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Human Skeletal Muscle Disuse Atrophy: Effects on Muscle Protein Synthesis, Breakdown, and Insulin Resistance—A Qualitative Review

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Abstract and Figures

The ever increasing burden of an aging population and pandemic of metabolic syndrome worldwide demands further understanding of the modifiable risk factors in reducing disability and morbidity associated with these conditions. Disuse skeletal muscle atrophy (sometimes referred to as " simple " atrophy) and insulin resistance are " non-pathological " events resulting from sedentary behavior and periods of enforced immobilization e.g., due to fractures or elective orthopedic surgery. Yet, the processes and drivers regulating disuse atrophy and insulin resistance and the associated molecular events remain unclear—especially in humans. The aim of this review is to present current knowledge of relationships between muscle protein turnover, insulin resistance and muscle atrophy during disuse, principally in humans. Immobilization lowers fasted state muscle protein synthesis (MPS) and induces fed-state " anabolic resistance. " While a lack of dynamic measurements of muscle protein breakdown (MPB) precludes defining a definitive role for MPB in disuse atrophy, some proteolytic " marker " studies (e.g., MPB genes) suggest a potential early elevation. Immobilization also induces muscle insulin resistance (IR). Moreover, the trajectory of muscle atrophy appears to be accelerated in persistent IR states (e.g., Type II diabetes), suggesting IR may contribute to muscle disuse atrophy under these conditions. Nonetheless, the role of differences in insulin sensitivity across distinct muscle groups and its effects on rates of atrophy remains unclear. Multifaceted time-course studies into the collective role of insulin resistance and muscle protein turnover in the setting of disuse muscle atrophy, in humans, are needed to facilitate the development of appropriate countermeasures and efficacious rehabilitation protocols.
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REVIEW
published: 25 August 2016
doi: 10.3389/fphys.2016.00361
Frontiers in Physiology | www.frontiersin.org 1August 2016 | Volume 7 | Article 361
Edited by:
Li Zuo,
Ohio State University, USA
Reviewed by:
Han-Zhong Feng,
Wayne State University School of
Medicine, USA
Fan Ye,
University of Florida, USA
Feng He,
California State University, Chico, USA
*Correspondence:
Iskandar Idris
iskandar.idris@nottingham.ac.uk
Philip J. Atherton
philip.atherton@nottingham.ac.uk
Equal last authors.
Specialty section:
This article was submitted to
Striated Muscle Physiology,
a section of the journal
Frontiers in Physiology
Received: 24 May 2016
Accepted: 08 August 2016
Published: 25 August 2016
Citation:
Rudrappa SS, Wilkinson DJ,
Greenhaff PL, Smith K, Idris I and
Atherton PJ (2016) Human Skeletal
Muscle Disuse Atrophy: Effects on
Muscle Protein Synthesis, Breakdown,
and Insulin Resistance—A Qualitative
Review. Front. Physiol. 7:361.
doi: 10.3389/fphys.2016.00361
Human Skeletal Muscle Disuse
Atrophy: Effects on Muscle Protein
Synthesis, Breakdown, and Insulin
Resistance—A Qualitative Review
Supreeth S. Rudrappa, Daniel J. Wilkinson, Paul L. Greenhaff, Kenneth Smith,
Iskandar Idris *and Philip J. Atherton *
Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, MRC-Arthritis Research UK Centre for
Musculoskeletal Ageing Research, Royal Derby Hospital, University of Nottingham, Derby, UK
The ever increasing burden of an aging population and pandemic of metabolic syndrome
worldwide demands further understanding of the modifiable risk factors in reducing
disability and morbidity associated with these conditions. Disuse skeletal muscle atrophy
(sometimes referred to as “simple” atrophy) and insulin resistance are “non-pathological”
events resulting from sedentary behavior and periods of enforced immobilization e.g.,
due to fractures or elective orthopedic surgery. Yet, the processes and drivers regulating
disuse atrophy and insulin resistance and the associated molecular events remain
unclear—especially in humans. The aim of this review is to present current knowledge
of relationships between muscle protein turnover, insulin resistance and muscle atrophy
during disuse, principally in humans. Immobilization lowers fasted state muscle protein
synthesis (MPS) and induces fed-state “anabolic resistance.” While a lack of dynamic
measurements of muscle protein breakdown (MPB) precludes defining a definitive role
for MPB in disuse atrophy, some proteolytic “marker” studies (e.g., MPB genes) suggest
a potential early elevation. Immobilization also induces muscle insulin resistance (IR).
Moreover, the trajectory of muscle atrophy appears to be accelerated in persistent IR
states (e.g., Type II diabetes), suggesting IR may contribute to muscle disuse atrophy
under these conditions. Nonetheless, the role of differences in insulin sensitivity across
distinct muscle groups and its effects on rates of atrophy remains unclear. Multifaceted
time-course studies into the collective role of insulin resistance and muscle protein
turnover in the setting of disuse muscle atrophy, in humans, are needed to facilitate the
development of appropriate countermeasures and efficacious rehabilitation protocols.
Keywords: skeletal muscle, disuse, immobilization, protein metabolism, diabetes
INTRODUCTION
Skeletal muscle tissue represents the largest protein/amino acid (AA) reservoir in the human
body (Bonaldo and Sandri, 2013). Skeletal muscles are not only crucial for locomotion but also
represent the body’s largest metabolically active tissue, glucose disposal site, and fuel reservoir
for other organs in fasting and pathological conditions (i.e., hepatic supply of amino acids for
gluconeogenesis). Loss of muscle mass occurs with many common illnesses (Evans, 2010) including
Rudrappa et al. Mechanisms of Disuse Atrophy in Humans
cancers (Stephens et al., 2010), renal/heart failure (Gordon et al.,
2013), sepsis (Gordon et al., 2013), muscle genetic diseases
(Sandri, 2010), and neurodegenerative disorders (Verdijk et al.,
2012). Muscle atrophy also occurs in otherwise healthy
individuals under situations of reduced neural input—such as
during immobilization, e.g., due to leg casting for fractures
(Phillips et al., 2009), bed-rest, spinal cord injury (Castro et al.,
1999) and during “aging” (i.e., sarcopenia; Evans and Lexell,
1995). The main environmental determinants of muscle mass in
adulthood are exogenous essential amino acids (AA) (needing
to be acquired through dietary protein intake), Newton’s gravity
and locomotion (DeFronzo and Tripathy, 2009). Indicative of
this, lack of energy intake during starvation (Rennie et al., 2010),
inactivity (Wall et al., 2013), spaceflight (Vandenburgh et al.,
1999), or limb immobilization (Phillips et al., 2009) all lead to
a reduction in muscle cross sectional area (CSA), an associated
loss of function, and muscle insulin resistance. Crucially, loss of
muscle mass is associated with greater morbidity and mortality
(Sasaki et al., 2007), reduced independence, especially in older
populations (Leenders et al., 2013) and this is accelerated in type
2 diabetes (Leenders et al., 2013).
Overview of Disuse Atrophy,
Countermeasures, and Muscle Metabolism
Disuse atrophy is often referred to as “simple atrophy” in
that atrophy is intrinsic to the muscle(s) specifically exposed
to disuse; that is, disuse atrophy is not a systemic condition.
Countermeasures for disuse atrophy are limited but include
forms of mechanical loading (Wilkinson et al., 2008) such
as exercise/electrical stimulation (Wall et al., 2012), passive
physiotherapy (Fowles et al., 2000) and harnessing the adjunct
anabolic effects of protein nutrition (Churchward-Venne et al.,
2012b). Ascertaining an understanding of the mechanisms
of disuse atrophy—particularly in relation to the regulation
of muscle protein synthesis (MPS) and breakdown (MPB)—
is important for designing countermeasures or rehabilitation
protocols (Reggiani, 2015). Furthermore, despite accumulating
evidence that physical inactivity plays a causative role in
development of non-communicable diseases such as obesity,
insulin resistance, type 2 diabetes and dyslipidemia (Atherton
et al., 2016), the mechanistic role of muscle insulin resistance
(IR) in driving muscle atrophy in the context of “simple disuse”
remains unclear (Atherton et al., 2016). This review will focus on
identifying different models of human disuse atrophy, the degree
of muscle and strength loss and the regulation of muscle protein
turnover and muscle IR. Future translational studies to mitigate
disuse atrophy will rely upon robust evidence being present in
humans of active mechanisms (often of putative mechanisms that
have been pre-identified in animals). As such, this review will
focus mainly on current evidence from clinical studies.
The Impacts of Experimental Disuse on
Muscle Mass and Strength Loss in Humans
A plethora of clinical studies have investigated the degree of
muscle loss in humans exposed to disuse. The most frequent
employed models to study disuse atrophy in humans are
unilateral limb suspension (ULLS) using a knee brace or cast,
and bed rest; other scenarios include spinal cord injury and
spaceflight. In terms of muscle mass, the observed rate of decline
in muscle size (CSA) for each day of ULLS in knee extensors
was 0.40% and 0.36% for plantar flexors following 42 days
of unloading (Hackney and Ploutz-Snyder, 2012). Other studies
have demonstrated losses of muscle strength and mass early
on in disuse, i.e., 5 days of cast immobilization lead to 3.5%
reductions in quadriceps CSA and 9% in strength (Dirks et al.,
2014). This had progressed to 8% reductions in CSA and 23%
reductions in strength by 14 days (Wall et al., 2013). Additionally,
Suetta et al. reported 10% reductions in myofibre area and
13% decreases in strength after just 4 days progressing to 20%
reductions in myofibre area and strength after 14 days of ULLS
(Suetta et al., 2012, 2013). A further study reported decreases in
mid-thigh CSA of 11% following 28 days of bed rest (Brooks
et al., 2008). Lastly, a study by Castro et al. showed muscle
CSA to be 45% less compared to able-bodied controls 6-weeks
after complete spinal cord injury (Castro et al., 1999). Adding
to the above constellation, Gibson et al. studied men who were
immobilized following tibial fracture (thus having 6-weeks of
casting) and reported reductions in quadriceps CSA of 17%.
Furthermore, Alkner et al. reported that 90 days bed rest led
to 10 and 16% reductions in quadriceps and triceps surae
mass after 29 days, with rates of weekly loss slowing during
the last 2 months to roughly half that observed during the first
month (Alkner and Tesch, 2004). Finally, muscle CSA decreased
by 5% (de Boer et al., 2007; Glover et al., 2010) at 14 days
and 10%, at 23 days, i.e., 0.5% day following ULLS (de Boer
et al., 2007). Collectively, these studies indicate a varying degree
of rates of disuse muscle atrophy, depending on the duration
and nature of immobilization but also measurement techniques,
i.e., MRI/DXA/ultrasound/ myofibre CSA; however, it appears
atrophy occurs more rapidly in first 3–14 days of unloading and
eventually reaching a nadir where further loss of muscle occurs
at a slower rate despite continued unloading of muscle (Bodine,
2013).
Differences in the rate of muscle atrophy have also been
observed according to different muscle and fiber types as well
as the mode of immobilization. For example, after prolonged
disuse (180 days of space flight), loss of fiber size and force
was reported in the soleus and gastrocnemius muscles with the
order of atrophy (greatest-least) being: soleus type I >soleus type
II >gastrocnemius type I >gastrocnemius type II (Fitts et al.,
2010). Similar effects of disuse on fiber type following 35 days of
bed rest was reported in the vastus lateralis (VL) muscle, i.e., the
loss of fiber CSA was greater in type 1 than type II fibers (Brocca
et al., 2012). Conversely, muscle fiber type specificity has not been
observed in other studies (Bamman et al., 1998; Trappe et al.,
2008; Hvid et al., 2010) where duration of immobilization was
shorter (<14 days). It is notable that, these studies have mainly
focused on a single muscle with muscle biopsy taken from a
single site in the periphery of the muscle. This is relevant because
muscles do not atrophy uniformly across the entire length of a
single muscle (Miokovic et al., 2012), with differential atrophy
across different muscles being observed following 27–60 days
head-down-tilt bed rest. The investigators also reported that the
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Rudrappa et al. Mechanisms of Disuse Atrophy in Humans
posterior calf muscles atrophied faster than the knee extensor
muscles (Vastus Lateralis) and ankle flexors (Tibialis anterior).
In another study where multiple muscles were examined using
MRI over 43 days disuse in the form of ankle immobilization,
the greatest rate of muscle loss was observed in soleus and
medial gastrocnemius muscle followed by lateral gastrocnemius
and tibialis anterior (Psatha et al., 2012). The aetiology driving
distinct fiber type, and individual muscle atrophy susceptibility is
poorly defined.
Regulation of Skeletal Muscle Mass in
Ambulated and Unloaded Human Muscle
Ambulated Regulation of MPS and MPB
Skeletal muscle mass is regulated by the balance between MPS
and MPB. Nutrients (i.e., AA) and nutrient derived hormones
(i.e., insulin) play a crucial role in regulating the balance
between MPS and MPB. In ambulated humans, intake of
dietary protein stimulates MPS due to the essential amino acids
(EAA) components of proteins (Atherton and Smith, 2012).
These anabolic responses are dose-dependent and saturable; at
a maximal stimulus, rates of MPS increase 200–300% for a
period of 2 h following 20 g protein (Cuthbertson et al.,
2005; Atherton and Smith, 2012). In contrast, insulin released
following intake of dietary protein and/or CHO, is neither
necessary nor sufficient to stimulate MPS (Greenhaff et al., 2008).
Reflecting this, the anti-catabolic effects of insulin upon MPB was
not recapitulated via AA infusions when insulin concentrations
were clamped at 5 µU.ml1(post absorptive; Greenhaff et al.,
2008). Instead, insulin concentrations of just 15 IU/ml (3×post
absorptive) are sufficient to maximally suppress MPB (Wilkes
et al., 2009). This anti-catabolic effect of insulin acting on MPB
was confirmed in a recent systematic review and meta-analysis of
44 human studies, which concluded insulin did not significantly
affect MPS but has a crucial role in reducing MPB (Abdulla et al.,
2016). Therefore, while EAA’s stimulate MPS, insulin suppresses
MPB (and stimulates muscle glucose uptake). On the basis,
EAA and insulin are so vital in maintaining muscle metabolic
homeostasis, failure of these mechanisms inevitably leads to
skeletal muscle atrophy and IR.
Impact of Disuse on MPS and Anabolic Pathways
Disuse of human skeletal muscle alters muscle metabolism
dynamics (Mallinson and Murton, 2013). For instance, early
work by Gibson et al. showed that young men exposed to ULLS
exhibited 30% slower rates of fasted state MPS compared
to the contralateral non-immobilized limb (Gibson et al.,
1987). Subsequent studies confirmed reductions in MPS; for
instance, 50% reductions in MPS following 2-weeks of bed
rest (Ferrando et al., 1996; Paddon-Jones et al., 2004) and ULLS
with braces/casting (Glover et al., 2008). In further agreement
with this, Kortebein et al. reported 30% reductions in post-
absorptive MPS during a 24 h period in older adults after 10
days of bed rest (Kortebein et al., 2007). Crucially, blunting
of MPS in response to muscle unloading is not restricted to
fasted periods. Glover et al. reported that ULLS led to 27%
reductions in postprandial rates of MPS at both low and high
doses of AA infusions (Glover et al., 2008). Similarly, Drummond
et al. reported that 7 days of bed rest blunted fed state MPS
following EAA ingestion (Drummond et al., 2012). Similarly,
14 days of ULLS led to a 30% reduction in MPS after
ingestion of 20 g dietary protein (Wall et al., 2013). On this
basis, available evidence strongly supports the notion that skeletal
muscle atrophy in humans during a period of disuse is driven by
blunting of both postabsorptive and postprandial MPS (Rennie
et al., 2010; Wall et al., 2013).
The mTOR (mechanistic target of rapamycin) pathway is
the major signal transduction network “hub” involved in the
regulation of mRNA translation. Cell and rodent based research
suggests this system senses important stimuli responsible
for the regulation of MPS, i.e., (1) insulin and insulin-like
growth factor-1 (IGF-1) through IRS (insulin receptor substrate)
and PI3K (Phosphatidylinositol-3) pathways (Wackerhage and
Ratkevicius, 2008); (2) AA through leucyl-tRNA/Rag-mTORc1
pathways (Wackerhage and Ratkevicius, 2008), (3) Energy stress
through AMPK-eukaryotic elongation factor (AMP activated
protein kinase/ eEF2) pathway (Wackerhage and Ratkevicius,
2008), and (4) mechanical stress, e.g., through mechano-sensory
pathways (Wackerhage and Ratkevicius, 2008). However, the
impact of these factors in the regulation of MPS in human
remains unclear. For example, under ambulated conditions, one
of the mTOR upstream effector, the class III PI3K hVps34
(human vacuolar protein sorting-34) was shown to be inhibited
in amino acid starved state (basal condition) and increased
activity of this protein appears to be concomitant with increased
S6K1, suggesting it to be key player in mTOR signaling pathway
(Dickinson and Rasmussen, 2011). Furthermore, intake of
dietary protein typically leads to increase in phosphorylation
status of class III PI3K hVps34 (Dickinson and Rasmussen,
2011), mTOR and its downstream substrates regulating mRNA
translation, such as p70S6K (Drummond et al., 2012). However,
in response to disuse, blunted phosphorylation of mTOR and
p70S6K was shown after 2-weeks of immobilization while, no
significant increases in phosphorylation of mTOR and p70S6K
were noted after immobilization (Wall et al., 2013). In contrast,
others have reported no decreases in Akt/mTOR signaling despite
reductions in MPS (de Boer et al., 2007; Wackerhage and
Ratkevicius, 2008). It is worthwhile noting that peak stimulation
of signaling proteins (mTOR and p70S6K) occurs 1–2 h after
protein intake while many studies have muscle biopsies taken
3–6 h after protein intake when the response would be attenuated
or perhaps absent (Glover et al., 2010; West et al., 2011;
Churchward-Venne et al., 2012a; Drummond et al., 2012).
Further studies are needed with frequent biopsy sampling to fully
determine a role for deactivation in mTORC1-related signaling
networks (or indeed other putative mechanisms) regulating
depressions in post-absorptive and post-prandial MPS.
Impact of Disuse on MPB and Catabolic Pathways
In contrast to the recognized deficits in MPS during
immobilization, the role of MPB in disuse atrophy is less
clear. This is partly confounded by the technical and clinical
challenges in measuring in vivo rates of MPB (e.g., arterio-venous
balance methods to measure rate of appearance i.e., breakdown
and pulse chase stable isotope approaches to measure fractional
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Rudrappa et al. Mechanisms of Disuse Atrophy in Humans
breakdown rates; Kumar et al., 2009; Atherton and Smith,
2012; Atherton et al., 2016). In the only study to our knowledge
to measure this, Symons et al. demonstrated no increase in
MPB in his study of healthy young volunteers exposed to 21
days of microgravity setting using a bed rest model (Symons
et al., 2009). Although not directly quantifying MPB, Wall
et al. reported that muscle free tracer enrichment over 4-h
post prandial period was >3 fold higher after immobilization
(Wall et al., 2013). Since MPS has consistently been shown to
be reduced with immobilization, a likely explanation is that
MPB is actually reduced (rather than increased) and hence
the less unlabeled phenylalanine efflux was diluting the muscle
free labeled tracer (L-(ring-2H5) phenylalanine pool (Wall
et al., 2013). Alternatively, an accumulation of free tracer could
simply be explained by established “anabolic resistance,” i.e.,
where a failure of AA incorporation into the muscle lead to its
accumulation (Glover et al., 2008; Phillips et al., 2009; Rennie,
2009; Wall et al., 2013). Nevertheless, regardless of the driving
force behind muscle atrophy (i.e., disuse, aging, cancer, organ
failure), blunted postabsorptive and postprandial MPS (Figure 1;
anabolic resistance) seem to be the major drivers of disuse
atrophy—rather than increases in MPB. Nonetheless, more work
is needed across the time-course of unloading to verify this.
In terms of the molecular regulation of MPB, the ubiquitin
proteasome system (UPS; Lecker et al., 2006) supplemented
by lysosomal and calcium activated calpain (ATP–independent)
and caspase dependent cleavage of actinomyosin complexes
(Glover et al., 2010) are the major catabolic pathways in
muscle. The identification of the “atrogenes” as genes that are
uniformly upregulated irrespective of the atrophy stimulus (e.g.,
denervation, disuse, thermal injury) has received much attention
as key drivers of atrophy programming (Bodine et al., 2001a;
Jones et al., 2004; Milan et al., 2015). This led to members
of the Forkhead Box (Fox) O family (Fox1, 3, and 4) being
identified as downstream targets of Akt pathway (Figure 2) and
as the main transcription factors regulating MAFbx/atrogin-
1expression (Sandri et al., 2004). In terms of disuse atrophy,
mRNA expression of two E3 ubiquitin ligases was initially found
to be crucial in immobilization, unloading and denervation
induced muscle atrophy (Bodine et al., 2001b). These genes,
MuRF-1 (Muscle Ring Finger-1), and MAFbx/atrogin-1 (Muscle
Atrophy F-box), are expressed in skeletal muscle at low levels
FIGURE 1 | Diagrammatic representation of the main mechanisms involved in disuse skeletal muscle atrophy in humans: Immobilization/disuse reduces
both postabsorptive and post prandial muscle protein synthesis (MPS) via the mammalian target of rapamycin (mTORC1) and Akt signaling. The role of MPS, muscle
protein breakdown (MPB) and insulin resistance (IR) in simple disuse atrophy remain poorly defined in humans. So the role of insulin resistance and MPB in the setting
of disuse atrophy needs further evaluation. Inflammation probably leads to IR. Recently, reactive oxygen species (ROS) has been implicated in development of muscle
atrophy in disuse setting, but the mechanism in human remains putative. Solid arrow shows positive association and broken arrow shows putative association. See
text for more details.
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Rudrappa et al. Mechanisms of Disuse Atrophy in Humans
FIGURE 2 | Diagrammatic representation of the overlap between insulin signaling pathway, reactive oxygen species (ROS), inflammatory cytokine
such as NF-κB and ubiquitin-proteasome system in insulin resistant (IR) states particularly diabetes: In IR state, PI3K activity is decreased, leading to
decreased activity of Akt, which in turn release the inhibition of FOXO and caspase-3 resulting in elevation of muscle ring finger-1 (MuRF-1) and muscle atrophy F-box
(MAFbx/atrogin-1) finally leading to increased proteolytic activity. Also, ROS and low grade inflammation via NF-κB pathway lead to muscle atrophy. See text for more
details.
but rapidly induced in response to unloading (Bodine et al.,
2001b). In humans, after 5 days (Dirks et al., 2014) and 2
weeks (Jones et al., 2004) of immobilization, MAFbx and MuRF1
mRNA were reported to be elevated. Nonetheless, while their
expression is thought to be regulated by transcription factors
such as FOXO1, FOXO3a, (Sandri et al., 2004) and NFκβ (p50
and Bcl-3)(Wu et al., 2011), no increase in mRNA expression
in FOXO’s were noted after 4 or 14 days of immobilization
(Suetta et al., 2012). De Boer et al reported the expression of
MuRF-1 but not MAFbx mRNA was increased after 10 days
of ULLS (Jones et al., 2004; de Boer et al., 2007), while both
had decreased by 21 days (de Boer et al., 2007). Furthermore,
increases in UPP components (particularly UBE2E) were up-
regulated 48 h following instigation of ULLS (Urso et al., 2006).
In contrast, a recent study by Brocca et al. found that muscle
atrophy following ULLS found no change in mRNA expression
of ubiquitin-proteasome and autophagy systems (Brocca et al.,
2015). Some work has been done in relation to autophagy
(and calpain-signaling) in relation to human disuse (Jones
et al., 2004). Autophagy is responsible for removing unfolded,
damaged and dysfunctional proteins and organelles via the
formation of autophagosomes for degradation by lysosomes
(Sandri, 2010). Interestingly, up-regulation of autophagy markers
such as Beclin-1 suggested increased autophagosome formation
and hence a higher activity of the macro-autophagy by 24 days
of bed rest; nonetheless, other autophagy markers such as P62,
LC3II/I ratio, and cathepsin-L were not up-regulated (Brocca
et al., 2012). While details of the pathways discussed above
are outside the scope of this review, the readers are referred
to reviews by Bonaldo and Sandri (2013) and Sandri (2010).
What is clear however is that without more clinical studies with
time-course acquisition, including dynamic measures of MPB in
tandem with molecular markers spanning different proteolytic
systems, no firm conclusions can be made surrounding the mixed
results regarding whether existing molecular data suggest MPB
is increased, decreased or unchanged in response to disuse in
humans.
In addition to the anabolic and catabolic pathways described
above, emerging evidence indicates that disturbed redox
signaling may also be an important regulator of MPS and MPB
in muscle disuse atrophy (Powers et al., 2012; Zuo and Pannell,
2015). Oxidative injury has been shown to occur in muscle fibers
during periods of disuse in locomotor skeletal muscles (Min et al.,
2011) and in non-load bearing muscle such as the diaphragm
during prolonged mechanical ventilation (Kavazis et al., 2009).
After 35 days of bed rest, vastus lateralis muscle showed 18%
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Rudrappa et al. Mechanisms of Disuse Atrophy in Humans
muscle fiber atrophy and increased protein carbonylation (Dalla
Libera et al., 2009). Furthermore, an inverse linear relationship
was observed between normalized levels of protein oxidation and
muscle fiber CSA (Dalla Libera et al., 2009). An analysis of gene
expression showed up-regulation of pathways involved in the
oxidative stress response (increase in mRNA for stress response
gene heme oxygenase-1) following 48 h of unilateral lower leg
suspension (ULLS; Reich et al., 2010). Conversely, in a limb
immobilization human model with 5.7% muscle and 11.8%
muscle fiber loss after 14 days of immobilization, no increase in
lipid peroxidation and protein oxidation in vastus lateralis was
observed (Glover et al., 2010). Although information on oxidative
stress and potential mechanisms explaining proteolysis in disuse
human muscle is still sparse (Pellegrino et al., 2011), these
findings support the extensive evidence available from animal
studies that oxidative stress inhibits MPS (Powers et al., 2011a)
and increases muscle MPB (via increased gene expression of key
proteins involved in the proteolytic pathways such as autophagy,
calpains and proteosomes; activation of both calpain and caspase-
3 and possibly by modification of myofibrillar proteins which
enhances their susceptibility to proteolytic processing; Powers
et al., 2011a). Interaction between ROS and insulin signaling
pathway has also been described, i.e., ROS may regulate Insulin
growth factor-1 (IGF-1) signaling either positively or negatively
depending on the amount of ROS produced (Papaconstantinou,
2010). Low levels of endogenous ROS due to their reversible
oxidative inhibition of protein tyrosine phosphatases induces
phosphorylation of tyrosine residue on the insulin receptor and
its substrates triggering IGF-1 signaling (Bashan et al., 2009).
In contrast, the IGF-1 signaling pathway is inhibited by higher
levels of ROS and recent evidence suggests ROS down regulates
the IGF-1 cascade and induces insulin resistance (Bashan et al.,
2009;Figure 2). For detailed discussion of the signaling pathways
linking ROS and muscle atrophy, the interested reader is referred
to recent reviews on oxidative stress and disuse muscle atrophy
(Pellegrino et al., 2011; Powers et al., 2011b, 2014; Zuo and
Pannell, 2015).
Impact of Disuse on Muscle IR and Links to Muscle
Mass in Persistent IR States
Insulin-mediated glucose uptake is also blunted with muscle
disuse (Mikines et al., 1991; Biensø et al., 2012); that is, unloaded
muscle becomes IR. This IR can be observed at a whole-body
level following bed-rest, but is most apparent at the muscle level
across the physiological range of insulin concentrations under
clamp conditions (Mikines et al., 1991). Recently, a 1 week bed-
rest study in young males by Dirks et al. revealed reduced muscle
mass (1.4 kg lean tissue and 3% quadriceps CSA) and whole-
body insulin sensitivity (29%)(Dirks et al., 2016). Thus, disuse
lowers MPS, induces anabolic resistance to nutrients and impairs
insulin-mediated muscle glucose uptake—even in healthy adults
(Fink et al., 1983).
The role of IR in driving muscle atrophy however is poorly
defined. Evidence from large cross-sectional and longitudinal
studies reports accelerated loss of muscle mass in individuals
with persistent IR (i.e., people with Type 2 Diabetes), perhaps
pointing to mechanistic links. For instance, declines in muscle
mass were inversely related to duration of diabetes or HbA1c
(Park et al., 2007, 2009; Kalyani et al., 2013) and attenuated
with insulin sensitizers (Kuo et al., 2009). Human muscle
tissue accounts for 80% of glucose uptake after food ingestion
and insulin resistance (HOMA-IR) is associated with reduced
quadriceps muscle strength (Kalyani et al., 2013; Leenders
et al., 2013), power (Kalyani et al., 2013) and muscle mass
(Leenders et al., 2013) in humans. Approximately a 50% more
rapid decline in knee extensor strength has been observed in
older patients with type 2 diabetes compared with patients
without diabetes over a 3 year period, suggesting that decreased
muscle strength may be accelerated in type 2 diabetes (Park
et al., 2007). In a further study, Volpato et al. reported
differences in walking speed, muscle strength, power and muscle
quality between individuals with and without diabetes were
independent of co-existing peripheral motor neuropathy or
peripheral vascular disease, suggesting a direct effect of diabetes
per se on muscle performance (Volpato et al., 2012). These
findings are important because in catabolic conditions such as
diabetes, atrophy in combination with reduced activity decrease
quality of life and increase mortality (Zinna and Yarasheski,
2003). Yet despite clear evidence linking accelerated muscle loss
in diabetes compared to non-diabetes, studies investigating the
direct effect of immobilization on muscle protein turnover in
patients with diabetes compared to those without diabetes are
scant. Furthermore, clear distinction between Type 1 and Type
2 diabetes needs to be made when investigating patients with
diabetes. This is because Type 1 diabetes is a condition with
severe depletion of energy stores and reduced mitochondrial
function resulting in accelerated muscle protein loss (Hebert
and Nair, 2010), which can be reversed by insulin replacement
(Workeneh and Bajaj, 2013). In contrast, muscle loss, whilst
accelerated in type 2 diabetes, is unaffected by insulin treatment
(Workeneh and Bajaj, 2013), possibly due to IR. Hence,
skeletal muscle mass loss whilst common, appears to occur less
predictably and to varying degree in Type 2 diabetes compared
with Type 1 diabetes (Workeneh and Bajaj, 2013). Collectively,
these data are consistent with the notion that diabetes causes
muscle mass loss possibly due to mechanisms driving muscle
IR, however there is lack of data regarding the effects of
immobilization or disuse on muscle mass in individuals with
diabetes.
The mechanistic regulation of muscle IR in driving muscle
atrophy in the setting of “simple disuse” remains vague (Atherton
et al., 2016). Early human studies by Shulman et al. showed
that, under steady state plasma concentration of both glucose
and insulin mimicking postprandial conditions, the mean rate
of muscle glycogen synthesis accounted for most of the whole
body glucose uptake and virtually all of non-oxidative glucose
metabolism in both healthy and diabetic subjects (Shulman,
2000), with defects in muscle glycogen synthesis playing a
major role in causing insulin resistance in type 2 diabetes
(Shulman, 2000). This may be explained by defects in the
insulin receptor substrate (IRS)-1/phosphatidylinositol (PI) 3-
kinase pathway, leading to reduced glucose uptake and utilization
in insulin target tissues (Draznin, 2006). Free fatty acids induce
muscle IR by inhibiting glucose transport/phosphorylation and
Frontiers in Physiology | www.frontiersin.org 6August 2016 | Volume 7 | Article 361
Rudrappa et al. Mechanisms of Disuse Atrophy in Humans
reductions in both the rate of muscle glycogen synthesis and
glucose oxidation (Roden et al., 1996). Additionally, many
other mechanisms have been postulated to explain free fatty
acid-induced muscle IR, including the Randle cycle, oxidative
stress, inflammation and mitochondrial dysfunction (Martins
et al., 2012). Full details regarding above mechanisms escape
the scope of this article and readers are referred to a review
by Martins et al. (2012). With regard to disuse induced muscle
atrophy, following (7 days) bed-rest healthy volunteers showed
reduced glucose infusion rate and leg glucose extraction (after
bed rest) along with reduced muscle GLUT4, hexokinase II,
protein kinase B/Akt1, and Akt2 protein levels, and a tendency
for reduced 3-hydroxyacyl-CoA dehydrogenase activity (Biensø
et al., 2012). Further in the same study, the ability of insulin to
phosphorylate Akt and activate glycogen synthase was reduced
post bed-rest (Biensø et al., 2012); but whether this observation
is causative or a consequence of immobilization is not clear.
However, a substantial decline in glucose uptake within 24
h of immobilization would argue against a causative effect
(Atherton et al., 2016). Recently, Vigelso et al. showed an
inverse association between the increase in muscle pyruvate
dehydrogenase complex (PDC) activation and leg lactate
release during contraction after 2 weeks unilateral lower limb
immobilization, suggesting PDC as a potential key regulator
of immobilization induced muscle IR (Vigelsø et al., 2016).
Overall the above data suggests that muscle disuse results in
development of whole body and muscle specific IR, fuelling
the argument that lack of muscle contraction per se may be
the main physiological driver for this dysregulation, however a
mechanistic explanation for this still remains unclear (Atherton
et al., 2016).
CONCLUSIONS
Disuse muscle atrophy causes many undesirable complications.
There seems to be complex interplay of numerous mechanisms
contributing to the aetiology of disuse muscle atrophy. During
muscle disuse, both post-absorptive and post-prandial MPS is
suppressed, with little evidence to support there being an increase
in “bulk” protein MPB. Moreover, animal models show increased
(2.5 times) rate of muscle protein turnover and are also very
sensitive to disuse, while exhibiting marked fiber-type-dependant
differences in rates of muscle protein turnover (type I fibers being
twice as great as type II fibers) when compared to humans. Due
to these inherent species-specific differences, pre-clinical findings
cannot easily be reconciled with nor extrapolated to humans.
So, further research quantifying MPS and MPB and their
temporal relationship during disuse in humans is warranted.
There is strong evidence that type 2 diabetes accelerates muscle
loss, possibly due to mechanisms innate to diabetes. Crucially,
muscle IR secondary to disuse appears to drive the procession
of disuse muscle atrophy independent of other mechanisms
known to cause muscle IR. Nonetheless, the mechanistic role
of muscle IR driving this atrophic response is poorly defined.
Because, common proteolytic mechanisms may exist across
“simple muscle atrophy” and other catabolic conditions (e.g.,
type 2 diabetes, inflammation, cachexia etc.), these two process
can rarely be seen as being mutually exclusive (Atherton et al.,
2016). Further, many questions remain unanswered especially
the molecular regulation of MPS and MPB and muscle insulin
resistance. This whole area of research has potential implications
for the wider clinical community as similar metabolic processes
occur during cancer cachexia, metabolic syndromes including
type 2 diabetes, aging (i.e., sarcopenia), sepsis and many
neurodegenerative disorders. Henceforth, further translational
research is necessary before this knowledge can be effectively
applied in developing targeted strategies to prevent this in the
setting of disuse muscle atrophy.
AUTHOR CONTRIBUTIONS
All authors listed, have made substantial, direct and intellectual
contribution to the work, and approved it for publication.
ACKNOWLEDGMENTS
The first author is a doctoral research student funded through
the University of Nottingham within the MRC-ARUK Centre
for Musculoskeletal Ageing Research. The MRC-ARUK Centre
for Musculoskeletal Ageing Research was funded through
grants from the Medical Research Council [grant number
MR/K00414X/1] and Arthritis Research UK [grant number
19891] awarded to the Universities of Nottingham and
Birmingham.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2016 Rudrappa, Wilkinson, Greenhaff, Smith, Idris and Atherton. This
is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) or licensor are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.
Frontiers in Physiology | www.frontiersin.org 10 August 2016 | Volume 7 | Article 361
... [2][3][4] It follows that a significant body of research has, and continues to focus upon, the mechanisms of muscle atrophy and mitigation strategies. 5 In terms of the temporal nature of muscle atrophy upon immobilization, there is growing evidence of rapidity over short time frames (<1 week). 4,6 The average UK NHS hospital stay is~4.5 days (Source: NHS HES 2018-2019) and time off work due to flu related illness~3 days. ...
... bed rest, casting, unilateral lower-limb suspension) that vary in mobility restrictions and clinical relevance, yet they all share common outcomes of decreased muscle mass and strength. 5 For instance, reductions in strength of 9% have been reported with 5 days of leg casting 4 and 25% with 14 days of unilateral lower-limb suspension. 10 Here, we report losses of~11% in just 4 days, adding to previous literature of rapid losses of muscle strength. ...
... of assessment and immobilization model used. 5 For instance, while~10% declines in fibre cross-sectional area (CSA) were reported after 4 days of knee-bracing, 29 5 days of leg-casting showed no measurable decline in fibre CSA, de-spite~3.5% decreases in whole muscle CSA. 4 In the present study, using independent measurement techniques, we show significant declines in DXA derived thigh lean mass loss of 1.7% which correlated with significant declines in VL MT of 4%. Our data therefore agree with previous literature highlighting that muscle mass loss occurs rapidly at the onset of immobilization. ...
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... Disuse leads to a decrease in the content of mitochondrial proteins, which occurs along with muscle mass decrease. In human skeletal muscle with a mixed fiber type compositions (for example, in vastus lateralis), muscle mass decrease is closely associated with a decrease in the rate of protein synthesis [90,91]. It may be suggested that during disuse, the decrease in the content of mitochondrial proteins is at least partially due to the suppression of translation, which occurs simultaneously with a decrease in the content of mRNA encoding some mitochondrial and ribosomal proteins [80]. ...
... During disuse, a decrease in the content of contractile proteins and muscle mass in the mixed vastus lateralis is predominantly associated with a decrease in the rate of protein synthesis rather than with an increase in the rate of their degradation [90,91]. However, a number of studies on models [110] and human muscle showed the activation of some elements of the ubiquitin-proteasome and autophage-lysosomal systems [72,75,77,79,80,[111][112][113][114]. ...
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Physical inactivity and disuse lead to a decrease in the functionality of skeletal muscles (oxidative capacity, insulin sensitivity, and performance), which is associated with a change in mitochondrial density. In contrast, aerobic exercise training is effective for maintaining/increasing skeletal muscle mitochondrial density and functionality. The review considers the effect of increasing and decreasing physical activity on the mitochondrial density of human skeletal muscles, as well as the main mechanisms responsible for these changes. It is discussed that the content of mitochondrial proteins can be regulated by changing the content of their mRNAs, changes in the rate of synthesis specific for mitochondrial proteins, as well as changes in the rate of degradation, transport, import, and stability of mitochondrial proteins. It has been shown that the mechanisms of regulation of the mitochondrial proteins content under various interventions are significantly different. At the same time, their contribution to the change in the content of mitochondrial proteins is characterized clearly insufficiently, which emphasizes the relevance of further research in this area.
... Maintenance of skeletal muscle mass relies on a strictly regulated balance of muscle protein synthesis (MPS) and breakdown (MPB) -or proteostasis. Muscle atrophy occurs when the rate of MPB exceeds that of MPS, so there is understandably great interest into elucidating the mechanisms underlying atrophy and any key factors that may influence the process [3][4][5]. ...
... First, in the liver, where 25-hydroxylase hydroxylates the C25 of vitamin D to form 25-hydroxyvitamin D (25 (OH)D 3 ; calcidiol) -the metabolite used clinically to determine an individual's vitamin D status [6]. Subsequently, 25(OH)D 3 is hydroxylated at its C1α site by the enzyme 1α-hydroxylase (CYP27B1) to form the biologically active hormone, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ; calcitriol) [7]. This second hydroxylation step was initially believed to be specific to the kidney, but current evidence indicates widespread extra-renal expression of CYP27B1 [8,9]. ...
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Muscle atrophy and sarcopenia (the term given to the age-related decline in muscle mass and function), influence an individuals risk of falls, frailty, functional decline, and, ultimately, impaired quality of life. Vitamin D deficiency (low serum levels of 25-hydroxyvitamin D (25(OH)D3)) has been reported to impair muscle strength and increase risk of sarcopenia. The mechanisms that underpin the link between low 25(OH)D3 and sarcopenia are yet to be fully understood but several lines of evidence have highlighted the importance of both genomic and non-genomic effects of active vitamin D (1,25-dihydroxyvitamin D (1,25(OH)2D3)) and its nuclear vitamin D receptor (VDR), in skeletal muscle functioning. Studies in vitro have demonstrated a key role for the vitamin D/VDR axis in regulating biological processes central to sarcopenic muscle atrophy, such as proteolysis, mitochondrial function, cellular senescence, and adiposity. The aim of this review is to provide a mechanistic overview of the proposed mechanisms for the vitamin D/VDR axis in sarcopenic muscle atrophy.
... Muscle atrophy is often associated with reduced quality of life, reduced mobility in general, and decreased individual independence (12,14). Furthermore, the decrement in muscle mass due to muscle atrophy is linked to a higher prevalence of several chronic diseases (e.g., type 2 diabetes mellitus, cardiovascular diseases, and depression) and even higher mortality (4,13,15). Consequently, understanding and preventing muscle atrophy has a critical role in human health. Skeletal muscle atrophy occurs via a coordinated dismantling of the myofibrillar protein lattice and the loss of organelles and cytoplasmic proteins (16)(17)(18). ...
... It has been recognized that decreased MPS is the primary driving process leading to loss of muscle protein over time in disuse muscle atrophy in humans (4,15,26,27,29) (Fig. 1). Declines of 50%-60% in both fasting (hypoaminoacidemia) and fed (hyperaminoacidemia) MPS during unloading support the theses that elevated MPB, and "bulk" proteolysis has little, if any, contribution to the decrement in muscle mass observed during simple muscle disuse (29). ...
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Decreased skeletal muscle contractile activity (disuse) or unloading leads to muscle mass loss, also known as muscle atrophy. The balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB) is the primary determinant of skeletal muscle mass. A reduced mechanical load on skeletal muscle is one of the main external factors leading to muscle atrophy. However, endocrine and inflammatory factors can act synergistically in catabolic states, amplifying the atrophy process and accelerating its progression. Additionally, older individuals display aging-induced anabolic resistance, which can predispose this population to more pronounced effects when exposed to periods of reduced physical activity or mechanical unloading. Different cellular mechanisms contribute to the regulation of muscle protein balance during skeletal muscle atrophy. This review summarizes the effects of muscle disuse on muscle protein balance and the molecular mechanisms involved in muscle atrophy in the absence or presence of disease. Finally, a discussion of the current literature describing efficient strategies to prevent or improve the recovery from muscle atrophy is also presented.
... According to the study of Dalle et al., IL-1 also palyed an important role in sarcopenia in RA 39 . In addition, rheumatoid musculoskeletal symptoms including pain, swelling and stiffness preclude patients from exercise and physical activity, leading to disuse atrophy of the skeletal muscles 40 , resulting in an imbalance between protein metabolism and catabolism; the persistent inflammation exhausted myocytes and impeded muscle growth; the nutritional deficit resulted from disuse and flammation further aggravated the impasse 41 . ...
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This study investigated the effect of poor balance and sarcopenia on vertebral spinal osteoporotic fracture (VOPF) in female rheumatoid arthritic (RA) patients. A total of 195 female RA and 126 normal subjects were enrolled, and the correlations between sarcopenia, poor balance and VOPF were analyzed. Furthermore, we explored the relationships between sarcopenia or poor balance with disease related indexes of female RA. Binary logistic regression analyses were performed to identify potential risk factors for VOPF in female RA. We found that female RA had an increased risk of sarcopenia, poor balance (Berg balance scale, BBS ≤ 40) and VOPF than controls (P < 0.0001). Female RA with VOPF were more likely to have poor balance and sarcopenia than those without VOPF (P < 0.0001–0.05). Meanwhile, female RA with sarcopenia and poor balance often had higher disease activity, more serious joint damage and worse joint function (P < 0.05) compared with those without sarcopenia and poor balance. Binary logistic regression analysis (LR backwald) revealed that age (OR = 1.112, 95% CI 1.065–1.160, P < 0.0001), OP (OR = 10.137, 95% CI 4.224–24.330, P < 0.0001) and GCs usage (OR = 3.532, 95% CI 1.427–8.741, P = 0.006) were risk factors, while SMI (OR = 0.386, 95% CI 0.243–0.614, P < 0.0001) and BBS (OR = 0.952, 95% CI 0.929–0.976, P < 0.0001) were protective factors for VOPF in female RA. Hence, sarcopenia and poor balance are associated with a higher risk for VOPF and are closely related to disease activity and joint structure damage of female RA.
... Muscle atrophy caused by unloading or decreasing neural activity is often classified as disuse and commonly seen in patients with limb immobilization and peripheral nerve injury due to fracture [1][2][3]. Although both limb immobilization and peripheral nerve injury eventually lead to muscular dysfunction, their treatment and rehabilitation are different [4]. ...
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Disuse atrophy, caused by situations of unloading such as limb immobilisation, causes a rapid yet diverging reduction in skeletal muscle function compared to muscle mass. While mechanistic insight into the loss of mass is well studied, deterioration of muscle function with a focus towards the neural input to muscle remains underexplored. This study aimed to determine the role of motor unit adaptation in disuse-induced neuromuscular deficits. 10 young, healthy male volunteers underwent 15 days of unilateral lower limb immobilisation. Intramuscular EMG (iEMG) was recorded from the vastus lateralis during knee extensor contractions normalised to maximal voluntary contraction (MVC) pre and post disuse-induced loss of function. Muscle cross-sectional area was determined by ultrasound. Individual MUs were sampled and analysed for changes in discharge characteristics and MU potential (MUP) shape and structure. Vastus lateralis CSA was reduced by approximately 15% which was exceeded by a two-fold decrease of 31% in muscle strength in the immobilised limb, with no change to the non-immobilised. Parameters of MUP size were largely reduced, while neuromuscular junction (NMJ) transmission instability increased at several contraction levels and MU firing rate reduced. All adaptations were observed in the immobilised limb only. These findings highlight impaired neural input following immobilisation reflected by suppressed MU discharge rate and instability of transmission at the NMJ which may underpin the disproportionate reductions of strength relative to muscle size.
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AIMS/HYPOTHESIS: We aimed to investigate the role of insulin in regulating human skeletal muscle metabolism in health and diabetes. METHODS: We conducted a systematic review and meta-analysis of published data that examined changes in skeletal muscle protein synthesis (MPS) and/or muscle protein breakdown (MPB) in response to insulin infusion. Random-effects models were used to calculate weighted mean differences (WMDs), 95% CIs and corresponding p values. Both MPS and MPB are reported in units of nmol (100 ml leg vol.)(-1) min(-1). RESULTS: A total of 104 articles were examined in detail. Of these, 44 and 25 studies (including a total of 173 individuals) were included in the systematic review and meta-analysis, respectively. In the overall estimate, insulin did not affect MPS (WMD 3.90 [95% CI -0.74, 8.55], p = 0.71), but significantly reduced MPB (WMD -15.46 [95% CI -19.74, -11.18], p < 0.001). Overall, insulin significantly increased net balance protein acquisition (WMD 20.09 [95% CI 15.93, 24.26], p < 0.001). Subgroup analysis of the effect of insulin on MPS according to amino acid (AA) delivery was performed using meta-regression analysis. The estimate size (WMD) was significantly different between subgroups based on AA availability (p = 0.001). An increase in MPS was observed when AA availability increased (WMD 13.44 [95% CI 4.07, 22.81], p < 0.01), but not when AA availability was reduced or unchanged. In individuals with diabetes and in the presence of maintained delivery of AA, there was a significant reduction in MPS in response to insulin (WMD -6.67 [95% CI -12.29, -0.66], p < 0.05). CONCLUSIONS/INTERPRETATION: This study demonstrates the complex role of insulin in regulating skeletal muscle metabolism. Insulin appears to have a permissive role in MPS in the presence of elevated AAs, and plays a clear role in reducing MPB independent of AA availability.
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Key points: This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms. Abstract: Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 20 ± 1 W (∼50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age- and immobilization-induced IMTG accumulation.
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