Therapeutic potential of C-peptide
Following its discovery in 1967 C-peptide was tested for insulin-like effects but none were found. The possibility that C-peptide may exert direct physiological effects of its own was re-evaluated in the early 1990’s. In a series of studies involving administration of the peptide to patients with type 1 diabetes, who lack C-peptide, it could be shown that replacement of C-peptide in physiological concentrations resulted in significant improvements of several diabetes-induced functional abnormalities.These findings prompted a renewed interest in C-peptide physiology and during the past 20 years a steadily increasing number of reports on new aspects of C-peptide physiology has emerged. The information available today includes studies of the peptide's interaction with cell membranes and its intracellular signaling properties. In vivo studies in animal models of type 1 diabetes have defined a beneficial influence of C-peptide on diabetes-induced functional and structural abnormalities of the kidney and the peripheral nerves. In addition, several clinical studies describing positive effects of C-peptide replacement therapy on nerve and kidney function in type 1 diabetic subjects have been reported. The wealth of information now available supports the hypothesis that C-peptide has therapeutic potential, as summarized below.
For a recent review of this topic, see 
A potential beneficial effect of C-peptide on impaired nerve function in type 1 diabetes has been evaluated in two clinical studies. A double-blind placebo-controlled trial involving 46 patients with type 1 diabetes and early stage neuropathy showed that C-peptide replacement over a 3 month period resulted in a gradual increase in sural nerve conduction velocity (SNCV) reaching 2.7 m/s, amounting to 80% correction of the initial nerve conduction deficit (Fig 1A) as well as improvements in vibration perception . These results have subsequently been confirmed and extended in a larger study involving 139 patients with type 1 diabetes and clinically manifest neuropathy .
Six months of C-peptide administration resulted in improvements in sensory SNCV, vibration perception and clinical scores of neuropathy impairment. C-peptide was administered either as a replacement dose (1.5 mg/day in four subcutaneous injections) or a dose 3 times higher. The higher dose tended to give more favorable results than the replacement dose for all variables, but the difference did not reach statistical significance. The levels of glycemic control were similar in C-peptide and placebo-treated subjects and unchanged during the study. A clinical trial involving 250 type 1 diabetes subjects with neuropathy receiving a long-acting form of C-peptide or placebo is underway; results will be presented early in 2015.
• Fig 1. C-peptide improves sensory nerve conduction velocity and decreases urinary albumin excretion in type 1 diabetes. A. Sensory nerve conduction velocity (SNCV) before and after 6 and 12 weeks of C-peptide therapy (red symbols, n=26) and placebo administration (blue symbols, n=20). Black square represents healthy controls. The asterisk indicates P<0.05 for difference between the C-peptide and placebo groups in change of SNCV from baseline. Data from . B. Urinary albumin excretion rate measured in type 1 diabetes subjects (n=21) receiving C-peptide plus insulin (red symbols) or placebo plus insulin (blue symbols) in a randomized crossover study design. The asterisks indicate P<0.01 for the difference between treatments after 3 months. Data from . [Click to Enlarge]
The mechanism behind the beneficial effect of C-peptide on nerve function may involve several factors. Direct measurements of nerve blood flow in diabetic animals have shown that endoneurial blood flow is substantially reduced in diabetes. Administration of C-peptide in replacement doses resulted in a marked improvement of the endoneurial perfusion deficit, probably as a result of C-peptide mediated stimulation of endothelial nitric oxide synthase (eNOS) and augmented NO availability . C-peptide effects on both endoneurial blood flow and nerve conduction velocity were abrogated by an eNOS blocker, indicating that C-peptide improves nerve function in type 1 diabetes via an NO-sensitive neurovascular mechanism, mediating dilation of the vasa nervorum. Decreased Na+,K+-ATPase activity in peripheral nerve tissue is also a characteristic abnormality in type 1 diabetes. It is associated with inactivation of Na+ channels, intra-axonal sodium accumulation and swelling of the paranodal region during the early phase of the disorder.
C-peptide in physiological concentrations has been shown to prevent or partially correct the diabetes-induced reduction in nerve Na+,K+-ATPase activity in animals with experimental diabetes, thereby contributing to diminished Na+ -retention and partial correction of nerve structural abnormalities, paranodal swelling in particular . Finally, recent studies have shown that C-peptide inhibits hyperglycemia-mediated formation of reactive oxygen species (ROS) in vascular endothelium via several mechanisms , thereby counteracting an important step in the pathogenesis of microvascular complications of diabetes.
Short-term effects of C-peptide administration on renal function have been studied in young type 1 patients without signs of nephropathy other than glomerular hyperfiltration, typical of the early stage of this disorder. C-peptide, infused for 2 h at rates sufficient to achieve physiological plasma concentrations, resulted in a decreased glomerular filtration rate and a slightly increased renal plasma flow. These observations were subsequently extended in a double-blind randomized study in young type 1 patients with incipient nephropathy as reflected by mild microalbuminuria . C-peptide was infused s.c. together with insulin for 4 weeks. The glomerular filtration rate decreased and there was a reduction in albumin excretion to approximately half the basal value after 4 weeks in the group receiving C-peptide.
The above findings have been further explored in a clinical trial involving C-peptide administration for 3 months. A double-blind, placebo-controlled, randomized, crossover study design was employed and patients with mild nephropathy received C-peptide plus insulin for 3 months and insulin plus placebo for 3 months. Pre-study urinary albumin excretion rates were on average 52 µg/min and all patients were normotensive. During the C-peptide treatment period, urinary albumin excretion decreased progressively to values 40% below those observed at baseline period (Fig 1B). When the patients received insulin only, albumin excretion did not change significantly and the difference between groups was significant (P<0.01). All patients remained normotensive throughout the study and glycaemic control improved slightly but to the same extent during the two treatment periods. Thus, the diminished albumin excretion during C-peptide therapy could not be ascribed to a reduction in arterial blood pressure or to an improvement in blood glucose control. Taken together, the evidence supports the view that C-peptide in replacement doses has the capacity to limit glomerular hyperfiltration and to reduce urinary albumin excretion in type 1 diabetes subjects with mild nephropathy.
Several studies in animals with experimental diabetes demonstrate that C-peptide is capable of exerting renoprotective effects. The peptide binds specifically to renal cell membranes and has the capacity to regulate the activity of renal glomerular and tubular Na+,K+-ATPase, endothelial nitric oxide synthase and several transcription factors (NF-kβ, PPARγ, ZEB, CREB, ATF1 and Bcl-2) of importance for cellular growth, inflammatory responses and cellular apoptosis, for an overview see . In addition, C-peptide has recently been shown to exert potent antioxidative effects by inhibiting ROS generation in endothelial cells exposed to hyperglycemia . Administration of replacement doses of C-peptide to rats with streptozotocin (STZ)-induced diabetic nephropathy has been shown to prevent glomerular hypertrophy and mesangial expansion typical of this disorder, abolish glomerular hyperfiltration, limit the transcription of the pro-fibrotic cytokine TGFβ-1 and markedly reduce the urinary excretion of albumin , thus providing an experimental basis for C-peptide´s renoprotective effects in subjects with type 1 diabetes.
No clinical trial has focused directly on the effect of C-peptide on diabetic retinopathy. Retinal function has, however, been evaluated in a study involving subcutaneous infusion of insulin and native C-peptide by pump for four weeks in type 1 diabetes subjects without signs of structural retinal damage. A double-blind, randomized study design with two parallel groups was used. It was found that fluorescein leakage across the blood-retinal barrier (BRB), thought to be an early indicator of retinal vascular damage, decreased by approximately 30% in the subjects receiving C-peptide (P<0.05) but not in those on insulin alone . In a subsequent study with a double-blind, randomized, cross-over design involving 21 type 1 diabetes subjects and 3 months treatment with C-peptide plus insulin or placebo plus insulin, fluorescein leakage across the BRB tended to improve but the difference between the groups receiving C-peptide and placebo did not reach statistical significance . Although not conclusive, the clinical findings show trends in agreement with experimental findings in diabetic animals.
An effect of the peptide on retinal vascular dysfunction in STZ diabetic rats has been reported following five weeks of C-peptide administration. Extravascular permeation of 125I-labelled albumin in the retina was increased in the diabetic animals and C-peptide administration was found to markedly attenuate the albumin permeation to levels comparable to those in the healthy control animals , indicating that C-peptide mediated a reduction of retinal vascular
• Fig 2. C-peptide prevents retinal vascular leakage in diabetic mice. Non-diabetic and streptozotocin diabetic mice were injected with 2 μl C-peptide (2.0 pmol) or saline intravitreally. 24 hours post-injection, fluorescein was injected and the retinas were examined for vascular leakage using confocal microscopy. Lower row graphs present a magnified view.  (Click to enlarge)leakage.
This finding has been confirmed and extended in a study involving STZ-diabetic mice. At six days after STZ injection, when hyperglycemia had become established, the animals were given an intravitreous injection of 2 pg C-peptide, allowing physiological C-peptide levels to be maintained within the vitreous space. Retinal vascular permeability was evaluated using fluorescein angiography and confocal microscopy at 24 h post C-peptide administration. Untreated diabetic animals showed marked retinal extravasation of fluorescent material, while C-peptide injection was found to completely prevent vascular leakage, rendering the angiography image in the C-peptide treated animals similar to that for non-diabetic animals (Fig.2) .
Several mechanisms may have contributed to C-peptide´s beneficial effects on retinal vascular function in the diabetic state. Vascular endothelial growth factor (VEGF) has been implicated as a primary mediator of augmented vascular permeability in diabetic retinopathy; the endothelial expression of VEGF being increased by high glucose, oxidative stress and inflammatory reactions. In line with this hypothesis, administration of a monoclonal anti-VEGF antibody almost completely prevented the vascular leakage seen in the STZ-diabetic mice. The negative influence exerted by VEGF is mediated by its stimulatory effect on endothelial generation of reactive oxygen species (ROS) which, in turn, leads to capillary wall stress fiber formation and interruption of vascular endothelial cadherin-based adherens junctions, resulting in augmented vascular permeability.
The beneficial effect observed for C-peptide may be mediated by its inhibitory influence on VEGF-elicited ROS formation. This is a direct effect on NADPH oxidase-2 and/or an effect via stimulation of AMPK . In addition, a 6-fold increase in leukocyte infiltration was observed in the retinas of diabetic mice. This infiltration was almost completely blocked following the intravitreal administration of C-peptide, probably reflecting an inhibitory influence of C-peptide on the endothelial expression of cell adhesion molecules and subsequent diminished leukocyte adhesion to the endothelial surface .
Overall, the clinical and experimental findings provide support for a beneficial effect of C-peptide on diabetic retinopathy, particularly its early stage. The available evidence indicates a clear need for a long-term clinical trial to define the potential role of C-peptide in the treatment of diabetic retinopathy.
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