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.

Can C-peptide mediated anti-inflammatory effects retard the development of microvascular complications of type 1 diabetes?

Hyperglycemia is considered to be the major cause of microvascular complications of diabetes. Growing evidence highlights the importance of hyperglycemia-mediated inflammation in the initiation and progression of microvascular complications in type 1 diabetes. We hypothesize that lack of proinsulin C-peptide and lack of its anti-inflammatory properties contribute to the development of microvascular complications. Evidence gathered over the past 20 years shows that C-peptide is a biologically active peptide in its own right. It has been shown to reduce formation of reactive oxygen species and nuclear factor-κB activation induced by hyperglycemia, resulting in inhibition of cytokine, chemokine and cell adhesion molecule formation as well as reduced apoptotic activity. In addition, C-peptide stimulates and induces the expression of both Na⁺, K⁺-ATPase and endothelial nitric oxide synthase. Animal studies and small-scale clinical trials in type 1 diabetes patients suggest that C-peptide replacement combined with regular insulin therapy exerts beneficial effects on kidney and nerve dysfunction. Further clinical trials in patients with microvascular complications including measurements of inflammatory markers are warranted to explore the clinical significance of the aforementioned, previously unrecognized, C-peptide effects.

Splanchnic balance of free fatty acids, endocannabinoids, and lipids in subjects with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Animal studies suggest that endocannabinoids could contribute to the development of nonalcoholic fatty liver disease (NAFLD). In addition, NAFLD has been shown to be associated with multiple changes in lipid concentrations in liver biopsies. There are no data on splanchnic free fatty acid (FFA), glycerol, ketone body, endocannabinoid, and lipid fluxes in vivo in subjects with NAFLD. METHODS: We performed hepatic venous catheterization studies in combination with [(2)H(2)]palmitate infusion in the fasting state and during a low-dose insulin infusion in 9 subjects with various degrees of hepatic steatosis as determined using liver biopsy. Splanchnic balance of endocannabinoids and individual lipids was determined using ultra performance liquid chromatography coupled to mass spectrometry. RESULTS: Concentrations of the endocannabinoid 2-arachidonoylglycerol were higher in arterialized (91 ± 33 µg/L basally) than in hepatic venous (51 ± 19 µg/L; P < .05) plasma. Fasting arterial (r = 0.72; P = .031) and hepatic venous (r = 0.70; P = .037) concentrations of 2-arachidonoylglycerol were related positively to liver fat content. Analysis of fluxes of 85 different triglycerides showed that the fatty liver overproduces saturated triglycerides. In the plasma FFA fraction in the basal state, the relative amounts of palmitoleate and linoleate were lower and those of stearate and oleate were higher in the hepatic vein than in the artery. Absolute concentrations of all nontriglyceride lipids were comparable in arterialized venous plasma and the hepatic vein both in the basal and insulin-stimulated states. CONCLUSIONS: The human fatty liver takes up 2-arachidonoylglycerol and overproduces triacylglycerols containing saturated fatty acids, which might reflect increased de novo lipogenesis.

Precise determination of glucose-d(2)/glucose ratio in human serum and plasma by APCI LC-MS/MS.

BACKGROUND: Determinations of glucose turnover based on infusion of deuterium-labelled glucose are frequently undertaken in studies involving the pathophysiology of diabetes or obesity or the metabolic response to physical exercise. METHODS: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for determination of the glucose-d(2)/glucose ratio in human serum and plasma following the intra-venous infusion of 6,6-d(2)-glucose. Atmospheric pressure chemical ionization measuring negative ions in selected reaction monitoring mode (product ions at m/z 179-->89 and m/z 181-->89) was used. The serum or plasma samples (50 microL) were prepared by protein precipitation with acetonitrile and the resulting supernatant was directly used for the LC-MS/MS analysis. The chromatography was performed on a Luna 3 micron NH2 100A column (100 x 2 mm) using a mobile phase containing 85% acetonitrile with 20 mmol/L of ammonium acetate at a flow rate of 400 microL/min and an oven temperature of 40 degrees C. RESULTS: A linear response was obtained for the glucose-d(2)/glucose ratio over the relative concentration range of 0-10%, r(2)>0.999. The peak area ratio was determined with an imprecision of 1.20-8.19% (coefficient of variation). The ion suppression from matrix compared to water was in the order of 55%. Chromatographic retention time was between 4 and 5 min and the total analysis time was 10 min. The validated method was successfully applied for the analysis of human serum samples from a clinical study involving infusion of 6,6-d(2)-glucose and evaluation of endogenous glucose production. CONCLUSIONS: It is concluded that the described method provides an easy and precise technique for the determination of serum and plasma glucose-d(2)/glucose ratios in clinical studies.

C-Peptide replacement therapy and sensory nerve function in type 1 diabetic neuropathy.

OBJECTIVE: C-peptide replacement in animals results in amelioration of diabetes-induced functional and structural abnormalities in peripheral nerves. The present study was undertaken to examine whether C-peptide administration to patients with type 1 diabetes and peripheral neuropathy improves sensory nerve function. RESEARCH DESIGN AND METHODS: This was an exploratory, double-blinded, randomized, and placebo-controlled study with three study groups that was carried out at five centers in Sweden. C-peptide was given as a replacement dose (1.5 mg/day, divided into four subcutaneous doses) or a dose three times higher (4.5 mg/day) during 6 months. Neurological examination and neurophysiological measurements were performed before and after 6 months of treatment with C-peptide or placebo. RESULTS: The age of the 139 patients who completed the protocol was 44.2 +/- 0.6 (mean +/- SE) years and their duration of diabetes was 30.6 +/- 0.8 years. Clinical neurological impairment (NIA) (score >7 points) of the lower extremities was present in 86% of the patients at baseline. Sensory nerve conduction velocity (SCV) was 2.6 +/- 0.08 SD below body height-corrected normal values at baseline and improved similarly within the two C-peptide groups (P < 0.007). The number of patients responding with a SCV peak potential improvement >1.0 m/s was greater in C-peptide-treated patients than in those receiving placebo (P < 0.03). In the least severely affected patients (SCV < 2.5 SD below normal at baseline, n = 70) SCV improved by 1.0 m/s (P < 0.014 vs. placebo). NIA score and vibration perception both improved within the C-peptide-treated groups (P < 0.011 and P < 0.002). A1C levels (7.6 +/- 0.1% at baseline) decreased slightly but similarly in C-peptide-and placebo-treated patients during the study. CONCLUSIONS: C-peptide treatment for 6 months improves sensory nerve function in early-stage type 1 diabetic neuropathy.

C-Peptide reverses nociceptive neuropathy in type 1 diabetes.

We examined the therapeutic effects of C-peptide on established nociceptive neuropathy in type 1 diabetic BB/Wor rats. Nociceptive nerve function, unmyelinated sural nerve fiber and dorsal root ganglion (DRG) cell morphometry, nociceptive peptide content, and the expression of neurotrophic factors and their receptors were investigated. C-peptide was administered either as a continuous subcutaneous replacement dose via osmopumps or a replacement dose given once daily by subcutaneous injection. Diabetic rats were treated from 4 to 7 months of diabetes and were compared with control and untreated diabetic rats of 4- and 7-month duration. Osmopump delivery but not subcutaneous injection improved hyperalgesia and restored the diabetes-induced reduction of unmyelinated fiber number (P < 0.01) and mean axonal size (P < 0.05) in the sural nerve. High-affinity nerve growth factor (NGF) receptor (NGFR-TrkA) expression in DRGs was significantly reduced at 4 months (P < 0.01). Insulin receptor and IGF-I receptor (IGF-IR) expressions in DRGs and NGF content in sciatic nerve were significantly decreased in 7-month diabetic rats (P < 0.01, 0.05, and 0.005, respectively). Osmopump delivery prevented the decline of NGFR-TrkA, insulin receptor (P < 0.05), and IGF-IR (P < 0.005) expressions in DRGs and improved NGF content (P < 0.05) in sciatic nerve. However, subcutaneous injection had only marginal effects on morphometric and molecular changes in diabetic rats. We conclude that C-peptide exerts beneficial therapeutic effects on diabetic nociceptive neuropathy and that optimal effects require maintenance of physiological C-peptide concentrations for a major proportion of the day.

Novel LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one in human plasma. Evidence for a significant extrahepatic metabolism.

A new isotope dilution LC-MS/MS method for assay of 7alpha-hydroxy-4-cholesten-3-one without need for derivatization is described. This method was used in catheterization experiments on healthy fasting volunteers. The levels of this generally used marker for bile acid synthesis were slightly but significantly higher in the hepatic vein than in the brachial artery. In contrast, the levels of the precursor to 7alpha-hydroxy-4 cholesten-3-one, 7alpha-hydroxycholesterol, were the same in the two vessels. It is concluded that there is a net extrahepatic metabolism of 7alpha-hydroxy-4-cholesten-3-one. The similarity and very high correlation between the levels in the two vessels (r=0.97) are consistent with the contention that 7alpha-hydroxy-4-cholesten-3-one is a suitable marker for the activity of the hepatic cholesterol 7alpha-hydroxylase and thus bile acid synthesis.

The contribution of visceral adipose tissue to splanchnic cortisol production in healthy humans.

Cortisol is regenerated from cortisone by 11beta-hydroxysteroid dehydrogenase type 1 (11HSD1), amplifying glucocorticoid action in adipose tissue and liver. 11HSD1 inhibitors are being developed for type 2 diabetes and may be most effective in obesity, where adipose 11HSD1 is increased. However, the magnitude of regeneration of cortisol in different tissues in humans is unknown, hindering understanding of the pathophysiological and therapeutic importance of 11HSD1. In eight healthy men, we infused 9,11,12,12-(2)H4-cortisol and measured tracer enrichment in the hepatic vein as an indicator of total splanchnic cortisol generation. Oral cortisone (25 mg) was then given to measure first-pass hepatic cortisol generation. In steady state, splanchnic cortisol production was 45 +/- 11 nmol/min when arterialized plasma cortisone concentration was 92 +/- 7 nmol/l. Extrapolation from hepatic cortisol generation after oral cortisone suggested that, at steady state, the liver contributes 15.2 nmol/min and extrahepatic splanchnic tissue contributes 29.8 nmol/min to the total splanchnic cortisol production. We conclude that tissues draining into the portal vein, including visceral adipose tissue, contribute substantially to the regeneration of cortisol. Thus, in addition to free fatty acids and adipokines, the portal vein delivers cortisol to the liver, and inhibition of 11HSD1 in visceral adipose tissue may indeed be valuable in ameliorating insulin resistance in obesity.

Long-Acting C-Peptide and Neuropathy in Type 1 Diabetes: A 12-Month Clinical Trial.

OBJECTIVE: Lack of C-peptide in type 1 diabetes may be an important contributing factor in the development of microvascular complications. Replacement of native C-peptide has been shown to exert a beneficial influence on peripheral nerve function in type 1 diabetes. The aim of this study was to evaluate the efficacy and safety of a long-acting C-peptide in subjects with type 1 diabetes and mild to moderate peripheral neuropathy. RESEARCH DESIGN AND METHODS: A total of 250 patients with type 1 diabetes and peripheral neuropathy received long-acting (pegylated) C-peptide in weekly dosages of 0.8 mg (n = 71) or 2.4 mg (n = 73) or placebo (n = 106) for 52 weeks. Bilateral sural nerve conduction velocity (SNCV) and vibration perception threshold (VPT) on the great toe were measured on two occasions at baseline, at 26 weeks, and at 52 weeks. The modified Toronto Clinical Neuropathy Score (mTCNS) was used to grade the peripheral neuropathy. RESULTS: Plasma C-peptide rose during the study to 1.8-2.2 nmol/L (low dose) and to 5.6-6.8 nmol/L (high dose). After 52 weeks, SNCV had increased by 1.0 ± 0.24 m/s (P < 0.001 within group) in patients receiving C-peptide (combined groups), but the corresponding value for the placebo group was 1.2 ± 0.29 m/s. Compared with basal, VPT had improved by 25% after 52 weeks of C-peptide therapy (Δ for combined C-peptide groups: -4.5 ± 1.0 µm, placebo group: -0.1 ± 0.9 µm; P < 0.001). mTCNS was unchanged during the study. CONCLUSIONS: Once-weekly subcutaneous administration of long-acting C-peptide for 52 weeks did not improve SNCV, other electrophysiological variables, or mTCNS but resulted in marked improvement of VPT compared with placebo.

Effects of proinsulin C-peptide in experimental diabetic neuropathy: vascular actions and modulation by nitric oxide synthase inhibition.

Proinsulin C-peptide treatment can partially prevent nerve dysfunction in type 1 diabetic rats and patients. This could be due to a direct action on nerve fibers or via vascular mechanisms as C-peptide stimulates the nitric oxide (NO) system and NO-mediated vasodilation could potentially account for any beneficial C-peptide effects. To assess this further, we examined neurovascular function in streptozotocin-induced diabetic rats. After 6 weeks of diabetes, rats were treated for 2 weeks with C-peptide to restore circulating levels to those of nondiabetic controls. Additional diabetic groups were given C-peptide with NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA) co-treatment or scrambled C-peptide. Diabetes caused 20 and 16% reductions in sciatic motor and saphenous sensory nerve conduction velocity, which were 62 and 78% corrected, respectively, by C-peptide. L-NNA abolished C-peptide effects on nerve conduction. Sciatic blood flow and vascular conductance were 52 and 41%, respectively, reduced by diabetes (P < 0.001). C-peptide partially (57-66%) corrected these defects, an effect markedly attenuated by L-NNA co-treatment. Scrambled C-peptide was without effect on nerve conduction or perfusion. Thus, C-peptide replacement improves nerve function in experimental diabetes, and the data are compatible with the notion that this is mediated by a NO-sensitive vascular mechanism.

C-peptide exerts beneficial effects on myocardial blood flow and function in patients with type 1 diabetes.

Myocardial dysfunction, perfusion abnormalities, and the extent to which these abnormalities may be reversed by C-peptide administration was assessed in type 1 diabetic patients. Eight patients were studied before and during a 0.84-mg/kg dipyridamole administration using a randomized double-blind crossover protocol with infusion of C-peptide (6 pmol x kg(-1) x min(-1)) or saline during 60 min on two different days. Myocardial function was measured as peak myocardial velocity during systole (Vs) and early diastole (Vd) by pulsed tissue Doppler imaging. Myocardial contrast echocardiography was used for assessment of myocardial blood volume (SI(max)) and myocardial blood flow index (MBFI) calculated from the relation between trigger interval and signal intensity. Eight age-matched healthy volunteers served as control subjects. In the basal state, Vd (13.8 +/- 0.6 vs. 15.6 +/- 0.5 cm/s, P < 0.04) and SI(max) (6.6 +/- 0.6 vs. 8.2 +/- 0.6 a.u. P < 0.04) were reduced in patients compared with control subjects. Dipyridamole administration significantly increased indexes of myocardial function and blood flow to a similar extent in patients and control subjects. During C-peptide administration, Vs and Vd increased by 12% (P = 0.03), SI(max) increased from 6.6 +/- 0.6 to 8.1 +/- 0.7 a.u. (P < 0.02), and MBFI increased from 3.3 +/- 0.4 to 5.3 +/- 0.9 (P < 0.05). The results demonstrate that type 1 diabetic patients have impaired myocardial function and perfusion in the basal state that can be improved by short-term replacement of C-peptide.

Amelioration of sensory nerve dysfunction by C-Peptide in patients with type 1 diabetes.

Studies have demonstrated that proinsulin C-peptide stimulates the activities of Na(+),K(+)-ATPase and endothelial nitric oxide synthase, both of which are enzyme systems of importance for nerve function and known to be deficient in type 1 diabetes. The aim of this randomized double-blind placebo-controlled study was to investigate whether C-peptide replacement improves nerve function in patients with type 1 diabetes. Forty-nine patients without symptoms of peripheral neuropathy were randomized to either 3 months of treatment with C-peptide (600 nmol/24 h, four doses s.c.) or placebo. Forty-six patients (15 women and 31 men, aged 29 years, diabetes duration 10 years, and HbA(1c) 7.0%) completed the study. Neurological and neurophysiological measurements were performed before and after 6 and 12 weeks of treatment. At baseline the patients showed reduced nerve conduction velocities in the sural nerve (sensory nerve conduction velocity [SCV]: 50.9 +/- 0.70 vs. 54.2 +/- 1.2 m/s, P < 0.05) and peroneal nerve (motor nerve conduction velocity: 45.7 +/- 0.55 vs. 53.5 +/- 1.1 m/s, P < 0.001) compared with age-, height-, and sex-matched control subjects. In the C-peptide treated group there was a significant improvement in SCV amounting to 2.7 +/- 0.85 m/s (P < 0.05 compared with placebo) after 3 months of treatment, representing 80% correction of the initial reduction in SCV. The change in SCV was accompanied by an improvement in vibration perception in the patients receiving C-peptide (P < 0.05 compared with placebo), whereas no significant change was detectable in cold or heat perception. In conclusion, C-peptide administered for 3 months as replacement therapy to patients with early signs of diabetic neuropathy ameliorates nerve dysfunction.

Large-surface biosensor technology for enhanced recovery in protein characterization.

A large-surface biosensor technique using surface plasmon resonance (SPR) was tested for protein purification by recovery of a monoclonal antibody against human proinsulin C-peptide. Notably, both reversible attachment/desorption and actual purification of the antibody from a multi-component protein mixture was shown. For initial chip attachment of the peptide ligand, C-peptide was biotinylated and attached to neutravidin on plastic chips with a large gold surface (effective area 26 mm(2)). Antibody binding and desorption was monitored in real-time SPR, and for elution different conditions were employed. Five percent formic acid (in contact with the chip surface for 3 min) in a 60-mul segment between air bubbles was efficient for subsequent analysis. In this manner, protein amounts up to 35 pmoles were recovered in a single capture/elution cycle. Evaluation by SDS-PAGE showed essentially no carryover between fractions in this elution process, and also not with other proteins in the mixture after purification. Compared to existing commercial instruments, this technique gives higher recovery and makes it possible to monitor monitor protein binding/desorption. Recovery of affinity partners at the multi-pmole level is demonstrated for protein purification in SPR approaches.

C-peptide: new findings and therapeutic possibilities.

Much new information on C-peptide physiology has appeared during the past 20 years. It has been shown that C-peptide binds specifically to cell membranes, elicits intracellular signaling via G-protein and Ca2+ -dependent pathways, resulting in activation and increased expression of endothelial nitric oxide synthase, Na+, K+ -ATPase and several transcription factors of importance for anti-inflammatory, anti-oxidant and cell protective mechanisms. Studies in animal models of diabetes and early clinical trials in patients with type 1 diabetes demonstrate that C-peptide in replacement doses elicits beneficial effects on early stages of diabetes-induced functional and structural abnormalities of the peripheral nerves, the kidneys and the retina. Much remains to be learned about C-peptide's mechanism of action and long-term clinical trials in type 1 diabetes subjects will be required to determine C-peptide's clinical utility. Nevertheless, even a cautious evaluation of the available evidence presents the picture of a bioactive endogenous peptide with therapeutic potential.

Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle.

To elucidate mechanisms regulating capillary transport of insulin and glucose, we directly calculated the permeability surface (PS) area product for glucose and insulin in muscle. Intramuscular microdialysis in combination with the forearm model and blood flow measurements was performed in healthy males, studied during an oral glucose tolerance test or during a one-step or two-step euglycemic hyperinsulinemic clamp. PS for glucose increased significantly from 0.29 +/- 0.1 to 0.64 +/- 0.2 ml/min.100 g after oral glucose tolerance test, and glucose uptake increased from 1.2 +/- 0.4 to 2.6 +/- 0.6 micro mol/min.100 g (P < 0.05). During one-step hyperinsulinemic clamp (plasma insulin, 1.962 pmol/liter), PS for glucose increased from 0.2 +/- 0.1 to 2.3 +/- 0.9 ml/min.100 g (P < 0.05), and glucose uptake increased from 0.6 +/- 0.2 to 5.0 +/- 1.4 micro mol/min.100 g (P < 0.05). During the two-step clamp (plasma insulin, 1380 +/- 408 and 3846 +/- 348 pmol/liter), the arterial-interstitial difference and PS for insulin were constant. The PS for glucose tended to increase (P = not significant), whereas skeletal muscle blood flow increased from 4.4 +/- 0.7 to 6.2 +/- 0.8 ml/min.100 ml (P < 0.05). The present data show that PS for glucose is markedly increased by oral glucose, whereas a further vasodilation exerted by high insulin concentrations may not be physiologically relevant for capillary delivery of either glucose or insulin in resting muscle.

In vivo biodistribution and pharmacokinetics of (18)F-labeled human C-peptide: evaluation in monkeys using positron emission tomography.

The recently observed beneficial effects exerted by C-peptide in insulin-dependent diabetes patients (IDDM) have instigated research into the mechanisms of C-peptide action as well as the location for it. Here we report in vivo biodistribution studies performed in monkeys using positron emission tomography (PET) and C-peptide labeled in the N-terminal with fluorine-18. Following iv injection of the radiotracer, dynamic decay data were collected over the chest and/or abdomens of the monkeys. The radioactivity distributed mainly to the kidneys, less to the heart and to some extent to the liver. Excretion of radioactivity into the urinary bladder was observed. Brain uptake was not detected in a static emission scan of the head performed at late times. Accumulation of radioactivity in the skeleton as a result of in vivo defluorination was not observed. Pharmacokinetic modeling of the regional concentrations of radioactivity over time resulted, for most organs, in two-compartment models. The organs with the highest radioactivity concentrations have been identified, enabling dose estimations for studies in humans with low or no C-peptide.

C-peptide: new findings and therapeutic implications in diabetes.

In contrast to earlier views, new data indicate that proinsulin C-peptide exerts important physiological effects and shows the characteristics of an endogenous peptide hormone. C-peptide in nanomolar concentrations binds specifically to cell membranes, probably to a G-protein coupled receptor. Ca(2+)- and MAP-kinase dependent signalling pathways are activated, resulting in stimulation of Na(+), K(+)-ATPase and endothelial nitric oxide (NO) synthase, two enzyme systems known to be deficient in diabetes. C-peptide may also interact synergistically with insulin signal transduction. Studies in intact animals and in patients with type 1 diabetes have demonstrated multifaceted effects. Thus, C-peptide administration in streptozotocin-diabetic animals results in normalization of diabetes-induced glomerular hyperfiltration, reduction of urinary albumin excretion and diminished glomerular expansion. The former two effects have also been observed in type 1 diabetes patients given C-peptide in replacement dose for up to 3 months. Peripheral nerve function and structure are likewise influenced by C-peptide administration; sensory and motor nerve conduction velocities increase and nerve structural changes are diminished or reversed in diabetic rats. In patients with type 1 diabetes, beneficial effects have been demonstrated on sensory nerve conduction velocity, vibration perception and autonomic nerve function. C-peptide also augments blood flow in several tissues in type 1 diabetes via its stimulation of endothelial NO release, emphasizing a role for C-peptide in maintaining vascular homeostasis. Continued research is needed to establish whether, among the hormones from the islets of Langerhans, C-peptide is the ugly duckling that--nearly 40 years after its discovery--may prove to be an endogenous peptide hormone of importance in the treatment of diabetic long-term complications.

The structure, molecular interactions and bioactivities of proinsulin C-peptide correlate with a tripartite molecule.

Many biological roles have been assigned to proinsulin C-peptide over the years. Some appear surprisingly disparate and sometimes even contradictory, like chaperone-like actions and depository tendencies. This review summarizes recently reported biomolecular interactions of the peptide and presents how they correlate with structural and functional aspects into a partitioned molecular architecture. At the structural level, the C-peptide sequence and fold can be subdivided into three distinct parts ('tripartite'). At the functional level, its chaperone-like abilities, self-assembly, and membrane interactions, as well as interactions with relevant proteins can be separately ascribed to these three segments. At the biological level, the assignments are compatible with the suggested roles of C-peptide in granular insulin storage, chaperone-like activities on insulin oligomers, possible depository tendencies, and proposed receptor interactions. Finally, the assignments give interesting parallels to further bioactive peptides, including glucagon and neurotensin. Provided pharmaceutical and clinical trials are successfully completed, the present interpretations should supply mechanistic explanations on C-peptide as a bioactive compound of importance in health and diabetes.