<title>1. INTRODUCTION</title> <p>Patients with diabetes mellitus type 1 present with an extensive
risk for microvascular complications like retinopathy, nephropathy, and
peripheral neuropathy. Although hyperglycemia is recognized as a major driver
in the development of these diabetic complications, the precise mechanism,
whereby diabetes precipitates these complications, is not fully understood.
Furthermore, also in type 1 diabetic patients with good metabolic control, the
risk for the development of microvascular complications is reduced but still
not abolished. In the DCCT trial, type 1 diabetic patients with sustained
C-peptide secretion showed a significant smaller risk for microvascular
complications compared with those patients totally lacking C-peptide secretion
from the beta cell [##REF##12610045##1##]. In this study, even
modest beta cell activity was associated with a decrease in the incidence of
microvascular complications.</p> <p>Regulation of vascular tone is a dynamic process, regulated by a
complex interaction of several balancing and counterbalancing forces. The
kinetics of postprandial insulin, C-peptide,
and blood glucose levels was
shown to interact in the regulation of microvascular blood flow in several
tissues like the skin or the heart [##REF##15998667##2##, ##REF##15866056##3##]. Although it is not
possible to separate the beneficial effects of residual C-peptide secretion
from those of residual insulin secretion, there is increasing evidence that
C-peptide might play a putative role in the physiology of microvascular blood
flow regulation.</p> <p>In type 1 diabetes mellitus, numerous functional alterations in
blood flow could be observed early after beta cell dysfunction has emerged [##REF##17254616##4##, ##REF##9616259##5##]. Early type 1 diabetes is characterised by increased microvascular
blood flow, increased shear stress, and tangential pressure on the
microvascular endothelium. In addition, increased leukocyte-endothelial adhesion [##REF##9710358##6##], increased blood viscosity [##REF##3770316##7##, ##UREF##0##8##], and changes in the haemodynamic properties of red blood cells [##REF##3025045##9##, ##REF##689301##10##] further affect microvascular blood flow. These early functional
disturbances proceed structural alterations in the vessel wall, including
basement membrane thickening as well as arteriolar hyalinosis [##REF##7789639##11##].</p> <p>The role of vascular endothelium for micro- and macrovascular blood
flow regulation has been extensively investigated within the last decade [##REF##11159815##12##, ##REF##2462367##13##]. The endothelial cells
coat the internal lumen of the vessels and serve as an interface between
circulating blood cells and the vascular smooth muscle cell. In addition to
serve as a physical barrier between the blood and the underlying smooth muscle
cells, the endothelial cell facilitates a complex array of signalling between
the vessel wall and the enclosed blood compartment. There are several
transmitters released from endothelial cells like nitric oxide (NO), endothelin
1, prostaglandins, thrombin, substance P, bradykinin, serotonin, and others
which impact the vascular tone [##REF##3057888##14##, ##REF##6253831##15##].</p> <p>Nitric oxide was identified
as the primary vasodilator released from the endothelium [##REF##3495737##16##]. As shown in ##FIG##0##Figure 1##, NO elicits vasodilatation through stimulation of endothelial NO-synthase (eNOS),
increasing the endothelial release of NO
and subsequent activation of the guanylcyclase in the vascular smooth muscle
cell [##REF##11159815##12##, ##REF##8582544##17##–##REF##8062516##19##].</p> <p>As shown in ##FIG##1##Figure 2##, the activity of eNOS could be stimulated or
suppressed by several signaling molecules, known to be altered in patients with
diabetes mellitus. Reduced levels of circulating NO contribute to vascular
injury by facilitating platelet-vascular wall interaction, increasing the
adhesion of circulating monocytes to the endothelial surface, and stimulation
of vascular smooth muscle proliferation [##REF##8080489##20##]. Impaired endothelial function and a reduction in endothelial NO
release are early features
of type 1 diabetes and thought to be principal causes of morbidity and mortality in these patients.</p>
<title>6. CONCLUSIONS</title> <p>Insulin depletion in type 1 diabetic patients results in
hyperglycaemia and the development of vascular complications of diabetes
mellitus. Treatment of type 1 diabetes mellitus with insulin replacement is an
effective tool for addressing glucose metabolism, but it seems conceivable that
the loss of C-peptide secretion from pancreatic beta cells might contribute to
the vascular complications in patients with diabetes mellitus type 1. As shown
in this review, recent studies showed that C-peptide is biologically active by
modulating endothelial function and microvascular blood flow. The underlying
mechanisms involve the activation of endothelial nitric oxide synthase and the
activation of Na<sup>+</sup>K<sup>+</sup>ATPase, which was shown to be
calcium-dependent and ouabain sensitive. The postulated mechanism by which
C-peptide interact with microvascular blood flow is illustrated in ##FIG##6##Figure 7##.</p> <p>Since the vascular effects of C-peptide could not be confirmed in
all tissues, it seems conceivable that there are tissue specific differences in
the mode of C-peptides vascular activities. Instead a specific binding of
C-peptide to the cell membrane could be demonstrated [##REF##10557318##67##], no specific receptor for C-peptide could be isolated neither from
endothelial cells nor from other cell systems. Therefore, there is still a
substantial need for the further investigation of the molecular effects of
C-peptide on cellular level.</p> <p>Nevertheless, the improvement in erythrocyte flexibility and
microvascular blood flow after C-peptide supplementation in type 1 diabetic
patients encourages the claim for further prospective interventional trials to
establish the clinical relevance for C-peptide supplementation in type 1
diabetic patients.</p>
<p>Recommended by George King</p> <p>During the recent years, the role of C-peptide, released from the pancreatic beta cell, in regulating microvascular blood flow, has received increasing attention. In type 1 diabetic patients, intravenous application of C-peptide in physiological concentrations was shown to increase microvascular blood flow, and to improve microvascular endothelial function and the endothelial release of NO. C-peptide was shown to impact microvascular blood flow by several interactive pathways, like stimulating Na<sup>+</sup>K<sup>+</sup>ATPase or the endothelial release of NO. There is increasing evidence, that in patients with declining beta cell function, the lack of C-peptide secretion might play a putative role in the development of microvascular blood flow abnormalities, which go beyond the effects of declining insulin secretion or increased blood glucose levels.</p>
<title>2. EFFECTS OF C-PEPTIDE ON NITRIC OXIDE (NO)</title> <p>C-peptide was shown to
significantly enhance the release of NO from bovine aortic endothelial cells (BAECs)
in a dose-dependent manner [##REF##14623175##21##, ##REF##10230651##22##]. The release of NO in this study
was dose dependent and already obtained within the physiological range of 1–6 nM. C-peptide
increased the intracellular Ca<sup>2+</sup> concentration in BAEC (see ##FIG##2##Figure 3##).
Since the endothelial eNOS is a Ca<sup>2+</sup>/calmodulin-regulated enzyme [##REF##1705708##23##], both the C-peptide-stimulated Ca<sup>2+</sup> signal and the NO release were abolished in Ca<sup>2+</sup>-free medium.
Therefore, the peptide is likely to stimulate eNOS activity by facilitating an
influx of Ca<sup>2+</sup> into BAEC.</p> <p>The NO release from BAEC declined from 2–30 minutes of
incubation, indicating a desensitization of the potential receptor, or the subsequent
signalling cascade, which has been also demonstrated for other peptide signals
for endothelial NO release [##REF##2448055##24##]. In conclusion, C-peptide is able
to stimulate an influx of Ca<sup>2+</sup> into endothelial cell, thereby
activating the Ca<sup>2+</sup>-sensitive endothelial NO synthetase and
stimulating the release of NO from the
endothelial cell.</p> <p>In a study by Kitamura et al.,
C-peptide was shown to stimulate NO production by enhancing the
mitogen-activated protein-kinase dependent transcription of endothelial nitric
synthase in aortic endothelial cells of Wistar rats [##REF##12059784##25##]. In this study, it was shown that C-peptide
increases NO release from aortic endothelial cells by enhancing eNOS expression
through an ERK-dependent transcriptional pathway.</p> <title>3. EFFECTS OF C-PEPTIDE ON ERYTHROCYTE Na<sup>+</sup>K<sup>+</sup>ATPASE</title> <p>Na<sup>+</sup>K<sup>+</sup>ATPase activity has been found to be
attenuated in various cell types under diabetic conditions [##REF##8393294##26##–##REF##9165217##28##]. It has also been
shown that hyperglycemia inhibits Na<sup>+</sup>K<sup>+</sup>ATPase activity by
an endothelium dependent mechanism [##REF##3003162##29##]. Na<sup>+</sup>K<sup>+</sup>-ATPase
is a plasma membrane-associated protein complex, expressed in most eukaryotic
cells. It couples the energy released from the intracellular hydrolysis of ATP
to the transport of cellular ions, a major pathway for the controlled
translocation of sodium and potassium ions across the cell membrane. Therefore,
Na<sup>+</sup>K<sup>+</sup>-ATPase controls directly or indirectly many
essential cellular functions, for example, cell volume, free calcium
concentrations, and membrane potential [##REF##8070076##30##]. Although there are tissue specific differences in the regulations
of Na<sup>+</sup>K<sup>+</sup>-ATPase activity, hyperglycemia and diabetes are
predominantly characterized by a decrease in ouabain-sensitive Na<sup>+</sup>K<sup>+</sup>-ATPase
activity. This would result in an
increase in intracellular calcium concentration and an increased vascular tone,
promoting the development of vascular complications in diabetes mellitus. Na<sup>+</sup>K<sup>+</sup>ATPase activity is
involved in vascular regulation based on a complex interaction between Na<sup>+</sup>K<sup>+</sup>-pump-activity
and an endothelium dependent increase of NO [##REF##2985409##31##, ##REF##8794828##32##]. On the other hand, NO
and cyclic-GMP have been shown to increase vascular Na<sup>+</sup>K<sup>+</sup>ATPase
activity, with subsequent vasorelaxation [##REF##7911280##33##, ##REF##1380379##34##].</p> <p>In order to hypothesize the potential mechanism of C-peptide
activity, previous studies concerning Na<sup>+</sup>K<sup>+</sup>ATPase
activity in erythrocytes and renal tubular cells are of considerable
interest [##REF##3025045##9##, ##REF##8635672##35##, ##REF##9541168##36##]. Ohtomo et al. were
able to show that the attenuated activity of Na<sup>+</sup>-K<sup>+</sup>-ATPase
activity in renal tubular segments of diabetic rats is restored by C-peptide.
On the other hand, an attenuation of Na<sup>+</sup>-K<sup>+</sup>-ATPase
activity has been demonstrated to correlate with decreased erythrocyte
deformability in type 1 diabetic patients [##REF##3025045##9##].</p> <p>In a recent study, erythrocyte Na<sup>+</sup>K<sup>+</sup>ATPase
activity was found to be reduced in type 1 diabetic patients, while in type 2
diabetic patients a wide range of individual Na<sup>+</sup>K<sup>+</sup>ATPase activities was observed, presenting some patients with
very low Na<sup>+</sup>K<sup>+</sup>ATPase activity and others with a normal Na<sup>+</sup>K<sup>+</sup>ATPase
activity. It appeared that erythrocyte Na<sup>+</sup>K<sup>+</sup>ATPase
activity was significantly lower in those type 2 diabetic patients treated with
insulin compared with those on oral treatment. Also in the former, Na<sup>+</sup>K<sup>+</sup>ATPase
activity was comparable to those in type 1 diabetic patients.</p> <p>In an in vitro study by Djemli-Shiplolye et al., incubation of
erythrocytes from type 1 diabetic patients with C-peptide normalized
erythrocyte Na<sup>+</sup>K<sup>+</sup>ATPase activity [##REF##10909997##37##]. In another study, intravenous
infusion of C-peptide was found to improve erythrocyte Na<sup>+</sup>K<sup>+</sup>ATPase
activity in type 1 diabetic patients [##REF##10677386##38##].</p> <title>4. EFFECT OF C-PEPTIDE ON RED CELL DEFORMABILITY</title> <p>Blood flow in larger vessels is determined by the vessel diameter,
blood viscosity, and vessel length according to the law of Hagen-Pouiseuille.
In the capillary bed, especially if the diameter of the vessel is below the
diameter of the erythrocytes, blood flow is predominantly determined by the
viscosity and deformability of the erythrocytes. Thus, reduced erythrocyte
deformability will reduce blood flow if the capillary diameter and blood
pressure remain constant [##UREF##2##39##].</p> <p>Several studies demonstrated that factors such as decreased
erythrocyte deformability, increased erythrocyte aggregation, and increased
cell membrane rigidity contribute to alterations in microvascular blood flow in
patients with diabetes mellitus [##REF##3770316##7##, ##REF##3025045##9##, ##REF##689301##10##, ##REF##3090107##40##–##REF##971793##44##].</p> <p>Concerning the possible mechanism of reduced erythrocyte
deformability, it is noteworthy that Na<sup>+</sup>-K<sup>+</sup>-ATPase
activity has been shown to be attenuated in several cell types, including
erythrocytes in diabetic patients [##REF##3025045##9##, ##REF##8635672##35##, ##REF##9541168##36##], and that it may be restored not only by insulin but C-peptide as
well [##REF##8635672##35##].</p> <p>The deformability of erythrocytes in type 1 diabetic patients was found
to be reduced compared to healthy controls [##REF##10230651##22##]. Both groups were matched concerning their glucose levels in order
to exclude a glucotoxic effect. Deformability was tested under physiological
(0.3 to 10 Pa) and supraphysiological (&gt;10 Pa) shear stress rates by means
of laser diffractoscopy. Incubation of erythrocytes from healthy controls and
type 1 diabetic patients with different concentrations of C-peptide restored
erythrocyte deformability in type 1 diabetic patients but was without any
effect in erythrocytes of nondiabetic controls (see ##FIG##3##Figure 4##).</p> <p>It is speculative to discuss the underlying mechanism based upon
these results, but impaired Na<sup>+</sup>K<sup>+</sup>ATPase activity may
contribute to the decrease in erythrocyte deformability by increasing the
intracellular sodium concentration with subsequent intracellular accumulation
of free calcium ions due to competition in transmembranous exchange [##REF##2162303##45##]. These abnormalities in calcium homeostasis are known to enhance
spectrin dimer-dimer interaction and spectrin-protein 4.1-actin interaction [##REF##3403710##46##, ##REF##7673158##47##]. The latter is being
promoted by adducin, a membrane-skeleton-associated calmodulin-binding protein [##REF##3511042##48##].</p> <p>Pretreatment of erythrocytes from type 1 diabetic patients with ouabain,
EDTA, or pertussis toxin completely abolished C-peptide effects on erythrocyte
deformability as shown in more detail in the paper of Hach et al. in the same
issue of Experimental Diabetes Research.</p> <title>5. EFFECTS OF C-PEPTIDE ON MICROVASCULAR BLOOD FLOW</title> <p>In several studies, C-peptide was shown to affect microvascular
blood flow and to improve nerve or renal function in animal models of type 1
diabetes and in humans with type 1 diabetes mellitus [##REF##12829651##49##–##UREF##3##52##]. In a study by
Lindstrom et al., C-peptide supplementation was shown to increase microvascular
blood flow and to enhance the recruitment of capillaries in isolated kidneys of
the rat [##REF##8866882##53##]. In another study, the
effect of C-peptides was investigated in skeletal muscle arterioles isolated
from rat cremaster muscles [##REF##10199846##54##]. In this study, C-peptide evoked a concentration independent
arteriolar dilatation in a range between 0.3 and 1000 ng/mL. Addition of
insulin at low concentrations, which had no vascular effect by its own,
enhanced the vascular effect of C-peptide, indicating a permissive role of both
pancreatic peptides in the regulation of microvascular blood flow. Inhibition
of the nitric oxide synthase by LNMA completely abolished the vasodilating
response to C-peptide, further stressing the role of NO in the transmission of
C-peptide vascular effects.</p> <p>In a study done by Ido et al., beneficial effects of C-peptide supplementation could be documented in
several vascular beds in diabetic rats [##REF##9228006##55##]. In their study, biosynthetic human C-peptide was given
subcutaneously twice daily for 5 weeks in control rats and streptozotocin-induced diabetic rats. Highly supraphysiological peak plasma C-peptide levels
between 9 and 10 nM were reached. C-peptide markedly reduced the diabetes
induced increase in blood flow in the anterior uvea, retina, and sciatic nerve.
In addition, C-peptide prevented increased <sup>125</sup>I-labeled albumin
permeation in retina, nerve, and in the aorta. The effect on microvascular
blood flow was accompanied by an increase in caudal motor nerve conduction
velocity. No effect of C-peptide, neither on microvascular blood flow nor on
motor nerve conduction velocity, could be observed in the healthy control rats.
Cotter et al. observed the vascular effects of C-peptide on sciatic endoneurial
blood flow in streptozotocin diabetic rats at physiological C-peptide
concentrations [##REF##12829651##49##]. In their study, C-peptide supplementation revealed an improvement
in endoneurial blood flow and vascular conductance by 57 and 66%, respectively.
The increase in endoneurial blood flow was accompanied by an improvement in
motor nerve conduction velocity by 62% and in sensory nerve conduction velocity
by 78%. Again, treatment with L-NNA abolished the effect of C-peptide on
endoneurial blood flow and nerve conduction velocity.</p> <p>In an investigation by Johanssen et al., the effect of C-peptides on
skeletal muscle blood flow was observed in type 1 diabetic patients and in
healthy controls during exercise [##REF##1478367##56##]. In the type 1 diabetic subjects, blood flow and capillary
diffusion capacity of the exercising forearm at baseline were approximately 30%
lower compared to the healthy control subjects. Intravenous administration of
C-peptide increased forearm blood flow by 27% and capillary diffusion capacity
by 52% to levels similar to those observed in the healthy controls. No
significant changes in blood flow could be observed in healthy controls
receiving C-peptide or in diabetic patients receiving placebo infusion. In
accordance with the observed improvements in muscle blood flow, forearm oxygen
and glucose uptake increased markedly after C-peptide administration in type 1
diabetic patients.</p> <p>Skin blood flow is affected early after the diagnosis of diabetes
mellitus [##UREF##4##57##–##REF##1600701##59##]. The skin capillary circulation is functionally situated in
parallel to the arteriovenous shunts and is thought to have the primary
function of tissue nutrition. It has been estimated that 80–90% of total skin
blood flow passes through thermoregulatory arteriovenous shunts and does not
enter the nutritive part of the capillary bed [##REF##7712701##60##–##REF##7060849##62##]. While total skin
perfusion is increased in diabetes mellitus, nutritional capillary skin blood
flow was shown to be reduced in diabetic patients [##REF##7712701##60##, ##REF##7796989##61##, ##REF##2271088##63##]. As shown in ##FIG##4##Figure 5##, short-term infusion of C-peptide in type 1 diabetic patients was
found to redistribute microvascular blood flow from the subpapillary
thermoregulatory blood flow into the nutritive capillary bed [##REF##9593759##64##]. At baseline, nutritive
capillary blood flow was significantly lower in type 1 diabetic patients
compared with the control group. C-peptide supplementation in type 1 diabetic
patients increased capillary blood flow to a level comparable to that observed
in the healthy control group. Thirty minutes after the termination of the
C-peptide infusion, capillary blood flow had declined to a level not different
from baseline levels. No such effect of C-peptide application on microvascular
skin blood flow could be observed in nondiabetic subjects. A linear
relationship was found between plasma C-peptide levels and the capillary blood
flow velocity (<italic>r</italic> = 0.401; <italic>P</italic> &lt; .0001).</p> <p>Fernqvist-Forbes
et al. studied the effect of C-peptide on flow-mediated vasodilatation (FMD) in
type 1 diabetic patients [##REF##11472302##65##]. In addition, the arterial
dilatation to glyceryl trinitrate, which is an endothelium independent marker
of vascular smooth muscle function, was investigated. When compared with the
healthy control group, the type 1 diabetic patients revealed a lower FMD.
Following C-peptide administration, blood flow in the brachial artery increased
by approximately 35%, and FMD was significantly improved. No effect of
C-peptide could be observed on the microvascular response to glyceryl trinitrate,
which further confirm the endothelium dependent pathway of C-peptide.</p> <p>As shown in ##FIG##0##Figure 1##, acetylcholine
elicits vasodilatation through a stimulation of endothelial NO-synthase (eNOS) with an increase in the endothelial
release of NO and a subsequent stimulation of the guanylcyclase in the vascular
smooth muscle cell. In a recent study, the effect of intravenous C-peptide
infusion on the acetylcholine induced increase in microvascular blood flow was
investigated in type 1 diabetic patients [##REF##10677386##38##]. Skin microvascular response was measured by laser Doppler fluxmetry,
and acetylcholine was applied to the dorsum of the foot using the technique of
iontophoresis. The microvascular response to acetylcholine increased by 133%
during short-term infusion of C-peptide, which was accompanied by a significant
increase in plasma cyclic GMP levels (see ##FIG##5##Figure 6##).</p> <p>In contrast, in a study of Polska et al., no effect of C-peptide
supplementation in type 1 diabetic patients could be observed on retinal blood
flow [##REF##16936149##66##].</p> <p>Therefore, it could be postulated that C-peptide affects microvascular blood flow in a tissue specific manner.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Mechanism of endothelial nitric oxide synthesis with stimulation of guanylcyclase in the vascular smooth muscle cell and subsequent vasorelaxation.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Substrates known to activate or reduce the endothelial nitric oxide synthase system.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>C-peptide induced calcium influx into endothelial cells. Effect of C-peptide on the Ca2+ signal in endothelial cells loaded with Fluo-3. Addition of C-peptide to bovine aortic endothelial cells yielded in a significant increase in fluorescence (above), which was not found in calcium free medium (b).</p></caption></fig> <fig id="fig4" position="float"><label>Figure 4</label><caption><p>
<italic>Representative analysis of erythrocyte deformability at 1.75 Pa</italic>. This graph shows the alterations of elongation index E at a shear stress of 1.75 Pa, which is frequently achieved in vivo. C-peptide did not modify the deformability of erythrocytes obtained from healthy controls, whereas the deformability of diabetic erythrocytes was restored to normal levels after administration of different concentrations of the peptide. Statistical analysis was performed by Student's <italic>t</italic> test.</p></caption></fig> <fig id="fig5" position="float"><label>Figure 5</label><caption><p>Skin capillary during intravenous application of C-peptide (8 pmol/kg/min) in type 1 diabetic patients (■) and non-diabetic controls (□) (mean ± SEM; * = <italic>P</italic> &lt; .05; *** = <italic>P</italic> &lt; .001).</p></caption></fig> <fig id="fig6" position="float"><label>Figure 6</label><caption><p>Cyclic guanyl monophosphate (cGMP) at baseline and after 60 and 120 minutes of C-peptide (■) or placebo (□) (mean ± SEM; $ = <italic>P</italic> &lt; .05 versus baseline; § = <italic>P</italic> &lt; .05 versus 60 minutes).</p></caption></fig> <fig id="fig7" position="float"><label>Figure 7</label><caption><p>Schematic presentation of the molecular mechanism of C-peptide activity on endothelial cells and microvascular blood flow.</p></caption></fig>
<graphic xlink:href="EDR2008-176245.001"/> <graphic xlink:href="EDR2008-176245.002"/> <graphic xlink:href="EDR2008-176245.003"/> <graphic xlink:href="EDR2008-176245.004"/> <graphic xlink:href="EDR2008-176245.005"/> <graphic xlink:href="EDR2008-176245.006"/> <graphic xlink:href="EDR2008-176245.007"/>
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Exp Diabetes Res. 2008 Jul 28; 2008:176245