C Peptide

Article Author:
Senthil Venugopal
Article Editor:
Ishwarlal Jialal
Updated:
10/27/2018 12:31:26 PM
PubMed Link:
C Peptide

Introduction

C-peptide (connecting peptide) connects alpha and beta chains of proinsulin, which are formed in the endoplasmic reticulum following removal of the signal peptide of pre-proinsulin. It is secreted from the beta cells of islets of Langerhans of the endocrine pancreas when proinsulin is cleaved into insulin and C-peptide. It plays an important role in the correct folding of insulin and the formation of disulfide bridges. C-peptide is removed in the Golgi apparatus from proinsulin resulting in the formation of the mature insulin molecule with both alpha and beta chains bound together by disulfide bonds.[1] Both insulin and C-peptide are stored in secretory vesicles and released in equimolar concentrations upon stimulation of beta cells by glucose and other secretagogues.[2] The most important indications for measurement of C-peptide levels include the differential diagnosis of fasting hypoglycemia with hyperinsulinism and as a measure of insulin secretory reserve. This brief review covers the relevant biochemistry, measurement, and clinical indications.

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

C-peptide is a 31-amino acid polypeptide and is negatively charged. In mammals, the 8 residues (position 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 the 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 the overweight individuals. C-peptide is catabolized in the kidneys, and only a small fraction is excreted in 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 a 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 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 post-prandial 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 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.


References

[1] Yosten GL,Maric-Bilkan C,Luppi P,Wahren J, Physiological effects and therapeutic potential of proinsulin C-peptide. American journal of physiology. Endocrinology and metabolism. 2014 Dec 1     [PubMed PMID: 25249503]
[2] Steiner DF,Cunningham D,Spigelman L,Aten B, Insulin biosynthesis: evidence for a precursor. Science (New York, N.Y.). 1967 Aug 11     [PubMed PMID: 4291105]
[3] Rigler R,Pramanik A,Jonasson P,Kratz G,Jansson OT,Nygren P,Stâhl S,Ekberg K,Johansson B,Uhlén S,Uhlén M,Jörnvall H,Wahren J, Specific binding of proinsulin C-peptide to human cell membranes. Proceedings of the National Academy of Sciences of the United States of America. 1999 Nov 9     [PubMed PMID: 10557318]
[4] Al-Rasheed NM,Willars GB,Brunskill NJ, C-peptide signals via Galpha i to protect against TNF-alpha-mediated apoptosis of opossum kidney proximal tubular cells. Journal of the American Society of Nephrology : JASN. 2006 Apr     [PubMed PMID: 16510765]
[5] Zhong Z,Kotova O,Davidescu A,Ehrén I,Ekberg K,Jörnvall H,Wahren J,Chibalin AV, C-peptide stimulates Na , K -ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells. Cellular and molecular life sciences : CMLS. 2004 Nov     [PubMed PMID: 15549182]
[6] Kitamura T,Kimura K,Jung BD,Makondo K,Okamoto S,Cañas X,Sakane N,Yoshida T,Saito M, 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. The Biochemical journal. 2001 Apr 1     [PubMed PMID: 11256956]
[7] Bhatt MP,Lim YC,Kim YM,Ha KS, C-peptide activates AMPK╬▒ and prevents ROS-mediated mitochondrial fission and endothelial apoptosis in diabetes. Diabetes. 2013 Nov     [PubMed PMID: 23884890]
[8] Luppi P,Cifarelli V,Tse H,Piganelli J,Trucco M, Human C-peptide antagonises high glucose-induced endothelial dysfunction through the nuclear factor-kappaB pathway. Diabetologia. 2008 Aug     [PubMed PMID: 18493738]
[9] Wahren J,Larsson C, C-peptide: new findings and therapeutic possibilities. Diabetes research and clinical practice. 2015 Mar     [PubMed PMID: 25648391]
[10] Leighton E,Sainsbury CA,Jones GC, A Practical Review of C-Peptide Testing in Diabetes. Diabetes therapy : research, treatment and education of diabetes and related disorders. 2017 Jun     [PubMed PMID: 28484968]
[11] Bowman P,McDonald TJ,Shields BM,Knight BA,Hattersley AT, Validation of a single-sample urinary C-peptide creatinine ratio as a reproducible alternative to serum C-peptide in patients with Type 2 diabetes. Diabetic medicine : a journal of the British Diabetic Association. 2012 Jan     [PubMed PMID: 21883437]
[12] McDonald TJ,Perry MH,Peake RW,Pullan NJ,O'Connor J,Shields BM,Knight BA,Hattersley AT, EDTA improves stability of whole blood C-peptide and insulin to over 24 hours at room temperature. PloS one. 2012     [PubMed PMID: 22860060]
[13] Okuno Y,Komada H,Sakaguchi K,Nakamura T,Hashimoto N,Hirota Y,Ogawa W,Seino S, Postprandial serum C-peptide to plasma glucose concentration ratio correlates with oral glucose tolerance test- and glucose clamp-based disposition indexes. Metabolism: clinical and experimental. 2013 Oct     [PubMed PMID: 23831440]
[14] Greenbaum CJ,Mandrup-Poulsen T,McGee PF,Battelino T,Haastert B,Ludvigsson J,Pozzilli P,Lachin JM,Kolb H, Mixed-meal tolerance test versus glucagon stimulation test for the assessment of beta-cell function in therapeutic trials in type 1 diabetes. Diabetes care. 2008 Oct     [PubMed PMID: 18628574]
[15] Arzamendi AE,Rajamani U,Jialal I, Pseudoinsulinoma in a white man with autoimmune hypoglycemia due to anti-insulin antibodies: value of the free C-Peptide assay. American journal of clinical pathology. 2014 Nov     [PubMed PMID: 25319986]