Biochemistry, C Peptide

Fundamentals

Once secreted, both insulin and C-peptide are routed through the liver. In the liver, insulin binds to its receptors and initiates glucose uptake, inhibits gluconeogenesis, glycogenolysis, and ketogenesis and is degraded within 5 to 10 minutes. C-peptide, on the other hand, has limited degradation in the liver and is degraded by the kidneys. Hence, the half-life of C-peptide is around 30 to 35 minutes. As a result, although both insulin and C-peptide are secreted in equimolar concentrations, the molar ratio of circulating insulin to C-peptide is less than 1. Several studies have shown that C-peptide can bind to the cell membranes and could exert biological effects; however, a specific receptor has not been identified, and this is described later.

Issues of Concern

The normal physiological C-peptide plasma concentration in a fasted state is 0.9 to 1.8 ng/ml.[1] A high level could indicate insulin resistance, insulinoma, or kidney disease. A low C-peptide is usually present in patients with type 1, or sometimes, type 2 diabetes.

Cellular Level

C-peptide is a 31-amino acid polypeptide and is negatively charged. In mammals, the 8 residues (positions 1, 3, 6, 11, 12, 21, 27, and 31) are conserved, and the C-terminal pentapeptide has been shown to interact with the cell membrane and elicit signaling pathways.[3] Although the exact mechanism of binding is not known, the binding characteristics and the intracellular effects could be modified by pertussis toxin, suggesting that the G-protein coupled receptors might be involved.[4] The binding of C-peptide was shown to elevate the intracellular calcium levels. It also can induce phospholipase C, protein kinase C isoforms, Rho A,  and p38 MAPK in renal tubular cells and fibroblasts.[5] Activation of the PI3 kinase, Akt, and PPAR-gamma is also observed in fibroblasts, myoblasts, renal tubular cells, and lymphocytes.[6] In endothelial cells, C-peptide was shown to induce nitric oxide release by enhancing the expression of eNOS mRNA and protein in aortic endothelial cells. It was also shown to stimulate Na, K-ATPase in renal tubular cells in vitro.[5] 

C-peptide was also shown to possess several anti-inflammatory, cytoprotective, and anti-apoptotic effects in various cell types. Under physiological conditions, C-peptide was shown to inhibit the formation of reactive oxygen species (ROS) via RAC1-mediated inhibition of NAD(P)H oxidase in endothelial cells of streptozotocin (STZ)-diabetic mice.[7] C-peptide could inhibit ROS-mediated activation of transglutaminase 2, thereby inhibiting apoptosis. It also exerted the anti-apoptotic effect by inhibiting caspase 3 activation and enhancing the anti-apoptotic protein, BCL-2 in endothelial cells and neuroblastoma cells. C-peptide was also shown to inhibit the inflammatory pathway by the downregulation of NF-kB. It was also shown that C-peptide could decrease the expression of high glucose-induced ICAM, VCAM, and P-selectin.[8] High glucose-induced, vascular, smooth muscle cell proliferation and migration may be inhibited by the presence of C-peptide, thereby causing the inhibition of atherosclerotic lesion formation.[9] There is considerable interest in the biology of C-peptide, its elusive receptor, and biological effects in humans. However, this body of data is largely experimental and needs much further research in the clinical area to gain mainstream support.

Testing

As C-peptide is released in the circulation along with insulin, it has widely been used as a measure of insulin secretion to assess the pancreatic beta-cell function.[10] It also has the advantage of bypassing clearance by the liver, unlike insulin, and hence, has a much longer half-life of around 30 minutes. The plasma concentration of C-peptide in a fasted state is 0.9 to 1.8 ng/ml, and the postprandial levels are 3 to 9 ng/m in healthy individuals.[1] The higher levels are observed in overweight individuals. C-peptide is catabolized in the kidneys, and only a small fraction is excreted in the urine. Modern ultrasensitive C-peptide immunoassays can detect plasma levels as low as 0.0045 to 0.0075 ng/ml. The presence of increased titers of anti-insulin antibodies that bind to both proinsulin and C-peptide could give false-positive results. Urinary C-peptide (UCP) measurement is a non-invasive test, and the urine can be collected in boric acid, where UCP is stable at room temperature for up to 3 days. With normal urinary function, the UCP excretion is reflective of 5% to 10% of total C-peptide secreted by the pancreas.[11]

Since C-peptide is a polypeptide and less stable due to its susceptibility to enzymatic proteolytic cleavage, the serum C-peptide measurements should be done immediately after the sample collection. Hence, gel tubes are required to collect samples on ice to transport the sample to the laboratory. The samples should be immediately centrifuged and stored under frozen conditions until the estimation is done. However, EDTA-prepared tubes can be used for plasma C-peptide determination, which increases its stability at room temperature for up to 24 hours, and hence, plasma is the preferred sample.[12]

The plasma C-peptide levels can be measured in random, fasting (8 to 10 hours) or stimulated state. Random non-fasting sampling (rCP) is the easiest method to test C-peptide (fCP) levels. The rCP has been shown to correlate with 90-minute mixed meal tolerance test (MMTT) C-peptide responses. The stimulation can be done using glucagon, intravenous/oral glucose, tolbutamide, sulfonylurea, and glucagon-like peptide 1, amino acids, or a mixed meal. Glucagon stimulation test (GST) is the most widely used test due to its high sensitivity in detecting residual insulin secretion using a dose of 1.0 mg. C-peptide can also be measured using the oral glucose tolerance test (75 g, OGTT) where the samples are collected at 0, 30, 60, 90, and 120 minutes.[13] This test significantly correlates with insulin secretion in type 2 diabetes patients. Both MMTT and GST provide sensitive and reproducible results for residual beta-cell function in type 1 diabetes with peak responses seen at 90 and 6 minutes, respectively.[14] Although the C-peptide levels are useful to classify diabetes, it always must be interpreted in the clinical context of disease duration, comorbidities, and family history. However, it is still a very valid measure in clinical research studies.

Clinical Significance

The 2 major indications for measuring C-peptide levels include fasting hypoglycemia and assessment of insulin secretory reserve in patients with diabetes. In patients with fasting hypoglycemia with concomitant hyperinsulinism, one needs to entertain a differential diagnosis comprising insulinoma, exogenous insulin administration (factitious), sulfonylurea therapy (factitious), insulin autoimmune syndrome due to endogenous anti-insulin antibodies (Hirata disease).[15] All of these conditions can result in hypoglycemia with elevated insulin levels. A C-peptide level is very useful in the differential diagnosis since it is only elevated with a beta cell tumor, insulinoma, and sulfonylurea therapy. A sulfonylurea drug screen can exclude the latter. C-peptide is not elevated with Hirata disease, which is confirmed by positive anti-insulin antibodies and is decreased with factious, exogenous insulin therapy. Hence, it is a very important test in the workup of fasting hypoglycemia with hyperinsulinism.

The other major indication is the assessment of insulin secretory reserve in patients with diabetes. Diabetes mellitus is characterized by hyperglycemia due to the lack of insulin secretion and/or insulin action. Insulin deficiency is associated with C-peptide-deficiency in type 1 diabetes due to beta-cell demise. A fasting C-peptide level of less than 0.6 ng/ml is consistent with beta-cell failure and predicts the requirement for insulin therapy. Although the origins of type 2 diabetes are insulin resistance, it only manifests clinically when there is beta-cell failure resulting in impaired insulin and C-peptide secretion culminating in fasting and postprandial hyperglycemia. Much more research is needed to define the biology of C-peptide and potential role in the pathogenesis of diabetic microvascular complications or as a novel therapeutic agent. 

Also, Medicare uses a C-peptide assessment of insulin reserve as a criterion for continuous subcutaneous insulin infusion therapy (insulin pump therapy).

Pearls

C-peptide is secreted in equimolar concentrations with insulin from the beta cells.

It is a valid measure of insulin secretion especially following challenges with glucagon or a mixed meal.

C-peptide is extremely useful in the differential diagnosis of hyperinsulinemic-hypoglycemia.