1 Department of Surgical Sciences, Section of Clinical Physiology, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden
2 Section of Clinical Neurophysiology, Karolinska Institutet, Stockholm, Sweden
3 Section of Neurology, Karolinska Institutet, Stockholm, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Diabetic neuropathy is defined by the presence of detectable sensory, motor, and autonomic deficits on clinical examination, with or without the presence of dysesthetic or paresthetic symptoms (1). Factors of importance for the pathogenesis of diabetic neuropathy are formation of advanced glycation end products and polyol pathway activation following hyperglycemia, reduced nerve Na+,K+-ATPase activity, and microvascular abnormalities, e.g., reduced endoneurial perfusion (2). Although several studies demonstrate that it is possible to retard the progression of diabetic complications by intensified insulin treatment, development of neuropathy cannot be prevented (35). Recently, interest has focused on the possibility that C-peptide, previously considered to lack biological activity, may play a beneficial role in the treatment and prevention of diabetic complications (6).
During the last decade, C-peptide has been found to exert a number of physiological effects in patients with type 1 diabetes (6). These effects are probably mediated via a G-protein-coupled membrane receptor (7,8). Upon stimulation, a Ca2+-dependent signaling pathway is activated, resulting in a stimulation of Na+,K+-ATPase and endothelial nitric oxide synthase (eNOS) activities (8,9). Both of these enzyme systems are known to be deficient in diabetes (10,11). C-peptide has been shown to exert beneficial effects on both diabetic nephropathy and neuropathy in animal models of type 1 diabetes and in patients with type 1 diabetes. These effects are partly related to the ability of C-peptide to stimulate blood flow and promote capillary recruitment in peripheral tissues, but possibly also to direct stimulation of Na+,K+-ATPase (8,12). In the early stages of diabetes C-peptide reduces glomerular hyperfiltration (13,14), and in patients with incipient diabetic nephropathy C-peptide replacement results in diminished urinary albumin excretion rate (15). Similar effects have been demonstrated during short- and long-term administration of C-peptide in streptozotocin (STZ)-induced diabetic rats (STZ rats), where it has been demonstrated that the beneficial effects of C-peptide are accompanied by corrections of morphologic abnormalities secondary to the diabetic state (16,17). C-peptide replacement in BB/Wor rats partially reverses acute and chronic metabolic, functional, and structural changes in peripheral nerves following the onset of diabetes (12). The diabetes-induced reduction in motor nerve conduction velocity was arrested by C-peptide, and the Na+,K+-ATPase activity of the nerves was partly restored in rats treated with replacement doses of C-peptide during 38 months. Furthermore, C-peptide treatment resulted in decreased paranodal swelling and demyelination, decreased axonal degeneration, and augmented regenerative activity (12). In addition to the stimulation of Na+,K+-ATPase activity, the C-peptide effects on nerve function and structure may be mediated by an improved nerve microcirculation secondary to stimulation of vasa nervorum eNOS activity. In STZ rats treated with C-peptide for 2 weeks, the improvement in motor and sensory nerve conduction velocity was accompanied by a near normalization of nerve blood flow (18).
Only little information is available with regard to the possible influence of C-peptide on nerve function in humans. However, C-peptide augments the capacity of the heart to adjust to fluctuations in venous inflow during deep breathing in patients with early signs of autonomic neuropathy, as indicated by improved heart rate variability during respiratory excursions (19). After only a few years of type 1 diabetes, asymptomatic patients present with reduced nerve conduction velocities (20). The aim of the present study was to examine whether C-peptide may exert a positive influence on early peripheral nerve function abnormalities in patients with type 1 diabetes. Specifically, the effect of 3 months of C-peptide treatment on peripheral nerve conduction velocity and other early signs of diabetic neuropathy were investigated.
![]() |
RESEARCH DESIGN AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the 54 patients, 2 did not fit the inclusion criteria, and 3 withdrew their consent to participate before start of trial medication. Thus, 49 patients started on trial medication. Three of these, all randomized to active treatment, were considered major protocol violators due to doubtful compliance, and their results were excluded from further analysis. Of the remaining 46 patients completing the study, 26 were randomized to C-peptide and 20 to placebo. All patients were informed of the nature, purpose, and possible risk of the study before consenting to participate. The protocol was approved by the institutional human ethics committee and by the Swedish Medical Product Agency, and the study was conducted in accordance with Good Clinical Practice guidelines and the principles of the Declaration of Helsinki.
For comparison, 15 healthy volunteers (10 men and 5 women) matched for age, sex, and body height were recruited. Their mean age was 30 ± 2 years and mean height was 169 ± 3 and 180 ± 2 cm for women and men, respectively. The control subjects underwent the same sensory function and neurophysiological examinations as the patients at baseline.
Protocol.
The study was carried out in a double-blind placebo-controlled randomized fashion with two study arms: treatment with either C-peptide or placebo for 3 months. After giving informed consent, patients underwent a physical examination, including a neurological evaluation, electrocardiogram at rest and during deep breathing, blood pressure measurement, and clinical chemistry laboratory testing. In addition, samples for C-peptide levels in plasma and urine were collected. Furthermore, a neurophysiological examination was carried out including measurements of nerve conduction velocities (NCV), and sensory function was assessed by quantitative sensory testing (QST). The patients found to fit the inclusion criteria were randomized to either of the treatment groups; 60% were assigned to treatment with C-peptide and 40% to placebo. The patients were instructed to take the trial medication four times daily as subcutaneous injections of 120 nmol C-peptide in connection with their regular insulin administration in the morning, at lunch, and at dinner and 240 nmol C-peptide at bedtime or an equivalent volume of C-peptide diluent (placebo). C-peptide was recombinantly produced and kindly provided by Schwarz Pharma, Monheim, Germany. This C-peptide dose regimen, 600 nmol/24 h, is equimolar to a dose of 100 IU insulin and is expected to result in C-peptide plasma levels within the physiological range during 1820 h per day. After 6 weeks of treatment, renewed assessments of the NCV were performed, and after 12 weeks the NCV and QST were measured again. Thereafter, the study was ended and the trial medication discontinued. Patient compliance with the treatment was checked by visual control of the returned medication vials and on the basis of analyses of C-peptide concentration in urine and plasma samples during and at the end of the study (evaluation performed by an independent analyst). Randomization and blinding of the trial medication was performed by the Karolinska Hospital Pharmacy, and source data verification was monitored by an external monitor (Vinkla Konsult, Sollentuna, Sweden).
Sensory function and neurophysiological assessments.
Motor nerve conduction velocity (MCV) and compound muscle action potential amplitude (CMAP) in the peroneal nerve and sensory nerve conduction velocity (SCV) and sensory nerve action potential amplitude (SNAP) in the sural nerve were measured bilaterally as previously described (20) using surface electrodes and digital equipment for stimulation and recording (Neuropak 2; Nihon-Kohden, Kyoto, Japan). The assessments were performed under strictly standardized conditions in a warm room, with the legs warmed with heat pads for at least 10 min before the nerve conduction measurements in order to obtain skin temperatures at 34°C. The coefficient of variation (CV) for MCV and SCV, respectively, was 2.7 and 4.9%, based on determinations made in the same individual on separate occasions. The QST included measurements of perception thresholds for vibration, heat and cold. A vibrating probe (Vibrameter; Somedic, Stockholm, Sweden) was applied over the first metatarsal to allow evaluations of the vibration perception. Temperature threshold determinations were done with the Marstock technique using a temperature-regulated probe (Thermotest, Somedic, Stockholm, Sweden) (21). The probe was applied over the dorsum of the feet, and the patient reported temperature sensations by pressing a button on perception of heat, cold, etc. All respective measurements of perception thresholds were done bilaterally and in triplicate, and the mean of all six measurements was used in the statistical analysis.
Analyses.
Clinical chemistry variables, including HbA1c, were determined according to the standard procedures at the Department of Clinical Chemistry at the Karolinska Hospital. C-peptide concentrations in plasma and urine were analyzed by radioimmunoassay using a commercial kit (Eurodiagnostica, Malmö, Sweden).
Statistical methods.
Power analysis was performed on the basis of the results published by Hylliemark et al. (20), reporting a significant decrease in both MCV and SCV in a cohort of 75 young type 1 diabetes patients. The sample size of the two groups in the present study, 20 taking placebo and 30 taking C-peptide, was estimated from the assumption that a two-group t test, with a 0.05 two-sided significance level, would have 80% power to detect the difference between a 5% change of NCV in the C-peptide-treated group and no change in the placebo group, assuming that the common standard deviation (SD) was 4.0 m/s.
All data are presented as means ± SE. Statistical tests were performed using nonparametrical methods. The Mann-Whitney two-sample test was applied in comparisons between groups. When comparing changes within groups, Wilcoxons signed-rank test was used. The data were analyzed on a per protocol basis, i.e., including only those subjects of the full analysis set who did not show major protocol violations. The decision whether a protocol deviation should be considered minor or major was made by a panel including the trial manager and the investigators before the randomization code was opened. The safety analysis data set included all subjects who received at least one dose of C-peptide.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analogous with the findings for NCV, there was a proportionate response in the QST results. C-peptide treatment for 3 months resulted in an improved vibration perception, which is in good agreement with the findings in SCV, since vibration perception in the feet is primarily mediated via the sural nerve. Again, there was a greater vibration threshold improvement in the patients showing the poorest response at baseline. This was also the case for the heat perception threshold, although it was not possible to detect an overall statistical significant improvement for either heat or cold perception thresholds.
It has previously been shown that C-peptide ameliorates autonomic nerve dysfunction in patients with type 1 diabetes (19), and the results in the present study may serve as a first indication of a potential effect of C-peptide on peripheral nerve dysfunction in type 1 diabetic patients. In animals, C-peptide has been shown to improve both functional and structural changes caused by diabetes (12). The BB/Wor rat represents a type 1 diabetes animal model that spontaneously develops diabetes. BB/Wor rats were treated with rat C-peptide for 38 months in controlled randomized studies. The C-peptide treatment prevented the progression of diabetic neuropathy as evaluated by sciatic tibial MCV measurements, and significant improvement in MCV was seen after 8 months of treatment. In addition, examination of sensory nerve (sural) morphology after C-peptide treatment demonstrated significantly less marked structural alterations (reduced paranodal swelling and demyelination, increased number of intercalated nodes, and regenerating fibers) compared with untreated diabetic rats. Furthermore, and in agreement with previous observations (24), the C-peptide treatment resulted in increased nerve Na+,K+-ATPase activity (12). Recently, it was demonstrated that improvements in MCV and SCV following C-peptide replacement in rats with STZ-induced diabetes was accompanied by a marked improvement of nerve blood flow (18). This improvement was completely abolished in the presence of a NOS blocker, and the effect is thus most likely mediated via C-peptides ability to stimulate endothelial nitric oxide production and subsequently blood flow, as previously demonstrated for other tissues (9,2528).
It is primarily in C-peptide-deficient patients that a series of different physiological effects of C-peptide have been demonstrated. Healthy subjects, with normal C-peptide plasma concentrations, show no detectable response to C-peptide administration. A possible explanation to this phenomenon derives from in vitro experiments, in which half-saturation of C-peptide binding was already demonstrated at 0.3 nmol/l and full saturation at 0.9 nmol/l (7). Because full saturation of C-peptide binding sites already occurs at the ambient C-peptide concentration in healthy subjects, further physiological effects cannot be expected when the concentration is increased above the saturation level. Dose-dependency for C-peptide effects have been demonstrated in vivo in animal models of diabetes and in type 1 diabetic patients (27,29). It is noteworthy that the patients included in the present study all had C-peptide plasma concentrations <0.2 nmol/l at baseline. However, when the patients are stratified according to baseline C-peptide plasma levels, it becomes apparent that the increase in SCV after 3 months of C-peptide tended to be greater in the patients with nondetectable C-peptide levels at baseline (7.1%) compared with those with baseline concentrations in the range of 0.130.20 nmol/l. In the latter group, no significant improvement could be seen. The importance of this seemingly marked difference is limited by the fact that only four patients presented with C-peptide plasma levels 0.130.20 nmol/l at baseline. Nevertheless, the observation suggests that the beneficial effect of C-peptide is most marked in the C-peptide-negative patients.
Diabetic neuropathy is a highly disabling condition for which there is no causal therapy available today. Maintenance of good metabolic control may delay the progression of diabetic neuropathy but will not restore nerve function (3). Most of the pharmaceutical agents tested aim to prevent or retard the metabolic consequences of hyperglycemia. Among these agents aldose reductase inhibitors (ARIs) have been the most promising. Although shown to have beneficial effects on neuropathy (30,31), several clinical trials of ARIs have been discontinued because of unacceptable adverse effects, e.g., skin rash, renal toxicity, and serious hepatic effects (32,33). In a recent study, a new well-tolerated ARI fidarestat was administered during 52 weeks to mostly type 2 diabetic patients (34). In contrast to the present findings following C-peptide, fidarestat did not improve SCV, but it significantly increased MCV by 0.9 m/s, as measured by F-wave conduction velocity in the median nerve. Another compound also aiming to minimize the effects of hyperglycemia is the specific protein kinase Cß inhibitor LY333531. After 58 weeks of treatment a statistically significant improvement of MCV could be demonstrated (35). This finding is in agreement with the argument presented above that motor and sensory nerve dysfunction in type 1 diabetes may have separate pathogenic backgrounds. The positive effect on sensory function following C-peptide replacement is most likely mediated via other mechanisms than influence on hyperglycemia, as discussed above. Irrespective of the mechanisms involved, it is intriguing to consider that lack of C-peptide, an endogenous peptide previously believed to be without biological activity of its own, may possibly play a role in the development of long-term diabetes complications. Further studies are warranted to investigate the possible role of C-peptide in the treatment and prevention of diabetic neuropathy.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Eva-Lena Forsberg, Monika Jurkiewicz, Agneta Reinholdsson, Alice Skogholm, and Ann-Marie Åberg for excellent technical assistance.
![]() |
FOOTNOTES |
---|
Received for publication 27 August 2002 and accepted in revised form 11 November 2002.
ARI, aldose reductase inhibitor; CMAP, compound muscle action potential; eNOS, endothelial nitric oxide synthase; MCV, motor nerve conduction velocity; SCV, sensory nerve conduction velocity; SNAP, sensory nerve action potential amplitude; STZ, streptozotocin
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|