Oleate protects against palmitate-induced insulin resistance in L6 myotubes
Dan Gao, Helen R. Griffiths* and Clifford J. Bailey
School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK
(Received 12 February 2009 – Revised 29 April 2009 – Accepted 26 May 2009 – First published online 22 July 2009)
Oleate has been shown to protect against palmitate-induced insulin resistance. The present study investigates mechanisms involved in the
interaction between oleate and palmitate on insulin-stimulated glucose uptake by L6 skeletal muscle cells. L6 myotubes were cultured for 6h
with palmitate or oleate alone, and combinations of palmitate with oleate, with and without phosphatidylinositol 3-kinase (PI3-kinase) inhibition.
Insulin-stimulated glucose uptake, measured by uptake of 2-deoxy-D-[3H]glucose, was almost completely prevented by 300mM-palmitate. Cells
incubated with oleate up to 750mmol/l maintained a significant increase in insulin-stimulated glucose uptake. Co-incubation of 50–300mM-oleate
with 300mM-palmitate partially prevented the decrease in insulin-stimulated glucose uptake associated with palmitate. Adding the PI3-kinase
inhibitors wortmannin (1027mol/l) or LY294002 (25mmol/l) to 50mM-oleate plus 300mM-palmitate significantly reduced the beneficial effect
of oleate against palmitate-induced insulin resistance, indicating that activation of PI3-kinase is involved in the protective effect of oleate.
Thus, the prevention of palmitate-induced insulin resistance by oleate in L6 muscle cells is associated with the ability of oleate to maintain insulin
signalling through PI3-kinase.
NEFA: Insulin resistance: L6 muscle cells: Phosphatidylinositol 3-kinase
Insulin resistance, which describes an impaired response to
physiological concentrations of insulin, is strongly associated
with obesity and type 2 diabetes, and contributes to
cardiovascular risk(1–3). Excess accumulation of saturated
lipid, especially in skeletal muscle which is the major site of
insulin-stimulated glucose uptake, is an important factor
in the development of insulin resistance(4–6). Since insulin
signalling to regulate glucose uptake in muscle is mediated
largely through a pathway dependent upon phosphatidyl-
inositol 3-kinase (PI3-kinase) and Akt phosphorylation, the
effects of lipids on this pathway provide an important focus
for the study of lipid-induced insulin resistance(7–9).
Insulin resistance caused by cellular lipid accumulation is
mainly associated with SFA(10). Palmitate, one of the most
abundant SFA, representing about 30% of the total NEFA
in human plasma, has been shown to induce insulin resistance
in cultured skeletal muscle cells and adipocytes(11,12). By con-
trast, oleate, representing about 90% of the monounsaturated
NEFA and 30% of the total NEFA in human plasma, has
not been reported to cause significant insulin resistance.
Recent studies have shown that combination of oleate with
palmitate can reverse palmitate-induced alterations in insulin
signal transduction(13–15). These studies also suggested that
the oleate:palmitate ratio may influence the protective effect
of oleate. However, these studies have not examined the
effect of oleate on palmitate-induced insulin resistance at
the level of glucose uptake.
The present study examines whether oleate protects against
palmitate-induced insulin resistance at the level of glucose
uptake in cultured rat L6 muscle cells. The study also investi-
gates whether the PI3-kinase pathway is involved, and
whether the morphology and viability of the cells are affected
by palmitate and/or oleate.
Materials and methods
The L6 rat skeletal muscle cell line was obtained from the
European Culture Collection (Porton Down, UK). Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum
(FBS), an antibiotic and trypsin-EDTA were purchased from
Cambrex (Verviers, Belgium). Sodium palmitate, sodium
oleate, fatty acid-free bovine serum albumin (BSA), sodium
pyruvate, PI3-kinase inhibitors (wortmannin and LY294002),
trypan blue solutionand
5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma (Poole, UK). 2-Deoxy-D-[3H]glucose (555GBq/mmol)
L6 myoblasts were grown in DMEM containing 5% heat-
inactivated FBS, 25mM-D-glucose, 1mM-sodium pyruvate,
*Corresponding author: Dr Helen R. Griffiths, fax þ44 121 359 0572, email email@example.com
Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide; PI3-kinase, phosphatidylinositol 3-kinase.
British Journal of Nutrition (2009), 102, 1557–1563
q The Authors 2009
British Journal of Nutrition
1mM-L-glutamine, penicillin (100U/ml) and streptomycin
(100mg/ml) as described previously(16). Cells were maintained
at 378C with humidified 95% air and 5% CO2. Experiments
were undertaken in twenty-four-well plates seeded from
preconfluent flasks with 5 £ 104cells in 1ml. The cells
were grown to 70–80% confluence and the medium was
changed to DMEM containing 0·5% FBS for 24h which
induces rapid differentiation and fusion of these myoblasts
Conjugation of fatty acids to bovine serum albumin
A stock solution of palmitate (200mmol/l) or oleate
(100mmol/l) was prepared by dissolving sodium palmitate
or sodium oleate into 70% ethanol and 0·1 M-NaOH as
described previously(17). Fatty acids were then complexed
with 5% fatty acid-free BSA to a concentration of 5mmol/l
at 378C, stirring for 4h and adjusted to pH 7·4. After sterilis-
ing through a 0·2mm filter, both solutions were stored at 48C
for no longer than 2 weeks. A control solution was prepared
by mixing 70% ethanol and 0·1 M-NaOH with 5% BSA in
the absence of fatty acids.
Incubation with fatty acids and phosphatidylinositol 3-kinase
L6 myotubes were incubated with various concentrations of
palmitate (50–300mmol/l) or oleate (50–750mmol/l) alone,
and combinations of 300mM-palmitate with 50–300mM-
oleate for 6h. Palmitate (300mmol/l) induces a time
(0–6h)-dependent reduction in insulin-stimulated glucose
uptake which is maximal at 6h and does not induce significant
toxicity(18). Further experiments to investigate any involve-
ment of the PI3-kinase pathway in the effects of fatty acids
were undertaken with and without the PI3-kinase inhibitors
wortmannin (1027mol/l) for 6h or LY294002 (25mmol/l)
for 1h 10min at 378C. The presence of wortmannin at
1027mol/l elicited a reduction of insulin-stimulated glucose
uptake by L6 muscle cells over 6h, from 140·5 (SEM 4·8) to
105·5 (SEM 3·2) % of non-insulin-stimulated glucose uptake
(P,0·001; n 8). Similarly, pre-incubation of L6 muscle
cells with LY294002 for 10min (25mmol/l) reduced insulin-
stimulated glucose uptake by L6 muscle cells from 150·1
(SEM 11·5) to 93·3 (SEM 4·6) % of non-insulin-stimulated
glucose uptake (P,0·001; n 9). Control cells received BSA
and/or other vehicles as appropriate.
2-Deoxy-D-glucose uptake was undertaken immediately after
completion of the 6h incubation with fatty acids in which
insulin (1026mol/l) had been added for the last 1h. L6
myotubes were then washed with glucose-free Krebs–Ringer
bicarbonate buffer at 228C and incubated with 0·5ml of this
buffer supplemented with 0·1mM-2-deoxy-D-glucose and
2-deoxy-D-[3H]glucose (3700Bq/ml) for 10min at 228C.
After washing cells three times with ice-cold Krebs–Ringer
1 M-NaOH and radioactivity was counted in 5ml Hi-safe 3
scintillant using a Packard 1900 TR liquid scintillation counter
(Packard, Chicago, IL, USA). Uptake of 2-deoxy-D-glucose
was expressed as the percentage compared with control
(100%), which was typically 5–8pmol/105cells per min for
basal uptake of 2-deoxy-D-glucose in the present studies as
Cell viability assays
Trypan blue exclusion assay.
assay, L6 myotubes in twenty-four-well plates were gently
washed with PBS, and trypsinised with 100ml trypsin-EDTA
for 2min and neutralised with 100ml DMEM with 10%
FBS. After mixing with 0·4% trypan blue solution, the live
cells were counted on a Neubauer haemocytometer (AO
Scientific, Buffalo, NY, USA). Cell viability was expressed
as percentage of live cells.
Caspase-3 activity assay. L6 myotubes in six-well plates
were gently washed twice with ice-cold PBS. After application
of lysis buffer (10mM-2-amino-2-hydroxymethyl-propane-
0·2mM-NaF, 0·2mM-Na3VO4, leupeptin (0·3mg/ml)) on ice
for 30min, cells were centrifuged at 10000g for 5min. The
supernatant fraction was collected and caspase-3 activity
was measured using a fluorescent caspase-3 substrate II
L-valyl-L-aspartic acid amide; Ac-DEVD-AMC)(20)and protein
content was measured by bicinchoninic acid protein assay(21).
mide assay. For the MTT assay, the medium from treated
and control L6 myotubes in twenty-four-well plates was
removed and replaced with 0·5ml fresh DMEM with 0·5%
FBS. Cells were then incubated with 100ml MTT solution
(10mg/ml in PBS) for 4h at 378C. Lysis buffer (100ml;
20% SDS in 50% dimethyl formamide, pH 4·7) was added
to each well and the plates were incubated for a further 16h
at 378C in a humidified 5% CO2air incubator. The absorbance
was read at 570nm using an MRX Microplate reader (Dynex
Technologies Limited, Worthing, West Sussex, UK). Blank
wells contained DMEM without cells.
For the trypan blue exclusion
To assess cell morphology, L6 myotubes were photographed
under a phase-contrast microscope (Olympus, Southend-
on-Sea, Essex, UK) at magnification £ 20.
Data are expressed as mean values with their standard errors,
and 2-deoxy-D-glucose uptake is expressed as percentage
compared with control (100%). Statistical analyses were
performed by one-way ANOVA with Tukey–Kramer post
hoc tests. P,0·05 was considered significant.
Effect of palmitate or oleate alone on glucose uptake
Initial experiments were conducted to characterise the effect
of palmitate and oleate alone on basal and insulin-stimulated
glucose uptake by L6 myotubes. Since palmitate was
D. Gao et al. 1558
British Journal of Nutrition
conjugated to BSA, the control and the oleate incubations
were conducted with the same concentration of BSA. L6
myotubes were incubated with palmitate (50–300mmol/l) or
oleate (50–750mmol/l) alone for 6h. Both palmitate and
oleate at the concentrations tested showed no significant
effect on basal glucose uptake compared with controls
(Fig. 1(a), (b)). When BSA-treated control cells were stimu-
lated with 1026M-insulin for 1h, this produced a significant
increase in glucose uptake by about 40–50% which is a
maximal insulin-induced effect for these cells over this time
period (data not shown). However, when L6 myotubes
were incubated with palmitate for 6h, there was a concen-
uptake. Insulin-stimulated glucose uptake was almost comple-
tely prevented by exposure to 300mM-palmitate (Fig. 1(a)).
Therefore, this concentration of palmitate was chosen to
induce insulin resistance in subsequent studies.
Oleate alone caused a small concentration-dependent
decrease in insulin-stimulated glucose uptake. The highest
oleate concentration tested (750mmol/l) did not completely
prevent insulin-stimulated glucose uptake (Fig. 1(b)).
Oleate protects L6 cells from palmitate-induced insulin
The effect of oleate on palmitate-induced insulin resistance was
examined by co-incubating increasing concentrations of oleate
(50, 150 and 300mmol/l) with 300mM-palmitate for 6h with
and without 1026
M-insulin stimulation for the last 1h.
Whereas 300mM-palmitate alone abolished insulin-stimulated
glucose uptake, co-incubation with oleate (50mmol/l) partially
restored (to 75%) insulin-stimulated glucose uptake in the
presence of 300mM-palmitate, but co-incubation with 50mM-
BSA (control) did not exert any protective effect against the
inhibition of insulin-stimulated glucose uptake by palmitate.
The protective effect of oleate against palmitate-induced
insulin resistance was not further affected by increasing the
oleate concentration to 300mmol/l (Fig. 2).
Effects of fatty acids on L6 cell viability
To test whether fatty acids have adverse effects on L6
myotubes, membrane integrity was assessed by trypan blue
exclusion, and mitochondrial-reducing capacity was measured
by an MTT assay.
Cells incubated with 300mM-oleate alone did not show
any significant difference in membrane integrity compared
with BSA controls. With 300mM-palmitate, there was a
slight reduction in membrane integrity compared with
BSA controls (viability of 74 (SEM 4) v. 88 (SEM 3) %;
prevented the slight reduction in membrane integrity with
300mM-palmitate (Fig. 3).
The overall pattern in mitochondrial-reducing activity was
similar to that of membrane integrity. Palmitate (300mmol/l)
decreased mitochondrial-reducing activity by more than
50% (P,0·001), whereas the same concentration of oleate
(300mmol/l) caused a 25% decrease in mitochondrial-
reducing activity compared with BSA controls. Addition of
150mM- and 300mM-oleate with 300mM-palmitate partially
Fig. 1. Effect of palmitate (a) and oleate (b) alone on basal (B) and insulin-
stimulated ( ) 2-deoxy-D-glucose (2-DG) uptake by L6 myotubes. L6
myotubes were incubated with bovine serum albumin (BSA) as control
or BSA with palmitate (50–300mmol/l) and oleate (50–750mmol/l) alone
for 6h at 378C. Insulin (1026mol/l) was added for the last 1h of incubation.
Data are the means of three independent experiments performed in triplicate,
with standard errors represented by vertical bars. Mean value was signifi-
cantly different from that of the same treatment without insulin addition:
**P,0·01, ***P,0·001. Mean value was significantly different from that of
the insulin-stimulated BSA-only-treated control (0mM-palmitate): †P,0·05,
Fig. 2. Effect of combinations of 300mM-palmitate with various concen-
trations of oleate on basal (B) and insulin-stimulated ( ) 2-deoxy-D-glucose
(2-DG) uptake by L6 myotubes. L6 myotubes were incubated with 300mM-
palmitate and oleate (50, 150, 300mmol/l) or 50mM-bovine serum albumin as
control for 6h at 378C. Insulin (1026mol/l) was added for the last 1h of incu-
bation. Data are the means of three independent experiments performed in
triplicate, with standard errors represented by vertical bars. *** Mean value
was significantly different from that of the same treatment without insulin
Fatty acids and muscle insulin sensitivity1559
British Journal of Nutrition
prevented the decrease in mitochondrial-reducing activity
associated with palmitate (P,0·001). A lower concentration
of oleate (50mmol/l) did not show a significant protective
effect against the palmitate-induced decrease in mitochon-
drial-reducing activity (P.0·05) (Fig. 4).
Oleate prevents the palmitate-induced alteration in L6 cell
There was a distinct difference between the effects of
palmitate and oleate alone on L6 myotube morphology.
When L6 myotubes were incubated with 300mM-palmitate,
the muscle cells lost their spindle shape (Fig. 5(b)), whereas
cells incubated with the same concentration of oleate
maintained a similar shape to BSA controls (Fig. 5(c)).
When 50mM-oleate was co-incubated with 300mM-palmitate,
the spindle shape was retained (Fig. 5(d)). This protective
effect of oleate was not further enhanced when the oleate con-
centration was raised to 150 and 300mmol/l (Fig. 5(e), (f)).
To exclude the possibility that BSA (a fatty acid carrier
protein) could contribute to the protection by oleate, it was
noted that the loss of spindle shape caused by palmitate was
not prevented by co-incubating with increasing concentrations
of BSA (Fig. 5(g), (h), (i)).
Phosphatidylinositol 3-kinase activity in palmitate- and
oleate-treated L6 cells
To investigate the underlying mechanism of the protective
effect of oleate against palmitate-induced insulin resistance
in L6 myotubes, the involvement of PI3-kinase activation
was examined by incubating L6 myotubes with two different
types of PI3-kinase inhibitors: wortmannin which binds to
the catalytic subunit (P110) and LY294002 which binds to
the regulatory subunit (P85) of PI3-kinase(22). Both wortman-
nin (1027mol/l) and LY294002 (25mmol/l) slightly reduced
basal glucose uptake by 10% (P,0·05) and prevented insu-
lin-stimulated glucose uptake by L6 myotubes (Fig. 6(a),
(b)). The reduction in insulin-stimulated glucose uptake by
300mM-palmitate was partially prevented by co-incubating
with 50mM-oleate as tested earlier (Fig. 2). With the addition
of wortmannin (1027mol/l) or LY294002 (25mmol/l), the
improvement in insulin-stimulated glucose uptake by 50mM-
oleate in 300mM-palmitate-treated cells was significantly
reduced (P,0·001) (Fig. 6(a), (b)).
In the present study, the effect of oleate on palmitate-induced
insulin resistance atthe
was investigated in L6myotubes.
with 300mM-palmitate for 6h almost completely abolished
insulin-stimulated glucose uptake. Oleate (up to 750mmol/l)
produced only a slight reduction in insulin-stimulated
Fig. 3. Effect of palmitate (Pa) and oleate (Oa) on viability measured as
(a) membrane integrity by trypan blue exclusion and (b) caspase-3 activity in
cell lysates. (a) L6 myotubes were incubated with 300mM-Pa and 300mM-Oa
alone and combinations of 300mM-Pa with various concentrations of Oa
(50, 150, 300mmol/l) or bovine serum albumin (BSA) for 6h at 378C. Viable
cells were counted after trypsinisation. (b) L6 myotubes were incubated with
BSA, 300mM-Pa and 300mM-Oa alone for 6h at 378C. Caspase-3 activity
in lysates was measured as the release of coumarin fluorescence from the
synthetic peptide 7-amino-4-methylcoumarin, N-acetyl-L-aspartyl-L-glutamyl-
L-valyl-L-aspartic acid amide (Ac-DEVD-AMC). Data are the means of
three independent experiments performed in triplicate, with standard errors
represented by vertical bars. *** Mean value was significantly different from
that of the Pa-only treatment (P,0·001).
Fig. 4. Effect of palmitate (Pa) and oleate (Oa) on mitochondrial-reducing
bromide (MTT) assay. L6 myotubes were incubated with 300mM-Pa
and 300mM-Oa alone and combinations of 300mM-Pa with various concen-
trations of Oa (50, 150, 300mmol/l) or bovine serum albumin (BSA) for 6h
at 378C. Data are the means of three independent experiments performed
in triplicate, with standard errors represented by vertical bars. ***Mean
value was significantly different from that of the Pa-only treatment
(P,0·001). ††† Mean value was significantly different from that of the BSA-
only treatment (P,0·001).
D. Gao et al. 1560
British Journal of Nutrition
glucose uptake. However, addition of 50mM-oleate reduced
the inhibitory effect of 300mM-palmitate on insulin-stimulated
glucose uptake. This protective effect was not further
increased with increasing concentrations of oleate up to
300mmol/l but required the activity of PI3-kinase.
Fatty acid-induced insulin resistance has been extensively
studied in vitro using skeletal muscle cells(13,20). In L6
glucose uptake acutely (6h), confirming previous studies
with these cells(23).
Two distinct fatty acid metabolites formed from oversupply
of lipids to skeletal muscle have been implicated in the
development of skeletal muscle insulin resistance: ceramide
and diacylglycerol(24). Palmitate is an important precursor
of de novo synthesis of ceramide(25), and ceramide strongly
inhibits insulin action(26,27), mainly by disrupting activation
of Akt (protein kinase B) and reducing translocation of
GLUT into the cell membrane(28–30). Studies of lipid-induced
insulin resistance in muscles of animals and human subjects
support a role of diacylglycerol in the activation of the
protein kinase Cu–NF-kB pathway(31). This involves serine
phosphorylation of insulin receptor substrate-1 and an associ-
ated reduction of PI3-kinase activity(32).
Although oleate alone (up to 750mmol/l) slightly reduced
insulin-stimulated glucose uptake by L6 myotubes, only
50mM-oleate was required to prevent 300mM-palmitate-
induced insulin resistance. This observation is consistent
with a recently published study in which 100mM-oleate
C2C12 myotubes; this effect involved altered phosphorylation
of Akt(15). Using the PI3-kinase inhibitors, wortmannin and
LY294002, we have demonstrated that the effect of oleate to
partially reverse palmitate-induced insulin resistance requires
PI3-kinase activity, which is an insulin-signalling intermediate
between insulin receptor substrate-1 and the 3-phosphoinosi-
tide-dependent kinase that regulates Akt. It is anticipated
that reactivation of this pathway will restore GLUT-4 translo-
cation and glucose transport(33). It has been reported
previously that long-term exposure to oleate can stimulate
cells(34). Since the present study involves only 6h exposure
to oleate, the effect of oleate on PI3-kinase is unlikely to be
a direct effect on gene expression. Also, oleate alone did not
increase insulin-stimulated glucose uptake.
It has been proposed that palmitate-induced insulin resist-
ance could be mediated via protein kinase Cu-induced
Fig. 5. Effect of palmitate (Pa), oleate (Oa), combinations of palmitate with various concentrations of Oa or bovine serum albumin (BSA) on L6 myotube
morphology. L6 myotubes were incubated with 300mM-Pa and 300mM-Oa alone and combinations of 300mM-Pa with various concentrations of Oa (50, 150,
300mmol/l) or BSA (8, 25, 50mmol/l) for 6h at 378C. The cell cultures were photographed using an inverted phase-contrast light microscope at magnification
£ 20. (a) BSA control; (b) 300mM-Pa; (c) 300mM-Oa; (d) Pa þ50mM-Oa; (e) Pa þ150mM-Oa; (f) Pa þ300mM-Oa; (g) Pa þ8mM-BSA; (h) Pa þ25mM-BSA;
(i) Pa þ50mM-BSA.
Fatty acids and muscle insulin sensitivity1561
British Journal of Nutrition
serine-phosphorylation to suppress insulin receptor substrate-1,
which then reduces PI3-kinase activity(32,35). Activation of
protein kinase Cu is promoted by diacylglycerol, which is
generated from palmitate or by inhibitor kB kinase b, a
serine kinase that normally prevents activation of NF-kB(23).
Oleate has been reported to inhibit the activation of NF-kB
in endothelial cells(36). Therefore, the protective effect of
oleate against palmitate could be due to suppression of
the protein kinase Cu and NF-kB pathways. Thus, oleate
protection against palmitate-induced insulin resistance may
result from the channelling of diacylglycerol into neutral
TAG which does not induce insulin resistance(13–15,37–39).
It remains to be investigated whether ceramide generation is
involved in the protection by oleate against palmitate-induced
Another detrimental effect of palmitate observed in the
present study was the loss of spindle shape of L6 myotubes.
Normal cell morphology was restored by co-incubation
with 50mM-oleate, and this was associated with a small
increase in membrane integrity. However, the reduction in
membrane integrity was much smaller than the reduction in
suggesting that the small loss in cell membrane integrity is
not the primary contributor to the palmitate-induced insulin
resistance in L6 myotubes. Mitochondrial dysfunction has
been implicated in the development of insulin resistance(40),
and palmitate diminished the mitochondrial-reducing capacity
of L6 cells. Nevertheless, the inability of 50mM-oleate to
restore mitochondrial function while reversing 300mM-palmi-
tate-induced insulin resistance suggests that improved mito-
chondrial function is not crucial for the protective effect of
oleate against palmitate-induced insulin resistance in muscle.
Addition of 150mM- and 300mM-oleate partially prevented
the palmitate-induced decrease in mitochondrial activity and
this protective effect is similar to the addition of increasing
concentrations of BSA. This suggests that the protection by
the higher concentrations of oleate may partly reflect the
increased amount of BSA. There was no effect of palmitate
or oleate on apoptosis under the same conditions as assessed
by caspase-3 assay on the adherent cells. However, apoptotic
cells can lose adherence, so additional measures of cell death
are required to exclude an effect on apoptosis.
In summary, the present study has demonstrated a protec-
tive effect of the MUFA oleate against insulin resistance
induced by the SFA palmitate in L6 muscle cells. This protec-
tive effect is associated with the ability of oleate to preserve
insulin-stimulated PI3-kinase activity in palmitate-treated
cells. The protective effect of oleate has potentially important
implications for the balance of dietary monounsaturated and
saturated fats in the development of insulin resistance and
these data suggest possible benefits of increasing plasma
oleate in vivo; an early meta-analysis of dietary fat intake
supported the hypothesis that monounsaturated fat may
improve diabetic control and this has been substantiated by
more recent studies(41–43). Whether dietary oleate offers a
strategy to improve glucose homeostasis in type 2 diabetes
mellitus requires further investigation through well-designed,
appropriately powered dietary intervention studies.
The authors gratefully acknowledge financial support for the
present study from Aston University and for D. G. from an
Overseas Research Students Awards Scheme (ORSAS) scho-
D. G. undertook all experiments and drafted the manuscript.
H. R. G. was principal investigator and supervisor to D. G. and
C. J. B. was associate supervisor.
The authors declare that there is no conflict of interest.
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