Molecular effects of proinsulin C-peptide

The proinsulin C-peptide has been held to be merely a by-product in insulin biosynthesis, but recent reports show that it elicits both molecular and physiological effects, suggesting that it is a hormonally active peptide. Specific binding of C-peptide to the plasma membranes of intact cells and to detergent-solubilised cells has been shown, indicating the existence of a cell surface receptor for C-peptide. C-peptide elicits a number of cellular responses, including Ca(2+) influx, activation of mitogen-activated protein (MAP) kinases, of Na(+),K(+)-ATPase, and of endothelial NO synthase. The pentapeptide EGSLQ, corresponding to the C-terminal five residues of human C-peptide, mimics several of the effects of the full-length peptide. The pentapeptide displaces cell membrane-bound C-peptide, elicits transient increase in intracellular Ca(2+) concentration and stimulates MAP kinase signalling pathways and Na(+),K(+)-ATPase. The Glu residue of the pentapeptide is essential for displacement of the full-length C-peptide, and free Glu can partly displace bound C-peptide, suggesting that charge interactions are important for receptor binding. Many C-peptide effects, such as phosphorylation of MAP-kinases ERK 1 and 2, stimulation of Na(+),K(+)-ATPase and increases in intracellular calcium concentrations are inhibited by pertussis toxin, supporting interaction of C-peptide with a G-protein-coupled receptor. However, all C-peptide effects cannot be explained in this manner, and it is possible that additional interactions are involved. Combined, the available observations show that C-peptide is biologically active and suggest a molecular model for its physiological effects.     

Molecular and Cellular Effects of C-peptide—New Perspectives on an Old Peptide

New results present C-peptide as a biologically active peptide hormone in its own right. Although C-peptide is formed from proinsulin and cosecreted with insulin, it is a separate entity with biochemical and physiological characteristics that differ from those of insulin. There is direct evidence of stereospecific binding of C-peptide to a cell surface receptor, which is different from those for insulin and other related hormones. The C-peptide binding site is most likely a G–protein–coupled receptor. The association constant for C-peptide binding is approximately 3 × 10⁹M^-1. Saturation of the binding occurs already at a concentration of about 1 nM, which explains why C-peptide effects are not observed in healthy subjects. Binding of C-peptide results in activation of Ca²⁺ and MAPK-dependent pathways and stimulation of Na⁺,K⁺-ATPase and eNOS activities. The latter 2 enzymes are both deficient in several tissues in type 1 diabetes. There is some evidence that C-peptide, and insulin may interact synergistically on the insulin signaling pathway. Clinical evidence suggests that replacement of C-peptide, together with regular insulin therapy, may be beneficial in patients with type 1 diabetes and serve to retard or prevent the development of long-term complications.     

IL-12 serum levels in patients with type 2 diabetes treated with sulphonylureas

Interleukin-12 (IL-12) has been identified as a pro-inflammatory cytokine which is thought to contribute to the development of atherosclerosis. However, to date, the various associations between factors related to the course of type 2 diabetes, like metabolic compensation, beta cell secretory dysfunction, insulin resistance and IL-12 serum levels, remain unclear. Our study involved 41 patients with type 2 diabetes, 19 patients with coronary artery disease (CAD), and 19 healthy controls. We measured serum levels of fasting glucose, HbA₁c, 1,5-anhydro-d-glucitol, and lipids. In addition, serum levels of C-peptide, insulin, proinsulin and IL-12 were assayed. HOMA_IR score was calculated. The serum concentrations of IL-12 were higher in diabetics than in either patients with CAD or healthy controls, and were correlated with BMI, C-peptide, insulin, HOMA_IR, proinsulin and HDL serum levels. Multiple regression analysis revealed that the IL-12 serum level in type 2 diabetics primarily is dependent upon fasting proinsulin concentration. Our results demonstrate that elevated IL-12 serum levels in type 2 diabetics treated with sulphonylureas are induced especially by peripheral insulin resistance and beta cells dysfunction, as expressed by fasting serum proinsulin levels. This finding gives us hope that treatment to decrease peripheral insulin resistance and to avoid excessive proinsulin secretion might be successful in the prevention of IL-12-induced atherosclerosis.     

Characterization of Antibodies to Products of Proinsulin Processing Using Immunofluorescence Staining of Pancreas in Multiple Species

The efficient processing of proinsulin into mature insulin and C-peptide is often compromised under conditions of beta cell stress, including diabetes. Impaired proinsulin processing has been challenging to examine by immunofluorescence staining in pancreas tissue because the characterization of antibodies specific for proinsulin, proinsulin intermediates, processed insulin and C-peptide has been limited. This study aimed to identify and characterize antibodies that can be used to detect products of proinsulin processing by immunofluorescence staining in pancreata from different species (mice, rats, dog, pig and human). We took advantage of several knockout mouse lines that lack either an enzyme involved in proinsulin processing or an insulin gene. Briefly, we report antibodies that are specific for several proinsulin processing products, including: a) insulin or proinsulin that has been appropriately processed at the B-C junction; b) proinsulin with a non-processed B-C junction; c) proinsulin with a non-processed A-C junction; d) rodent-specific C-peptide 1; e) rodent-specific C-peptide 2; and f) human-specific C-peptide or proinsulin. In addition, we also describe two ‘pan-insulin’ antibodies that react with all forms of insulin and proinsulin intermediates, regardless of the species. These antibodies are valuable tools for studying proinsulin processing by immunofluorescence staining and distinguishing between proinsulin products in different species.     

Bioactivity and pharmacokinetics of human proinsulin in comparison to human insulin after intravenous and subcutaneous injection

The hypoglycemic actions of human insulin (1 IU/kg b.w.) and biosynthetic human proinsulin in about equimolar amounts were studied after intravenous and subcutaneous injection in rabbits. Blood samples were taken up to four hours after injection for the determination of blood glucose and immunoreactive levels of both insulin and human C-peptide. For the determination of human C-peptide, serum taken after proinsulin injection was divided into two fractions. One was examined directly by the human C-peptide radioimmunoassay and the other after incubation with a protein-A-sepharose coupled insulin antibody to find "free human C-peptide". Proinsulin in amounts equimolar to 1 IU insulin/kg b.w., exerted a 34% stronger hypoglycemic action after subcutaneous injection than after intravenous administration (area under curve estimation). Proinsulin-induced hypoglycemia did not last longer after intravenous administration than that induced by intravenous insulin. Although subcutaneous proinsulin did not show the same maximum decrease of blood glucose compared to subcutaneous insulin, its action was significantly prolonged (up to 180 min). Specific measurement of free human C-peptide showed no evidence of conversion of proinsulin to insulin and C-peptide.     

Proinsulin C-peptide interaction with protein tyrosine phosphatase 1B demonstrated with a labeling reaction

Based on nickel-catalyzed cross-labeling where binding partners become biotinylated, we have studied molecular interactions with an N-terminally fused GGH-tag proinsulin C-peptide. Since C-peptide has been reported to influence phosphatase activity in intact cells, we employed this method to study possible binding of the peptide to protein tyrosine phosphatase 1B (PTP-1B). C-peptide was found to interact with PTP-1B (and for control, also with antibodies to C-peptide), as did also the N- and C-terminal fragments of C-peptide which have sequence similarities with PTP-1B binding proteins. The labeling data combined with enzyme activity analysis indicate a functional interaction between acidic regions of C-peptide and specific sites of PTP-1B. Results highlight the importance of possible phosphatase/C-peptide roles in diabetes, and the usefulness of the cross-labeling reaction also for acidic peptides like C-peptide.     

The intracellular handling of insulin-related peptides in isolated pancreatic islets. Evidence for differential rates of degradation of insulin and C-peptide.

Islets of Langerhans isolated from adult rats were maintained in tissue culture for 3 days in the continued presence of [3H]leucine. Labelled proinsulin, C-peptide and insulin were measured by quantitative h.p.l.c., a method which also allowed for resolution of C-peptide I and II, and of insulin I and II (the products of the two rat insulin genes). The results showed that: (1) at early times, proinsulin was the major radiolabelled product; with progressive time in culture, intra-islet levels of [3H]proinsulin decreased, despite continuous labelling with [3H]leucine, indicating that the combined rates of proinsulin conversion into insulin and of proinsulin release, exceeded the rate of synthesis; (2) insulin I levels were always greater than those of insulin II, both in the islets and for products released to the medium; (3) the molar ratio of [3H]insulin I and II to their respective 3H-labelled C-peptides increased with time for products retained within islets, reaching a value close to 3:1 by 3 days; by contrast, for products released to the medium during the culture period, the ratio was always close to unity; (4) when islets were incubated with [3H]leucine for 2 days, and then left for a further 1 day without label (chase period), the intra-islet [3H]insulin/[3H]C-peptide ratios rose to values as high as 9:1. Again, for material released to the medium, the values were close to 1:1; (5) it is concluded that C-peptide is degraded more rapidly than insulin within islet cells, thereby accounting for the elevated insulin/C-peptide ratios. The difference between the ratios observed in the islets and those for material released to the medium is taken to indicate that degradation occurs in a discrete cellular compartment and not in the secretory granule itself.     

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 improves adenosine-induced myocardial vasodilation in type 1 diabetes patients

Patients with type 1 (insulin-dependent) diabetes show reduced skeletal muscle blood flow and coronary vasodilatory function despite intensive insulin therapy and good metabolic control. Administration of proinsulin C-peptide increases skeletal muscle blood flow in these patients, but a possible influence of C-peptide on myocardial vasodilatory function in type 1 diabetes has not been investigated. Ten otherwise healthy young male type 1 diabetic patients (Hb A1c 6.6%, range 5.7-7.9%) were studied on two consecutive days during normoinsulinemia and euglycemia in a double-blind, randomized, crossover design, receiving intravenous infusion of C-peptide (5 pmol.kg-1.min-1) for 120 min on one day and saline infusion on the other day. Myocardial blood flow (MBF) was measured at rest and during adenosine administration (140 microg.kg-1.min-1) both before and during the C-peptide or saline infusions by use of positron emission tomography and [15O]H2O administration. Basal MBF was not significantly different in the patients compared with an age-matched control group, but adenosine-induced myocardial vasodilation was 30% lower (P < 0.05) in the patients. During C-peptide administration, adenosine-stimulated MBF increased on average 35% more than during saline infusion (P < 0.02) and reached values similar to those for the healthy controls. Moreover, as evaluated from transthoracal echocardiographic measurements, C-peptide infusion resulted in significant increases in both left ventricular ejection fraction (+5%, P < 0.05) and stroke volume (+7%, P < 0.05). It is concluded that short-term C-peptide infusion in physiological amounts increases the hyperemic MBF and left-ventricular function in type 1 diabetic patients.     

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.     

Insulin receptors on rat pancreatic islets: specificity of 125 I-insulin binding

Binding sites of isolated rat pancreatic islets have been shown to interact with insulin. Employing various species-insulins, insulin analogues and substances not being structurally related to insulin, structure-specificity as well as pH- and temperature-dependence of insulin binding to rat pancreatic islets have been studied. Rat insulin displaced 125 I-insulin from its binding sites in the same concentration-dependent manner as pork insulin did, whereas the insulin analogue des-(phe-val-asp)B1-3-p-glu B4-insulin was less effective. Pork C-peptide hardly competed for binding and pork proinsulin did not compete at all. Both the species' insulins inhibited glucose (16.7 mM)-induced insulin secretion. The inhibitory effect was less when des-(phe-val-asp)B1-3-p-glu B4-insulin was employed and no inhibition of insulin secretion was observed by the use of pork C-peptide or proinsulin. Glucagon and somatostatin did not affect insulin binding. pH optimum of insulin binding appears to be in the range between 7.0 and 8.0. Binding was augmented with increasing temperature up to 37 degrees C. It is concluded that rat pancreatic islets possess insulin because binding and biological potency of substances related to insulin were in harmony. Moreover pH- and temperature-optimum of insulin binding are in a physiological range.     

The Role of Insulin C-Peptide in the Coevolution Analyses of the Insulin Signaling Pathway: A Hint for Its Functions

As the linker between the A chain and B chain of proinsulin, C-peptide displays high variability in length and amino acid composition, and has been considered as an inert byproduct of insulin synthesis and processing for many years. Recent studies have suggested that C-peptide can act as a bioactive hormone, exerting various biological effects on the pathophysiology and treatment of diabetes. In this study, we analyzed the coevolution of insulin molecules among vertebrates, aiming at exploring the evolutionary characteristics of insulin molecule, especially the C-peptide. We also calculated the correlations of evolutionary rates between the insulin and the insulin receptor (IR) sequences as well as the domain-domain pairs of the ligand and receptor by the mirrortree method. The results revealed distinctive features of C-peptide in insulin intramolecular coevolution and correlated residue substitutions, which partly supported the idea that C-peptide can act as a bioactive hormone, with significant sequence features, as well as a linker assisting the formation of mature insulin during synthesis. Interestingly, the evolution of C-peptide exerted the highest correlation with that of the insulin receptor and its ligand binding domain (LBD), implying a potential relationship with the insulin signaling pathway.     

Preserved Proinsulin Production in Homozygous Protein Tyrosine Phosphatase Nonreceptor Type 22 C1858T Variant Type 1 Diabetes: A Possible Explanation for Absence of Overt Ketoacidosis Despite Omission of Exogenous Insulin

ObjectiveTo report a postulated mechanism for resistance to overt ketoacidosis due to prolonged insulin omission in a severely hyperglycemic woman with a 14-year history of autoimmune type 1 diabetes (T1D).MethodsHistory, physical examination, laboratory testing, and genotyping were performed. We also review the medical literature pertinent to this patient’s phenotype and genotype.ResultsProinsulin levels remained within the normal range (suppressed with hypoglycemia) despite simultaneous almost unmeasurable C-peptide levels during hyperglycemia. We confirmed a homozygous (TT) variant of protein tyrosine phosphatase nonreceptor type 22 (PTPN22) 1858T, a T1D susceptibility gene associated with higher proinsulin levels.ConclusionThe extraordinarily preserved proinsulin biological activity may explain the unusual resistance to overt ketoacidosis despite omission of exogenous insulin administration for extended periods of time. The role of the associated PTPN22 1858TT variant remains speculative. (Endocr Pract. 2013;19:426-430)     

C-peptide inhibits leukocyte-endothelium interaction in the microcirculation during acute endothelial dysfunction.

C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte-endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte-endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte-endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte-endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte-endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte-endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium.     

Glucagon-like peptide-1 improves insulin and proinsulin binding on RINm5F cells and human monocytes

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.     

Acidic residues on the N-terminus of proinsulin C-Peptide are important for the folding of insulin precursor

To investigate the role of C-peptide in the folding of insulin precursor, a series of C-peptide mutant proinsulin genes were constructed, overexpressed in Escherichia coli and the proteins purified. Correct disulfide linkages of these proteins were confirmed by both tryptic peptide mapping and insulin receptor binding analyses. In vitro refolding experiments were performed with the purified proteins and showed that mutations on the glycine-rich middle segment of C-peptide, GGGPGAG, and deletion of the C-terminal pentapeptide, EGSLQ, as well as mutations on the two pairs of dibasic residues at the two ends of C-peptide did not significantly affect the refolding yields. However, both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N-terminus of the C-peptide resulted in serious aggregation during refolding. The results indicate that the highly conserved acidic N-terminal part of C-peptide is very important for insulin precursor folding, and that C-peptide may have some intramolecular chaperone-like function in the folding of insulin precursor.     

Improved meal-related insulin processing contributes to the enhancement of B-cell function by the DPP-4 inhibitor vildagliptin in patients with type 2 diabetes.

The aim of this study was to evaluate the contribution of insulin processing to the improved meal-related B-cell function previously shown with the DPP-4 inhibitor vildagliptin. Fifty-five patients with type 2 diabetes (56.5+/-1.5 years; BMI=29.6+/-0.5 kg/m(2); FPG=9.9+/-0.2 mmol/l; HbA1c=7.7+/-0.1 %) were studied: 29 patients were treated with vildagliptin and 26 patients with placebo, both added to an ongoing metformin regimen (1.5-3.0 g/day). A standardized breakfast was given at baseline and after 52 weeks of treatment, and proinsulin related to insulin secretion was measured with C-peptide in the fasting and postprandial (over 4 h post-meal) states to evaluate B-cell function. The between-treatment difference (vildagliptin-placebo) in mean change from baseline in fasting proinsulin to C-peptide ratio (fastP/C) was -0.007+/-0.009 (p=0.052). Following the standard breakfast, 52 weeks of treatment with vildagliptin significantly decreased the dynamic proinsulin to C-peptide ratio (dynP/C) relative to placebo by 0.010+/-0.008 (p=0.037). Importantly, when the P/C was expressed in relation to the glucose stimulus (i.e., the fasting glucose and glucose AUC(0-240 min), respectively), the P/C relative to glucose was significantly reduced with vildagliptin vs. placebo, both in the fasting state (p=0.023) and postprandially (p=0.004). In conclusion, a more efficient B-cell insulin processing provides further evidence that vildagliptin treatment ameliorates abnormal B-cell function in patients with type 2 diabetes.     

C-peptide binding to human cell membranes: importance of Glu27

In addition to its established role in proinsulin folding, C-peptide has a function in regulation of cellular activity. The 31-residue peptide influences renal, vascular, and metabolic functions in patients with insulin-dependent diabetes mellitus. Binding to cells has been demonstrated for C-peptide, which can be displaced by its C-terminal pentapeptide. We have now used fluorescence correlation spectroscopy to investigate structural requirements on the pentapeptide part for C-peptide binding. All pentapeptide residues, E(27)GSLQ(31), were individually replaced with Ala and the capacity of the resulting peptides to displace rhodamine-labelled full-length human C-peptide from human renal tubular cell membranes was determined. This showed that Glu27 is essential for displacement, while replacement of Gly28 with Ala has little effect, and replacement of any of the three most C-terminal residues had intermediate effects. Morevover, free Glu displaces full-length C-peptide to about 50%, while free Ala, C-peptide(1-26), and the truncated pentapeptide, corresponding to the tetrapeptide G(28)SLG(31), have no displacing capacity. The peptides EVARQ (corresponding to the rat C-terminal pentapeptide) and ELGGGPGAG (corresponding to positions 11-19 of human C-peptide) do not displace human C-peptide. These results indicate that Glu27 of C-peptide is critically involved in binding to cellular targets.     

Protective effects of either C-peptide or l-arginine on pancreatic β-cell function,proliferation, and oxidative stress in streptozotocin-induced diabetic rats

Diabetes and cardiometabolic risk factors including hypertension and dyslipidemia are the major threats to human health in the 21st century. Apoptosis in pancreatic tissue is one of the major causes of diabetes type 1 progression. The aim of this study was to investigate the effects of C-peptide or l -arginine on some cardiometabolic risk factors, pancreatic morphology, function and apoptosis, and the mechanisms of their actions. Forty adult male albino rats were divided into four equal groups: 1—control nondiabetic, 2—diabetic (no treatment), 3—diabetic + C-peptide, and 4—diabetic + l -arginine. Diabetes was induced by a single intraperitoneal injection of high dose streptozotocin. At the end of the experiment, sera glucose, insulin levels, total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO), and pancreatic MDA, TAC, and B-cell lymphoma 2 were measured. The morphology and proliferating activity of the pancreas were examined by hematoxylin and eosin histological stain, proliferative cell nuclear antigen (PCNA), and insulin antibodies. Our results showed that induction of diabetes caused hyperglycemia, dyslipidemia, and oxidative stress. However, administration of C-peptide or l -arginine significantly improved the pancreatic histopathology with a significant increase in the area % of insulin immunoreactivity, the number of PCNA immunopositive cells, the number of islets, and the diameter of islets compared with the diabetic group. C-peptide treatment of the diabetic rats completely corrected these errors, while l -arginine partially antagonized the above diabetic complications. So the administration of C-peptide as an adjuvant therapy in type 1 diabetes can significantly decrease apoptosis of pancreas and subsequent progression of diabetes complication.