Proinsulin C-peptide-mediated signalling and the search for its receptor

Proinsulin connecting peptide (C-peptide) joins the A and B-chain of proinsulin and plays an important role in coordinating the folding of insulin. For many years this peptide was simply considered an inert by-product of insulin biosynthesis and was used mainly as an alternative marker for insulin secretion. Recent evidence, however, demonstrates convincingly that C-peptide has biological function and establishes C-peptide as an attractive therapeutic agent to provide protection against chronic diabetic complications. Little is known about C-peptide signalling in pancreatic β cells with studies focussing on antioxidant effects. C-peptide could have protective roles in these cells. For example, pancreatic β cells exposed to immune complexes in type 2 diabetes require protection possibly via C-peptide. Data presented here demonstrate that C-peptide induced a concentration-dependent phosphorylation (activation) of rpS6, at S235/S236 and S240/244, as well as phosphorylation of components of the upstream signalling pathways of rpS6 (ERK1/2, Akt and S6K) in the pancreatic β cell line, INS-1E. C-peptide also caused concentration-dependent increases in phospo-ERK1/2 and phospho-rpS6 (S240/244) in HEK293 and SH-SY5Y, but not in HEK293A. Stimulatory effects of C-peptide on two important intracellular signalling pathways, ERK1/2 and rpS6, can deliver cytoprotective effects and may, therefore, be of potential importance in the treatment of diabetes. A major limitation of current work on C-peptide is that the receptor(s) have not been identified convincingly. Some recent evidence suggests that the orphan GPCR, GPR146, may be the C-peptide receptor. Here, stable overexpression of C-terminally EGFP-tagged GPR146 in HEK293 and HEK293A cells showed predominant membrane localisation of the receptor. However, C-peptide responses in these were unaffected and C-peptide-evoked internalisation/co-localisation of GPR146-EGFP was not observed. An expression cloning approach in Xenopus oocytes was used to screen pools of cDNA library clones for Gαi-evoked responses of C-peptide given the pertussis toxin sensitivity of responses. This approach did not, however, reveal any C-peptide-evoked responses in any of the approximately 130,000 clones screened. Furthermore, the published C-peptide receptor candidates, GPR146 and α-enolase did not respond to C-peptide when expressed in oocytes. Taken together, signalling events in a pancreatic β cell line suggest relevance of C-peptide to events such as cell survival and proliferation. However, the present work provides no evidence that GPR146 is the C-peptide receptor, which still remains elusive.