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The effect of exercise induced cytokines on insulin stimulated glucose
transport in C2C12 cells
Stuart Robert Gray and Torkamol Kamolrat
Institute of Medical Sciences, University of Aberdeen, Aberdeen
Keywords: Cytokines, Glucose Uptake, Insulin Sensitivity
Running Title: Cytokines and glucose transport
Corresponding Author:
Stuart R Gray
Institute of Medical Sciences
University of Aberdeen
Foresterhill
Aberdeen
UK
AB25 2ZD
Tel: +44 (0)1224 555894
s.r.gray@abdn.ac.uk
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Abstract
Skeletal muscle contractile activity increases the production of the myokines
interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-15 (IL-15) and also skeletal
muscle glucose transport. Previous work has revealed a role for IL-6 in
mediating glucose uptake, while research on the physiological roles of IL-8
and IL-15 is not so abundant. In the present study we investigated the effects
of different concentrations and combinations of IL-6, IL-8 and IL-15 on insulin
stimulated glucose transport in C2C12 cells. Furthermore, we also measured
AMPK Thr172 and Akt Ser473 phosphorylation via Western blotting.
Exposure to 20pg/ml of individual cytokines had no affect on glucose transport
while 1ng/ml enhanced (P<0.05) glucose uptake with IL-6, Il-8 and IL-15,
respectively. Moreover, the combinations of IL-8+IL-6 and IL-15+IL-6 at both
20pg/ml and 1ng/ml stimulated (P<0.05) glucose transport with IL-8+IL-15 and
IL-8+IL-6+IL-15 only increasing (P<0.05) glucose transport at 1ng/ml with no
affect observed of these combinations at 20pg/ml. The changes in glucose
transport were all associated with an increase (P<0.05) in AMPK Thr172
phosphorylation with no changes in Akt Ser473 phosphorylation. These
findings demonstrated that the exercise induced myokines IL-6, IL-8 and IL-15
enhance glucose transport at 1ng/ml, with changes only seen at 20pg/ml with
certain myokine combinations. Furthermore these changes in insulin
stimulated glucose transport were associated with increased AMPK
phosphorylation.
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Introduction
Following from the work of Northoff and Berg
30,
who demonstrated an
increase in systemic levels of IL-6 after exercise, research has revealed that
skeletal muscle is capable of producing and releasing a variety of cytokines
(“myokines”) in response to muscular contraction and that these myokines
can act in both an endocrine or paracrine fashion
12.
As the increase in
systemic levels of IL-6 was the earliest and largest cytokine increase
31
observed the majority of research has thus far focussed on the source and
physiological function of this cytokine.
Initial efforts focussed on immune cells as the source of exercise induced IL-6
with Starkie et al finding that after exercise there was an increase in the
number of monocytes producing IL-6, with the amount of cytokine in each cell
was reduced 40. It then became clear that contracting skeletal muscle per se is
the major source of IL-6 in response to exercise. This was established by the
findings that skeletal muscle IL-6 mRNA and the skeletal muscle nuclear
transcription rate for IL-6 increased in response to exercise
22.
Further
research, utilising arterial-femoral venous difference over the exercising leg, it
was also demonstrated that IL-6 is released from exercising limbs
been found to have roles in fatigue
36,
hepatic glucose production
41.
IL-6 has
11,
lipolysis
and fat oxidation 42 and skeletal muscle glucose transport 8.
Exercise has also been found to result in the production of several other
myokines, including IL-8 and IL-15
32.
Systemic and skeletal muscle levels of
IL-8 have been shown to increase in response to a 3-h run with skeletal
muscle levels attenuated by concomitant carbohydrate ingestion
29.
A small
transient net release of IL-8 from exercise limbs has also been demonstrated
1.
Research into the function of IL-8 has highlighted a possible role of IL-8 in
the stimulation of angiogenesis after exercise
13,
although its precise role in
this is by no means clear. IL-15 is highly expressed in skeletal muscle
16
and
skeletal muscle mRNA levels are known to increase in response to resistance
exercise
27.
This myokine has been found to be negatively associated with
trunk fat mass suggesting a role for IL-5 in muscle to fat cross talk
28.
Indeed
over-expression of IL-15 has been demonstrated to reduce body fat, increase
Page |4
bone mineral content and also induce skeletal muscle hypertrophy
33;34.
Further work has also demonstrated, in a cell culture model, that IL-15 can
stimulate glucose transport and oxidation 7.
The signalling pathways involved in the control of glucose transport in
response to IL-6 have been studied in relative depth, with Kelly et al
24
demonstrating that in IL-6 knockout mice phosphorylation of AMP-activated
protein kinase (AMPK) was reduced and that exposure of EDL muscles to IL6 stimulates phosphorylation of AMPK. This pleiotropic protein, amongst other
effects, is known to stimulate glucose transport through translocation of
GLUT4 to the muscle membrane
e.g. 19.
However the signalling through which
IL-8 and IL-15 may stimulate glucose transport has yet to be addressed.
The role of myokines in metabolism, and particularly in skeletal muscle
glucose metabolism, is important as during exercise and in the recovery
period after exercise there is a significant rise in peripheral glucose uptake
and hepatic glucose output
10.
Furthermore the identification of the
mechanisms controlling skeletal muscle glucose uptake have not been fully
elucidated and may also improve pharmacological treatments in conditions
such as the metabolic syndrome and type 2 diabetes. Moreover, to our
knowledge, no authors have investigated the effect of combined exposure of
myokines, which is potentially more reflective of the “exercise environment” on
glucose transport in skeletal muscle. The aim of the current study, therefore,
is to investigate the effect of combinations of myokines on glucose transport
and phosphorylation of Akt and AMPK in a murine skeletal muscle cell line.
Page |5
Methods
Chemicals and Materials
All chemicals and materials were purchased from Sigma-Aldrich, Poole,
United Kingdom unless otherwise stated.
C2C12 Culture
C2C12 myoblasts (Health Protection Agency Culture Collections, Salisbury,
United Kingdom) were cultured in growth medium (DMEM with 4.5g/l glucose,
4 mM glutamine and 10 % vol/vol foetal calf serum). At ≈90% confluency
medium was changed to differentiation medium (DMEM with 4.5g/l glucose, 4
mM glutamine and 2 % vol/vol horse serum) to stimulate myotube formation.
Cells were maintained in differentiation medium for 4 days before
experimentation.
Glucose Transport
After 4 days of differentiation myotubes were serum deprived for 3 hours in
DMEM with 1g/l glucose for 3 hours prior to cytokine treatment. Cells were
then treated with the following combinations of cytokines, with 100nM insulin,
for 2 hours: IL-8 (20pg/ml), IL-8 (1ng/ml), IL-6 (20pg/ml), IL-6 (1ng/ml), IL-15
(20pg/ml), IL-15 (1ng/ml), IL-8 (20pg/ml)+IL-6 (20pg/ml), IL-6 (20pg/ml)+IL-15
(20pg/ml), IL-8 (20pg/ml)+IL-15 (20pg/ml), IL-8 (20pg/ml)+IL-6 (20pg/ml)+IL15 (20pg/ml), IL-8 (1ng/ml)+IL-6 (1ng/ml), IL-6 (1ng/ml)+IL-15 (1ng/ml), IL-8
(1ng/ml)+IL-15 (1ng/ml), and IL-8 (1ng/ml)+IL-6 (1ng/ml)+ L-15 (1ng/ml).
Cells were rinsed with HEPES-buffered saline (140 mM NaCl, 5 mM KCl, 2.5
mM MgSO4, 1.0 mM CaCl2, and 20 mM HEPES-Na, pH 7.4, 295 ± 5 mOsm)
and glucose transport was determined by the addition of [3H] 2-deoxy-Dglucose (0.5µCi/ml – American Radiolabeled Chemicals, Saint Louis, MO)
and 10µM 2-deoxyglucose. Cytochalasin B (10µM) was included in some
wells of the uptake assay to block transporter-mediated radiolabeled 2-deoxyD-glucose was included in some wells to measure background glucose
transport. This was subtracted from the total uptake to calculate transporter-
Page |6
mediated glucose uptake. After 5 min incubation the medium was aspirated
quickly and cells washed 3 times with ice cold PBS. Cells were then lysed
with 0.05 N NaOH, scintillation fluid added and samples measured on a
scintillation counter.
Protein Extraction and Western Blotting
C2C12 cells were washed in ice-cold PBS and lysed in lysis buffer (50 mM
Tris-HCL, 1 mM EDTA, 1 mM EGTA, 1 % (vol/vol) Triton X-100, pH 7.5)
supplemented with protease inhibitor cocktail, 10 mM β-glycerophosphate, 50
mM NaF and 0.5 mM sodium orthovanadate). C2C12 lysates were
homogenised on ice and then centrifuged at 13,000 g for 10 min. The protein
concentration in the supernatant was measured using a bicinchoninic acid
assay kit (Thermo Fisher Scientific, Northumberland, United Kingdom). The
supernatant was then diluted in 3x Laemmli SDS buffer and heated for 5 min
at 95 °C.
For electrophoresis 40 µg protein from each sample was loaded onto a 10%
gel and run for approximately 20min at 100V and 40min at 200V. The proteins
were transferred to polyvinylidene diflouride (PVDF) membraneand blocked
with 5% non-fat milk powder in TBS-T for 2 hours and exposed to the primary
antibody overnight at 4°C. The primary antibodies from New England Biolabs,
Beverly, MA, USA were used: p- Akt Ser463, p-AMPK Thr172 and β-actin.
The following day the PVDF membranes were incubated for 1 h at ambient
temperature with the secondary antibody, horse-radish peroxidise (HRP)
linked anti-rabbit IgG New England Biolabs, Beverly, MA, USA) before
detection using enhanced chemiluminescence (ECL) detection reagent
(Amersham Biosciences, Buckinghamshire, United Kingdom).
Statistical Analyses
Differences were analysed via an ANOVA followed by Tukeys post-hoc test.
Data are presented as mean (S.D.) with significance set at P<0.05.
Page |7
Results
20pg/ml concentrations individual cytokines do not alter glucose
transport
In myotubes exposed to 20 pg/ml of IL-6, IL-8 and IL-15 there was no change
(P<0.05) in levels of insulin stimulated glucose transport (Fig. 1A).
1ng/ml concentrations of individual cytokines stimulate glucose
transport
In contrast to the findings with 20pg/ml concentration of cytokines 1ng/ml of
individual cytokines were found to alter glucose transport (Fig. 2A). Indeed, 2h
exposure to 1ng/ml IL-8, IL-6 or IL-15 resulted in an increase (P<0.05) in
glucose transport, with IL-15 having the largest effect.
The effects of cytokine combinations of glucose transport
At 20pg/ml there was no effect (P<0.05) of the combination of IL-8+IL-15 or
the combination of IL-8+IL-6+IL-15 on glucose transport. However there was
an increase (P<0.05) in glucose transport after exposure to the combination of
20pg/ml IL-8+IL-6. Similarly the combination of 20pg/ml IL-6+IL-15 resulted in
a rise (P<0.05) in glucose transport (Fig. 3A). Exposure to the 1ng/ml
combinations of cytokines leads to an increase (P<0.05) in glucose transport
for all combinations (Fig. 4A).
The effects of cytokines on phosphorylation of Akt and AMPK
In all of the individual and combined treatments there was no effect on the
phosphorylation of Akt compared to the cells treated with insulin alone (data
not shown). Similarly when IL-6, IL-8 and IL-15 were applied to the cells
individually at 20pg/ml there was no change in the phosphorylation of AMPK
(Fig 1B). Similarly to the effects of 1ng/ml of individual cytokines on glucose
transport 2 hours exposure to IL-8, IL-6 and IL-15, individually, resulted in
increases (P<0.05) in AMPK phosphorylation (Fig 2B). Again following the
effects seen in glucose transport at 20pg/ml the combinations of IL-8 + IL-6
and IL-15 + IL-6 lead to increases (P<0.05) in AMPK phosphorylation, with no
Page |8
changes when myotubes were exposed to IL-8 + IL-15 and all three cytokines
simultaneously (Fig 3B). At 1ng/ml AMPK phosphorylation was increased
(P<0.05) by all combinations of cytokines and exposure to all three cytokines
at the same time (Fig 4B).
Page |9
Discussion
The present study has demonstrated that 20pg/ml individual of myokines has
no effect on skeletal muscle glucose transport. However during exercise,
when the expression of these cytokines increases
31,
skeletal muscles are
exposed to a milieu of cytokines and not a single cytokine. With that in mind,
we therefore investigated the effects of combinations of cytokines on glucose
transport and found that the combinations of IL-8+IL-6 and IL-6+IL-15
stimulate
glucose transport.
At
1ng/ml
all individual cytokines and
combinations thereof lead to increases in glucose transport. Observed
changes in glucose transport were associated with increases in AMPK
phosphorylation.
Prior to entering a discussion on the effects of cytokines on glucose transport
it is prudent to relate the 20pg/ml and 1ng/ml concentrations of cytokines to
levels seen in body fluids. The 20pg/ml concentration of cytokines was
chosen as this closely reflects the post-exercise systemic levels of cytokines
e.g. 31.
The levels of cytokines to which the muscles are exposed are, however,
likely to be higher, both intracellularly and in the interstitial fluid. For example,
using the microdialysis technique it has been demonstrated, in young healthy
males, that immediately after repetitive low force exercise skeletal muscle
interstitial IL-6 concentrations reach ~ 1.2 ng/ml and continue to rise up to 2.1
ng/ml in the recovery period
38.
Interstitial levels up to 3-4 ng/ml have also
been reported in females after exercise
37.
To our knowledge, there is no data
on post-exercise interstitial levels of IL-8 and IL-15, although resting interstitial
levels of IL-8 have recently been shown to be around 0.3pg/ml in patients with
polymyalgia rheumatic
25.In
the current study, therefore, the concentration of
1ng/ml was chosen in an attempt to replicate the levels of cytokines that
skeletal muscles are exposed to after exercise.
Circulating levels of IL-6 have previously been found to be positively
associated with insulin resistance
2;3;20.
Similarly circulating levels of IL-8 have
been found to be elevated in obese patients and correlate with measures of
insulin resistance 6. Furthermore in the work of Rotter et al
39
it was revealed
that both IL-6 and IL-8 were ~15 fold elevated in adipocytes taken from
P a g e | 10
individuals with insulin resistance. The association of IL-6 and IL-8 with insulin
resistance seems counterintuitive and paradoxical, since both are produced
and released from skeletal muscle during exercise
in increased skeletal muscle glucose uptake
10.
41
a stress known to result
The potential for IL-6 and IL-8
to be involved in the aetiology of insulin resistance is also confounded by
further research investigating the role of these cytokines in glucose
metabolism. Pioneering work by Wallenius et al 43 developed an IL-6 knockout
mouse which developed mature onset obesity, decreased glucose tolerance
and increased systemic leptin concentration. Furthermore it has been
demonstrated that IL-6 can increase glucose uptake in humans using muscle
samples collected via biopsy
15
and using whole body stable isotopes 8. The
current study demonstrated that 20pg/ml IL-6 has little effect on glucose
transport and only when the concentration is increased to 1ng/ml does
glucose transport increase, similar to previous studies 14;17.
With respect to IL-8 previous work has demonstrated that, similarly to the IL-6
response, when exercise is performed when glycogen availability is low there
is an increased skeletal muscle IL-8 gene expression 9. Whilst previous work
has focussed on a potential role of IL-8 and its receptor CXCR2 in
angiogenesis
13,
it is surprising no studies have investigated a potential
metabolic role for IL-8. The current study found that at 20pg/ml there was no
effect of IL-8 on glucose transport in C2C12 cells, however when 1ng/ml used
there was a significant increase in glucose transport. This is the first study to
identify a potential metabolic role for IL-8 in stimulating glucose transport
although as this effect was only seen at 1ng/ml, levels similar to those seen in
the interstitial fluid, its effect during exercise is likely to be local only, although
further work is required to clarify this.
Skeletal muscle levels of IL-15 have been shown previously to increase
during exercise
28.
27;29
and have a potential role in cross talk with adipose tissue
Further work has supported this and demonstrated that overexpression of
skeletal muscle IL-15 levels, with a subsequent increase in serum IL-15,
causes a decrease in fat mass without any changes in lean mass or other
cytokines
28;33.
Specifically investigating the role of IL-15 in glucose
P a g e | 11
metabolism Busquets et al
7
demonstrated that IL-15 stimulates glucose
uptake into rat EDL muscles and C2C12 cells. The current study confirmed
the findings of Busquets and colleagues by demonstrating that 1ng/ml IL-15
stimulates glucose transport and shows that 20 pg/ml of IL-15, individually,
has no effect on glucose transport. This highlights a potential role for IL-15 in
glucose transport, although as very little data is available on intramuscular
and interstitial concentrations of IL-15 the potential role of this during exercise
requires further examination.
When considering the physiological roles of the myokines IL-6, IL8 and IL-15
it is prudent to acknowledge that there is not only an increase in one cytokine
but a simultaneous increase in all, exposing skeletal muscle to a milieu of
cytokines during and after exercise. While there are several other cytokines
that increase in response to exercise the current study focussed on the
combined effects of IL-6, IL-8 and IL-15 on glucose transport. We have
demonstrated that when combined at 20pg/ml there was no effect of IL-8+IL15 on glucose transport. However, when IL-6+IL-8 and IL-6+IL-15 are utilised
in experiments there is a significant increase in glucose transport in both
treatments. On the other hand, somewhat surprisingly, when all three
cytokines were included in the medium these effects were abolished. The
mechanism behind this observation is at present not clear and requires further
work. In experiments when the same combinations of cytokines were
employed at 1ng/ml increases in glucose transport, generally to a greater
extent compared to 20pg/ml, were observed with all combinations of
cytokines. With the combination of 1ng/ml IL-6+IL-15 there was no further rise
in glucose transport compared to 20pg/ml concentrations. The combination of
IL-6+IL-8+IL15 still having the lowest glucose transport, again suggesting
some form of inhibition when all three cytokines are present in the medium.
One limitation of the current study, when attempting to relate the current
investigation to the role of these myokines during exercise, is accounting for
the contribution of their respective receptors. It is known that IL-6 signals
through membrane bound IL-6R and gp130 receptors, with gp130
ubiquitously expressed and IL-6R expression more limited
21.
Both these
P a g e | 12
receptors also exist within body fluids in soluble forms as sIL-6R and sgp130.
Exercise is known to increase both skeletal muscle membrane bound IL-6R,
sIL-6R and sgp130
18;23.
It is therefore possible that the stimulation of these
receptors, occurring during exercise, would further enhance glucose transport
e.g. 17.
There are two homologous chemokine receptors (CXCR1 and CXCR2)
that bind IL-8 with high affinity
4
and CXCR2 expression has been
demonstrated to double after a bout of exercise
13.
Similarly to IL-6 it is,
therefore, possible that the effects of IL-8 on glucose transport may be
enhanced by the exercise induced rise in CXCR2 expression. Whilst genetic
variation in the IL-15 receptor-α gene have been found to account for the
variability in adaptations to resistance exercise
35,
there is no available
information on the response of the IL-15 receptor isoforms to exercise.
Further work is required to ascertain the effects of exercise on IL-15 receptors
and subsequently investigate to roles that each cytokines receptors, both
membrane bound and soluble, play in glucose transport.
Previous studies have demonstrated that IL-6 can stimulate phosphorylation
of AMPK at Thr172
24
and Akt at Ser473
44.
The physiological roles of AMPK
are numerous and importantly for this study its phosphorylation is increased
during contractile activity and can stimulate glucose transport
26.
On the other
hand Akt is well known for its role in increasing insulin stimulated glucose
transport but has little involvement in the insulin independent contraction
mediated stimulation of glucose transport
5.
Furthermore while previous
studies have found that increases in IL-6 stimulated glucose transport are
associated with increased AMPK phosphorylation
been found in Akt phosphorylation
15;17.
8;14;15;17
no differences have
The current study supports these
assertions as 1ng/ml concentrations of IL-6 stimulated glucose transport
through AMPK phosphorylation, with no changes in Akt. Both IL-8 and IL-15
were also demonstrated to increase glucose transport through an increase in
AMPK phosphorylation, with this being the first study to demonstrate that
these myokines can activate AMPK, with no changes in Akt. Furthermore all
combinations of myokines that resulted in an increase in glucose transport
were also associated with increase AMPK phosphorylation.
P a g e | 13
In conclusion the current study has demonstrated that at levels similar to
those seen in plasma after exercise the myokines IL-6, IL-8 and IL-15 have
little effect on glucose transport into murine skeletal muscle cells. However at
levels similar to those seen in the interstitial fluid after exercise the current
study has revealed that each cytokine can stimulate glucose transport and
may, therefore, have pharmacological potential in the treatment of diabetes.
We also showed that certain combinations of cytokines can stimulate glucose
transport although as each cytokines response was not always additive (e.g.
IL-6+IL-8+IL-15 do not increase glucose transport) and dose response
relationships were not clear caution should be taken when interpreting the
effects of the exercise induced milieu of cytokines on glucose transport.
P a g e | 14
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Figure Legends.
Figure 1. The effect of 20pg/ml individual cytokines on insulin stimulated
glucose transport (A) and AMPK (Thr 172) phosphorylation (B) in C2C12
cells. Data are presented as mean (SD) (A n=6; B n=4)). *denotes a
significant difference from control (insulin stimulated levels); p<0.05.
Figure 2. The effect of 1ng/ml individual cytokines on insulin stimulated
glucose transport (A) and AMPK (Thr 172) phosphorylation (B) in C2C12
cells. Data are presented as mean (SD) (A n=6; B n=4)). *denotes a
significant difference from control (insulin stimulated levels); p<0.05
Figure 3. The effect of 20pg/ml combined cytokines on insulin stimulated
glucose transport (A) and AMPK (Thr 172) phosphorylation (B) in C2C12
cells. Data are presented as mean (SD) (A n=6; B n=4)). *denotes a
significant difference from control (insulin stimulated levels); p<0.05
Figure 4. The effect of 1ng/ml combined cytokines on insulin stimulated
glucose transport (A) and AMPK (Thr 172) phosphorylation (B) in C2C12
cells. Data are presented as mean (SD) (A n=6; B n=4)). *denotes a
significant difference from control (insulin stimulated levels); p<0.05
P a g e | 23
Figure 1
A
4.0
3.5
Glucose Transport (A.U)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control
Insulin (100um)
Insulin + IL-8 (20pg/ml)
Insulin + IL-6 (20pg/ml)
Insulin + IL-15 (20pg/ml)
Treatments
B
p-AMPK
Insulin + IL-15 (20pg/ml)
Insulin + IL-6 (20pg/ml)
0.4
Insulin + IL-8 (20pg/ml)
0.45
Insulin (100μM)
Control
Β-actin
0.35
pAMPK/B-Actin (A.U)
0.3
0.25
0.2
0.15
0.1
0.05
0
Control
Insulin (100um)
Insulin + IL-8 (20pg/ml)
Treatments
Insulin + IL-6 (20pg/ml) Insulin + IL-15 (20pg/ml)
P a g e | 24
Figure 2.
A
5.0
*
*
4.5
*
4.0
Glucose Transport (A.U)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Control
Insulin (100um)
Insulin + IL-8 (1ng/ml)
Insulin + IL-6 (1ng/ml)
Insulin + IL-15 (1ng/ml)
Treatments
B
p-AMPK
Insulin + IL-15 (1ng/ml)
Insulin + IL-6 (1ng/ml)
*
0.5
Insulin (100μM)
0.4
Control
0.45
Insulin + IL-8 (1ng/ml)
Β-actin
*
*
Insulin + IL-6 (1ng/ml)
Insulin + IL-15 (1ng/ml)
pAMPK/B-Actin (A.U)
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Control
Insulin (100um)
Insulin + IL-8 (1ng/ml)
Treatments
P a g e | 25
Figure 3.
A
6.0
*
5.0
*
Glucose Transport (A.U)
4.0
3.0
2.0
1.0
0.0
Insulin (100um)
Insulin + IL-8 + IL-6 (20pg/ml)
Insulin + IL-15 + IL-6
(20pg/ml)
Insulin + IL-8 + IL-15
(20pg/ml)
Insulin + IL-8 + IL-15 + IL-6
(20pg/ml)
Treatments
*
*
Insulin + IL-8 + IL-15 + IL-6 (20pg/ml)
0.45
Insulin + IL-8 + IL-15 (20pg/ml)
0.50
Insulin + IL-15 + IL-6 (20pg/ml)
Insulin (100μM)
Β-actin
Insulin + IL-8 +IL-6 (20pg/ml)
p-AMPK
B
0.40
pAMPK/B-Actin (A.U)
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Insulin (100um)
Insulin + IL-8 + IL-6
(20pg/ml)
Insulin + IL-15 + IL-6
(20pg/ml)
Treatments
Insulin + IL-8 + IL-15
(20pg/ml)
Insulin + IL-8 + IL-15 +
IL-6 (20pg/ml)
P a g e | 26
Figure 4.
A
7.0
*
6.0
*
*
Glucose Transport (A.U)
5.0
*
4.0
3.0
2.0
1.0
0.0
Insulin (100um)
Insulin + IL-8 + IL-6 (1ng/ml) Insulin + IL-15 + IL-6 (1ng/ml) Insulin + IL-8 + IL-15 (1ng/ml) Insulin + IL-8 + IL-15 + IL-6
(1ng/ml)
Treatments
B
*
0.5
Insulin + IL-8 + IL-15 + IL-6 (1ng/ml)
*
Insulin + IL-8 + IL-15 (1ng/ml)
0.6
Insulin + IL-15 + IL-6 (1ng/ml)
Insulin (100μM)
Β-actin
Insulin + IL-8 +IL-6 (1ng/ml)
p-AMPK
*
pAMPK/B-Actin (A.U)
*
*
0.4
*
0.3
0.2
0.1
0.0
Insulin (100um)
Insulin + IL-8 + IL-6
(1ng/ml)
Insulin + IL-15 + IL-6
(1ng/ml)
Treatments
Insulin + IL-8 + IL-15
(1ng/ml)
Insulin + IL-8 + IL-15 +
IL-6 (1ng/ml)