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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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2017 The Authors. The Journal of Physiology C
2017 The Physiological Society DOI: 10.1113/JP274462
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 kg−1min−1, and 155 (8) g l−1, 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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J Physiol 595.17 Subcellular lipid droplet distribution in human skeletal muscle 5783
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 l−1. 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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5784 H.-C. E. Koh and others J Physiol 595.17
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
(μm3μm−3myofibrillar 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
(μm3μm−2fibre 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 (μm3μm−3
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
(μm3μm−3myofibrillar space ×103)
127 (108–143) 80 (66–95)††† 111 (98–125)∗∗ 67 (44–84)∗∗,†††
Subsarcolemmal mitochondrial volume fraction
(μm3μm−2fibre area ×103)
440 (295–628) 366 (240–485)†374 (310–436) 156 (94–258)∗,††
Total mitochondrial volume fraction (μm3μm−3
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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J Physiol 595.17 Subcellular lipid droplet distribution in human skeletal muscle 5787
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
fibres(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
(µm3µm−3
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 µm−3
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
(µm3µm−2
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 µm−2
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 µm−3 myofibrillar space × 10 3)
Intermyofibrillar lipid droplet v olume fraction decline
(Pre - Post µm3 µm−3 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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5790 H.-C. E. Koh and others J Physiol 595.17
Pre Post Pre Post
0
5
10
15
20
25
30
Subsarc olemmal lipid droplet volume f raction
(µm3 µm−2 fibre area 103)
Type 1 Type 2
Pre Post Pre Post
0
100
200
300
400
500
600
700
Subsarcolemmal lipid droplet numbers
(number µm−2
fibre area 103)
Type 1 Type 2
Pre Post Pre Post
0
5
10
15
20
25
30
Intermyofibrillar lipid droplet volume fraction
(µm3 µm−3 myofibrillar space 103)
Type 1 Ty pe 2
Pre Post Pre Post
0
50
100
150
200
250
300
Intermyofibrillar lipid droplet numbers
(number µm−3
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 cm3cm−3)
and type 2a (0.0027 cm3cm−3) 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.
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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.
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2017 The Authors. The Journal of Physiology C
2017 The Physiological Society
