# C-Peptide and Atherogenesis: C-Peptide as a Mediator of Lesion Development in Patients with Type 2   Diabetes Mellitus?

Authors: Nikolaus Marx; Daniel Walcher
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Patients with insulin resistance and early type 2 diabetes exhibit an increased propensity to develop a diffuse and extensive pattern of arteriosclerosis. Typically, these patients show increased levels of C-peptide and over the last years various groups examined the effect of C-peptide in vascular cells as well as its potential role in lesion development.  While some studies demonstrated beneficial effects of C-peptide, for example, by showing an inhibition of smooth muscle cell proliferation, others suggested proatherogenic mechanisms in patients with type 2 diabetes. Among them, C-peptide may facilitate the recruitment of inflammatory cells into early lesions and promote lesion progression by inducing smooth muscle cell proliferation. The following review will summarize the effects of C-peptide in vascular cells and discuss the potential role of C-peptide in atherogenesis in patients with type 2 diabetes.


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## Body

## 1. Introduction

Patients with diabetes and insulin resistance exhibit an increased
propensity to develop arteriosclerosis with its sequelae acute myocardial
infarction and stroke [1]. Due to peripheral insulin resistance, these patients
temporarily demonstrate elevated levels of the proinsulin cleavage product C-peptide.
For a long time, C-peptide has been considered to be biological inert until
recent work in kidney cells suggest that C-peptide can activate intracellular signaling pathways such as NA-K-ATPase [2, 3]. In
addition, experimental data have shown activation of MAP kinase in fibroblasts
and lung endothelial cells as well as activation of protein kinase C and
PI3-kinase through C-peptide [4, 5]. These data suggested that C-peptide may be
biologically active. Moreover, various groups demonstrated that C-peptide
administration in patients with type 1 diabetes mellitus ameliorates
diabetes-induced renal and nerve dysfunction [6, 7]. Wallerath et al.
demonstrated that C-peptide stimulates the release of NO in endothelial cells
in vitro and that this effect is mediated by an induction of Ca2+ influx into the cells [8]. Such mechanism may also explain some of the
beneficial effects of C-peptide in type 1 diabetes.In addition, recent work addressed effects of C-peptide in vascular cells; the following review will
focus on these effects and discuss the potential relevance for atherogenesis in patients with type 2 diabetes.


## 2. Atherogenesis

According to our current understanding, atherogenesis is an inflammatory process in the
vessel wall with different phases and stages [9]. The early phase, before any
appearance of microscopic or macroscopic changes, is characterized by
endothelial dysfunction. Under the influence of cardiovascular risk factors,
the endothelium looses its integrity and becomes permeable for plasma compounds
like low-density lipoprotein (LDL) which can enter the vessel wall and deposit
in the subendothelial space. In addition, during endothelial dysfunction the
endothelium releases cytokines and chemotactic proteins and expresses adhesion
molecules on the cell surface. Such mechanisms facilitate the recruitment of
monocytes and CD4+ lymphocytes, important inflammatory cells during
lesion development [10]. Once these cells have entered the vessel wall,
monocytes differentiate to macrophages and express scavenger receptors on their
surface to promote the uptake of oxidized LDL. These lipid-loaden cells then
become foam cells, the classical cellular substrate of fatty-streaks. Foam
cells release various kinds of proinflammatory and prothrombotic mediators and
play a critical role in plaque progression. CD4+ lymphocytes are
also attracted by chemokines and enter the vessel wall as naïve TH0 cells. In the subendothelial space, these cells then encounter
antigens like oxidized LDL and differentiate towards TH1 cells which release
proinflammatory cytokines such as IFNγ, TNFα, and IL-2. Some of these cytokines
then enhance endothelial expression and release of T-cell specific chemokines,
creating a vicious cycle of cell activation and cell recruitment [11]. In
addition, these TH1 cytokines activate other cells in the vessel wall like
macrophages and smooth muscle cells (SMCs), thus orchestrating the inflammatory
response in the vessel wall. With increased recruitment of these inflammatory
cells fatty streaks develop and SMCs from the media start to proliferate and
migrate into the intima. As lesion formation progresses, advanced and
potentially complicated lesions are formed. These lesions may lead to a
progressive narrowing of the vessel wall thus potentially creating stable
angina if located in the coronary artery. Alternatively, these plaques may
become vulnerable and upon rupture can cause an acute coronary syndrome [12].


## 3. C-Peptide Deposition in Early Arteriosclerotic Lesions

Since endothelial dysfunction with increased permeability occurs in patients with
insulin resistance and early type 2 diabetes, a group of patients with temporarily
high C-peptide serum levels, it has been hypothesized that C-peptide might deposit
in the vessel wall in these patients in early atherogenesis.
Immunohistochemical analyses of early arteriosclerotic lesions of patients with
diabetes from the PDAY study (pathobiological determinants of atherosclerosis
in youth) revealed C-peptide deposition mainly in the subendothelial space and
the intima. Some of the diabetic subjects also exhibited C-peptide deposition
in the media. In contrast, only very little C-peptide deposition has been found
in early arteriosclerotic lesions of nondiabetic subjects. Computer-assisted analyses
revealed significantly higher C-peptide deposition in lesions from diabetic
individuals compared to lesions of age, sex, and risk factor matched
nondiabetic subjects [13]. Interestingly, no deposition of insulin or
proinsulin was detectable in diabetic or nondiabetic subjects. Staining of
parallel sections as well as immunofluorescence techniques demonstrated
colocalisation of C-peptide with intimal monocyte/macrophages and CD4+ lymphocytes in some of the diabetic
individuals [14]. In the studies cited above, C-peptide deposition has been
found in 100% of the 21 diabetic individuals examined, while monocyte
infiltration was only present in 77%, and CD4+ lymphocyte
infiltration only in 57%. These data suggested that C-peptide deposition may precede
monocyte and T-cell migration into the vessel wall. Based on this observation, the
hypothesis was raised that C-peptide may deposit in the vessel wall during
early atherogenesis and then—through chemotactic effects—promote the recruitment of monocytes and CD4+ lymphocytes.
Still, it remains unclear to what extent other peptides may
also deposit in the subendothelial space and exhibit similar effects.


## 4. Chemotactic Activity of C-Peptide Towards Monocytes and Cd4+ Lymphocytes

In vitro migration assays, employing a modified Boyden chamber, revealed that
C-peptide induces the migration of both monocytes and CD4+ lymphocytes
in a concentration- dependent manner. Interestingly, insulin did not have such
an effect. The chemotactic effects of C-peptide on these cells were within the
range of very well-established chemokines like MCP-1 and RANTES. In addition,
checkerboard analyses showed that C-peptide induces chemotaxis rather than
chemokinesis [13, 14]. Interestingly, C-peptide does not exhibit any
chemotactic activity towards neutrophils, cells which are not present in
arteriosclerotic lesions.Inhibition migration assays as well as western blot analyses and activity assays demonstrated
that C-peptide mediates its chemotactic activity through an as of yet
unidentified pertussis toxin sensitive G-protein coupled receptor with
subsequent downstream activation of PI3-kinaseγ.In summary, these data suggest that—during endothelial dysfunction with increased
endothelial permeability—C-peptide may deposit in the arterial intima
in patients with insulin resistance and early type 2 diabetes mellitus and
subsequently attract monocytes and CD4+ lymphocytes intothe vessel wall (Figure 1). Such
mechanisms may promote lesion development and potentially explain why patients
with diabetes develop such a diffuse and extensive pattern of arteriosclerosis
at a very early time point.Figure 1
Potential role of C-peptide in early
atherogenesis in patients with insulin resistance and early type 2 diabetes
mellitus. During endothelial dysfunction with increased endothelial
permeability, C-peptide could deposit in the intima and through its chemotactic
activity on monocytes and CD4+ lymphocytes facilitate the recruitment of these inflammatory cells into the
vessel wall.In addition to these proatherogenic effects, interesting data in monocyte-like
THP1 cells showed that C-peptide increases the expression of CD36, an important
scavenger receptor for the macrophage uptake of oxidized LDL in
arteriosclerotic lesions [15]. These data suggest that C-peptide—in addition to its chemotactic effects on
monocytes—may also promote the differentiation of
monocyte/macrophages towards foam cells, thus representing another potential
proatherogenic effect of C-peptide.


## 5. C-Peptide and Smooth Muscle Cell Proliferation

Since C-peptide also colocalized
with SMCs in the media of early arteriosclerotic lesions in some diabetic
subjects, it has been suggested that C-peptide could also exhibit biological
activity in these cells [16]. SMCs play a crucial role in the development of
arteriosclerotic lesions and the formation of fatty streaks. In addition, SMCs
are of critical importance in the formation of restenosis after coronary
intervention: after vascular injury these cells start to proliferate and then
migrate into the developing neointima, becoming the major cellular substrate of
the restenotic tissue [17]. These mechanisms seem to be accelerated in patients
with diabetes and insulin resistance, thus contributing to their increased risk
of restenosis after coronary intervention [18, 19]. Several mechanisms like the
release of growth factors from activated platelets as well as the secretion of stimulatory
mediators from inflammatory cells have been shown to induce SMC proliferation
during atherogenesis and restenosis formation [9]. Conflicting data exist on
the role of C-peptide in SMC proliferation. A recent report coming from
Kobayashi et al. demonstrated an inhibition of rat SMC proliferation after 3 days
of treatment with human C-peptide under high glucose concentrations [20]. These
effects observed at high C-peptide concentrations (100 nmol/L) were mediated by
an inhibition of PDGF-beta receptor expression as well as by a reduction in
p42/p44 MAP-kinase phosphorylation. These data are in contrast to results from
our own group demonstrating an induction of SMC proliferation upon C-peptide
stimulation [16]. In our experimental setting, human C-peptide induced the proliferation
of human SMCs while rat C-peptide exhibited similar effects in rat SMCs. These mitogenic
effects of C-peptide on SMCs were inhibited by a specific inhibitor of
Src-kinase as well as transfection of cells with short interference RNA (siRNA)
against Src. Similarly, inhibition of PI3-kinase and MAP-kinase reduced
C-peptide-induced SMC proliferation. Moreover, C-peptide induced Src phosphorylation
as well as activation of PI3-kinase and ERK 1/2, promoting the concept that these
signalling molecules are involved in C-peptide induced SMC proliferation.
Moreover, C-peptide increased cyclin D1 expression as well as phosphorylation of
the retinoblastoma protein (Rb). These data suggest that C-peptide induces SMC
proliferation and demonstrate another mechanism how C-peptide may contribute to
plaque development and restenosis formation in patients with insulin resistance
and early type 2 diabetes. Different experimental conditions may account for
the discrepant results between the two studies cited and future work is
warranted to further elucidate this issue.


## 6. C-Peptide and Microvascular Thrombus Formation in Mice

Interesting experimental data demonstrated a delay of microvascular thrombus formation in
normal and diabetic mice upon high-dose C-peptide treatment. Such mechanisms
were most likely mediated by a reduction in endothelial plasminogen activator
inhibitor 1 (PAI-1) expression in arterioles and venules, but not by an effect
on platelet activation. Interestingly, concomitant treatment with insulin
abolished the effect of C-peptide on microvascular thrombus formation [21]. These
data suggest that C-peptide could exhibit antithrombotic actions in vivo.Additional effects of C-peptide on endothelial function as well as on endothelial-leukocyte interaction are discussed by Kunt
and Pfutzner in the same issue of this journal.


## 7. Summary and Future Prospects

The majority of data described above suggest that C-peptide may promote lesion
development in patients with type 2 diabetes mellitus and insulin resistance,
while the application of C-peptide in type 1 diabetic patients who lack C-peptide
has been shown to improve diabetic microvascular complications such as diabetic
neuropathy. Is the potential proatherogenic action of C-peptide in contrast to such
clinical benefits of C-peptide treatment in patients with type 1 diabetes?—Definitely not. When one compares the
situation of C-peptide in type 1 and type 2 diabetic patients with the clinical
presentation of hypo- and hyperthyroidism, C-peptide's effects are not
contradictory. L-thyroxine treatment in patients with hypothyroidism is without
any doubt beneficial, but elevated levels of L-thyroxine in those with hyperthyroidism
can cause serious clinical manifestations. Similar mechanisms may apply for
C-peptide: supplementation of C-peptide in type 1 diabetic patients may be
beneficial while an increase in C-peptide levels in patients with insulin
resistance and type 2 diabetes may be harmful.Further studies in animal models of arteriosclerosis are warranted to examine whether
the hypothesis of C-peptide’s proatherogenic effects holds true in vivo. Moreover,
additional work is needed to identify the C-peptide receptor. Such a receptor could
be an interesting therapeutical target for both, patients with type 1 or type 2
diabetes. Activating such a receptor could be beneficial in type 1 diabetic
patients, while blocking of C-peptide receptors in patients with insulin
resistance and early type 2 diabetes may help to prevent vascular complications
from early on.



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