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Pronounced limb and fibre type differences in subcellular lipid droplet content and distribution in elite skiers before and after exhaustive exercise

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Key points: Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete. Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle - one beneath the sarcolemma and the other between myofibrils. At rest, well-trained leg muscles of cross-country skiers contain 4- to 6-fold more lipid droplets than equally well-trained arm muscles, with a 3-fold higher content in type 1 than in type 2 fibres. During exhaustive exercise, lipid droplets between the myofibrils but not those beneath the sarcolemma are utilised by both type 1 and 2 fibres. These findings provide insight into compartmentalisation of lipid metabolism within skeletal muscle fibres. Abstract: Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm and leg muscles before and after 1 h of exhaustive exercise. Intermyofibrillar lipid droplets accounted for 85-97% of the total volume fraction, while the subsarcolemmal pool made up 3-15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal lipid droplets were 4- to 6-fold higher in leg than in arm muscles (P < 0.001). Furthermore, the volume fraction of intermyofibrillar lipid droplets was 3-fold higher in type 1 than in type 2 fibres (P < 0.001), with no fibre type difference in the subsarcolemmal pool. Following exercise, intermyofibrillar lipid droplet volume fraction was 53% lower (P = 0.0082) in both fibre types in arm, but not leg muscles. This reduction was positively associated with the corresponding volume fraction prior to exercise (R2 = 0.84, P < 0.0001). No exercise-induced change in the subsarcolemmal pool could be detected. These findings indicate clear differences in the subcellular distribution of lipid droplets in the type 1 and 2 fibres of well-trained arm and leg muscles, as well as preferential utilisation of the intermyofibrillar pool during prolonged exhaustive exercise. Apparently, the metabolism of lipid droplets within a muscle fibre is compartmentalised.
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J Physiol 595.17 (2017) pp 5781–5795 5781
The Journal of Physiology
Pronounced limb and fibre type differences in subcellular
lipid droplet content and distribution in elite skiers before
and after exhaustive exercise
Han-Chow E. Koh1,JoachimNielsen
1,2 ,BengtSaltin
4, Hans-Christer Holmberg3
and Niels Ørtenblad1
1Department of Sports Science and Clinical Biomechanics, SDU Muscle Research Cluster (SMRC), University of Southern Denmark,
Odense M, Denmark
2Department of Pathology, SDU Muscle Research Cluster (SMRC), Odense University Hospital, Odense C, Denmark
3School of Sport Sciences, UiT Arctic University of Norway, Tromsø, Norway
4Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark
Key points
rAlthough lipid droplets in skeletal muscle are an important energy source during endurance
exercise, our understanding of lipid metabolism in this context remains incomplete.
rUsing transmission electron microscopy, two distinct subcellular pools of lipid droplets can be
observed in skeletal muscle – one beneath the sarcolemma and the other between myofibrils.
rAt rest, well-trained leg muscles of cross-country skiers contain 4- to 6-fold more lipid droplets
than equally well-trained arm muscles, with a 3-fold higher content in type 1 than in type 2
fibres.
rDuring exhaustive exercise, lipid droplets between the myofibrils but not those beneath the
sarcolemma are utilised by both type 1 and 2 fibres.
rThese findings provide insight into compartmentalisation of lipid metabolism within skeletal
muscle fibres.
Abstract Although the intramyocellular lipid pool is an important energy store during prolonged
exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron
microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres
of the arm and leg muscles before and after 1 h of exhaustive exercise. Intermyofibrillar lipid
droplets accounted for 85–97% of the total volume fraction, while the subsarcolemmal pool
made up 3–15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal
lipid droplets were 4- to 6-fold higher in leg than in arm muscles (P<0.001). Furthermore,
the volume fraction of intermyofibrillar lipid droplets was 3-fold higher in type 1 than in type 2
fibres (P<0.001), with no fibre type difference in the subsarcolemmal pool. Following exercise,
intermyofibrillar lipid droplet volume fraction was 53% lower (P=0.0082) in both fibre types
in arm, but not leg muscles. This reduction was positively associated with the corresponding
volume fraction prior to exercise (R2=0.84, P<0.0001). No exercise-induced change in the
subsarcolemmal pool could be detected. These findings indicate clear differences in the subcellular
distribution of lipid droplets in the type 1 and 2 fibres of well-trained arm and leg muscles, as
well as preferential utilisation of the intermyofibrillar pool during prolonged exhaustive exercise.
Apparently, the metabolism of lipid droplets within a muscle fibre is compartmentalised.
(Received 5 April 2017; accepted after revision 19 June 2017; first published online 22 June 2017)
Corresponding author H.-C. E. Koh: Department of Sports Science and Clinical Biomechanics, University of Southern
Denmark, Campusvej 55, 5230 Odense M, Denmark. Email: hckoh@health.sdu.dk
Abbreviations TEM, transmission electron microscopy; ˙
VO2max, maximum oxygen uptake.
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5782 H.-C. E. Koh and others J Physiol 595.17
Introduction
Cytosolic lipid droplets composed of a lipid core
surrounded by a phospholipid monolayer (Martin &
Parton, 2006; Fujimoto & Parton, 2011) are no longer
considered to be simply inert deposits in adipocytes, but
are ubiquitous and dynamic organelles with a variety of
functions, including a key role in fatty acid trafficking
(Martin & Parton, 2006; Fujimoto & Parton, 2011). Such
lipid droplets in skeletal muscle provide a ready source of
energy, especially during exercise, with their content being
reduced 20–70% following endurance exercise (van Loon,
2004; Shaw et al. 2010).
Quantification of lipid droplets in muscle fibres can
be achieved by, for example, biochemical extraction,
magnetic resonance spectrometry, histochemical staining
with immunofluorescence microscopy, and transmission
electron microscopy (TEM) (van Loon, 2004; Kiens,
2006). Indeed, using the TEM method, lipid droplets
are observed in two distinct subcellular compartments
of a skeletal muscle fibre, namely the intermyofibrillar
and subsarcolemmal regions (Crane et al. 2010; Nielsen
et al. 2010). In a recent study, Chee and co-workers (2016)
showed that the content of intermyofibrillar, but not sub-
sarcolemmal, lipid droplets was reduced by 40% following
a 1 h bout of moderate-intensity exercise in lean young
individuals. Additionally, in the subsarcolemmal region,
overweight old individuals had larger lipid droplets than
their lean counterparts and the content of these lipid
droplets increased following the 1 h exercise (Chee et al.
2016). Interestingly, the size of individual lipid droplets
was unchanged, in line with an earlier report that 90 min of
moderate-intensity exercise attenuatedthe overall content,
but not the size, of lipid droplets (Devries et al. 2007).
Collectively, these reports imply that a comprehensive
understanding of lipid droplet metabolism in skeletal
muscle requires detailed analyses of both the subcellular
localisation and the size of the lipid droplets.
From the few studies directly comparing arm and leg
muscles, it appears that arm muscle is less oxidative
metabolically than leg muscle. Blood flow in arm muscles
varies more, and when exercising they are less capable
of extracting oxygen from the circulation, regardless of
training status (Calbet, 2005). Moreover, limited evidence
points to a lower fat oxidation in exercising arm muscle
when compared to leg muscle (Calbet, 2005; Helge et al.
2008). However, further research is needed to confirm
these findings and direct determination of lipid droplet
content in arm muscle could provide valuable insights in
this regard.
Despite the importance of the arms in sports like
swimming, rowing and cross-country skiing, biological
research on the arms is under-represented and warrants
more attention. Moreover, reports comparing the arms
and the legs could be confounded if the training status of
each limb is not closely matched. Thus, direct comparisons
of the highly trained arm and leg muscles of elite
cross-country skiers, can be made unequivocally.
Each mammalian skeletal muscle contains a
heterogeneous and distinct mixture of muscle fibres
of different fibre types. Phenotypically, type 1 fibres are
better equipped than type 2 fibres to utilise lipid substrate
for energy metabolism, especially during endurance
exercise. Accordingly, type 1 muscle fibres are found to
have a greater intramyocellular lipid content (van Loon
et al. 2004; De Bock et al. 2005; Shepherd et al. 2012), and
the content reduction following prolonged exercise has
mainly been observed in type 1 muscle fibres (van Loon
et al. 2004; De Bock et al. 2005; Shepherd et al. 2012).
Therefore, considering the divergence in lipid metabolism
between muscle fibre types, it is essential to discriminate
between fibre types when assessing substrate utilisation
by skeletal muscle.
By employing quantitative electron microscopy, this
study aimed to determine limb-dependent and fibre
type-dependent subcellular lipid droplet content and
distribution in elite cross-country skiers before and after
a 1 h skiing time trial. We hypothesised that at rest the
volumes of both intermyofibrillar and subsarcolemmal
lipid droplets are higher in leg than in arm muscles, as
well as in type 1 than in type 2 muscle fibres. In addition,
we predicted that during acute exercise lipid droplets in
the intermyofibrillar, but not the subsarcolemmal region
would be utilised.
Methods
Ethical approval
The project was approved by the Regional Ethical Review
Board in Ume˚
a, Sweden (no. 07–076M). The experiments
conformed to the standards set by the Declaration
of Helsinki. Prior to giving their written consent to
participate, the subjects were fully informed about the
project, the risks involved, discomfort associated with
the experiments and that they could withdraw from the
project at any time.
Subjects
Ten male elite cross-country skiers participated in the
study. Their mean (SD) age, height, weight, maximum
oxygen uptake ( ˙
VO2max) and haemoglobin concentration
were 22 (1) years, 182 (8) cm, 80 (9) kg, 68
(5) ml kg1min1, and 155 (8) g l1, respectively.
Their percentage myosin heavy chain (MHC) isoform
compositions (mean (SD)) in the arms and legs were MHC
I, 40% (8%), MHC II: 60% (8%), and MHC I, 58% (6%);
MHC II, 42% (6%), respectively, as described in detail in
a companion paper (Ørtenblad et al., 2011). This study
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was part of a larger project described in part previously
(Nielsen et al. 2011, 2016; Ørtenblad et al. 2011).
Experimental protocol
The skiers were instructed to eat according to their usual
intake and not to exercise the day before the time trial.
They were provided standard meals the evening prior and
in the morning of time trial day. After performing their
own warm-up and ski preparation, the skiers performed a
20 km cross-country skiing time trial (classic style) with
an average finishing time of 57 min (range: 52–63 min),
with mean (SD) post-exercise blood lactate level of 10.4
(2.4) mmol l1. The skiers did not consume any food
or fluid during the time trial. Snow conditions and the
profile of the course favoured usage of the diagonal
stride technique and a high degree of double poling,
i.e. upper-body exercise. The skiers’ ˙
VO2max values were
determined in the laboratory within 2 weeks of the time
trial (for further details, see Ørtenblad et al. 2011).
Muscle biopsies
Muscle biopsies were taken from the arm and leg before
breakfast of time trial day (Pre) and within 1–2 min
after (Post) the time trial. All biopsies were obtained
in a randomized order from the left- and right-hand
side, with one biopsy in each arm and leg, respectively.
All Post biopsies were taken contralateral to the Pre
biopsies. Only exercised muscle biopsies were taken as
lipid stores in resting muscle are not expected to change
(Sacchetti et al. 2004). All biopsies from individual sub-
jects were taken by the same person to minimise any
variation in the location and depth of the biopsy. After
local anaesthesia (2–3 ml 2% lidocaine (lignocaine)), an
incision was made through the skin and fascia and the
muscle biopsy was taken from m. vastus lateralis (leg) and
the distal part of the lateral head of m. triceps brachii
(arm), using a modified Bergstr¨
om needle with suction.
These muscles were preferred because they are highly
active during cross-country skiing (Holmberg et al. 2005).
We divided each muscle biopsy into multiple portions
for different purposes, including one portion being fixed
in glutaraldehyde for transmission electron microscopy
analysis. Due to insufficient tissue from a Post leg biopsy,
only a total of 39 muscle biopsies were collected from the
10 subjects.
Transmission electron microscopy
Muscle biopsy specimens were prepared for TEM as
described previously (Nielsen et al. 2011). In brief,
specimens were fixed with 2.5% glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.3) for 24 h and sub-
sequently rinsed four times in 0.1 Msodium cacodylate
buffer. Subsequently, we post-fixed muscle specimens
with 1% osmium tetroxide (OsO4) and 1.5% potassium
ferrocyanide (K4Fe(CN)6)in0.1Msodium cacodylate
buffer for 90 min at 4°C. After post-fixation, the muscle
specimens were rinsed twice in 0.1 Msodium cacodylate
buffer at 4°C, dehydrated through a graded series of
alcohol at 4–20°C, infiltrated with graded mixtures of
propylene oxide and Epon at 20°C, and embedded in
100% Epon at 30°C. We made ultra-thin (60 nm) sections
(using a Leica Ultracut UCT ultramicrotome) in three
depths separated by 150 μm, in order to have more muscle
fibres to choose for imaging. The sections were contrasted
with uranyl acetate and lead citrate before we examined
and captured electron micrographs in a pre-calibrated
Jeol-1400plus transmission electron microscope (Jeol Ltd,
Tokyo, Japan) with a CCD camera (Quemesa, EMSIS
GmbH, M¨
unster, Germany).
We made electron micrographs of eight to nine
longitudinally oriented muscle fibres per biopsy. From
each fibre, 80 electron micrographs were obtained at
×10,000 magnification in a systematic random order,
including 40 from the subsarcolemmal region, 20 from
the superficial region and 20 from the central region of the
myofibrillar space. This was based on the minimal electron
micrographs required, whereby no further improvement
in the precision of estimating volume by point-counting
could be achieved with additional micrographs. Details
on the precision of estimates are provided below. Each
electron micrograph covers an area of 5.7 μmby3.8μm,
which is the minimum interval between micrographs.
Volume fraction estimation
To make robust and unbiased volume estimates of lipid
droplets and mitochondria (for fibre-typing, see below),
we used point-counting techniques, with reference to
Cavalieri’s principle and the Delesse principle (Weibel,
1979), to estimate areas of mitochondria and lipid droplets
in the electron micrographs (Fig. 1). Identification of
mitochondria in micrographs was based on previously
reported work on mammalian skeletal muscles (Hoppeler
et al. 1973). Our criteria for identifying lipid droplets
included having a circular white-greyish appearance with
a fuzzy border (absence of distinct membrane) and a
minimum diameter of 200 nm.
A point grid (135 nm grid size) was overlaid onto each
micrograph, generating a total of 1107 points per micro-
graph for point-counting. Two subcellular localisations
of lipid droplet and mitochondria volume fractions were
defined: (i) intermyofibrillar volume fraction and (ii)
subsarcolemmal volume fraction. The volume fractions
of intermyofibrillar mitochondria and lipid droplets per
myofibrillar volume were estimated by point-counting
micrographs from the superficial and central regions of
the myofibrillar space. Muscle fibres are assumed to have
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a cylindrical shape. As the superficial region of a cylinder
occupies 3 times more volume than the central region,
volume estimates from the superficial myofibrillar space
were weighted 3 times more than those from the central
myofibrillar space.
The volume fraction of subsarcolemmal lipid droplets
and mitochondria per fibre surface area was estimated
by point-counting micrographs from the subsarcolemmal
region. We measured the fibre length at the base of the
subsarcolemmal region seen in the 40 micrographs. Fibre
surface area was calculated as the length of the muscle fibre
multiplied by the thickness of the fibre (60 nm).
The volume fractions of total lipid droplets and total
mitochondria were computed by adding intermyofibrillar
and subsarcolemmal volume fractions per myofibrillar
space. The subsarcolemmal volume fraction per myo-
fibrillar space were converted from the subsarcolemmal
volume fraction per fibre surface area by multiplying the
subsarcolemmal volume fraction per fibre surface area by
a factor of 20. Assuming a muscle fibre to be cylindrical
with a diameter of 80 nm, the volume beneath a surface
area of 1 μm2is 20 μm3based on the following formula:
V=r×0.5Awhere ris fibre radius and Ais fibre surface
area.
The relative distribution of subcellular lipid droplet
volume fractions was calculated after the volume fraction
of total lipid droplets was obtained.
The precision of stereological estimates, represented by
the estimated coefficient of error (CEest), for the sub-
cellular mitochondrial and lipid droplet volume fractions
was calculated as proposed by Howard & Reed (2005). All
point-counting was conducted using a commercial TEM
imaging software, iTEM (EMSIS).
Muscle fibre-typing
We fibre-typed all muscle fibres before estimating lipid
droplet volume fractions. Muscle fibres were categorised
as type 1 or 2, based on a distinct differentiation in terms
of intermyofibrillar mitochondrial volume fraction and
Z-line width, which was shown to relate to myofibrillar
ATPase properties (Sj¨
ostr¨
om et al. 1982). Intermyofibrillar
mitochondrial volume fraction was first plotted against
Z-line width for all the fibres (n=6–9) obtained from
each biopsy. The fibres with the highest intermyofibrillar
mitochondrial volume fraction and thickest Z-line width
were categorised as type 1 fibres and vice versa for type
2fibres(n=2–3 fibres of each type per biopsy). These
fibres were used for point-counting. Uncategorised fibres
(n=3–5 per biopsy) were put aside. The mean (SD)
intermyofibrillar mitochondrial volume percentage and
Z-line width for type 1 fibres were 12.5% (1.7%) and 88
(9) nm, and for type 2 fibres were 7.4% (1.4%) and 77
(8) nm.
ss
SMF
CMF
x 40
x 20
x 20
**
*Mi
Mi
Mi
Mi
Z
Z
AB
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Figure 1. Transmission electron microscopy and point-counting of subcellular lipid droplets
A, acquisition of electron micrographs was systematically randomized to cover the subsarcolemmal (SS), superficial
myofibrillar (SMF) and central myofibrillar regions (CMF) of each longitudinally oriented fibre. The arrow indicates
the sarcolemma. (original magnification ×600, scale bar: 20 µm). B, subcellular localisations of lipid droplets. The
arrow indicates the sarcolemma. C, close-up view of point-counting of subsarcolemmal lipid droplet volume fraction
with grid (size 135 nm) overlay. The open circles indicate intersections touching a lipid droplet. , a subsarcolemmal
lipid droplet; ∗∗, an intermyofibrillar lipid droplet; Mi, mitochondria; Z, Z-line. (Original magnification ×10,000,
scale bar: 1 µm.)
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Table 1. Subcellular volume fractions of lipid droplet and mitochondria in skeletal muscle at Pre
Arm Leg
Typ e 1 Typ e 2 Typ e 1 Typ e 2
Intermyofibrillar lipid droplet volume fraction
(μm3μm3myofibrillar space ×103)
2.1 (1.5–4.3) 1.3 (0.5–1.6)8.6 (5.0–12.1)∗∗ 3.5 (1.3–6.8)∗∗,
Subsarcolemmal lipid droplet volume fraction
(μm3μm2fibre area ×103)
2.2 (1.2–3.0) 0.9 (0.0–3.3) 8.2 (5.6–14.9)∗∗ 10.7 (4.8–17.4)∗∗
Total lipid droplet volume fraction (μm3μm3
myofibrillar space ×103)
2.2 (1.6–4.4) 1.4 (0.5–1.7)†† 9.3 (5.3–13.0)∗∗ 4.2 (1.9–7.1)∗∗,††
Intermyofibrillar mitochondrial volume fraction
(μm3μm3myofibrillar space ×103)
127 (108–143) 80 (66–95)††† 111 (98–125)∗∗ 67 (44–84)∗∗,†††
Subsarcolemmal mitochondrial volume fraction
(μm3μm2fibre area ×103)
440 (295–628) 366 (240–485)374 (310–436) 156 (94–258),††
Total mitochondrial volume fraction (μm3μm3
myofibrillar space ×103)
144 (132–171) 102 (84–112)††† 129 (116–142)∗∗ 76 (48–97)∗∗,†††
Values are medians (interquartile range). n=95 fibres. Limb difference: P<0.001; ∗∗P<0.0001. Fibre type difference: P=0.025;
††P<0.001; †††P<0.0001.
Lipid droplet size and number estimations
Lipid droplet size was estimated by measuring and
averaging the major and minor diameters of each
lipid droplet with the iTEM imaging software. The
aspect ratio of the lipid droplets, calculated as (major
diameter)/(minor diameter), reflects the ‘length-to-width
ratio’. The median (IQR) aspect ratio at Pre, 1.31
(1.24–1.36), was not statistically different from the Post
value, 1.31 (1.26–1.37).
Lipid droplet numbers were derived by dividing the
subcellular lipid droplet volume fraction by the mean
individual droplet volume of the respective subcellular
localisation. We assumed an individual lipid droplet to be
a sphere and its volume was calculated using the formula:
V=4/3πr3,whereris the radius based on the average of
the major and minor diameters of each lipid droplet.
Lipid droplet–mitochondria contact, represented by
percentage of lipid droplets in contact with surrounding
mitochondria, was estimated by counting the number of
lipid droplets located next to (<15 nm) surrounding
mitochondria, out of the total number of whole lipid
droplets observable in each electron micrograph.
Point-counting of mitochondrial content for the
purpose of fibre-typing was conducted by three
blinded investigators, where the electron micrographs
from the different time points and fibre types were
distributed equally between the investigators. Analyses
of lipid droplets were conducted by a single blinded
investigator. Inter-investigator and intra-investigator
intraclass correlation coefficients for all analyses were
>0.98.
A total of 14,576 electron micrographs from 190 muscle
fibres (95 Pre, 95 Post) were analysed in this study.
The median (IQR) CEest for the subsarcolemmal lipid
droplet volume fraction was 0.36 (0.26–0.46) at Pre, 0.40
(0.34–0.57) at Post, and for the intermyofibrillar lipid
droplet volume fraction it was 0.22 (0.17–0.30) at Pre,
0.27 (0.20–0.40) at Post.
All analyses on lipid droplet volume fraction and
numbers were conducted with each observation based on
estimates from 2–3 fibres.
Statistical analysis
All interactions or main effects were tested using a
linear mixed-effects model with subjects and time
as random effects and with time, limb, fibre type
and subcellular location as fixed effects. Variables
with skewed distributions were square root-transformed
or log-transformed before analysis. Non-parametric
Pre–Post comparison and differences across subcellular
locations, limbs and fibre types at Pre were tested with lipid
droplet–mitochondria contact data. Associations between
variables were evaluated using Pearson’s correlation
coefficient. Values are presented as medians with inter-
quartile range (IQR), unless stated otherwise. Significance
level was set at α=0.05. Statistical analyses were
performed using Stata 14 (StataCorp LP, College Station,
X, USA) and Prism 6 (GraphPad Software Inc., San Diego,
CA, USA).
Results
Subcellular volume fraction of mitochondria at
baseline (Pre)
Theabsolutebaselinevolumefractionsofinter-
myofibrillar and subsarcolemmal mitochondria are shown
in Table 1. When expressed as mitochondrial volume
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5786 H.-C. E. Koh and others J Physiol 595.17
percentage, total mitochondria (i.e. intermyofibrillar and
subsarcolemmal) content in arm type 1 and type 2
fibres were 14.4% and 10.2%, respectively, while in leg
type 1 and type 2 fibres, values were 12.9% and 7.6%,
respectively. The intermyofibrillar and total mitochondrial
volume fractions were 10–11% higher in arm than in
leg muscles (P<0.0001). Furthermore, subsarcolemmal
mitochondrial volume fraction was 2-fold higher in arm
type 2 fibres than in leg type 2 fibres (P<0.001), while
no difference was found between the limbs in type 1 fibres
(P=0.13).
Subcellular lipid droplet distribution at baseline (Pre)
Volume fraction. The baseline lipid droplet volume
fraction, size and density in the different locations are
shown in Fig. 2, with the absolute values of the lipid droplet
volume fractions shown in Table 1. The intermyofibrillar
Type 1 Type 2 Type 1 Type 2
0
3
6
9
12
15
Lipid droplet volume fraction
(µm
3
µm
-3
myofibrillar space x 10
3
)
Lipid droplet volume fraction
Arm Leg
*
Type 1 Type 2 Type 1 Type 2
0
100
200
300
400
500
600
700
Lipid droplet diameter (nm)
Lipid droplet size
Arm Leg
Intermyofibrillar
Subsarcolemmal
*
Type 1 Type 2 Type 1 Type 2
0
20
40
60
80
100
120
140
160
180
Lipid droplet numbers
(number µm
-3
myofibrillar space
x
10
3
)
Lipid droplet numbers
Arm Leg
*
A
B
C
Figure 2. Baseline subcellular lipid droplet distributions in arm (m. triceps brachii) and leg (m. vastus
lateralis) muscle fibres (n=20 observations)
A, subcellular lipid droplet volume fractions. P<0.0001 main limb effect in intermyofibrillar and subsarcolemmal
regions; P=0.0002 main fibre type effect in intermyofibrillar region. B, subcellular lipid droplet size in diameter.
P<0.001 main limb effect in type 2 fibres; P=0.007 main fibre type effect in leg muscle. C, subcellular lipid
droplet numbers. P<0.0001 main limb effect in intermyofibrillar and subsarcolemmal regions; P<0.0001
main fibre type effect in intermyofibrillar region. Bars and lines represent medians with interquartile range (Aand
C), or means with 95% confidence intervals (B).
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Table 2. Relative distribution of intermyofibrillar and sub-
sarcolemmal lipid droplets in type 1 and 2 human skeletal muscle
fibres before (Pre) and after (Post) exercise
Intermyofibrillar Subsarcolemmal
Pre Post Pre Post
Arm
Type 1 95 (92–97) 91 (84–93) 5 (3–8) 9 (7–16)
Type 2 97 (87–100) 90 (75–94) 3 (0–13) 10 (6–25)
Leg
Type 1 95 (93–97) 95 (94–97) 5 (3–7) 5 (3–6)
Type 2 85 (65–95) 91 (80–92) 16 (5–35)9 (8–20)
Values are medians (interquartile range), n=190 fibres.
P=0.003 vs.Pre.P=0.0001 vs. Leg Type 1.
and total lipid droplet volume fractions were 4-fold higher
in leg than in arm muscles, and the subsarcolemmal lipid
droplet volume fraction was 6-fold higher in leg than
in arm muscles (P<0.0001). Furthermore, the inter-
myofibrillar (P=0.0002) and total (P=0.0003) lipid
droplet volume fractions were 3-fold higher in type 1
fibres than in type 2 fibres, while no difference was found
between the fibre types (P=0.6) in the subsarcolemmal
lipid droplet volume fraction.
In general, the subsarcolemmal lipid droplet volume
fractions in type 1 and 2 fibres of the arm, and also
in type 1 fibres of the leg, each respectively contributed
3–5% towards the total fibre lipid droplet volume fraction
(Table 2). However, the subsarcolemmal lipid droplet
volume fraction represented 16% of the total fibre lipid
droplet volume fraction in type 2 fibres of the leg, which
was 3-fold higher than type 1 fibres of the leg (P=
0.0001).
Lipid droplet size. At baseline, there was no difference in
lipid droplet size between the intermyofibrillar and sub-
sarcolemmal regions (Fig. 2B). However, in type 2 muscle
fibres, lipid droplet diameter was found to be 24% larger in
thelegsthaninthearms(P<0.001),w hile in type 1 muscle
fibres, lipid droplet diameter tended to be 12% larger in
thelegsthaninthearms(P=0.05). Furthermore, lipid
droplet diameter was 8% larger in type 2 than in type 1
fibres (P=0.007) in leg muscle, but no difference between
fibre types was observed in arm muscle.
Lipid droplet numbers. Intermyofibrillar and sub-
sarcolemmal lipid droplet numbers were found to be
59–60% higher in leg than in arm muscles (P<0.001)
(Fig. 2C). Comparing fibre types in upper and lower limbs,
intermyofibrillar lipid droplet numbers were observed to
be 68% higher in type 1 than in type 2 fibres (P<0.0001),
while there was no difference between subsarcolemmal
lipid droplet numbers (P=0.2).
Effect of exercise on lipid droplet volume fractions,
size and numbers in distinct subcellular locations
Lipid droplet volume fraction. In the arms, the inter-
myofibrillar lipid droplet volume fraction decreased
by 53% across muscle fibre types following exercise
(P=0.0082) (Fig. 3A), and this decline was positively
associated with the intermyofibrillar lipid droplet volume
fraction at baseline (Pre) (R2=0.84, P<0.0001)
(Fig. 4). Between fibre types, the reduction in type 1
bres(58%)tendedtobelargerthantype2fibres(51%)
(P=0.09). Although no difference was observed in
type 1 fibres (Fig. 3B), the subsarcolemmal lipid droplet
volume fraction in type 2 fibres tended to be 1.5-fold
higher following exercise (P=0.09). Furthermore, the
contribution of the subsarcolemmal lipid droplet volume
fraction to the total fibre lipid droplet volume fraction
was 2-fold to 3-fold higher in both fibre types following
exercise (P=0.0026, Table 2).
In leg muscle, no difference in lipid droplet volume
fractions was found across localisations following exercise
(Fig. 5Aand B). Correspondingly, there was no difference
in the relative distributions of the subsarcolemmal lipid
droplet volume fraction in leg muscle (Table 2).
Lipid droplet size and numbers. No difference was
observed in lipid droplet size across all localisations in arm
and leg muscles prior to and following exercise (Figs 5C
and Dand 6).
Although there was no difference in subsarcolemmal
lipid droplet numbers across fibre types in arm muscle,
intermyofibrillar lipid droplet numbers decreased by 36%
following exercise (P=0.012, Fig. 3Eand F). However,
the opposite was observed in leg muscle (Fig. 5Eand F),
where subsarcolemmal lipid droplet numbers decreased
by 31% (P=0.019), while no difference was found in
intermyofibrillar lipid droplet numbers across fibre types
following exercise (P=0.3).
Lipid droplet–mitochondria contact before and after
exercise. The proportions of lipid droplets in contact
with the surrounding mitochondria across subcellular
locations, limbs and fibre types before and after exercise are
shown in Table 3. At baseline, no difference was detected
at most locations except a higher percentage in the inter-
myofibrillar than in the subsarcolemmal regions of type 2
fibres in the arms (P=0.03). No difference was detected
following exercise at most locations except a small increase
in the intermyofibrillar region of type 1 fibres in the legs
(P=0.02).
Discussion
By leveraging on the high-resolution characteristics of
electron microscopy, we were able to apply stereological
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Pre Post Pre Post
0
2
4
6
8
10
Intermyofibrillar lipid droplet volum e fraction
(µm3µm3
myof ibrillar s pace 103)
Type 1 Type 2
Pre Post Pre Post
0
20
40
60
80
100
120
140
Intermyofibrillar lipid droplet numbers
(number µm3
myofibrillar space 103)
Type 1 Type 2
*
*
Pre Post Pre Post
0
2
4
6
8
10
12
14
Subsarc olemmal lipid droplet volume f raction
(µm3µm2
fibre area 103)
Type 1 Type 2
Pre Post Pre Post
300
400
500
600
Subsarc olemmal lipid droplet diameter (nm)
Type 1 Type 2
Pre Post Pre Post
0
50
100
150
200
250
Subsarc olemmal lipid droplet numbers
(number µm2
fibre area 103)
Type 1 Type 2
Pre Post Pre Post
300
400
500
600
Intermyofibrillar lipid droplet diameter (nm)
Type 1 Type 2
AB
Lipid droplet volume fraction
CD
Lipid droplet size
Lipid droplet numbers
EF
x
x
x
x
Figure 3. Lipid droplet content in intermyofibrillar and subsarcolemmal regions of arm skeletal muscle
(m. triceps brachii) before (Pre) and after (Post) approximately 1 h cross-country skiing time trial
Values were observations (type 1, Pre: n=10, Post: n=10; type 2, Pre: n=10, Post: n=10) estimated in 2–3
fibres of each fibre type. Pre- and post-exercise intermyofibrillar (A,Cand E) and subsarcolemmal (B,Dand F) lipid
droplet volume fraction (Aand B), lipid droplet size (Cand D) and lipid droplet numbers (Eand F). Lines shown are
medians and interquartile range. P=0.0082 main time effect. P=0.012 main time effect.
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J Physiol 595.17 Subcellular lipid droplet distribution in human skeletal muscle 5789
techniques to discriminate between intermyofibrillar and
subsarcolemmal lipid droplets and semi-quantitatively
estimate the lipid droplet volume fractions in these
distinct subcellular locations. Our key observations on
highly trained endurance athletes (elite cross-country
skiers) were as follows: (i) at baseline, before exercise,
higher volume fractions of both intermyofibrillar and sub-
sarcolemmal lipid droplets were observed in leg muscle
than in arm muscle, even though both upper and lower
limbs were highly trained; (ii) at baseline, the volume
fraction of intermyofibrillar, but not subsarcolemmal,
lipid droplets, was higher in type 1 than in type 2 muscle
fibres; (iii) in arm muscles, the volume fraction of inter-
myofibrillar lipid droplets decreased following 1 h of
exhaustive exercise, with no change in the subsarcolemmal
pool; and (iv) the intermyofibrillar lipid droplets were
utilised selectively by both type 1 and 2 fibres, but lipid
dropletsizeappearednottochange.
Limb-dependent and fibre type-dependent
subcellular lipid droplet differences at baseline
Direct comparisons between arm and leg metabolic
profiles may be confounded by an unequal training
status of the limbs. However, exercise studies on
cross-country skiers have revealed possible inherent
02468
−2
0
2
4
6
8
Intermyofibrillar lipid droplet volume fraction at Pre
(µm3 µm3 myofibrillar space × 10 3)
Intermyofibrillar lipid droplet v olume fraction decline
(Pre - Post µm3 µm3 myofibrillar spac e × 103)
R2 = 0.84
P < 0.0001
Type 1
Type 2
Figure 4. The relationship between the volume fraction of
intermyofibrillar lipid droplets in arm (m. triceps brachii)
skeletal muscle fibres before exercise (Pre) and the
subsequent net decline in this parameter following 1 h
cross-country skiing time trial
The decline in the volume fraction of lipid droplets was positively
associated with the corresponding volume at Pre. The open triangles
represent type I fibres (n=10 observations), and open circles type II
fibres (n=10 observations). The line was fitted linearly.
Table 3. Percentage of lipid droplets in contact with
mitochondria in type 1 and 2 human skeletal muscle fibres
before (Pre) and after (Post) exercise
Intermyofibrillar Subsarcolemmal
Pre Post Pre Post
Arm
Type 1 83 (75–97) 86 (72–100) 67 (50–100) 71 (50–100)
Type 2 78 (60–100) 75 (50–89) 60 (25–100)67 (50–100)
Leg
Type 1 89 (85–93) 93 (87–100)81 (67–100) 90 (75–100)
Type 2 80 (65–91) 80 (65–91) 78 (50–88) 89 (58–100)
Values are medians (interquartile range). n=39–249 lipid
droplets (arm) and 147–872 lipid droplets (leg). Wilcoxon’s
matched-pairs signed-rank tests were performed between time
points and across subcellular locations, limbs and fibre types at
Pre. P=0.02 vs.Pre.P=0.03 vs. Intermyofibrillar.
qualitative differences in physiological and metabolic
parameters between the limbs (Helge, 2010). Many of
these studies have demonstrated a lower fat oxidation in
arm than in leg exercises (Calbet, 2005; Helge et al. 2008),
a higher lactate release during arm than in leg exercises
at equal intensity (Jensen-Urstad & Ahlborg, 1992), and
a different ventilatory response between exercise mostly
involving upper body muscle (double poling) and lower
body exercise (leg only) of comparable metabolic demand
(Calbet, 2005; Holmberg & Calbet, 2007). Furthermore,
the fibre type distribution in muscles may be different,
with arm muscle having a higher proportion of type 2
fibres (m. triceps brachii) than leg muscle (m. vastus
lateralis). Here we examined possible intrinsic limb
differences in the metabolic profile, using trained leg and
arm muscles, and demonstrate that leg and arm muscles
exhibit a clear difference in their lipid droplet volumes
and distribution with a 4-fold higher volume fraction of
intermyofibrillar lipid droplets and 6-fold higher volume
fraction of subsarcolemmal lipid droplets in leg than in
arm muscles. This is evident in both type 1 and t ype 2 fibres
from highly trained arm and leg muscles. This marked
difference in lipid droplet volumes between the limbs
could not be fully explained by the training status of the
muscles involved, or the different fibre type distribution
in the limbs, and it is therefore evident that the limbs have
inherently different lipid storage characteristics that are
independent of fibre type differences, and as a result a
possible difference in metabolic regulation.
In accordance with the long-held tenet regarding the
oxidative capacities of muscle fibre types, the type 1 fibres
areconsideredtobemoreoxidativethanthetype2
fibres in this study, because they are differentiated based
on intermyofibrillar mitochondria volume fractions and
Z-line width. Interestingly, while the intermyofibrillar
lipid droplet volume fraction was indeed 3-fold higher
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Pre Post Pre Post
0
5
10
15
20
25
30
Subsarc olemmal lipid droplet volume f raction
(µm3 µm2 fibre area 103)
Type 1 Type 2
Pre Post Pre Post
0
100
200
300
400
500
600
700
Subsarcolemmal lipid droplet numbers
(number µm2
fibre area 103)
Type 1 Type 2
Pre Post Pre Post
0
5
10
15
20
25
30
Intermyofibrillar lipid droplet volume fraction
(µm3 µm3 myofibrillar space 103)
Type 1 Ty pe 2
Pre Post Pre Post
0
50
100
150
200
250
300
Intermyofibrillar lipid droplet numbers
(number µm3
myofibrillar space 103)
Type 1 Ty pe 2
Pre Post Pre Post
300
400
500
600
Intermyofibrillar lipid droplet diameter (nm)
Type 1 Ty pe 2
Pre Post Pre Post
300
400
500
600
700
Subsarc olemmal lipid droplet diameter (nm)
Type 1 Type 2
Lipid droplet volume fraction
Lipid droplet size
Lipid droplet numbers
AB
CD
EF
x
x
x
x
Figure 5. Lipid droplet content in intermyofibrillar and subsarcolemmal regions of leg skeletal muscle
(m. vastus lateralis) before (Pre) and after (Post) approximately 1 h cross-country skiing time trial
Values were observations (type 1, Pre: n=10, Post: n=9; type 2, Pre: n=10, Post: n=9) estimated in 2–3
fibres of each fibre type. Pre- and post-exercise intermyofibrillar (A,Cand E) and subsarcolemmal (B,Dand F) lipid
droplet volume fraction (Aand B), lipid droplet size (Cand D) and lipid droplet numbers (Eand F). Lines shown are
medians and interquartile range. P=0.019 main time effect.
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J Physiol 595.17 Subcellular lipid droplet distribution in human skeletal muscle 5791
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
10
20
30
Lipid droplet diameter (nm)
Percent age
Type 1
Pre
Post
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
10
20
30
40
Lipid droplet diameter (nm)
Percent age
Type 1
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
10
20
30
Lipid droplet diameter (nm)
Percentage
Type 2
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
10
20
30
40
Lipid droplet diameter (nm)
Percentage
Type 2
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
5
10
15
20
Lipid droplet diameter (nm)
Percent age
Type 2
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
5
10
15
20
Lipid droplet diameter (nm)
Percentage
Type 1
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
5
10
15
20
Lipid droplet diameter (nm)
Percent age
Type 1
100
200
300
400
500
600
700
800
900
1000
1100
1200
0
5
10
15
20
Lipid droplet diameter (nm)
Percent age
Type 2
Arm
Leg
Intermyofibrillar Subsarcolemmal
AB
CD
EF
GH
Figure 6. The diameters of intermyofibrillar and subsarcolemmal lipid droplets in arm (m. triceps brachii)
and leg (m. vastus lateralis) skeletal muscle fibres before (Pre) and after (Post) 1 h cross-country skiing
time trial
Values were estimated from intermyofibrillar arm (Aand C:n=648) and leg (Eand G:n=2121) lipid droplets,
and subsarcolemmal arm (Band D:n=227) and leg (Fand H:n=771) lipid droplets. There were no significant
differences between Pre and Post.
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in type 1 than in type 2 fibres, the subsarcolemmal lipid
droplet pool was not found to be different across fibre
types in both upper and lower limbs, indicating that the
distribution of lipid droplets in the subsarcolemmal region
may not be dependent on the oxidative characteristic
of muscle fibres in highly trained individuals. Notably
in the legs, the indifferent subsarcolemmal lipid droplet
distribution across fibre types could be partly explained
by the presence of a second pool of lipid droplets (Fig. 6H),
with diameters of 600–650 nm, which could be regulated
differently from the main pool of lipid droplets (400 nm
diameter). However, more evidence is still needed to verify
our observations.
As muscle fibre type discrimination is rarely conducted
in studies that have examined ultrastructural features of
muscle cells using TEM, the direct comparison of results
between this study and others is very much limited. A
previous report was identified (Howald et al. 1985) and
we could see that the absolute values of baseline total lipid
droplet volume fractions estimated in this study (Table 1)
are higher than those observed in type 1 (0.0065 cm3cm3)
and type 2a (0.0027 cm3cm3) fibres of young sedentary
males in that report. This is probably due to the highly
trained status of the cross-country skiers in this study.
Subcellular lipid droplet volume fraction changes
following exercise
As the debate on whether intramuscular lipids serve
as an energy source during exercise can be explained
by methodological concerns regarding the biochemical
extraction of lipids, it is now recognised that there is
indeed a substantial decline in intramuscular lipid content
following acute exercise (van Loon, 2004; Kiens, 2006;
Shaw et al. 2010). Our findings in arm muscle (53%
reduction in the volume fraction of intermyofibrillar lipid
droplets) are in line with previous studies, including
those using immunofluorescence staining and detection
of intramuscular lipids, which have reported 50–70%
reduction, mainly in type 1 muscle fibres, following acute
enduranceexercise(vanLoonet al. 2004; De Bock et al.
2005; Stellingwerff et al. 2007; Shepherd et al. 2012).
Importantly, we also showed that lipid droplets located
in the intermyofibrillar region were utilised during 1 h
of exhaustive exercise, while lipid droplets located in the
subsarcolemmal region appeared untouched. This would
imply a compartmentalised energy demand for the use of
lipid droplets within skeletal muscle fibres during exercise
and that the spatial arrangement of lipid droplets dictates
their roles in energy metabolism. Moreover, our results
areinagreementwitharecentstudyshowingthatafter
1 h of moderate-intensity cycling by young subjects, the
intermyofibrillar lipid content decreased by 40%, while
the subsarcolemmal lipid content did not change (Chee
et al. 2016). Furthermore, in this study, the magnitude of
intermyofibrillar lipid droplet utilisation during exercise
was observed to be positively correlated with the amount
of intermyofibrillar lipid droplets at baseline, which has
also been reported by other studies (Zehnder et al. 2006;
Stellingwerff et al. 2007). This implies that a larger lipid
droplet pool is associated with higher lipolytic machinery.
It has been speculated that subsarcolemmal
mitochondria provide ATP mainly for the requirements
of myonuclei and membrane function, while inter-
myofibrillar mitochondria support ATP production
for contracting myofibrils (Hood, 2001). Furthermore,
energy-consuming processes might not be high in the
subsarcolemmal region during muscular contractions,
as compared to the intermyofibrillar region. Hence, this
might explain the utilisation of intermyofibrillar but not
subsarcolemmal lipid droplets during exercise in this
study.
Lipid droplet size changes following exercise
In general, there was no observed change in lipid droplet
size following exercise. Additionally, no large leftward
shift (towards smaller diameters) in the diameters of lipid
droplets across locations following exercise was present in
this study (Fig. 6). This implies that the number of lipid
droplets is a more likely factor for the exercise-induced
decline in the volume fraction of intermyofibrillar lipid
droplets in arm muscle. This is in line with previous
findings. Devries and co-workers (2007) reported that
90 min of moderate-intensity cycling led to a decrease in
intramuscular lipid content, with concomitant reduction
in lipid droplet density but not lipid droplet size in males
and females. More recently, intramuscular lipid droplet
density in type 1 muscle fibres was found to be decreased
by exhaustive knee-extensor exercise (Prats et al. 2013).
Limb differences in lipid droplet utilisation during
exercise
The present study examined the effects of 1 h of
cross-country skiing exercise, which required a varying
magnitude of concurrent and alternating upper/lower
body movement in a field condition. While clearly there
was lipid droplet utilisation in arm muscle, we did not
find a corresponding use in leg muscle during exhaustive
exercise. It should be noted that a substantial decrease
in both arm and leg intramuscular glycogen levels was
evident in the same subjects as previously reported by our
group (Nielsen et al. 2011), indicating that leg muscles
were adequately recruited for the exercise as well. The
elite cross-country skiers have well-trained arm and leg
muscles and if we exclude the influence of inherent
limb differences, the absence of lipid droplet volume
fraction decline in leg muscle after exercise could be partly
explained by laboratory observations made in another
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J Physiol 595.17 Subcellular lipid droplet distribution in human skeletal muscle 5793
group of elite cross-country skiers during skiing exercise
that consisted of diagonal striding and double-poling
phases (van Hall et al. 2003). In that study, plasma fatty
acid uptake by arm muscle was found to be low (near
zero) during diagonal striding, while there was a consistent
plasma fatty acid uptake by leg muscle throughout the
whole exercise period, inferring that there was lower
plasma fatty acid availability for arm muscle during
classic style cross-country skiing. Furthermore, studies
have shown enhanced utilisation of intramuscular lipids
during exercise when plasma fatty acid availability is low
(Watt et al. 2004; van Loon et al. 2005). Taken together, the
leg muscle probably did not have as great a need as arm
muscle to utilise subcellular lipid droplets during exercise
due to a more consistent energy source from circulating
fatty acids.
Fibre-type-dependent reduction of lipid droplets
following exercise
While other studies had demonstrated that intramuscular
lipid decreased predominantly in only type 1 fibres
following exercise, we could observe a substantial decline
in both type 1 (58%) and type 2 (51%) fibres, with no
difference in the relative reduction of lipid droplet content
between fibre types. This is most likely due to the very
well-trained type 2 muscle fibres in these elite skiers. Type
2a muscle fibres typically make up more than half of
the fibre composition in triceps brachii muscles (Mygind,
1995; Terzis et al. 2006), and both fibre types are heavily
recruited when triceps brachii muscles extend the elbows
during double poling in classical cross-country skiing
(Holmberg et al. 2005). Moreover, the type 2 fibres of the
elite cross-country skiers in this study are comparatively
oxidative, as the mean intermyofibrillar mitochondrial
volume percentage (7.4%) is higher than that observed
in type 1 and 2 fibres (3.2–5.6%) of untrained individuals
(Hoppeler et al. 1973; Bylund et al. 1977). It is also possible
that the intense nature of the time trial was able to recruit
the type 2 fibres of the skiers to a greater extent than the
moderate-intensity exercise trials used in other studies.
Recent in vitro and human in vivo studies have
demonstrated that intracellular lipid droplets are
‘dynamic’ hubs with free fatty acids trafficking between
systemic circulation, lipid droplets and mitochondria
in different metabolic conditions (e.g. cell starvation)
(Kanaley et al. 2009; Rambold et al. 2015). Moreover, intra-
muscular triacylglycerol (the main constituent of lipid
droplets) was reported to have a high turnover rate in
post-absorptive males (Sacchetti et al. 2004). Thus, using
the present quantitative electron microscopy method, any
absence of a change in lipid droplet content (i.e. volume
fraction, size) following exercise would not exclude the
presence of fatty acid metabolic flux between systemic
circulation, lipid droplets and mitochondria.
Lipid droplet–mitochondria contact following exercise
Higher volume fractions of mitochondria were found,
together with higher volume fractions of lipid droplets,
in type 1 than in type 2 muscle fibres, indicating a clear
distinction in oxidative capacity between muscle fibre
types. However, we observed lower volume fractions of
lipid droplets in the arm muscles than in the leg muscles,
despite higher fractions of mitochondria found in the arm
muscles than in leg muscles. This could imply that lipid
metabolism is regulated differently in well-trained arm
and leg muscles. Lipid droplets are known to be located
close to mitochondria in skeletal muscle (Hoppeler 1986;
Vock et al. 1996). This intimacy between lipid droplets
and surrounding mitochondria could facilitate substrate
supply for oxidative metabolism in the mitochondria.
We examined the contact between lipid droplets and the
surrounding mitochondria across the different subcellular
locations, limbs, fibre types at baseline and following acute
exhaustive exercise. However, differences could only be
observed between subcellular locations in arm type 2
fibres at baseline and in the intermyofibrillar region of
leg type 1 fibres following exercise. Since we observed
a clear utilisation of lipid droplets in the arms but not
in the legs, the proportion of lipid droplets in contact
with the surrounding mitochondria does not seem to
correspond to lipid droplet utilisation during exercise in
the current study. Indeed, our results are in line with pre-
vious findings which did not show an increase in lipid
droplet–mitochondria contact following acute exercise
despite a reduction in intramyocellular lipid area density
in men (Devries et al. 2007). Interestingly, chronic training
was demonstrated to be able to increase the contact
between the two organelles (Tarnopolsky et al. 2007).
In conclusion, employing quantitative electron micro-
scopy, we demonstrate here that the well-trained leg
muscles of elite cross-country skiers contain 4- to
6-fold more lipid droplets than the equally well-trained
arm muscles, and that intermyofibrillar but not sub-
sarcolemmal lipid droplets are utilised during exhaustive
exercise. Importantly, these findings indicate that the
distributions of lipid droplets in the muscles of upper and
lower limbs differ, and that the utilisation of lipid droplets
depends on their subcellular localisation.
References
Bylund AC, Bjur¨
o T, Cederblad G, Holm J, Lundholm K,
Sj¨
ostro¨
om M, Angquist KA & Scherst´
en T (1977). Physical
training in man. Skeletal muscle metabolism in relation to
muscle morphology and running ability. Eur J Appl Physiol
Occup Physiol 36, 151–169.
Calbet JAL (2005). Why do arms extract less oxygen than legs
during exercise? Am J Physiol Regul Integr Comp Physiol 289,
R1448–R1458.
C
2017 The Authors. The Journal of Physiology C
2017 The Physiological Society
5794 H.-C. E. Koh and others J Physiol 595.17
Chee C, Shannon CE, Burns A, Selby AL, Wilkinson D, Smith
K, Greenhaff PL & Stephens FB (2016). The relative
contribution of intramyocellular lipid to whole body fat
oxidation is reduced with age, but subsarcolemmal lipid
accumulation and insulin resistance are only associated with
overweight individuals. Diabetes 65, 840–850.
Crane JD, Devries MC, Safdar A, Hamadeh MJ & Tarnopolsky
MA (2010). The effect of aging on human skeletal muscle
mitochondrial and intramyocellular lipid ultrastructure.
J Gerontol A Biol Sci Med Sci 65A, 119–128.
De Bock K, Richter EA, Russell AP, Eijnde BO, Derave W,
Ramaekers M, Koninckx E, L´
eger B, Verhaeghe J & Hespel P
(2005). Exercise in the fasted state facilitates fibre
type-specific intramyocellular lipid breakdown and
stimulates glycogen resynthesis in humans. JPhysiol564,
649–660.
Devries MC, Lowther SA, Glover AW, Hamadeh MJ &
Tarnopolsky MA (2007). IMCL area density, but not IMCL
utilization, is higher in women during moderate-intensity
endurance exercise, compared with men. Am J Physiol Regul
Integr Comp Physiol 293, R2336–R2342.
Fujimoto T & Parton RG (2011). Not just fat: the structure and
function of the lipid droplet. Cold Spring Harb Perspect Biol
3, a004838.
Helge J (2010). Arm and leg substrate utilization and muscle
adaptation after prolonged low-intensity training. Acta
Physiol 199, 519–528.
Helge J, Damsgaard R, Overgaard K, Andersen J, Donsmark M,
Dyrskog S, Hermansen K, Saltin B & Daugaard J (2008).
Low-intensity training dissociates metabolic from aerobic
fitness. ScandJMedSciSports18, 86–94.
Holmberg H-C & Calbet JAL (2007). Insufficient ventilation as
a cause of impaired pulmonary gas exchange during
submaximal exercise. Respir Physiol Neurobiol 157, 348–359.
Holmberg H-C, Lindinger S, St¨
oggl T, Eitzlmair E & M¨
uller E
(2005). Biomechanical analysis of double poling in elite
cross-country skiers. Med Sci Sports Exerc 37, 807–818
Hood DA (2001). Invited review: contractile activity-induced
mitochondrial biogenesis in skeletal muscle. JApplPhysiol
90, 1137–1157.
Hoppeler H (1986). Exercise-induced ultrastructural changes
in skeletal muscle. Int J Sports Med 7, 187–204.
Hoppeler H, L ¨
uthi P, Claassen H, Weibel ER & Howald H
(1973). The ultrastructure of the normal human skeletal
muscle. A morphometric analysis on untrained men, women
and well-trained orienteers. Pflugers Arch 344, 217–232.
Howald H, Hoppeler H, Claassen H, Mathieu O & Straub R
(1985). Influences of endurance training on the
ultrastructural composition of the different muscle fiber
types in humans. Pflugers Arch 403, 369–376.
Howard CV & Reed MG (2005). Unbiased Stereology:
Three-Dimensional Measurement in Microscopy. Bios
Scientific Publishers, Oxford, UK.
Jensen-Urstad M & Ahlborg G (1992). Is the high lactate release
during arm exercise due to a low training status? Clin Physiol
12, 487–496.
Kanaley JA, Shadid S, Sheehan MT, Guo Z & Jensen MD
(2009). Relationship between plasma free fatty acid,
intramyocellular triglycerides and long-chain acylcarnitines
in resting humans. JPhysiol587, 5939–5950.
Kiens B (2006). Skeletal muscle lipid metabolism in exercise
and insulin resistance. Physiol Rev 86, 205–243.
Martin S & Parton RG (2006). Lipid droplets: a unified view of
a dynamic organelle. NatRevMolCellBiol7, 373–378.
Mygind E (1995). Fibre characteristics and enzyme levels of
arm and leg muscles in elite cross-country skiers. Scand J
Med Sci Sports 5, 76–80.
Nielsen J, Gejl KD, Hey-Mogensen M, Holmberg H-C, Suetta
C, Krustrup P, Elemans CPH & Ørtenblad N (2016).
Plasticity in mitochondrial cristae density allows metabolic
capacity modulation in human skeletal muscle. JPhysiol595,
2839–2847.
Nielsen J, Holmberg H-C, Schrøder HD, Saltin B & Ørtenblad
N (2011). Human skeletal muscle glycogen utilization in
exhaustive exercise: role of subcellular localization and fibre
type. JPhysiol589, 2871–2885.
Nielsen J, Mogensen M, Vind BF, Sahlin K, Hojlund K,
Schroder HD & Ørtenblad N (2010). Increased
subsarcolemmal lipids in type 2 diabetes: effect of training
on localization of lipids, mitochondria, and glycogen in
sedentary human skeletal muscle. Am J Physiol Endocrinol
Metab 298, E706–E713.
Ørtenblad N, Nielsen J, Saltin B & Holmberg H-C (2011). Role
of glycogen availability in sarcoplasmic reticulum Ca2+
kinetics in human skeletal muscle. JPhysiol589, 711–725.
Prats C, Gomez-Cabello A, Nordby P, Andersen JL, Helge JRW,
Dela F, Baba O & Ploug T (2013). An optimized
histochemical method to assess skeletal muscle glycogen and
lipid stores reveals two metabolically distinct populations of
type I muscle fibers. PLoS One 8, e77774.
Rambold AS, Cohen S & Lippincott-Schwartz J (2015). Fatty
acid trafficking in starved cells: regulation by lipid droplet
lipolysis, autophagy, and mitochondrial fusion dynamics.
Dev Cell 32, 678–692.
Sacchetti M, Saltin B, Olsen DB & van Hall G (2004). High
triacylglycerol turnover rate in human skeletal muscle.
JPhysiol561, 883–891.
Shaw CS, Clark J & Wagenmakers AJ (2010). The effect of
exercise and nutrition on intramuscular fat metabolism and
insulin sensitivity. Annu Rev Nutr 30, 13–34.
Shepherd SO, Cocks M, Tipton KD, Ranasinghe AM, Barker
TA, Burniston JG, Wagenmakers AJM & Shaw CS (2012).
Preferential utilization of perilipin 2-associated
intramuscular triglycerides during 1 h of moderate-intensity
endurance-type exercise. Exp Physiol 97, 970–980.
Sj¨
ostr¨
om M, Angquist KA, Bylund AC, Frid´
en J, Gustavsson L
&Scherst
´
en T (1982). Morphometric analyses of human
muscle fiber types. Muscle Ner ve 5, 538–553.
StellingwerffT,BoonH,JonkersRAM,SendenJM,SprietLL,
Koopman R & van Loon LJC (2007). Significant
intramyocellular lipid use during prolonged cycling in
endurance-trained males as assessed by three different
methodologies. Am J Physiol Endocrinol Metab 292,
E1715–E1723.
Tarnopolsky MA, Rennie CD, Robertshaw HA,
Fedak-Tarnopolsky SN, Devries MC & Hamadeh MJ (2007).
Influence of endurance exercise training and sex on
intramyocellular lipid and mitochondrial ultrastructure,
substrate use, and mitochondrial enzyme activity. Am J
Physiol Regul Integr Comp Physiol 292, R1271–R1278.
C
2017 The Authors. The Journal of Physiology C
2017 The Physiological Society
J Physiol 595.17 Subcellular lipid droplet distribution in human skeletal muscle 5795
Terzis G, Stattin B & Holmberg H-C (2006). Upper body
training and the triceps brachii muscle of elite cross country
skiers. ScandJMedSciSports16, 121–126.
van Hall G, Jensen-Urstad M, Rosdahl H, Holmberg H-C,
Saltin B & Calbet JAL (2003). Leg and arm lactate and
substrate kinetics during exercise. Am J Physiol Endocrinol
Metab 284, E193–E205.
van Loon LJC (2004). Use of intramuscular triacylglycerol as a
substrate source during exercise in humans. JApplPhysiol
97, 1170–1187.
van Loon LJC, Thomason-Hughes M, Constantin-Teodosiu D,
Koopman R, Greenhaff PL, Hardie G, Keizer, HA, Saris
WHM & Wagenmakers AJM (2005). Inhibition of adipose
tissue lipolysis increases intramuscular lipid and glycogen
useinvivoinhumans.Am J Physiol Endocrinol Metab 289,
E482–E493.
van Loon LJC, Koopman R, Stegen JHCH, Wagenmakers AJM,
Keizer HA & Saris WHM (2004). Intramyocellular lipids
form an important substrate source during moderate
intensity exercise in endurance-trained males in a fasted
state. JPhysiol553, 611–625.
Vock R, Hoppeler H, Helgard C, Wu DXY, Billeter R, Weber
J-M, Taylor CR & Weibel ER (1996). Design of the oxygen
and substrate pathways. VI. Structural basis of intracellular
substrate supply to mitochondria in muscle cells. JExpBiol
199, 1689–1697.
Watt MJ, Holmes AG, Steinberg GR, Mesa JL, Kemp BE &
Febbraio MA (2004). Reduced plasma FFA availability
increases net triacylglycerol degradation, but not GPAT or
HSL activity, in human skeletal muscle. Am J Physiol
Endocrinol Metab 287, E120–E127.
Weibel ER (1979). Stereological methods. Volume 1: Practical
methods for biological morphometry. Academic Press,
London.
ZehnderM,ChristER,IthM,AchesonKJ,PouteauE,KreisR,
TreppR,DiemP,BoeschC&D
´
ecombaz J (2006).
Intramyocellular lipid stores increase markedly in athletes
after 1.5 days lipid supplementation and are utilized during
exercise in proportion to their content. Eur J Appl Physiol 98,
341–354.
Additional information
Competing interests
The authors declare that they have no competing interests.
Author contributions
The experiments were performed at the Department of Sports
Science and Clinical Biomechanics, University of Southern
Denmark (muscle analysis, electron microscopy analysis) and
Department of Pathology, Odense University Hospital (electron
microscopy), and the Swedish Winter Sports Research Centre,
Mid Sweden University (exercise and testing). All authors
contributed to the conception and design of the experiments,
collection and analysis of data. H.C.E.K., J.N., H.C.H. and
N.Ø. contributed to the interpretation of data, drafting and/or
revision of manuscript, and approval of the final version of
manuscript. B.S. passed away on 12 September 2014 before this
manuscript was drafted. All authors agree to be accountable for
all aspects of the work in ensuring that questions related to the
accuracy or integrity of any part of the work are appropriately
investigated and resolved. All persons designated as authors
qualify for authorship, and all those who qualify for authorship
are listed.
Funding
This study was supported by grants from the Danish Diabetes
Academy funded by the Novo Nordisk Foundation and the
Lundbeck Foundation (R208-2015-3220 and R211-2015-3223).
Acknowledgements
We thank Karin Trampedach and Susan Bøgebjerg for
their excellent technical assistance in transmission electron
microscopy.
C
2017 The Authors. The Journal of Physiology C
2017 The Physiological Society
... In addition, the site of lipid storage, with athletes having more lipid droplets in the intramyofibrillar area than individuals with type 2 diabetes, spatially and functionally matches a high lipid dropletderived fat oxidative capacity. Indeed, reduction in lipid droplet number and content in the intramyofibrillar area upon acute exercise is observed [8,16], suggesting a preferential utilisation of intramyofibrillar lipid droplets during exercise. ...
... Data on changes in lipid droplet-mitochondria tethering during exercise are only available for endurance-trained athletes. In male elite cross-country skiers, lipid dropletmitochondria interactions increase upon an acute exercise bout despite unaltered IMCL content [16]. In endurancetrained women, lipid droplet-mitochondria tethering increases during exercise, with a concomitant reduction in IMCL content [23]. ...
... The latter study suggests that lipid droplet-mitochondrial interaction upon exercise promotes fatty acid oxidation. The seemingly contradictory finding that an exercise-mediated increase in lipid dropletmitochondria interaction is paralleled by reduced IMCL content in women [23] but not in men [16] might originate from sex differences, as reviewed recently [24]. A lack of a reduction in IMCL upon exercise (as observed in the male elite cross-country skiers) may also be reflective of a high IMCL turnover (IMCL utilisation during exercise matches fatty acid incorporation into lipid droplets). ...
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Fatty acids are an important energy source during exercise. Training status and substrate availability are determinants of the relative and absolute contribution of fatty acids and glucose to total energy expenditure. Endurance-trained athletes have a high oxidative capacity, while, in insulin-resistant individuals, fat oxidation is compromised. Fatty acids that are oxidised during exercise originate from the circulation (white adipose tissue lipolysis), as well as from lipolysis of intramyocellular lipid droplets. Moreover, hepatic fat may contribute to fat oxidation during exercise. Nowadays, it is clear that myocellular lipid droplets are dynamic organelles and that number, size, subcellular distribution, lipid droplet coat proteins and mitochondrial tethering of lipid droplets are determinants of fat oxidation during exercise. This review summarises recent insights into exercise-mediated changes in lipid metabolism and insulin sensitivity in relation to lipid droplet characteristics in human liver and muscle. Graphical abstract
... First, we investigated fibre type and region (peripheral and central)-specific differences in IMTG content detected using BODIPY and ORO in muscle biopsies obtained after an overnight fast (study 1). In this context, subcellular distribution is an important consideration because IMTG use during exercise occurs primarily in the central (intermyofibrillar) region of type I fibres of the cell (Jevons et al. 2020;Koh et al. 2017), and IMTG deposited in the peripheral (subsarcolemmal) region is inversely related to insulin sensitivity Nielsen et al. 2010). We also critically reviewed several steps in the immunohistochemical protocol and analytical procedure that can affect the visualisation and quantification of IMTG content. ...
... White bar = 30 µm be considered to more accurately reflect subcellular differences in IMTG content. In this regard, studies employing transmission electron microscopy to investigate subcellular lipid content support this assertion, because they also demonstrate IMTG content to be greater in the central (or intermyofibrillar) region (Koh et al. 2017). Thus, it appears that the choice of lipid dye will have implications for the conclusions that are drawn from studies investigating regional differences in IMTG content and LD morphology in human skeletal muscle in relation to health and disease. ...
... While IMTG utilisation was of a similar magnitude in the central and peripheral regions when using BODIPY, utilisation of IMTG was specific to the peripheral region when examined using ORO. This latter finding is in direct contrast with a recent study employing transmission electron microscopy which reported that IMTG utilisation during high-intensity exercise occurred exclusively in the intermyofibrillar region (Koh et al. 2017). Whilst we acknowledge that using BODIPY may also not entirely replicate the observations made using high-resolution electron microscopy (because we observed exercise-induced decreases in IMTG content in both subcellular regions), our data suggest subcellularspecific IMTG utilisation during exercise should not be investigated using ORO. ...
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Despite over 50 years of research, a comprehensive understanding of how intramuscular triglyceride (IMTG) is stored in skeletal muscle and its contribution as a fuel during exercise is lacking. Immunohistochemical techniques provide information on IMTG content and lipid droplet (LD) morphology on a fibre type and subcellular-specific basis, and the lipid dye Oil Red O (ORO) is commonly used to achieve this. BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra. Here we provide the first quantitative comparison of BODIPY and ORO for investigating exercise-induced changes in IMTG content and LD morphology on a fibre type and subcellular-specific basis. Estimates of IMTG content were greater when using BODIPY, which was predominantly due to BODIPY detecting a larger number of LDs, compared to ORO. The subcellular distribution of intramuscular lipid was also dependent on the lipid dye used; ORO detects a greater proportion of IMTG in the periphery (5 μm below cell membrane) of the fibre, whereas IMTG content was higher in the central region using BODIPY. In response to 60 min moderate-intensity cycling exercise, IMTG content was reduced in both the peripheral (- 24%) and central region (- 29%) of type I fibres (P < 0.05) using BODIPY, whereas using ORO, IMTG content was only reduced in the peripheral region of type I fibres (- 31%; P < 0.05). As well as highlighting some methodological considerations herein, our investigation demonstrates that important differences exist between BODIPY and ORO for detecting and quantifying IMTG on a fibre type and subcellular-specific basis.
... To the best of our knowledge, a relationship between PFO or Fat max and performance has not been elucidated in endurance sports dominated by upper body exercise. Interestingly, there are limb-specific differences in fiber-type composition, glycolytic and oxidative capacity, oxygen extraction, and storage of intramuscular triacylglycerol in highly trained cross-country (CC) skiers with equally trained upper and lower bodies, [13][14][15][16] which implies that the relationship between fat oxidation and performance may be different in upper body compared with lower body exercise. Furthermore, even at similar exercise intensities different fat oxidation rates have been reported between whole-body exercise modalities, [17][18][19] but it remains unknown whether this affects the potential relationship between peak fat oxidation and prolonged endurance performance. ...
... The reliance on intramuscular triacylglycerol (IMTG) is higher in trained compared with untrained individuals, 47,48 and in a study on elite CC-skiers, a 53% reduction in IMTG volume fraction in m. triceps brachii together with a solid positive correlation between IMTG degradation during a 20-km CC-skiing time trial and baseline IMTG volume fraction was reported. 16 This implies that the higher peak fat oxidation in ES compared with RS might derive from superior IMTG utilization. ...
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The peak fat oxidation rate (PFO) and the exercise intensity that elicits PFO (Fatmax) is associated with endurance performance during exercise primarily involving lower body musculature, but it remains elusive whether these associations are present during predominant upper body exercise. The aim was to investigate the relationship between PFO and Fatmax determined during a graded exercise test on a ski‐ergometer using double poling (GET‐DP) and performance in the long‐distance cross‐country skiing race, Vasaloppet. Forty‐three healthy men completed GET‐DP and Vasaloppet and were divided into two subgroups; recreational (RS, n=35) and elite skiers (ES, n=8). Additionally, RS completed a cycle‐ergometer GET (GET‐Cycling) to elucidate whether the potential relationships were specific to exercise modality. PFO (r²=0.10, p=0.044) and Fatmax (r²=0.26, p<0.001) were correlated with performance, however, V̇O2peak was the only independent predictor of performance (adj. R²=0.36) across all participants. In ES, Fatmax was the only variable associated with performance (r²=0.54, p=0.038). Within RS, DP V̇O2peak (r²=0.11, p=0.047) and ski specific training background (r²=0.30, p=0.001) were associated with performance. Between the two GETs Fatmax (r²=0.20, p=0.006) but not PFO (r²=0.07, p=0.135) was correlated. Independent of exercise mode, neither PFO nor Fatmax were associated with performance in RS (p>0.05). These findings suggest that prolonged endurance performance is related to PFO and Fatmax but foremost to V̇O2peak during predominant upper body exercise. Interestingly, Fatmax may be an important determinant of performance among ES. Among RS, DP V̇O2peak and skiing experience appeared as performance predictors. Additionally, whole‐body fat oxidation seemed specifically coupled to exercise modality.
... There is growing interest in elucidating how contracting muscles use lipid droplets (LD) in order to sustain exercise metabolism and/or for unknown purposes. For instance, during high-volume, high-intensity exercise (i.e., 57 min and 11 mmol/L blood lactate), LD within myofibrils but not those LD located close to the sarcolemma are used [58]. This is surprising as this kind of exercise mostly relies on oxidative metabolism mainly through fatty acid and glucose oxidation [59]. ...
... Furthermore, during innate immune response, LD increase their size [62]. This may also reveal different effects of LDs within skeletal muscle fibers, for instance, type 2 fibers have lower LD density, but they seems to have higher size than those LD form type 1 fibers in the leg skeletal muscle [58]. Although the current evidence is scarce, it would be interesting to test whether exercise impacts innate immune function by altering LD-mitochondria contact. ...
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The incidence and severity of metabolic diseases can be reduced by introducing healthy lifestyle habits including moderate exercise. A common observation in age-related metabolic diseases is an increment in systemic inflammation (the so-called inflammaging) where mitochondrial reactive oxygen species (ROS) production may have a key role. Exercise prevents these metabolic pathologies, at least in part, due to its ability to alter immunometabolism, e.g., reducing systemic inflammation and by improving immune cell metabolism. Here, we review how exercise regulates immunometabolism within contracting muscles. In fact, we discuss how circulating and resident macrophages alter their function due to mitochondrial signaling, and we propose how these effects can be triggered within skeletal muscle in response to exercise. Finally, we also describe how exercise-induced mitochondrial adaptations can help to fight against virus infection. Moreover, the fact that moderate exercise increases circulating immune cells must be taken into account by public health agencies, as it may help prevent virus spread. This is of interest in order to face not only acute respiratory-related coronavirus (SARS-CoV) responsible for the COVID-19 pandemic but also for future virus infection challenges.
... It has recently been reported that~35% of isolated rat cortical astrocytes 24 h after the seeding (~6 × 10 4 cells/coverslip) contain multiple LDs with an average diameter of~450 nm (ranging between 0.2 and 1 µm; [37]), comparable with the measurements performed on hepatocytes [22,43], skeletal muscle [44,45], and tumor cell lines [46]. In contrast, adipocytes normally contain a single LD, occupying most of the cytoplasm and ranging up to 100 µm in diameter [47]. ...
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In recent years, increasing evidence regarding the functional importance of lipid droplets (LDs), cytoplasmic storage organelles in the central nervous system (CNS), has emerged. Although not abundantly present in the CNS under normal conditions in adulthood, LDs accumulate in the CNS during development and aging, as well as in some neurologic disorders. LDs are actively involved in cellular lipid turnover and stress response. By regulating the storage of excess fatty acids, cholesterol, and ceramides in addition to their subsequent release in response to cell needs and/or environmental stressors, LDs are involved in energy production, in the synthesis of membranes and signaling molecules, and in the protection of cells against lipotoxicity and free radicals. Accumulation of LDs in the CNS appears predominantly in neuroglia (astrocytes, microglia, oligodendrocytes, ependymal cells), which provide trophic, metabolic, and immune support to neuronal networks. Here we review the most recent findings on the characteristics and functions of LDs in neuroglia, focusing on astrocytes, the key homeostasis-providing cells in the CNS. We discuss the molecular mechanisms affecting LD turnover in neuroglia under stress and how this may protect neural cell function. We also highlight the role (and potential contribution) of neuroglial LDs in aging and in neurologic disorders.
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Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells. Copyright © 2015 Elsevier Inc. All rights reserved.
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The lipid droplet (LD)-associated protein perilipin 2 (PLIN2) appears to colocalize with LDs in human skeletal muscle fibres, although the function of PLIN2 in the regulation of intramuscular triglyceride (IMTG) metabolism is currently unknown. Here we investigated the hypothesis that the presence of PLIN2 in skeletal muscle LDs is related to IMTG utilisation during exercise. We therefore measured exercise-induced changes in IMTG and PLIN2 distribution and changes in their colocalization. Muscle biopsies from the vastus lateralis were obtained from seven lean, untrained men (22 ± 2 years old, body mass index 24.2 ± 0.9 kg m(-2) and peak oxygen uptake 3.35 ± 0.13 l min(-1)) before and after 1 h of moderate-intensity cycling at ~65% peak oxygen uptake. Cryosections were stained for perilipin 2, IMTG and myosin heavy chain type I and viewed using wide-field and confocal fluorescence microscopy. Exercise induced a 50 ± 7% decrease in IMTG content in type I fibres only (P < 0.05), but no change in PLIN2 content. Colocalization analysis showed that the fraction of PLIN2 associated with IMTG was 0.67 ± 0.03 before exercise, which was reduced to 0.51 ± 0.01 postexercise (P < 0.05). Further analysis revealed that the number of PLIN2-associated LDs was reduced by 31 ± 10% after exercise (P < 0.05), whereas the number of PLIN2-null LDs was unchanged. No such changes were seen in type II fibres. In conclusion, this study shows that PLIN2 content in skeletal muscle is unchanged in response to a single bout of endurance exercise. Furthermore, the PLIN2 and IMTG association is reduced postexercise, apparently due to preferential utilization of PLIN2-associated LDs. These results confirm the hypothesis that the PLIN2 association with IMTG is related to the utilization of IMTG as a fuel during exercise.
Article
Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, = 68 ± 5 ml kg-1 min-1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. © 2011 The Authors. Journal compilation