Specific Binding of Leptin to Red Blood Cells Delivers a Pancreatic Hormone and Stimulates ATP Release.

Since its discovery in 1994, leptin continues to have new potential physiological roles uncovered, including a role in the regulation of blood flow. Leptin's role in regulating blood flow is not completely understood. Red blood cell (RBC)-derived ATP is a recognized stimulus of blood flow, and multiple studies suggest that C-peptide, a hormone secreted in equimolar amounts with insulin from the pancreatic ß-cells, can stimulate that release when delivered by albumin and in combination with Zn2+. Here, we report leptin delivers C-peptide and Zn2+ to RBCs in a saturable and specific manner. We labeled leptin with technetium-99 m (99mTc) to perform binding studies while using albumin to block the specific binding of 99mTc-leptin in the presence or absence of C-peptide. Our results suggest that leptin has a saturable and specific binding site on the RBC ((Kd = 1.79 ± 0.46) × 10-7 M) that is statistically equal to the binding affinity in the presence of 20 nM C-peptide ((Kd = 2.05 ± 0.20) × 10-7 M). While the binding affinity between leptin and the RBC did not change with C-peptide, the moles of bound leptin did increase with C-peptide, suggesting a separate binding site on the cell for a leptin/C-peptide complex. The RBC-derived ATP increased in the presence of a leptin/C-peptide/Zn2+ addition, in a concentration-dependent manner. Control RBCs ATP release increased (71 ± 5.6%) in the presence of C-peptide and Zn2+, which increased further to (94 ± 5.6%) in the presence of Zn2+, C-peptide, and leptin.

C-peptide enhances glucagon secretion in response to hyperinsulinemia under euglycemic and hypoglycemic conditions.

Several studies have associated the presence of residual insulin secretion capability (also referred to as being C-peptide positive) with lower risk of insulin-induced hypoglycemia in patients with type 1 diabetes (T1D), although the reason is unclear. We tested the hypothesis that C-peptide infusion would enhance glucagon secretion in response to hyperinsulinemia during euglycemic and hypoglycemic conditions in dogs (5 male/4 female). After a 2-hour basal period, an intravenous (IV) infusion of insulin was started, and dextrose was infused to maintain euglycemia for 2 hours. At the same time, an IV infusion of either saline (SAL) or C-peptide (CPEP) was started. After this euglycemic period, the insulin and SAL/CPEP infusions were continued for another 2 hours, but the glucose was allowed to fall to approximately 50 mg/dL. In response to euglycemic-hyperinsulinemia, glucagon secretion decreased in SAL but remained unchanged from the basal period in CPEP condition. During hypoglycemia, glucagon secretion in CPEP was 2 times higher than SAL, and this increased net hepatic glucose output and reduced the amount of exogenous glucose required to maintain glycemia. These data suggest that the presence of C-peptide during IV insulin infusion can preserve glucagon secretion during euglycemia and enhance it during hypoglycemia, which could explain why T1D patients with residual insulin secretion are less susceptible to hypoglycemia.

A C-peptide complex with albumin and Zn2+ increases measurable GLUT1 levels in membranes of human red blood cells.

People with type 1 diabetes (T1D) require exogenous administration of insulin, which stimulates the translocation of the GLUT4 glucose transporter to cell membranes. However, most bloodstream cells contain GLUT1 and are not directly affected by insulin. Here, we report that C-peptide, the 31-amino acid peptide secreted in equal amounts with insulin in vivo, is part of a 3-component complex that affects red blood cell (RBC) membranes. Multiple techniques were used to demonstrate saturable and specific C-peptide binding to RBCs when delivered as part of a complex with albumin. Importantly, when the complex also included Zn2+, a significant increase in cell membrane GLUT1 was measured, thus providing a cellular effect similar to insulin, but on a transporter on which insulin has no effect.

Insights into the physiology of C-peptide.

Current knowledge suggests a complex role of C-peptide in human physiology, but its mechanism of action is only partially understood. The effects of C-peptide appear to be variable depending on the target tissue, physiological environment, its combination with other bioactive molecules such as insulin, or depending on its concentration. It is apparent that C-peptide has therapeutic potential for the treatment of vascular and nervous damage caused by type 1 or late type 2 diabetes mellitus. The question remains whether the effect is mediated by the receptor, the existence of which is still uncertain, or whether an alternative non-receptor-mediated mechanism is responsible. The Institute of Endocrinology in Prague has been paying much attention to the issue of C-peptide and its metabolic effect since the 1980s. The RIA methodology of human C-peptide determination was introduced here and transferred to commercial production. By long-term monitoring of C-peptide oGTT-derived indices, the Institute has contributed to elucidating the pathophysiology of glucose tolerance disorders. This review summarizes the current knowledge of C-peptide physiology and highlights the contributions of the Institute of Endocrinology to this issue.

Intranasal Administration of Proinsulin C-Peptide Enhances the Stimulating Effect of Insulin on Insulin System Activity in the Hypothalamus of Diabetic Rats.

In type 1 diabetes mellitus, the levels of insulin and C-peptide decrease at the periphery and in CNS. C-peptide potentiates the regulatory effects of insulin. We studied the effects of single and repeated (over 7 days) individual and combined nasal administration of C-peptide (10 µg/day) and insulin (20 µg/day) on activity of Akt kinase and kinase-3ß-glycogen synthase (GSK3ß), the components of 3-phosphoinositide pathway, in the hypothalamus of intact rats and rats with mild streptozotocin-induced type 1 diabetes mellitus. Phosphorylation of Akt kinase at Thr308 and Ser473 (stimulation) and GSK3ß at Ser9 (inhibition) was evaluated. In diabetes, phosphorylation of Akt kinase and, to a lesser extent, GSK3ß, is reduced. A single injection of insulin or C-peptide and insulin increased this process. Long-term combined treatment with C-peptide and insulin normalized activity of Akt kinase and GSK3ß in diabetic rats, treatment with insulin alone produced less pronounced effect; monotherapy with C-peptide was ineffective. Intranasal co-administration of C-peptide and insulin effectively stimulates the insulin system in the hypothalamus that is weakened at diabetes mellitus type 1, which can be used in the treatment of this disease.

The pharmacokinetics of porcine C-peptide after intraperitoneal injection.

BACKGROUND: Previously, we have demonstrated that there were very low C-Peptide concentrations and normal blood glucose levels when we transplanted encapsulated islets in the abdominal cavity of diabetic nude mice. In addition, the C-peptide concentration in the ascites fluid of the peritoneal cavity was 40 times higher than in the peripheral blood. In this study, we investigated the pharmacokinetics of intraperitoneal porcine C-peptide. METHODS: To assess the pharmacokinetics of porcine C-peptide, a synthesized porcine C-peptide solution was injected into the peripheral circulation through the tail vein or into the peritoneal cavity in rats at low or high doses of either 200 or 2000 pmol/kg, respectively. Arterial blood samples were collected at time intervals of 1-120 minutes after injection to calculate the terminal elimination half-life (t1/2 ) and area under the time-concentration curve (AUC0-t ). RESULTS: After intraperitoneal C-peptide injection, the highest porcine C-peptide concentration in peripheral blood was only one-fortieth compared to after intravenous injection. The AUC0-t for the intraperitoneal injection was 78% at the low dose and only 39% at the high dose compared to the intravenous injection. This finding indicates that C-peptide remains in the abdominal cavity when intraperitoneally transplanted islets release C-peptide via high glucose stimulation. CONCLUSIONS: Porcine C-peptide injected into a peritoneal cavity slowly and incompletely entered peripheral circulation, which resulted in very low concentration in peripheral blood.

[Co-administration of intranasally delivered insulin and proinsulin C-peptide to rats with the types 1 and 2 diabetes mellitus restores their metabolic parameters.]

The C-peptide, the product of proinsulin proteolysis, not only is a signal molecule, but also, forming a complex with insulin, is able to modulate the signaling functions of insulin. The signaling systems sensitive to insulin in the hypothalamus and other brain areas are among the targets of insulin. We hypothesized that in systemic deficiency of insulin and C-peptide in the type 1 diabetes mellitus (DM) and in severe forms of the type 2 DM, the increase in the level of C-peptide in the CNS will improve central effects of insulin, including its influence on peripheral metabolism. To verify this, the influence of separate and co-administration of intranasal insulin (II) and C-peptide (IP) on their metabolic parameters and sensitivity to insulin in rats with acute and mild type 1 DM induced by the treatment with streptozotocin at the doses of 60 and 35 mg/kg and in rats with neonatal type 2 DM corresponding to severe long-term form of type 2 DM in human was studied. The treatment of animals with II and IP was carried out for 7 days in the daily doses of 20 and 10 µg/rat, respectively. The co-administration of II and IP leading to an increase of insulin and C-peptide levels in the brain was most effective. In rats with type 1 DM treated with the combination of II plus IP, hyperglycemia was decreased and weight loss was prevented. In rats with type 2 DM, co-administration of II and IP led to the normalization of glucose homeostasis and the increase in insulin sensitivity, as shown by glucose-tolerance and insulin-glucose tolerance tests, and to improvement of lipid metabolism, as demonstrated by the decrease in the atherogenic index. The effectiveness of monotherapy with II was lower than in the case of a combination of II+IP, while monotherapy with C-peptide had little effect on the indicators studied. Thus, the simultaneous increase of insulin and C-peptide levels in the brain in the conditions of their deficiency in diabetic pathology can be considered as one of the promising approaches to restore the central insulin-dependent regulation of peripheral metabolism and to improve the utilization of glucose in different forms of DM.

Long-Acting C-Peptide and Neuropathy in Type 1 Diabetes: A 12-Month Clinical Trial.

OBJECTIVE: Lack of C-peptide in type 1 diabetes may be an important contributing factor in the development of microvascular complications. Replacement of native C-peptide has been shown to exert a beneficial influence on peripheral nerve function in type 1 diabetes. The aim of this study was to evaluate the efficacy and safety of a long-acting C-peptide in subjects with type 1 diabetes and mild to moderate peripheral neuropathy. RESEARCH DESIGN AND METHODS: A total of 250 patients with type 1 diabetes and peripheral neuropathy received long-acting (pegylated) C-peptide in weekly dosages of 0.8 mg (n = 71) or 2.4 mg (n = 73) or placebo (n = 106) for 52 weeks. Bilateral sural nerve conduction velocity (SNCV) and vibration perception threshold (VPT) on the great toe were measured on two occasions at baseline, at 26 weeks, and at 52 weeks. The modified Toronto Clinical Neuropathy Score (mTCNS) was used to grade the peripheral neuropathy. RESULTS: Plasma C-peptide rose during the study to 1.8-2.2 nmol/L (low dose) and to 5.6-6.8 nmol/L (high dose). After 52 weeks, SNCV had increased by 1.0 ± 0.24 m/s (P < 0.001 within group) in patients receiving C-peptide (combined groups), but the corresponding value for the placebo group was 1.2 ± 0.29 m/s. Compared with basal, VPT had improved by 25% after 52 weeks of C-peptide therapy (Δ for combined C-peptide groups: -4.5 ± 1.0 µm, placebo group: -0.1 ± 0.9 µm; P < 0.001). mTCNS was unchanged during the study. CONCLUSIONS: Once-weekly subcutaneous administration of long-acting C-peptide for 52 weeks did not improve SNCV, other electrophysiological variables, or mTCNS but resulted in marked improvement of VPT compared with placebo.

A 9-Month Toxicity and Toxicokinetic Assessment of Subcutaneous Pegylated Human C-peptide (CBX129801) in Cynomolgus Monkeys.

C-peptide is formed in the biosynthesis of insulin and is therefore deficient in patients with type 1 diabetes mellitus. A pegylated form of human synthetic C-peptide (CBX129801) has been developed to extend the half-life of the native peptide and is undergoing clinical investigation as replacement therapy to treat diabetic peripheral neuropathy. This monkey study was conducted to evaluate the toxicity of CBX129801 with weekly subcutaneous dosing for 39 weeks at dose levels of 0 (vehicle), 0.4, 1.33, and 4.0 mg/kg/wk. No systemic adverse effects were observed at any dose with maximal CBX129801 plasma concentrations of 735 to 1050 nmol/L during the dosing period (physiological range is 1-3 nmol/L). CBX129801-related effects were limited to minimal macrophagic vacuolization at the injection sites and in the associated draining (axillary) lymph nodes; these local effects largely resolved by the end of a 7-week recovery period. No systemic macrophagic vacuolization was observed. Additionally, there was no histological evidence for plaque formation in the major arteries of these nondiabetic animals.

C-peptide and zinc delivery to erythrocytes requires the presence of albumin: implications in diabetes explored with a 3D-printed fluidic device.

People with type 1 diabetes (T1D) must administer insulin exogenously due to the destruction of their pancreatic ß-cells. Endogenous insulin is stored in ß-cell granules along with C-peptide, a 31 amino acid peptide that is secreted from these granules in amounts equal to insulin. Exogenous co-administration of C-peptide with insulin has proven to reduce diabetes-associated complications in animals and humans. The exact mechanism of C-peptide's beneficial effects after secretion from the ß-cell granules is not completely understood, thus hindering its development as an exogenously administered hormone. Monitoring tissue-to-tissue communication using a 3D-printed microfluidic device revealed that zinc and C-peptide are being delivered to erythrocytes by albumin. Upon delivery, erythrocyte-derived ATP increased by >50%, as did endothelium-derived NO, which was measured downstream in the 3D-printed device. Our results suggest that hormone replacement therapy in diabetes may be improved by exogenous administration of a C-peptide ensemble that includes zinc and albumin.

Enhanced insulin action following subcutaneous co-administration of insulin and C-peptide in rats.

BACKGROUND: This study was undertaken to examine if C-peptide (C) may interact with hexameric insulin and facilitate its disaggregation into the physiologically active monomeric form. METHODS: Regular insulin (I) or an insulin analogue (IA) were injected s.c. in rats together with C or its C-terminal pentapeptide (PP). I or IA and C or PP were administered either as a physical mixture or into two separate s.c. depots. Whole body glucose utilization was evaluated using the euglycemic clamp technique. Phosphorylation of Akt/PKB and GSK in liver and skeletal muscles and 86Rb⁺ uptake by L6 cells were measured. RESULTS: S.c. injection of a mixture of I and C or I and PP resulted in a 30-55% greater (P < 0.01-0.001) and 15-27% (P < 0.05-0.001) longer stimulation of whole body glucose utilization than after separate injections. Insulin-stimulated phosphorylation of Akt/PKB in liver increased 35% more after injection of I and C in mixture compared with after separate injections. Phosphorylation of GSK3 was augmented by 50% (P < 0.05) following the injection of I and C in mixture compared with separate injections. Stimulation of myotubes with premixed I and C (1 nM) elicited 20% additional increase in ouabain-sensitive 86Rb⁺ uptake (P < 0.05) in comparison with the effect when I and C were added separately. CONCLUSIONS: Subcutaneous co-administration of insulin and C results in augmented insulin bioactivity at the level of tissue glucose uptake, intracellular signalling, and enzyme activation. These effects may be attributed to augmented C mediated disaggregation of hexameric insulin into its physiologically active monomeric form.

Renoprotective effects of C-peptide in the Dahl salt-sensitive rat.

Previous studies have demonstrated that renoprotective effects of C-peptide in experimental models of diabetes-induced renal disease may be mediated via lowering blood glucose. The present study examined the renoprotective effects of C-peptide in a model of nondiabetic renal disease, the Dahl salt-sensitive (SS/jr) rat. SS/jr rats were placed on a 2% NaCl diet for 2 wk (HS2, resulting in mild to moderate renal injury) or 4 wk (HS4, resulting in advanced renal injury) and then received either vehicle (veh) or C-peptide (Cpep) for additional 4 wk. Urine albumin (UAE) and protein (UPE) excretion rates were measured at baseline (i.e., before initiation of veh or Cpep treatment) and 4 wk later (i.e., at the time of death). Glomerular permeability, indexes of glomerulosclerosis and tubulointerstitial fibrosis, the presence of inflammatory cells, and protein expression of transforming growth factor-ß (TGF-ß) and podocin were measured at the time of death. In HS2 + veh rats, UAE and UPE increased by 74 and 92%, respectively, from baseline and the time of death. While HS2 + Cpep attenuated this increase in UAE and UPE, HS4 + Cpep had no effect on these parameters. Similarly, HS2 + Cpep reduced glomerular permeability, tubulointerstitial fibrosis, renal inflammation, TGF-ß, and podocin protein expression, while HS4 + Cpep had no effect. These studies indicate that C-peptide is renoprotective in nondiabetic experimental models with mild to moderate renal injury.

Human insulin secreted from insulinogenic xenograft restores normoglycemia in type 1 diabetic mice without immunosuppression.

In the present study, we examined the therapeutic potential of human amnion-derived insulin-secreting cells for type 1 diabetes. Human amniotic mesenchymal stem cells (hAMs) were isolated from amnion and cultivated to differentiate into insulin-secreting cells in vitro. After culture in vitro, the differentiated cells (hAM-ISCs) were intensively stained with dithizone and secreted insulin and c-peptide in a high-glucose-dependent manner. They expressed mRNAs of pancreatic cell-related genes, including INS, PDX1, Nkx6-1, NEUROG3, ISL1, NEUROD1, GLUT1, GLUT2, PC1/3, PC2, GCK, PPY, SST, and GC, and were positive for human insulin and c-peptide. Transplantation of hAM-ISCs into the kidneys of mice with streptozotocin-induced diabetes restored body weight and normalized the blood glucose levels, which lasted for 210 days. Only human insulin and c-peptide were detected in the blood of normalized mice after 2 months of transplantation, but little mouse insulin and c-peptide. Removal of graft-bearing kidneys from these mice resulted in causing hyperglycemia again. Human cell-specific gene, hAlu, and human pancreatic cell-specific genes, insulin, PDX1, GLUT1, GLP1R, Nkx6-1, NEUROD1, and NEUROG3, were detected in the graft-bearing kidneys. Colocalization of human insulin and human nuclei antigen was also observed. These results demonstrate that hAMs could differentiate into functional insulin-secreting cells in vitro, and human insulin secreted from hAM-ISCs following transplantation into type 1 diabetic mice could normalize hyperglycemia, overcoming immune rejection for a long period.

Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide.

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in long-term treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low-cost platform for oral and injectable delivery of cleavable proinsulin.

C-peptide improves neuropathy in type 1 diabetic BB/Wor-rats.

BACKGROUND: The spontaneously diabetic BB/Wor-rat is a close model of human type 1 diabetes and develops diabetic polyneuropathy (DPN) similar to that seen in type 1 patients. Here we examine the therapeutic effects of C-peptide, delivered as continuous infusion or once daily subcutaneous injections on established DPN. METHODS: Diabetic rats were treated from four to seven months duration of diabetes with full continuous replacement dose of rat C-peptide via (a) osmopumps (OS), (b) full replacement dose (HSC) or (c) one-third of full replacement dose (LSC) by once daily injections. RESULTS: Diabetic rats treated with OS showed improvements in motor nerve conduction velocity (p < 0.001), sural nerve myelinated fibre number (p < 0.005), size (p < 0.05), axonal area (p < 0.001), regeneration (p < 0.001) and overall neuropathy score (p < 0.001). The progressive decline in sensory nerve conduction velocity was fully prevented. The frequencies of Wallerian degeneration were decreased (p < 0.005). HSC-treated rats showed prevention of further progression of DPN (p < 0.001), whereas LSC-treated rats showed a milder progression of DPN (p < 0.001) compared to untreated rats as assessed by neuropathy score. CONCLUSION: We conclude that (1) C-peptide is effective in the treatment of established DPN, (2) its effect is dose-dependent and (3) replacement by continuous infusion is the most effective administration of C-peptide.

C-peptide does not affect ocular blood flow in patients with type 1 diabetes.

OBJECTIVE: The aim of the present study was to investigate the effect of intravenous C-peptide infusion on ocular blood flow in patients with type 1 diabetes under euglycemic conditions. RESEARCH DESIGN AND METHODS: The study was performed in a randomized, placebo-controlled, double-masked, two-way, crossover design in 10 type 1 diabetic patients. C-peptide was intravenously administered at two different dosages (dosage 1: 25 pmol . kg(-1) . min(-1) bolus followed by 5 pmol . kg(-1) . min(-1) continuous infusion; dosage 2: six times higher than dosage 1), each for 60 min. Physiologic saline solution was used as a control for C-peptide on a different study day. On both study days, euglycemic clamps were performed. To assess retinal blood flow, laser Doppler velocimetry (blood flow velocities) and retinal vessel analyzer (vessels diameters) measurements were performed. Laser interferometric measurements of fundus pulsation were used to assess pulsatile choroidal blood flow. Blood velocities in the ophthalmic artery were measured using color Doppler imaging. RESULTS: Eight patients (two female and six male) completed the study according to the protocol and without adverse events. One patient developed an anaphylactic reaction to C-peptide, which resolved without sequelae. The following results originate from the remaining eight subjects. Systemic hemodynamic parameters remained stable during both study days. Infusion of C-peptide did not affect any ocular hemodynamic parameter. CONCLUSIONS: The data of the present study indicate that exogenous C-peptide exerts no effect on ocular hemodynamic parameters in type 1 diabetic patients under euglycemic conditions. The maximum detectable change in these parameters was <25%.

Intracerebroventricular injection of proinsulin C-peptide does not influence food consumption in male Long-Evans rats.

Insulin and C-peptide are released in equimolecular concentrations in the circulation after food ingestion as they result from the enzymatic cleavage of proinsulin. In the brain, insulin inhibits food intake through hypothalamic receptors. In the present study, we tested the ability of C-peptide to modulate food intake when injected in the brain lateral ventricle of Long-Evans rats. For this purpose, 10 adult male rats (BW 320 - 350 g) were deprived of food overnight. They were intra-cerebroventricularly injected with 10 microg C-pepitde or vehicle (artificial cerebrospinal fluid [CSF]) during the first hour of the light period and chow intake was measured 1, 3, 6 and 24 hours after injection. Chow availability immediately triggered food consumption. Food intake was not different between CSF- and C-peptide-injected rats either after one hour (5.7 +/- 0.6 g [CSF] vs. 6.7 g +/- 0.5 g; ns) or after 24 hrs (23.3 +/- 1.4 g [CSF] vs. 25.1 g +/- 1.4 g; ns). In addition, a higher dose (20 microg/rat) had no effect at all in satiated rats one hour after injection or later contrary to the 100 % increase measured after injection of 2 microg of neuropeptide Y. Thus, we conclude that contrary to insulin, C-peptide does not regulate feeding behaviour in normal rats whatever their insulin status.

Proinsulin C-peptide activates vagus efferent output in rats.

The aim of this study was to examine the effect of proinsulin C-peptide on the autonomic nervous systems in rats. Intravenous administration of C-peptide gradually increased electrophysiological activity of the vagus nerves into the stomach and pancreas for at least 90 min. It also slightly increased gastric acid secretion that was suppressed by the treatment with atropine. Intraperitoneal injection of C-peptide did not affect the basal and stress-induced norepinephrine (NE) turnover rate, a biochemical index of sympathetic nerve activity. These results indicate that C-peptide increases parasympathetic nerve activity without affecting sympathetic nerve activity. This could explain, at least in part, the ameliorating effects of C-peptide on impaired cardiac autonomic nerve functions in patients with type 1 diabetes.

C-peptide: much more than a byproduct of insulin biosynthesis.

OBJECTIVES: During the past decade, numerous studies in both humans and animals have demonstrated that C-peptide, although not influencing blood sugar control, might play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. The aim of this paper is to present an up-to-date review of C-peptide, focusing on its role in insulin biosynthesis and in the classification of diabetes mellitus, as well as its potential clinical applications. METHODS AND RESULTS: The relevant literature cited in the MEDLINE database shows that the measurement of C-peptide production combined with screening for the presence of islet-cell and other autoantibodies seems to exert an important role in the accurate differentiation between patients with type 1 and type 2 diabetes. Also, both experimental and clinical data provide evidence suggesting that combined replacement of insulin and C-peptide has potential therapeutic value in patients with type 1 diabetes. CONCLUSIONS: Further study in this area is warranted, but the findings that pancreas transplants promote the reversal of diabetic neuropathy and stabilization of diabetic retinopathy and that both pancreas and islet transplants lead to the reversal of diabetic nephropathy lend credence to the concept that combined replacement of insulin and C-peptide may more effectively mitigate the inexorable progression of diabetes-related complications.