PROGLYCOGEN AND MACROGLYCOGEN IN HUMAN SKELETAL MUSCLE
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
April, 1998
OKristi B. Adamo ,1998
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ABSTRACT
PROGLYCOGEN AND MACROGLYCOGEN IN HUMAN SKELETAL MUSCLE
Kristi Bree Adarno
University of Guelph, 1998
Advisor:
Dr. Terry E. Graham
This thesis \vas designed to investigate the existence of two pools of glycogen.
proplucogen(PG) and macroglycogen(MG) in human skeletal muscle. First it was
necessary to develop a measurement technique to isolate the two f o m s and then compare
the new method to the traditional methods of acid hydrolysis(AC) and en-atic
hydrolysis(E2) for measuring muscle glycogen. It was found that PG and MG do exist in
human muscle and the measurement technique for (PG+MG) total glycogen was accurate
and precisr. The second part of this thesis was designed to study the synthesis pattern of
the two pools. ~Malevolunteers completed exhaustive exercise triaIs on two occasions in
ordçr to deplete muscle glycogen. Over the following 48h they ingested either a high
carbohydnte(HC) or a low carbohydrate(LC) diet. and muscle biopsies were taken frorn
the izrsrics kcret-dis at 4 time points. In the HC trial it was found that the PG pool
dominated the synthesis over the tirst 24h and then it'j synthesis slowed over the second
48h while MG synthesis remained steady at a slower rate. The synthesis in the LC trial
was considerably less and at a slower rate. Over the final 24h of the HC data collection
period the amount of MG synthesized was much greater than PG. and it represented
about 4O0/b of the total glycogen concentration. The major findings of this study were that
MG and PG are metabolized differently both in timing and in terms of magnitude and
both are sensitive to nutritional intake.
TABLE OF CONTENTS
.I\cknowledgements
...................................................... i
..
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III
...
1.0 WTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Glycogenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Classic Hypothesis of Glycogen Synthesis Regulation . . . . . . . . . . . . . . . . . . . . . 8
Newer Concepts of Glycogen Bioqnesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Supercompensation and CHO supplementation . . . . . . . . . . . . . . . . . . . . . . . . . 19
Esperimental Objectives: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .II
2.0 STUDY 1: METHOD COMPARISON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Cornparison1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Cornparison II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Cornparison III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .- 2 7
CornpansonIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
CornparisonV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Xnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
ComparisonI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
CornparisonII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
ComparisonlII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
CornpansonIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
ComparisonV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.0 STUDY II:
The Efféct of dietary CHO on the resynthesis of macroglycogen and progiycogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
blethods: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Subjects: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Experhental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 0
prr-trial procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
.
VO: mau testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .- 5 0
Dietary Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 0
HabituationRide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Dietary Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1
Expenmental Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 2
..
Analyses: .......................................................
33
Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 6
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Muscle : MG and PG concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 7
Net Glycogen Synthrsis Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
PG s~nthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
MG synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 6 3
Blood Glucose and Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 6 7
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
Future Directions and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
APPENDIX.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90
Subjrct Screening Questionnaire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
iNFORhdED CONSENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
MEDICAL ASPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Bioodsampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Il.1uscle Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93
[NFORiMED CONSENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 9 5
EXPERIMENTAL PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . YS
FiNAL RELEASE - . . + . . . . . - . . - . - . . - - . . . . . - . . . . - . - - - - . - - - - .96
.......
Acknowledgements
1 riauld fn t and foremost like to thank my advisor Terry Graham without whose
euidance and advice I'd never have reached this stage. He allowed me to choose rny orvn
path and ga1-eme the freedom 1 needed as well as the answers 1 required. Through the
countless opponunities he has provided for me. 1 have gained the confidence I so badly
needed and made my graduate expenence much more enjoyable than 1 ever imapinrd.
b
1 rnust aIso thank m? t'riends and confidants Premila Sathasivam and Kim
Robertson for al1 of their never-snding patience. support and knowledge. The nonthreatening laboratory atmosphere Prern and Kim together have created is second to none.
They have cach in their own way helped me tremendously.
Cyndy McLran. Fel icia Greer and Farah Thong have all contributed significantl?.
to this rhesis not only through their technical assistance but through fnendship as well. 1
appreciate al1 that they have done for my grow-th both as a student and an individual.
1 would like to thank my CO-advisorMitch Kanter. not only for his financial
support through the Gatorade Sports Science lnstitute but for his help in brainstorming
and experimrntal design. I must also extend thanks to Mark Tmopolsky who aided in
designing the resynthesis study as well as sacrificed a great deal of time and effort in
performinp muscle biopsies during my trials.
MJparents have bern a constant source of love and support and their belidin rny
abilities helpsd me overcome problems with self-doubt. 1 am forever indebted to thrm for
al1 of thrir contributions to my education . for backing me up and for never questioning
mj.academic decisions.
Lastly. 1 have to thank my future husband Rob McMullin for more than 1 can put
in rvords. He has been unselfish in his giving. while his humour and calrn nature hart
kept me sane in times of chaos. For his love and tolerance 1 thank him from the bottom of
rn? heart.
LIST OF TABLES
Table 1 : Past Findings of Two Foms o f Glycogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 4
Table 2: Cornparison of EZ. .AC.and MGlPG for rodent muscle glycogen . . . . . . . . . . JJ
i1
Table 3: Cornparison of EZ.AC . and MGRG for human muscle glycogen . . . . . . . . . - 3 5
Table 4 : Subject Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 4 9
Table 5 : Dista- Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
-3
2-
Table 6: Xluscle giycogen concentrations HC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 8
Table 7: Muscle glycogen concentrations LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5 8
Table 8: Net Synthesis of MG over 18h recovery for HC and LC trials . . . . . . . . . . . . . 65
Table 9: Synthesis of PG over 48h recovery for HC and LC trials . . . . . . . . . . . . . . . . - 6 5
Table 10: Blood Glucose and Serum Insulin Concentrations . . . . . . . . . . . . . . . . . . . . . 68
LIST OF FIGURES
figure 1 : Krisrnan's mode1 for the structure and biogenesis of glycogen . . . . . . . . . . . . . 7
tipure 2: Pathways of glucose metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
tiyure 3: GI- logen synthesis involves the addition of glucose . . . . . . . . . . . . . . . . . . . . 1-7
figure 4: The role of glycogenin in the biogenesis of muscle glyco-n
. . . . . . . . . . . . . . 14
figure 5: The new biogenesis hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
figure 6: MG and PG Extraction Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
tigure 7: MG+PG vs AC Pooled Muscle Sarnples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
figure 8: AC vs MG+PG Individual Muscle Samples . . . . . . . . . . . . . . . . . . . . . . . . . . - 3 7
C
Figure 9: EZ vs . MGtPG Individuai Muscle Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
tigure 10: EZ vs AC Cornparison of Traditional Measurements. . . . . . . . . . . . . . . . . . .39
figure 1 I : MG vs PG over a widr range of total glycogen concentrations. . . . . . . . . . . - 4 1
t i g r e 12: MG vs MG+PG ovrr a wide range of total glycogen concentrations . . . . . . . 41
tiyure 1 3 EZ vs MG+PG . Cornparison to independent laboratos measurernents . . . . - 4 2
figure 14: Esprrimcntal Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .- 5 4
figure 15: Total Giycogen Values(MG+PG) for the HC condition . . . . . . . . . . . . . . . . . 59
tipure 16: Total Glycogen Values(MG+PG) for the LC condition . . . . . . . . . . . . . . . . . . 60
tigurc 17: The amount of MG and PG synthesized over time for the HC condition . . . . 61
figure 18: The amount of MG and PG synthesized over time for the LC condition . . . . .62
figure 19: The net rate of PG sythesis for HC and LC over 48h of recovery . . . . . . . . . 64
tigure 20: The net rate of MG s>mhesis for HC and LC over 1 8 h of recovev . . . . . . . - 6 6
figure 7 1 : Blood Glucose Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -70
figure 22: Insulin Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1
figure 23: The relationship between MG and PG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
1.0 INTRODUCTION
Background
The structure and rnetabolism of glycogen have been intensely investipated sincr
it's discoven in dog liver bp Claude Bernard in 1 857. Glycogen is a readily mobilizrd
storage form of glucose and is hund in abundance in nearlp al1 types of animal cells. It is
a large branchrd( 1.4 a and 1.6 a ) polyrner of glucose residues that is built upon a protein
'-backbonr--. There are two main sites of glycogen storage. liver and muscle. The liver
contains the highest concentration of glycogen. because of its role in the maintenance and
regulation of blood glucose. Skeletal muscle. althou& having a lower relative
concentration of glycogen than liver. contains the larger amount due to it's greater mas.
The synthrsis and degradation of muscle glycogen and the hormonal replation of these
procrssrs have been studied in depth.
Recentty there have been advances in O u r
understanding of the structure and tk novo biosynthesis of glycosen(Lomako et al.. 1 990.
1991. 1993. Alonzo et al.. 19953- Rodriguez and Whelan. 1985). The discovery of the
muscle glyogen protein --backbone". glycogenin. by Rodnguez and Whelan( 1973) is one
of these ndvancrs. While isolating the glycogenin protein. Lornako and colleagues
obserwd two forms of muscle plycogen in rodent muscle. liver and heart. These two
pools of elvcontin are now referred to as proglycogen(PG)and macroglycogen(MG).
C I
C
The idra of two forms ofglyogen is not by any means novel: there is literature
dating back to the earlp 1 900's discussing the possibility of two pools of glycogen.
Willstaetter and Rhodewald reported the presence of 14.0- and desmoglycogen each
having differential responses to physiological stress(Wil1staetter and Rhodewald. 1934).
They were found to be separable by tnchloroacetic acid(TC A) solubility . Lyoglycopn
(what is now referred to as MG) was soluble and believed to be protein-free or -unbound0
M
hile desmosglycogen was insoluble or *residualglycogen*and thought to be in
combination with protein(perhaps the portion that is rekrred to as PG). It was noted that
the proportion of glycogen in the lyo form depended upon the nutritional state of the
animal(Wi l lstartter and Rhodewald. 1934). These rexarchers illustrated that aHer fasting.
coose l ivrr showrd a drcrrasr in the soluble Form(MG). According to Pfluger and
C
Loeschke(Van Heijningen et al.. 1955) the protein-tixed fraction(PG) is produced by the
enclosure of part of the glycogen by the proteins in the course of their precipitation. and
should be considered an artifact. Thus. the hypothesis of two types of glycogen was
rejected at that time on this basis. In the 1960's Stetton et al. noted that past findings
revealrd a metabolic inhomoyenrity of glycogen at an intrrmolecular. as well as ai an
intnmolecular Ievel. They found that on fractionalization there was glycogen of differing
mean molrcular weights. The!. also reported that three hours afier injection of glucose Cl4
into rats. consistently highrr sprcific activities were associated with the residual glyogcn
(potentially PG) than with the fraction of muscle soluble in TCA(potential1p MG). This
Isd them to belirve that the residual(PG) might be available for immediate metabolic use.
In 1980 Jansson publishrd a study demonstrating that there was a portion of
elvcogen
ihat was acid soluble(MG) and a portion that was acid insoluble(PG). Jansson
-
C
was attempting io demonstrate that using a PCA extraction in which one uses the
supernatant for other metabolic analyses and then using only the precipitate for glycogen
mesurement would give an underestimate of total glycogen(TotG). She found this to be
tmr as the supernatant contained a significant portion of glycogen. Jansson also observed
that up to a TotG concentration of 300 mrnoVkg dw the acid soIuble(MG fraction)
compked -23%. and at greater TotG concentrations the increase was predominantly in
the soluble(MG) pool. This \vas supported in 1 989 by Friden et al. u-ho discoverrd that
when using rlsctron rnicroscopy there were two different size glycogen particles located
in human sksletal muscle and suggested they could be the two foms of glycogen reportrd
by Jansson. There have b e n a few isolated reports ofacid soluble and insoluble glpcopttn
in human muscle and the concept of two forms/pools has not been studied physiologicall~
(Jansson. 1980. Gaitanos et al. 1 993. Cheetharn et al.. 1996. Tsintzas et ai.. 1996). Rather
this concept has been cmployrd as an alternative way to get a total sum of glycogen. and
little metabolic investigation has bren conducted.
Tablc 1: Past findin~sof Two Pools of Glvcogen
Author
Tissue
date
terms used
beliefs: findingv
WiIlstaettçr
and
Rhodewald.
Goose liver
t 934
lyog\ycogn/
lyoglycopn(MG)-no protein and desn~oglycogen(PG)
bound to protein: proportion dependent on nutritional state.
Fast ing 1lyoglycogen(MG)
dcsiiioglyc»geii
Stelton et al. Rodeii t
iiiuscle &
liver
readi ly extractable/
residunl
bouiid
l-iuniaii
niuscle
1980
acid soliible/ ncid
insoliible
residunl(PG) concentration more constant, and
metabolically active, extractable(MG) more variable
-higher specific activities associated with residual glycogen
thaii the soluble(MG) fraction ~ A e 3r h of
injection
acid soluble(MG) represented up to 25% below 300
mmollkg then increased at higher total concentration.
Friden et al.
I-iuman
niuscle
1989
large granules/ sinall
grandes
granules were found in 5 distinct subcellular locations. The
small granules(PG) were more plenti ful. Ratio 4: 1
1,omako et
al.
Rodent
liver &
muscle
Huang et al.
Rodent
muscle
1997
MG/1%
-concentration of each was dependent on metabolic(insulin,
glucose) condition. PG more dynamic, usable portion
Adamo and
Graham.
Rodent
muscle,
human
muscle
1998
MG1 PG
PG in excess of MG in al1 conditions but MG starts to
become > contributor at higher total concentration.
-molecules of 2 different molecular weights PG(400kDa)
and ~ ~ ( 1 Da)
0 ' -PO found to be the precursor of MG
Ratio: 18mol MG to 82 ni01 PG.
With the added knowledge from molecular biology techniques it appean that the
acid-soluble lyoglycogen is representative of the larger MG. and that desmogiycogen is
actuall>.the srnaller but insoluble PG. According to Lomako the two forms difier not in
the presrnce or abxncr of protein(as in the Willstaetter i-f>pothesis)but in relative
proportion of protrin to CHO. Both molecular forms contain a single glycognin but
different amounts of CHO. thus MG consists 0t'O.35% protein while PG contains about
10°/o protttin. PG is the smallsr molecute(400kDa).and is insoluble in PCA. while MG
which is much larger( 107Da)and having a srnaller protein component. is soluble(A1onzo
et al.. 1995a).
GLYCOGENIN
It has brrn recognized for nearly 30 years that the de novo synthesis of glycogen
rrquires a priming mrchanism since the glycogen synthase(GS)enzyme cannot transfer
ylucosyl residues from uridine-diphosphate-glucose(UDP-sic) without a disaccharide
riIrrad! present(Ca1der. 1991). Work bp Krisman et al.. 1975 suggested that the glucosyl
units are transkrrsd to a protttin primer. not a carbohydrate. by a -glycogen initiator
synthase' before elongation by GS( figure I ). Rodriguez and Whelan. 1985 temed the
muscle glyogen protein backbone glycogenin and the moleciilar mass of approx imately
38 000 Da has been confirmed by Pitcher et al. 1987. Glycogenin is not only the protein
backbone on which glycogen is synthesized but is also a divalent-cation-dependent
glucosyltransferase which bnngs about its own glucosylation as an essentiai prerequisite
for de noiw bios>nthrsisof glycogen(Smytheand Cohen. 1991 ). Self-glucosylation of
gl>cogenin is stimulated by ~ n 'and requires UDP-glc as a substrate. Such discoveries
indicate that Knsman's 'glycogen initiator synthase' is glycogeenin itself.
It has been shown that glycogenin is a subunit of skeletai muscle GS(Pitcher et al..
1987) and is present in a 1 :1 rnolar ratio with the catalytic subunit of the enzyme. Earlier.
Cohen and çolleagucts had shown this protein cornplexed with GS yet dismissed it as an
irnpurity(Smythe and Cohen. 1991 i. Later the) puritied rabbit muscle gipcogen.
con tim~ingthat the glycogcn-protein linkage \vas a glucose-tyrosine(ty) bond and. in
muscle. e w p . fi! cocen
+
is bound to protein(Campbel1and Cohen. 1989). By drtermining
the çntire 333 amino acid srquence the tyr-194 was reported as the sole site of
glucos>Mon. When tyr kvas replaced with phenylalanine(phe).Alonzo et ai. round that
the glycogenin would no longer self-glucosylate(Aionzoet al.. 1995ab). Hence this site is
nrcrssan tor the activity and function of glycogenin and serves as the anchor for glucose
transfer(Caoand Roach. 1993 1. It has been reported that the glucosylation of t y - 194 is
iiot rate-Iimiting(Alonzo et al.. 1 995a).
UDPG t
Glycogen initiaior
synthase
-
Glysogsn synthasr<m)
+ Branching enzyme
+~ ~ P G \ c
t
UDPGk
e-
Protein primer
Protein primer
centraining oligosaccharide
( 1, 4-a-glucanl chains
Glycogen molecules are
buil1 onto h a proteici
backboiie
figure 1: Krisman's mode1 for the structure and biogenesis of glycogen. Glycogen
biogenesis was proposed to require the presence of 3 distinct proteins. The f h t was the
protein primer on which glycogen molecule is constmcted. A glycogen-initiator synthase
would then add glucose units from UDP-glc to protein primer at multiple sites to form
oligosaccharides which wlien long enough woidd be capable of undergoing elongation by
GS.(Krismanand Barerigo, 1975)
Glycogenin has a specitication for a pyrimidine base and UDP-glc is it's natunl
substrate. howver ATP can compete for the binding site and has been found to be an
inhibitor(Lomako et al.. 1991). Trsting UDP-xylose as a substrate. it was found that the
e n q me
N
il1 ont! add one xylosc: residue and is unabte to add another(Lomako et al..
199 1 ). They suggested that in vivo the presence of UDP-xylose might serve io limir
glyogen synthrsis. Other pyrimidine bases such as CDP and TDP-glc are used less
ctYectivei> and cannot be usrd as substrate for GS(Alonzo et al.. 1995b). Although
nlvcogenin
is able to catalyse the formation of alpha- 1..Cglucosidic linkages. it is still
-
C
unknown ho\\ plycogenin recruits the first glucose residue to a tyrosol group in order to
start the oligosaccharide chain. Recently the cDNA For human muscle glycogenin has
been sequenced and the gene coding for glycogenin is localized to human chromosome 3.
band q74 and is 97% identical to rabbit glycogenin(Lomako et al.. 1996).
Clrssic Hypothesis of Glycogen Synthesis Regulation
The rate at which glyogen synthesis occurs has been considered to revolvs
entirel>-around the statrs of activation or covalent modification of the enzymes GS and
phospho~.lasc..which control s>nthesis and breakdown resprctively( Bloch et al .. 1 994.
Kochan et al.. 1979. Lomako et al.. 1991 ). Synthesis and degradation are k n o ~ nto occur
sirnultaneousl>.and the balance dictates the net response. Sqnthesis and degradation of
si! cogen i nvo Ive separate enzyme cascades where phosphorylation has opposite et'fects
on GS and phosphorylase. Glycogen synthase. responsible for the transfer of glucose
from UDP-glc io an amylose chain. has been regarded as the rate limiting enzyme in the
process of glyçogen formation(Zachwiejaet al.. 1991 ). It has an inactive(D)/active(1)
contiguration. and the 1 form(independent of G-6-P) of GS is activated by
dephosphorylation of a speci fic senne residue(B1och et al.. 1994. Kochan et al.. 1979).
lnsulin is postulatcd to initiate the cascade of events by releasing the activity of protein
phosphatase 1(PP I ) acting on the senne residue and leading to the activation of GS(Beck'iieisen et ;il., 1992. Shulman et al.. 1990).
Howxer there continues to be a controversy over whether it is GS that lirnits the
formation of plycogen or if it is glucose transport. Given the hi& activiv of
hexokinase(HK) in muscle. an increase in glucose transport c m stimulate glycogen
tonnation(Friedman et al.. 1991 ). As soon as glucose enters the cell. it is phosphorylated
via HK to t o m gIucose-6-phosphate(G6P)(Vestergaardet al.. 1994).Since reversal of the
HK reaction is thermodynamically unfavourable and glucose-6-phosphatase activity is
negligible in muscle. phosphop.lation 'waps" glucose inside the ce11 where it must be
utilized b.! gl>-colysisand oxidation or stored as glycogen. Glucose-&phosphate is k n o w
to inhibit HK allosterically(Connett and Sahlin. 1996) hence iFG6P accumulates. HK
would become rate lirniting and intracellular free glucose should theoretically accumulatt.
a-ithin the muscle. However. it has been shown that over a &ide range or glucose uptakr
rates frcx glucose does not accumulate(1~yet al.. 1988) implying that glucose transport is
rate limiting for glucose utilization in muscle.
It has been suggested that the muscle glucose transport is affected by eccentric
muscle activity while GS activity is not(Friedman et al.. 1991 ). It has been proposcd that
decreased GLUT-l content may be an important factor in the delayed glycogen resynthesis
pattern afier this type of esercise(Hickner et al.. 1997). However. Asp et al. 1997
demonstrated that total GLUT4 protein content is unaltered in the gas~ocnemiusmuscle
following a marathon and that the slow recovery of muscle glycogen after the race
apparently involves factors other than changes in total content of this protein. Yet this
m q be specitic to the gastrocnrmius since these same researchen found delayed changes
in GLLiT4 in vastus lateralis muscle afier eccentnc contractions(Aspet al. 1995). In
addition. the absence of changes in the total GLUT4 content does not exclude that the
translocation of GLUT-l from intracsllular store to sarcolrmrna was impaired(Aspet al..
1997). The muscle darnage from the marathon could very well have affected the
contraction stimulateci nitric oxide(N0) migration or insulin stimuiated P-1-3 kinase
r lucose transport.
C
In 1988. Ivy et al. demonstrated that delaying the ingestion of carbohydrate (CHO)
b>. 2 hours alter esercise resulted in a significantly lower glycogen synthesis rate than
ingestion imrnediately atier essrcise. Glycogen synthase activity was not different
brtwern treatrnents and could not account for the differences in resynthesis rate. It is
likely that glucose transport \vas accelerated during the initial 2 hours afier exrrcise with
immediatt: frcding. accounting for the enhanced resynthesis rate(1k-y et al.. 1988). Studirs
have also indicated that the dcfect in glucose disposal in nonobese NIDDM patients is
o\.ercomr b'. hypzrglycaemia. which. by mass action. increases muscle glucose uptake
(Henry et al.. 1990) and results in increased nonoxidative glucose disposal. presumably
through increased formation ofplgcogen. Thus. glucose transport appears to play an
integral role in the rate of glycogen synthesis in a number of physiological
situations(tigure 2).
Numerous studies have shown that die activity of GS varies inversely with muscle
-glvcogen content(B1och et al..
d
1994. Gaesser and Brooks. 1980. I w a al. 1988. Kochan et
al. 1979). Small changes in the activity ratio(96 I tom) of GS can lead to large changes in
the rate of glycogeen sqnthesis. but GSI appears to increase veiy little in response to
exercise induced glycogen depletion. and renims to preexrmise levels as glycogen levels
r e t m to normal(Kochan et al.. 1979). Thus attempts to explain glycogen resynthctsis
based on change in cither total GS activity or increases in the active I form of GS have
been unsuccesstùl. Although still inconclusive as to whether CS or glucose transport is
the prirnary controller of glycogen sythesis- it is more likely to be a combination of the
two po tential regulators that govems synthesis.
( Glucose-t -phosphate
Hexakrnase
ATP
ACP
1
figure 2: Pathways of glucose metabolism(Fnedman et al.. 1991)
11
1
-
Newer Concepts of Glycogen Biogenesis
It is well known that glycogen synthesis occurs as giucose binds together to forrn
a storage polysaccharide(figure 3 )
P hosphoglucornutase
Glucose 6-P p
G~ucose1-P
Glucose 1-P
UDP-glucose pyrophosphorylase
,f-~~'+\
UOP-glucose
=
UDP +(glucose),
PPi
UTP
UOP-glucose + (glucose),
=
Glycogen synthase
~
g
~
+
,
'
figure 3: Glycogen synthesis involves the addition of glucose units in the fom of UDP4 c to a pre-existing glycogen primer molecule shown as (glucose),, Since one ATP is
=
consumrd to rnake G6P. the enerw cost to store one giucose unit as glycogen would be 2
energ-rich phosphates: one ATP and one UTP.(Houston. 1995)
Rccent discoveries have increased the understanding of these pathways. It is now
knowm that the biogenesis of glycogen involves a specific initiation sequence mediated b!
glycogenin. Glycogenin can be obtained from tissues by d e p d i n g the glycogen to which
it is bound(Lomako et al.. 1991 ). As stated previously. Krisrnan in 1975 proposed that the
enzyme GS catalyses the transfer of many glucose residues to glycogenin and glycogenin
functions as an accrptor when complexed to GS(figure 1 ).
In the absence of glycogen. glycognin is complexed to glycogen synthase and
this complrv is the starting point for the putative -giycogen initiator sythase' that links
the tirst glucose residues to glycogenin. Glycogenin binds 2 or more 14 linked glucose
residues and appears to be autocatalytic. Autocatalysis consists of apposition of
additionai GDP-glc residues to generate an oligosaccharide(malto-octose)which serves as
a primer for gl'cogen synthesis(A1onzoet al.. 1995a Lomako et al.. 1991. Smyth and
Cohen. 1991 ). The priming reaction involving the attachment of about 6 additional
clucosr residues to glycogrnin requires UDP-glc and it is entirely dependent on the
c.
presencr of Mn '-( Lornako et al.. 1991. 1995). Although it was originally proposrd that
GS attaches funhrr glucose residurs to plycogrnin(Pitcher et al.. 1988). it has been show n
that plycogrnin is in fact self glucosylating(Lornakoet al.. 1988). Consequently it would
apprar that two distinct enq-matic activities are required to form the primer for glycogen
synthrsis: the tint being the covalent attachent of a single sugar residue to the
4vcogenin
catalysed by the --glycogeninitiator synthase"(Krisman and Barengo. 1975).
-
2
and the second step is an autoglucosylation reaction catalyzed by glycogenin itself.
Howe\w. it is now known that the glycogen initiator synthase is also glycogenin itself
and so here are two steps but only one enzyme. The length of the oligosacchaEde p h a
is miportant for glycogen synthase action and the fUy primed ch& is 7-8 glucose
residues long. Once the polysaccharide has reached this critical level the GS may
dissociate fiom glycogenin and in conjunction with bmchmg enzyme, complete the
synthesis of the rnolecule(Pitcher et al.,l987)(figure 4).
GGT
uowC
figure 4: The role of giycogenin in the biogenesis of muscle giycogen.
Following de novo biosynthesis of glycogenin(G), the first step in glycogen biogenesis
involves the covalent aitachment of glc to tyr- 194 in glycogenin catalyzed by an as yet
unidentified protein tyr glucosyltransfzrase(GGT). Glycogenin and GS fom a ûght 1:1
cornplex, ensuring that the glucan primer generated by the giycogenin catalyzed Mn
dependent autoglucosylation reaction can be elongated by GS when conditions for
glycogen synthesis prevail. GS and glycogaili can ody synthesize a glycogen molecuie
fiom such primed giycogenin when the 2 proteins are complexed together. The
progressive transfer of many glucose nits to the growing glycogen molecule by GS
results in its dissociation 6om giycogenin. The growth of glycogen molecules then
continues by the combined action of branching enzyme and CS.
GIycogenin rernains attached to the carbohydrate in the mature glycogen
molecule(Lomako et al.. 1995). Glycogenin has the potential to be the key rate-iimiting
e n e me in glycogen synthesis and its activity may well be subject to rigid control. thereb>.
dictating the ovcrall Pace of glycogen synthesis.
The discowry of PG and glycognin led to insights into the regdation of glycogen
metabolism tnditionally believed to be regulated at glycogen phosphorylase and glycoycn
synthase by cot-alent modification(Alonzo et al. 199%). Studies of rodent muscle have
shown that PG is fomed through glucosylation by a synthase-like enzyme(not the
autocatai'ic
protein) and this strong protein glucosylating activity is not stimulated by
Mn" but is stimulated by G6P(Alonzo et al.. 1995a Lornako et al.. 1991. 1993). The
G6P stimulated activity characteristic of GS was observed at a substrate concentration of
2 . d which is 3 times below the Kmof 'classical' synthase for UDP-glc using glycogen as
a primer which is iq the millimolar range(A1onzo et al.. 1995a). It has been proposed that
there are t ~ enzymes
o
that catalyze glucosylation. one self-glucosylating protein which is
stimulated by Mn' and carries out the autocatalysis of glycoeenin to PG(prog1ycogen
synthase(PGS)) and a GS-likt enzyme that glucosylates PG to MG and is referred to as
macroplycogen synthase(MGS )( Lomako et al.. 1 993).
The relationship between glycogenin. PG and MG was fmher established bp a
study of glycogen synthesis in rat brain astrocytes(Lomako et al.. 1993). Astrocytes
provide a versatile rneans of studying glycogen metabolism due to the rapid stimulation
of plycogrn synthesis and the marked limitation of synthesis. in that it only occurs in cells
previouslj. esposed to ammoniurn(NH,~).Glycogen is spnthesized rapidly whrn NH,'
treated astrocytes are exposed to glucose(Lornako et al.. 1993). PG synthesis frorn a
dvcogenin primer occurred by a glycogen synthase-like UDP-pic transglucosy lase
C
I
activity. and s~nthesisstopped at this point. This enzyme was distinguished from well
recognized GS by it's strong affinity for UDP-glc. It \vas found to be more inhibited by
XTP than was GS. and hencr
not used.
\as
narned proglycogen synthase(PGS). M e n NH,. was
glucose shoa-edan immediate appearance in PG followed 30 minutes latcr
by it's presence in .MG(Lomako et al.. 1993). The coincidental hl1 in PG label
rstablishes it as the putative precursor between the two. The process linking PG to MG is
catal yzrd b ) what has been termed as macroglycogen synthase(MGS). There is a rapid
increase in PGS activity especially in the presence of G6P but the rapid synthesis of PG is
not parallelled bu a significant change in MGS activity. MGS. measured with glycogen
as a primer. is active before the ceils receive glucose and changes linle during the period
of rapid PG gl~cogensynthesis. demonstrating that the initiation of glycogen synthesis is
not relatçd to a change in MGS activity(Lomako et al.. 1993). Therefore in the synthesis
of MG from gI!-cogenin via PG. the step between glycogenin and PG appears to he rate
lirnitingt figure 5).
-- BIOGENESIS OF GLYCOGEN
Macroglycogen Synthase
+ Branching Enzyme
w
Proglycogen Syn
+ Branching enzyme,
Macroglycogen
Initial
Glycogenin
--%/
Autocatalysis
Glucosyfation Self-glucosylating
Protein (SGP)
Mt 37 kDa
Mako-octaosylglycogenin
(Primer for proglycogen
synthesis)
figure 5: The new biogenesis hypothesis
From glycogenin-->PG-->MG.Glycogeiin autocatalytically adds glucose from UDP-glc
to its tyr- 194 and then an average of 7 residues to form protein bound maltosaccharides
that serve as the primer for the synthesis of PG by PUS distinct in properties h m MGS
that takes PG to MG. The priming chah of 8 glc residiias corresponds to the average
length of elongation but in achiality rcuiges tioiii 7 to 1 1 glc residues(l,omako, 1993).
In a similar study using rodent muscle. Huang et al. 1997 discovered that labelled
glucose first appeared in the PG fraction and later. with increases in glycogen s~ithesis.
appeared in the MG fraction. These results are equivalent to those of Stetton in i 960. In
quail ernbpo muscle it has been dernonstrated that proglycogen also functions as an
intermediate in MG degradation and. in one set of circumstances. represents an arrest
point in gl>-cogenbreakdotm which does not continue to glycogenin(A1onzo et al.. 199%.
Lomako et al.. 1993). Phenviephrine brought about a rapid degradation of MG to PG but
not bevond. suggcsting that. as in synthesis. separate processes control the conversion of
MG into PG and the conversion of PG into glycogenin(Lomako et al.. 1995). From such
studies. PG has bren proposed to be the stable intermediate for the synthesis and
breakdown of depot MG and that total glycogen oscillates. according to glucose supply
and energ! dernand- between the MG and PG(A1onzo et al.. 1995a. Lomako et al.. 1993).
It is presumrd that in the nutritionally dependent turnover of glyco_eenin tissues the
molecules cycle between PG and MG and are not normally degraded to glycogenin.
It could be argued that PG is not a distinct entity. rather it represents the TCA-
soluble fraction of glycogen molecules in vanous stages of growth. However. studies
have show-nthat this is not the case. In fresh resting muscle from a rabbit there are no
detectable glycogen molecules having Mr 4 0 0 000(Lomako et al.. 1993). As welt, the
elcctron microscop!. evidencr of only two sizes of glycogen granules. one large and one
small. supports the hypothesis of two distinctly different types of glycogen
molecules(Friden et al.. 1989). Furthemore this study has suggested up to 5 subcellular
locations for granules and has also provided data showing that sorne locations are not
depleted when others are. These concepts of different subcellular locations or pools
suggest a significantly more complex glpcogen metabolism pathway. It is unknow if
these ideas apply to human muscle or if MG and PG c m be rneasured reliably. Hence
sprcitic assays for these distinct components must be devised and tested.
Supercompensation and CHO supplementation
Studiss have provided direct evidence that muscle glycogen synthesis represents
the prima^. pathuay for non-osidative glucose disposa1 in both normal and diabetic
subjects(Beck-Nielsenet al.. 1992. Shulrnan et al., 1990). The main substrate for
repletion of the muscle glyogen store is blood glucose derived from hepatic glucose
output as wII
as from CHO consumption during the post exercise period(Shu1man et al..
1990). Muscle glycogen resynthesis takes precedence over restoration of liver glycogen.
and rvcn in the absence of CHO intake afier exercise it occurs at a low rate( I -2rnrnoVkg
i n v l h ) (Burke. i 996 ). It is known that there is a fibre type difference in both glycogctn
synthesis and depletion. Fast twitch(FT) muscle fibres generally contain more glycogen.
synthesize glyogen faster. and deplete during intense exercise while moderate to low
intensity ssercise depletes glycogen from slow twitch(ST)(Vollestadet al.. 1989).
Muscle glycogen stores are quite variable depending on training status. exercise
patterns and prwious diet. In untrained. sedentary individuals muscle glycogen stores
range between 80- 1 00 mmolkg ww( Hultman. 1967). Training individuals who are
esrrcising dail!. and consuming a normal diet(50% CHO) ~picallyhave muscle glycogen
stores of about 130-1 35 mmolkg rw(Hu1tman. 1967). The depletion of body CHO stores
pla>.sa major rols in fatigue during prolonged moderate-to-high intensity exercise. hcnce
sufticient stores of muscle glycogen are essential for optimum performance during
intense prolongrd exercise. and repeated bouts of exercise of an anaerobic nature(1~.
1991 ). Studies have reported improvements in endurance capacity and esercise
performance whrn muscle glycogen stores are enhanced prior to high intensity
rxrrcise( Bergstrorn et al.. 1967). This practice referred to as glycogen loading, is
common in the athlrtic population. Because of the paramount importance of muscle
cI~.cogsnduring prolonged. intense exercise. a considerable amount of research has been
C
conducted in an attempt to design the best regimen to rlevate muscle glycogen stores
prior to competition and to determine the most effective means of rapidly replenishing the
muscle glycogen stores afer exercise.
It is widely accepted that CHO supplementation irnmediatelp post exercise
increases glycoyen resynthesis(Gaesser and Brooks. 1980. I w . 1991. Zachwieja et al..
199 1 ). With the cessation of rxercise. glyogen repletion takes place rapidly in skeletal
muscle and c m result in glycogen lcwls higher than those present before
csrrcise( Friedman et al.. 199 1. Hultrnan. 1967). Previous resrarch suggests that providing
~
body weightih is sufficient to mavimize glycogen
approximatcl! 0 . 7 0 ~glucosekg
synthesis rates.( Friedman et al.. 1991. Blom et ai.. 1986). Maximal rates of post-exercise
muscle glycogen storage reported dunng the first 12 hours of recovery range from 5-1 0
rnmolikg uwlh ( Blom et al.. 1986. IV. 199 1 ). Coyle has suggested that at a mean rate of
5-6 mmolkg uwlh. the time required for muscle glycogen to reach normal resting levels
of 300-350 mrnol glucosyl unitskg dw after prolonged moderate intensity exercise is 14
hours provided approximately 500 to 700 g of CHO is ingested(Friedman et al.. 1991 ).
Generally 48-71 h are required to reach supercornpensation(Saltin and Karlsson. 1971 ).
It has been dernonstrated that muscle glycogen increases very graduallp to abovenormal lei-els(supercompensation ) in the days following depleting exercise. providrd a
high CHO diet is followcd(1vy et al.. 1988). It is not known what happens to the relative
concentrations of .MG and PG with supercompensation. Not onlp would this information
b r practical tiom a training perspective but it is useful to studp the metabolic regdation
of the two pools as altering the substnte may result in ditTerent rares of synthesis. This
would not only allow investigation of 2 pools/foxms but also would give insight into the
concept of 2 GS enzymes. Although Jansson. 1980 attempted to measure changes in
soluble glyogen(MG) with diet and exercise her rneasurements were not made
consistently and didn't include corresponding measurements for the insoIuble(PG) form
as e l . These two forrns. as potrntial distinct metabolic pools. have not been esaminrd
in human muscle and generally the rodent methods have used large tissue sainples. which
are unreasonable xhen w o r k i n with human muscle. Albeit indirectly. Jansson and others
have reported acid soluble!insoluble fractions and Fnden was able to demonstrate using
clectron microscopy that therr were two different size glycogen particles. Consctquently.
it is highly unlikely that one of these forms is simply an extraction procedure artifact.
Esperimen ta1 Objectives:
The primary objectives of this thesis were to develop an accurate measurement
technique for the two pools of muscle glycogen. and to test if the pools are measurable in
human muscle. The research in this area is quite limited. hence there are a varictty of
fundamental questions that have not previously been addressed. The major goal was to
determine if a relationship benveen MG and PG exists in human muscle. This potential
relationship could be examineci in many ways and it was felt a particularly useful way
would bs to study the respnthesis of the two glycogen pools using different levels of
dirtary CHO. The specitic intcnt and purpose of each study is further clarified in the
upcoming chapters.
2.0 STUDY 1: METHOD COMPARISON
Introduction
The biopsy technique has been used for many years to measure muscle
metabolites including glycogen and it is vital that the analyses employed to masure
dycogrn give an accunte representation of the muscle glycogen stores. Glycogen is
C
found in granules and ihus there is the potential for a hi& degree of variability beiwetrn
biopsy sarnplrs. An electron microscopy study of glycogen granules by Fnden et al.
dçmonstrated that there are two dit'ferent particle size populations in human skeletal
muscle(Fridrn et al.. 1989). These two separate populations of glycogen particles were
stored in h r different locations within the muscle ce11 and the authon speculated that
difkrent granules could be two t o m s of the glycogen molecule that were utilized at
different timrs from distinct locations. Despite these potential sources of variation. Essen
and Henriksson, 1974. Harris et al., 1974 and Hultrnan, 1967 have shown that in human
muscle. the determination of total glycogen is very reliable and that muscle biopsy
samplss are representative of the whole muscle.
Traditionally there have been tvo methods for mrasuring glyogen. either by acid
hydrolpis(AC ) ( Passoneau and Lauderdale. 1971. Passoneau and Lowy. 1993) or bu
snzymatic(EZ)hydrolysis(Bergmeyer. 1974). Unile it has been stated that the variability
of the anal'ical
technique is about 210- 13 mm01 glucosyl unitskg dry weight(dw)
(Harris et al.. 1974). no detailed cornparison of AC vs. EZ has been published
determining if the methods are comparable. Bergmeyer has suggrsted that EZ of purified
plycogen yives values which are 5% higher than those obtained with AC(Bergmeuer.
1974). ahile Passomeau claimed that the two methods give equivalent
results(Passonneau and Lauderdale. 1974). Neither author provided data to substantiate
their claim. In the only direct cornparison of AC and EZ that we are aware of.
Jansson(Jansson. 1980) performed the AC and EZ methods in hurnan muscle over a wide
concentration range and stated rhat there was no systematic difference between the values
obtainrd b!- eithrr method. Hou-ever. no data were provided to validate this tinding.
Prim to Jansson's study(Jansson. 1980) it was cornmon for glycogrn
mrasurements to be made on the precipitate that had bern pretreated with perchloric
acid(PCA) ( Karlsson. 1971. Passomeau and Lauderdale. 1974. Passomeau and Low-ry.
1993. Van Heijningen et al.. 1955). This method was thought to make the most efficient
use of a muscle sampie by allowing for the precipitation of proteins and extraction of
PCA soluble metabolites while also permitting the determination of glycogen on the same
piece of tissue. However. it has been reported that only a portion of the total glycogen
\vas rxtracted during PCA treatment(Hermansen and Vaage. 1977. Hultman. 1967.
Karlsson. 1971. Roe et ai.. 1961 ). Jansson compared this method to AC and EZ and
documentcd that about 1 5 - 3 % of the plycogen was PCA soluble and hence an>mrasurement of glycogen only from the precipitate would underestimate the total. These
findings of Jansson discouraged the analysis of PCA precipitate to determine total
glycogen but did not stimulate further research regarding the possibility that these two
fractions represrnted two physiological pools.
Rrcrntly. while identit'ying and studying glycogenin. the protein core OFthe
glycogen rnolctcule. investigators(Alonzo et al.. 19953. Lomako et al.. 1991. 1993)
realized that there were two pools of glycogen in rodent skeletd muscle and other tissues
such as liver and hem. This work was performed solely on rodent resting muscle and did
not measure esercise metabolism or human tissue.
No one has systernatically compared the two traditional methods nor have these
methods e \ w bren compared to the MGPG determination for glycogen. Furthemore PG
and MG concentrations have not been esamined in human muscle under any
circumstanccs. Bçfore studies into the physiological response and regulation of these
pools can be undertaken a thorough rvaluation of these methods is required.
Purposes
The purposes of this study were to:
( A)compare AC
and EZ determinations for total glycogen
iB)establish if MG and
PG exist in human muscle and if they can be measured reliably in
a biops>-s i x sample
( C kvaluatr the reproducibility of the AC. EZ. PG and MG determinations
(D)comparethe AC. EZ and PG+MG values for total muscle glycogen.
Methods
In order to evaluate precision and variability and compare the various rnethods
(AC. EZ and PG+MG separation) of measuring muscle glycogen. a varie- of approaches
u-rre employed. The muscle sarnples were collected dunng a number of difTerent studies
and conditions ranging from exhaustive exercise to resting muscle. Every samplr in each
cornparison was i -3 mg dw( ie. biopsy size sampie) and rneasured in duplicate.
COMPARISON 1
The first cornparison used rodent muscle since most of the initial MG. PG data are
dcrived from this species and because large muscle samples could be obtained. Muscle
samples of various known glycogen concentrations from rodent hindlimb were fieeze
drird. combined. and mixed thoroughly in order to create three large pools for repeated
measures. One low concentration pool and two normal pools whose total glycogen values
uere averagrd. .A total of 45 sarnples were analysed. Independent samples (n=15) of the
low pool and each of the normal pools were measured for glycogen by AC(n=j) or
EZ(n=5) or .LlG+PG(n=5).
COMP.4RISON 11
Human muscle has a much wider range of muscle glycogen concentration than
rodent muscle. Hence human muscle samples. which from previous determination werr
known to have a wide range of total glycogen concentration. were combined and mixed
thoroughly to givr three pools of distinctly different glycogen concentration. A total of
18 samples w r r analysed for this cornparison. Independent samples n=6 of each pool
n w r measured for glycogen using AC(n=3) or MG+PG(n=3).
COMPARISON III
Individual human muscle samples were anaiysed using each of the methods.
These were studied to compare the AC. EZ and MGRG determinations when measuring
total plycogen concentration in small biopsy samples. Due to limited size, not al1 sarnples
could be analysed with ail 3 methods. MGPG was performed on every sample (n=50).
EZ on 43 samples. AC on 20 samples. and of the latter. 15 were analysed by EZ as well.
COMPARISON N
Human muscle samples (n=26) were obtained from an independent laboratos
where they had previously been analysed for total glycogen by EZ(Tarnopo1sky et al..
1997). These samples were subsequently measured for MG and PG in blind fashion. and
then compared to the pnor independent EZ data.
COMPARISON V
Rat muscle was used to determine the percent recovery of a given amount of
exogenous glycogen using the MG+PG separation. A single pool of muscle was used and
a portion was analysed for MG+PG. A glycogen solution was made fiom oyster
elycogen(Sigma G-8571) and a portion of this solution was analysed for glucosyl units.
C
Muscle samples had a known quantity of this exogenous oyster glycogen solution added
dunng the PCA extraction procedure(n=9). After the samples were assayed for glucosyl
units the previously detemined MG+PG concentration was subtracted from the total
concentration determined for the exogenous plus muscle sarnple. In order to find the
percent recovery. this value was divided by the exogenous gly-cogen concentration and
then multiplied by 100.
Analysis
The AC glycogen method used was adopted fiom Passonneau. 1974 and is as
follows. Freeze dned muscle samples of between 2 and 3 mg were hydrolysed w-ith ZM
HCI and then heated for 2 h at 85-90'C followed by neutnlization with 2M NaOH. The
rstncts were then analysed in duplicate fluorometrically using the Ber-gneyer. 1971
rnethod for determinine glucosy1 units.
The EZ method used was the amyloglucosidase method: 0.1 M NaOH was added
to 2-3 mg d q muscle and samples were incubated for I O min. at 80'C to destroy
'background' glucose and hexose monophosphates. Then the samples were neutralized
by a combination of 0.1 M HCI. 0.2M citric acid and 0.2M Na,-. HPO,. Amyloglucosidase
was added and samples incubated for 1 h at room temperature while glycogen degradation
took place. The samples were analysed in duplicate spectrophotometrically at 34Onrn
using the method of Passonneau and Lowry. 1993.
The rnethod for determination of PG and MG fractions was based on that
descnhed by .\lonzo et al.. 1995a and Lomako et al.. 1993 and is also similar to that
described by Jansson. 1980 for acid soluble and. insoluble glycogen. (ce cooled 1 . j M
PCA(ZO0 ul) was added to 1. W m g freeze dried muscle samples in 5ml Pyrex tubes. The
muscle was pressed against the glass tubes with a plastic rod to ensure al1 the muscle was
esposed to acid. The extraction continued on ice for 70 min. The samples were
centrifùgrd at 3000 rpm for 15 min afier which 100 u1 of the PCA supernatant was
removed. placed in Pyrex tubes and used for the determination of MG. The remaining
PCA was discarded and the pellet was kept for the determination of PG. 1ml of 1M HCI
\vas added to the MG and to the PG sarnple: the former was vortexed. while the pellet of
the latter was pressed against the g l a s with a plastic rod. The tube weights were then
recorded. The tubes were sealrd with fitted glass stoppen and a11 of the samples were
placed in the water bath( 100' C ) for 2 h after which they were re-weighed and an- change
>50 u1 was rectified with the addition of dH,O. The samples were then neutralized with
2M trisma base. vortexed. centrifuged at 3000 rpm for 5 min and transferred to rppendorf
tubes for analysis of glucosyl units using the method of Bergmeyer. 1974 or stored at -
80°C.Subsrquent determination of muscle glycogen on frozen MG or PG extracts gave
the same values as rresh extract.
Statistics
The total glycogen for the MGPG detemination was obtained by surnming the 2
%actions.The fraction of MG to PG was determined by dividing the concentration of the
MG by the summed total (MGIMG+PG) and reported as a percent (X100%). The
coefficient of variation(CV) was obtained by the formula SD/mean. Linear regression
analysis was pertonned within cornparison sections and 95% confidence intervals wrre
used to determine agreement between analysis methods. T-tests were used to determine
difference between means of total glycogen for each of the 3 methods. In al1 cornparisons
di fferences were accepted as signiticant at the 0.05 probability levzl.
Results
COMPARISON 1
The important findings of this section were that the three measurements were
reproduciblct u-ith rat muscle and PG+MG \vas reliable for -biopsy-sized' samples.
Generally the coefficients of variation were within the published range. although the CV
increased with lower total glycogen concentration(Table 2). However. the standard m o r s
were small and the higher CV was a reflection of the low glycogen concentration. One of
the *normal' glycogen pools had a high CV for AC due to the value for one of the 5
sarnples being unusually high.
When cornparhg the AC and EZ rnethods a positive. linear relationship was
found( r=l .O) and there was no signifiant difference between the values detemined by
the two methods. The MG+PG detemination was not significantly different from the
values from either the EZ or the AC.
Table 2: Cornoarison of EZ. AC. and MGlPG for rodent muscle glvcopen*
Measure
I
Total Glycogen Pool
MG
LOW
3.9
0.9
0.4
23.0
NORM
1 11.6
1 1.4
1 0.6
1 10.7
LOW
(54.1
1 3.3
1 1.5
( 6.1
NORM
116.0
13.4
I
5.5
10.6
-
MG+PG
EZ
LOW
58.0
4.0
1.8
6.9
NOM
139.2
13.9
5.8
10.0
LOW
58.3
4.3
1.9
7.4
NORM
121.3
3.7
1.7
3.1
NORM
1 122.8
1 29.6
1 13.2
1 24.1
* ic. SD. SE. and CV represent the mean. standard deviation and coefficient of variation
for each value. n=5 for the LOW pool and n=IO for the N0RIL.I pool. Each determination
being performed in duplicate. All values are mmol/kg dw except CV which is reported as
a percentage.
COMPARISON II
With the pooled huma. muscle the repeatability was similar to that in comparison
I (Table 3 ) . Over a wide range of total glycogen concentrations the SD and CV of the
MG and PG determination of human muscle were within the range reponed by Harris et
al.. 1974. The MGiPG detenination demonstrated a strong correlation with the AC
methoc!( r= 1 ) and the line of identity with dope equal to one Fe11 within the 95%
contidencr interval. illustrating that there was no difference between methods(Rgure 7).
There was no significant difference in the total glycogen values determined by AC and by
MG+PG.
COMPARISON III
This comparison of muscle glycogen for individual biopsy sarnples of Iiurnan
muscle present rvidence that the MGPG determination is comparable to the AC and EZ
mrthods( tigures 8.9.10). The correlation coetlïcients as well as the line of identity and
regression Iinr not being signiticuitly diffierent. demonstrate how similar the three
methods w r e in giving a precise measurement of total glycogen. The correlation
coeftkient for EZ vs MG+PG(n=43). AC vs. MG+PG(n=r>O)and EZ vs. AC(n=15) are
0.96. 0.99. and 0.97 respectiveiy. The ratio of MG to PG varied greatly in these individual
samples with MG representing frorn 6 to 38% depending on total glycogen concentration.
The percentage of MG to PG is not constant. as the total concentration of muscle
-
dvcogen(MG+PG) increased so did the percentage of MG( figure 12). The relationship is
C
curvilinear. There appears to be a cluster of MG concentrations ranging fiorn 4 to about
50 mm01 glucosyl unitskg du. when the total glycogen concentration is less than 300. At
highçr glycogen concentrations the PG concentration isn't increasing a great deal but the
MG is the fraction that continues to grow and becornes a greater contributor. yet never
equals that of the PG fiaction(figure 1 1 ).
Table 3: Cornparison of EZ. AC. and MGmG for human muscle dycogen*
Total Gtycogen Pool
MG
LOW
iMED
HIGH
IIMG+PG
LOW
143.8
LOW
1 48.8
* 2. SD. SE. and CV represent the mean. standard deviation and coefficient of variation
for each value. n=j with each determination being performed in duplicate. Ail values are
mmollkg da- rxcept CV which is reported as a percentage.
regression
95% CI
line of identity
O
100
200
300
400
MG+PG(rnmol glucosyl unitsikg dw)
figure 7: MG+PG vs AC Pooled muscle Samptes. A cornparison of Total Muscle
Glÿcogen in pooled hurnan muscle of 3 distinct concentrations usine AC and MG+PG
methods(Comparison II) y 1.O 1 x-8.38- r=1 .O. n=9
regression
95% CI
line of identib
O
t/
O
-'
I
I
1
l
1
1
700
200
300
400
500
600
AC(mmol glucosyl unitslkg dw)
figure 8: AC vs MG+PG Individual Muscle Samples. A cornparison of total muscle
d y c o g n in human muscle using AC and MG+PG methods(Comparison III)
.y=0.92x+3.8. ~0.99.
n=X.
C
100
200
300
400
500
600
EZ(mmol glucosyl unitsikg dw)
figure 9:EZ vs. MG+PG IndMdual Mususde Samples. A cornparison oftotal muscle
glycogen in h m a n muscle using EZ and MGtPG(Comparison III)
y=û.84~+30.0,&I.96,n=43.
regression
95% CI
line of identity
100
200
300
400
500
600
EZ(mmol glucosyl unitsikg dw)
figure 10: EZ vs AC Cornparison of Traditlonal Measurements. A cornparison of total
muscle glycogen in h m a n muscle using the traditional measurement methods, EZ and
AC(Compa&on III). y=0.87x+14.6,~û.97,~ 1 5
COMPARISON IV
In this cornparison human biopsy samples were analysed for MGRG and then
compared to EZ determinations made by an independent laboratory. Again the MG+PG
total glycogen correlated well(r=.97) with the previously rneasured values based on the
EZ mrthod( tigure 13 ). Again there was no signiticant di fference between the line of
identit! and regression line.
COMPARISON V
This part of the study was designed to test how much of a given arnount of
glycogen is actuallp recovered from the MG and PG extraction procedure. A 10 ul sample
of a 43 uM solution of exogenous glycogen was added to a measured amount of rat
muscle and the glycogen concentration was compared to the concentration of an
independent aliquot of the same pool. The percent recovery avenged 105 28.5% (n=9)
and the rxogenous glycogen was entirely recovered in the MG portion.
O
50
1 O0
150
200
250
MG(mmol glucosyl unitskg dw)
figure 11: MG vs PG over a wide range oftotal glycogen concentrations. Relationship
-
between MG and PG.n=50
MG(mmol glucosyl unitskg dw)
figure 12: MG vs MGtPG over a wide range oftotalglycogen concentrations.
Relationship between MG and total glycogen. n=50
&/y
regression
--
1O0
200
line of identity
300
EZ(mrnol glucosyl unitslkg dw)
figure 13: EZ vs MG+PG. Cornparison to independent laboratory measuremmts. A b h d
cornparison of previously determined EZ total glycogen measurements from an
independent laboratory and MGtPG rneasurments of total glycogen.(ComparisonIV)
y=û.93x+19.2,r=0.97,1~26.
Discussion
This study was designed to compare the 2 well-accepted methods of measuring
the total glycogen of a muscle biopsy sample over a wide range of total glycogen
concentrations. It was also conducted to determine if MG and PG c m be measured in
biopsp s i x samples and if it can be applied to human tissue. The precision of this new
technique was also evaluated and compared to that of the 2 accepted methods. This study
yoests
presents the fnt deteminations of MG+PG for human muscle and it strongly su&,
that there are t a o foms and probably two metabolic pools of glycogen in human muscle.
In nt rnuscle(cornparison 1) there was no statistical significant differences in total
glycogen between the AC and EZ methods. ln addition. the AC and EZ methods were
found to be rquivalent in reproducibility within a pool(Tab1e 2). Similady with human
muscle we found that there was no systematic difference in the two rneasurements as the
line of identity with slope of 1 was within the limits of the 95% confidence intenal(tigurr:
10). This is in agreement with a comment made by Jansson. 1980. Harris et al.. 1974
deterrnined that in the measurrment of muscle glycogen. there was a variation of 8-43
mm01 glucosyl unitskg dw dur to analytical procedure error alone. The standard
deviation for routine analysis of muscle glycogen sarnples taken during exercise
experiments was found to be between 1 O and 13 mm01 glucosyl unitskg dw(Han-ïset al..
1974). Our data for rodent and human muscle glycogen are in agreement with these
tindings. Therefore each of the 3 methods give low and similar CV which are comparable
with the literature.
The reproducibility for the pooled human muscle samples is in agreement with
other studies and the variances(Tab1e 3) in the present study were within the published
range(Harris et al.. 1974). The repeated measurement of MG. PG and MG+PG on hurnan
rnuscle(n=9) had a CV range from 0.2% to 2 1%. The majority of the CV values
displa)çd in Table 3 lie betwern 3 and 6%. Essen and Henriksson. 1974 found that there
u s a CV of about 6.3% when rneasuring glpcogen uithin single muscle tibres. When
comparinp the human muscle total glycogen for the MG/PG determination for the low.
medium or high concentrations to those determined by the .AC method thçre was no
statistical signi ficance between the values. The correlation between these methods was
1 .O with slope=l .O1 and the line of identity almost identical to the regression line .
demonstrating that the MG+PG technique is as precise as the traditional AC and EZ
methods. The MGPG detemination for individual muscle samples(n=20)compared to
AC also gave extremely high correlations. Thus the 3 rnethods are comparable.
The MG:PG percentages calculated from cornparison II suggest that as the total
d y c o ~ r nconccntntion rises so does the percentage of MG. We found that at a MG+PG
C
concentration of 43.8 mm01 glucosyl unitdicg dw the PUIG:PG ratio was 1387. -4s the
total increased to 1 8 1.8 mm01 glucosy1 unitskg dw the ratio increased to 19:8 1 and to
2 5 7 5 as the total concentration reached 340.2 mm01 glucosyl unitskg dw. The
percentage of MG in the high concentration pool was significantly higher than the
percentage in the low pool. This is in accord with Jansson's results which displayed an
increasing percentage of soluble glycogen as the total concentration increased. She found
that PCA soluble muscle glycogen constituted 25% of the total glycogen content \vas
<
350 mmol/kg dw and increases in total glycogen above that concentration seemed to be
mainly due to the soluble tvpe(MG). In the present study. the individuai muscle sample
with the highest total glycogen (519.6 mm01 glucosyl unitskg dw) the ratio %as 38:6Z.
Alonzo et al.. 1995a also showed a greater percentage of PG relative to MG(mo1ar
percentage:8j0/o PG to 15% MG) in resting rabbit muscle. Interestingly. Fiden et al.
reported that two populations of l y c o p n particles were clearly distinguished and of the
IUpaniclrs counted in a pool in the intermyofibrillar space about 76% of them werr of
the srnaller particle size(Friden et al. 1989). This consensus suggests that MG+PG can br
detected by rlrctron rnicroscop).
Glucose analyses on the PCA extracrs of hurnan muscle pnor to hydrolysis
vielded low values(0.11-3.6 1 mmolkg dw)(Jansson. 1980). This demonstrated that
glycogen was not hpdrolysed into glucose residues dunng the PCA extraction procedure.
Jansson also dernonstrated that the relationship between PCA soluble and insoluble
-
alvcogen kvas not influenced by strrngth of the PCA in the O . M M range or the type of
C
acid(PCA vs. TC.4). Nor was it affected by the freeze drying procedure or the weight of
the samplcs in the range 0.2-?mg. The benefit of using the MCcPG determination is in
the ability to separate the two fractions of muscle glycogen and hence study the
mrtabolism of each one individually.
There are reports of di fferent pools of glycogen dating back to 1900 whtn
Nrrking(Stetton and Stetton. 1960) and later Willstaetter in 1934 concluded that tissue
glycogrn appeared in two forms: an acid-extractable or free form and an acid-nonestractable or protein- fixed fom. It was not resolved whether the glycogen-protein
comple~rswere artifacts or whether the? constituted a physiological entity. Since then.
investigators have shown different sizes. different concentrations and different solubilities
of glycogen molecules. Integrating the results of past studies suggests that data have
consistently pointed to the existence of two different pools of muscle glycogen with the
smallrr molecular/granular form being most common.
From the lirnited human data in the present study. it is speculated that the PG is
the srnall and dynamic. intermediate t o m of muscle glycogen while MG is the larger
storagr f o m which appears to increase on a relative basis as the total glycogen increaxs.
It is possible that when the glucose environment is favourable and the PG has reached a
cntical limit. a portion is synthesized into MG and it is conceivable that the 2 pools ma?
eventually equilibrate. With this new separation technique it is now possible to study
each pool of glycogen exclusively in ordrr to understand how the fractions change under
conditions of breakdown and resynthesis.
In surnmary the AC and EZ deteminations are not systematically different. IMG
and PG do exist in human skelrtal muscle and c m be measured accurately in biopsy size
sarnplcs. The .AC. EZ and MG-PG deteminations are reproducible and give the same
values for total muscle glycogen.
3.0 Study II:
The Effect of dietary CHO on the resyntbesis of macroglycogen and proglycogen
in traduction
In the past it has been demonstrated that it is possible to increase muscle glyogen
stores to abow normal concentration ie. supercompensation(Bergstromet al.. 1967).
This has b e n s h o w to be beneficial to athletes who are involved in multiple dail?
training bouts. or compete in long term. intense sxercise such as marathon running(Saltin
and Karlson. 1971 ). The most important prerequisites for achieving glycogen
supercompensation by the classic method are having the subjects deplete the glycogen in
the involved muscle groups and then eat a high CHO diet(Goforth et al.. 1997). The
glycogen repletion process is biphasic. exhibiting a rapid early phase (-34h)and a slow
phase lasting for several days(Gofonh et al.. 1997. Bloch. et al.. 1994. Gunderson et ai.
1996). It has been s h o w that it takes 24 h for muscle glycogen to return to normal levels
after exhaustive exercise and if a high CHO diet is continued the supercompensated Ievel
çan be achievrd in the following days. It has been proposed that GS activity plays a key
role in detemining the rates of glucose uptake and glycogen synthesis in muscle(Bak and
Pedersen, 1990. Bloch et al.. 1994 and Blom et al.. 1986). However. the increased GS
activity O bserved immediately aller exercise has retumed to normal before this
supercomprnsation has occurred(B1och et ai. 1994. and Kochan et al.. 1979 ).
The discovery of the increasing concentration of the MG pool and the relatively
small growth of the PG pool at high concentrations in Study 1 lead to the belief that there
uas a point at which syithesis was solely in the MG pool and that perhaps the synthesis
in these pools correlates with the biphasic repletion pattern. Based on these findings from
snidy I and the observations of Jansson. it is proposed that supercompensation is due to
increased synthesis of MG. It is hypothesized that the smaller PG form will dominate the
intital resynthesis phase and will be the predominant form until the concentration reaches
the normal resting range of350-400 mm01 glucosyl unitsikg dw. In order to address these
hypotheses . the resynthesis of MG and PG was examined for 48h following gly-cogen-
depleting escrcise and employed high and low CHO diets to manipulate the repletion
rates.
Methods:
Su biects:
The experimental prot oc01 was approved by the University of Guelph's Human
Subjrcls Cornmittee and nine male volunteers gave their wntten consent to partic i patr in
the studt. This consent was precrded by a subject screening questionnaire. as well as a
verbal and witten esplmation of the expenmental protocol and medical
procedures(Appendix A ). The nine males were al1 recreationally active. The range and
mean 2 srm of the subjects age \vas 19-28 yr. ( 23.12 1 .Z ). height 170-191 cm. ( 180.6+
1.9 ). weight 68-89 kg. (77.61 2.2) and V02max 53-68 mI/kg/min(58.7
1.7).
Table 4 : Subiect Characteristics
Subject
age(yrs)
39
--
height(crn)
weight(kg)
33
--
180
183
175
89
73
77
19
180
19
27
183
183
77
--
191
28
180
21
1 70
73
80
73
85
80
68
28
V0,max
Power
(m
Output(?O%)
230
59.6 1
6 1.52
219
64.18
240
67.86
54.60
53.O6
250
58.20
230
54.56
54.8 1
328
184
180
151
I
The nine. male subjects completed 2.5-day trials separated by 2 weeks. with the
percentage of diet-
carbohydrate ingested during a 2 day recovery as the only parameter
changed. The diets were assigned using a randomized cross-over design: high
carhohydrate(HC) diet(75% of rncrgy intake) and low carbohydntr(LC) diet(3??/6of
e n e r g intaks ).
PRE-TRIAL PROCEDURES
V 0 : mas testing
The subjects undement an incremental maximum oxygen consumption (VO,
m m )test on a Quinton Excaiiber electronically braked cycle ergorneter. Following a 3
minute warmup. the workload was increased every 2 min until the subject was unable to
maintain the required pedal frequency for greater than 15 seconds or volitional e'rhaustion
was reached. The subjects ivere encouraged by the researcher and respiratory variables
wrre drtrrrnined at each exercise intensity. Expired gas samples were analyzed for
fractions of 0, and CO, with an applied Electrochemical S-3A O>analyzer and a
Sensormrdics LB-2 CO, analyzer. Expired volume was determined with a ParkinsonC o w n volumeter. The analyzers were calibrated with gases of knom-n concentrations.
The volumemeter was calibrated with a 3 1 calibrated syringe. From this test. a pou-er
output equivalent to 70% VO: mêu was determined for the experimentd triais.
Dietary Analysis
The subjects were required to keep a 3 day diet record on 2 weekdays and 1
weekend da! in order for the inwstigator to complete a full analysis of their daily dietary
intake(habitua1intake: Table 5 ) . This information was later utilized for designing
individual diets ensuring that intake was as usual and foods normally consumed were
included.
Habituation Ride
A practice ride or habituation ride at approximately 70% of VO: mau kvas
cornpleted 2 d e s prior to each of the two data collection trials(tigure 14). This ride
s e n r d 3 purposrs: tirstly. exercise workload intensity. secondly. familiarization to the
workload and esprrimental protocol. and thirdly. to lower the muscle glycogen in
preparation for the subsequent data collection period.
Dietaty Control
For the two days following the habituation ride and prior to the data collection. a
weighed. prepackaged rnixed diet(contro1 diet: jj%CHO. 30% fat. 15% protein) was
supplied to the subjects. This dirtary control in combination with the practice ride
rnsured that they would be starting the trials with similar levels of glycogen. This diet
was identical berween trials and was isoenergetic with each individuals habitua1 intakr. In
addition. the subjects recorded and followed the same recreational exercise regimc during
the 2 days before rach data collection. Subsequentlp. following a prolonged exhaustive
sxercise(drscribed below) they werr provided prepackaged meals for the ensuing 48h and
Kerr instructed to eat solely the food supplied. Each subject received a high CHO diet
providing 75% of enrrgy fiom CHO in one trial and in the other trial a low CHO diet
which provided 32% of energy from CHO(Tab1e 5).
Table 5: Dietarv Sumrnarv*
HABITUAL
LOW CHO
HIGH CHO
Energy(kcal/d)
3 109.7 2 183.0
3211.8264.7
3263 + 95.9
CHO(g)
404.5 2 33.9
261.628.3
640.2 + 18.5
PROTEM(g)
130.1 + 15.1
155.9 2 4.8
77.6 2 3.0
FAT(g)
104.1 + 8.2
173.7 2 3.3
53.1 22.1
*Al1 data are presented as mean SEM
Experirnenta1 Protocol
The entire experimental protocol is illustrated in figure 14. Subjects arrived in the
morning to the testing centre and completed an exhaustive glycogen depletion ride at 70%
of their VO- ma\. Exhaustion was determined at the point when subjects could no longer
maintain the prdalling frequenc). at a constant resistance. This exhaustive exercise kvas
rmployd because of the data that cxists demonstrating that muscle glycogen stores must
be depletrd tint in order to sec muscle glycogen concentrations rise above normal or
"suprrcomprnsate"(Bergstrom et al.. 1967. Hultman. 1967). Cycling esercise uas
çhosen on account of the liieraturr demonstrating that plycogen supercompensation is
b a t achirvçd when the esercise is largely concentric and the mode of exercise does not
disrupt the mechanisrns of glycogen synthesis(Goforth et al.. 1997).
At exhaustion, a muscle biopsy(EXH)was taken from the Cirstzts lateruli.~
muscle
using the percutaneous needle biopsy technique. A catheter was placed percutaneously
into a media! antecubital vein from which a blood sampie was taken and a nomal saline
drip(0.9%: 308rnOsdl) was started. Afier the first sample. blood was drawn every 30
minutes for 4 hours. During the 4 hour penod the subjects were allowed water [id
fihirirrn. however those who were on the high C C 0 trial were given 5OOml o f a
maltodextrin/dextrose solution containing 70g of CHO(Gatorlode@:QuakerOats.
Barrington. Illinois) every hour for the first 3 hours.
.A second biopsy(4B) \vas taken at the 4 h post recovery. The subjects returntrd the
nest day approximately W h(24B) attrr the EXH biopsy for a third sample and again
approsimately 48 h afier the EXH biopsy for a final biopsy sample(48B). Again during
this 48 h period the subjects maintained a strict HC or LC diet. The trial was reprated 2
rvecks latrr wi th the same subjects ingesting opposite diets.
Analyses:
Blood
Blood sarnples were imrnediately separated into 2 aliquots: 3 ml were transferred
to a non-heparinized tube. ailowed to dot. and s e m \vas stored for subsequent insulin
msasurements. The other aliquot was transferred into a sodium heparinized tube and a
100 ul sample of heparinized blood \vas added to 1 ml of 0.6 M perchloric acid and
crntrihged with the supernatant collected for whole blood glucose analysis. Ail smples
were stored at -80' C. The whole blood extracts were analyzed in duplicate(Bergermeyer.
1974) for glucose with a Perkin Elmer LS-50 fluororneter. Serurn insulin concentration
was rneasured quantitatively using an "'1 radioirnmunoassay kit.(Coat-A-Countll. DPC:
Los Angeles. CA)
Muscle
Muscle biopsies were stored at -80' C until they were fieeze dried and dissrcted
tiec of visible blood. comective tissue and other non-muscle elements. A 1.5-3 mg
portion of freeze drkd muscle was extracted following the MG and PG extraction
procedure descn bed previousl y followed bp e n m a t i c measurement of glucosyl
units(Bergmeyrr. 1974) and reported as mrnol glucosyl unitskg dw.
Statistical Anaiysis
Total Glycogen(TotG) was reported as the sum of MG+PG while the percentage
of MG (and PG) was determined by dividing the concentration of each pool by the TotG
and multiplying bu IOO%(MG/TotG X 100%). The net spnthesis raies were calculated b j
taking the diffrrence between the concentration at two time points and dividing b!. the
time intend in hours(eg. 4B-EXHA h). Two-way (time and treatment) analysis of
variance(ANOV.4) for repeated rneasures were used to compare the blood glucose.
insulin and MG. PG and TotG concentration data from the two trials. When these
analyses revealed significant differences. a Tukey post-hoc test was used to locatr the
di fference. Di merences were considered to be significant if p<O.Oj. Al1 muscle glpcogen
results are presented as means 2 SEM for 9 HC and 8 LC subjects. as one subject a-as
sick on his LC trial day. Blood glucose and insulin concentrations are also reported as
means 2 SEM. There is one less subject in these data than in the glycogen dur to the
inability to attain blood samples tiom one of the subjects.
Results
.Muscle : MG and PG concentrations
The TotG at EXH was 7921 5.6 and 1 l3+20.1 mm01 giucosyl unitskg dw for the
HC and LC respectively( tigure 15. 16). At exhaustion the MG concentration representrd
1 ;?/O of the TotG in the HC condition and 1 1% in the LC.
During the flrst 4 h after exercise there was a sipificant increase in PG of 68
mm01 glucosy1 units/kp dw for the HC trial. and no significant change in PG in LC or
MG in either diet(p<0.05). From J-Xh the concentration of PG increased by 152 and 65
mmol glucosyl unitskg dw for the HC and LC respectively. while the MG pool rose onl?
in the HC(p<O.Oj)(tigure 1 7.1 8 ).
Between 2448h. the significant elevation of TotG in HC was due to the MG
concentration uhich increased by 79 mm01 glucosyl units while the PG concentration did
not change signiiicantly(p<O.O5)(figure 15.1 7). For the LC condition there was no
significant change in the PG or MG concentration from 2448h(figure 16.1 8). Both the
MG and PG concentrations were much greater for HC than LC at 24 and 48h(p<0.05). At
48h the MG represented 40% of the TotG for the HC diet but only 2 1% for the LC.
(figure 15.16) The 4 subjects with the highest TotG concentrations had MG
concrntrations representing about 50% of the total.
Table 6: Muscle ~ l y c o ~ econcentrations
n
HC
-
-
-
-
EXH
9.8613.30
68.7011 3.06
77.2021 5.59
4h
22.8StJ.30
132.33218.62 a
155.18222. 14
24 h
1 12.35129.48ab*
25 1.78228.78 ab*
364.13+50.11
48 h
190.34fr23.27abc*
289-6421 1.72 ab*
479.98223-69
Table 7: Muscle glycogen concentrations LC
MG?SEM
PG~SEM
Al! values are reponed as mm01 glucosyl unitskg dw.
a=p<O.Oj vs EXH. b=p<O.O5 vs 41. c=p<O.O5 vs 24h and *=p<O.Oj vs LC.
ab*
T
time
figure 15: Total Glycogen Vaiues(MG+PG) for the HC condition. The histogam
represents total glycogen: the open section is representative of PG while the shaded is
MG. The ratios are representative of %MG:PG. Statistics are summarized for PG and
MG. but not TotG as follows: a=p<0.05 vs EXH. b =<0.05 vs 4h. c=p<O.O5 vs 241 and
*=p<O.OS vs LC
FXH
24h
4h
48h
time
figure 16: Total Glycogen Vahies(MG+PG) for the LC condition. The ratios are
representative of %MG:PG. a=p<0.05 vs EXH, and b=p<0.05 vs 4h-
time
figure 17: The amount of MG and PG synthesized over t h e for the HC condition. The
dark bars represent MG while the lighter ones represent PG.a=p<0.05 vs EXH-4h,
b =<O.OS vs 4-24h, LpC0.05 vs LC,and #=p<O.O5 vs MG
time
figure 18: The amount of MG and PG synthesized over fime for the LC condition
a=p<0.05 vs EXH-4h, and #+p<0.05 vs MG.
Net Giyeoyn Svnthesis Rates
PG .siwltu.sis
During the tïrst 4h after the exhaustive exercise there was a 3- fold difference in
the net rate of PG synthesis between the HC and LC condition. The rates of PG synthesis
w r e 15-92 1.7 and 5.42 1.8 mm01 glucosyl unitsikg dwlh for HC and LC
respectively(figure 19). There was a significant decrease in the net rate of synthesis of PG
from 4-24h for HC to 6 . 3 1.1
~ mm01 glucosyl unitsjkg dw/h(p<0.05).
There was no net change in the net synthesis rate of PG over the 48h LC trial
penod(p<O.Oj). The average net PG spnthssis rate over the course of the LC trial was
3 - 3 5 1.1 mm01 glucosyl unitskg dwlh. This was not significantly different from the net
PG synthesis rate between 2448h in the HC(0.4~0.7mmolglucosyl unitskg duih)
(Figure 19).
MG svnthrsis
The net rate of MG synthesis did not change significantly over 48h within sach
trial. howevsr between trials the net rate of MG synthesis was significantly greater during
~ mm01 glucosyl
the HC. The net rate of synthesis for the HC trial was steady at 3 . 7 0.4
unitskg d w h kvhile for the LC condition the rate tvas only 0.72 0.5 mmol glucosyl
unitskg dw/h(tigure >O). Dunng the 24-48h period the net MG synthesis rate of 3.5i 0.8
mmol glucos)-1unitskd d w h was 8-fold greater than that of PG corresponding with the
significantly greater synthesis of MG during this tirne.
18
1
Ta*
+ High CHO
+ Law CHO
i
figure 19: The net rate of PG synthesis for HC and LC over 43h of recovery.
The solid circles represent HC and the open circles represent the LC condition. Values
with the same letter are not different within a trial and *p<0.05 vs LC.
Table 8: Net Svnthesis of MG over 43h recove- for HC and LC trialstn=9)
'I-IME
EXH-4 h
4-24 h
'MG svnthesis+ SEM
3.25 2 0.5 1 a*
4.29 + 1 N a *
WC Svnthesis 5 SEM
11.16 0.73a
10.86 5 0.78a
Table 9: Svnthesis of PG over 48h recovery for HC and LC triats(n=8)
HC
TIME
EXH-4 h
4-24 h
2448 h
PGsynthesis + SEM
15.91 r 2.86 a*
LC
b~ synthesis + SEiM
6.34 2 1 .O8 b*
5.38 2 1.78a
2.69 2 1.90a
0.42 f 0.74 c
1.82
+ 2.06a
Values are reported as mm01 glucosyl u n i t s k g h dw. Values with the s a m r letter are not
different nithin a trial and *=p<O.Oj vs LC.
+High CHO
* Low CHO
4-24h
time
figure 20: The net rate of MG synthesis for HC and LC over 48h of recovery. Values with
the same letter are not diffkrent within a trial and *<0.05vs LC .
Blood Glucose and Insulin
Blood glucose was not significantly different at EXH between the Z trials. The
blood glucose increased significantly within 30 min in the HC trial after ingestion of the
CHO drink and remained elevated throughout the first 3.5 h(figure 2 1 ). Sirnilarly the
insulin concentrations at EXH were not significantly different and the HC treatmrnt
increased the insulin concentration. howevrr this didn't reach significance until 1 .Sh of
recovr?. The insulin concentration \vas still elevated above EXH at the 4 h mark of the
HC trial \\.hile the concentration remained unchanged in the LC trial(figure I I ).
Table 10: Blood Glucose and Serum Insulin Concentrations
Blood Glucose(m M)
Serum Insulin(uIU/rnl)
tirne
EXH
0.Sh
* al1 data are presrntrd as mean= SEM.
a=p<O.Oj vs
EXH and b=p<O.Oj vs corresponding data for LC
Blood Glucose
10
figure 21 : Blood Glucose Concentrations. a=p<O.Oj vs EXH. b=p<O.Oj vs LC
figure 22: Insulin Concentrations. a=p<0.05 vs EXH. b==<O.OS vs LC
Discussion
The purpose of this study was to determine ifthe two structural forms of muscle
glycogen function as different metabolic pools under physiological conditions. The major
findings of this stud!. were that MG and PG are metabolized differently both in timing
and in terms of magnitude and both are sensitive to nutritional intake. At exhaustion. M G
reprrsrnted only I 1 - 13% of the TotG and during the first 4h of recovery the synthesis \*as
predominantl~in the PG pool dut-ing the HC trial. As well. for the first 2Jh of the HC
treatment the synthesis was predominantly PG with a modest increase in MG. In contrast.
during the second 24h the synthesis of MG was significantly greater than PG. The PG
synthrsis uas the most dynamic varying approximately 40-fold. Sirnilar responses
occured with LC but the changes were more rnodest. particularly in the synthesis of MG.
Our data show that the PG pool is especially sensitive to dietary CHO availability.
It appears that the PG pool is the major contributor during the earlp recovery phase with
ctlevated blood glucose and insulin concentrations. The synthesis of MG is slower and
more constant: MG contributes up to 25% of the TotG after 24 h when a HC diet is
provided. The increase in TotG beyond this time point is predorninantlp in the MG pool
and it represrnts 40-50% of the TotG afier 48h.
The sum of the net PG and MG synthesis rates over the first 4 h a f e r exhaustive
rsercise wi th HC was approximately 19 mm01 glucosyl unitskg dwih which matches
well with the 20 mmol/kg dw/h reported over 4 h of CHO supplementation following
rxercise(1vy et al.. 1988). It has also been demonstrated that without supplementation.
the glycogen synthesis rates are about 6-8 mm01 glucosyl unitskg dw/h(Connett and
Sahlin. 1996). u-hich is in agreement with our value of 6 mm01 glucosyi unitskg dw/h for
TotG for the sarne time fiame with LC. However the two pools follow very different
resynthesis patterns.
The average net PG resynthesis rate of 16 mm01 glucosy1 unitsjkg dw/h for the
tïrst I h when a HC diet is provided is much highrr than any other synthesis data in this
study. but the net synthesis rate is probably men higher in the tirst 1-2 hours following
clycogen depletion. The net PG synthesis rate is much slower when CHO are restricted.
t
It is also wonh pointing out that even when there is no dietaq provision of nutrients. as in
LC. the PG synthesis rate still tended to be higher in the first 4h than later in recovery
when CHO and other nutrients were ingested. The net MG synthesis rates art: much
slower following esercise and stay relatively constant. It also was affected by the dietary
CHO sincr the spnthesis rate is greater for the HC as cornpared to the LC condition. Over
the tirst 4h in the HC condition the net PG synthesis rate was 5-fold greater than that ssrn
in the MG pool. Hence glycogrn resynthesis is tint generated in PG and the later increasr
and uitirnatel) the supercompensation in TotG occurs predominantly in MG(figure 15.
17).
The reiationship between PG and MG is summarized in figure 73. PG appears to
increase to approximately 250-300 mm01 glucosyl unitskg dw and subsequentlp EvfG
continues to increase while PG concentration remains relatively constant. This transition
from PG to MG occurs at a TotG concentration of about 300-350 mm01 giucosyl unitsjkg
dw. This apress with Janssen's finding where glycogen synthesis above the 350 mm01
concentration was due to the acid soluble(MG) form(Jansson. 1980).This apparent
threshold of 350 mm01 glucosyl unitskg dw corresponds to the normal resting
concentration of muscle glycogen(Harris et al.. 1974. Hultman. 1967). We had previously
obsen-eda similar pattern among biopsies in our methods cornparison study. Hou-ever the
data in the uppcr concentration range were limited and samples were obtained from
various protocols. Hence hrther investigation was required to elucidate this relationship.
These human data correlate with work by Lomako et al. 1993 and Huang et al..
1997 who used labelled glucose to study glycogen synthesis in resting rodent muscle.
These investigators demonstrated that a set amount of the label first appeared in the PG
pool and then later in the MG pool with it's coincidental disappearance from the PG pool.
suggesting that PG is the precursor for MG.
The relationships illustrated in figures 19 and 20 are consistent with the proposa1
by Lornako et al.. 1993. that there are two forms of glycogen synthasr(GS).The)-
speculated that there is one form proglycogen synthase(PGS) controlling the synthesis of
PG and a second forrn. macroglycogen synthase(MGS). controlling the synthesis of MG.
Currently. there is no evidence that there are two isoforms in humans. but this data
drmonstntes that functionally they could very well exist. Cheetham. 1986. Gaitanos.
1993 and Bogdanis. 1995. 1996 rneasured acid soluble(MG) and insoluble(PG) glycogen
as a means of attaining total plycogen and have unintentionally s h o w that there is
differential regulation of the rate of use of the two pools during short sprinting exercise.
Their data H hich was acquired through personal communication provides evidence of
these fùnctional pools responding differently to exercise. The rate of PG use was much
grrater than MG and this is similar to what is seen in synthesis rates in this thesis. As
rvell Tsintzas. 1996 ~vhileusing a CHO supplement during exercise %as able to show a
buffering of the PG pool. The CHO supplied went into the PG pool preserving it's
concentration while the muscle continued using MG. The use of the MG pool was
unc hanged during supplementation.
Previously it has been shown that GS is highly active irnmediately afier exercise
when inactive glucose-6-phosphate dependent glycogen synthase(GSD) has been
convened to active glucose-6-phosphate independent GS(GSI)(Blochet al. 1994. Blom et
al.. 1986. Friedman et al.. 1991. Ivy et al.. 1988). GS activity has been s h o w to be
invtrsrly correlatcd with glycopn levels. The postexercise increase in GSI is rewnçd
atier 24h when a normal resting TotG has been reached. yet the slow rise in muscle
gl~~cogen(supercornpensation)
still occurs(Connett and Sahlin. 19%). This data
demonstrates that the GS associated with PG has a huge range: early in recovery. the
synthase is 5 times more active than that associated with MG. It is dramatically down
regulated during the last 24h and its activity is only one eighth of the rate of MGS.
Whether the- are stnicturally different enzymes or not. the GS associated with PG and
MG are obviously regulated vety differently. This proposed two enzyme system would
help to explain this supercompensation phenomenon when the conventional regulator.
GSI activity. is no longer elevated.
The data from this study demonstrate the usual biphasic synthesis of muscle
glycopen with a npid initial synthesis and then a subsequent slower phase. These phases
have been referred to as insulin dependant and independent stages(Price et al.. 1994.
Bloch et al.. 1994). It appears that the PG is synthesized rapidly especidly when glucose
and insulin are high and then declines over the next 48 h. while the MG synthrsis
remains lower and steady. T'he synthesis rate of MG is apparently unaffected by the
insulin concentration since it's net synthesis rate doesn't change over the 48h of rither
trial. The concentration of MG continues to increase when PG synthesis has declinsd
between 2-1-18h(figure 19). Thus not only does the PGS appear to be insulin sensitive. but
also later in recovery it declines to a very low rate while MGS does not change. Whether
these are trul>.two structurally different f o m s of synthase or the same t o m under
different regulation is unknown.
To our knowledge this is the only study of human muscle identiliing the
rdationship bcrwern MG and PG during recovery and certainly the first using txercise
and diet manipulation to alter the stores in order to examine the synthesis of MG and PG.
ln conclusion. the results from this study show that at exhaustion. the MG store shows the
ereatest relative depletion and that PG is most sensitive to dietary CHO and is
C
synthrsized most rapidly following glycogen depletion. This study also demonstrates that
the PG reaches a plateau at I 4 h while the MG pool continues to expand and is
responsible for the supercompensation seen in the days following exhaustive exercise in a
high CHO condition.
4.0 Summary:
The primary objectives of this thesis were to develop an accurate rneasurement
technique for the two foms of muscle glycogen. determine if the foms are measurable in
human muscle and to study the relationship between MG and PG dunng respthcsis.
Thrse two studirs successfull~~
accomplished our goals by demonstrating that the t o m s
c m be measurrd in human muscle and that a particular pattern
of resynthesis rsists( ie. the
2 foms are ditfrrent metabolic pools). One major finding of this thèsis was that the MG
and PG extraction procedure is accurate. precise and not systematically different than the
traditional glpcogen measurement techniques. AC and EZ.
Secondly. this thesis illustrated that the two pools in human muscle are
synthesized differently in both timing and magnitude. It has also been shown that both
pools are dirt sensitive. with a HC diet having a positive influence on the rate and arnount
of glycogen synthesis. This thesis demonstmted that the PG pool is the major contnbutor
to glycogrn synthesis early in recovery while the MG synthesis was slow and steady and
dominated in the later stages of recovery. It u-as demonstrated that when the TotG
concentration reachsd a normal resting concentration. the sythesis of PG decreased
tremendously and the MG continued to be synthesized. resulting in the
supercompensation seen in the days following glycogen depletion. The PG pool appeared
to be most dynarnic ranging from 68.7 to 289.6 mm01 glucosyl unitskg dw within 2 d of
recovery ~ i i t ha HC diet.
Future Directions and Applications
Future studies should certainly address the use of the two pools during exercise.
and steps should be taken to isolate the putative MGS and PGS e q m e s . It appears from
the PG and MG synthesis relationship seen in the second study that the PGS controls the
rate and overall amount of glycogen synthesis and hence this e n q m e would b r rate
limiting. The control of these putative enzymes requires fürther research. Perhaps these
are the samc enzyme with different regulation or they could be different isoforms.
Considering the soluble and insoluble glycogen data received from a research group at
Loughborough University. England(Cheetharn. Bogdanis. and Tsintzas) there seerns to be
simiiar diferences in glycogen catabolism. and therefore different pools of phosphorylasc
may also exist.
The traditional regulators of glycogen synthesis need to be reconsidered. The
glycogenin molecule. which functions both as an initiating enzyme and as the protein core
of the glycogrn. map be shown to be the ovemding factor in glycogen regulation. Sincr
&cogen synthesis depends on the autocatal'ic
ability of glycogenin to self-glucosylate
providing the primer molecule and acting as the substrate of GS. it is possible that the
amount of glycogenin protein available for synthesis determines the overall amount of
glycogen that an individual ma- produce. This may be genrtically determined. howevrr.
the potrntial to manipulate the avaiiable glycogenin proteins via training or diet rnust also
be considered. This may be directlp applicable to competirive athletes who wish to
increase their capacity to store glycogen prior to cornpetition.
It seems logical that in synthesis or in degradation. there would be glycogen
particles of varying sizes. There must exist intermediate stages of PG and MG in human
skeletal muscle and this could be visudized using EM. EM work should provide
evidence of glycogen particles in different stages of growh or deterioration as well as in
di tTerent companments.
Glucose uptalir in human muscle is stimulated by insulin as well as esrrcix. both
of which recruit GLUT4 vesicirs from intracellular sites to plasma rnembranes(Codsrrt:et
al.. 19951. GLUT-l transporters are e.uclusively found between m)-otibrils within the tnad
(t-tubules)(Friedman et al.. 1991 ). The HK enzyme and insulin receptors are also found in
close proxirnity to the t-tubles between myofibrils. The stimulation of glucose transport
by both exercise and insulin are rnutually exclusive and recruit GLUTS vesicles tiom
diffrrent fractions. Coderre. 1995 has suggested that GLUT4 vesicles may be found in
association with glycogen particles. They have suggested that the association is govemed
by the amount of glycogen in the muscle. Depletion of muscle glycogen correlates \sith
the translocation of GLUTS and it has been demonstrated that aftsr exercise rats are more
sensitive u ith respect to insulin-stimulated glucose transport(Coderre et al.. 1995).
Speculatively. it could be glycogenin or intermediate foms of PG that are associated nith
the GLUT-I and with depletion these glycogenin or primer molecules are transiocatrd to
the plasma membrane with the transporter. This availability of glycogrnin could
potentially allow for the increased synthesis of the PG pool that we see in this study.
Knowing that glycogen is synthesized at different rates in different tibre types.
(Vollestad et al.. 1989, Essen and Henriksson. 1974) it would also be benefkial to look at
ditterences in MG and PG concentration between fast twitch(FT)and slow twitch(ST)
muscle fibres. After prolonged exhaustive exercise. glycogen synthesis is more rapid in
FT muscle in the first 90 min of recovery(Vol1estad et al.. 1989). There is a delayed
acceleration of glycogen synthesis in ST. and hence ST have a faster resynthesis during
the subsequent 90 min. of recovery(Vollestad et aI.. 1989). Studies could determine if
there is a tibre type difference in the arnount of MG and PG and also look at the MGS and
PGS activity pattern in the two muscle fibre types.
1s thrrr anp metabolic advantage to the patterns observed in the two glycogen
pools during resynthesis? If we link this idea to the work of Cheetharn and Bogdanis it
seems that the PG pool needs to be in greater supply and made faster in preparation h r
it's irnmediate use during exercise. Wlat are the implications of these two pools for the
normal individual, for the athlete. for the diabetic and for women who have been shoun
previously to lack the ability to glyogen supercompensate(Tarnopolsky et al.. 1995).
How dors the importance of glycogenin in glycogen s-ynthesis relate to protein needs'? 1s
the glycogenin ~ n binding
"
site a possible site of regulation and c m the concentration of
Mn-" limit the activity of glycogenin in vivo? There are man- unanswered questions in
the study of proglycogen and macroglycogn and al1 require Further investigation.
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APPENDIX A- MEDICAL QUESTIOhNL\IRE AND NFORMED CONSENT
Subject Screening Questionnaire
YOUR RESPONSES TO THIS QUESTIONNAIRE ARE CONFIDENTIAL AND YOU
ARE ASKED TO COMPLETE IT FOR YOUR O \ W HEALTH AND SAFETY-IFYOU
ANSWER "YES" TO .4NY OF THE FOLLOWMG QUESTIONS PLEASE GIVE
MORE DETAIL M THE SPACE PROVIDED AFTER THE QUESTION.
1 Have !ou s w r bern told that )-ou have a heart problem? YES
NO
2 Have you rver been told that !.ou have a breathing problern such as asthma'?
NO
3 Have you ever bern told that you sometimes experience seizures?
YES
NO
4 Have >-ourvrr had any major joint instability or ongoing chronic pain such as in
the knee or back?
YES
NO
5 Have you evrr been toid that you have kidney problems? YES NO
6 Have you had an>-allergies to medication? YES NO
7 Haïe >ou had an!. allergies to food or environmental factors?
8 Have you had any stomach problems such as ulcen? YES NO
YES NO
YES
9 When you experience a cut do you take a long time to stop bleeding?
YES NO
10 When >-oureceive a blow to a muscle do o u develop bruises easil??
YES NO
1 1 Arc: you currently taking an! medication (including aspirin) or have you taksn an>
medication in the last two days? YES NO
12 1s there any major medical condition with which you have been diagnosed and
are under the care of a physician eg diabetes. high blood pressure. YES NO
1 3 Because Our investigation invo Ives the handling of blood and other hurnan tissues. if
o u have ever tested positive for hepaiitis or HIV please contact Dr Mark Tarnopolskp.
Dr Tem- Graham or Premila Sathasivarn in order that we c m taks the proper precautions
a-hen handling the sarnples.
I N F O M E D CONSENT
MEDICAL ASPECTS
In the study in which o u are invited to participate there are two procedures which
have mrdical involvement. namely blood sampling and the taking of muscle biopsies.
Prior to your participation in the study you must have completed the Subject Sçreening
questionnaire which is designed to identiQ an? medical reason why you should not be a
subjrct.
BIood sampling:
On the day of the exhaustive exercise trial a small catheter will be placed in a rein
in your forearm by Prernila Sathasivam. a medically trained and approved technician.
The Teflon catheter is inserted with the assistance of a srnaIl needle and subsequently the
nredle is rernoved. The discornfort ofthis procedure is transient and is very similar to
having an injection by a needle. Once the needle is removed there should be no sensation
from the catheter. During the course of the 4 hours after O u r exhaustive ride we will
withdraw blood samples from time to time via the catheter. In this particular experirnent
the total blood loss is less than 50 ml and this is Iess than one tenth of the blood loss
when you donate blood for the Red Cross. It minor amount of blood loss wilI not effect
your physical performance in any way. After the taking of a blood sample the catheter is
'.flushed" with a sterile saline solution. This is a salt solution that is very similar to your
ou-n blood and i< will not affect ?ou. This procedure prevents blood from clotting in the
catheter.
The insertion of a venous catheter is a very common medical practice and it
in\-olvrs few risks. However. there rnay be intemal bleeding from the vein if adequate
pressure is not maintained upon removal of the catheter. This map cause bmising and
somr minor discornfort. In rare occasions. injury of the intemal wall of the vein ma?
occur causing the formation of a small blood dot which will normally dissolve soon aftw
the rxprriment. There is a remotr chance of inkction in the area of the venipuncture.
although this is vrry rare.
LWuscleBiopsy :
Small pieces of muscle will be sarnpled from your thigh by means of a procedure
rrferred to as a muscle biopsy. A medical doctor wil1 clean an area of your thigh. inject a
local anaesthetic into and under the skin. Then he mil1 make a smali incision (about 0.5
cm) in the skin to create an opening through which the biopsy needle c m enter the thigh
muscle. One incision is made for each biopsy. There is a small amount of bleeding from
the incision. but this is minimal. At those times in the experiment when a biopsy is
required ive will take the bandage off the area of the incision and the medical doctor will
insert the nerdle into O u r thigh. He will then quickly (approximately 5 seconds) cut o f f a
very small piece of muscle (it is about the size of the eraser on the end of a pencil) and
remove it. During this time you may feel pressure in o u r thigh and o n some occasions it
is moderately painful. The discornfort very quickly passes and pou are quite capable of
walking and continuing your daily routine.
Follou.ing the biopsy your leg will feel tight and o f e n there is the sensation of a
d r r p bruisr or "Charlie Horse". You should not take any medicine that contains .ASA for
24 hours follou-inp the experimrnt as this can promote bleeding in the muscle. The
follou-ing da? you will probably k e l uncornfortable going down stain. The tightnrss in
the muscle disappears in 1-7 days and subjects can exercise moderately the nest da)-.
Subjects resume normal exercise programs in 2-3 days. Swimming should be avoided
until the incisions heal - normally 2-3 days.
The technique very rarely has any complication associated with it. On occasion a
srnaIl lump of fibrous tissue may form under the site of the incision. but this disappears in
2-3 months. Interna1 bleeding in the area c m cause discoIouration of the skin and as with
an? incision there is a srnaIl risk of infection. If the incision does not heal quickly. o r if
the area appears inflarned and infected contact us irnmediately. In very rare occasions
there c m be damage to a superticial sensory neme which will result in temporary
numbness in the area.
The blood. muscle tissues and the associated data will not be used for an!
purposrs other than those outlined in the study. You will have complete access to your
data as urll as the s u m m q data that represents a11 of the subjects in the study.
1 acknowledge that 1 h a w completed the "Subject Screening Questionnaire" and
have discussrd the esperimrntal and medical protocol with Dr. T Gnham or Premila
Sathasivarn and understand the risks involved. If I do have any concems about the
medical risks described above. 1 understand that 1 can discuss these concerns with the
Medical Director. Dr. Mark Tarnopolsky. pnor to my participation in this study.
Subject's signature:
Investigator's Signature:
Date:
Date:
INFORMED CONSENT
EXPERIMENTAL PROTOCQL
This rxprrimentai design and conditions has received the approval of the University of
Guelph Human Subjects Cornmittee. The study for which you have volunterred is
designed to study the resynthesis of two muscle glycogen pools: proglycogen and
rnacrogl~cogrnaHer depletion. The use of 2 different diets a high carbohydrate diet with
a CHO supplement and a normal carbohydrate diet without supplement rvill allow the
investigator to compare the glycogen resynthesis within the pools.
The experiment entails 2 trials as follows:
.4)A preiiminary test of VO, max and a 3 day diet record.(this only needs to b r dont: once)
B)A habituation ride 2 days prior to the triai and following a controlled dict and rserciss
regime for 2 days pnor to the glycogen depletion ride. Subjects will also be required to
follow a particulai- control diet for the 48hours afier the depletion ride.
C)An exhaustive exercise ride on a cycle ergorneter( 1.Wh) on July 19th and .4ugust 8th.
D)4 muscle biopsies during each trial. 1 immediately following essrcise. 1 4 hours a k r
depletion. 1 24 hours afier the I st biopsy and finally 1 at 48 hours afier the depletion
ride. Blood samples will be talcen every 3Omin for the 4 hours following the exhaustive
ride.
wiil perfom a maximal osrgen uptake test on a cycle egomrtrr. This rttquires
that y011 rxercisr at progressivrl> higher powrr outputs until we measure o u r maximal
ability to consume osupen. This is a measure of aerobic fitness. As well the 3 da'. diet
record would be kept during 2 weekdays and 1 weekend dap in order for us to customizr
a control dict according to your eating habits. This is a ven; detailed record of everything
)-ou eat during thcse days and how much of each food.
a: You
b: You u-il1be asked to corne into lab 2 days before the trial to habituate yourself with the
exhaustive ride. This will make it easier for us to judge how long you will require on the
test day to reach rxhaustion so we are prepared. You will also need to follow a specific
diet for 2 days prior to the biopsy procedure. You will be piven instructions and supplied
food to eat for this period. This is an attempt to minimize differences in resting muscle
dvcogen on the day of the biopsies. A specific diet will also be assigned and provided for
the 48 h period afier the exhaustive ride.
C
4
c: The biopsies will be performed by Dr. Mark Tamopolsky from the McMaster
University Hospital. The blood will be taken from a forearm vein by Premila Sathasivam
and your thigh will be prepared for muscle biopsies by Dr. Tamopolsky. These
procedures are descnbed in the accompanying form entitled "Informed Consent-Medical
.Aspectsw.Muscle samples are taken immediatelp following exercisr. at 1 h. 14 h and 48
h.
To assist you in the expense associated with having to follow the specitic diet and to
compensate you for your time and effort we will give you an honourarium of $300 as well
as supplying -ou with the food. Your decision to be a subject in this study is voluntap.
and you are frec to withdraw from the study at any rime including during the experimental
trial.
FINAL RELEASE
-
1 hereby release and discharge the University of Guelph. Dr. M. Tamopolsky. Dr T
Graham. Premila Sathasivam. Kristi Adamo and the Department of Human B i o l o ~
and
Nutritional Sciences fiom any and al1 actions, causes of actions. claims. and demands for
charges. loss or injury howsoever arising which may hereafter be sustained by me as a
consequence of my participation as an experimental subject in the research to which 1
have giwn m! informed consent to participate in. Furthemore I understand that the
information obtained as a result of this expenment may be used for educational purposss.
including publication of the results.
Subject Signature:
In\-estigator'sSignature:
Date:
Date:
IMAGE EVALUATION
TEST TARGET (QA-3)
----
APPLIED i lIN1AGE. lnc
= 1653 East Main Street
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Rochester. NY 14609 USA
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Phone: i f 6/482-O3ûO
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