REVIEW

Physiological aspects of the subcellular localization of glycogen inskeletal muscleJoachim Nielsen and Niels Ørtenblad

Abstract: Glucose is stored in skeletal muscle fibers as glycogen, a branched-chain polymer observed in electronmicroscopy imagesas roughly spherical particles (knownas�-particles of 10–45nm indiameter),which are distributed indistinct localizationswithin themyofibers andarephysically associatedwithmetabolic and scaffoldingproteins. Although the subcellular localizationof glycogenhasbeen recognized for more than 40 years, the physiological role of the distinct localizations has received sparse attention. Recently,however, studies involving stereological, unbiased, quantitative methods have investigated the role and regulation of these distinctdeposits of glycogen. In this report, we review the available literature regarding the subcellular localization of glycogen in skeletalmuscle as investigated by electron microscopy studies and put this into perspective in terms of the architectural, topological, anddynamic organization of skeletal muscle fibers. In summary, the distribution of glycogen within skeletal muscle fibers has beenshown to depend on the fiber phenotype, individual training status, short-term immobilization, and exercise and to influence bothmuscle contractility and fatigability. Basedonall these data, the available literature strongly indicates that the subcellular localizationof glycogenhas to be taken into consideration to fully understand and appreciate the role and regulation of glycogenmetabolism andsignaling in skeletal muscle. A full understanding of these phenomenamay prove vital in elucidating themechanisms that integratebasic cellular events with changing glycogen content.

Key words: glycogen metabolism, muscle physiology, subcellular localization, type 2 diabetes, muscle disuse, muscle training,muscle function, exercise physiology, fiber types, muscle fatigue.

Résumé : Le glucose est stocké dans les fibresmusculaires squelettiques sous formede glycogène, un polymère a chaîne ramifiée qui,sous le microscope électronique, se présente sous forme de particules grossièrement sphériques (connues sous le nom de particulesbêta d'un diamètre de 10 a 45 mm), distribuées dans des régions distinctes des fibres musculaires et associées physiquement auxprotéines du métabolisme et d'échafaudage. Même si on connait depuis plus de 40 ans la région infracellulaire où se trouve leglycogène, on sait très peu de choses au sujet du rôle physiologique des régions distinctes. Néanmoins, des études récentes réaliséesaumoyende techniques stéréologiques quantitatives ne présentant aucunbiais examinent le rôle et la régulation des dépôts distinctsde glycogène. Ce rapport présente d'une part une analyse documentaire des études disponibles réalisées parmicroscopie électroniqueet traitantde la localisation infracellulaireduglycogènedans lemuscle squelettique et d'autrepart, situe les donnéesdansuncontexted'organisation architecturale, topologique et dynamiquedesfibresmusculaires. En résumé, la répartitionduglycogènedans les fibresmusculaires dépend, données a l'appui, du phénotype des fibres, du degré d'entraînement individuel, de l'immobilisation de courtedurée, de l'exercice; elle exerce de plus une influence sur la contractilité et la fatigabilité du muscle. En regroupant tous les faits,l'analyse des documents disponibles révèle clairement qu'il faut tenir compte de la localisation infracellulaire du glycogène pour biencomprendre et mesurer l'importance du rôle et de la régulation du métabolisme et de la signalisation du glycogène dans le musclesquelettique. C'est en comprenant parfaitement ces phénomènes qu'on pourra mieux élucider les mécanismes reliant les actionscellulaires fondamentales a la modification du contenu en glycogène. [Traduit par la Rédaction]

Mots-clés : métabolisme du glycogène, physiologie musculaire, localisation infracellulaire, diabète de type 2, inactivité muscu-laire, entraînement musculaire, fonction musculaire, physiologie de l'exercice physique, types de fibres, fatigue musculaire.

IntroductionGlycogen is a branched polymer of glucose that serves as an

energy reserve for ATP production and is found principallywithin muscle and liver cells. In skeletal muscle, the key im-portance of glycogen as an energy reserve lies in its rapid mo-bilization in response to elevated energy requirements, itscapacity to serve as an energy source in the absence of oxygen,and the fact that it and glycolytic enzymes are widely distrib-uted within the cell in close proximity to sites of energy con-sumption, thereby creating optimal conditions for efficientregulation.

The importance of glycogen in skeletal muscle became clear asa consequence of the now classic studies conducted by Hultman

and colleagues in the late 1960s that showed a strong correlationbetween muscle glycogen depletion and fatigue during medium- tohigh-intensity cycling exercise (Bergström et al. 1967; Hermansenet al. 1967). This findingwas subsequently confirmed in several stud-ies (see Green 1991) and it is nowwell established that fatigue duringprolonged, exhaustive exercise has a glycogen-dependent compo-nent (Allen et al. 2008). Although this has been acknowledged formore than 20 years, fatigue's underlyingmechanism is yet to be fullyelucidated.

During this same period, the concept of intracellular compart-mentalization of metabolites developed (see Ovadi and Saks2004), which points out not only that a high concentration ofmolecules in the cytosol results in macromolecular crowding and

Received 16 May 2012. Accepted 15 August 2012.

J. Nielsen and N. Ørtenblad. SDU Muscle Research Cluster (SMRC), Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Campusvej 55,DK-5230 Odense, Denmark; Swedish Winter Sports Research Centre, Department of Health Sciences, Mid Sweden University, 83125 Östersund, Sweden.

Corresponding author: Joachim Nielsen (e-mail: jnielsen@health.sdu.dk).

91

Appl. Physiol. Nutr. Metab. 38: 91–99 (2013) dx.doi.org/10.1139/apnm-2012-0184 Published at www.nrcresearchpress.com/apnm on 9 November 2012.

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

poor diffusibility (Srere 1967), but also that metabolic pathwayscan be compartmentalized (Saks et al. 2008); this may be impor-tant in cells with a high and fluctuating energy turnover, such asskeletal muscle fibers. Indeed, evidence for compartmentalizedglycogen metabolism comes from studies showing simultaneoussynthesis and utilization of glycogen (Shulman and Rothman2001), lactate production during fully aerobic conditions (Connettet al. 1986), and a preferential requirement for glycolytic ATP byNa+-K+ pumps in several cell types, including skeletal muscle fi-bers (James et al. 1999; Dhar-Chowdhury et al. 2007). Therefore,the difficulties in explaining, mechanistically, how glycogen de-pletion affects muscle function may reside partly in the generalassumption of “soluble” metabolic pathways and in gaps in ourunderstanding of metabolic organization in vivo (see Welch andClegg 2010).

In addition to a crowded cytosol, the components of skeletalmuscle fibers are highly organized in terms of localization. Themain constituents of fibers are the contractile filaments, whichserve as the molecular motors of muscle contraction and are ar-ranged in myofibrils and occupy approximately 80% of the fibervolume. These myofibrils are intermingled with mitochondria,the Ca2+-containing sarcoplasmic reticulum (SR), and a tubular (t-)system in a highly ordered arrangement, which together createdistinct compartments and microenvironments with high localATPase activity and restricted diffusion of metabolites (Han et al.1992). Intriguingly, within this organization, glycogen particlesare found in distinct locations, including just beneath the plasmamembrane, close to the SR and mitochondria, and within themyofibrils between the contractile filaments (Figs. 1 and 2). Recentstudies have shown that the distribution of glycogen is influencedby several factors, such as muscle work (Marchand et al. 2007;Nielsen et al. 2011) and disuse (Nielsen et al. 2010b), and that dif-ferent localizations of glycogen are related to distinct processes inskeletalmuscle function (Nielsen et al. 2009; Ørtenblad et al. 2011).

Therefore, the limited diffusibility in the cytosol and the or-dered arrangement of organelles and inclusions necessitate a con-sideration of spatial organization to fully understand the role ofglycogen in muscle function.

Aon and colleagues (2007) considered the topic of subcellularnetworks and originated the term “structural organization” tomean the architecture of cellular networks based onmorphology.In addition, networks should be analyzed based on their topologyand dynamic organization, which refer to the pattern of organi-zation and the ongoing activity in the network, respectively (Aonet al. 2007). In glycogen metabolism, a topological view involvesthe channeling of glycolytic intermediates and metabolites fromthe glycogen particle to distant localizations and protein–proteinand protein–membrane interactions, whereas the dynamic orga-nization accounts for alterations in the architecture and topologyof glycogen metabolic networks during changing environmentalconditions (calcium transients, oxygen and hormone levels, etc.),as well as fluctuations in the networks themselves (i.e., glycogenparticle size and associated proteins).

This review focuses on the role and regulation of the subcellularlocalization of glycogen in skeletal muscle, mainly from an architec-tural viewpoint basedonelectronmicroscopy studies, althoughbothtopological and dynamic views are also considered. In particular, wediscuss the current knowledge and interpretations of fiber-type dif-ferences and the roles of exercise, training, immobilization, and type2 diabetes, based on quantitatively conducted studies.

Definitions of glycogen localizationElectron microscopy studies have revealed 3 distinct subcellular

localizations of glycogen (Fig. 1): (i) intermyofibrillar (IMF) glycogen,which is located between themyofibrils in close proximity to the SRand mitochondria; (ii) intramyofibrillar (Intra) glycogen, which islocated in the myofibrils interspersed within the contractile fila-

ments, and most often in the I-band of the sarcomere; and(iii) subsarcolemmal (SS) glycogen, which is distributed just beneaththe surface membrane in close association with mitochondria, lip-ids, golgi apparatus, andnuclei. In relative terms, IMFglycogen is themajor site of glycogendeposition, constituting approximately 75%ofthe cell's total store, whereas Intra and SS glycogen each accountonly for 5% to 15% of the total. However, the relative distributiondepends on various factors, as discussed in the following sections. Itis important to keep in mind that our understanding of glycogenlocalization is confined to thesedefinitions,which, in turn, arebasedon a 2-dimensional anatomical view of the muscle cell. Thus, theremaybe subfractionsof glycogenwithin thedefined localizations thatshowdifferential regulatory properties and roles inmuscle function.However, in this review, and in most available literature for thatmatter, the glycogen is considered and discussed in terms of the 3distinct localizations we have mentioned.

Fiber typesVertebrate skeletal muscle is composed of muscle fibers that

exhibit different characteristics with respect to their myofibrillarprotein composition; metabolic enzymes; and ionic transmem-brane fluxes; these are mainly manifested as diverse speeds ofshortening and resistance to fatigue (reviewed in Schiaffino andReggiani 2011). Based on the isoform of the myofibrillar protein,myosin heavy chain (MHC), muscle fibers can be categorized into4 major types: (i) type I fibers, which have a high oxidative capac-ity and are suitable for prolonged repetitive contractions; (ii) typeIIa fibers, which are less oxidative but more glycolytic and have ahigh speed of contraction, making them suitable for short burstsof powerful actions; (iii) type IIx fibers, which in humans are al-most exclusively glycolytic and easily fatigable but can producehigh power outputs (in rodents, type IIx fibers are more oxida-tive); and (iv) type IIb fibers, which are found only in rodents andresemble the type IIx fibers found in humans. Electron micros-copy studies have highlighted the fact that several ultrastruc-tural parameters correlate with fiber types. In particular, Z-linewidth and mitochondria content in combination have beenfound to be a reliable predictor of fiber type and are easilyapplicable using standard stereological methods on electronmicrographs (Sjöström et al. 1982b). The low number of fibers(�6–15) obtained per biopsy in electron microscopy restrictsthe fiber-type distinction to type I and II, where the definedgroup of type II fibers most likely represents a mix of type IIaand IIx fibers.

The first researchers to address the distribution of glycogen interms of fiber type were, to the best of our knowledge, Schmalbruchand Kamieniecka (1974), who conducted a qualitative study andshowed that the main localization of glycogen in type I and IIfibers was in the Intra and IMF spaces, respectively. Further, thedistribution of Intra glycogen particles seemed to differ betweenthe 2 fiber types, with a primary location in the I-band of type Ifibers and in the A-band of type II fibers. Later, Ekblom and col-leagues found that there seemed to be no difference in IMF and SSglycogen content between fiber types but they could confirm thefiber-type difference in the distribution of Intra glycogen betweenthe I- and A-bands (Sjöström et al. 1982a; Fridén et al. 1989). Usinga quantitative method, Marchand et al. (2002) confirmed in a pre-liminary examination in recreationally active subjects the findingof Schmalbruch and Kamieniecka (1974) that type I fibers havemore Intra glycogen than do type II fibers. Recently, we showed insedentary obese subjects (Nielsen et al. 2010a) and in recreation-ally active young and elderly men (Nielsen et al. 2010b) that thedistribution of glycogen was not different between type I and IIfibers. However, in elite endurance-trained cross-country skiers,we found that type I fibers had 82% more Intra glycogen and 31%more SS glycogen than did type II fibers (Nielsen et al. 2011). Incontrast, type II fibers had 11% more IMF glycogen than did type I

92 Appl. Physiol. Nutr. Metab. Vol. 38, 2013

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

Fig. 1. Transmission electron microscopy images illustrating the subcellular localization of glycogen in skeletal muscle. Images obtainedfrom the cross-section of a muscle fiber showing a large part of the fiber (A), several myofibrils (B), and 1 myofibril (C). (B) Nonperfectperpendicular cross-section revealing the higher concentration of glycogen particles in the I-band of the sarcomere compared with theA-band. (C) Typical distribution of glycogen particles within a myofibril. (D–F (longitudinal sections)) The 3 defined localizations of glycogen inassociation with selected components involved in skeletal muscle physiology. Glycogen particles are seen as black dots. Arrow, sarcolemma.IMF, intermyofibrillar glycogen; Intra, intramyofibrillar glycogen; N, nucleus; Mi, mitochondria; SS, subsarcolemmal; SR, sarcoplasmicreticulum; Z, Z-line; RyR, ryanodine receptor calcium release channel.

Nielsen and Ørtenblad 93

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

fibers (Nielsen et al. 2011). In summary, fiber-type differences ap-pear to be present in trained muscle only, with results suggestingthat the increased accumulation of Intra and SS glycogen may bean important adaptation for improved endurance capacity andthat increased IMF glycogen may be associated with improvedspeed of contraction (Table 1). The physiological aspect underlyingthis fiber-type-specific localization of glycogen requires furtherinvestigation.

The fiber-type-specific training adaptation in the distribution ofglycogen may be associated with other known physiological ad-aptations. Higher Intra and SS glycogen content in type I fibersmay be related to Na+-K+ pump activity in the t-system and sarco-lemma, respectively. Several findings point to an association be-tween Na+-K+ pump activity and Intra glycogen: (i) duringrepetitive contractions, Na+-K+ pumps are more active in type Ifibers than in type II fibers (Everts and Clausen 1992); (ii) Na+-K+

pumps prefer glycolytic-derived ATP (James et al. 1999; Dhar-Chowdhury et al. 2007; Dutka et al. 2007), and patients lackingglycogen phosphorylase have fewerNa+-K+ pumps and suffer froma K+ imbalance during exercise (Haller et al. 1998); (iii) Intra glyco-gen may be associated with Na+-K+ pump activity, at least in me-chanically skinned rat fibers (Nielsen et al. 2009); (iv) im-mobilization is accompanied by a decrease in both Intra glycogenand Na+-K+ pumps (Clausen 2003; see Effects of immobilization inrecreationally active men section). Collectively, the increased In-tra and SS glycogen content in type I fibers compared with type IIfibers may be an adaptation to meet higher Na+-K+ pump energyrequirements.

A muscle fiber's ability to have a high speed of contraction is theresult of several factorsworking inharmony. In addition to fastMHCisoforms, myosin ATPase, and troponin C in type II fibers, their fast

shortening velocity is accommodated by fast action potential propa-gation and fast calcium kinetics. Regarding the former, a tight con-nectionbetween theneuromuscular junctionat the sarcolemmaandthe myofibrils may be crucial for rapid activation (Mantilla et al.2007) and, in turn, may explain the small number of organelleslocated just beneath the sarcolemma in type II fibers. Further, alimitation may be present regarding Intra glycogen, which occu-pies space in the myofibrils. This is likely to push the contractilefilaments closer to each other and to diminish the shorting veloc-ity (Metzger and Moss 1987). Consequently, an upper limit of SSand Intra glycogen storage may exist to facilitate a high speed ofcontraction.

Fast calcium kinetics in type II fibers require a well-developed SRcontaining a high number of ryanodine receptor calcium releasechannels (RyRs) and calcium-transporting ATPases (SERCAs). RyRsplay a fundamental role in skeletal muscle excitation-contractioncoupling by mediating calcium release from the SR in response tot-system depolarization, whereas SERCAs serve as the active trans-port mechanism drawing calcium into the SR, giving rise to musclerelaxation. In mechanically skinned fibers, IMF glycogen content ispositively correlated with a faster relaxation time of tetanic contrac-tions in unfatigued fibers (Nielsen et al. 2009). This suggests a rolefor compartmentalized energy delivery in SERCA activity and re-fines the findings of previous studies that SERCA activity is relatedto glycolysis-derived ATP in mouse skinned ventricular fibers(Boehm et al. 2000) and to the glycogen content of rat purifiedskeletal muscle SR (Lees and Williams 2004) and of human SRvesicles (Duhamel et al. 2006). Thus, a higher IMF glycogen con-tent in type II fibers may be an adaptation to satisfy high energyrequirements by SERCAs during high-speed contractions. Collec-tively, the high speed of contraction of type II fibers may be in-

Fig. 2. Schematic representation of the 3 defined subcellular localizations of glycogen in relation to intracellular structures. SS glycogenis located just beneath the sarcolemma, IMF glycogen between the myofibrils in close association with the SR and mitochondria, andIntra glycogen within the myofibrils interspersing the contractile filaments (actin and myosin filaments). Intra, intramyofibrillar;SS, subsarcolemmal; IMF, intermyofibrillar; SR, sarcoplasmic reticulum; T, tubular.

94 Appl. Physiol. Nutr. Metab. Vol. 38, 2013

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

volved in the regulation of glycogen localization not only byrequiringmore glycogen in the IMF space, but also by limiting theaccumulation of glycogen in Intra and SS deposits to facilitatethe high speed of contraction. Future experiments are neededto verify these roles of glycogen localization in different fibertypes.

Exercise

Utilization and fatigueEndogenous glycogen stores are the main fuel supply for work-

ing skeletal muscle. The rate of glycogen utilization during exer-cise increases with the intensity of the exercise (Gollnick et al.1974; Vøllestad and Blom 1985) and decreases with the depletionof the glycogen store itself (Blomstrand and Saltin 1999; Hespeland Richter 1992). Thus, glycogen utilization is tightly regulatednot only to match the cell's specific energy requirements, but alsoto be balanced according to the cell's energy state. Interestingly,there is a close association between glycogen depletion and fa-tigue during prolonged moderate- to high-intensity exercise(Bergström et al. 1967; Hermansen et al. 1967), which ismanifestedin patients lacking glycogen phosphorylase (McArdle's disease)who are unable to withstand exercise at moderate to high inten-sities (Das et al. 2010). This demonstrates that the presence ofadequate amounts of glycogen in skeletal muscle is crucial foroptimalmuscle function. Although this fundamental relationshipin skeletal muscle has been long recognized, elucidation of theunderlying mechanism is needed (Allen et al. 2008).

Studies in the 1970s and 1980s using electron microscopy dem-onstrated that distinct subcellular deposits of glycogen were uti-lized to different degrees during exercise (Oberholzer et al. 1976;Sjöström et al. 1982a; Fridén et al. 1985, 1989). Such findingsstrongly suggest that the difficulties in explaining why low glyco-gen levels impair muscle function may be attributed to a disre-garded role of cell compartmentalization. Oberholzer et al. (1976)

estimated by quantitative stereological methods the humanmyo-fibrillar and SS content of glycogen before and after running100 km (average of 522min at 72%ofmaximal oxygen consumption).They found an unequal utilization of glycogen characterized by an�80% decrease in the myofibrillar content of glycogen (IMF andIntra) compared with a statistically nonsignificant decrease of�50% in the SS depot of glycogen. Unfortunately, they did notdiscriminate between IMF and Intra glycogen. Ekblom and col-leagues conducted a series of observational but more-detailedanalyses of glycogen localization in human vastus lateralis biopsyspecimens obtained after exercise and compared the results withnonexercising control subjects. Their main findings were that(i) after a 30-km cross-country run, SS and IMF glycogen werealmost completely depleted, whereas Intra glycogen particleswere observed relatively frequently (Sjöström et al. 1982a); (ii) SSglycogen content appeared to be lower after high-intensity inter-mittent exercise than that seen after marathon running (Fridénet al. 1985); and (iii) SS and Intra glycogen were preferentiallydepleted compared with IMF glycogen in type II fibers followingrepeated sprint exercise (Fridén et al. 1989). Together, these earlypioneering studies suggested that SS and Intra glycogen may berelatively more important during high-intensity exercise com-pared with prolonged medium-intensity exercise. Moreover, thefindings indicated that a better understanding of glycogen utili-zation during exercise requires knowledge of the subcellular lo-calization of the glycogen, in conjunction with the effects ofdifferent types of exercise and the fiber phenotypes involved.

More recently, a quantitative and more conclusive approachwas taken by Graham and colleagues (Graham et al. 2010), whoestimated the volume fractions of the 3 distinct localizations ofglycogen and reintroduced the use of potassium ferrocyanide toenhance the staining of glycogen particles for subsequent analysisby electron microscopy (Marchand et al. 2002, 2007). The validityof this transmission electron microscopy (TEM) method was con-

Table 1. Summary of main findings from quantitative studies on the subcellular localization of glycogen in skeletal muscle.

Parameter Findings

Fiber types In endurance-trained athletes, type I fibers have 80% more Intra glycogen and 30% more SS glycogen than dotype II fibers, which, in contrast, have 10% more IMF glycogen than do type I fibers (Nielsen et al. 2011). Nofiber-type differences are seen in sedentary obese subjects (Nielsen et al. 2010a) or recreationally activeyoung and elderly men (Nielsen et al. 2010b).

Endurance-trained athletesvs. untrained subjects

Elite cross-country skiers have 23% and 63% more IMF and SS glycogen, respectively, in both type I and IIfibers than do untrained subjects and 60% more Intra glycogen content in type I fibers only (Nielsen et al.2010a, 2010b, 2011).

Endurance training 10 weeks of aerobic training mediates a greater increase in SS glycogen content (90%) compared with theincrements in IMF and Intra glycogen content (15%) (Nielsen et al. 2010a).

Exercise and fatigue �1 h of exhaustive whole-body exercise (cross-country skiing) mediates a preferential utilization of Intraglycogen, which decreases by 89%, compared with 75% decrements in the IMF and SS glycogen contents oftriceps brachii-type I fibers (Nielsen et al. 2011). Similar preferential utilization is found in triceps brachii-type II fibers (Nielsen et al. 2011).

IMF and SS glycogen particles show a grouping appearance in the most glycogen-depleted fibersimmediately after exercise (Nielsen et al. 2011).

In mechanically skinned rat extensor digitorum longus fibers, Intra glycogen content correlates positivelywith fatigue resistance capacity, whereas IMF glycogen content correlates negatively with the relaxationtime of tetanic contractions in unfatigued fibers (Nielsen et al. 2009).

In isolated human SR vesicles, Intra glycogen content correlates with Ca2+ release rate (Ørtenblad et al. 2011).Recovery after exercise There is an initial (4-h) preferential resynthesis of Intra glycogen after high-intensity (but not modest)

glycogen-depleting exercise (Marchand et al. 2007; Nielsen et al. 2011, 2012).In the recovery period after a soccer match, resynthesis of Intra glycogen content is impaired during the

second day of recovery, whereas Intra glycogen increases exclusively from the second to the fifth day ofrecovery (Nielsen et al. 2012).

Type 2 diabetes The relative distribution of glycogen is no different in type 2 diabetic patients than in BMI-, age-, and sex-matched control subjects, with both groups showing similar adaptations to 10 weeks of aerobic training(Nielsen et al. 2010a).

Immobilization Intra glycogen content decreases by 50% after 2 weeks of immobilization, whereas IMF and SS glycogenremain unchanged (Nielsen et al. 2010b).

Note: SS, subsarcolemmal; IMF, intermyofibrillar; SR, sarcoplasmic reticulum; BMI, body mass index; Intra, intramyofibrillar.

Nielsen and Ørtenblad 95

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

firmed by 2 independent studies showing a strong concordancebetween the TEM-estimated glycogen volume fraction and thebiochemically determined glycogen concentration (Marchandet al. 2002; Nielsen et al. 2011). In biopsy specimens obtained im-mediately after prolonged (>4 h) medium-intensity (70% of maxi-mal oxygen consumption) cycling exercise, the Intra glycogencontent appeared to be preferentially depleted compared withvalues obtained in the subsequent recovery period (Marchandet al. 2007) and with respect to nonexercising control subjects(Marchand et al. 2002). This was subsequently confirmed in astudy inwhich biopsies undertaken before and after 1 h of exhaus-tive cross-country skiing clearly demonstrated that Intra glycogenwas broken down to relatively lower levels than were IMF and SSdepositions (Nielsen et al. 2011). Taken together, the recent quan-titative and the early observational studies suggest that Intra gly-cogen is preferentially utilized during both prolonged medium-intensity and intermittent high-intensity exercise. In the study byNielsen et al. (2011) on cross-country skiers using both arm and legmuscles, the higher utilization of Intra glycogen was apparent inboth type I and II fibers of the arms and type I fibers of the legs (noglycogen utilization was observed in type II fibers of the legs).Furthermore, in the most depleted fibers (type I fibers in thearms), SS glycogen decreased more than did IMF glycogen. Thus,in these highly endurance-trained subjects, glycogen utilizationby arm type I fibers was found to depend on subcellular localiza-tion in the order Intra > SS > IMF glycogen. Future quantitativestudies should address the role of subjects' training status, exer-cise mode, and intensity of the work performed in the phenome-non of localization-dependent glycogen utilization.

A detailed mechanistic explanation of the differential utiliza-tion of distinct glycogen deposits is yet to be determined. In theabove-mentioned study on cross-country skiers, an interestinggrouping of small IMF and SS glycogen particles was observed inthe most glycogen-depleted fibers after the exercise, and in rarecases these groupings colocalized with crystal-like structures alsofound exclusively in the most glycogen-depleted fibers (Nielsenet al. 2011). This shows that the utilization of IMF glycogen iscompartmentalized, as characterized by the almost complete de-pletion of glycogen in some areas while at the same time thesomewhat considerable retention of glycogen in others. Whetherthe grouping of particles is involved in such a retention mecha-nism needs to be clarified, along with the potential interactionbetween the grouping of particles and the crystal-like structures.That these groupings of IMF and SS glycogen particles may beinvolved in a retention (or safety) mechanism is supported by thefinding that glycogen synthase (GS) is directed to the crystal-likestructures in response to energy shortage in the cell (Prats et al.2009). GS is active during muscle work (Nielsen et al. 2003; Shul-man and Rothman 2001), which may be a compartmentalizedmechanism to prevent the muscle fibers from complete glycogendepletion and thereby impairment of crucial cellular functions.Indeed, results frommechanically skinned rat muscle fibers haveindicated that prevention of the total depletion of IMF glycogenmay ensure that necessary energy remains available for Ca2+ ho-meostasis, and thus cell survival, as suggested by the correlationbetween IMF glycogen and the half-relaxation time of tetanic con-tractions in unfatigued fibers (r2 = 0.25, Nielsen et al. 2009).

A higher relative utilization of Intra glycogen compared withthat of IMF and SS glycogen suggests that exhaustion of Intraglycogen may be implicated in muscle fatigue (i.e., the signal tocease forceful muscle contractions (Nielsen et al. 2011)). Two find-ings support this hypothesis. First, in mechanically skinned mus-cle fibers, the capacity to respond to repeated, electricalstimulated-evoked tetanic contractions correlates with the Intraglycogen content (r2 = 0.32, Nielsen et al. 2009). A partial rapidrecovery of force after exhaustion indicated a role of transmem-brane ion fluxes, pointing to a relationship between energy pro-vision from the breakdown of Intra glycogen and t-system Na+-K+

pumps. Second, the Intra glycogen content correlates with the SRCa2+ release rate in human SR vesicles (Ørtenblad et al. 2011),indicating that the in vivo breakdown of Intra glycogen mediatesan inhibitory signal, either direct or indirect, to the SR Ca2+ re-lease channels. An interesting aspect of these 2 findings is that thecolocalization in the diffusion-restricted triad, consisting of theSR terminal cisternae and t-system, integrates energy consump-tion (Na+-K+ pumps) with an early event of excitation-contractioncoupling (SR Ca2+ release) via Intra glycogen. In support of this, ithas been demonstrated that glycogen, some of its associated pro-teins, and glycolytic intermediates are able to physically interactwith the SR membrane (Wanson and Drochmans 1972; Entmanet al. 1980; Cuenda et al. 1995; Xu and Becker 1998; Kruszynskaet al. 2001) and, moreover, to affect the Ca2+ release properties ofthe SR (Hirata et al. 2003; Zima et al. 2006).

RecoveryThe resynthesis of biochemically determined glycogen in skel-

etal muscles after exercise was first demonstrated by Hultmanand colleagues (Bergström and Hultman 1966). It has been shownthat the time course of full replenishment depends on a list offactors, including, but not limited to, the training status of thesubjects (Hickner et al. 1997), their diet (Bergström et al. 1967), thetype of muscle work performed (degree of eccentric component)(Widrick et al. 1992), and the muscle's glycogen levels (Price et al.2000). Interestingly, during optimal conditions, glycogen levelscan increase to levels above pre-exercise levels, a phenomenonreferred to as “supercompensation” (Bergström and Hultman1966; Holloszy and Kohrt 1996). Although glycogen biosynthesisby the concerted action of GS and associated proteins has beenwell described biochemically, it remains largely unknown towhatextent and where the individual glycogen particle is synthesized(reviewed in Prats et al. 2011). Nevertheless, Graham and col-leagues (Graham et al. 2010; Marchand et al. 2007) demonstratedthat the initial 4 h of glycogen resynthesis in the vastus lateralisafter glycogen-depleting (<10% of 48-h recovery value) cycling ex-ercise was highly confined to the Intra space compared with theIMF and SS spaces. The relative distribution of glycogen located inthe Intra space increased from 3% immediately after the exerciseto 12% 4 h thereafter. These results are supported by a study show-ing that GS translocates to Intra glycogen particles in response toexercise-induced glycogen depletion (Prats et al. 2009) and corrob-orate the notion that Intra glycogen may play a profound roleduring muscle work (Nielsen et al. 2009, 2011; Ørtenblad et al.2011).

Recent studies (Nielsen et al. 2011, 2012) indicate that factorssuch as glycogen level, carbohydrate provision or availability, andmuscle damage may influence the localization-dependent recov-ery of muscle glycogen after exercise. These studies indicate thatexercise resulting in very low levels of glycogen (<10% of pre-exercise values or �36 mmol glycosyl units·kg−1 dw) induces apreferential initial replenishment of Intra glycogen (Marchandet al. 2007), whereas moderate to high levels of depletion (>10% ofpre-exercise values or 167–200mmol glycosyl units·kg−1 dw) are notfollowedbya localization-dependent initial replenishment (Nielsenet al. 2011, 2012). In the study by Nielsen et al. (2011), subjectsperformed exhaustive exercise for approximately 1 h and werethen divided into 2 groups, one of which consumed a high-carbohydrate-content food and the other water only during thefirst 4 h of recovery. Findings revealed that the absence of carbo-hydrate intake during this period of recoverymostly inhibited theresynthesis of Intra glycogen (Nielsen et al. 2011), indicating alocalization-dependent presence of some of the influential factorsinvolved in glycogen resynthesis. Likely areas of research requir-ing particular attention in future studies concern the differencebetween contraction- and insulin-mediated glucose uptake andthe role of the subcellular localization (and dynamic behavior) ofgluconeogenic enzymes in the resynthesis of distinct deposits of

96 Appl. Physiol. Nutr. Metab. Vol. 38, 2013

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

glycogen. It has been shown in skeletal and cardiac myocytes thatfructose-1,6-bisphosphatase translocates toward and away fromthe Z-line in response to high and low cytosolic-free Ca2+ concen-trations, respectively (Dzugaj 2006; Majkowski et al. 2010). Thissuggests that glyconeogenesis is directed towards IMF and SS gly-cogen (and away from Intra glycogen) in the resting condition (i.e.,during recovery with a low cytosolic-free Ca2+ concentration).

It was suggested earlier that an absence of insulin-mediatedglucose uptake in skeletal muscle mostly impairs the resynthesisof Intra glycogen. In line with this, we recently showed that theresynthesis of Intra glycogen after a soccer match consisting ofmany eccentric contractions accompanied by muscle damage(Krustrup et al. 2011) was impaired compared with the resynthesisof IMF and SS glycogen during the second day of recovery (Nielsenet al. 2012). Eccentric contractions are known to impair insulin-mediated glycogen resynthesis (Widrick et al. 1992; Doyle et al.1993), underscoring the likelihood of a tight connection betweenIntra glycogen and insulin-mediated glucose uptake. Another par-allel is the decrease in Intra glycogen following immobilization(Nielsen et al. 2010b), which suggests that the mechanisms in-volved in the delayed resynthesis of Intra glycogen followingmus-cle damage share similarities with immobilization-inducedalterations.

Interestingly, from the second to the fifth day of recovery afterthe soccer match, glycogen exclusively increased within the myo-fibrils (Intra glycogen), demonstrating that the muscle has thepotential for increasing glycogen content in a specific localizationlate in the recovery period. Unfortunately, because a prematchglycogen reading was not taken, it is unknownwhether there wasa delayed full replenishment or a supercompensation of Intraglycogen following the soccer match.

Taken together, the prevailing knowledge on the resynthesis ofdistinct deposits of glycogen following exercise gives rise to sev-eral questions and hypotheses in relation to insulin action, mus-cle damage, and glyconeogenesis: Does insulin-mediated glucoseuptake preferentially restore Intra glycogen? Does muscle dam-age transiently impair the resynthesis of Intra glycogen or does itdown-regulate Intra glycogen via pathways similar to those ofimmobilization? Does glyconeogenesis take place inside the myo-fibrils (Intra glycogen) duringmuscle work and high cytosolic-freeCa2+ concentrations, and does the opposite take place in the IMFspace during rest with a low cytosolic-free Ca2+ concentration?

Effects of immobilization in recreationally activemen

Changing environmental conditions in terms of periods ofphysical training and of muscle disuse are powerful experimentalapproaches that can provide new insights into skeletal muscleplasticity. With regard tomuscle disuse, we were surprised to findthat 2 weeks' immobilization of the vastus lateralis muscle re-sulted in a 50% decrease in the Intra glycogen content, whereasIMF and SS glycogen remained unchanged (Nielsen et al. 2010b).Convincingly, the Intra glycogen content of all the fibers investi-gated after immobilization was lower than the median value be-fore immobilization, indicating that most fibers, if not all,underwent this adaptation. The average size of the remainingIntra glycogen particles was not smaller after immobilization,indicating that the decreased Intra glycogen content was due to adecrease in the number of particles. Thus, adaptation to an almostcomplete absence of muscle activation is seen as a lowering of thecapacity for glycogen storage within the myofibrils.

Although no studies have been conducted to elucidate howIntra glycogen is lost following immobilization, a few findings arestriking. In the recovery period after pronounced glycogen-depleting exercise, Intra glycogen is resynthesized preferentially(Marchand et al. 2007) and GS is translocated to Intra glycogen

particles (Prats et al. 2009), suggesting that contractile activitymediates a shift in the distribution of glycogen towards the Intraspace. Muscle disuse may therefore induce a reverse effect, sug-gesting that GS is translocated away from the Intra glycogen par-ticles. Another potential mechanism, which requires furtherinvestigation, is insulin sensitivity. Rat skeletal muscle denerva-tion has been shown to decrease insulin-mediated glucose uptakeby the t-system, but not by the sarcolemma (Lauritzen et al. 2008),indicating that muscle disuse may induce a localization-specificadaptation in basal glucose uptake, which, in turn, may affect thedistribution of glycogen. Indeed, we have shown that resynthesisof Intra glycogen during the second day of recovery from a soccermatch is impaired (Nielsen et al. 2012), which appears to corre-spond temporally withmuscle-damage-induced insulin resistance(Widrick et al. 1992; Doyle et al. 1993). Thus, differences betweent-system and sarcolemmal glucose uptake may play a role in gly-cogen localization.

Effects of training in type 2 diabetic patientsType 2 diabetes is characterized by impaired insulin-mediated

skeletal muscle glucose uptake, glucose phosphorylation, andbasal and insulin-mediated GS activation (reviewed in Abdul-Ghaniand DeFronzo 2010). Interestingly, the architecturalorganization of the muscle cell interior has proven to be influen-tial in all these aspects. Firstly, the impaired insulin-dependentglucose uptake is associated with a defect in glucose transporter 4(GLUT4) translocation from intracellular storage sites to T-tubulesand the sarcolemma (Marette et al. 1992); in mice at least, thisdefect is primarily localized to T-tubules and not to the sarco-lemma (Lauritzen et al. 2008). Second, GLUT4 intrinsic activityseems to be influenced by glyceraldehyde 3-phosphate dehy-drogenase binding activity (reviewed in Klip 2009), providing astructural basis for coupling among glycogen, glycolysis, and glu-cose uptake. Third, phosphorylation of glucose following uptakeis accomplished by hexokinase II, which is found in both cytosolicand mitochondrial fractions (Sigel and Pette 1969), suggestingthat hexokinase II activity may play a role in glycogen localiza-tion. Fourth, GS has the ability to translocate between distinctlocalizations of glycogen and the cytoskeleton, depending on site-specific phosphorylation and the cell's energy status (Prats et al.2005, 2009). Finally, the activation of GS depends partly ondephosphorylation by protein phosphatase 1, which in skeletalmuscle interacts with glycogen particles by glycogen-associated regulatory subunits, of which some forms have beenreported to be influenced by insulin (Munro et al. 2005) andothers to be involved in regulating glycogen particle size andsubcellular distribution (Montori-Grau et al. 2011). Interest-ingly, GLUT4 translocation, hexokinase activity, and GS local-ization and activation depend on the glycogen level of the cell(Derave et al. 1999; Nielsen et al. 2001; Danforth 1965). Given thespecific subcellular localizations of these processes, it is mostlikely that the effect of glycogen depends on its subcellularlocalization.

To investigate whether the distribution of glycogen may dif-fer in the skeletal muscles of type 2 diabetics with respect tocontrol subjects and hence be related to at least one of theglucose metabolic disorders, we estimated the distribution ofglycogen in distinct subcellular localizations of thigh skeletalmuscle preparations from type 2 diabetic patients and fromage-, sex-, and body mass index (BMI)-matched control subjects(Nielsen et al. 2010a). No difference between the 2 groups wasobserved, and all showed similar adaptations to 10 weeks of aero-bic training. Indeed, all 3 defined subfractions of glycogen in-creased in response to training in parallel with improved insulinsensitivity, implying that the glycogen content of distinct local-izations is not involved in insulin resistance per se (Nielsen et al.2010a). However, the study by Nielsen et al. (2010a) did not inves-

Nielsen and Ørtenblad 97

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

tigate whether any of the key molecular players in insulin signal-ing were related to any of the different glycogen storage sites. Inboth type 2 diabetic patients and BMI-matched control subjects,12 weeks of aerobic training gave rise to a greater increase in SSglycogen (>90%) compared with the increments in IMF (14%) andIntra glycogen (15%) (Nielsen et al. 2010a). Increase in the latter2 glycogen contents may be explained by an increase in the size ofexisting particles (the mean particle volume increased by 23%),whereas the increase in SS glycogen necessitates an increase inthe number of particles (Nielsen et al. 2010a). This suggests that atraining-induced biosynthesis of new glycogen particles takesplace in the SS space.

Concluding remarks and perspectivesAlthough the relationship between glycogen and the endurance

capacity of skeletal muscle is a fundamental concept in exercisephysiology, it is still not clear how and why low glycogen levelsimpair muscle function. Consideration of compartmentalizedmeta-bolic pathways in the muscle, together with the observation of spa-tially distinct subcellulardepots of glycogen,hasproven,however, tobe an important advance in developing a more complete under-standing of the role and regulation of glycogen stores.

A recurring finding is that a minor depot located within themyofibrils (Intra glycogen) seems to be very susceptible to envi-ronmental alterations and apparently plays an essential role inmuscle function. Intra glycogen is preferentially utilized duringexercise and, if almost completely depleted, is subsequently re-synthesized during the recovery period. In combination with thefindings that Intra glycogen is correlated with both the SR Ca2+

release rate in SR vesicles and the endurance capacity in mechan-ically skinned fibers (Table 1), this strongly suggests that theassociation of glycogen with fatigue may be via Intra glycogenthrough as yet unknownmechanisms. Subsequently, determin-ing how type I fibers attain more Intra glycogen than do type IIfibers and how Intra glycogen particles are lost following im-mobilization are important advances to be made to better un-derstand the factors underlying optimal muscle function.Additionally, in biopsy specimens obtained immediately afterexercise, most of the glycogen was located in the IMF space, anda grouping appearance of the remaining glycogen particles sug-gested a differential breakdown of particles within the IMFspace during exercise. We suggest that a subfraction of IMFglycogen may be retained at the end of glycogen-depleting ex-ercise to serve as a safety energy store for Ca2+ reuptake by theSR, this being a crucial mechanism for cell survival.

AcknowledgementsWe thank The Lundbeck Foundation, the Danish Ministry of

Culture Committee on Sports Research, and the Swedish NationalCentre for Research in Sports for research funding.

ReferencesAbdul-Ghani, M.A., and DeFronzo, R.A. 2010. Pathogenesis of insulin resistance

in skeletal muscle. J. Biomed. Biotech. 2010: 476279.Allen, D.G., Lamb, G.D., and Westerblad, H. 2008. Skeletal muscle fatigue: cellu-

lar mechanisms. Physiol. Rev. 88: 287–332. doi:10.1152/physrev.00015.2007.PMID:18195089.

Aon, M.A., Cortassa, S., and O'Rourke, B. 2007. On the network properties ofmitochondria. In Molecular system bioenergetics. Edited by V. Saks. Wiley-VCH Verlag GmbH and Co., KGaA, Weinheim, Germany. pp. 111–136.

Bergström, J., andHultman, E. 1966. Muscle glycogen synthesis after exercise: anenhancing factor localized to the muscle cells in man. Nature, 210(5033):309–310. doi:10.1038/210309a0. PMID:5954569.

Bergström, J., Hermansen, L., Hultman, E., and Saltin, B. 1967. Diet, muscleglycogen and physical performance. Acta Physiol. Scand. 71: 140–150. doi:10.1111/j.1748-1716.1967.tb03720.x. PMID:5584523.

Blomstrand, E., and Saltin, B. 1999. Effect of muscle glycogen on glucose, lactateand amino acidmetabolism during exercise and recovery in human subjects.J. Physiol. 514: 293–302. doi:10.1111/j.1469-7793.1999.293af.x. PMID:9831734.

Boehm, E., Ventura-Clapier, R., Mateo, P., Lechène, P., and Veksler, V. 2000.Glycolysis supports calcium uptake by the sarcoplasmic reticulum in

skinned ventricular fibres of mice deficient in mitochondrial and cytosoliccreatine kinase. J. Mol. Cell. Cardiol. 32: 891–902. doi:10.1006/jmcc.2000.1130.PMID:10888244.

Clausen, T. 2003. Na+-K+ pump regulation and skeletal muscle contractility.Physiol. Rev. 83: 1269–1324. PMID:14506306.

Connett, R.J., Gayeski, T.E.J., and Honig, C.R. 1986. Lactate efflux is unrelated tointracellular Po2 in a working red muscle in situ. J. Appl. Physiol. 61: 402–408. PMID:3745033.

Cuenda, A., Nogues, M., Henao, F., and Gutierrez-Merino, C. 1995. Interactionbetween glycogen phosphorylase and sarcoplasmic reticulum membranesand its functional implications. J. Biol. Chem. 270: 11998–12004. doi:10.1074/jbc.270.20.11998. PMID:7744850.

Danforth,W.H. 1965. Glycogen synthase activity in skeletal muscle: interconver-sion of two forms and control of glycogen synthesis. J. Biol. Chem. 240:588–593. PMID:14275108.

Das, A.M., Steuerwald, U., and Illsinger, S. 2010. Inborn errors of energy metab-olism associated with myopathies. J. Biomed. Biotech. 2010: 340849.

Derave, W., Lund, S., Holman, G.D., Wojtaszewski, J., Pedersen, O., andRichter, E.A. 1999. Contraction-stimulated muscle glucose transport andGLUT-4 surface content are dependent on glycogen content. Am. J. Physiol.Endocrinol. Metab. 40: E1103–E1110.

Dhar-Chowdhury, P., Malester, B., Rajacic, P., and Coetzee, W.A., 2007. The reg-ulation of ion channels and transporters by glycolytically derived ATP. CellMol. Life Sci. 64: 3069–3083. doi:10.1007/s00018-007-7332-3. PMID:17882378.

Doyle, J.A., Sherman, W.M., and Strauss, R.L. 1993. Effects of eccentric and con-centric exercise on muscle glycogen replenishment. J. Appl. Physiol. 74:1848–1855. PMID:8514702.

Duhamel, T.A., Perco, J.G., and Green, H.J. 2006. Manipulation of dietary carbo-hydrates after prolonged effort modifies muscle sarcoplasmic reticulum re-sponses in exercisingmales. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291:R1100–R1110. doi:10.1152/ajpregu.00858.2005. PMID:16690765.

Dutka, T.L., and Lamb, G.D. 2007. Na+-K+ pumps in the transverse tubular systemof skeletal muscle fibers preferentially use ATP from glycolysis. Am. J.Physiol. Cell. Physiol. 293: 967–977.

Dzugaj, A. 2006. Localization and regulation of muscle fructose-1,6-bisphosphatase, the key enzyme of glyconeogenesis. Adv. Enzyme Regul. 46:51–71. doi:10.1016/j.advenzreg.2006.01.021. PMID:16857246.

Entman, M.L., Keslensky, S.S., Chu, A., and Van Winkle, B. 1980. The sarcoplas-mic reticulum-glycogenolytic complex in mammalian fast twitch skeletalmuscle. J. Biol. Chem. 255: 6245–6252. PMID:6446555.

Everts, M.E., and Clausen, T. 1992. Activation of the Na-K pump by intracellularNa in rat slow- and fast-twitch muscle. Acta Physiol. Scand. 145(4): 353–362.doi:10.1111/j.1748-1716.1992.tb09375.x. PMID:1326854.

Fridén, J., Seger, J., and Ekblom, B. 1985. Implementation of periodicacid-thiosemicarbazide-silver proteinate staining for ultrastructural assess-ment of muscle glycogen utilization during exercise. Cell Tissue Res. 242:229–232. PMID:2994885.

Fridén, J., Seger, J., and Ekblom, B. 1989. Topographical localization of muscleglycogen: an ultrahistochemical study in the human vastus lateralis. ActaPhysiol. Scand. 135: 381–391. doi:10.1111/j.1748-1716.1989.tb08591.x. PMID:2467521.

Gollnick, P.D., Piehl, K., and Saltin, B. 1974. Selective glycogen depletion patternin human muscle fibres after exercise of varying intensity and at varyingpedalling rates. J. Physiol. 241: 45–57. PMID:4278539.

Graham, T.E., Yuan, Z., Hill, A.K., andWilson, R.J. 2010. The regulation ofmuscleglycogen: the granule and its proteins. Acta Physiol. (Oxford), 199: 489–498.

Green, H.J. 1991. How important is endogenous muscle glycogen to fatigue inprolonged exercise? Can. J. Physiol. Pharmacol. 69: 290–297. doi:10.1139/y91-045.

Haller, R.G., Clausen, T., and Vissing, J. 1998. Reduced levels of skeletal muscleNa+K+ -ATPase in McArdle disease. Neurology, 50: 37–40. doi:10.1212/WNL.50.3_Suppl_2.37. PMID:9443454.

Han, J.W., Thieleczek, R., Varsanyi, M., and Heilmeyer, L.M., Jr. 1992. Compart-mentalized ATP synthesis in skeletal muscle triads. Biochemistry, 31: 377–384. doi:10.1021/bi00117a010. PMID:1731894.

Hermansen, L., Hultman, E., and Saltin, B. 1967. Muscle glycogen during pro-longed severeexercise.ActaPhysiol. Scand.71: 129–139. doi:10.1111/j.1748-1716.1967.tb03719.x. PMID:5584522.

Hespel, P., and Richter, E.A. 1992. Mechanism linking glycogen concentrationand glycogenolytic rate in perfused contracting rat skeletalmuscle. Biochem.J. 284: 777–780. PMID:1622395.

Hickner, R.C., Fisher, J.S., Hansen, P.A., Racette, S.B., Mier, C.M., Turner,M.J., andHolloszy, J.O. 1997. Muscle glycogen accumulation after endurance exercisein trained and untrained individuals. J. Appl. Physiol. 83: 897–903. PMID:9292478.

Hirata, Y., Atsumi, M., Ohizumi, Y., and Nakahata, N. 2003. Mastoparan binds toglycogen phosphorylase to regulate sarcoplasmic reticular Ca2+ release inskeletal muscle. Biochem. J. 371: 81–88. doi:10.1042/BJ20021844. PMID:12519071.

Holloszy, J.O., and Kohrt, W.M. 1996. Regulation of carbohydrate and fat metab-olism during and after exercise. Annu. Rev. Nutr. 16: 121–138. doi:10.1146/annurev.nu.16.070196.001005. PMID:8839922.

James, J.H., Wagner, K.R., King, J.-K., Leffler, R.E., Upputuri, R.K., Balasubramanian, A.,

98 Appl. Physiol. Nutr. Metab. Vol. 38, 2013

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.

etal. 1999. Stimulation of both aerobic glycolysis andNa+-K+-ATPase activity inskeletal muscle by epinephrine or amylin. Am. J. Physiol. Endo. Metab. 277:E176–E186.

Klip, A. 2009. The many ways to regulate glucose transporter 4. Appl. Physiol.Nutr. Metab. 34: 481–487. doi:10.1139/H09-047. PMID:19448718.

Krustrup, P., Ørtenblad, N., Nielsen, J., Nybo, L., Gunnarsson, T.P., Iaia, et al.2011. Maximal voluntary contraction force, SR function and glycogen resynthesisduring thefirst 72hafter ahigh-level competitive soccer game. Eur. J. Appl. Physiol.111(12): 2987–2995. doi:10.1007/s00421-011-1919-y. PMID:21448723.

Kruszynska, Y.T., Ciaraldi, T.P., and Henry, R.R. 2001. The endocrine system. InHandbook of Physiology. A critical, comprehensive presentation of physio-logical knowledge and concepts. Edited by Jefferson, et al. Oxford UniversityPress, New York, N.Y., USA. pp. 579–607.

Lauritzen, H.P., Ploug, T., Ai, H., Donsmark, M., Prats, C., and Galbo, H. 2008.Denervation and high-fat diet reduce insulin signaling in T-tubules in skele-tal muscle of living mice. Diabetes, 57: 13–23. PMID:17914033.

Lees, S.J., and Williams, J.H. 2004. Skeletal muscle sarcoplasmic reticulum gly-cogen status influences Ca2+ uptake supported by endogenously synthesizedATP. Am. J. Physiol. Cell. Physiol. 286: C97–C104. PMID:12967914.

Majkowski, M., Wypych, D., Pomorski, P., and Dzugaj, A. 2010. Regulation ofsubcellular localization of muscle FBPase in cardiomyocytes. The decisiverole of calcium ions. Acta Biochim. Polonica, 57(4): 597–605.

Mantilla, C.B., Rowley, K.L., Zhan, W.Z., Fahim, M.A., and Sieck, G.C. 2007. Syn-aptic vesicle pools at diaphragm neuromuscular junctions vary with mo-toneuron soma, not axon terminal, inactivity. Neuroscience, 146: 178–189.doi:10.1016/j.neuroscience.2007.01.048. PMID:17346898.

Marchand, I., Chorneyko, K., Tarnopolsky, M., Hamilton, S., Shearer, J.,Potvin, J., and Graham, T.E. 2002. Quantification of subcellular glycogen inresting human muscle: granule size, number, and location. J. Appl. Physiol.93: 1598–1607. PMID:12381743.

Marchand, I., Tarnopolsky, M., Adamo, K.B., Bourgeois, J.M., Chorneyko, K., andGraham, T.E. 2007. Quantitative assessment of humanmuscle glycogen gran-ules size and number in subcellular locations during recovery from pro-longed exercise. J. Physiol. 580: 617–628. PMID:17272352.

Marette, A., Burdett, E., Douen, A., Vranic, M., and Klip, A. 1992. Insulin inducesthe translocation of GLUT4 from a unique intracellular organelle to trans-verse tubules in rat skeletal muscle. Diabetes, 41(12): 1562–1569. doi:10.2337/diabetes.41.12.1562. PMID:1446797.

Metzger, J.M., and Moss, R.L. 1987. Shortening velocity in skinned single musclefibers. Influence of lattice spacing. Biophys. J. 52: 127–131.

Montori-Grau, M., Guitart, M., Garcis-Martinez, C., Orozco, A., andGomez-Foix, A.M. 2011. Differential pattern of glycogen accumulation afterprotein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression,compared to PPP1R3C and PPP1R3A, in skeletal muscle cells. BMC Biochem.12: 57. doi:10.1186/1471-2091-12-57.

Munro, S., Ceulemans, H., Bollen, M., Diplexcito, J., and Cohen, P.T.W. 2005. Anovel glycogen-targeting subunit of protein phosphatase 1 that is regulatedby insulin and shows differential tissue distribution in humans and rodents.FEBS J. 272: 1478–1489. doi:10.1111/j.1742-4658.2005.04585.x. PMID:15752363.

Nielsen, J.N., and Richter, E.A. 2003. Regulation of glycogen synthase in skeletalmuscle during exercise. Acta Physiol. Scand. 178: 309–319. doi:10.1046/j.1365-201X.2003.01165.x. PMID:12864735.

Nielsen, J.N., Derave, W., Kristiansen, S., Ralston, E., Ploug, T., and Richter, E.A.2001. Glycogen synthase localization and activity in rat skeletal muscle isstrongly dependent on glycogen content. J. Physiol. 531: 757–769. doi:10.1111/j.1469-7793.2001.0757h.x. PMID:11251056.

Nielsen, J., Schrøder, H.D., Rix, C.G., and Ørtenblad, N. 2009. Distinct effects ofsubcellular glycogen localization on tetanic relaxation time and endurancein mechanically skinned rat skeletal muscle fibres. J. Physiol. 587: 3679–3690. doi:10.1113/jphysiol.2009.174862. PMID:19470780.

Nielsen, J., Mogensen, M., Vind, B.F., Sahlin, K., Højlund, K., Schrøder, H.D., andØrtenblad, N. 2010a. Increased subsarcolemmal lipids in type 2 diabetes:effect of training on localization of lipids, mitochondria, and glycogen insedentary human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 298:E706–E713. doi:10.1152/ajpendo.00692.2009. PMID:20028967.

Nielsen, J., Suetta, C., Hvid, L.G., Schrøder, H.D., Aagaard, P., and Ørtenblad, N.2010b. Subcellular localization-dependent decrements in skeletal muscle gly-cogen and mitochondria content following short-term disuse in young andold men. Am. J. Physiol. Endocrinol. Metab. 299: E1053–E1060. doi:10.1152/ajpendo.00324.2010. PMID:20858747.

Nielsen, J., Holmberg, H.C., Schrøder, H.D., Saltin, B., and Ørtenblad, N. 2011.

Human skeletal muscle glycogen utilization in exhaustive exercise: role ofsubcellular localization and fibre type. J. Physiol. 589: 2871–2885. doi:10.1113/jphysiol.2010.204487. PMID:21486810.

Nielsen, J., Krustrup, P., Nybo, L., Gunnarsson, T.P., Madsen, K., Schrøder, et al.2012. Maximal voluntary contraction force, SR function and glycogen resyn-thesis during the first 72 h after a high-level competitive soccer game. Eur. J.Appl. Physiol. 112(10): 3559–3567. doi:10.1007/s00421-012-2341-9. PMID:22323299.

Oberholzer, F., Claassen, H., Moesch, H., and Howald, H. 1976. Ultrastrukturelle,biochemische und energetische Analyse einer extremen Dauerleistung(100 Km-Lauf). Schweiz Z. Sportmed. 24: 71–98. PMID:1013723.

Ørtenblad, N., Nielsen, J., Saltin, B., and Holmberg, H.C. 2011. Role of glycogenavailability on SR Ca2+ kinetics in human skeletal muscle. J. Physiol. 589:711–725. doi:10.1113/jphysiol.2010.195982. PMID:21135051.

Ovadi, J., and Saks, V. 2004. On the origin of intracellular compartmentation andorganized metabolic systems. Mol. Cell Biochem. 256/257: 5–12.

Prats, C., Cadefau, J.A., Cussó, R., Qvortrup, K., Nielsen, J.N.,Wojtaszewski, J.F.P.,et al. 2005. Phosphorylation-dependent translocation of glycogen synthase toa novel structure during glycogen resynthesis. J. Biol. Chem. 280: 23165–23172. doi:10.1074/jbc.M502713200. PMID:15840572.

Prats, C., Helge, J.W., Nordby, P., Qvortrup, K., Ploug, T., Dela, F., andWojtaszewski, J.F. 2009. Dual regulation of muscle glycogen synthase duringexercise by activation and compartmentalization. J. Biol. Chem. 284: 15692–15700. doi:10.1074/jbc.M900845200. PMID:19339242.

Prats, C., Gomez-Cabello, A., and Hansen, A.V. 2011. Intracellular compartmen-talization of skeletal muscle glycogen metabolism and insulin signalling.Exp. Physiol. 96(4): 385–390. doi:10.1113/expphysiol.2010.052860. PMID:21239465.

Price, T.B., Laurent, D., Petersen, K.F., Rothman, D.L., and Shulman, G.I. 2000.Glycogen loading alters muscle glycogen resynthesis after exercise. J. Appl.Physiol. 88: 698–704. PMID:10658040.

Saks, V., Beraud, N., and Wallimann, T. 2008. Metabolic compartmentation – asystem level property of muscle cells. Int. J. Mol. Sci. 9: 751–767. doi:10.3390/ijms9050751. PMID:19325782.

Schiaffino, S., and Reggiani, C. 2011. Fiber types inmammalian skeletal muscles.Physiol. Rev. 91: 1447–1531. doi:10.1152/physrev.00031.2010. PMID:22013216.

Schmalbruch, H., and Kamieniecka, Z. 1974. Fiber types in human brachialbiceps muscle. Exp. Neurology, 44: 313–328.

Shulman, R.G., and Rothman, D.L. 2001. The “glycogen shunt” in exercisingmuscle: A role for glycogen in muscle energetic and fatigue. Proc. Natl. Acad.Sci. U.S.A. 98(2): 457–461. doi:10.1073/pnas.98.2.457. PMID:11209049.

Sigel, P., and Pette, D. 1969. Intracellular localization of glycogenolytic andglycolytic enzymes in white and red rabbit skeletal muscle. J. Histochem.Cytochem. 17(4): 225–237. doi:10.1177/17.4.225. PMID:4240499.

Sjöström, M., Fridén, J., and Ekblom, B. 1982a. Fine structural details of humanmuscle fibres after fibre type specific glycogen depletion. Histochemistry, 76:425–438. doi:10.1007/BF00489899. PMID:7166509.

Sjöström, M., Angquist, K.A., Bylund, A.C., Fridén, J., Gustavsson, L., andSchersten, T. 1982b. Morphometric analyses of human muscle fiber types.Muscle Nerve, 5: 538–553. doi:10.1002/mus.880050708. PMID:6292711.

Srere, P.A. 1967. Enzyme concentrations in tissues. Science, 158(3803): 936–937.doi:10.1126/science.158.3803.936. PMID:6054167.

Vøllestad, N.K., and Blom, P.C. 1985. Effect of varying exercise intensity onglycogen depletion in human muscle fibres. Acta Physiol. Scand. 125: 395–405. doi:10.1111/j.1748-1716.1985.tb07735.x. PMID:4083044.

Wanson, J.C., and Drochmans, P. 1972. Role of the sarcoplasmic reticulum inglycogen metabolism. J. Cell. Biol. 54: 206–224. doi:10.1083/jcb.54.2.206.PMID:5040859.

Welch, G.R., and Clegg, J.S. 2010. From protoplasmic theory to cellular systemsbiology: a 150-year reflection. Am. J. Physiol. Cell Physiol. 298: C1280–C1290.doi:10.1152/ajpcell.00016.2010. PMID:20200206.

Widrick, J.J., Costill, D.L., McConell, G.K., Anderson, D.E., Pearson, D.R., andZachwieja, J.J. 1992. Time course of glycogen accumulation after eccentricexercise. J. Appl. Physiol. 72: 1999–2004. PMID:1601811.

Xu, K.Y., and Becker, L.C. 1998. Ultrastructural localization of glycolytic enzymeson sarcoplasmic reticulum vesticles. J. Histochem. Cytochem. 46: 419–427.doi:10.1177/002215549804600401. PMID:9575039.

Zima, A.V., Kockskämper, J., and Blatter, L.A. 2006. Cytosolic energy reservesdetermine the effect of glycolytic sugar phosphates on sarcoplasmic reticu-lum Ca2+ release in cat ventricular myocytes. J. Physiol. 577(Pt1): 281–293.doi:10.1113/jphysiol.2006.117242. PMID:16945967.

Nielsen and Ørtenblad 99

Published by NRC Research Press

App

l. Ph

ysio

l. N

utr.

Met

ab. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y U

NIV

CA

LG

AR

Y o

n 03

/13/

13Fo

r pe

rson

al u

se o

nly.