Image Anal Stereol 2020;39:25-32  doi: 105566/ias.2356 
Original Research Paper 
25 
 
INCREASE IN SUBCELLULAR GSK-3 CLUSTERS IN INSULIN- AND 
ADRENALINE-TREATED DIFFERENTIATED RAT SKELETAL MUSCLE 
FIBRES 
KATJA FINK
1*
, MATEJA LOBE PREBIL
1*
, NINA VARDJAN
1,2
, JØRGEN JENSEN
3
,  
ROBERT ZOREC

,1,2
 AND MARKO KREFT

,1,2,4
 
1
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of 
Medicine, University of Ljubljana, Zaloška 4, Ljubljana, Slovenia, 
2
Celica Biomedical, Tehnološki park 24, 
Ljubljana, Slovenia, 
3
Department of Physical Performance, Norwegian  School of Sport Sciences, Postboks 
4014 Ullevål stadion, Oslo, Norway, 
4
Department of Biology, Biotechnical Faculty, University of Ljubljana, 
Ljubljana, Slovenia 
*
These authors have equally contributed to the work. 
e-mail: katja.fink@mf.uni-lj.si, matejaprebil@gmail.com, nina.vardjan@mf.uni-lj.si, jorgen.jensen@nih.no, 
robert.zorec@mf.uni-lj.si, marko.kreft@mf.uni-lj.si 
(Received February 25, 2020; revised March 9, 2020; accepted March 10, 2020) 
ABSTRACT 
Glycogen synthase kinase 3 (GSK-3) plays an important role in metabolic regulation in skeletal muscles, 
and both insulin and adrenaline stimulate GSK-3 phosphorylation. The aim of the present study was to 
study the effect of insulin and adrenaline on GSK-3 localisation in skeletal muscles. We characterized 
subcellular localization of GSK-3 signal protein in fully differentiated muscle fibre by immunofluores-
cence and confocal microscopy. We stimulated muscle fibres with insulin and/or adrenaline. Images were 
analysed by segmentation of single central optical section of the muscle. We found GSK-3 to be localised 
in clusters. The number of GSK-3 clusters and their average size were increased after stimulation with 
insulin and/or adrenaline. Average GSK-3 particle size is linearly related to their quantity. We conclude 
that subcellular GSK-3 in isolated skeletal muscle fibres is localized in clusters and clustering increased 
after stimulation with insulin and/or adrenaline. 
Keywords: adrenaline, GSK-3 (glycogen synthase kinase 3), insulin, skeletal muscle fibre.  
INTRODUCTION  
Skeletal muscles play a key role in regulating the 
whole body glucose metabolism. During insulin 
stimulation, more than 80 % of the glucose disposal 
o c c u r s i n s k e l e t a l m u s c l e s w h e r e g l u c o s e i s 
incorporated into glycogen, as shown by hyperglyce-
mic-hyperinsulinemic clamp studies (Shulman et al., 
1990). Note that disposition in skeletal muscle of 75-g 
of ingested glucose in nondiabetic subjects is found to 
be 22 % (Meyer et al., 2004). Insulin increases the rate 
of glucose uptake into the skeletal muscle and activates 
glycogen synthase (GS) (Shepherd et al., 1998, Chiang 
et al., 2003, Sano et al., 2003, Chowdhury et al., 2005, 
Mancinelli et al., 2017). Insulin resistance in type 2 
diabetes is an outcome of several deficiencies in signal 
transduction, which is proposed to start with a 
discrepancy in insulin action on receptor tissue, such as 
skeletal muscle, and then loss of function in β-cells 
because of hyperinsulinemia (DeFronzo and Tripathy, 
2009). The molecular targets and intracellular 
signalling systems that are modified during insulin 
resistance have received great attention in the past 
(Pessin and Saltiel, 2000, Björnholm and Zierath, 
2005). 
Insulin binds to its receptor and causes tyrosine 
phosphorylation of the insulin receptor substrate-1 
(IRS-1) (White, 1998). Tyrosine phosphorylated IRS-1 
binds and activates class-IA PI 3-kinase, a lipid kinase 
which phosphorylates phosphatidylinositol 4,5-
bisphosphate (PIP2) to phosphatidylinositol 3,4,5-
trisphosphate (PIP
3
). When PIP
3
 is present in the 
membrane, protein kinase B (PKB) becomes activated 
by phosphorylation. Activated PKB phosphorylates and 
inactivates glycogen synthase kinase 3 (GSK-3), which 
is a major protein kinase involved in the regulation of 
glucose metabolism occurring in two known isoforms 
GSK-3α and GSK-3β (Medina and Castro, 2008). 
Inactivated GSK-3 results in less phosphorylated and 

  FINK K ET AL: GSK-3 cluster formation in insulin and adrenaline treated muscle fibres 
26 
 
thus more active GS (Cross et al., 1995, Lawrence and 
Roach, 1997, McManus et al., 2005). In addition is GS 
regulated by allosteric factors, primarily by glucose 6-
phosphate (Jensen & Lai, 2009). 
Inactivation of GSK-3 by phosphorylation has 
revealed GSK-3 ability to allow glycogen synthesis 
(Bouskila et al., 2008). GSK-3 is also involved in 
numerous cellular processes, including cell growth, 
proliferation, and differentiation (Cohen and Frame, 
2001, Neary and Kang, 2006). GSK-3 is constitutively 
active, and multiple regulatory mechanisms affect 
GSK-3 inhibition in mammals (Cohen and Frame, 
2001). The mechanism of these inhibitory signals 
affecting GSK-3α and GSK-3β specifically are not 
known (Tanji et al., 2002). 
Adrenaline has well characterised effects in 
muscle, a generally regarded counter-regulator of the 
insulin action. Adrenaline and insulin were believed to 
act via distinct pathways and the complex interactions 
between β-adrenergic and insulin signalling in skeletal 
muscles is not well understood (Brennesvik et al., 
2005, Jensen et al., 2008). The individual responses 
generated by insulin and adrenaline do not predict the 
responses mediated by combined stimulation with these 
agonists (Jensen et al., 2007). Interestingly, adrenaline 
also regulates GSK-3 phosphorylation, but still 
inactivates GS activity (Jensen et al., 2008). 
The aim of this study is to characterise the 
intracellular distribution and cluster formation of GSK-
3 in isolated skeletal muscle cells before and after 
stimulation with insulin and/or adrenaline. The results 
revealed that both hormones increase GSK-3 cluster 
size as well as their quantity in insulin- and adrenaline-
treated skeletal muscle fibres.  
 
MATERIAL AND METHODS  
EXPERIMENTAL ANIMALS 
Adult Wistar rats were used for the study. The 
animals were euthanized with carbon dioxide in 
accordance to the following ethical codes and 
directives: International Guiding Principles for 
Biomedical Research Involving Animals developed by 
the Council for International Organizations of Medical 
Sciences, and the Animal Protection Act (Official 
Gazette RS, No. 38/13). The experimental protocol was 
approved by The Administration of the Republic of 
Slovenia for Food Safety, Veterinary and Plant 
Protection (Republic of Slovenia, Ministry of 
Agriculture, Forestry and Food, Ljubljana), Document 
No. U34401-47/2014/7. 
 
EXPERIMENT ON ISOLATED MUSCLE 
FIBERS AND IMMUNOFLUORES-
CENCE  
Single muscle fibres were obtained from the flexor 
digitorum brevis muscle as described previously 
(Bekoff and Betz, 1977, Zorec et al., 1992). Briefly, 
the muscles were dissected from the animal and placed 
in 35 mm culture dishes, in 2 ml Eagle’s Minimal 
Essential Medium (EMEM; Sigma) supplemented with 
horse serum (5% by volume; Sigma), penicillin (100 
units ml
-1
; Sigma), streptomycin (100 µg ml
-1
; Sigma) 
and 2 mM L-glutamine (Sigma). To this medium, 
collagenase (GIBCO, USA) was added (3 mg ml
-1
). 
The muscles were incubated for 3 hours at 36°C in an 
air-5% CO
2
 incubator at 95% humidity, washed in 
fresh EMEM and mechanically dissociated into single 
muscle fibres by repeated passages through Pasteur 
pipettes. The isolated muscle fibres were cultured 
overnight on 22 mm glass coverslips with 10 g/cm
2
 
collagen type I (Sigma) and 1 g/cm
2
 laminin (Sigma). 
Two hours before experiments, cultures were 
transferred into cell culture medium without serum and 
antibiotics. For stimulation, insulin (70 nM, Novo 
Nordisk A/S, Denmark) and/or adrenaline (1 µM, 
Sigma, USA) was added to the cell culture medium for 
15 minutes.  
For immunofluorescence single muscle fibres were 
washed with PBS (phosphate-buffered saline) and then 
fixed for 15 minutes in 4% paraformaldehyde in PBS. 
The following 10 minutes muscle fibres were kept in 
fixative containing 0.1% of Triton X-100 and then four 
times washed with PBS. Non-specific staining was 
reduced by incubating muscle fibres in 3% BSA 
(bovine serum albumin) and 10% normal goat serum in 
PBS. Muscle fibres were then incubated with primary 
a n t i b o d i e s f o r 2 h o u r s a t 3 7 ° C . W e u s e d r a b b i t 
polyclonal primary antibodies (Chemicon International, 
U S A ) a n t i b o d i e s d i l u t e d 1 : 5 0 0 f o r l a b e l l i n g G S K - 3 . 
Muscle fibres were then washed and incubated in PBS 
containing Alexa Fluor 488- or Alexa Fluor 546-
labelled goat anti-rabbit (1:500) and 3% BSA for 45 
minutes. Cells were washed and mounted in SlowFade 
Kit (Invitrogen, CA, USA) as described previously 
(Vardjan et al., 2007).  
 
 

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0;39:25-32 
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  FINK K ET AL: GSK-3 cluster formation in insulin and adrenaline treated muscle fibres 
28 
 
GSK-3 particles in control fibres was: 0.21 ± 0.01 µm
2
 
(n=25; Fig. 2b), whereas in fibres treated with 70 nM 
insulin the average size was 0.37± 0.04 µm
2
 (n=30). 
 
Fig. 2: The above-threshold area of GSK-3 positive 
signal in confocal images of control and stimulated 
isolated muscle fibres, measured as the number (a) of 
GSK-3 particle profiles and their average size (b). a) In 
images of control muscle fibres the number of GSK-3 
particle profiles above intensity threshold level was 68 
± 8.85. In images of muscle fibres, treated with insulin 
(+Ins; 70 nM), adrenaline (+Adr; 1 µM) or insulin and 
adrenaline (+Ins +Adr), the number of particle profiles 
were 141.87 ± 13.36, 108.21 ± 11.17, 169.27 ± 13.96, 
respectively. The insulin and/or adrenaline treatment 
significantly increases the GSK-3 positive area in 
isolated muscle fibres (p-value obtained by one-way 
ANOVA followed by all pairwise multiple comparison 
procedure Holm-Sidak method). b) In images of control 
muscle fibres, the average size of GSK-3 particle 
profiles (in µm
2
) was 0.21 ± 0.01. In images of muscle 
fibres, treated with insulin (+Ins), adrenaline (+Adr) 
or insulin and adrenaline (+Ins +Adr), the relative 
area above the threshold level were 0.37 ± 0.04, 0.28 ± 
0.03, 0.48 ± 0.05, respectively. The numbers by the 
bars indicate the number of analysed muscle fibres. 
The insulin treatment and combined insulin/adrenaline 
treatment significantly increases the GSK-3 positive 
area in isolated muscle fibres (p-value obtained by 
Kruskal-Wallis one-way ANOVA on ranks followed by 
all pairwise multiple comparison procedure (Holm-
Sidak method).  
Similarly, when muscle fibres were treated with 1 
µM adrenaline the average size of particle profiles was 
0.28 ± 0.03 µm
2 
(n=29) The muscle fibres, which were 
stimulated with insulin and adrenaline simultaneously, 
display the GSK-3 particle size of 0.48 ± 0.05 µm
2
 
(n=26). These results show a significantly increased 
(see below) clustering of GSK-3 in muscle fibres after 
stimulation with insulin and/or adrenaline, which is 
marked by the increased number of particle profiles as 
well as by the increased average size of GSK-3 
particle. 
We have performed One-way analysis of variance 
(ANOVA) test (p=0.001) for normally distributed data, 
followed by all pairwise multiple comparison 
procedure (Holm-Sidak method). We found the number 
of GSK-3 particle profiles in muscle fibres when 
stimulated with insulin (p<0.001), adrenaline (p=0.023) 
and combined insulin and adrenaline (p<0.001) are 
significantly different from control muscle fibres. 
Further, we have found that combined treatment with 
insulin and adrenaline has larger effect on particle 
count than treatment with adrenaline alone (p=0.045).  
We have performed Kruskal-Wallis one-way 
ANOVA on ranks, where data was not normally 
distributed, followed by all pairwise multiple 
comparison procedure (Dunn's Method). We have 
found, that the average size of GSK-3 particle profiles 
in muscle fibres when stimulated with insulin (p=0.05), 
a n d c o m b i n e d i n s u l i n a n d a d r e n a l i n e ( p < 0 . 0 5 ) a r e 
significantly different from control muscle fibres. 
Moreover, the average size of GSK-3 particle profiles 
in muscle fibres differ when stimulated with adrenaline 
alone vs. combined stimulation with insulin and 
adrenaline (p<0.05). We have found that combined 
treatment with insulin and adrenaline has larger effect 
on particle size than treatment with adrenaline alone 
(p<0.05). 
INSULIN AND/OR ADRENALINE STIMU-
LATION OF MUSCLE FIBRE EXCITES 
CORRELATED INCREASE IN GSK-3 
PARTICLE NUMBER AND SIZE  
We tested next whether the labelled GSK-3 
particle number in a tested muscle fibre is correlated 
with the particle size. We found that larger GSK-3 
particle appear only in muscle fibres with the number 

Image Anal Stereol 2020;39:25-32 
29 
 
of particle profiles higher than 107 in the tested region 
of interest (Fig. 3). The latter coincides with the 
observation that the number of GSK-3 particle profiles 
in control muscle fibres is lower than this threshold.  
 
Fig. 3: Size of GSK-3 particle profiles is correlated to 
their number and is dependent on insulin and/or 
adrenaline stimulation. The average size of labelled 
GSK-3 particle is correlated (r=0.719, p=4.3 ꞏ10
-6
) to 
their number and the slope of linear regression is 
further increased after stimulation with insulin (white), 
adrenaline (red) or both (green). The broken 
regression line has its node at value 107 particle 
profiles per region of interest. At the node the average 
size of particle corresponds to 0.242 µm
2
.  
 
Consequently, their average size is largely limited 
to 0.35 µm
2
. We found significant overall correlation 
between the number of particle profiles in a muscle 
fibre and the average particle size (Pearson correlation 
coefficient r = 0.719, p: 4.3ꞏ10
-6
). We fitted the broken-
line regression to the data and found two slopes, first 
with parameters size (in m
2
)= 0.162 + 0.0007 ± 
0.0002 ꞏN  and second with parameters size (in m
2
)= -
0.0761 + 0.0030 ± 0.0005 ꞏN, where N denotes the 
average number of particle profiles per region of 
interest (p<0.0001). When stimulating muscle fibres 
with insulin and/or adrenaline, the number of GSK-3 
particle profiles correlatively increases, which is 
evident from the increased regression slope on the 
broken line fit (Fig. 3). The broken regression line has 
its node at value 107 particle profiles per region of 
interest. At the node the average size of particle 
corresponds to 0.24 µm
2
. 
DISCUSSION 
W e c h a r a c t e r i s e d f o r t h e f i r s t t i m e s u b c e l l u l a r 
localisation of signal protein GSK-3 in fully 
differentiated muscle fibre by immunofluorescence and 
confocal microscopy. We report that clusters of GSK-3 
exist in muscle without hormonal stimulation. Insulin 
and adrenaline are known to stimulate GSK-3 
phosphorylation, which inactivates GSK-3 (Bouskila et 
al., 2008; Jensen et al., 2007), in our study increased 
the number and size of GSK-3 clusters in skeletal 
muscle. 
Skeletal muscles are the specialised tissue for 
movement and the contractile proteins myosin and 
actin are localised in myofibrils surrounded by 
sarcoplasmic reticulum, mitochondria, glycogen 
particles, and other organelles (Nielsen and Ørtenblad, 
2013), and it is unknown how signalling from surface 
receptors are transferred into the muscle fibres, which 
have diameters of 30 µm or more. Our data show that 
GSK-3 exists in clusters that are homogenous 
distributed in the muscle fibres.  
GSK-3 modulates the function of a diverse series 
of proteins, as well as being associated with a wide 
variety of human disorders, including neurodegenera-
tive diseases, stroke, bipolar disorder, diabetes, and 
cancer. It controls microtubule and actin dynamics 
(Meijer et al., 2004, Zhou and Snider, 2005). Unlike 
many other kinases, GSK-3β is active in resting cells 
(Ciaraldi et al., 2007) and phosphorylation by PKB 
decreases GSK-3 activity. Interestingly, insulin which 
increases GSK-3 phosphorylation increased GSK-3 
clustering. Although we did not use phosphorspecific 
antibodies to study localisation, it is tempting to 
speculate that it is the phosphorylated GSK-3 that 
accumulates in the clusters. Importantly, these clusters 
are localised throughout the muscle fibre. These data 
suggest that in unstimulated cell, the active GSK-3 
molecules are diffusely distributed and phosphorylates 
target proteins. However, after GSK-3 inactivation by 
insulin stimulation, the inactive GSK-3 accumulates in 
clusters.  
Adrenaline has similarly well characterized effects 
in muscle which via binding to β–adrenergic receptor, 
production of cAMP and activation of PKA results in 
breakdown of glycogen (Brennesvik et al., 2005). This 
mechanism results in raised insulin-sensitive glucose 
uptake in muscle (Kolnes et al., 2015). On the other 
hand, insulin dependent signalling phosphorylates 
GSK-3 and activates GS (Bouskila et al., 2008). Hence 
glycogen is essential in body glucose metabolism, 
specifically in skeletal muscle, where exercise induces 
insulin sensitization and enhances glucose uptake in 
part to enable glycogen supercompensation in the post 
exercise period (Hingst et al., 2018). This is due to 
increased sensitivity of muscle to insulin and higher GS 

  FINK K ET AL: GSK-3 cluster formation in insulin and adrenaline treated muscle fibres 
30 
 
activity during and after exercise (Hingst et al., 2018). 
Importantly, the individual responses generated by 
insulin and adrenaline do not predict the responses that 
will be generated by combined stimulation with these 
agonists (Jensen et al., 2007). It was reported 
previously that the adrenaline alone does not have any 
effect on the phosphorylation state or activity of PKB 
in skeletal muscle however, adrenaline potentiates 
insulin's effects on PKB (Brennesvik et al., 2005). 
Moreover, adrenaline alone stimulates GSK-3 Serine 9 
phosphorylation via cAMP and PKA. Insulin and 
adrenaline are targeting distinct intracellular pools of 
PKB and GSK-3 suggesting that compartmentalization 
of different signalling molecules is likely to play an 
important role in dictating the responses that will be 
generated in muscle following stimulation with 
multiple agonists (Jensen et al., 2007).  
Adrenaline induces phosphorylation of a pool of 
GSK-3 that is not involved in the regulation of 
glycogen metabolism (Brennesvik et al., 2005). The 
combination of adrenaline and insulin may activate 
novel signalling molecules rather than just summing up 
their effects on linear pathways. Adrenaline is a strong 
activator of PKB despite that it has no effect when 
insulin is absent (Jensen et al., 2008). Insulin-
stimulated glucose uptake increases following 
adrenaline despite prolonged exposure to hypergly-
caemia in vivo. Increased insulin-stimulated glucose 
uptake and GS activation after adrenaline infusion 
cannot be explained by a reduction in glycogen content 
or an increase in PKB phosphorylation. Desensitization 
of β-adrenergic signalling pathway represents a 
potential mechanism for increased insulin action after 
adrenaline infusion (Jensen et al., 2005). Also, 
adrenaline does not influence basal or insulin-
stimulated PKB and GSK-3β phosphorylation in 
muscles, but completely blocks insulin-mediated GS 
activation and Serine 641 dephosphorylation. Still, 
insulin normalizes adrenaline-mediated hyperglycae-
mia (Jensen et al., 2010). 
Insulin and adrenaline are regulating the PKB and 
GSK-3 in muscle, but the regulation is not uniform for 
the whole pool of molecules, but is having a specific 
effect on distinct pools of these signalling molecules 
(Jensen et al., 2007). Compartmentalisation can occur 
either via localisation to different organelles or by 
colocalisation of signalling molecules in specific 
complexes regulated by scaffold proteins. It has been 
shown, for example, that GSK-3β and PKA are both 
colocalised on AKAP-220 and that cAMP reduces the 
activity of GSK-3 in this complex to a greater degree 
than activity of the total cellular pool of GSK-3 and a 
protein phosphatase inhibitor inhibits the activity of 
GSK-3β bound to AKAP-220 more strongly than the 
total GSK-3β activity (Tanji et al., 2002, Jensen et al., 
2007). AKAP-220, GSK-3β, PKA, and type 1 protein 
phosphatase (PP1) were suggested to form a quaternary 
complex. PKA and PP1 regulate the activity of GSK-
3β efficiently by forming a complex with AKAP-220 
(Tanji et al., 2002). It has become clear that scaffold 
proteins form complexes of signalling proteins to 
obtain specificity in signalling. Only a few PKB-
binding proteins have been described (Brazil et al., 
2002). Signalling seems to be ordered in modules and 
large protein complexes appear to direct signalling to 
organelles and regulate specific physiological functions 
for which software tool to analyse such complexes in 
fluorescent confocal images has been previously 
presented (Kreft et al., 2010). This is in line with our 
finding that GSK-3 is localised in skeletal muscle fibre 
in clusters (Fig. 1).  
As discussed above, GSK-3 has complex regula-
tion both upstream and downstream. In addition to 
insulin and adrenaline, Wnt signalling is well defined 
regulator of GSK-3 (Patel and Woodgett, 2017). It has 
been challenging to understand the complex interaction 
between insulin and adrenaline regulating GSK-3 and 
physiological processes, and the finding in the present 
study that GSK-3 clustering increases during hormonal 
stimulation that causes phosphorylation and 
inactivation will allow more in-depth studies of GSK-3 
signalling. We did some attempts to study co-
l o c a l i s a t i o n o f t h e G S K - 3 c l u s t e r a n d P K B , w h i c h 
showed no co-localisation. Future studies should find 
organelles and proteins associated with the clusters. 
Furthermore, it will be important to confirm that GSK-
3 phosphorylation is the mechanism initiating GSK-3 
cluster formation.  
I n c o n c l u s i o n , G S K - 3 f o r m s c l u s t e r s i n s k e l e t a l 
muscle fibres and clustering increases upon stimulation 
with insulin and adrenaline. This finding shows that it 
will be possible to dissect GSK-3 signalling in more 
detail in skeletal muscles. Furthermore, it will be 
important to characterise the physiological role of the 
cluster formation.   
 
ACKNOWLEDGEMENTS 
This work was supported by the grant #P3 310 of 
Slovenian Research Agency.  
 
 

Image Anal Stereol 2020;39:25-32 
31 
 
CONFLICT OF INTEREST 
Authors declare no conflict of interests. 
 
ABBREVIATIONS 
AKAP-220 - A-Kinase Anchoring Protein 220; 
cAMP – cyclic adenosine monophosphate; GS - 
glycogen synthase; GSK-3 -glycogen synthase kinase 
3 ; I R S - 1 - i n s u l i n r e c e p t o r s u b s t r a t e - 1 ; P I P 3  - 
phosphatidylinositol 3,4,5-trisphosphate; PKA – 
protein kinase A; PKB - protein kinase B; PP1 - type 1 
protein phosphatase; Wnt - wingless-related integration 
site signal transduction pathways. 
 
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