Biology of C-peptide
In the course of insulin biosynthesis, proinsulin is cleaved into insulin and C-peptide. The two are stored in the secretory granules of the pancreatic beta cells and C-peptide fulfills an important function in facilitating correct insulin synthesis. Following its discovery in 1967 , several attempts were made to detect insulin-like effects of C-peptide but the results were negative and, gradually, the view prevailed that C-peptide was without physiological effects. However, in the early 1990’s, the possibility that C-peptide may exert physiological effects of its own was re-evaluated. A series of studies were undertaken involving administration of the peptide to patients with type 1 diabetes, who lack C-peptide. It soon became apparent that replacement of C-peptide in physiological concentrations resulted in significant improvements of several diabetes-related functional abnormalities. These findings prompted a renewed interest in C-peptide physiology and during the past 20 years a steadily increasing number of reports on new aspects of C-peptide physiology have emerged. The information available today includes studies of the peptide's interaction with cell membranes, its intracellular signaling properties and its cellular end effects, for an overview, see .
The human C-peptide incorporates 31 amino acid residues. Historically, the peptide has been regarded as being biologically inert, serving primarily as a linker between the A- and B-chains of the insulin molecule, thus enabling the correct folding and interchain disulfide bond formation of insulin. C-peptide facilitates the intracellular transport, sorting, and proteolytic processing of proinsulin into biologically active insulin in the maturing secretory granules of the β-cells. After cleavage of proinsulin, the intact C-peptide is stored with insulin in the soluble phase of the secretory granules and is subsequently released by exocytosis to the circulation in equimolar amounts with insulin , providing a clinically useful independent indicator of insulin secretion.
C-peptide shows considerable interspecies variability but several residues are conserved among mammalian species. Thus, the glutamic acid residues in positions 3, 11 and 27 are well conserved in mammals as are the glutamine residues 6 and 31 and the leucines in 12 and 26 positions. The C-terminal pentapeptide segment shows membrane binding interactions and and biological activity; the pentapeptide elicits an intra- cellular Ca2+-increase and MAPK phosphorylation for which it requires the presence of its N-terminal glutamic acid residue (corresponding to the 27 position of C-peptide) . Conversely, des (27-31) C-peptide is without biological activity. Similar well defined functional C-terminal segments are also reported for other hormones such as gastrin and cholecystokinin.
Specific binding of C-peptide to cell membranes at low nanomolar concentrations has been shown for a number of different cell types, e.g. insulinoma cells, endothelial cells, renal tubular cells, fibroblasts, and nerve cells. The binding can be displaced by excess C-peptide and the C-terminal pentapeptide but not by insulin, IGF-1, IGF-2, proinsulin or randomly sequenced C-peptide . The binding characteristics suggest the possibility that C-peptide may interact with two or more receptor molecules. Following membrane binding, C-peptide is internalized to early endosomes and can be detected in the nucleus. Pretreatment of cells with pertussis toxin abolishes the binding and all downstream effects, indicating that a G-protein coupled receptor (GPCR) is involved in C-peptide signaling. This is in contrast to the receptor tyrosine kinase signaling effected by insulin. Studies have indicated that a Gαi –protein is activated following binding of C-peptide to the cell membrane. The nature of the GPCR involved has remained elusive for long and has delayed the recognition of C-peptide as a bioactive peptide in its own right. A recent study, however, involving a stepwise knock-down techique has demonstrated that the effect of C-peptide on intracellular signaling is blocked following siRNA- mediated knockdown of GPR146, but not several other orphan GPCRs . In addition, stimulation with C-peptide caused internalization of GPR146 and preliminary evidence suggests co-localization of C-peptide and GPR146 on cell membranes. Further studies are needed but these observations make it likely that GPR146 is involved C-peptide mediated signaling.
Cellular effects of C-peptide
Fig 1. (Click to enlarge) Schematic overview of C-peptide´s anti-oxidant, anti-inflammatory and anti-apoptotic effects. The peptide reduces high glucose induced ROS-formation via inhibition of NAD(P)H oxidase, inhibits proinflammatory gene activation by downregulation of NF-κB activity, diminishes pro-inflammatory cytokine and chemokine secretion, and reduces the expression of cellular adhesion molecules as well as VEGF, TGF-β and PAI-1. In addition, caspase-3 activity is inhibited and apoptotic activity retarded. C-peptide also elicits activation and induction of both eNOS and Na+,K+-ATPase. Finally, the peptide reduces vascular smooth muscle proliferation and migration induced by high levels of glucose.
A number of cellular effects have been demonstrated for C-peptide, as summarized in Fig. 1. Importantly, several anti-inflammatory, cytoprotective and anti-apoptotic effects of C-peptide have been reported, for recent reviews, see . Thus, C-peptide at physiological concentrations mediates a reduction in the formation of reactive oxygen species (ROS) in endothelial cells following exposure to stressful conditions. This effect is elicited via RAC-1 mediated inhibition of NAD(P)H oxidase, the principal source of ROS generation in endothelial cells after exposure to elevated levels of glucose. In addition, an inhibitory effect by C-peptide on NAD(P)H oxidase is exerted via stimulation of AMPKα, which in turn inhibits NAD(P)H oxidase. Moreover, C-peptide is known to limit the negative effects of ROS accumulation in endothelial cells by blocking ROS-mediated activation of the pro-apoptotic enzyme transglutaminase 2. C-peptide is also reported to exert further anti-apoptotic effects by inhibiting caspase 3 and stimulating the activity of the anti-apoptotic protein Bcl-2, as demonstrated for endothelial cells, neuroblastoma cells and β-cells.
It is also known that C-peptide mediates an inhibitory influence on the nuclear factor kappa β (NF-κβ) pathway, known to regulate cellular inflammatory responses. Thus, C-peptide elicits cytoprotective effects on vascular cells by reducing high glucose induced plasma concentrations of pro-inflammatory cytokines and chemokines (IL-1, IL-6, IL-8, MIP-1α, MCP-1, and TNFα). It also down-regulates the expression of VEGF, TGF-β and PAI-1. Moreover, by decreasing the expression of cell adhesion molecules such as ICAM, VCAM and P-selectin following exposure to elevated glucose levels. C-peptide contributes to diminished leukocyte-endothelial interactions and prevents or limits the development of endothelial impairment. Finally, it is noted that while exposure to high glucose may cause vascular smooth muscle cells to proliferate and migrate to the subendothelial space, C-peptide has been found to inhibit these early steps in the direction of atherosclerotic lesion formation.
Endothelial nitric oxide synthase (eNOS) and Na+,K+-ATPase are both stimulated and induced in a concentration dependent manner by C-peptide. Since both enzymes are known to be decreased in experimental models of diabetes and in human diabetes, C-peptide may offer a possibility to improve regional microcirculation and endothelial function in diabetes. These vascular effects of C-peptide are observable not only under in vitro conditions but also in vivo. Thus, C-peptide administration to rats with experimental diabetes results in improved endoneurial blood flow in peripheral nerve tissue and increased levels of nerve Na+,K+-ATPase . Likewise, C-peptide is known to diminish glomerular hyperfiltration by regulating intraglomerular capillary eNOS in streptozotocin- diabetic animals.
Role of C-peptide in normal physiology and diabetes – a hypothesis
In view of the anti-oxidant, anti-inflammatory and anti-apoptotic effects that have been documented for C-peptide, it can be speculated that its physiological role may be to prevent or diminish the formation of ROS and other oxidant species that accompanies also the modest elevations of blood glucose that result from ingestion of a carbohydrate-rich meal. In short, C-peptide may be viewed as an endogenous antioxidant, counteracting any ROS generation secondary to the rise in blood glucose that occurs despite normal insulin-mediated stimulation of tissue glucose uptake following carbohydrate ingestion.
From the above, it becomes apparent that type 1 diabetes subjects may be particularly vulnerable to blood glucose elevations since, due to lack of C-peptide, part of their normal defence against toxic effects of hyperglycemia is diminished. In addition, they are likely to be exposed to repeated episodes of hyperglycemia, despite insulin therapy. This formulation is in line with the “unifying hypothesis”  that seeks to explain the pathogenesis of microvasular complications of diabetes. According to this hypothesis, it is the elevated blood glucose levels and lack of insulin in diabetes that precipitates formation of ROS in vascular endothelial cells, generating oxidative stress and a series of downstream detrimental effects, eventually including capillary circulatory changes, endothelial impairment and apoptosis, cornerstones of what is clinically known as microcirculatory complications. It is in this context that the recently discovered antioxidant and cytoprotective effects of C-peptide should be considered; even a cautious evaluation of the available evidence presents the picture of a bioactive endogenous peptide with therapeutic potential.
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