C-peptide Receptor Research Articles

The role of C-peptide in regulation of the insulin signaling system (systematic review)

The C-peptide is a fragment of proinsulin, the cleavage of which forms active insulin. In recent years, new evidence has emerged indicating that C-peptide is involved not only in the processing of insulin in the secretory granules of pancreatic β-cells, but also has an important regulatory effect on the functions of many organs and tissues. C-peptide mediates physiological effects through signaling pathways by binding to a specific receptor on the cell membrane. Intracellular signaling occurs through the G-protein and Ca 2+ - dependent pathways, which leads to the activation and increased expression of endothelial nitric oxide synthase, Na + /K + -ATPase, and important transcription factors involved in apoptosis, anti-inflammatory and other intracellular defense mechanisms. One of the most important physiological effects of C-peptide is the regulation and modification of insulin signaling mechanisms. The nature of the effect of C-peptide on the insulin signaling system depends on the concentration of insulin. It is assumed that under conditions of low insulin levels, the C-peptide receptor binds to G i/o -proteins and leads to increased activation of processes induced by insulin. In this case, the C-peptide acts as insulin-mimetic peptide. Under high insulin conditions, the C-peptide receptor binds to G q/11 -proteins and leads to activation of protein kinase C with subsequent weakening of insulin-related signaling cascades. This review presents new facts indicating the participation of C-peptide in the regulation of the insulin signaling system.

Is GPR146 really the receptor for proinsulin C-peptide?

Proinsulin C-peptide has previously been proposed to interact with a G-protein coupled receptor (GPCR), specifically the orphan receptor GPR146. To investigate the potential of C-peptide in treating complications of diabetes, such as kidney damage, it is necessary to understand its mode of action. We used CHO-K1 cells expressing human GPR146 to study human and murine C-peptide in dynamic mass redistribution and GPCR β-arrestin assays, as well as with fluorescence confocal microscopy. Neither assay revealed any significant intracellular response to C-peptide at concentrations of up to 33 µM. We observed no internalisation of C-peptide by fluorescence microscopy. Our results do not support GPR146 as the receptor for C-peptide, but suggest that further investigations of the mode of action of C-peptide should be undertaken.

A Novel Model of Long‐term Human Microvascular Endothelial Cell Serum Starvation

Microvascular endothelial cell injury and progressive loss of microvasculature in the dermis, kidney, retina and perineural beds is caused by hyper‐and hypoglycemic insults in individuals with Diabetes mellitus (DM). The consequence of microvascular injuries occurring over years to decades is the onset of DM‐associated co‐morbidities such as delayed wound healing, renal failure, vision loss and peripheral neuropathy. The Diabetes Control and Complications Trial noted a correlation between baseline endogenous C‐peptide serum levels and the delay in onset of DM‐associated co‐morbidities. In immortalized human microvascular endothelial cells (HMEC) and primary human dermal microvascular endothelial cells treatment with 1.5nM C‐peptide and 0.5nM insulin prevented hyperglycemia‐induced oxidative injury. In studies of these cells using immunofluorescence to track C‐peptide and the C‐peptide receptor, G‐protein coupled receptor 146 (GPR146), endothelial cells repeatedly demonstrated cytosolic stores of C‐peptide. Fetal bovine serum, a component of almost all cell culture growth media was determined to have high concentrations of C‐peptide. The aim of this study was to develop a model of long term endothelial serum starvation that would result in depleted internal stores of C‐peptide, but would prevent endothelial cells transformation to smooth muscle cells or fibroblasts, as endothelial progenitor cells are capable of differentiating into one of these three cell types. We hypothesized that cytosolic C‐peptide would reduce over time with serum starvation. To test this hypothesis, HMEC were incubated for up to two months in MCDB13 media supplemented with human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, basic fibroblast growth factor, insulin‐like growth factor‐1, ascorbic acid, and L‐glutamine. Serum starved HMEC were harvested at various time points and stained for C‐peptide (MAB80561 R&D Systems clone 915612) and DAPI or lysed with lysis buffer with 2‐mercaptoethanol followed by RNA isolation, cDNA preparation and RT‐PCR evaluation of targets. Cytosolic C‐peptide fluorescent signal diminished over time in a linear manner but was never completely abolished. HMEC serum starved for 30 days retained endothelial markers CD31, CD34, VEGFR2, had no change in smooth muscle markers Smoothelin, or Smooth muscle actin, and similarly, had no change in expression Vimentin a fibroblast marker relative to un‐serum starved HMEC controls as determined by RT‐PCR. The ability to serum starve HMEC long‐term and substantially reduce internal C‐peptide stores has the potential to enhance research to elucidate the pathways employed by C‐peptide in the prevention of hyperglycemia‐induced microvascular injury.Support or Funding InformationNIDDK

Investigating the Binding of Albumin and C‐peptide to Red Blood Cells

The pancreatic β‐cells of healthy individuals secrete insulin and C‐peptide, a 31‐amino acid peptide, in a 1:1 mole ratio. However, the β‐cells of people with type 1 diabetes (T1D) are damaged or destroyed. Therefore, exogenous insulin is required for people with T1D to survive. Even with regular insulin treatments, patients develop complications such as retinopathy, neuropathy, and nephropathy. Research from our group demonstrates that C‐peptide increases microvascular blood flow, therefore, it could be a useful tool to decrease these complications due to poor blood flow. However, the unknown C‐peptide receptor and mechanism has been a major roadblock in utilizing C‐peptide as a therapeutic. Utilizing ELISA, our group has demonstrated that approximately 1,800 C‐peptide molecules bind per red blood cell (RBC) in the presence of albumin. Without albumin, there was no detectable C‐peptide binding per RBC. Rather than C‐peptide solely binding to a RBC receptor, we hypothesize that C‐peptide binds to RBCs through an albumin/C‐peptide complex receptor. Bovine serum albumin (BSA) binding per RBC was detectable through an attached gamma decay radiolabel (99mTc). A binding saturation experiment was conducted to examine the specific binding of BSA to RBCs with and without C‐peptide. The specific binding curves revealed that albumin saturates at 14,021 ± 1489 BSA molecules/RBC with a Kd of 1.07 (±0.19) × 10−7 M and a Bmax of 1.94 (±0.06) × 10−8 M, or 13,900 receptor molecules/RBC. Whereas, in the presence of C‐peptide and zinc, albumin saturates at 16,696 ± 1479 BSA molecules/RBC with a Kd of 1.91 (±0.09) × 10−7 M and a Bmax of 2.50 (±0.03) × 10−8 M, or 17,900 receptor molecules/RBC. At saturation, the additional 2,700 BSA molecules binding per RBC in the presence of C‐peptide and zinc indicates that an albumin receptor exists on RBCs as well as a separate albumin/C‐peptide complex receptor.Support or Funding InformationNIH

Red Blood Cells Specifically Bind a C‐peptide/Albumin Complex

People with Type 1 diabetes (T1D) require exogenous insulin due to the destruction of insulin‐secreting pancreatic β‐cells. However, even with insulin treatments, people with T1D develop complications such as retinopathy, neuropathy, and nephropathy that are often associated with poor microvascular blood flow. C‐peptide, a 31‐amino acid length peptide co‐secreted with insulin from the pancreatic β‐cells in a 1:1 mole ratio has been shown to improve microvascular blood flow in T1D rats and small‐scale human studies. C‐peptide indirectly enhances endothelium‐derived nitric oxide (NO) by stimulating the release of red blood cell (RBC) derived ATP, a recognized stimulus of endothelial NO synthase. Unfortunately, a receptor for C‐peptide has never been reported. Previously, using ELISA, we reported C‐peptide uptake by RBCs was approximately 1800 C‐peptide molecules/cell, but only when in the presence of albumin. In the absence of albumin, there was no measurable C‐peptide binding to the RBCs. We also report C‐peptide binding to albumin with a Kd of 5.7 ± (2.1) × 10−6 M; therefore, we hypothesize that C‐peptide binds to RBCs via a receptor that is specific for an albumin/Cpeptide complex. The binding of 99mTc labeled bovine serum albumin (BSA) to isolated RBCs, in the presence and absence of C‐peptide, was quantitatively determined by automatic gamma counting after repeated washing with a physiological salt solution. Sample counts per minute (CPM) were compared to a standard curve to determine the 99mTc‐BSA molecules per RBC. Results suggest BSA total binding to the RBCs saturates at approximately 1600 BSA molecules/RBC, whether or not C‐peptide is present, but that the BSA‐99mTc is specifically binding to RBCs in the presence of C‐peptide. Collectively, our data strongly suggest that the Cpeptide receptor has remained elusive because the receptor specifically binds an albumin/Cpeptide complex, as opposed to C‐peptide alone.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

C‐peptide and Insulin Work in Concert to Mitigate Hyperglycemia‐induced Oxidative Stress and Facilitate Cellular Recovery

The role of C‐peptide in the prevention and resolution of diabetes‐induced vascular injuries is not fully understood. C‐peptide is a 31 amino acid peptide that is co‐produced and co‐secreted with insulin by pancreatic beta islet cells. In insulin‐dependent diabetics C‐peptide levels correlate with the delayed onset of comorbidities that arise as a consequence of vascular compromise. These comorbidities include increased incidence of cardiovascular disease, retinopathies, renal failure, and compromised wound healing. In 2013, G‐protein‐coupled‐receptor 146 was identified by our group as the receptor for C‐peptide. GPR146 is highly expressed on endothelial cells, fibroblasts, red blood cells, and lymphocytes. We hypothesized that C‐peptide and insulin act in concert to regulate and reduce hyperglycemia‐induced oxidative stress and normalize cellular function in hyperglycemia. In this study, primary human dermal microvascular endothelial cells (HDMEC), primary normal human dermal fibroblasts (NHDF), and human microvascular endothelial cells (HMEC), were used to investigate cellular response to incubation in normal glucose(NG) or high glucose (25 mM)(HG) alone or with the addition of C‐peptide (1.5 nM), insulin (0.5 nM), or both C‐peptide and insulin. All cell lines were pre‐screened for the presence of the insulin receptor and GPR 146 by qPCR and the responses to C‐peptide and insulin by relative expression of cFOS mRNA. In cells incubated with 25 mM glucose for 3 days, the ratio of phosphorylated ERK1–2/ERK 1–2 was not significantly increased by C‐peptide or insulin alone but was significantly increased by incubation with both. Cells incubated in HG for 3 days demonstrated phosphorylated AKT/AKT levels which were not altered by C‐peptide alone, but were increased by insulin and markedly enhanced by the presence of both. In wound healing assays cells in HG demonstrated impaired migration relative to those in NG and the greatest improvement in migration was in the presence of paired C‐peptide and insulin. Endothelial cell nitric oxide (NO) production was significantly elevated in HG, and while there was no significant inhibition by C‐peptide or insulin alone, incubation with both resulted in complete restoration to normal levels. NO generated by ECs in response to HG is produced in conditions of mitochondrial oxidative stress and production of radical oxygen species (ROS), in this environment NO complexes with ROS to form peroxynitrites. Peroxynitrites are potent mediators of cellular damage, forming complexes with proteins, enzymes, and lipids. ROS and superoxide (SO) levels were increased in cells exposed to high glucose conditions, and incubation with both C‐peptide and insulin resulted in the reduction of ROS and SO levels. This study demonstrated the cytoprotective potential of C‐peptide when paired with insulin. These results support the hypothesis that co‐administration of C‐peptide with insulin to insulin‐dependent diabetics may reduce the onset of comorbidities.Support or Funding InformationHJ121456This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Physiology of Proinsulin C-Peptide: Unanswered Questions and a Proposed Model.

C-peptide is produced, processed, and secreted with insulin, and appears to exert separate but intimately related effects. In this review, we address the existence of the C-peptide receptor, the interaction between C-peptide and insulin, and the potential physiological significance of proinsulin C-peptide.

Physiological effects and therapeutic potential of proinsulin C-peptide

Connecting Peptide, or C-peptide, is a product of the insulin prohormone, and is released with and in amounts equimolar to those of insulin. While it was once thought that C-peptide was biologically inert and had little biological significance beyond its role in the proper folding of insulin, it is now known that C-peptide binds specifically to the cell membranes of a variety of tissues and initiates specific intracellular signaling cascades that are pertussis toxin sensitive. Although it is now clear that C-peptide is a biologically active molecule, controversy still remains as to the physiological significance of the peptide. Interestingly, C-peptide appears to reverse the deleterious effects of high glucose in some tissues, including the kidney, the peripheral nerves, and the vasculature. C-peptide is thus a potential therapeutic agent for the treatment of diabetes-associated long-term complications. This review addresses the possible physiologically relevant roles of C-peptide in both normal and disease states and discusses the effects of the peptide on sensory nerve, renal, and vascular function. Furthermore, we highlight the intracellular effects of the peptide and present novel strategies for the determination of the C-peptide receptor(s). Finally, a hypothesis is offered concerning the relationship between C-peptide and the development of microvascular complications of diabetes.

Low O2-induced ATP release from erythrocytes of humans with type 2 diabetes is restored by physiological ratios of C-peptide and insulin.

ATP release from erythrocytes in response to reduced oxygen (O2) tension stimulates local vasodilation, enabling these cells to direct perfusion to areas in skeletal muscle in need of O2. Erythrocytes of humans with type 2 diabetes do not release ATP in response to low O2. Both C-peptide and insulin individually inhibit low O2-induced ATP release from healthy human erythrocytes, yet when coadministered at physiological concentrations and ratios, no inhibition is seen. Here, we determined: that 1) erythrocytes of healthy humans and humans with type 2 diabetes possess a C-peptide receptor (GPR146), 2) the combination of C-peptide and insulin at physiological ratios rescues low O2-induced ATP release from erythrocytes of humans with type 2 diabetes, 3) residual C-peptide levels reported in humans with type 2 diabetes are not adequate to rescue low O2-induced ATP release in the presence of 1 nM insulin, and 4) the effects of C-peptide and insulin are neither altered by increased glucose levels nor explained by changes in erythrocyte deformability. These results suggest that the addition of C-peptide to the treatment regimen for type 2 diabetes could have beneficial effects on tissue oxygenation, which would help to ameliorate the concomitant peripheral vascular disease.

Evidence for an interaction between proinsulin C-peptide and GPR146

Microvascular diseases, such as retinopathies, neuropathies, and nephropathies, are a devastating consequence of type 1 and type 2 diabetes. The etiology of diabetes-associated microvascular dysfunction is poorly understood, and, likewise, treatment modalities for these disorders are limited. Interestingly, proinsulin C-peptide has been shown to play a protective role against diabetes-associated complications in experimental animals and in diabetic humans and is thus an attractive therapeutic target. However, an important step in the development of C-peptide-based therapeutics is identification of the C-peptide receptor, which is likely a G protein-coupled receptor (GPCR). Using a unique Deductive Ligand-Receptor Matching Strategy, we sought to determine whether one of the known orphan GPCRs is essential for C-peptide signaling. Knockdown of GPR146, but not GPR107 or GPR160, blocked C-peptide-induced cFos expression in KATOIII cells. Furthermore, stimulation with C-peptide caused internalization of GPR146, and examples of punctate colocalization were observed between C-peptide and GPR146 on KATOIII cell membranes. These data indicate that GPR146 is likely a part of the C-peptide signaling complex and provide a platform for the elucidation of the C-peptide signalosome.

Proinsulin C-peptide activates α-enolase: implications for C-peptide–cell membrane interaction

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.

Inflammatory Cardiovascular Risk Biomarkers: Update on Novelties and Limitations

Inflammatory Cardiovascular Risk Biomarkers: Update on Novelties and Limitations

C-Peptide and its Intracellular Signaling

Although long believed to be inert, C-peptide has now been shown to have definite biological effects both in vitro and in vivo in diabetic animals and in patients with type 1 diabetes. These effects point to a protective action of C-peptide against the development of diabetic microvascular complications. Underpinning these observations is undisputed evidence of C-peptide binding to a variety of cell types at physiologically relevant concentrations, and the downstream stimulation of multiple cell signaling pathways and gene transcription via the activation of numerous transcription factors. These pathways affect such fundamental cellular processes as re-absorptive and/or secretory phenotype, migration, growth, and survival. Whilst the receptor remains to be identified, experimental data points strongly to the existence of a specific G-protein-coupled receptor for C-peptide. Of the cell types studied so far, kidney tubular cells express the highest number of C-peptide binding sites. Accordingly, C-peptide exerts major effects on the function of these cells, and in the context of diabetic nephropathy appears to antagonise the pathophysiological effects of major disease mediators such as TGFbeta1 and TNFalpha. Therefore, based on its cellular activity profile C-peptide appears well positioned for development as a therapeutic tool to treat microvascular complications in type 1 diabetes.

C-Peptide is Relevant in Type 1 Diabetes and its Complications: Summary and Conclusions to the Special Issue

It has been very satisfying to put together this Special Edition of The Review of Diabetic Studies on C-peptide and diabetes. We are greatly encouraged by the advances being made in this research area, and especially to see the beneficial effects of C-peptide making their way into clinic. Once regarded as a redundant waste product of insulin synthesis, C-peptide is now recognized as an important regulatory hormone in its own right. Our understanding of C-peptide physiology is expanding rapidly. It is abundantly clear that C-peptide deficiency in type 1 diabetes plays important pathogenetic roles in the development of various complications, with consequent beneficial effects upon C-peptide replacement. Over the last decade and a half, a wealth of new information has accumulated regarding C-peptide's interaction with a variety of cell types, its intracellular signaling mechanisms, and its impact on specific cell functions, as well as growth and cell death. It also emerges from these reviews that several questions remain, e.g. the conundrum of C-peptide's interaction with the cell membrane remains unresolved. Is there a C-peptide receptor which, to date, has escaped detection? It is clear that C-peptide binds to cell membranes and accumulates in endosomes in the cytoplasm. Also, it exerts effects on major signaling pathways such as the MAPK and PI-3 kinase pathways with fundamental influences on Na+/K+-ATPase and eNOS acivities. These activities affect renal, peripheral nerve, and vascular functions. Another effect of C-peptide is the regulation of a variety of transcription factors, with impacts on gene and protein expression of structural and functional proteins in the central and peripheral nervous systems, and in renal tissues. Such effects have led to the suggestion that C-peptide may interact with insulin-signaling activities, which in certain cell-systems can be abolished with Wortmannin, but not in others. There is also evidence showing that C-peptide exerts its cellular signaling via a G-protein-coupled receptor, since its signal can be abolished in the presence of pertussis toxin. Interestingly, some of the pepide's activities are operative in the absence of insulin, as in red blood cells and rheological functions. Instead, C-peptide seems to exert such effects by binding to Zn2+-ions, suggesting a charge-specific interaction. Recent observations demonstrate that the peptide is capable of disaggregating insulin hexamers. This effect may be important in relation to exocytosis of insulin from the beta-cells. One of the transcription factors regulated by C-peptide is NF-κB with downstream effects on inflammatory properties in the vasculature and the central nervous system. Such effects occur in the absence of any effect on hyperglycemia. Perturbations of several transcription factors influence oxidative stress and apoptotic activities. Hence, C-peptide exerts anti-inflammatory and anti-apoptotic effects in several target tissues of type 1 diabetes complications. The major thrust of the research activities regarding C-peptide physiology has concerned its effects on microvascular complications of type 1 diabetes. In the type 2 diabetic situation, C-peptide effects have not yet been fully explored. Thus, elevated levels of C-peptide may possibly have adverse effects on atherogenesis and on red blood cell ATP synthesis with downstream effects on platelet and endothelial functions. On the other hand, reports also show that C-peptide is capable of preventing intimal hyperplasia and occlusion of vascular grafts. The beneficial effects of C-peptide on the chronic complications in type 1 diabetic patients have been repeatedly documented with respect to microvascular circulation, as well as renal and peripheral nerve complications. The mechanisms underlying these beneficial effects appear to vary in different tissues, and insulin is not always necessary for C-peptide to function. Furthermore, the physiological effects of C-peptide are conveyed within a narrow concentration range. Based on present knowledge, we can envisage that replacement of C-peptide, the second missing hormone in type 1 diabetes, may have a major impact in the prevention and treatment of the dreaded complications of diabetes. Proof of concept has been demonstrated. The principle of replacement therapy is not new, but is the basic rationale for the treatment of a number of endocrinological disorders. There are compelling reasons to call for responsible national agencies, patient interest groups, and the pharmaceutical industry to engage, and to promote large scale clinical trials. This would provide an early opportunity for the increasing type 1 diabetes population to benefit soon from therapy with this peptide. Disclosures: The authors report no conflict of interests.

The C-peptide signaling.

For years an assumption was made that C-peptide, a byproduct of insulin biosynthesis, possessed no appreciable physiologic role. As other contributions in this volume amply testify, the time has come to re-evaluate that notion. C-peptide either directly through interaction with its specific cell-surface receptor or indirectly through an interaction with a related membrane entity, exerts a unique effect on several intracellular processes.We review here results of studies attempting to elucidate such molecular effects of C-peptide in different cell systems and tissues. Lacking a purified C-peptide receptor, we also demonstrate C-peptide effects on distinct elements of the insulin signal transduction pathways.

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.

Proinsulin C-peptide rapidly stimulates mitogen-activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3-kinase and pertussis toxin-sensitive G-protein

It has been demonstrated that proinsulin C-peptide possesses several biological activities and that its specific binding sites are present on the surface of cell membranes. However, the molecular and cellular mechanisms of C-peptide actions are poorly known. In the present study we examined the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in C-peptide effects. C-peptide induced the phosphorylation of MAPK [p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2] in Swiss 3T3 and 3T3-F442A fibroblasts but not in 3T3-L1 fibroblasts and some other cell lines such as L6E9 muscle cells. In Swiss 3T3 cells, C-peptide-induced phosphorylation of MAPK was dependent on time and concentration, being maximal at 1min and at 1nM C-peptide and was accompanied by an increase in MAPK activity and MAPK kinase (MEK) phosphorylation. The MAPK phosphorylation by C-peptide was abolished by treatment with pertussis toxin (PTX) and also with a MEK inhibitor, PD 98059. In addition, MAPK phosphorylation was attenuated by treatment with a phosphoinositide 3-kinase (PI-3K) inhibitor, wortmannin, and with a protein kinase C (PKC) inhibitor, GF109203X, and by down-regulation of PKC by prolonged treatment with PMA. Similar effects of the inhibitors and PTX were found on the MAPK phosphorylation induced by neuropeptide Y. These results suggest that C-peptide activates MAPK through a putative Gi/Go-linked receptor for C-peptide and through PI-3K-dependent and PKC-dependent pathways.

Proinsulin C-peptide rapidly stimulates mitogen-activated protein kinases in Swiss 3T3 fibroblasts: requirement of protein kinase C, phosphoinositide 3-kinase and pertussis toxin-sensitive G-protein.

It has been demonstrated that proinsulin C-peptide possesses several biological activities and that its specific binding sites are present on the surface of cell membranes. However, the molecular and cellular mechanisms of C-peptide actions are poorly known. In the present study we examined the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in C-peptide effects. C-peptide induced the phosphorylation of MAPK [p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2] in Swiss 3T3 and 3T3-F442A fibroblasts but not in 3T3-L1 fibroblasts and some other cell lines such as L(6)E(9) muscle cells. In Swiss 3T3 cells, C-peptide-induced phosphorylation of MAPK was dependent on time and concentration, being maximal at 1 min and at 1 nM C-peptide and was accompanied by an increase in MAPK activity and MAPK kinase (MEK) phosphorylation. The MAPK phosphorylation by C-peptide was abolished by treatment with pertussis toxin (PTX) and also with a MEK inhibitor, PD 98059. In addition, MAPK phosphorylation was attenuated by treatment with a phosphoinositide 3-kinase (PI-3K) inhibitor, wortmannin, and with a protein kinase C (PKC) inhibitor, GF109203X, and by down-regulation of PKC by prolonged treatment with PMA. Similar effects of the inhibitors and PTX were found on the MAPK phosphorylation induced by neuropeptide Y. These results suggest that C-peptide activates MAPK through a putative G(i)/G(o)-linked receptor for C-peptide and through PI-3K-dependent and PKC-dependent pathways.