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)
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vii, 113 leaves : ill. ; 28 cm.
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McCoy, Jerry, 1958-
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Glucose   ( lcsh )
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Zoology thesis Ph.D
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non-fiction   ( marcgt )

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

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Full Text










THE REGULATION AND METABOLISM OF GLUCOSE IN THE
GREEN ANOLE (ANOLS. CAROLINENSIS)








By

JERRY MCCOY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1987































ACKNOWLEDGE.hNTS

The author wihes to thank the members of his advisory

committee, Dr. Harvey Lillywhite (chairman),

Dr. John Anderson, Dr. Brian McNab, Dr. Micheal Collopy,

and Dr. F. hayne King, for helpful suggestions during the

course of the research and preparation of the manuscript.

I would also like to thank Dr. Donald Allison of the

Biochemistry Department for helpful suggestions with the

enzyme assays.














TABLE OF CONTENTS



ACKNOWLEDGEMENTS..............***.......****.*........ ii

ABSTRACT....,V... ..,,............. .... v

CHAPTERS

I BACKGROUND AND PERPESPECTIVE......*......... 1

The Problem.................................. 1
Approach. .**********************.* 7

II SEASONAL VARIATION IN BLOOD GLUCOSE AND
GLYCOGEN STORES................*..**... 9

Introduction................................. 9
nethodsuctio....***************************** 9
ReSUltS************************************* 11
Discussion**.*****.*.******,..****.****. .**.* 17

III ACTIVITIES OF REGULATORY METABOLIC ENZYMES... 22

Introduction..***...*.*.*....*.....0*..* .***. 22

Me thodso********************************** o 3027

Discussion0********************************** 33

IV THE EFFECTS OF ACTIVITY AND FASTING ON TISSUE
GLYCOGEN AND PLASMA GLUCOSE LEVELS...... 42

Introduction, ..s *....... *.....***..** .... .** 42

ReSUltS*e,*********************************** 47
Discussion,*********************************. 69

V GLUCOSE TOLERANCES AND hORMONAL INFLUENCES IN
GLUCOSE REGULATION...*.................. 78

Introduction**.................*... *b.0*.+*. 78
MethodsRAs............ ....................... 79

Discussion*********************************** 94

VI SUMMARY AND CONCLUSIONS.*...* ,..** ....r.,.. 98














Page


LITERATURE CITED*********************..* ***.......... 102


APPENDIX

CALCULATIONS OF ENERGY EXPENDITURES FOR THE
OXIDATION OF LACTATE AND GLUCONEOGENESIS...... 111

BIOGRAPHICAL SKETCH................ >.*...,.,* .,,,.. 113














Abstract of Dissertation Presented to the
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 (ANOLIS CAROLINENSIS)

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, Anolis carolinensis, 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 and 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.












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.












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


XThe Problem


Vertebrate ectothermic poikilotherms 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,

Warnock 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),











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 al. 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).











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 gluconeogenesis 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










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 Psammophis sibil.ans, 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 pipieng,

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).












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 1982, 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 (overcompensation).

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 Saltin 1971).

Because anaerobiosis has such an important role in the

metabolism of activity in ectothermic vertebrate the

following questions arise, %Ihat 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 mechanismss? Is body glucose a limiting

factor in the metabolism of activity in reptiles? If so,

how?



Approach


This study examines ways in which glucose may be

regulated, metabolized and stored in a terrestrial

vertebrate ectotherm, the green anole, Analis carolinensis,

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 because 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












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 examined

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 19b4

to April 1987. They were returned to the laboratory after

capture. The lizards were kept in a constant temperature

cabinet set at the ambient 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.m.












and 12:00 p.m. 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 micropipette centrifuge for 3 minutes. Plasma glucose

concentration was measured colorimetrically 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/dl 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 -50C 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 the 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 ale method (1933). The glucose












was then measured using the phenol-sulfuric 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.



Results


Data on seasonal variation in plasma glucose and

tissue glycogen are summarized in Figures II-1 and

II-2. Plasma glucose levels are highest during the months

of April (223 A 6.9 mg/dl) and May (230 7.5 mg/dl). They

are lowest during August (125 t 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 (419 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).



























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Table II-1. Statistical correlations of seasonal data.
Accepted level of confidence equals 0.05.


Correlations

plasma glucose
vs
muscle glycogen


plasma glucose
vs
liver glycogen


liver glycogen
vs
muscle glycogen


-0.2d




0.049


> 0.05




0.40


0.567 0.001











Discussion


These data show that season affects both plasma

glucose and 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 Anolis

carolinensis (Dessauer 1953, Gleeson 1982, Patterson

et al. 1978). 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 Anolis is

mainly tropical in its distribution* The species

carolinensis 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*











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, Moore 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

vivipara (Patterson et al, 1978). In these two species












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 tend to reduce the catabolism of amino

acids which are the last resort to supply glucosyl residues*

Blood glucose continues to rise in the spring, which

may be due to carbohydrate intake through feeding. In

amphibians no correlation exists between feeding and blood

glucose concentration (Wright 1959). However, in Eumeces

obsoletus blood glucose concentrations increase with an

increase in feeding (Miller and Wurster 1958). In

Anolis during the summer, when temperatures are highest

and presumably the animals are operating at their

eccritic temperatures, blood 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.

Anolis is typical of other small iguanid 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 feeding, and can be

metabolically taxing (Pough and Andrews 1985). From late

winter through early autumn liver glycogen reserves are












low (Figure II-2)4 This period overlaps the reproductive

season of Anolis 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*

Anal.is carolinensis has a very peculiar form of

ovulation, termed monoallochronic 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. 1983, 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











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

stansburiana (Hahn and Tinkle 1965). A mechanism for

triacylglycerol degradation involving plasma glucose

reduction in A. carolinensis 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-1). The major pathway during

activity in reptiles is the oxidation of glucose or

glycogen to lactate by the Embden-Neyerhof pathway

(Figure III-2). High levels of glycolytic enzymes and

regulation of those that are rate limiting are adaptations














GLYCOGEN
HEXOSE
MONOPHOSPHATE
I SHUNT
GLUCOSE GLUCOSE 6-PHOSPHATE



TRIOSE PHOSPHATE GLYCEROL


PHOSPHOENOLPYRUVATE


PYRUVATE )> LACTATE


\ .-ACETYL-Co A


r OXALOACETATE


FUMERATE


SUCCINYL-Co A-------- (-KETOGLUTERATE


Figure III-1. Schematic of pathways of glucose and glycogen
catabolism* Double headed arrows indicate reversible reactions
utilizing the same enzyme. Multiple arrows indicate multiple
pathways.


CITRATE















HEXOSE MONOMERS


TRIOSE PHOSPhATE <-


PHOSPHORYLATED HEXOSES




TRIOSE PHOSPhATE


PYRUVATE

I
LACTATE


Figure III-2. Simplified schematic of the Embden-Meyerhof
pathway.


HEXOSE POLYMERS











allowing for high rates of substrate 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 examined 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-?-phosphate. Hexokinase controls the entry of free

glucose into the glycolytic pathway through the

phosphorylation of glucose. Unaer 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-limiting

enzyme, high levels are usually associated with high

anaerobic scopes* These four enzymes are all found in the

soluble fraction (cytosol) of the c-ell,

The mitochondrial enzymes, nicotinamide adenine

dinucleotiae (NAD)-linked isocitrate dehydrogenase and

succinic dehydrogenase are both involved in aerobic

metabolism and the generation of electron transporting













PHOSPHORYLASE B
(active R form)

AMP GLUCOSE 6-PHOSPHATE
V ATP



PHOSPHORYLASE B
(inactive T form)
4 1


2 H20\


2 ATP


ATASE KINA

2 Pi 2 ADP


--10


PHOSPHORYLASE A
(inactive T form)


1 ,


PhOSPHORYLASE A
(active R form)


SE


Figure 111-3. 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.


PLiOSPH












molecules. Thus, both are linked to the production 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 oC) 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 46 hours prior to being assayed. Lizards were

killed by freezing in liquid nitrogen. After freezing the

lizards were assayed immediately or stored frozen until

assayed at a later date. To assay, the entire liver was

removed along with the musculature of both fore and hind

limbs. All the dissected tissue was weighed and placed in

an ice bath. All preparations were performed in an ice bath.

Muscle and liver samples were homogenized separately

in ice cold buffered Ringer's solution (1 gram tissue

in 4 ml solution, Guillette 1982) with a mortar and

pestle. Triton X-100 was added to give a final

concentration of 1%, Triton X-100 increases the soluble

hexokinase activity (Burleigh and Schimke 1969). After

the Triton X-100 addition, the homogenate was incubated on

ice for 30 minutes followed by centrifugation at 17000

rpms at 4 oC.











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 o 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 albumin solutions were used as

standards (Sigma Chemical Company). All means are given

with their standard errors.


Hexokinase (E.C.2.7.1..1)

The reaction mixture contained 0.75 mM NADP, 3 mMi ATP,

7.5 mM magnesium chloride, 0.2 mM glucose, 1.5mM potassium

chloride, 2 IU glucose-6-phosphate dehydrogenase,

5 mM mercaptoethanol, and 50 mM sodium phosphate buffer

pH 7.4 (Baldwin and 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 Dehvdrogenase (E.C.1.1t1.27)

The reaction mixture contained 33 mh phosphate buffer

pH 7.4, 0.067 mM NADH, 0.33 mM sodium pyruvate and

homogenate. The reaction was started by the addition of











pyruvate. The specific activity was measured from the

decrease in optical density at 340 nm.


Phosohofructokinase (E.C.2.7.1.11)

The reaction mixture contained 5 mM magnesium

chloride, 200 mM potassium chloride, 50 mM Tris HC1 pH 7.4,

0.1 mM NADH, 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.


NAD-linked spocitrate Dehydrogenase (E.C.1.1.1.41)

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 dichlorophenol 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 Dehvdrogenase (IEC,1, .199.1)

The reaction mixture contained 40 mM phosphate

buffer, ph 7.5, 3.6 mM buffered potassium cyanide, pH 7.5,

0.040 mM dichlorophenol indophenol, 0.13 mM phenazine












methosulphate, 26 mM buffered sodium succinate, pH 7.5

and homogenate (Bennett 1972). Succinate was added to

start the reaction. Activity was determined 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, phosphorylase 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. Both liver

mitochondrial enzyme activities of the mammals were

higher than the reptilian values.






31






Table III-1. Comparisons of anaerobic and aerobic enzyme
activities between skeletal muscle of mammals and green
anoles, Values taken from Crabtree and Newsholme were
converted to nmoles/mg protein min assuming a ratio of 29:1
muscle tissue to muscle protein nitrogen and 108:1 liver
tissue to liver protein nitrogen. Assay temperature and
pH equals 33 0 C and 7.4 respectively.



MUSCLE


MAMMAL LIZARD


Hexokinase


Lactate Dehydronase

Phosphofructokinase

Phosphorylase

Isocitrate Dehydrogenase

Succinic Dehydrogenase


65..5*


47300**

238**

413.8*

25.6**


0.013

74200

1051

2522


5.6


22.4


114**


* From Crabtree and Newsholm 1972.
** From Bennett 1972.
*** From Prichard and Schofield 1968.















Table 111-2., Comparisons of anaerobic and aerobic enzyme
activities in liver tissue of mammals and green anoles.
Values taken from Crabtree and 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 o C and 7.4 respectively*


LIVER

MAMMALS LIZARDS


Hexokinase


Lactate Dehydrogenase

Phosphofructokinase


1.2***

16ti00**

93**


3.92

27600

56.6

105a.4


Phosphorylase


Isocitrate Dehydrogenase

Succinic Dehydrogenase


87-8**

261**


24.4

73.6


* From Crabtree and Newsholme 1972.
** From Bennett 1972.
*** From Prichard and Schofield 1966















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,

Phosphorylase in its active form, phosphorylase-a (Figure

111-3), catalyzes the breakdown of glycogen at a very high

rate to glucose-1-phosphate. Glucose-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












phosphorylase-b is inactive because of the inhibitory

effects of ATP and glucose-6-phosphate (Stryer 1981). The

greater activity of phosphorylase 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 and 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

phosphorylase to reach peak activity in the liver

(Lehninger 1982). Because of the inherent amplification

involved with each reaction step, hepatic phosphorylase 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 reduced. 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*

















STIMULUS --ADRENAL MEDULLA


EPINEPHRINE
_4 _--CELL MEMBRANE

ADENYLATE CYCLASE
ATP > cAMP + PPi

PROTEIN KINASE PROTEIN KINASE + CAMP
(inactive) Ca++ (active)


ATP + DEPHOSPHO-PHOSPHORYLASE --->PHOSPHO-PHOSPHORYLASE + ADP
KINASE Ca++ KINASE
(inactive) (active)


ATP + PHOSPhORILASE B ------ PHOSPhORYLASE A + ADP
(inactive) (active)


GLYCOGEN + Pi --- GLUCOSE 1-PHOSPHATE

I
GLUCOSE 6-PHOSPHATE


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 release into the blood increases its
concentration by 5 mM. The multiplier effect produced is
about 3 million times.











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 (Newsholme 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,

phosphofructokinase and phosphorylase 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 non-equilibrium or not. If the value of t is much

smaller than K', the reaction is displaced far from

equilibrium. Comparisons between t and K' in mammalian

tissues show hexokinase, phosphofructokinase and

phosphorylase all catalyze non-equilibrium reactions

(Newsholm and Start 1973). Similar results were also

found in spiders (Prestwich 1982).











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 al* 1977, Bennett 1972, Macleod

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 poikilotherms, 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

phosphorylated by hexokinase at the expense of one ATP,

By phosphorylating 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

through 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 continuously flood the intracellular

environment with glucose. Maintaining high levels of

glucose intracellularly may allow the low levels of

hexokinase to phosphorylate large numbers of glucose

molecules. This is discussed further in Chapter IV.





































Figure III-54 The glucose sodium symport. The sodium gradient
produced powers the transport of glucose. Adapted from Stryer
1981,







Cell membrane


,--ATP



Na ADP


Na+
r > Glucose
Symport


^Glucose












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











different tissues of a reptile and 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


PoiKilothermic ectotherms are generally regarded as low

energy systems. This is especially true when they are

compared metabolically to endothermic hoiaeotherms

(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 anaerobiosis (Bennett and Licht

1972, Gleeson 1982, Gratz and Hutchison 1977, Pough and

Andrews 1983). Although glycolysis is less efficient than

aerobiosis (i.e, Kreb'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 Gooanight 1964, Gaesser and Brooks 1980, Gleeson












1982, 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 and glycogen

replacement is also evaluated.


Methods


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 o 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 blood assayed

for glucose or lactate. Glucose and glycogen

concentrations were measured at rest, 30 seconds after

the onset of exercise, at the point of exhaustion and at











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 OC to 50 oC,

12:12 light:dark cycle). All means are expressed with

their standard errors.


Lactate Analysis

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 N perchloric acid equivalent to 12 times body mass.

To study compartmentilization 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 OC for 10

minutes* The supernatant was filtered and recentrifuged

at 10000 rpms at 4 OC for 10 minutes. The resulting

supernatant was assayed with an enzymatic test kit (Sigma











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.


Oxygen Consumption

Oxygen consumption (V02) was measured manometrically

using a Gilson Differential Respirometer during July and

August 1986. Lizards (n = 5) were placed in 125 ml

respirometer flasks* Each flask contained approximately

12 grams of soda lime to absorb CO2. All determinations

were made at 33 oC (ioe., eccritic temperature). Resting

rates were measured at 10:00 p.m E.S.T.

After several hours of acclimation in the flasks V02'

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 02/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.


Fasting

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.











Results


Oxygen consumption and whole body lactate at rest,

during exercise and recovery are summarized in Figure IV-1.

Maximum V02 occurred during exercise

(1.65 t 0.201 ml/g-h). With a resting V02 of 0.262

0.038 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). Table IV-1 summarizes the net recovery

oxygen consumption and the change in lactate

concentration during recovery.

Whole body lactate increased 8.4 times from resting

values, 0.29 0*01 mg/g, to exercise values

2.43 a 0.21 mg/g. Over the first 10 minutes of

recovery lactate declined very little (2.36 a 0.26 mg/g).

Sixty minutes into recovery, lactate had dropped

drastically (0.68 a 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

compartmentalization. All compartments show the same trend.

Maximal lactate concentrations occur during exercise. Muscle

and blood lactate levels after 2 hours of recovery were
































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Table IV-1, Resting and post exercise lactate and glycogen
levels* Units are mg lactate/glycogen per g tissue.





NOhFASTING FASTING


Muscle


REST


Lactate 0.62


POSTEXERCISE


REST POSTEXERCISE


2.53


Glycogen


Liver

Lactate 0.15

Glycogen 7.1


1 44

3.9


5.4 2.3


4i.1


1.6


3,9


1.9

































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(S/Sw) N011Vy1N33N03











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 t 0.07 mg/g and 0.62 :L 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 A 0.17 mg/g and 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, INo

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

































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CONCENTRATION


(mg/g)

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1.44 a 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 a 0.008 mg/g and

0.15 A 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 a 14.3 mg/dl to 149,6 a 12.3

mg/dle The plasma glucose level reaches its maximum

concentration, 239.4 a 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 a 0.07 mg/g

and 4.6 a 0.04 mg/g, P > 0.05, T-test). Compensation

















Table IV-2. Oxygen consumption requirements for lactate
oxidation and gluconeogenesis from lactate* Numbers are based
on data taken from Figure IV-2.







01YGEN REQUIRED (ml)


TIME LACTATE NET RECOVERY
(MIN) (mM) OXYGEN CONSUMPTION (ml)


0-4


0.072


4-15 0.070

15-45 0.020

45-75 0.019

75-120 0.009


0.098

0.074

0.029

0.010


LACTATE TO
GLUCOSE 6-P


7.6 x 10 "7

7.4 x 10 -7

2.0 x 10 -7

2.0 x 10 "7


0.002 9.5 x 10 -9


LACTATE
OXIDATION


4.838

4.704

1.344

1.277

0.605






























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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 8.4 : 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 A 18.1 mg/dl. Glucose levels

declined rapidly, and after 60 minutes of recovery, plasma

levels were equal to resting levels (151.2 A 10.3 mg/dl

and 149.6 A 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.


























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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, 1980b,

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).












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-labeled compounds. The fate of lactate can be

suggested by calculating the amount of oxygen required to

resynthesize glucose from lactate (i.e., gluconeogenesis),

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 2% 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 gluconeogenesis. This differs from the

situation in mammals, in which the primary fate of

lactate is oxidation (Brooks et al. 1973, Drury and wick

1956, Gaesser and Brooks 1980). The production of

glycogen from lactate appears to occur in other reptiles

(Gleeson 1985, Moberly 1968). Gleeson (1985), using




















Table IV-2* Oxygen consumption requirements for lactate
oxidation and gluconeogenesis from lactate. Numbers are based
on data taken from Figure IV-2.


TIME LACTATE NET RECOVERY
(MIN) (mM) OXYGEN CONSUMPTION (ml)


0-4 0.072

4-15 0.070

15-45 0.020

45-75 0.019

75-120 0.009


0.098

0.074

0.029

0,010


LACTATE TO
GLUCOSE 6-P


7.6 x 10 -7

7.4 x 10 -7

2.0 x 10 -7

2.0 x 10 -7


0.002 9.5 x 10 -9


LACTATE
OXIDATION


4.838

4.704

1.344

1.277

0.605












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).












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 phosphorylated 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 Anolis

(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-6). 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).












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. Anolis 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 60$) 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 Anolis.

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













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 Anolis 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 variable. Insulin is least effective in the

lizards examined, and shows the most marked 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.












Methods


Glucose Tolerance

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.












Glucaion

Mammalian glucagon was obtained from ICN biomedical Inc.

The hormone was injected intraperitoneally at

concentrations of 10 ug/kg and 100 ug/kg (n = 106).

Animals were assayed at the same intervals as in the

insulin experiment, results are expressed as previously

described.


results

Glucose Tolerance

Results are summarized in Figures V-1 and V-2. Plasma

glucose levels peaked between 2 and 3 hours after

ingestion (492 A ng/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).


Insulin

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




































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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.


Glucagon

Glucagon responses are summarized in Figures V-5 and

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 48 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 returned to normal (Figure V-6).



























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