Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein.

Abstract

Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide.
Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance.
Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days).
Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.

Full text

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ARTICLE
Cellular mechanisms by which proinsulin C-peptide
prevents insulin-induced neointima formation
in human saphenous vein
R. S. Mughal & J. L. Scragg & P. Lister & P. Warburton &
K. Riches & D. J. O’Regan & S. G. Ball & N. A. Turner &
K. E. Porter
Received: 5 February 2010 /Accepted: 25 February 2010 /Published online: 12 May 2010
# The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract
Aims/hypothesis Endothelial cells (ECs) and smooth mus-
cle cells (SMCs) play key roles in the development of
intimal hyperplasia in saphenous vein (SV) bypass grafts.
In diabetic patients, insulin administration controls hyper-
glycaemia but cardiovascular complications remain. Insulin
is synthesised as a pro-peptide, from which C-peptide is
cleaved and released into the circulation with insulin;
exogenous insulin lacks C-peptide. Here we investigate
modulation of human SV neointima formation and SV-EC
and SV-SMC function by insulin and C-peptide.
Methods Effects of insulin and C-peptide on neointima
formation (organ cultures), EC and SMC proliferation (cell
counting), EC migration (scratch wound), SMC migration
(Boyden chamber) and signalling (immunoblotting) were
examined. A real-time RT–PCR array identified insulin-
responsive genes, and results were confirmed by real-time
RT–PCR. Targeted gene silencing (siRNA) was used to
assess functional relevance.
Results Insulin (100 nmol/l) augmented SV neointimal
thickening (70% increase, 14 days), SMC proliferation
(55% increase, 7 days) and migration (150% increase, 6 h);
effects were abrogated by 10 nmol/l C-peptide. C-peptide
did not affect insulin-induced Akt or extracellular signal-
regulated kinase signalling (15 min), but array data and
gene silencing implicated sterol regulatory element binding
transcriptionfactor1(SREBF1).Insulin(1–100nmol/l)didnot
modify EC proliferation or migration, whereas 10 nmol/l
C-peptide stimulated EC proliferation by 40% (5 days).
Conclusions/interpretation Our data support a causative
role for insulin in human SV neointima formation with a
novel counter-regulatory effect of proinsulin C-peptide.
Thus, C-peptide can limit the detrimental effects of insulin
on SMC function. Co-supplementing insulin therapy with
C-peptide could improve therapy in insulin-treated patients.
Keywords C-peptide.Endothelial cell.Human.
Insulin.Migration.Neointima.Proliferation.
Saphenous vein.Signalling.Smooth muscle cell.
Sterol regulatory element binding transcription factor 1
Abbreviations
EC
ERK
HPRT1
IGF1R
IMA
PI3K
SMC
SORBS1
Endothelial cell
Extracellular signal-regulated kinase
Hypoxanthine phosphoribosyltransferase 1
IGF 1 receptor
Internal mammary artery
Phosphoinositide 3-kinase
Smooth muscle cell
Sorbin and SH3 domain containing protein 1
Electronic supplementary material The online version of this article
(doi:10.1007/s00125-010-1736-6) contains supplementary material,
which is available to authorised users.
R. S. Mughal:J. L. Scragg:P. Lister:P. Warburton:K. Riches:
S. G. Ball:N. A. Turner:K. E. Porter (*)
Division of Cardiovascular and Neuronal Remodelling,
University of Leeds,
Worsley Building,
Leeds LS2 9JT, UK
e-mail: medkep@leeds.ac.uk
D. J. O’Regan
The Yorkshire Heart Centre, Leeds General Infirmary,
Leeds, UK
R. S. Mughal:J. L. Scragg:P. Warburton:K. Riches:
D. J. O’Regan:S. G. Ball:N. A. Turner:K. E. Porter
Multidisciplinary Cardiovascular Research Centre,
University of Leeds,
Leeds, UK
Diabetologia (2010) 53:1761–1771
DOI 10.1007/s00125-010-1736-6

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SREBF1Sterol regulatory element binding transcription
factor 1
Saphenous veinSV
Introduction
Adults with diabetes have an increased prevalence of
coronary heart disease compared with non-diabetic indi-
viduals [1–4]. Intensive control of hyperglycaemia retards
diabetic microvascular complications (e.g. retinopathy,
nephropathy and neuropathy) [5], but beneficial macro-
vascular effects (coronary atherosclerosis) are less apparent
[6]. Long-term outcomes of revascularisation are disap-
pointing, irrespective of the mode: angioplasty, stenting or
coronary artery bypass grafting [1, 7, 8].
The autologous saphenous vein (SV) is used routinely as
a conduit for coronary artery bypass grafts. Occlusions
caused by intimal hyperplasia are common [9–11], with
only ∼50% of grafts patent after 10 years and fewer in
diabetic patients [12]. Implantation of the SV graft
immediately exposes it to arterial pressure, with profound
effects on endothelial cell (EC) and smooth muscle cell
(SMC) biology. Loss of homeostatic regulation due to EC
damage promotes SMC proliferation and migration in the
graft wall, culminating in intimal hyperplasia and graft
occlusion [13].
Insulin effectively controls blood glucose but can itself
stimulate SMC proliferation and migration [14], increasing
neointima formation in balloon-injured arteries of normal
and diabetic rats [15–17]. Insulin also reportedly enhanced
neointima formation in organ-cultured SV in tissue from
five patients [18]. Insulin acting on SV-SMCs may
therefore contribute to the pathogenesis of intimal hyper-
plasia in insulin-dependent diabetic patients.
Human proinsulin C-peptide is a 31-amino-acid peptide
that links the A and B chains of proinsulin, ensuring its
correct folding [19–21]. C-peptide is cleaved from pro-
insulin and released into the circulation with insulin in
equimolar concentrations. Early studies reported no bio-
logical function of C-peptide [22], an observation supported
by lack of interspecies conservation of the C-peptide
sequence, both in chain length and amino acid composition.
However, it is now apparent that human proinsulin C-
peptide is biologically active and modulates cellular
function [20]. Type 1 diabetic patients receive insulin
therapy from an early stage, and the majority of type 2
patients over time also require insulin. Exogenously
administered insulin does not contain C-peptide. Low
sustained concentrations of C-peptide are beneficial [23–25],
and chronic administration of replacement C-peptide with
insulin ameliorates microvascular complications in animal
models and type 1 diabetic patients [19, 20, 26]. A
combination of insulin and C-peptide may therefore provide
a more effective treatment for diabetic patients than insulin
alone.
Here we explored the interplay between human insulin
and proinsulin C-peptide on intimal hyperplasia in organ-
cultured SVs from patients without diabetes and on the two
key cell types involved in neointima formation, ECs and
SMCs, to resolve their independent and joint cell-type-
specific effects.
Methods
Materials Cell culture reagents were purchased from
Invitrogen (Paisley, UK), except FBS from LabTech
International (Ringmer, UK). Proinsulin C-peptide, insulin
and PDGF-BB were from Sigma (Poole, UK). PD98059
and LY294002 were from Calbiochem (Nottingham, UK).
Migration assay chambers were from BD Biosciences
(Oxford, UK).
Tissue and cell culture Samples of SV and internal
mammary artery (IMA) were obtained from non-diabetic
patients undergoing elective coronary artery bypass grafting
at the Leeds General Infirmary, with local ethical committee
approval and informed patient consent. The study was
carried out in accordance with the principles of the
Declaration of Helsinki as revised in 2000. Organ cultures
were performed using undistended SVas we have described
previously [27] in the absence or presence of insulin, alone
or with C-peptide. Segments were fixed and processed to
10 μm. Neointimal thickness was determined using
computational analysis on high field magnification at
60 μm intervals along each section by two independent
observers.
SMCs were cultured from human SV, IMA and aortic
root by an explant technique as we have described
previously [28]. SMCs were maintained at 37°C in DMEM
containing 25 mmol/l glucose and 10% FBS in a humidified
atmosphere of 5% CO2in air. SV-ECs were harvested by
collagenase digestion and cultured as described previously
[29]. Experiments were performed on cells of passage
number 2–6 from different patients.
Proliferation assays SV-EC proliferation assays were per-
formed by seeding cells from different patients in 24-well
culture plates at a density of 10,000 per well in full
endothelial growth medium (20% FBS). After 30–32 h
incubation cells were quiesced in medium containing 1%
FBS for 16 h. Cells were then exposed to control growth
medium (1% or 10% FBS as appropriate) and selected
concentrations of insulin, C-peptide or in combination.
1762 Diabetologia (2010) 53:1761–1771

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Medium and drugs were replaced on days 2 and 4 and
viable cell number determined in triplicate wells on day 5
using Trypan Blue and a haemocytometer.
SV-SMC and IMA-SMC proliferation assays were
performed essentially as described previously [28]. After
plating (10,000 cells per well), cells were incubated overnight
in full growth medium, then quiesced in serum-free medium
for 3 days before transfer to control growth medium (DMEM
with 0.2% FBS and 10 ng/ml platelet-derived growth factor)
alone or with appropriate supplements (insulin and/or
C-peptide). Medium and drugs were replaced on days 2 and
4 and viable cell number determined in triplicate wells on the
appropriate day (usually day 7).
Migration assays SV-EC migration was investigated in six-
well plates using a modification of a ‘scratch wound’
method [30]. Briefly, duplicate scratches were made with a
sterile 1 ml pipette tip in confluent endothelial monolayers,
reference points etched in the dishes and images were
captured (0 h). Cells were then exposed to the relevant
media with appropriate stimuli in a tissue-culture incubator
for an additional 24 h. Further images were then captured
by aligning the dishes with the reference point made at time
0 h, and a second image acquired. Distances of each scratch
closure were determined by measuring the cell-denuded
area at equal intervals along the wound at each time point
for each treatment condition.
SV-SMC migration was studied using a modified Boyden
chamber technique, as we have described previously [28].
Cells (100,000) from different patients were loaded in the
upper chamber in medium supplemented with 0.4% FBS.
The lower chamber contained 0.4% FBS with appropriate
supplements (insulin and/or C-peptide). After incubation for
6 h at37°Cina tissue-culture incubator, duplicate membranes
were processed and evaluated by counting migrated cells on
the underside ofthe membranein10randomfieldsunder high
power (×400) light microscopy [28].
Immunoblotting Serum-starved cells were exposed to serum-
free medium containing insulin and/or C-peptide for 15 min
beforepreparingwholecellhomogenatesandimmunoblotting
as described previously [31]. Phosphorylation of Akt
(Ser473) and extracellular signal-regulated kinase (ERK)
(Thr202/Tyr204) was determined using phospho-specific
and expression antibodies (Cell Signaling Technology,
Hitchin, UK). Immunolabelled bands were visualised by
chemiluminescence and densitometric analysis performed
as previously described [32].
PCR arrays Serum-deprived SV-SMCs were exposed to
0.4% FBS alone or supplemented with 100 nmol/l insulin
for 6 h before extracting RNA and performing first-strand
cDNA synthesis [33]. The cDNA was confirmed to be of
suitable quality for array analysis using the SuperArray QC
PCR array (SABiosciences) and the ABI 7500 Real-Time
PCR System. Samples of cDNA were then analysed using
the Insulin Signalling Pathway Array (SABiosciences).
Cycle threshold (Ct) values were obtained using Sequence
Detection Software SDS v1.2.3 (Applied Biosystems) and
data analysed by comparing Ct values of 84 insulin
pathway genes with those of five different housekeeping
genes using the SuperArray PCR array analysis software
(SABiosciences).
Real-time PCR Real-time RT–PCR performed as described
previously [33] using the ABI 7500 Real-Time PCR
System with the following human Taqman primer/probe
sets: sterol regulatory element binding transcription factor 1
(SREBF1) (product number Hs01088691_m1), IGF 1
receptor(IGF1R)(Hs00541255_s1),sorbinandSH3domain
containing protein 1 (SORBS1) (Hs00908953_m1) and
GLUT4 (Hs00168966_m1). Data were expressed as a
percentage of hypoxanthine phosphoribosyltransferase 1
(HPRT1) mRNA levels (Hs99999909_m1 primers) using
the formula 2?$Ct? 100, or normalised to control sample
using the formula 2?$$Ct.
SREBF1 gene silencing SV-SMCs were transfected with a
combination of four different SREBF1-targeted siRNA
oligonucleotides (100 nmol/l) (SMARTPool; Dharmacon,
Cramlington, UK) using Lipofectamine 2000 transfection
reagent (Invitrogen), as described previously [34]. Control
cells were transfected in the absence of siRNA (mock
transfected). Effects on protein levels were determined by
immunoblotting with mouse monoclonal anti-SREBF1
antibody (ab3259, Abcam), using the method described
above. The effects of SREBF1 gene silencing on SV-SMC
migration were determined 36 h after transfection.
Statistical analysis All data are expressed as means ± SEM,
with n representing the number of experiments on cells/
tissue from different patients. Data were analysed as ratios
using repeated measures one-way ANOVA with the
Newman–Keuls post hoc test, using GraphPad Prism
software (www.graphpad.com). p<0.05 was considered
statistically significant.
Results
Neointimal thickening in organ-cultured human SV The
ability of insulin to modulate neointima formation was
investigated using organ-cultured human SV from ten
different patients (Fig. 1). Mean neointima thickness was
10.4±1.4 µm (median 11.7 µm, range 5.2–16.6 µm) after
14 days’ incubation and increased by 70% (p<0.01)
Diabetologia (2010) 53:1761–1771 1763

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following supplementation with 100 nmol/l insulin (mean
17.7±2.6 µm, median 16.7 µm, range 6.5–31.4 µm).
Treatment of SV cultures with C-peptide (10 nmol/l)
together with insulin (100 nmol/l) prevented the increase
in neointima (mean 8.8±1.36 µm, median 8.9 µm, range
4.8–15.5 µm).
Proliferation and migration of ECs promote re-
endothelialisation of the damaged vessel wall, thereby
limiting neointimal development. By contrast, SMC prolifer-
ation and migration contribute to neointimal development.
We therefore investigated the effects of insulin and C-peptide
on SV-EC and SV-SMC proliferation and migration.
Endothelial cell proliferation Figure 2a shows SV-EC
counts from ten different patients cultured in 10% FBS
with no change in cell number in response to 1–100 nmol/l
insulin after 5 days. Similarly, no effect of insulin was seen
in cells exposed to 1% FBS (data not shown). When cells
were exposed to C-peptide (10 nmol/l) alone, a significant
increase in cell number of ∼40% was observed, which was
not affected by insulin (Fig. 2b). Further studies revealed
that C-peptide increased SV-EC proliferation at concen-
trations as low as 0.1 nmol/l (Fig. 2c).
Endothelial cell migration In the scratch wound assay, SV-
ECs at the wound edge migrate to repopulate a denuded
area created by the scratch (Fig. 2d). Cells exposed to 1%
FBS (Fig. 2e) had half the migratory capacity of cells in
10% FBS (Fig. 2f). No modulatory effects were observed
with either insulin, C-peptide or both together in either 1%
or 10% FBS-supplemented media (Fig. 2e, f).
Smooth muscle cell proliferation SV-SMCs from four
different patients cultured in control growth medium
exhibited a 2.5-fold increase in cell number after 7 days
compared with day 0 (Fig. 3a). C-peptide did not affect cell
number observed in the absence of insulin, but supplemen-
tation with 10–100 nmol/l insulin further increased SV-
SMC proliferation in a concentration-dependent manner
(ANOVA p<0.0001), with 100 nmol/l insulin stimulating a
55% increase in cell number (Fig. 3a). These mitogenic
effects of insulin were prevented by co-incubation with
10 nmol/l C-peptide (Fig. 3a). The mean number of
detached cells observed throughout the proliferation assays
was <1% of the total and was unaffected by any treatment
(data not shown).
IMA-SMCs cultured from the same four patients as the
SV-SMCs, displayed a 2.1-fold increase in cell number
after 7 days treatment in control growth medium (Fig. 3b),
which was lower than that observed for SV-SMCs. In stark
contrast to SV-SMCs, insulin (10–100 nmol/l) had no effect
on IMA-SMC proliferation, and C-peptide (10 nmol/l) did
not modulate proliferation in the presence or absence of
insulin (Fig. 3b).
A time course of the effects of insulin and C-peptide on
SV-SMC proliferation revealed that the mitogenic effects of
insulin, and the attenuating effects of C-peptide, were
evident over a 3–7 day period (Fig. 3c). Comparison of
areas under the proliferation curves revealed that cells
exposed to insulin proliferated at a higher rate than control
cells (p<0.01) or cells cultured in the presence of both
insulin plus C-peptide (p<0.05). There was no difference in
proliferation rates between control cells and those cultured
with insulin plus C-peptide. Further studies revealed that C-
peptide attenuated insulin-induced SV-SMC proliferation at
concentrations as low as 0.1 nmol/l (Fig. 3d). In contrast to
C-peptide, a scrambled version of C-peptide (31-mer with
the same amino acid content as C-peptide but in a random
order) had no effect on proliferation of SV-SMCs in the
absence or presence of insulin (Fig. 3e).
In view of C-peptide’s lack of effect on arterial vs venous
SMCs, and a previous report that it increased aortic SMC
proliferation in the absence of insulin [35], we investigated
the effects of insulin and C-peptide on SMCs cultured from
ControI Insulin
ControlInsulin
Insulin +
C-peptide
0
5
10
15
20
25
30
35
NS
*** ***
Neointima thickness (µm)
NI
I
M
Insulin+ C-peptide
a
b
Fig. 1 Effect of insulin and C-peptide on neointimal thickness in
organ-cultured human SV. SV segments were cultured for 14 days in
30% FBS alone (control) or supplemented with 100 nmol/l insulin
and/or human C-peptide (10 nmol/l). a Representative sections of
14-day cultured segments, stained with haematoxylin and eosin. Scale
bars, 30 μm. I, intima; M, media; NI, neointima. b Image analysis of
full-length SV sections to calculate mean neointima thickness. Scatter
plot shows individual values from ten different patients, with data
from the same patients linked. ***p<0.001 (n=10)
1764 Diabetologia (2010) 53:1761–1771

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human aortic root (Fig. 3f). C-peptide increased prolifera-
tion of human aortic SMCs (35% increase) in agreement
with Walcher et al. [35]. Insulin alone also stimulated a 28%
increase in aortic SMC number. Insulin and C-peptide
together showed no additive proliferative response com-
pared with either agent alone (Fig. 3f), and scrambled C-
peptide had no effect (data not shown).
Smooth muscle cell migration SMC migration was assessed
using Boyden chambers. A significant increase (2.5-fold)
was observed in migration towards a 100 nmol/l insulin
stimulus (Fig. 4a). Consistent with C-peptide’s effects on
neointima formation and SV-SMC proliferation (Figs 1, 3),
inclusion of 10 nmol/l C-peptide with insulin fully
prevented insulin-induced migration of SV-SMCs (Fig. 4a,
n=11 patients). Pre-treatment with inhibitors of the ERK
pathway (PD98059) or phosphoinositide 3-kinase (PI3K)/
Akt pathway (LY294002) fully prevented insulin-induced
migration (Fig. 4b), indicating essential roles for these
signalling pathways.
Smooth muscle cell signalling We next investigated whether
C-peptide could be acting by inhibiting theERK or PI3K/Akt
pathways, the two major pathways that lie downstream of the
insulin receptor. Insulin stimulated phosphorylation of Akt
(Fig. 4c), but ERK phosphorylation was not markedly
increased, probably owing to the high basal level of ERK
activation in these cells (Fig. 4d). C-peptide alone or in
combination with insulin did not modulate Akt or ERK
signalling (Fig. 4c,d). Thus, C-peptide ablates the effects of
insulin on SV-SMC function independently of ERK or Akt
signalling pathways.
Smooth muscle cell gene expression To identify insulin-
responsive genes modulated by C-peptide, we used a
SYBR-Green-based real-time RT–PCR array to monitor
expression of 84 genes involved in insulin signalling,
including insulin-receptor-associated proteins, components
of the ERK and Akt pathways, insulin-responsive genes,
transcription factors and genes involved in carbohydrate
and lipid metabolism. SV-SMCs from six patients were
0
50
100
150
200
250
Ins (nmol/l):
C-pep (nmol/l):
**
***
**
***
0 0 10 10 1010
0 0 0110 100
Cell number (×103)
00110100
0
20
40
60
80
100
120
Insulin concentration (nmol/l)
Cell number (×103)
0
200
400
600
800
Distance migrated (µm)
Ctrl InsC-Pep Ins+C-Pep
0
200
400
600
800
Distance migrated (µm)
Ctrl InsC-Pep Ins+C-Pep
Day 0
Day 5
Day 0 Day 5
0 h
24 h
00.1110
0
20
40
60
80
**
**
*
C-peptide concentration (nmol/l)
Cell number (×103)
bc
a
efd
Fig. 2 Effectof insulin and C-peptideonhumanSV-ECproliferation and
migration. a SV-EC proliferation. Quiesced cells treated with 10% FBS
supplemented with 0–100 nmol/l insulin. Cells counted on day 0 and
after 5 days with insulin and/or C-peptide treatment. ANOVA: p=0.599
for effect of insulin on day 5 (n=10). Post hoc: all non significant.
b SV-EC proliferation. Medium supplemented with 10 nmol/l C-peptide
alone or in combination with 1–100 nmol/l insulin for 5 days.
***p<0.001, **p<0.01 (n=4). c SV-EC proliferation. Medium supple-
mented with 0.1–10 nmol/l C-peptide for 5 days. ANOVA: p<0.001 for
effect of C-peptide. Post hoc: **p<0.01, *p<0.05 (n=6). d SV-EC
migration. Representative images of wounded endothelial cell
monolayers in 10% FBS at 0 h and 24 h. Migration distances of
SV-EC in 1% FBS (e) or 10% FBS (f) with insulin (100 nmol/l),
C-peptide (10 nmol/l) or both (n=4). ANOVA with post hoc: all NS
Diabetologia (2010) 53:1761–17711765

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treated with or without 100 nmol/l insulin for 6 h before
extracting RNA. Concomitant migration assays were
performed to confirm the functional effects of insulin and
C-peptide on SV-SMC migration (Fig. 5a).
Data are presented in Fig. 5b as a volcano plot with full
data included in the Electronic supplementary material
(ESM) Table 1. We selected four genes for further study
based on changes in transcript levels and reproducibility
(statistical significance): namely IGF1R, SORBS1, GLUT4
and SREBF1.
Taqman primer/probe sets were utilised to confirm array
data using the same RNA samples. The insulin-induced
increase in SREBF1 mRNA expression and decrease in
IGF1R mRNA expression were confirmed by RT–PCR
(Fig. 5c,d). However, the array data could not be confirmed
for SORBS1 or GLUT4 (data not shown).
Insulin-induced SREBF1 mRNA expression was fully
prevented by co-incubation with C-peptide (Fig. 5c). By
contrast, the effect of insulin on IGF1R expression was not
modulated by C-peptide (Fig. 5d). The acute temporal
profiles of SREBF1 and IGF1R mRNA expression in
response to insulin, with or without C-peptide, were then
determined over the 6 h period (Fig. 5e,f). Insulin-induced
SREBF1 mRNA expression was evident after 3 h and rose
thereafter, reaching a 40% increase after 6 h (ANOVA, p<
0.01) (Fig. 5e). No increase in SREBF1 mRNA levels was
apparent when C-peptide was present with insulin (ANOVA,
p=0.713) (Fig. 5e). In contrast to SREBF1, the insulin-
induced decrease in IGF1R mRNA expression after 2–6 h
(ANOVA, p<0.05) was not modulated by C-peptide (Fig. 5f).
Effect of SREBF1 gene silencing on SV-SMC migration
The above data implicated SREBF1 as a molecular target
for the antagonistic effects of C-peptide on insulin-induced
SV-SMC proliferation and migration. To confirm that
SREBF1 expression was involved in insulin-induced SV-
SMC migration, we employed a gene-silencing approach.
00 1025
Day 7
50100
0
20
40
60
80
100
***
***
††
Insulin (nmol/l):
Day 0 Day 0Day 7
††
†††
Cell number (×103)
00 1025 50100
0
20
40
60
80
100
Cell number (×103)
Cell number (×103)
Insulin (nmol/l):
Cell number (×103)
00.11 10
0
20
40
60
80
*
NS
NS
NS
C-peptide (nmol/l)
Cell number (×103)
Control
+C-pep +Scrambled
0
10
20
30
40
50
*
*
NS
Cell number (×103)
0
10
20
30
40
50
CtrlC-pep Ins C-pep+Ins
*
*
*
01234567
0
10
20
30
40
50
60
70
Day
bac
edf
Fig. 3 Effect of insulin and C-peptide on human SMC proliferation.
Quiesced SMCs incubated in medium containing 0.2% FBS+10
ng/ml platelet-derived growth factor supplemented with insulin and/or
C-peptide. Cells counted on day 0 (before additions) and after 7 days
unless stated otherwise. a SV-SMCs exposed to 0–100 nmol/l insulin
alone (white bars) or insulin together with 10 nmol/l C-peptide (black
bars). ANOVA: p<0.0001 for effect of insulin (***p<0.001 post hoc);
p=0.3084 for effect of insulin+C-peptide (n=4).
0.001 for effect of C-peptide in presence of insulin. b IMA-SMCs
from the same patients as (a) exposed to 0–100 nmol/l insulin alone
(white bars) or insulin together with 10 nmol/l C-peptide (black bars).
ANOVA: p=0.3743 for effect of insulin; p=0.7783 for effect of
insulin+C-peptide (n=4). Post hoc: all NS. c SV-SMCs exposed to
control basal medium (black squares) or basal medium supplemented
††p<0.01,
†††p<
with 100 nmol/l insulin (white squares) or 100 nmol/l insulin plus
10 nmol/l C-peptide (black circles) for 7 days. Cells counted on days
0–4 and 7. Area under curve analysis: control vs insulin, p<0.01;
control vs insulin+C-peptide, p>0.05; insulin vs insulin+C-peptide,
p<0.05 (n=4). d SV-SMCs exposed to 0–10 nmol/l C-peptide alone
(white bars) or together with 50 nmol/l insulin (black bars). *p<0.05;
NS, not significant for effect of insulin (n=4). e SV-SMCs exposed to
medium supplemented without (white bars) or with (black bars)
100 nmol/l insulin in absence (‘control’) or presence of 10 nmol/l
C-peptide or 10 nmol/l scrambled C-peptide. *p<0.05 (n=5).
f Human aortic SMCs exposed to control basal medium (‘Ctrl’) or
basalmediumsupplementedwith10nmol/lC-peptide,100nmol/linsulin,
or both together for 7 days. *p<0.05 (n=3)
1766Diabetologia (2010) 53:1761–1771

Page 7

SREBF1 protein production was markedly reduced 24–72 h
following siRNA transfection, although a return of produc-
tion was evident at the 96 h time point (Fig. 6a). SREBF1
gene silencing had no effect on basal SV-SMC migration
but specifically prevented insulin-induced migration, to a
level comparable with the inhibition achieved with C-
peptide (Fig. 6b). SREBF1 knockdown had no modulatory
effect on insulin-induced cell migration in the presence of
C-peptide, suggesting that C-peptide and SREBF1 siRNA
may be acting on the same target i.e. reducing SREBF1
expression. Thus, insulin-induced SV-SMC migration
requires SREBF1 expression, and the ability of C-peptide
to prevent insulin-induced migration may be explained
through a reduction in SREBF1 expression.
Discussion
The principal finding of our study was that neointima
formation in organ-cultured human SVs was increased in
the presence of insulin, an effect abrogated by proinsulin
C-peptide. Neointima formation requires SMC migration
and proliferation, both of which were induced by insulin
and fully inhibited by C-peptide. The effects of C-peptide
were not a result of inhibition of insulin-mediated Akt and
ERK signalling, but were related to reduced insulin-induced
SREBF1 expression.
In agreement with earlier studies [15, 18] we revealed a
causative role for insulin in neointima formation but
importantly demonstrated a counter-regulatory effect of
C-peptide. Our model mimics the situation of insulin-treated
diabetes, with the presence of insulin but not C-peptide, and
suggests that co-supplementing insulin with C-peptide could
provide a better treatment than insulin alone.
Damage to the SVs during harvesting and implantation
inevitably induces endothelial dysfunction, a feature
associated with neointima formation. As effective re-
endothelialisation is known to inhibit neointimal hyperplasia
[36], we investigated whether insulin and/or C-peptide
could modulate EC migration and/or proliferation. Insulin
did not alter EC proliferation, whereas C-peptide induced
an increase, an effect maintained in the presence of insulin.
Migration was not affected by either insulin or C-peptide.
Thus, C-peptide acts directly on SMCs to prevent neointima
formation while increasing EC proliferation to promote
re-endothelialisation [36].
In agreement with a previous study [37], insulin increased
migration of SV-SMCs via ERK and PI3K/Akt pathways.
However, C-peptide’s counter-regulation was not due to
inhibition of these pathways. The mode of action of C-
peptide is unknown, although it binds to human cell
membranes [38] and its target may be a G-protein-coupled
receptor in some tissues [39]. C-peptide has been shown to
internalise in Swiss 3T3 and HEK 293 cells [40], and a very
recent study in human aortic ECs and umbilical artery
SMCs indicated that C-peptide was internalised by early
endosomes [41]. Whether the endosomal pathway is
functional in human SV-SMCs and required for C-peptide
trafficking will require elucidation in future studies. Impor-
tantly, our data do not support speculation that C-peptide
binds to the insulin receptor [42]. Thus, in human SV-
SMCs, C-peptide acts independently of the insulin receptor
and its immediate downstream signalling pathways.
InsulinincreasedSV-SMCproliferationinaconcentration-
dependent manner. C-peptide even at very low concentration
abolished these effects, consistent with its activity at nano-
molar concentrations found in plasma [43] and full saturation
of C-peptide binding sites at low nanomolar concentrations
[38].
In the vasculature, the reported effects of C-peptide are
at variance and potentially explained by different experi-
mental approaches. Two previous studies have claimed that
C-peptide reduces SMC proliferation. One report used rat
ControlIns Ins+PD Ins+LY
0
2
4
6
8
10
12
14
*********
Migration (cells/HP field)
ControlIns Ins+C-pep
SFMIns Ins+C-pep
0
10
20
30
40
*** ***
Migration (cells/HP field)
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
***
NS
***
NS
p-Akt/Akt ratio
p-ERK
ERK
p-Akt
Akt
Insulin
+C-Pep
SFM C-Pep
NS
NS
NS
NS
p-ERK/ERK ratio
Insulin
Insulin
+C-Pep
SFMC-Pep Insulin
C-pep SFM Ins Ins+C-pepC-pep
ba
dc
Fig. 4 Effect of insulin and C-peptide on SV-SMC migration and
signalling. a Migration of SV-SMCs from 11 patients towards basal
medium (control), or medium supplemented with 100 nmol/l insulin
alone, or 100 nmol/l insulin plus 10 nmol/l C-peptide. ***p<0.001
(n=11). b SV-SMC migration towards 100 nmol/l insulin stimulus
following pretreatment with 30 μmol/l PD98059 (ERK pathway
inhibitor) or 10 μmol/l LY294002 (PI3K/Akt pathway inhibitor). The
bar chart depicts the mean number of migrated cells per high power
(HP) field. ***p<0.001 (n=4). c, d SV-SMCs exposed to serum-free
medium (SFM) alone or supplemented with 50 nmol/l insulin and/or
10 nmol/l C-peptide for 15 min before preparing homogenates and
immunoblotting for phosphorylated and total Akt (c) or ERK (d). The
bar charts depict densitometric ratios of phospho- to total Akt/ERK
from five separate experiments. ***p<0.001 (n=5)
Diabetologia (2010) 53:1761–17711767

Page 8

SMCs and human C-peptide and saw a modest effect on
high glucose-induced (25 mmol/l) proliferation, but only
after 3 weeks [44]. A more recent study using human
arterial SMCs also observed increased proliferation with
high glucose culture that was attenuated by C-peptide [45].
By contrast, our data differ from those reporting a
mitogenic effect of C-peptide on human and rat aortic SMCs
[35], in which C-peptide was coupled to activation of PI3K/
Akt and ERK pathways, which were not modulated by C-
peptide in our study using SV-SMCs. This raises the
intriguing possibility that C-peptide has opposing effects
on SMCs from different vascular beds. To test this, we
cultured human aortic SMCs and found that C-peptide was
pro-proliferative, in agreement with the findings of Walcher
et al. [35]. However, when aortic SMCs were cultured in
the presence of insulin, itself a mitogen, no additive effect
of C-peptide was observed. Unlike our present study, in
none of the above studies was the effect of insulin
investigated. It is also important to note that in individuals
without diabetes C-peptide is only ever present with
insulin. The findings of all these reports can therefore be
reconciled by taking into account species and source of
SMCs, together with insulin, C-peptide and glucose
concentrations.
ANGANG
IRS2IRS2
INSRINSR
FOSFOS
FRS2GAB1 FRS2GAB1
FASN
FASN
DOK3
DOK3
IGF1R
DOK1
SORBS1
SREBF1
GLUT4
CtrlInsIns+C-Pep
0.0
2.5
5.0
7.5
10.0
12.5
******
Migrated cells (per HP field)
012
Time (h)
346
0.6
0.8
1.0
1.2
1.4
1.6
*
+
SREBF1 mRNA expression
012
Time (h)
346
0.4
0.6
0.8
1.0
1.2
*
*
IGF1R mRNA expression
ControlIns Ins+C-pep
30
40
50
60
70
** **
NS
SREBF1 mRNA (% HPRT1)
ControlInsIns+C-pep
10
20
30
40
***
***
NS
IGF1R mRNA (% HPRT1)
ba
dc
fe
–0.78
0.43
0.68
1.18
1.43
1.68
0.93
0.18
–0.53
–Log2 (Group 1/Control Group)
–Log10 (p value)
–0.03–0.280.220.47 0.720.97
Fig. 5 Effect of insulin and
C-peptide on SV-SMC gene
expression. a SV-SMC migration
towards 100 nmol/l insulin in the
absence or presence of C-peptide
(10 nmol/l). ***p<0.001 (n=6).
b SV-SMCs from the same six
patients with 100 nmol/l insulin
for 6 h before extracting RNA
and performing PCR array. Data
expressed as volcano plot in
which the x-axis depicts changes
in mRNA levels (increases to the
right of the origin, decreases to
the left), and y-axis depicts
statistical significance. Full data
are available as ESM (Table 1).
c, d Real-time RT–PCR data of
SREBF1 or IGF1R mRNA levels
following 6 h treatment of
SV-SMCs with 100 nmol/l
insulin alone or together with
10nmol/lC-peptideaspercentage
of HPRT1 housekeeping gene
mRNA levels. **p<0.01,
***p<0.001 (n=6). e, f Real-time
RT–PCR of SREBF1 or IGF1R
mRNA levels following 1–6 h
treatment of SV-SMC with
100 nmol/l insulin alone (white
bars) or together with 10 nmol/l
C-peptide (black bars) (n=3).
Data are expressed relative to
HPRT1 levels and normalised to
0 h sample. SREBF ANOVA:
p=0.004 for effect of insulin
alone (*p<0.05 post hoc);
p=0.713 for effect of insulin+
C-peptide.+p<0.05 for effect of
C-peptide in presence of insulin.
IGFR1 ANOVA: p=0.018 for
effect of insulin alone (*p<0.05
post hoc); p=0.026 for effect of
insulin+C-peptide (*p<0.05 post
hoc). There was no significant
effectofC-peptideinthepresence
of insulin
1768Diabetologia (2010) 53:1761–1771

Page 9

In contrast to the SVs, IMA grafts provide better long-
term patency even in diabetic patients [46], indicating
resistance to restenosis. This remarkable difference in
patency between conduits is clearly multifactorial, but we
have previously shown that SV-SMCs are inherently
more proliferative than IMA-SMCs from the same
patients [32]. In direct contrast to SV cells, insulin did
not promote IMA-SMC proliferation, which may help
explain the similar patency rates of IMA grafts in insulin-
treated patients compared with those in non-diabetic
patients [46]. The inability of insulin to induce IMA-SMC
proliferation was not due to lack of insulin signalling, as both
Akt and ERK phosphorylation were evident in insulin-
treated IMA-SMCs (data not shown). These data highlight
the importance of using relevant cell types and add to the
concept of cell-type-specific effects of both insulin and
C-peptide.
Having established the effects of insulin and C-peptide
on SV-SMC migration and proliferation, we used an insulin
signalling array to identify potential molecular mechanisms.
SREBF1 mRNA expression was increased in response to
insulin; an effect attenuated by C-peptide. SREBF1 gene
silencing selectively attenuated the insulin-induced compo-
nent of SV-SMC migration, strongly suggesting that the
ability of C-peptide to reduce SV-SMC migration is due to
inhibition of insulin-induced SREBF1 expression. SREBF1
(SREBP1) is a member of the basic helix-loop-helix-leucine
zipper (bHLH-Zip) family of transcription factors that
regulate lipid and carbohydrate metabolism [47]. A role for
SREBF1 in proliferation of murine aortic SMCs is reported
[48]. SREBF1, by regulating lipid and cholesterol biosyn-
thesis, may play a role in plasma membrane synthesis and
could optimise membrane properties for SV-SMC migration
and proliferation in response to insulin. Importantly, these
effects of insulin can be abrogated by C-peptide.
Vascular SMC proliferation is induced by a plethora of
growth factors and cytokines, and additionally hyperinsu-
linaemia has been implicated as an important risk factor for
atherosclerotic lesion formation in diabetic patients through
increased SMC proliferation [49]. Here we have shown that
insulin and its C-peptide differ in their effects on cells from
different vascular beds. These peptides are normally secreted
in equimolar amounts but this relationship is perturbed when
exogenous, recombinant insulin is administered to replace
natural synthesis.
A potential limitation of our study was the use of
100 nmol/l insulin: a supraphysiological concentration
when compared with that observed in vivo after a meal in
normal (∼0.3–0.4 nmol/l) or insulin-resistant (∼1.4–
1.5 nmol/l) individuals. Relatively high concentrations of
insulin are often required in vitro to observe functional
effects, probably reflecting the numerous differences
between in-vitro functional assays and the situation in vivo.
Lower concentrations of insulin (e.g. 25 nmol/l) also
resulted in increased SV-SMC proliferation in our experi-
ments (Fig. 3a), but we selected the 100 nmol/l concentration
based on the reproducibility and magnitude of its effects. One
strength of our study was the use of cells from multiple
patients. Interestingly, C-peptide had maximal effects on
SV-EC and SV-SMC proliferation at concentrations as low
as 0.1 nmol/l (Figs 2c, 3d): levels of C-peptide that are
physiologically relevant.
During the last decade there has been a wealth of new
information regarding the impact of C-peptide on a variety of
cell types and specific cell functions. However, until recently
the major focus has been on the microvascular complications
of type 1 diabetes [50], with its effects in type 2 diabetes
being less well explored. It is generally perceived that
C-peptide is beneficial (for reviews see [51, 52]), although
the mechanisms underlying such effects appear to be tissue-
and cell-type specific, and insulin is not always necessary for
C-peptide to function. Indeed, our own data, for example,
indicate a differential effect of C-peptide on SMCs from
aorta and SV. In this respect, elucidation of the specific
mechanisms underlying the functional effects of C-peptide in
relevant cell types is of key importance.
In summary, our study is the first to report a direct
modulatory effect of proinsulin C-peptide on the potentially
deleterious effects of insulin on human SV neointima
Mock-transfected
SREBF1 siRNA
0
5
10
15
20
*** **
NS NS
Migrated cells (per HP field)
24 h
m si m si m si m si
48 h 72 h96 h
150 -
100 -
75 -
50 -
a
b
Fig. 6 Effect of SREBF1 gene silencing on insulin-induced SV-SMC
migration. Cells were mock-transfected or transfected with SREBF1-
targeted siRNA before measuring SREBF1 protein and SV-SMC
migration. a Immunoblot showing SREBF1 protein production 24–
96 h after transfection with or without SREBF1 siRNA. The arrow
indicates the position of the SREBF1 band (125 kDa). b Migration
assay showing effects of SREBF1 gene silencing (36 h) on SV-SMC
migration towards basal medium (control; white bars), or medium
supplemented with 100 nmol/l insulin alone (black bars) or together
with10nmol/lC-peptide (hatchedbars).**p<0.01, ***p<0.001 (n=6)
Diabetologia (2010) 53:1761–17711769

Page 10

formation,througheffectsonbothSMCandECfunction.Our
data implicate SREBF1 as a candidate for further investiga-
tion. Co-administration of C-peptide with insulin may
improve SV bypass graft patency rates in insulin-treated
diabetic patients. Clinical trials will be necessary to test this
hypothesis.
Acknowledgements
British Heart Foundation and the University of Leeds, UK. S.G. Ball
is a British Heart Foundation Professor of Cardiology. N.A. Turner is
the recipient of a Research Councils UK Academic Fellowship. We
are grateful to J. Kaye and C. Bauer for cell culture expertise.
We gratefully acknowledge funding from The
Duality of interest
interest associated with this manuscript.
The authors declare that there is no duality of
Open Access
Commons Attribution Noncommercial License which permits any
noncommercial use, distribution, and reproduction in any medium,
provided the original author(s) and source are credited.
This article is distributed under the terms of the Creative
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