteins.
Reducing blood glucose may also have secondary effects
on other forms of stored energy, such as lipids. In
mammals lipid degradation can result from hormone sensitive
lipases or may depend on the availability of glucose.
Glucose is required for the reesterification of free
fatty acids through the generation of glycerol-3-
phosphate. Therefore, if glucose availability to the
adipose cells is reduced, it results in the release of
free fatty acids (Marschall and Gist 1973). Estrogen
treatment results in fatty acid release in Uta
stans Duriana (Hahn and Tinkle 1 965 ). A mechanism for
triacylglycerol degradation involving plasma glucose
reduction in JL*. caro 1 inensis may occur. Such a
mechanism was suggested by Marschall and Gist (1973)
because even when presented with a glucose load, the
incorporation of labeled glucose carbon into fat bodies
was reduced during estrogen treatment.

CHAPTER III
ACTIVITIES OF REGULATORY METABOLIC ENZYMES
Introduction
Carbohydrates usually make up a large proportion of the
diet of most animals. Some of the carbohydrate intake
may be converted to, and metabolized as, fat» However
the major function of carbohydrates is a source of energy
through cellular oxidation. The major form of carbohydrate
for oxidation is the hexose glucose. Other principal
monosaccharides which may be utilized include fructose, the
major form found in fruits, and galactose, which is found in
dairy products» Fructose is only important if the diet
includes large amounts of sucrose, a disaccharide composed
of glucose and fructose. Both fructose and galactose can
be converted into glucose through hepatic mechanisms.
Carbohydrate metabolism can occur through several
pathways (Figure III-I), The major pathway during
activity in reptiles is the oxidation of glucose or
glycogen to lactate by theEmbden-Meyerhof pathway
(Figure III-2)» High levels of glycolytic enzymes and
regulation of those that are rate limiting are adaptations
22

23
GLUCOSE
x^OX ALO ACETATE
PHOSPHOENOLPYRUVATE
1
PYRUVATE
> LACTATE
ACETYL-Co A
FUMERATE
SUCCINYL-Co A<-
CITRATE
V
OC-KETOGLUTERATE
Figure III-1, Schematic of
catabolism. Double headed
utilizing the same enzyme,
pathways.
pathways of glucose and glycogen
arrows indicate reversible reactions
Multiple arrows indicate multiple

24
PHOSPHORYLATED HEXOSES
TRIOSE PHOSPhATE RUVATE
TRIOSE PhOSPHATE
V
LACTATE
Figure III-2. Simplified schematic of the Embden-Meyerhof
pathway.

25
allowing, for high rates of suDstrate passage through
glycolytic pathways. If anaerobiosis represents the major
pathway for energy production during activity, one would
expect to find high activities of anaerobic enzymes.
Enzymes examinee were hexokinase, phosphorylase,
lactate dehydrogenase and phosphofructokinase, key
regulatory catalysts in glycolysis. These four enzymes are
used to estimate the maximum rates of glycolysis in
muscle (Crabtree and Newsholme 1972). Phosphorylase Is
not an enzyme of the Embden-Meyerhof pathway proper
(Figure III-3). Phosphorylase is involved in the
degradation of glycogen through the mobilization of
glucose-1-phosphate, hexokinase controls the entry of free
glucose into the glycolytic pathway through the
phospnorylation of glucose. Unoer physiological conditions
this reaction can be considered to be irreversible
because of the considerable loss of free energy as heat.
Although lactate dehydrogenase is not a rate-1imiting
enzyme, high levels are usually associated with high
anaerobic scopes. These four enzymes are all found in the
soluble fraction (cytosol) of the cell.
The mitochondrial enzymes, nicotinamide adenine
dinucleotioe (NAD)-linkeo isocitrate dehydrogenase and
succinic dehydrogenase are both involved in aerobic
metabolism and the generation of electron transporting

26
PHOSPHORYLASE B
(active R form)
Á
AMP
GLti COSE b-PHOSPHATE
ATP
PHOSPHORYLASE B
(inactive T form)
(inactive T form)
PhOSPKORYLASE A
(active R form)
Figure II1-3* Control of the activity of phosphorylase in
hepatic and skeletal muscle tissue* The enzyme can change from
an inactive T (tense) form, to active R (relaxed) form. The
proportion of the active conformation is determined mainly by
the rates of phosphorylation and dephosphorylation. Adapted
from Stryer 1961»

27
molecules* Thus, both are linked to the proauction of ATP
via the Kreb's cycle and they are also key regulatory steps
in the Kreb's cycle.
Methods
Green anoles were obtained from a dealer in Louisiana
and held in captivity for 7 days in a thermal gradient
(22 - 50 °C) on a 12:12 light and dark cycle. The lizards
were fed crickets and mealworms daily. Five animals
were used to measure each enzyme activity. Animals were
fasted for 48 hours prior to Deing assayea. Lizards were
killed by freezing in liquid nitrogen. After freezing the
lizards were assayed immediately or storea frozen until
assayed at a later date. To assay, the entire liver was
removed along with the musculature of both fore ana hind
limbs. All the dissected tissue was weighed and placed in
an ice bath. All preparations were performed in an ice oath.
Muscle and liver samples were homogenized separately
in ice cola bufferea Ringer's solution (1 gram tissue
in 4 ml solution, Guillette 1 982) with a mortar and
pestle. Triton X-1G0 was added to give a final
concentration of 1$. Triton X-100 increases the soluble
hexokinase activity (Burleigh and Schimke 19b9). After
the Triton X-100 addition, the homogenate was incubated on
ice for 30 minutes followed by centrifugation at 17000
rpms at 4 °C.

28
Enzyme activities were measured according to the methods
outlined for each enzyme* Concentrations are expressed in
the total volume of the final reaction mixture.
All measurements were made with a model 6/20 Coleman Junior
2 spectrophotometer. Samples were kept at 33 0 C
(eccritic temperature) in a water bath and the optical
density measured every 60 seconds. Mean specific activities
are expressed in n-moles of product formed per minute per
milligram protein nitrogen* Protein was estimated by the
biuret method according to Gornall et al. (1949)*
Purified bovine serum aloumin solutions were used as
standards (Sigma Chemical Company)* All means are given
with their standard errors.
fle.SQMn.a.sg. I
The reaction mixture contained 0.76 m M N A D P , 3 ni H ATP,
7*5 mM magnesium chloride, 0*2 mM glucose, 1«5mM potassium
chloride, 2 IÜ g1ucose-6-phosphate dehydrogenase,
5 mM mercaptoethano1, and 50 mM sodium phosphate buffer
pH 7.4 (Baldwin ano Seymour 1977). The reaction was
started with the addition of homogenate* Enzyme activity
was measured by following the reduction of NADP at 340 nm.
Lactate Dehvdroaenase ( E . C* 1 * 1 . .1*27.)
The reaction mixture contained 33 mM phosphate buffer
pH 7*4, 0*067 mM NADh, 0*33 mM sodium pyruvate and
homogenate. The reaction was started Dy the addition of

29
pyruvate. The specific activity was measured from the
decrease in optical density at 340 nm.
£.j}Q.s_B.ii0.1 f.u.S t.P.k.1ng,g,e .( ñ . C , 2.7 , 1 , 1 1 )
The reaction mixture contained 5 mM magnesium
chloride, 200 mM potassium chloride, 50 mM Tris hCl pH 7.4,
0*1 mM N A D H , 1 mM ATP, 2 mM AMP, 0*3 mM sodium cyanide,
2 mM glucose-6-phosphate, 2 IU alpha-glycerophosphate
dehydrogenase and homogenate (Baldwin and Seymour 1977).
The reaction was started by the addition of glucose-6-
phosphate. The decrease in optical density at 340 nm was
used to calculate specific activity.
tqp-i;Uke.a Xg.os.Á.tfa.tg. p.g.hY.dr.pggnaag (¿. c. i. i * i .41)
The reaction mixture contained 20 mM phosphate
buffer pH 6.5, 1*7 mM magnesium chloride, 0*17 mM
manganese chloride, 0.50 mM buffered potassium cyanide,
pH 7*5, 0.030 mM dich1oropheno1 indophenol, 0.17 mM NAD,
6.7 mM sodium DL-isocitrate and homogenate (Bennett 1972).
The reaction was started by adding the isocitrate, and the
decrease in optical density at 600 nm was used to
calculate specific activity.
Succinic Dehydrogenase .(
The reaction mixture contained 40 mM phosphate
buffer, pH 7.5, 3*o mM buffered potassium cyanide, pH 7.5,
0.040 mM dichlorophenol indophenol, 0.13 mM phenazine

30
methosu1phate, 26 ah buffered sodium succinate, pH 7,5
and homogenate (Bennett 1972). Succinate was added to
start the reaction. Activity was determinea from the
decrease in optical density at 600 nm.
Results
The specific activities of hexokinase, phosphorylase,
lactate dehydrogenase, phosphofructokinase, NAD-linked
isocitrate dehydrogenase and succinic dehydrogenase
compared with those of mammalian values in Tables III-1
and III-2 (Bennett 1972, Lamb et al. 1969)»
Of the four skeletal muscle glycolytic enzyme
comparisons, only hexokinase showed greater activities in
the mammals* Lactate dehydrogenase, phosphory1 ase and
phosphofructokinase were greater in the reptile, However,
in comparing the activities of mitochondrial enzymes,
mammalian activities were greater. Reptilian hexokinase
and lactate dehydrogenase activities were greater in the
hepatic comparisons. Phosphofructokinase activities of the
reptile were lower than those of the mammals, botn liver
mitochondrial enzyme activities of the mammals were
higher than the reptilian values.

31
Table III-1. Comparisons of anaerobic ana aerobic enzyme
activities between skeletal muscle of mammals and green
anoles, Values taken from Craotree and hewsholme were
converted to nmoles/mg protein min assuming a ratio of 29:1
muscle tissue to muscle protein nitrogen and 10tí:1 liver
tissue to liver protein nitrogen. Assay temperature and
pH equals 33 ° C and 7.4 respectively.
MlSC-LE
Hexokinase
Lactate Dehydronase
Phosphofructokinase
Phosphorylase
Isocitrate Dehydrogenase
Succinic Dehydrogenase
LIZARD
65,5*
0.013
473GÜ**
74200
238**
1051
4 1 3 « b *
2522
25.6**
5.6
114**
22.4
* From Crabtree and Newsholm 1972.
** From Bennett 1972,
*** From Prichard and Schofield 1968.

32
Table III-2, Comparisons of anaerobic and aerobic enzyme
activities in liver tissue of mammals and green anoles.
Values taken from Crabtree ana hewsholme were converted to
nmoles/mg protein min assuming a 29:1 ratio of muscle tissue
to muscle protein nitrogen and a 108:1 ratio of liver tissue
to liver protein nitrogen, Assay temperature and pH
equals 33 0 C and 7.4 respectively.
um
LIZARDS
Hexokinase
1.2***
3.92
Lactate Dehydrogenase
18800**
27600
Phosphofructokinase
93**
56.6
Phosphorylase
1058.4
Isocitrate Dehydrogenase
87.8**
24.4
Succinic Dehydrogenase
261 **
73.6
* From Crabtree and Newsholme 1972.
** From Bennett 1972.
*** From Prichard and Schofield 1968

33
Discussion
The comparatively higher activities of glycolytic
enzymes in the lizard suggest a greater capacity for
anaerobic metabolism than in mammals* Likewise, the higher
activities of aerobic enzymes in the mammal reflect a
greater aerobic capacity. Utilizing the less efficient
glycolytic pathway for energy production has the
advantage of the rapid production of ATP* Other
ectothermic species examined reveal the same pattern
(Baldwin 1975, Baldwin and Seymour 1977, Baldwin et al.
1977, Bennett 1972, 1974, Crabtree and Newsholm 1972).
All glycolytic enzymes excluding hexokinase exhibit
greater activities in skeletal muscle than in the liver.
The greater activity is expected because of rapid depletion
of available oxygen in skeletal muscle during activity
and subsequent anaerobiosis*
Phosphorylase activity in skeletal muscle is
approximately 2.5 times higher than its activity in liver,
Phosphory1 ase in its active form, phosphory1ase-a (Figure
III-3), catalyzes the breakdown of glycogen at a very high
rate to glucose-1-phosphate. G1ucose-1-phosphate can then
be shuttled through glycolysis after isomerization into
glucose 6-phosphate. This mobilization of glucose-1 -
phosphate from glycogen is very important in the liver
and skeletal muscle. Under most physiological conditions

34
phosphorylase-b is inactive because of the inhibitory
effects of ATP and g1ucose-6-phosphate (Stryer 1961), The
greater activity of phosphory1 ase in skeletal muscle may
be due to the speed at which the end product is used.
Phosphorylase is the control point in an amplification
cascade in hepatic and extrahepatic tissues (Figure III-
4). In liver tissue the end result of this cascade is the
release of free glucose into the bloodstream* Skeletal
muscle lacks glucose-6-phosphatase ana cannot produce free
glucose. Thus all glycogen stored in skeletal muscle is
only used by muscle: skeletal muscle does not act as a
glycogen reserve for other tissues,
Several minutes may be required for glycogen
phosphory1 ase to reach peak activity in the liver
(Lehninger 19d2). Because of the inherent amplification
involved with each reaction step, hepatic phosphory1 ase may
possess lower activity because of the tremendous production
from each subsequent step in the cascade. The amplification
is estimated to be some 25 million fold, The cascade in
skeletal muscle is lower and, therefore, the amplification
is reauced» In addition, under certain conditions,
the energy required by the muscles must be instantaneous.
In situations where rapid escape behavior or prey ambush
is required, any delay in glucose utilization may be
deleterious

35
STIMULUS >ADRENAL MEDULLA
I
EPINEPHRINE
V
ADENYLATE CYCLASE
CELL MEMBRANE
ATP-
PROTEIN KINASE
(inactive)
-> CAMP + PPi
C a + +
PROTEIN KINASE + CAMP
(active)
ATP + DEPHOSPHO-PHOSPHORYLASE
KINASE Ca++
(inactive)
>PHOSPHO-PHOSPHORYLASE + ADP
KINASE
(active)
ATP + PHOSPHORYLASE B
(inactive)
PHOSPhORYLASE A + ADP
(active)
GLYCOGEN + Pi
-^GLUCOSE 1-PHOSPHATE
1
GLUCOSE 6-PHOSPHATE
I
GLUCOSE + Pi
CELL MEMBRANE
BLOOD GLUCOSE
Figure III-4. Epinephrine produced amplification cascade in
hepatic cells. Epinephrine arrives at the receptor sites on
the cell membrane at a concentration of approximately one nM.
The free glucose released into the blood increases its
concentration by 5 mM• The multiplier effect produced is
about 3 milllion times.

36
Low hexokinase activities in skeletal muscle may
reflect the inability of this tissue to use free glucose
from the blood as a readily available energy source. Low
hexokinase activities have been reported for other
poikilotherms (Baldwin and Seymour 1977, Baldwin et al.
1977, Bennett 1972, 1974, Crabtree and Newsholm 1972)»
Enzymes with low specific activities that form part of
the chain of a metabolic pathway usually catalyze
non-equilibrium reactions (hewsholme and Start 1973).
This is indicative of fewer active sites in the
enzyme molecule than is required to reach equilibrium*
The greater catalytic capacities of proceeding and
subsequent enzymes form substrates and remove products
faster than the non-equilibrium enzyme can inter-convert
them* Thus, enzymes that control non-equilibrium reactions
form a bottleneck in metabolic pathways, Hexokinase,
pnosphofructokinase and phosphory1 ase are prime
candidates for catalyzing non-equilibrium reactions,
By comparing mass-action ratios (t) with apparent
equilibrium constants (K') a reaction can be evaluated as
being no n-e q u i 1 i b r i um or not. If the value of t is much
smaller than tC', the reaction is displaced far from
equilibrium. Comparisons between t and K' in mammalian
tissues show hexokinase, phosphofructokinase and
phosphory1 ase all catalyze non-equilibrium reactions
(Newsholm and Start 1973)* Similar results were also
found in spiders (Prestwich 1962),

37
Mass action ratios were not measured here» However,
the similar enzyme activities found here and in studies
of other ectotherms, indicate that very few if any
differences in the glycolytic pathway exists (Baldwin and
Seymour 1977, Baldwin et a 1. 1977, Bennett 1972, hacleod
et al, 1963)» If no differences exist in the descriptive
aspects of the pathway, the same bottlenecks will occur at
the same locations. However, the higher activities of
certain key steps in poiki1otherms, enhance the output of
this pathway.
In emergency situations or during periods of fasting the
liver, mobilizing glycogen stores, releases free glucose
into the blood. Glucose molecules enter cells by means of
a symport (Figure III-5). Once inside the cell glucose is
phospnorylated by hexokinase at the expense of one ATP.
By phosphory1ating all the glucose that enters the cell,
a large glucose concentration gradient is maintained
between the blood and the intracellular compartment. Low
hexokinase activity reduces this potential gradient
througn the reduction of glucose-6-phosphate formation.
One potential solution to low hexokinase activity is
maintaining high blood glucose levels* High blood glucose
levels may serve to continously flood the intracellular
environment with glucose. Maintaining high levels of
glucose intracellularly may allow the low levels of
hexokinase to phosphory1 ate large numbers of glucose
molecules. This is discussed further in Chapter IV.

Figure III-5* The glucose sodium symport* The sodium graaient
produced powers the transport of glucose* Adapted from Stryer
1961*

Cell membrane
ATP
v
rU—* k +
Na+
^ADP+ P
Symport
Na +
^Glucose
^Glucose

40
Seasonal variation may be involved in enzyme activities,
as may the rate of metabolism* All enzyme assays were
performed on tissue samples from resting animals during late
summer when plasma glucose levels are declining and tissue
glycogen levels are low. During this time enzymes are
expected to be most effective. If true, muscle hexokinase
activities would be even more reduced during times when
glucose and glycogen levels are high* Ozand and Narahara
(1964) reported increases in the phosphofructokinase
reaction rate in contracting muscle. They suggested that
under conditions in which the phosphofructokinase of the
muscle is saturated with fructose-6-phosphate, an
increase in Vmax of the reaction may involved. If we
assume that regulatory enzyme function follows a
conservative pattern, hexokinase activity may also show
an increase with an increase in metabolism.
The nigher activities of the mitochondrial enzymes in
the mammal may be due to either or both of two reasons,
First it may be simply due to a more rapid conversion of
substrate with no difference in the concentration of
enzyme between the anole and the rat. However the amount
of enzyme present in the mammalian tissue may be greater
due to either a greater proportion of mitochondria,
greater relative membrane surface area, or both. In
comparing the activity of cytochrome oxidase in four

41
different tissues of a reptile ana a mammal, Else and
Hulbert (1981) reported higher activities in the latter in
all four tissues. They attributed this to greater
mitochondrial volume densities and mitichondrial membrane
surface areas in the mammal, A greater mitochondrial
volume density in mammals is probably related to the use
of fatty acids as a metabolic substrate (Bennett 1972,
Drummond 1971)» Because of the preferential use of glucose
as an energy substrate for activity in reptiles, fatty
acid metabolism is only utilized during specific times
such as fasting and reproduction (Dessauer 1955,
Smith 1968).

CHAPTER IV
THE EFFECTS OF ACTIVITY AND FASTING ON
TISSUE GLYCOGEN AND PLASMA GLUCOSE LEVELS
Introduction
Poikí1othermic ectotherms are generally regarded as low
energy systems» This is especially true when they are
comparea metabo 1ica1ly to endothermic homeotherms
(Bennett 1972)» Reptiles generally are not as active as
are mammals or birds. The basis for this less active
lifestyle is the preferential use of anaerooic,
glycolytic pathways instead of aerobiosis during intense
activity» More than 50% of the total energy produced from
activity is derived from anaerooiosis (Bennett and Licht
1972, Gleeson 1982, Gratz and Hutchison 1977, Pough and
Andrews 1 983)* Although glycolysis is less efficient than
aerobiosis (i»e«, Kreo's cycle), the former produces ATP's
at a much faster rate.
Reliance on glycolysis for energy production suggests
that glucose is a limiting factor in metabolism during
activity in poikilotherms. Muscle glycogen depletion
occurs in most vertebrates after intense activity (Dean
and Goodnight 1964, Gaesser and Brooks I960, Gleeson
42

43
1582, Gratz and Hutchison 1977, Johnston and Goldspink
1973» MacDougall et al* 1977, Piehl 1974), During the
recovery period following depletion of muscle glycogen,
small reptiles are particularly susceptible to predation.
Their susceptibility increases with the time course of
glycogen replenishment.
This chapter examines the effects of burst activity on
tissue glycogen, blood glucose, lactate production and the
effects of fasting on carbohydrate utilization before,
during and after exercise. Additionally the
relationship between carbohydrate loading ana glycogen
replacement is also evaluated.
Glucose and glycogen analyses were performed during
July and August 1986, as described in Chapter II, Animals
were exercised in a water-driven treadmill at their
eccritic temperature (33 0 C), until exhausted.
Exhaustion is defined as loss of the righting response.
Immediately upon exhaustion the animals were either
dropped into liquid nitrogen and assayed for tissue
glycogen and lactate or decapitated and the cilood assayed
for glucose or lactate. Glucose and glycogen
concentrations were measured at rest, 3U seconds after
the onset of exercise, at the point of exhaustion and at

44
various times up to 720 minutes during recovery* Recovery
here and in all subsequent assays is defined as the time
from the end of exercise to when pre-exercise (resting)
levels of the parameter in question are reached. Plasma
glucose is expressed as mg/dl. Tissue glycogen is
expressed as mg glycogen/g of tissue. All animals were
maintained in a photothermal gradient (22 °C to 50 °C,
12:12 light:dark cycle). All means are expressed with
their standard errors.
Lactate ft.n.a.ly.si.g.
Ten groups of five animals each were analyzed at rest,
after exhaustion, or 10, 60 or 120 minutes during the
recovery period. One half of the groups were usea to analyze
whole body lactate concentrations. Lizards were
decapitated and dropped into liquid nitrogen. After
freezing, the bodies were homogenized in volumes of cold
0.6 h perchloric acid equivalent to 12 times body mass.
To study compartment!1ization of lactate, samples of
blood, liver and leg muscle from the animals of the other
five groups were weighed, pooled in cold perchloric acid
(5 times the sample weight) and homogenized* All
homogenates were centrifuged at 3000 rpms at 4 °C for 10
minutes. The supernatant was filtered and recentrifugea
at 10000 rpms at 4 °C for 10 minutes. The resulting
supernatant was assayed with an enzymatic test kit (Sigma

45
Chemical Company)* Samples were read on a model 6/20
Coleman Junior 2 spectrophotometer at 340 nm. Lactate
concentrations are expressed as mg lactate/g tissue
weight*
Q.xy.fig.p, G.Q.nsvupp.tJ-pn
Oxygen consumption (Vq2) was measured manometrically
using a Gilson Differential Respirometer during July and
August 19Ó6* Lizards (n = 5) were placed in 125 ml
respirometer flasks. Each flask contained approximately
12 grams of soda lime to absorb CO^. All determinations
were made at 33 °C (i.e., eccritic temperature). Resting
rates were measured at 10:00 p»m» E*S.T*
After several hours of acclimation in the flasks Vq2's
were measured, To measure oxygen consumption after
exhaustion and during recovery, animals were exercised at
9:00 p.m. in the respirometry flasks by the motorized
shaking of the flasks. To increase the activity of the
lizards in the flasks, small glass spheres were added,
The movement of the spheres agitated the lizards thereby
causing activity. During the recovery period rates were
measured every 2 minutes. Rates are expressed as
ml G 2 /g h.

46
Carbohydrate Loading
The experiments were performed in August, the time when
body sugar levels are lowest» The role of these experiments
was to explore the relationship between glucose
supplementation and glycogen resynthesis» Lizards were fasted
for 48 hours (n = 120). After exercising to exhaustion,
half the lizards were fed a glucose solution, 20 ul/g body
weight at a concentration of 1 mg/4 ul by stomach tube
and allowed to recover for 12 hours. This volume and
concentration is equivalent to 5 mg glucose per g body
weight. The other lizards were given an equivalent volume
of distilled water via a stomach tube. After 12 hours one
half of the glucose supplemented lizards were assayed for
tissue glycogen as previously described. One half of the
control group were assayed for tissue glycogen. The other
glucose supplemented lizards were given a second glucose
feeding and were assayed 12 and 24 hours later. The rest
of the control lizards were also assayed at these times.
These experiments examined the effect of fasting on
glucose concentrations at rest, during exercise and
during recovery. Lizards fasted for 2 weeks during August.
After fasting the animals were exercised as previously
described until exhaustion. Glucose and glycogen levels
were measured at the same intervals as previously
described

47
Results
Oxygen consumption and whole body lactate at rest,
during exercise and recovery are summarized in Figure 1V-1.
9
Maximum Vq2 occurred during exercise
( 1.65 + 0*20 1 ml/g h). With a resting VQ2 of 0.262 +
0.036 ml/g h, the aerobic scope equals 6.3. Oxygen
consumption declined steadily throughout the recovery
period. At 75 minutes of recovery no statistically
significant difference existed in the recovery and
resting rates of oxygen consumption (P > 0*05, T-test
for paired data). TaDle IV-1 summarizes the net recovery
oxygen consumption and the change in lactate
concentration during recovery.
Whole body lactate increased 6.4 times from resting
values, 0.29 ±. 0.01 mg/g, to exercise values
2*43 ± 0.21 mg/g* Over the first 10 minutes of
recovery lactate declined very little (2.36 + 0.26 mg/g).
Sixty minutes into recovery, lactate had dropped
drastically (0.68 +. 0.14 mg/g). By 120 minutes the
lactate levels were not statistically different from those
of resting values (P > 0.05, paired T-test).
Figure IV-2 summarizes the data for lactate
compartmenta1ization. All compartments show the same trend.
Maximal lactate concentrations occur during exercise. Muscle
and blood lactate levels after 2 hours of recovery were

Figure 1V-1* Lactate concentration and oxygen comsumption
before, during and after exercise. Circles = oxygen
consumption» Triangles = lactate. Each symbol represents
the mean of 5 lizards.

TIME (minutes)
LACTATE CONCENTRATION (mg/g)
6t7

50
Table IV-1. Resting ana post exercise lactate and glycogen
levels» Units are mg lactate/glycogen per g tissue»
E.AS.XIM.
Muscle
REST.
g.aaxEm£i&E
EES.X
mXE&EEC.I£E
Lactate
0.62
2.53
—
—
Glycogen
4.1
1 .6
3.9
1 .9
Liver
Lactate
0.15
1 .44
—
—
Glycogen
7.1
3.9
5.4
2.3

Figure IV-2. The compartraenta1ization of lactate befor
during and after exercise. Squares = plasma lactate.
Triangles = skeletal muscle lactate. Circles = liver
lactate. Each symbol represents the mean of 5 lizards.

TIME (minutes)
CONCENTRATION (mg/g)
29

53
statistically indistinguishable from resting values
(P > 0*05, T-test). Liver lactate levels during recovery
were the same as resting levels by 2*5 hours. Resting
muscle lactate levels were significantly greater than
those of the liver and blood (P > 0.05, T-test). Resting
liver and blood lactate levels are very similar
(0.15 mg/g and 0.12 mg/g respectively).
Muscle glycogen and lactate levels show the expected
trends (Figure IV-3). As exercise proceeds glycogen levels
drop and lactate concentrations increase. As activity
ceases lactate begins to decrease with a concomitant
increase in muscle glycogen. At 120 minutes into recovery
lactate levels are not statistically different from resting
levels (0.59 ± 0.07 mg/g and 0.62 ± 0.05 mg/g,
respectively, P > 0.05, T-test). Muscle glycogen returns
to resting levels 6 h after the cessation of activity
(4.3 +. 0.17 mg/g ana 4.1 ± 0.33 mg/g, respectively,
P > 0.05, T-test).
Liver glycogen and lactate concentrations also show
opposite changes (Figure IV-4). Liver glycogen levels do
not drop significantly until the end of exercise, ho
significant change in liver glycogen occurs after one
minute into exercise (7.1 ± 0.83 mg/g and 7.0 ± 0.67 mg/g
respectively, P > 0.05, T-test). At the end of activity,
the glycogen level drops to 3.9 +. 0.44 mg/g. Liver lactate
increased from a resting level of 0.15 ±. 0.02 mg/g to

Figure IV-3» Muscle glycogen and lactate before, during and
after exercise. Circles = glycogen. Triangles = lactate.
Each symbol represents the mean of 5 lizards. The vertical
bars are one standard error.

LACTATE CONCENTRATION (mg/g)
h5
TIME (minutes)
GLYCOGEN CONCENTRATION (mg/g)

Figure 1V-4. Liver lactate and glycogen before, during and
after exercise. Triangles = lactate. Circles = glycogen.
Each symbol represents the mean of 5 lizards.

LACTATE CONCENTRATION (mg/g
-8.0
IME (minutes)
T
cn
GLYCOGEN CONCENTRATION (mg/g)

58
1.44 +. 0.11 mg/g at the end of exercise. Lactate levels
continue to rise after exercise reaching a peak value of
1.47 +. 0*14 mg/g after 10 minutes of recovery. Lactate
levels then declined to 0*78 +. 0.03 mg/g after 60 minutes
of recovery. After 2.5 hours of recovery lactate levels
returned to resting levels (0.13 +. 0*008 mg/g and
0*15 +. 0.02 mg/g respectively, P > 0.05, T-test).
Resting and peak lactate and glycogen concentrations in
muscle and liver with time to recovery are summarized in
Table IV-2.
Plasma glucose concentrations before, during and after
exercise are summarized in Figure IV-5. Plasma glucose in
the post-exercise period reached higher levels than
pre-exercise levels. During exercise plasma glucose
values fell from 179*6 ± 14.3 mg/dl to 149.6 ± 12.3
mg/dl« The plasma glucose level reaches its maximum
concentration, 239*4 +. 12.1 mg/dl two hours into recovery.
This level remains fairly constant for the next three
hours. After 12 hours, plasma glucose levels decline to
134*1 ± 6.7 mg/dl,
Carbohydrate loading during recovery produced different
results for muscle and liver glycogen replenishment. The
initial glucose supplement had no effect on muscle
glycogen replenishment (Figure IV-6). After 12 hours control
and experimental values were not different (4.5 +. 0.07 mg/g
and 4.6 + 0.04 mg/g, P > 0.05, T-test). Compensation

59
Table IV-2* Oxygen consumption requirements for lactate
oxidation and gluconeogenesis from lactate* Numbers are based
on data tasen from Figure IV-2.
TIME
(MIN )
LACTATE NET
(mm) OXYGEN
RECOVERY
CONSUMPTION (ml)
LACTATE TO
GLUCOSE b-P
LACTATE
OXIDATION
0-4
0.072
0*098
7.6
X
10 "7
4.838
4-1 5
0.070
0.074
7.4
X
10 -7
4.704
15-45
0.020
0.029
r\)
#
o
X
10 "7
1.344
45-75
0.019
0.010
2.0
X
10 -7
1 .277
75-120
0.009
0.002
9.5
X
10 "9
0.605

Figure IV-5* Plasma glucose concentration before, during and
after exercise. Triangles = fasting animals. Squares =
nonfasting animals. Each symbol represents the mean of 5
lizards. The vertical bars are one standard error.

250
200
150
100
50
R Ex
60
r~
120
300
TIME (minutes)
720
cr>

Figure IV-6. Effects of post exercise carbohydrate loading on
muscle glyocgen. Squares = controls» Triangles = animals
receiving glucose load. N = 120. The vertical bars are
one standard error. Glucose is given at the end of
exercise.

TIME (hours)
MUSCLE GLYCOGEN (mg/g)
£9

64
occurred only in muscle after the second glucose feeding*
After the second feeding muscle glycogen levels increased
almost 2 times (4*6 ± 0*04 mg/g to b.4 +. 0*23 mg/g*
Overcompensation occurred after both glucose feedings in
liver tissue (Figure IV-7)% Liver glycogen had increased
10 times over resting levels after the second feeding
(7.0 +. 0.66 mg/g to 70.1 +. 4.08 mg/g).
Plasma glucose is affected differently during exercise
in fasting and nonfasting animals (Figure IV-5). Plasma
glucose of fasting lizards increased from
149.6 ± 13.1 mg/dl to 177.1 ± 16.1 mg/dl. Glucose levels
declined rapidly, and after 60 minutes of recovery, plasma
levels were equal to resting levels (151*2 ± 10.3 mg/dl
and 149.6 +. 13*1 mg/dl respectively, P > 0.05, T-test).
Plasma levels then dropped and remained slightly below
resting levels.
Resting tissue glycogen concentrations are lower in
fasting animals than nonfasting animals (Figure IV-8),
The same patterns during and after activity are
produced among fasted lizards as seen among nonfasted
individuals (Figures IV-3, IV-4). however glycogen levels
are generally depressed among fasted animals. Peak
resting and recovered muscle and liver glycogen values
are summarized in Table IV-1.

Figure IV - 7 • Effects of post exercise carbohydrate loading on
liver glycogen. Squares = controls* Triangles = animals
receiving glucose load* N = 120. The vertical bars are
one standard error* Glucose Is given at the end of
exercise.

80
70
60
50
40
30
20
10
12
TIME (hours)
TEST
R Ex
T~
24
36
cr>
os

Figure IV-b. Tissue glycogen before, during ana after
exercise in fasted lizards. Squares = muscle glycogen.
Triangles = liver glycogen. Each symbol represents the
mean of 5 lizards. The vertical bars are one standard
error

TIME (minutes)
cr>
Co

69
Discussion
A characteristic prolonged elevation of oxygen
consumption occurs after exercise in vertebrates.
Hill et al* (1924) originally termed this the oxygen
debt. This elevated oxygen consumption was interpreted as
the oxidation of lactic acid produced as the result of
tissue glycogen catabolism, The original hypothesis
stated that only a fraction of the lactate, approximately
20%, was actually oxidized. The lactate oxidized provided
the energy required to resynthesize glycogen from the
remaining lactate. Results from several studies have
questioned the validity of this hypothesis (Bennett and
Licht 1973, Brooks et al, 1971, 1973, Gleeson 1980a, 19&0b,
Gratz and Hutchison 1977, Hutchison et al* 1977)* Results
of this study also appear to refute the classic oxygen
debt hypothesis*
Seventy-five minutes into the post-exercise recovery
period oxygen consumption has returned to resting values
(Figure IV-1). Post exercise lactate levels require two
hours to return to resting levels* These data show that
net lactate removal is still occurring after oxygen
consumption has returned to normal, however, muscle and
liver glycogen levels return to pre-exercise levels
without any carbohydrate intake* The time course for this
tissue glycogen replenishment is several hours (Figures
IV-3 , IV -4 )•

70
Lactate removal appears, in Figure IV — 1, not to be the
result of elevated post-exercise oxygen consumption
(EPOC), however, the temporal uncoupling between lactate
removal and EPOC is misleading. The metabolic pathway(s)
used for lactate removal cannot be determined without
using radio-1 a be 1ed compounds. The fate of lactate can be
suggested by calculating the amount of oxygen required to
resynthesize glucose from lactate (i.e«, g1uconeogenesis),
or to completely oxidize lactate to carbon dioxide and
water (Appendix), and by comparing these estimates with
those actually measured. Table IV-2 is such a summary.
Obviously the increase in oxygen consumption during
recovery cannot supply the energy required to completely
oxidize lactate (Table IV-2). The net recovery oxygen
consumption only accounts for approximately 0»3 - of
the energy required for complete lactate oxidation.
However, the net recovery oxygen consumptionis more than
adequate for the resynthesis of glucose from lactate.
These comparisons suggest that most if not all of the
lactate produced during forced activity is reconverted
into glycogen via g1uconeogenesis. This differs from the
situation in mammals, in which the primary fate of
lactate is oxidation (Brooks et al* 1 9 7 3» Drury and Vvick
1956, Gaesser and Brooks 1980). The production of
glycogen from lactate appears to occur in other reptiles
(Gleeson 1985, Moberly 1968). Gleeson (1985), using

71
Table IV-2. Oxygen consumption requirements for lactate
oxidation and gluconeogenesis from lactate. Numbers are based
on data taken from Figure IV-2.
Sim REQUIRED (ml)
TIME
(MIN )
LACTATE NET
(mM) OXYGEN
RECOVERY
CONSUMPTION (ml)
LACTATE TO
GLUCOSE 6-P
LACTATE
OXIDATION
0-4
0.072
0.098
7.6
X
10 "7
4.838
4-15
0.070
0.074
7.4
X
10 “7
4.704
15-45
0.020
0.029
2.0
X
10 "7
1.344
45-75
0.019
0.010
2.0
X
10 "7
1.277
75-120
0.009
0.002
9.5
X
10 "9
0.605

72
inferential data reported seasonal variation in the
ability of red muscle to produce glycogen from lactate,
He suggested that muscle gluconeogenesis may account for
approximately 20% of the lactate removed during recovery
from exhaustive activity, because many reptiles rely on
burst activity in many of their behaviors, utilizing
the gluconeogenic pathway for lactate removal allows them
to resynthesize the much needed substrate for its reuse.
The compartmentalization of lactate also suggests the
possibility of a gluconeogenic fate for lactate
(Figure IV-2). Liver lactate levels during recovery
remain elevated after blood and muscle lactate levels
have returned to pre-activity levels, which suggests that
the liver may be responsible for the reduction of lactate
levels. The liver has been shown to possess relatively
high specific activities of lactate dehydrogenase
(Chapter III), an enzyme that is required to convert
lactate back into pyruvate before it is metabolized. This
conversion is necessary to regenerate NAD+ so the
glycolytic pathway remains active as long as possible. As
such the metabolic burden of energy production does not
fall on the skeletal muscle alone. Part is shifted from
muscle to the liver (Stryer 19 6 T)*
The potential for the liver as the primary site of
gluconeogenesis is strengthened when one examines the
plasma glucose levels during recovery (Figure IV-5).

73
Plasma glucose levels remain elevated for at least 5
hours following forced activity, which may be due to the
reconversion of lactate to glucose by the liver, After
the conversion of lactate to glucose by the liver, free
glucose enters the bloodstream from which it is stored by
various tissues as glycogen. Glycogen then serves as the
primary substrate for catabolic energy production.
Plasma glucose levels are probably not rapidly reduced
during activity due to the low hexokinase activity
(Chapter III), A high concentration would then be
required to establish a suitable concentration
gradient between the plasma and intracellular
compartments. Such a gradient permits the passage of
glucose into the cell. Once glucose moves inside, the
gradient prevents the leakage of glucose to the outside
until it can be phosphory1ated by hexokinase and acted
upon by various other enzymes for storage as glycogen.
The liver glycogen level requires more time to return
to resting levels after exercise than is required for
repletion of muscle glycogen (Figures IV-3, IV-4),
suggesting that the primary course of glycogen
replenishment is first in muscle and then in the liver,
this sequence is logical because muscle glycogen is utilized
first during episodes of intense activity. The glycogen
stored in the muscles is for use by muscles only,

74
whereas the liver acts as a storage site for glycogen to
be shuttled to extrahepatic tissues at the onset of
activity*
During intense activity the liver apparently releases
free glucose into the bloodstream. However, the total
amount of glycogen stored in muscles limits the amount of
anaerobically produced work that occurs during intense
activity. If an anaerobically dependent animal depletes
part of its glycogen store through activity or fasting,
it then reduces the amount of work that it can perform*
The same pattern of tissue glycogen utilization occurs
during activity and recovery in fed and fasted Ano 1 is
(Figure IV-8). Fasted lizards, however, exhausted much
faster than those that were fed. During activity, plasma
glucose levels increase and then decline (Figure IV-5), a
pattern during periods of glycogen depletion that
produces a concentration gradient for glucose uptake by
skeletal muscles. Muscle glycogen stores are not replaced
more quickly in fasting versus nonfasting lizards (Figure
IV - b ) * However the establishment of a plasma
intracellular glucose gradient may allow for anaerobiosis
to occur after a shorter recovery period (i*e«, the free
glucose entering the cell is used immediately for
glycolysis ) .

75
Otner studies have shown that repetitive bouts of
activity result in decreased times to exhaustion
(Putnam 1979a( 1979b)* Decreased glycogen stores are part
of the reason for this reduction in performance. Lactate
buildup would play a major role in performance reduction
if lactate levels were still elevated. Ano 1 is possesses
the ability to quickly replace reduced muscle glycogen
stores compared to mammals (Conlee et al, 1978). This
ability may involve a mechanism that minimizes any
potential reduction of activity resulting from fatigue
or fasting. Muscle glycogen repletion appears to occur
without the need for dietary carbohydrate intake.
A large part (approximately 6 0 %) of an oral load of
glucose is taken up preferentially by the liver in resting
humans (Maehlum et al, 1977). Less than 15% ends up as
muscle glycogen. However during post exercise recovery,
a greater percentage of splanchnic glucose ends up as
muscle glycogen. Muscle glycogen repletion has a higher
priority over hepatic retention during the recovery period.
The picture is different during recovery in Ano 1 is.
Carbohydrate loading appears to increase glycogenesis. The
ingestion of glucose after exercise causes an increase in
hepatic glycogen. An oral glucose load has no initial
effect on muscle glycogen. Skeletal muscle only shows an
increase in storage after a second glucose feeding. This
preferential overcompensation effect in liver tissue

76
suggests that muscle cells are storing glycogen at or near
their maximum rate and further strengthens the
conclusion that these animals rely heavily on
stored glycogen in muscle for activity metabolism.
Muscle tissue increases its storage of glycogen
apparently only after saturating the hepatic glycogen
storage rate. Whether this increase in storage is temporary
is not known» If so, the only way the glycogen
concentration can be reduced is through the catabolic
activities of the muscle» Muscle cells do not possess
glucose-6-phosphatase. Therefore, muscle glycogen cannot
be utilized as a precursor to fat storage and can only be
used by the muscle.
The storage of glycogen increases the time a lizard
may be engaged in vigorous activity, but it will occur
only if the lactate produced is not the maximum amount
that can be tolerated. If not, the upper limit for lactate
tolerance would prevent any increase in the time a lizard
may engage in intense activity, regardless of the amount
of glycogen stored.
The cost of using anaerobiosis coupled to
gluconeogenesis is unfavorable. Consider that three high
energy phosphate bonds are produced per glucose molecule
utilized and derived from tissue glycogen. Six high energy
phosphate bonds are used in the reconversion of glucose
from pyruvate. Consequently, a net loss of three high energy

77
phosphate bonds results from coupling glycolysis with
gluconeogensis. The loss in bond energy is equivalent to
21,9 kcal/mole of phosphate bond hydrolyzed»
The evolutionary benefits derived from an anaerobic
system outweigh its costs* The metabolic machinery for
aerobic metabolism is present although its performance is
somewhat reduced (Chapter III)» By utilizing glycolysis
for the production of energy during activity Ano 1 is is
limited in the length of time it may engage in burst
activity. Because of this limitation a large amount of
stored energy is not required to engage in short term
bouts of intense activity.

CHAPTER V
GLUCOSE TOLERANCE AND HORMONAL INFLUENCES
IN GLUCOSE REGULATION
Introduction
The effects of insulin and glucagon have been examined
in every order and suborder of reptiles except for
rhyncocephalians (Penhos and Ramey 1973)* The physiological
effects are variaDle. Insulin is least effective in the
lizards examined, and shows the most markea effect in
alligators. Resting levels of plasma glucagon have not
been determined in ectothermic vertebrates, however,
injected glucagon raises the blood sugar level in all
reptile species examined.
Glucose tolerance tests allow the effects of an added
glucose load on body sugar levels to be evaluated. The
usual procedure involves the intraperitoneal injection of
a glucose solution and subsequent monitoring of blood
glucose and/or tissue glycogen levels over time.
Alligators show the greatest deviations from glucose
homeostasis. This study is an attempt to characterize
glucose homeostatis in green anoles and compare them to
other species previously examined. Glucose tolerance,
insulin and glucagon responses were used as criteria
for this characterization,
78

79
Methods
G-iü.g.P.gg, 1 ole range
Gastric glucose tolerance was examined during August,
Large adult lizards (S-V length ¿ 600 mm), were
purchased from an animal dealer. The lizards fasted for
two days prior to testing (n = 70), Glucose was
administered by gastric intubation at a concentration of
2 and 5 mg/g body weight. Plasma glucose and tissue
glycogen were analyzed as described in Chapter II. Sugar
concentrations were measured hourly for 10 hours, then
24, 4b and 72 hours after intubation. Plasma glucose is
expressed as mg/dl +. 1 S.E,M, Skeletal muscle and liver
glycogen are expressed as mg glycogen/g tissue ± 1 S»E,M,
Insulin
Mammalian insulin was obtained as a zinc salt from
ICN Biomedical Inc, The insulin was injected
intraperitoneally at concentrations of 1000 units/kg and
2000 units/kg. Animals were assayed for plasma glucose,
muscle and liver glycogen at 10, 20, 30, 60, 90, 120, 300
minutes and 24 hours after the injection (n = 108),
The results are expressed as previously described.

80
Glucagon
Mammalian glucagon was obtained from ICN Biomedical Inc.
The hormone was injected intraperitoneally at
concentrations of 10 ug/kg and 100 ug/kg (n = "108).
Animals were assayed at the same intervals as in the
insulin experiment, Results are expressed as previously
described.
hesults
Glucose Tolerance
Results are summarized in Figures V-1 ana V-2. Plasma
glucose levels peaked between 2 and 3 hours after
ingestion (492 +. mg/dl). The plasma glucose levels
were back to resting levels after 48 hours
(135 +. 4.7 mg/dl). Muscle glycogen showed little effect
of added glucose. Liver glycogen levels peaked between 10
and 24 hours (52.3 ± 3.33 mg/g).
Ins.y.i-in.
Insulin responses are summarized in Figures V-3 and V-4.
In both cases there is a rapid onset of hypoglycemia. Plasma
glucose levels dropped from 132 +. 2*1 mg/dl to
99 ± 1.4 mg/dl and 134 +. 1.7 mg/dl for the low and high
insulin concentrations respectively. Within 60 minutes
after the onset of hypoglycemia plasma glucose levels
returned to normal values. At 90 minutes after
administering insulin, the lizards showed a plasma

Figure V-1. Glucose tolerance for plasma glucose. Each
symbol represents the mean of five animals.

PLASMA GLUCOSE (mg/dl)
500
400
300
200
100
1
1—i—i—i—i—i—i i i i r
0 1 5 10
TIME (hours)
24 48 72
00
inj

Figure V-2. Glucose tolerance for muscle and liver glycogen.
Triangles = muscle glycogen. Circles = liver glycogen.
Each symbol represents the mean of five animals.

TIME (hours)
GLYCOGEN CONCENTRATION (mg/g)
t/8

Figure V-3. The effect of insulin on plasma glucose, muscle,
and liver glycogen» Insulin concentration = 1000 units/kg,
Squares = plasma glucose» Triangles = muscle glycogen»
Circles = liver glycogen» Insulin was injected at time
zero*

GLYCOGEN CONCENTRATION (mg/g)
0 10 20
60 90 120 300
TIME (minutes//hours)
24
00
cn
PLASMA GLUCOSE (mg/dl)

Figure V -4. The effects of insulin on plasma glucose,
and liver glycogen» Insulin = 2000 units/kg# Squares
glucose. Triangles = muscle glycogen. Circles = liver
glycogen. Insulin was injected at time zero.
muscle,
p1 asma

TIME (minutes //hours)
CO
00
PLASMA GLUCOSE (mg/di)

89
compensatory hyperglycemia which ended at 120 minutes.
Muscle glycogen levels showed a rapid increase after the
insulin injection. Within 10-20 minutes glycogen
reached peak levels. Liver glycogen concentrations show
the same trend, a rapid increase in stored glycogen
followed by the maintenance of a relatively constant level,
both dosage groups produced the same results.
G.ims.agpp.
Glucagon responses are summarized in Figures V-5 ana
V-6. Animals injected with the higher concentration
(100 ug/kg) showed the greatest effect. Plasma glucose
concentration increased rapidly in both groups In the
lower concentration group (10 ug/kg), this
hyperglycemia continued for 6 hours. After 24 hours
plasma glucose levels returned to pre-injection levels.
In the 100 ug/kg group plasma glucose levels remained
elevated for 46 hours. After 72 hours plasma glucose levels
returned to normal. No effect of glucagon on muscle
glycogen levels appeared in either group. In the liver
stored glycogen levels decreased. In the 10 ug/kg group
this reduction continued for 5 hours* Liver glycogen
returned to resting levels after 24 hours (Figure V-5).
In the higher concentration group, the reduction in liver
glycogen continued for 24 hours. After 72 hours the
glycogen levels returnea to normal (Figure V-6).

Figure V-5. The effects of glucagon on plasma glucose, and
muscle and liver glycogen. Glucagon concentration = 10 ug/kg.
Squares = plasma glucose. Triangles = muscle glycogen.
Circles = liver glycogen. Glucagon was injected at time
zero •

GLYCOGEN CONCENTRATION (mg/g)
9
1—i—rn 1 1 i 1 “r-
O 10 30 60 90 120 300 24
TIME (minutes//hours)
PLASMA GLUCOSE (mg/g)

Figure V-6. The effects of glucagon on plasma glucose and
muscle and liver glycogen» Glucagon concentration = 100
ug/kg. Squares = plasma glucose» Triangles = muscle glycogen.
Circles = liver glycogen» Glucagon was injected at time
zero

GLYCOGEN CONCENTRATION (mg/g)
9
TIME (minutes-hours)
CjO
PLASMA GLUCOSE (mg/dl)

94
Discussion
Reptiles have a lower glucose turnover rate than do
amphioians (Coulson and hernandez 1953, 1964, houssay and
Penhos 1960, Penhos and Ramey 1973, Wright 1959, Miller
ana burster 1956) probably because amphibians are more
sensitive to insulin (Penhos and Ramey 1973)» Andes
appear to be similar to other reptiles. The relatively
slow decline of plasma glucose after glucose loading may
reflect a low hexokinase activity. However, other factors
may be important including a low sensitivity to insulin.
Insulin does not appear to have a very significant
effect in lowering plasma glucose. In fact, lizards as a
group appear to be the least insu1in-sens itive of all
reptiles examined (Penhos and Ramey 1 973)* Andes require
a relatively high aosage of insulin to show the minimal
signs of an insulin effect. This was also reported in
other lizards (Miller and burster 1956). One interesting
note is that although 1000 units/kg produced slight
effects, 2000 units/kg caused typical insulin overdose
symptoms, including trembling of the entire soma, muscle
spasms and loss of muscle coordination.
Anoles have either an inherently low insulin production
or they are not particularly sensitive to bovine insulin.
The available literature has conflicting reports. Miller
and burster (1956) reported that pancreatic beta cells
are the site of insulin production in the lizard

95
Eumeces obsoletus» These animals like Jinolis. were very
glucagon sensitive, Pancreatectomized lizards become
severely hypoglycemic, which suggests that lizards are
very similar to birds in their dependence on glucagon
for maintaining plasma glucose homeostasis.
Miller and burster (1956) reported beta cell destruction
and a concomitant increase in blood glucose after alloxan
treatment, suggesting that insulin production by these
cells help to maintain blood glucose homeostasis. They
reported earlier (1956) that glucose loading resulted in
beta cell activation. However the hyperglycemia contined
for at least 5 hours. They did not indicate how long it
took for resting blood glucose levels to be achieved.
Here it took 46 hours for resting blood glucose levels to
be reached. If we assume the same or a similar situation
metabolically, it is obvious that insulin sensitivity or
proauction is low in lizards.
Consequently the combination of low hexokinase
activity, low insulin production and low insulin
sensitivity, a large glucose load cannot be efficiently
handled. Confirmation must be made using radioimmunoassay,
Ozand and Narahara (1964) reported that insulin can
increase the rate of glycolysis in the grass frog
Rana pjpjens. even in the absence of extracellular
glucose. In addition to increasing cell membrane
permeability, they suggested that insulin enhances the

96
activity of phosphofructokinase. If insulin can increase
the rate of glycolysis, either directly or indirectly,
then the low insulin production that occurs may be
produced mainly for enhancement of the glycolytic
pathway. I suggested in Chapter III that the regulation of
high plasma glucose levels may be an adaptation allowing
for the maintenance of a large blood-cell concentration
gradient. In Ano 1is insulin may mainly increase
glycolysis, out it will also aid the passage of glucose
into the cells, regardless of how little insulin is produced.
neptiles are very glucagon sensitive. After glucagon
injections reptiles respond with hyperglycemia that may last
from 1 to 1o days. Houssay and penhos (I960) reported blood
sugar levels increasing over 7 times resting levels after
glucagon injections in a snake. A high sensitivity to
glucagon may be an adaptation that allows lizards such as
Ano 1is to maintain fairly high blood glucose levels.
This view is supported by a histological examination of the
lizard pancreas: lizards have a large proportion of alpna
cells (Miller and burster 1956). Houssay and Penhos
(1960) also reported that completely pancreatectomized
lizards become severly hypoglycemic and hypothesized
that after an insulin injection, pancreatectomized
lizards should become hypoglycemic. Actually, such
lizards showed an increase in blood glucose, which may

97
have been due to the use of an insulin preparation
contaminated with glucagon. Nevertheless, glucagon has a
much greater effect in lizards than does insulin.
The green anoles's reliance on burst anaerobic
activity coupled with their inability to utilize glucose
rapidly probably necessitate the need for glucagon
sensitivity. If true, then lizards probably use glucagon
as the primary means of plasma glucose homeostasis.

CHAPTER VI
SUMMARY AND CONCLUSIONS
Glucose regulation in Ano 1is is similar only in some
respects to glucose regulation in mammals. Seasonal
variation in liver glycogen and plasma glucose is greater
in Ano 1is than in mammals. Much of the seasonal variation
may be attributed to ultimately to hormones. Although
plasma glucose shows a lot of variation, the levels
maintained in Ano 1 is are still higher than the levels
maintained in mammals. The high plasma glucose levels are
probably compensation for low hexokinase activity. Ano 1 is
probably cannot utilize glucose rapidly because of this
low hexokinase activity. Maintaining high plasma
glucose levels produce a large glucose concentration
gradient between the outside and inside of the cells.
Ano 1 is apparently maintains this large gradient through
a high glucagon sensitivity and a high glucagon production.
Enzyme activities reflect the use of anaerobiosis
for energy production during activity. Anaerobiosis severely
limits the duration of activity and is inefficient
because of lactate production, but produces energy rapidly.
Ano 1is most likely utilizes lactate by converting it to
tissue glycogen. Because Ano 1is relys in many cases on
burst activity, conversion of lactate to pyruvate for
98

99
aerobic catabolism would be inefficient as, aerobiosis
occurs, but not at the level found in mammals. The lower
level of aerobiosis is probably due to lower
mitochondrial volume densities and membrane surface
areas.
Carbohydrate loading after activity results in hepatic
overcompensation. Only after supersaturating the hepatic
system does muscle tissue start to store more glycogen
than normal. Therefore muscle glycogen storage probably
occurs at some preset rate very close to maximum. During
the active times of the year, fasting is associated with
a reduction in tissue glycogen. Glycogen is then more
important as an energy source than fats. During the
winter, wnen the animal may become torpid over parts of
its range, fat reserves are more important than stored
carbohydrates.
Glucose loading produces a long lasting hyperglycemia.
This effect is most likely due to a combination of low
insulin sensitivity, and/or production, and low hexokinase
activity. Insulin only produces a response when
administered in very high concentrations. The
hypoglycemia produced is quickly compensated by a
resulting hyperglycemia, probably as a result of
glucagon secretion

100
In other reptiles examined, the sensitivity to insulin
is variable* Alligators are the most sensitive and
possess low plasma glucose levels, while lizards are the
least insulin sensitive and have high plasma glucose
levels. In amphibians, anurans show insulin responses
(i.e„, long lasting hypoglycemia, Penhos and Ramey 1973).
Anurans also have resting plasma glucose levels that are
very low (ca. 25 mg/dl). Snakes are somewhat insulin
sensitive and tend to have fairly low resting plasma
glucose levels, 25 - 70 mg/dl (Penhos and Ramey 1973)»
It appears that insulin sensitivity is correlated with
inherently low plasma glucose levels.
It seems certain that other terrestrial vertebrate
ectotherms use variations on the themes presented here.
Snakes possess low plasma glucose levels but tend to be
less active than lizards. Anaerobic enzyme activities in
snakes are similar to those of Ano 1is (Baldwin and
Seymour 1977)» Interesting enough, sluggish snakes with a
high oxygen affinity appear better adapted for aerobic
metabolism in muscle, while active snakes with lower
oxygen affinity are better adapted for high rates of
anaerobic energy production (Baldwin and Seymour 1977).
ivithin a taxon, ectothermic vertebrates display
variability in anaerobic and aerobic energy production.

The oalance between aerobic and anaerobic metabolism
needs to be examined further in other groups of
ectotherms. The data presented here will hopefully
provide the impetus for further research into the
interrelationships involved in glucose regulation in
lower vertebrates

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APPENDIX
CALCULATIONS OF ENERGY EXPENDITURES FOR THE
OXIDATION OF LACTATE AND GLUCONEOGENESIS
&Iy.£P.ne p.ag.D.g.s.jL a
The following relationships are used to calculate the
amount of phosphate bond energy and oxygen required to
convert lactate to glucose* The costs of polymerizing
glucose to glycogen are ignored* Three high energy
phosphate bonds are required per lactate to make
g1ucose-6-phosphate» The oxygen required for this process
would be 10.6 ul per mole of lactate,
Q.y.l&Latl&n.
To completely oxidize one mole of lactate would
require 3 moles of oxygen. This is equivalent to 67.2
ml per mmole of lactate.
G-iy.s.ei.y s.j,g.
Two ATPs are synthesized per glucose molecule routed
through glycolysis. Three ATPs are produced for each hexose
derived from glycogen going through glycolysis. Therefore
there are 1*5 moles of phosphate bonds produced per mole of
lactate produced,
111

112
Agxg.fei-.p.s.is,
Produces a maximum number of 3d phosphate bonds per
glucose cleaved from glycogen» This phosphate bond energy
produced requires 6 oxygen molecules or 0»283 mmoles
phosphate bond per ml oxygen used»

BIOGRAPHICAL SKETCH
Jerry McCoy was born on September 2, 1958, in Ocala,
Florida* During his youth he became fascinated witn
animal life, especially reptiles. After graduating from
Ocala Forest High School in 1978, he attended Central
Florida Community College on a basketball scholarship*
During the spring and summer of his freshman and
sophomore years in college he worked at the Silver
Springs Reptile Institute as a lecturer and venom lab
technician* This experience reinforced nis intrigue with
herpetology, he received his B.S* in zoology from the
University of Florida in 1981* Since then he has
continued his studies on reptile physiology and ecology*
113

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
a dissertation for the degree of Doctor of Philosophy*
A
lOMJtVl )
L
Dr.^Harvdy Liliywhiti
Professor of Zoology'
p, Chairman
as
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
a dissertation for the degree of Doctor of Philosophy*
Dr/ John Anderson
Associate Professor of Zoology
as
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
a dissertation for the degree of Doctor of Philosophy*
as
Dr. Brian McNab
Professor of Zoology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degres of Doctor of Philosophy.
Dr, F. Wayne Kini
Professor of ZoqM/ogy
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope ana quality,
a dissertation for the degree of Doctor of Philosophy.
as
Dr* Micheál Collopy^
Associate Professor of Forest
Resources and Conservation

This dissertation was submitted to the Graduate Faculty
of the Department of Zoology in the College of Liberal
Arts and Sciences and to the Graduate School and was
accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
December 1987
Dean, Graduate School

UNIVERSITY OF FL opina
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