# C-Peptide and Its C-Terminal Fragments Improve Erythrocyte Deformability in Type 1 Diabetes Patients

Authors: Thomas Hach; Thomas Forst; Thomas Kunt; Karin Ekberg; Andreas Pfützner; John Wahren
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Aims/hypothesis. Data now indicate that proinsulin C-peptide exerts important physiological effects and shows the characteristics of an endogenous peptide hormone. This study aimed to investigate the influence of C-peptide and fragments thereof on erythrocyte deformability and to elucidate the relevant signal transduction pathway.
Methods. Blood samples from 23 patients with type 1 diabetes and 15 matched healthy controls were incubated with 6.6 nM of either human C-peptide, C-terminal hexapeptide, C-terminal pentapeptide, a middle fragment comprising residues 11–19 of C-peptide, or randomly scrambled C-peptide. Furthermore, red blood cells from 7 patients were incubated with C-peptide, penta- and hexapeptides with/without addition of ouabain, EDTA, or pertussis toxin. Erythrocyte deformability was measured using a laser diffractoscope in the shear stress range 0.3–60 Pa. Results. Erythrocyte deformability was impaired by 18–25% in type 1 diabetic patients compared to matched controls in the physiological shear stress range 0.6–12 Pa (P<.01–.001). C-peptide, penta- and hexapeptide all significantly improved the impaired erythrocyte deformability of type 1 diabetic patients, while the middle fragment and scrambled C-peptide had no detectable effect. Treatment of erythrocytes with ouabain or EDTA completely abolished the C-peptide, penta- and hexapeptide effects. Pertussis toxin in itself significantly increased erythrocyte deformability. Conclusion/interpretation. C-peptide and its C-terminal fragments are equally effective in improving erythrocyte deformability in type 1 diabetes. The C-terminal residues of C-peptide are causally involved in this effect. The signal transduction pathway is Ca2+-dependent and involves activation of red blood cell Na+,K+-ATPase.


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## Body

## 1. Introduction

During
the past decade, several studies have provided evidence that C-peptide is a
biologically active endogenous  peptide. It binds specifically in nanomolar
concentration to cell membranes [1], possibly to a G-protein-coupled
receptor, resulting in internalisation  of the peptide [2] and activation of Ca2+- and MAPK-dependent signalling pathways [3–6]. Cellular end-effects
include stimulation and induction of both Na+,K+-ATPase,
and eNOS as well as activation of a series of transcriptional fators [3, 7–10]. Studies in
patients with type 1 diabetes who lack endogenous C-peptide production show
that administration of C-peptide in replacement doses results in increased
regional blood flow in several tissues [11–13] and amelioration of
diabetes-induced functional and structural abnormalities of the peripheral
nerves and the kidneys [14–16]. Not only the
full-length native C-peptide but also its C-terminal pentapeptide fragment is
reported to exert physiological effects [1, 4, 6, 17, 18].It is
well established that diabetes is associated with reduced deformability of red
blood cells [19–21]. This abnormality
is of clinical significance in that it compromises the ability of red blood
cells to pass through capillaries, reduces tissue perfusion, and impairs tissue
oxygen supply [4, 22]. The mechanism
underlying the decreased red cell compliance in diabetes is not well understood,
but it has been shown that C-peptide is capable of restoring red cell
deformability to normal levels, most likely via stimulation of red cell Na+,K+-ATPase
[23]. The extent to which
C-peptide fragments will do the same has not been determined. Consequently, the
aim of this study was to examine the influence of C-peptide and its  C-terminal fragments, as well as middle segments on the abnormal
deformability of red blood cells  from type 1 diabetes patients. In addition,
aspects of a possible signal transduction pathway were evaluated.


## 2. Subjects, Materials, and Methods

### 2.1. Subjects

Blood samples were taken from 23 type 1
diabetes patients and 15 matched healthy controls (Table1). Inclusion criteria
were C-peptide serum level <0.2 nmol/L, diabetes duration >2 years, serum
creatinine <2 mg/dL, and no clinically relevant diabetic long-term complications.
Hematocrit values were
in the normal range for all patients and controls.Table 1
Clinical characteristics of study subjects.
Healthy controlsn=15Type 1 diabetes
patientsn=23Sex (male/female)8/713/10Age (years)34 ± 436 ± 3Height (m)1.76±0.021.75±0.02Weight (kg)78.0±2.877.5±2.6Diabetes duration (years)0 ± 024 ± 3Serum glucose (mmol/L)4.94±0.227.44±0.67HbA1c (%)5.7±0.37.3±0.2Serum C-peptide (nmol/L)0.77±0.090.01±0.003


### 2.2. Materials and Methods

Laser diffractoscopy was performed
using a Rheodyn SSD shear stress diffractometer (Myrenne GmbH, Roetgen, Germany).
The method of laser diffractoscopy has been described in detail previously [21, 23]. In summary, a He-Ne-Laser detects
deformation of erythrocytes  between two parallel glass discs, one of which
rotates resulting in defined shear stresses as per the following equation:
(1)τ=2πr⋅ηt⋅h,
where τ= shear stress, r= laser beam distance from rotation center, η= viscosity, h= height of gap
between discs, and t= time.Adjusting for equipment-specific values (r= 25 mm, h= 0.5 mm, and η= 24 mPa), the equation is condensed to(2)τ=7.536Pa⋅rpm,
where rpm = revolutions per minute.The applied shear stress range is
electronically regulated and includes 8
levels (0.3 Pa; 0.6 Pa; 1.2 Pa; 3 Pa; 6 Pa; 12 Pa; 30 Pa; 60 Pa).The erythrocyte deformability measurement detects
scattered-light intensities along orthogonal axes (A, B) of red blood cells
within the laser diffraction light cone. The erythrocyte  elongation index (EI)
is calculated by the following equation:(3)EI(%)=(A−BA+B)⋅100.All experiments were carried out at 23°C, which
was thermostatically regulated. Blood samples were collected using
ammonium-heparinated vials and subsequently distributed to dextran medium (30 μL blood per 2 mL dextran). Stability tests were performed to exclude
significant effects of storage time and/or serum glucose concentration on
erythrocyte deformability measured by laser diffractoscopy (data not shown). All
measurements of erythrocyte deformability were carried out before incubation
with active ingredients as well as at 1, 30, and 60 minutes after incubationC-peptide and C-peptide Fragments
Human C-peptide and all C-peptide
fragments were provided by Creative Peptides (Stockholm, Sweden)
at a purity of>98%. C-peptide and its fragments were dissolved in phosphate buffer
and incubations were made at 6.6 nmol/L. The incubation and mixing of probes
were done smoothly to avoid external shear stress. The fragments used in this
study were C-terminal hexapeptide, C-terminal pentapeptide, a
middle fragment including residues 11–19, and randomly scrambled C-peptide;
their amino acid sequences are presented in Table 2.Table 2
Amino acid structure of C-peptide and
C-peptide fragments used in this study.
Amino acid
structurePositionC-PeptideEAEDLQVGQVELGGGPGAGSLQPLALEGSLQ(1–31)MFELGGGPGAG(11–19)HPLEGSLQ(26–31)PPEGSLQ(27–31)Ouabain
Ouabain was obtained from Sigma-Aldrich (St. Louis, Mo, USA). Treatment with ouabain was titrated to
achieve a concentration of 1 μmol/L. Incubations with and without C-peptide or
fragments lasted 30 minutes at 23°C.EDTA
Incubations with EDTA (1.6 mg/mL) with and
without C-peptide or fragments lasted 30
minutes at 23°C. Treatment with EDTA alone did not alter erythrocyte
deformability (data not shown).Pertussis toxin
Pertussis toxin was obtained from
Sigma-Aldrich. Treatment with pertussis toxin (1 μg/mL) was conducted for 60 minutes at 37°C.Statistical analysis
Results are
expressed as the means ± SD. Gaussian distribution was checked by the
Kolmogoroff-Smirnoff test. Statistical analysis were done by two-site ANOVA and
Student'st test. A P-value of
less than .05 was regarded as statistically significant.


## 2.1. Subjects

Blood samples were taken from 23 type 1
diabetes patients and 15 matched healthy controls (Table1). Inclusion criteria
were C-peptide serum level <0.2 nmol/L, diabetes duration >2 years, serum
creatinine <2 mg/dL, and no clinically relevant diabetic long-term complications.
Hematocrit values were
in the normal range for all patients and controls.Table 1
Clinical characteristics of study subjects.
Healthy controlsn=15Type 1 diabetes
patientsn=23Sex (male/female)8/713/10Age (years)34 ± 436 ± 3Height (m)1.76±0.021.75±0.02Weight (kg)78.0±2.877.5±2.6Diabetes duration (years)0 ± 024 ± 3Serum glucose (mmol/L)4.94±0.227.44±0.67HbA1c (%)5.7±0.37.3±0.2Serum C-peptide (nmol/L)0.77±0.090.01±0.003


## 2.2. Materials and Methods

Laser diffractoscopy was performed
using a Rheodyn SSD shear stress diffractometer (Myrenne GmbH, Roetgen, Germany).
The method of laser diffractoscopy has been described in detail previously [21, 23]. In summary, a He-Ne-Laser detects
deformation of erythrocytes  between two parallel glass discs, one of which
rotates resulting in defined shear stresses as per the following equation:
(1)τ=2πr⋅ηt⋅h,
where τ= shear stress, r= laser beam distance from rotation center, η= viscosity, h= height of gap
between discs, and t= time.Adjusting for equipment-specific values (r= 25 mm, h= 0.5 mm, and η= 24 mPa), the equation is condensed to(2)τ=7.536Pa⋅rpm,
where rpm = revolutions per minute.The applied shear stress range is
electronically regulated and includes 8
levels (0.3 Pa; 0.6 Pa; 1.2 Pa; 3 Pa; 6 Pa; 12 Pa; 30 Pa; 60 Pa).The erythrocyte deformability measurement detects
scattered-light intensities along orthogonal axes (A, B) of red blood cells
within the laser diffraction light cone. The erythrocyte  elongation index (EI)
is calculated by the following equation:(3)EI(%)=(A−BA+B)⋅100.All experiments were carried out at 23°C, which
was thermostatically regulated. Blood samples were collected using
ammonium-heparinated vials and subsequently distributed to dextran medium (30 μL blood per 2 mL dextran). Stability tests were performed to exclude
significant effects of storage time and/or serum glucose concentration on
erythrocyte deformability measured by laser diffractoscopy (data not shown). All
measurements of erythrocyte deformability were carried out before incubation
with active ingredients as well as at 1, 30, and 60 minutes after incubationC-peptide and C-peptide Fragments
Human C-peptide and all C-peptide
fragments were provided by Creative Peptides (Stockholm, Sweden)
at a purity of>98%. C-peptide and its fragments were dissolved in phosphate buffer
and incubations were made at 6.6 nmol/L. The incubation and mixing of probes
were done smoothly to avoid external shear stress. The fragments used in this
study were C-terminal hexapeptide, C-terminal pentapeptide, a
middle fragment including residues 11–19, and randomly scrambled C-peptide;
their amino acid sequences are presented in Table 2.Table 2
Amino acid structure of C-peptide and
C-peptide fragments used in this study.
Amino acid
structurePositionC-PeptideEAEDLQVGQVELGGGPGAGSLQPLALEGSLQ(1–31)MFELGGGPGAG(11–19)HPLEGSLQ(26–31)PPEGSLQ(27–31)Ouabain
Ouabain was obtained from Sigma-Aldrich (St. Louis, Mo, USA). Treatment with ouabain was titrated to
achieve a concentration of 1 μmol/L. Incubations with and without C-peptide or
fragments lasted 30 minutes at 23°C.EDTA
Incubations with EDTA (1.6 mg/mL) with and
without C-peptide or fragments lasted 30
minutes at 23°C. Treatment with EDTA alone did not alter erythrocyte
deformability (data not shown).Pertussis toxin
Pertussis toxin was obtained from
Sigma-Aldrich. Treatment with pertussis toxin (1 μg/mL) was conducted for 60 minutes at 37°C.Statistical analysis
Results are
expressed as the means ± SD. Gaussian distribution was checked by the
Kolmogoroff-Smirnoff test. Statistical analysis were done by two-site ANOVA and
Student'st test. A P-value of
less than .05 was regarded as statistically significant.


## 3. Results

Erythrocyte deformability was significantly
decreased in type 1 diabetes patients compared to healthy controls over the
full range of shear stress tested (Figure1). In the physiological shear stress
range (≤12Pa), the difference between diabetic patients and healthy controls amounted
to 18–25%  (P<.01–.001). Incubation
of the red cells from type 1 diabetes patients with C-peptide completely normalized the erythrocyte defortmability at
all tested shear stress levels (Table 3). Incubation with the penta- and
hexapeptides also resulted in significant improvements in erythrocyte deformability
over the range of shear stress tested; the responses to C-peptide and the
C-terminal peptides were similar and there were no statistically significant differences
between these three treatment groups. In contrast, the middle fragment exerted
no significant effect on the diabetes-induced abnormal erythrocyte
deformability. Likewise and as expected, scrambled C-peptide with a random amino acid sequence had no
beneficial effect on erythrocyte deformability.Table 3
Erythrocyte elongation index (EI) (%) in the
shear stress range 0.6–12 Pa for
different treatment groups,n=23.
Shear
stress0.6 Pa1.2 Pa3 Pa6 Pa12 PaHealthy controls5.87±0.72*14.37±0.61*29.08±0.79*39.19±1.02*45.93±0.85*Diabetes patients4.38±0.6311.77±0.4926.01±0.6035.48±0.8443.34±0.92C-peptide5.40±0.82*14.11±0.85*28.34±1.58*39.07±1.46*45.82±1.43*HP5.77±0.81*14.18±1.17*28.61±1.37*38.49±1.62*45.82±1.81*PP6.12±0.93*14.02±1.37*28.23±1.46*38.85±1.54*45.20±1.74*MF4.42±0.8911.39±1.0725.82±1.2435.79±0.9843.39±1.17SCR4.10±0.9111.25±1.0725.58±1.0735.64±0.9143.48±1.00P<.01* versus type 1 diabetes
patients.Figure 1
Erythrocyte elongation index (EI) (%) ± SD for
erythrocytes from healthy controls (open columns) and type 1 diabetes patients
(filled columns) at different levels of shear stress.P<.05.*P<.01.**P<.001***
versus controls.Pretreatment of erythrocytes from patients with
type 1 diabetes with  ouabain or EDTA completely abolished the C-peptide-, penta- and hexapeptide-induced improvements in deformability as shown in Figure2 for the pentapeptide
at the shear stress 1.2 Pa. Similar results were obtained for all levels of
shear stress. Pretreatment
of erythrocytes with pertussis toxin in itself increased erythrocyte
deformability significantly (P<.05) in the shear stress range of 0.6–12 Pa.
Therefore, the
possible influence of G-protein inactivation on the effects mediated by C-peptide and its fragments could not be evaluated.Figure 2
Erythrocyte elongation index(EI) (%) ± SD for erythrocytes from healthy controls and type 1 diabetes
patients. The latter cells were incubated with C-peptide, hexapeptide (HP), and
pentapeptide (PP) from the C-terminal region of C-peptide as well as a middle fragment (MF)
and scrambled peptide (SCR, control). Results for combined incubation with PP
and oubain or EDTA are also shown. Data are obtained at the shear stress level 1.2 Pa. P<.001*** versus untreated
erythrocytes from type 1 diabetes patients.


## 4. Discussion

The present results confirm and
extend previous observations indicating that red blood cell deformability is
compromised in diabetes [19–21] and that this abnormality can be
corrected by C-peptide [23]. Thus, using whole blood samples
and laser diffractoscopy, a method with high reproducibility (CV < 1%),
erythrocyte deformability measured in blood samples from type 1 diabetes
patients was found to be reduced by 18–25% over the
physiological shear stress range (0.6–12 Pa), in
keeping with previous results using the same methodology [23]. Exposing blood cells from the
diabetic patients to 6.6 nM, C-peptide was found to fully normalize membrane
deformability. We now show that not only the native, full length C-peptide but
also its C-terminal penta- and hexapeptides possess this capability. In
contrast, a middle segment comprising residues 11–19 of C-peptide had no
effect. The specificity of the observed effects is attested by the finding that scrambled C-peptide,
a control peptide with its residues assembled in random order, had no
detectable effect.The current results for the pentapeptide
are in line with previous reports that it competes with C-peptide for cellular
binding [1], that it elicits an increase in
intracellular Ca2+-concentration [4], activates PKC isoforms, causes
phosphorylation of ERK1/2 and JNK, and stimulates Na+,K+-ATPase
activity [6, 17, 24]. Studies evaluating the
pentapeptide's cellular binding/competition characteristics, its stimulatory
effect on intracellular Ca2+-concentrations and ERK1/2 phosphorylation
demonstrate that it is the N-terminal Glu-residue of the pentapeptide (Glu 27
of C-peptide) that is of primary importance for its bioactivity; alanine
substitution of the other four residues has little or no effect on the
pentapeptide’s bioactivity [4, 25, 26]. Its bioactivity has been found to
be similar to that of the native peptide both in the above in vitro studies and
in invivo experiments evaluating its stimulatory effect on whole-body glucose
utilization [18] and its inhibition of
diabetes-induced glomerular hyperfiltration [27] in streptozotocin-diabetic rats.The C-terminal hexapeptide was found
to be equally potent as the pentapeptide and the native C-peptide with regard
to its amelioration of diabetes-induced abnormal red blood cell deformability (Figure2). Only little information is available with regard to the physiology
of the hexapeptide but the current findings are in line with previous
observations that it is capable of stimulating Na+,K+-ATPase
in renal tubular segments with potency similar to that of the pentapeptide [17]. In contrast to the findings for
the two C-terminal segments, there was no indication that the middle fragment
(residues 11–19) exerted a measurable effect on diabetes-induced abnormal red
cell deformability. Variable results have been reported previously for middle
fragments; mild stimulation of Na+,K+-ATPase in renal
tubule segments [17] but no effect on glucose
utilization under in vivo conditions [18] have been observed.Exposing red blood cells from
patients with type 1 diabetes to either oubain or EDTA resulted in complete
abrogation of the normalizing effect by the penta- and hexapeptides or
C-peptide on erythrocyte deformability. Evaluation of a G-protein involvement
in the signalling pathway could not be carried out since exposure of the
erythrocytes to pertussis toxin in itself resulted in alteration of red cell
deformability in contrast to previous findings for rabbit erythrocytes [28]. Nevertheless, the results indicate
that the beneficial effects of the peptides are mediated via a Ca2+-dependent
stimulation of erythrocyte Na+,K+-ATPase, in analogy with
the previously established signal transduction pathway for C-peptide and Na+,K+-ATPase
in human renal tubular cells [24], even though the EDTA results may
suggest that other metal ions also are of importance for the signal
transduction [29].Exposure of erythrocytes to C-peptide and its
C-terminal fragments is thus likely to result in augmented generation and
release of ATP, as has been observed directly in the case of C-peptide and both
normal and diabetic red blood cells [29]. ATP is a recognized stimulus of
nitric oxide synthase of platelets and erythrocytes [30, 31]. Nitric oxide, besides being a
potent vasodilator, is also effective in eliciting improved red blood cell
deformability as a result of direct action of nitric oxide on the erythrocyte
membrane [32, 33]. In addition, C-peptide-related
improvement of red cell deformability may be mediated not via ATP but by direct
stimulation of red cell nitric oxide synthase. The latter is functionally
similar to that of endothelial cells [31], and C-peptide and most likely also
its C-terminal fragments are known to stimulate and cause induction of
endothelial nitric oxide synthase [8, 10]. The above mechanisms are likely to
contribute to the maintenance of normal erythrocyte membrane plasticity and the
extent to which they are modified in diabetes is unknown. However, irrespective
of the exact mechanism involved, the present findings demonstrate that both
C-peptide and its C-terminal fragments are capable of effectively normalizing
the diabetes-induced reduction in red blood cell deformability. It is probable
that this effect contributed importantly to the observed improvement in
regional blood flow of skin, muscle, myocardium, and peripheral nerve in
animals and type 1 diabetes patients, following administration of C-peptide to
physiological concentrations [11–13, 34]. Furthermore, the beneficial
clinical effects of C-peptide on early-stage neuropathy [14] and nephropathy [15] in type 1 diabetes may in part be
related to correction of the abnormal red cell deformability and subsequently
improved microcirculation. Further clinical trials are warranted document and
define the role of C-peptide and its C-terminal fragments in the treatment
and/or prevention of microvascular complications of type 1 diabetes.



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*Source: 730594-2008-05-06.xml*