# Effect of C-Peptide on Diabetic Neuropathy in Patients with Type 1 Diabetes

Authors: Karin Ekberg; Bo-Lennart Johansson
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Recent results indicate that proinsulin C-peptide, contrary
to previous views, exerts important physiological effects and
shows the characteristics of a bioactive peptide. Studies in
type 1 diabetes, involving animal models as well as patients,
demonstrate that C-peptide in replacement doses has the
ability to improve peripheral nerve function and prevent or
reverse the development of nerve structural abnormalities.
Peripheral nerve function, as evaluated by determination of
sensory nerve conduction velocity and quantitative sensory
testing, is improved by C-peptide replacement in diabetes type
1 patients with early stage neuropathy. Similarly, autonomic
nerve dysfunction is ameliorated following administration of C
peptide for up to 3 months. As evaluated in animal models of
type 1 diabetes, the improved nerve function is accompanied by
reversal or prevention of nerve structural changes, and the
mechanisms of action are related to the ability of C-peptide
to correct diabetes-induced reductions in endoneurial blood
flow and inNa+,K+-ATPase activity and modulation of neurotrophic
factors. Combining the results demonstrates that C-peptide may be
a possible new treatment of neuropathy in type 1 diabetes.


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## Body

## 1. Introduction

Neuropathy is one of the most common long-term complications accompanying diabetes mellitus. It affects
patients with both type 1 and type 2 diabetes, but it progresses more rapidly and its manifestations are more severe in type 1
diabetes [1, 2].
Diabetic neuropathy is defined by the presence of detectable sensory,
motor, and autonomic deficits on clinical examination, with or without the presence of symptoms [3, 4]. As many as 50% of the patients may be asymptomatic, diagnosis
may only be made on examination or, in some cases, when the patient presents with a painless foot ulcer [5]. Other patients may not volunteer symptoms
but on inquiry admit that their feet feel numb or dead.
A thorough neurological examination of the lower limb usually
reveals sensory loss of vibration, pressure, pain, and temperature
perception mediated by small and large fibers, and
absent ankle reflexes. In addition to manifestations of autonomic neuropathy, for
example, impaired cardiovascular and gastrointestinal functions, signs of
peripheral sympathetic autonomic dysfunction are
also frequently seen in patients with diabetes and may include a warm or cold foot,
sometimes with distended dorsal foot veins, dry skin, and the presence
of calluses under pressure-bearing areas. Diabetic neuropathies may present as
rapidly reversible hyperglycemic neuropathy and focal or multifocal
neuropathies, but the most relevant clinical form is the persistent distal
symmetric polyneuropathy (DSPN) [4].The DSPN is characterized as a gradual progression in structural changes consisting of distal axonal
degeneration of “dying-back” type [6, 7]
most prominent in the lower limbs, but involves also small fiber sensory
dysfunction early in the course of the condition [7].
The prevalence of DSPN is approximately 30% for diabetes patients in general [8], but the number varies greatly in the literature related to the definition
chosen for presence of diabetic neuropathy and the methodology chosen to assess
its presence. Clinical examinations and patients' symptom assessment are
considered important tools in the evaluation of neuropathy status, but both
techniques rely greatly on subjective components and have thus poor
reproducibility and specificity. Assessments using more objective markers of
polyneuropathy, such as especially nerve conduction velocity (NCV) but also
vibration perception threshold (VPT), may serve not only as reliable methods
for detection of neuropathy, but the result may also be used for the prediction
of mortality in diabetic patients [9, 10]. The pathogenesis of diabetic neuropathy
involves metabolic effects mediated directly and indirectly by hyperglycemia,
resulting in oxidative stress, accelerated polyol pathway metabolism and
generation of advanced glycation endproducts [11–13]. Furthermore, diabetic neuropathy is accompanied by reduced nerve
Na+,K+-ATPase activity, and microvascular abnormalities (e.g., reduced endoneurial perfusion) [14]. Type 1 diabetes is associated with specific structural nerve abnormalities that
are not frequent in type 2 diabetes. These abnormalities include axonal atrophy
and characteristic nodal and paranodal changes that contribute to the
progressive deterioration of nerve conduction velocity [15–17]. In contrast, in type 2
diabetes the axonal degeneration is milder and no or only minimal nodal and paranodal
abnormalities occur [17, 18]. However, after several years
type 2 diabetes often become insulin and C-peptide deficient, and at this stage
it is most likely that the type 2 DSPN will start presenting with characteristics
similar to that of type 1 neuropathy.In the case of type 1 diabetes, available data suggest that C-peptide deficiency is an important
contributing factor to the characteristic structural abnormalities [21]. In
conformity with this hypothesis several studies have demonstrated
that it is possible to retard the progression of diabetic complications by intensified insulin treatment and improved metabolic
control, but development of neuropathy cannot be prevented [22–24]. Thus, other factors, such as
C-peptide deficiency, are likely to be of importance for the progression of
diabetic neuropathy in type 1 diabetes. Evidence documenting
significant physiological effects of C-peptide has been presented during the
latest decades. It shows that C-peptide, in contrast to previous belief,
possesses the characteristics of a bioactive peptide. C-peptide binds
specifically to various cell membranes, including endothelial, renal and nerve
cells [25],
with subsequent activation of an intracellular signaling cascade resulting in
stimulation of endothelial nitric oxide synthase (eNOS) and Na+,K+-ATPase
[26].
Moreover, recent data indicate that C-peptide stimulates several
transcriptional factors, as well as several neurotrophic factors [27].
Thus, it has been demonstrated that exogenous administration of C-peptide in
replacement dose to patients lacking endogenous C-peptide results in
restoration of reduced blood flow in several tissues [28–31] and improvement of renal [32] and
nerve function, the latter reviewed below. It is also a long-standing clinical
observation that compared with type 1 diabetes patients in whom beta-cell
secretion ceases totally, those patients who retain a low-endogenous C-peptide
and insulin secretion are less prone to develop microvascular long-term
complications involving the kidneys, the eyes, or the nervous system [33–35].


## 2. Clinical Studies on C-Peptide and  Nerve Function

To date, only a few studies on the clinical
effects of C-peptide on nerve function have been performed, but several preclinical
studies indicate significant effects on diabetes-induced nerve dysfunction and
structural abnormalities [36–38]. The available clinical results indicate beneficial effects of C-peptide on both peripheral and autonomic nerve
function in type 1 diabetes patients. Thus, in a double-blind,
placebo-controlled study including 46 type 1 diabetes patients, with an average
age of 29 years and approximately 10 years of diabetes duration, and with reduced
sensory nerve and motor nerve conduction velocities (NCV) but no other signs of
neuropathy, C-peptide replacement (1.8 mg/day) or placebo was given for 3 months together with the
patients’ regular insulin therapy [19]. Sensory nerve conduction velocity assessed in
the sural nerve bilaterally, but not motor NCV (peroneal nerve), increased
gradually during the study. The increase after 3 months amounted to 2.7 m/s,
corresponding to an 80% correction of the initial conduction velocity deficit
in these patients. This change was accompanied by an improvement in vibration
perception assessed on the dorsum of the feet, although these patients had
essentially normal perception thresholds already at baseline. This improvement
is consistent with an improved sural nerve function since vibration perception
in this anatomic region is primarily mediated via the large fibers of the sural nerve.The improvement in nerve function demonstrated in this early patient population is now confirmed and extended in a recently
completed clinical trial including patients with diabetic neuropathy [20]. The study was a double-blind,
placebo-controlled, randomized multicenter study, including 161 type 1 diabetes
patients, with an average age of 44 years and 29 years of diabetes duration and
defined DSPN (according to the San Antonio criteria [3]). At baseline their sensory nerve conduction
velocity assessed in the sural nerve (SCV) was on average 2.6 SD below normal,
and following 6 months of C-peptide replacement treatment there was a statistically
significant improvement in SCV for the patients receiving C-peptide, amounting to 0.48±0.19 m/s assessed as peak
velocities and 0.93±0.29 m/s for the initial response (the velocity change from
baseline for the fastest axon in the nerve). However, these changes were not
statistically significantly different from the change in the placebo group. The
number of responders, defined as patients with an improvement in peak SCV > 1 m/s,
was statistically significantly greater in the group receiving C-peptide as compared to those receiving placebo (37% versus
19%, resp., p < .032). It is noteworthy though that among the included
patients some had substantial nerve conduction deficits at baseline and with
the study duration of no more than 6 months it is conceivable that the patients
who were relative less affected at baseline may have a greater potential for
improvement. Thus, a subgroup analysis was performed in the subset of patients
with the least affected SCV at baseline (half of the patient population). In
this group, C-peptide administration for 6 months resulted in an improvement that was 1.03 m/s
greater as compared to the change in the corresponding placebo treated patients
(P < .014). Analyzing the number of responders in this half of the patient population
revealed that among patients receiving C-peptide, 39% demonstrated an improvement in
SCV >1 m/s whereas only 5% among the placebo treated patients showed a similar change
P < .004. Accompanying these changes in sural nerve conduction velocity there
where, however, no statistically significant change in motor nerve conduction
velocity, but there was an improvement within the C-peptide treated patients for vibration perception.
There was also a trend towards an improvement in neurological examination
scores following C-peptide. Combining these data provides evidence of a therapeutic improvement of diabetes-induced
peripheral nerve dysfunction following C-peptide administration in patients with type 1
diabetes; 3–6 months of C-peptide replacement to patients with early stage
neuropathy resulted in approximately 1.5 m/s in sensory nerve conduction
velocity (see Figure 1) accompanied by other signs of nerve function
improvements.Figure 1
Change in peak sensory nerve conduction
velocity in the sural nerves following 3–6 months of C-peptide
replacement treatment (red bar) or placebo (gray bar) to patients with type 1 diabetes. The figure presents pooled
data from [19, 20].There is also evidence of beneficial effects of C-peptide administration to
type 1 patients with signs of autonomic neuropathy. Deficient autonomic nerve
function may be evaluated in patients as reduced heart rate variability (HRV)
during deep breathing, a measurement that, with a high degree of
reproducibility, primarily reflects vagal function. Patients were studied twice
under normoglycemic conditions and during 3-hours intravenous infusion of
either human C-peptide
or saline in a double-blind study. At baseline HRV was reduced13±1% (normal
reference value 24%) and during the C-peptide infusion which restored plasma concentrations
to physiological levels, the HRV improved to 20±2%, while no change was seen
after saline infusion (p < .001) [39]. The heart rate brake index after a tilting
maneuver was also improved after C-peptide for 3 hours in the patients showing reduced
index before the study. In agreement with these results, a 20% improvement in
HRV was seen after 3 months of C-peptide replacement in type 1 diabetes patients
whereas no change or a slight deterioration was observed in the same patients
during a placebo treatment period [32].


## 3. Treatment of Diabetic Neuropathy

There is no effective pharmaceutical therapy
available for diabetic neuropathy today. The onset and the progression of the
diabetes-induced abnormalities may be delayed by maintenance of good glycemic
control [22–24]. In the DCCT, the incidences of diabetes neuropathy were substantially
lower in patients on intensive insulin treatment as compared to conventional
insulin therapy [23]. For the patients in the DCCT secondary
prevention group, with an average age of 28 years and a diabetes duration of 9
years, the 2% improvement in HbA1c seen following 5 years of
intensified insulin treatment was accompanied by an improved SCV of 1.5 m/s
whereas patients with unchanged metabolic control in the conventional treatment
group experienced a 2.2 m/s reduction in SCV. Interestingly, the patients in
this group are directly comparable to the patients in the first C-peptide intervention study (average age 29 years and
diabetes duration of 10 years) where 3 months of C-peptide replacement resulted in 2.7 m/s improvement in
SCV, and it is of note that these patients were already on an intensified
insulin treatment regimen [19]. The magnitude of the response following C-peptide replacement treatment occurred completely
independent of improved glycemic control, and in fact in top of an already good
glycemic control. This suggests that the C-peptide is in fact acting on DSPN disease-modifying
mechanisms. Impaired nerve blood flow secondary to perturbed nitric oxide
metabolism [36, 38] and reduced levels of nerve Na+,K+-ATPase
activity [40, 41] are both factors that have been implicated in
the pathogenesis of the DSPN [7]. The ability of C-peptide to improve endoneurial
blood flow and Na+,K+-ATPase activity as well as its stimulation of
neurotrophic factors as demonstrated in several animal models of type 1
diabetes [27, 37, 40] is thus likely to contribute to the positive effects of the peptide.In addition to improved insulin therapy, most
therapeutic interventions previously evaluated for DSPN have been directed toward
correction of the adverse effects of hyperglycemia. For example, one approach
involves the reduction of the intracellular sorbitol accumulation by aldose
reductase inhibitors (ARI). Although shown to have beneficial effects on
neuropathy [42, 43], several clinical trials involving ARIs have
been discontinued because of unacceptable adverse effects, for example, skin
rash, renal toxicity, and serious hepatic effects [44]. A new and apparently well-tolerated ARI,
ranirestat, is currently in development, and phase II data indicate that its
administration for 60 weeks to mostly type 2 diabetes patients has beneficial
effects on nerve function [45, 46]. Another
compound also aiming to minimize the effects of hyperglycemia is
the specific protein kinase C beta inhibitor, ruboxistaurin, but recent
clinical development has not documented impressive clinical effect [47].It has become increasingly apparent that DSPN
presents a clear unmet medical need, and the health authorities and
representatives for patient associations have expressed their concern [5]. Previously, the regulatory agencies have
required efficacy on symptom relief and reduced wound and amputation frequency
in order to accept a new drug application. However, it is now becoming increasingly
recognized that treatment should be started well, before the deterioration of
nerve function has reached the stage of severe symptoms and wounds. Moreover,
the treatment should not only be directed at a symptom relief but to modify the
underlying disease mechanisms. In the case of peripheral neuropathy
accompanying type 1 diabetes, the beneficial effects on
nerve function following C-peptide replacement therapy may indicate a new potential
treatment paradigm, even though extended clinical trials will be needed to
finally elucidate its usefulness.



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