Human C-peptide antagonises high glucose-induced endothelial dysfunction through the nuclear factor-κB pathway
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- Luppi, P., Cifarelli, V., Tse, H. et al. Diabetologia (2008) 51: 1534. doi:10.1007/s00125-008-1032-x
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Endothelial dysfunction in diabetes is predominantly caused by hyperglycaemia leading to vascular complications through overproduction of oxidative stress and activation of the transcription factor nuclear factor-κB (NF-κB). Many studies have suggested that decreased circulating levels of C-peptide may play a role in diabetic vascular dysfunction. To date, the possible effects of C-peptide on endothelial cells and intracellular signalling pathways are largely unknown. We therefore investigated the effect of C-peptide on several biochemical markers of endothelial dysfunction in vitro. To gain insights into potential intracellular signalling pathways affected by C-peptide, we tested NF-κB activation, since it is known that inflammation, secondary to oxidative stress, is a key component of vascular complications and NF-κB is a redox-dependent transcription factor.
Human aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence of C-peptide (0.5 nmol/l) for 24 h and tested for expression of the gene encoding vascular cell adhesion molecule-1 (VCAM-1) by RT-PCR and flow cytometry. Secretion of IL-8 and monocyte chemoattractant protein-1 (MCP-1) was measured by ELISA. NF-κB activation was analysed by immunoblotting and ELISA.
Physiological concentrations of C-peptide affect high glucose-induced endothelial dysfunction by: (1) decreasing VCAM-1 expression and U-937 cell adherence to HAEC; (2) reducing secretion of IL-8 and MCP-1; and (3) suppressing NF-κB activation.
During hyperglycaemia, C-peptide directly affects VCAM-1 expression and both MCP-1 and IL-8 HAEC secretion by reducing NF-κB activation. These effects suggest a physiological anti-inflammatory (and potentially anti-atherogenic) activity of C-peptide on endothelial cells.
KeywordsAtherosclerosis C-peptide Cytokines Endothelial cells Inflammation Monocytes NF-κB Nuclear factor κB Vascular smooth muscle cells
endothelial basal medium-2
human aortic endothelial cells
monocyte chemoattractant protein-1
mean fluorescence intensity
reactive oxygen species
vascular cell adhesion molecule-1
Diabetes is a well-established risk factor for vascular diseases. Vascular disease in diabetes originates from common functional and structural changes in the tunica media of small (microangiopathy) as well as large vessels (macroangiopathy). In large vessels, these changes increase the probability of developing atherosclerosis, which is one of the major complications affecting diabetic patients. As a result of the diabetic state, the vascular compromise at small vessels level principally affects the eye, kidney and both peripheral and autonomic nerves, and this dysfunction contributes significantly to the morbidity associated with diabetes .
Diabetes causes vascular compromise secondary to endothelial dysfunction, measured by in vivo studies of flow-mediated vasodilation  and increased circulating levels of biochemical markers, such as, but clearly not limited to vascular cell adhesion molecule-1 (VCAM-1) [3, 4]. Generally, VCAM-1 is expressed at a low level on endothelial cells and is upregulated upon cellular activation, such as that observed after exposure to inflammatory stimuli or high glucose [5, 6]. VCAM-1 binds to the leucocyte integrin α4β4 (also called very late antigen-4; CD49d) and has a principal role in the early stages of monocytes adhesion to the vascular endothelium, one of the first steps in atherosclerosis plaque formation. A major hallmark of diabetes is an abnormally elevated blood glucose level, i.e. hyperglycaemia, which has been proposed as one factor causing endothelial dysfunction in diabetes. In endothelial cells, acute and chronic hyperglycaemia works through reactive oxygen species (ROS) production [5, 7, 8] that leads to activation of the transcription factor nuclear factor-κB (NF-κB) [5, 9, 10] and ultimately the production of inflammatory mediators .
In the unstimulated state, NF-κB exists in its canonical form as a heterodimer composed of p50 and p65 subunits bound to IκB. Upon activation, IκB is phosphorylated and degraded causing the release of p50/p65 components of NF-κB . The active p50/p65 heterodimer translocates to the nucleus and initiates the transcription of a gamut of genes involved in the inflammatory response, such as those encoding pro-inflammatory cytokines, cell surface adhesion molecules and chemokines, including IL-8 and monocyte chemoattractant protein-1 (MCP-1) [5, 11, 13, 14, 15]. IL-8 and MCP-1 production is present in human atherosclerotic plaques  and participates in the development of atherosclerosis by recruiting monocytes into the subendothelial cell layer .
It has been suggested that proinsulin C-peptide may possess cytoprotective effects on the microvasculature during inflammatory events . In line with this, it has been reported that type 1 diabetic patients with circulating levels of C-peptide closer to the physiological level of 0.5 nmol/l  or receiving whole pancreas  or allogeneic islet transplantation  show a reduced incidence of microvascular complications. The mechanisms able to produce the beneficial effects of C-peptide on vascular dysfunction in diabetes remain largely unknown. One study performed in vivo in a rat inflammatory model of vascular dysfunction showed that a single i.v. dose of C-peptide significantly inhibited leucocyte–endothelium interaction via decreased expression of endothelial cell adhesion molecules , a phenomenon associated with release of nitric oxide [22, 23], which in turn has been shown to inhibit NF-κB . Similar results were obtained in isolated ischaemic and reperfused rat hearts, where addition of C-peptide attenuated polymorphonuclear cell adherence to the vascular endothelium . To date, no data are available on the effects of C-peptide on human endothelial cells exposed to the damaging insult of hyperglycaemia, a common condition in diabetes.
We therefore initiated a study on the direct effects of C-peptide, testing VCAM-1 expression on the cell surface, monocyte adherence and secretion of IL-8 and MCP-1 by human aortic endothelial cells (HAEC) exposed to short-term high glucose. Since activation of the transcription factor NF-κB is involved in these pro-inflammatory responses, we also investigated the direct effect of C-peptide on nuclear translocation of the NF-κB subunits p50/p65 in HAEC. We hypothesised that physiological concentrations of C-peptide protect HAEC from high glucose-induced cellular dysfunction by decreasing NF-κB activation, thus inhibiting NF-κB-dependent genes, such as those encoding VCAM-1, IL-8 and MCP-1.
Cell culture of HAEC
HAEC were obtained from Cambrex (Cambrex Bioscience Walkersville, Walkersville, MD, USA) and grown into 75 cm2 culture flasks (250,000 per flask) (Corning, Corning, NY, USA) at 37°C, 5% CO2 and in the presence of endothelial basal medium-2 (EBM-2) supplemented with endothelial growth media SingleQuots (Cambrex). HAEC were used at passages two to six.
EBM-2 containing 25 mmol/l glucose (Sigma Chemical, St Louis, MO, USA) was used as a high glucose condition in all the experiments, while regular EBM-2, which contains 5.6 mmol/l glucose, was used as normal glucose condition. In all experiments, HAEC were used when having reached an 80% to 90% confluency. On the day of the experiment, cells were washed with fresh EBM-2 and then replaced with EBM-2 containing 25 mmol/l glucose in the presence or absence of physiological concentrations of human C-peptide (0.5 and/or 1 nmol/l) (Sigma Chemical)  for 4 to 24 h in an incubator at 37°C and 5% CO2. As a control for C-peptide activity, C-peptide was heat-inactivated by boiling it for 1 h and then added to the culture. Human recombinant TNF-α (10 ng/ml; R&D Systems, Minneapolis, MN, USA), which activates HAEC, was used as a positive control. The effect of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 10 μmol/l; Sigma) was also tested on HAEC in certain experiments. This inhibitor was added to EBM-2 with 25 mmol/l glucose. Separate sets of experiments were performed in which C-peptide was added to regular EBM-2 containing 5.6 mmol/l glucose.
VCAM-1 detection by RT-PCR
HAEC were grown into 75 cm2 culture flasks (250,000 per flask; Corning) and exposed to the treatment conditions mentioned above. After 4 and 24 h, cells were trypsinised and frozen at −80°C. RNA extraction was performed using the RiboPure-Blood kit (Ambion, Austin, TX, USA). For RT-PCR, 1 μg RNA was used together with Oligo(d)T (RETROscript; Ambion) and 1 μl of cDNA was used to amplify VCAM-1. Human GAPDH, 18S ribosomal RNA and β-actin were amplified and served as internal controls . Sequences of the oligonucleotides used to amplify these genes and PCR conditions are reported as Electronic supplementary material (ESM). Three independent experiments were performed. Densitometry was performed with UN-SCAN-IT gel software (Silk Scientific, Orem, UT, USA). Data are expressed as median ± SD.
Quantification of VCAM-1 by flow cytometry
HAEC (50,000 per well) were maintained in 6-well plates (Corning) until confluent. On the day of the experiment, cells were exposed to the treatment conditions mentioned above for 24 h. Determination of VCAM-1 expression by surface staining was performed on paraformaldehyde-fixed HAEC monolayers following a methodology shown to preserve single cell integrity . A phycoerythrine-conjugated anti-human monoclonal antibody to CD106 (VCAM-1) or corresponding isotype control (BD Pharmigen, San Diego, CA, USA) were used for staining. Cells were run on a Becton Dickinson FACSCalibur and analysed at a later time (Becton Dickinson, San Jose, CA, USA). For a more detailed description of methodology and data analysis, see ESM. Three sets of independent experiments were performed. Within each experiment, each condition was tested in triplicate. Data are expressed as median ± SD.
Monocyte adhesion assay
HAEC were grown on 48-well plates (12,000 per well; Corning) and exposed to the treatment conditions mentioned above for 4 h. Human monocytic U-937 cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and grown in RPMI 1640 (Cambrex) containing 10% FCS, 100 μl/ml streptomycin, 100 IU/ml penicillin, 250 ng/ml fungizone, 1 mmol/l sodium pyruvate, 10 mmol/l HEPES (all from Gibco Invitrogen, Carlsbad, CA, USA) at 37°C and 5% of CO2. On the day of the experiment, medium was removed from each well, cells were washed with PBS and fresh medium containing U-937 cells (1 × 106 cells/ml, 500 μl) was added to each well and incubated for 1 h at room temperature on a rocking plate. Non-adherent U-937 cells were removed and adherent cells fixed in 1% glutaraldehyde. The number of adherent cells was evaluated by counting three random 40× fields per well by a blinded investigator, avoiding areas of non-confluence and cell clusters. Three experiments were performed. Within each experiment, each condition was tested in triplicate. Results are showed as median ± SD.
IL-8 and MCP-1 detection in culture supernatant fraction by ELISA
HAEC were maintained in 6-well plates (50,000 per well; Corning) in EBM-2 (Cambrex). On the day of the experiment, cells were exposed to the treatment conditions mentioned above for 4 h. The supernatant fraction was collected and kept at −20°C until tested by ELISA (Quantikine; R&D Systems). Three independent experiments were performed, in which each condition was tested in triplicate. Concentration of the chemokines (pg/ml) was assessed by calculating values according to the values obtained in the standard curve. Results from three separate experiments are shown as median ± SD.
NF-κB analysis assays
HAEC were cultured in 75 cm2 culture flasks (250,000 per flask; Corning) and exposed to the treatment conditions as indicated above. Cells were collected at 4 and 24 h and pretreated with 25 μl of protease inhibitor cocktail (Pierce, Rockford, IL, USA). Nuclear and cytoplasmic fractions were separated using a kit (NE-PER Nuclear and Cytoplasmic Extraction; Pierce). Protein content of the extract was measured using a bicinchoninic acid assay kit (Pierce Biotechnology). For detection of NF-κB p65 subunit by western blot, 10 μg of nuclear protein extracts were used as previously described . Densitometry analysis of the bands was performed with UN-SCAN-IT gel software (Silk Scientific). Activation of the NF-κB p50 subunit was detected on 3 μg of nuclear protein extracts using a kit (EZ-Detect Transcription Factor Kit; Pierce Technology). For each set of data, a minimum of three experiments was performed. Data were averaged and expressed as means ± SD.
Paired t test (two-tailed) was used to analyse differences between 5.6 and 25 mmol/l glucose. ANOVA with the Dunnett’s post hoc test was used to assess differences between 25 mmol/l glucose, C-peptide and PDTC using GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA). Values of p < 0.05 were considered to be statistically significant.
C-peptide reduces VCAM-1 on HAEC exposed to high glucose
When C-peptide was added to low glucose medium, it did not cause a significant change in VCAM-1 expression (Fig. 3b).
Because C-peptide at a dose of 0.5 nmol/l demonstrated a significant reduction in high glucose-induced VCAM-1 expression, we conducted all further experiments using this dose only.
C-peptide inhibits U-937 cells adhesion to endothelial cells
When we evaluated the adherence of U-937 to HAEC exposed to normal glucose and with C-peptide added, we did not observe a significant change as compared with normal glucose alone (ESM Fig. 1).
C-peptide inhibits IL-8 and MCP-1 secretion by HAEC
C-peptide added to regular medium containing low glucose failed to significantly modify IL-8 and MCP-1 secretion compared with basal medium without C-peptide (ESM Fig. 2).
C-peptide decreases high glucose-induced NF-κB translocation in HAEC
Activation of the NF-κB p50 subunit in glucose-stimulated HAEC was assessed by ELISA. As shown in Fig. 7b, high glucose significantly induced NF-κB p50 activity in contrast to normal glucose (p = 0.002). High glucose-induced NF-κB p50 binding activity was efficiently ablated by the addition of C-peptide (p < 0.01).
Type 1 diabetes patients have an increased risk of developing atherosclerosis and microvascular complications compared with the non-diabetic population. This risk is in part associated with the difficulty in maintaining euglycaemic conditions even in the context of an appropriate exogenous insulin treatment . Recombinant insulin does not contain C-peptide, a product of insulin protein biosynthesis that exerts beneficial effects on some of the microvascular complications associated with diabetes [30, 31, 32].
In this study, we investigated the impact of human C-peptide specifically in the early process of atherogenesis. The few studies available on the topic have tested the effect of C-peptide on low and high glucose-induced proliferative activities of vascular smooth muscle cells, one major component involved in the formation of atherosclerotic plaque [33, 34]. Here, we wanted to expand upon these studies by evaluating the potential effects of C-peptide on the endothelial cell component of the vessel wall during hyperglycaemia.
The adhesion and migration of circulating monocytes into the subendothelial space is one of the key events in the early stages of atherogenesis . This process is in part regulated by the expression of adhesion molecules, such as VCAM-1, on the surface of endothelial cells , and by the release of chemotactic factors, including IL-8 and MCP-1 . We found that in vitro C-peptide exerts an inhibitory effect on high glucose-induced upregulation of the adhesion molecule VCAM-1 on HAEC. C-peptide at the physiological concentration of 0.5 nmol/l reduced high glucose-induced expression of VCAM-1 to basal levels observed under normal glucose conditions. This effect was observed as early as 4 h after C-peptide addition to the high glucose medium and was still detected after 24 h incubation. The decrease in high glucose-induced VCAM-1 expression by C-peptide on HAEC was detected both at the mRNA and protein level. Conversely, when C-peptide was added to the medium containing normal glucose levels, it failed to significantly reduce VCAM-1 expression. These results are in line with findings from another group demonstrating that C-peptide reduced expression of the adhesion molecules P-selectin and intercellular adhesion molecule-1 on the rat microvascular endothelium during acute endothelial dysfunction in vivo . In another model of vascular injury, C-peptide was shown to decrease polymorphonuclear leucocyte infiltration into the myocardium thereby improving cardiac dysfunction . Overall, these data seem to point to an anti-inflammatory effect of C-peptide on the endothelium, especially in conditions of insult. This hypothesis is supported by recent in vivo data showing that survival rates of mice following endotoxic shock is improved after C-peptide administration . In these mice, plasma levels of the pro-inflammatory cytokines TNF-α and MCP-1 were also decreased, suggesting a decreased generalised inflammatory response . In the context of type 1 diabetes patients, upregulation of endothelial VCAM-1 and inflammation are early events in the course of the disease [2, 38, 39, 40]. These patients are insulin-dependent and take exogenous insulin to manage their blood glucose levels. It might well be that addition of physiological levels of C-peptide to the traditional exogenous insulin therapy could be a means of ‘counteracting’ the insult of high glucose on the endothelial cells of diabetic patients.
Another component of endothelial dysfunction affected by C-peptide is the secretion of IL-8 and MCP-1, chemokines that facilitate leucocyte-endothelial interactions. In support of other investigators [5, 6, 16], we observed an increased secretion of both chemokines in the supernatant fraction of endothelial cells under high glucose. Unique to this study, however, is the finding that C-peptide reduced high glucose-stimulated IL-8 and MCP-1 secretion by endothelial cells to near or below the basal levels measured under normal glucose concentrations. Based on our findings, it seems that C-peptide might exert its most meaningful biological effects on the endothelium in conditions of insult, as C-peptide did not significantly change chemokine secretion by HAEC when added to normal glucose-containing medium. In addition, adhesion of U-937 cells to high glucose-stimulated HAEC decreased after addition of C-peptide, an effect not detected when C-peptide was heat-inactivated. C-peptide at 0.5 nmol/l suppressed U-937 attachment to HAEC exposed to 25 mmol/l glucose by 50% due to a C-peptide-mediated inhibitory effect on VCAM-1, IL-8 and MCP-1 secretion by endothelial cells. Although in this study we focused on the effects of C-peptide on high glucose-induced endothelial dysfunction, another likely cellular target of C-peptide action in diabetes could be the immune cells. Previous studies from our laboratory  and others  have shown that phenotypic changes suggestive of cellular activation are present in circulating monocytes of recently diagnosed type 1 diabetes patients. It is tempting to speculate that in conditions of hyperglycaemia and the underlying inflammation typical of diabetes, C-peptide might exert beneficial effects on both endothelial and immune cell dysfunction, thereby decreasing the overall risk of developing vascular lesions. The biological effect of C-peptide on immune cells is currently under investigation in our laboratory.
The mechanisms underlying the effects of C-peptide on the human vasculature, specifically on endothelial cells, are still largely unknown. Nevertheless, the signal transduction pathways that lead to the enhanced expression of genes encoding adhesion molecules and inflammatory cytokine secretion in endothelial cells require translocation of the transcription factor NF-κB . Therefore, this study investigated C-peptide effects on NF-κB activation in high glucose-stimulated HAEC. In support of a previous study , we confirm that short-term high glucose exposure of endothelial cells stimulates NF-κB activation. However, our work has moved the paradigm forward by demonstrating that exogenous addition of C-peptide significantly reduced high glucose-induced nuclear translocation of canonical components of NF-κB, p65 and p50. The suppressive effect on NF-κB activation and high glucose-induced VCAM-1 expression as well as IL-8 and MCP-1 secretion in HAEC was specific for C-peptide, since heat-inactivated C-peptide was not able to elicit the same phenotype. Although we did not investigate the precise mechanism of action of C-peptide on the inhibition of NF-κB nuclear translocation in HAEC, evidence of cellular internalisation and binding to intracellular components has been recently demonstrated in Swiss 3 T3 and HEK-293 cells . In the same study, interestingly, C-peptide was also shown to localise within the nuclei . We can therefore speculate that the inhibitory action of C-peptide on NF-κB activation in HAEC could result from an effect on the phosphorylation of protein substrates in the cytoplasm and/or of a direct interaction of C-peptide with NF-κB p65/p50 subunits at the nuclear level, preventing DNA binding. In the lung of endotoxin-treated mice, C-peptide inhibited phosphorylation of extracellular signal-regulated kinase-1/2 followed by upregulation of nuclear levels and DNA binding of the nuclear transcription factor peroxisome proliferator-activated receptor-γ, which plays an important role in the modulation of inflammation . Currently, we are exploring which NF-κB-dependent upstream signalling events are affected by C-peptide in endothelial cells; examples are ROS generation and IκB kinase, an enzyme that elicits phosphorylation of the cytosolic NF-κB inhibitor IκBα. This latter upstream event regulates NF-κB translocation from the cytoplasm to the nucleus. In vascular smooth muscle cells we found that C-peptide reduced high glucose-induced phosphorylation of IκBα , a pathway likely to be also targeted in HAEC.
Inhibition of NF-κB would be suggestive of an anti-inflammatory effect of physiological concentrations of C-peptide at the endothelial cell level [19, 22] and would be consistent with a potential anti-atherosclerotic effect in type 1 diabetes. Higher supra-physiological levels of circulating C-peptide, such as those measured in type 2 diabetic patients with the hyperinsulinaemia associated with insulin resistance, might have deleterious effects on the vasculature. This view is supported by Walcher et al. , who found that higher concentrations of C-peptide, mimicking those found in the circulation of type 2 diabetic patients, produced maximal stimulation of lymphocyte chemotaxis in vitro. Future studies are required to further elucidate these issues.
Although this evidence in human endothelial cells is reported here for the first time, a protective effect of C-peptide on high glucose-induced vascular dysfunction has been invoked by other groups, who tested the efficacy of C-peptide in small clinical trials of type 1 diabetic patients [30, 31, 32, 46]. In addition to endothelial cells, vascular smooth muscle cells also appear to be the target of beneficial effects of C-peptide on the vasculature in conditions of hyperglycaemia [33, 44]. Physiological concentrations of C-peptide attenuate glucose-induced hyperproliferation of vascular smooth muscle cells [33, 44], a phenomenon associated, at least in part, with a specific inhibitory effect on NF-κB .
In conclusion, our findings support the hypothesis that C-peptide has major physiological effects on the inhibition of endothelial dysfunction under high glucose conditions. It does this by interfering with NF-κB activation and its effect on the reduced production of pro-inflammatory cytokines and chemokines. These findings underscore a role of C-peptide in endothelial cell functions, especially in conditions of diabetic insult to the vasculature. Our results support the idea of prolonged administration of physiological quantities of C-peptide to type 1 diabetes patients in an effort to lessen endothelial dysfunction and complications that may potentially arise during the course of the disease.
This study was supported by the Henry Hillman Endowment Chair in Pediatric Immunology (to M. Trucco) and by grants DK 024021-24 from the National Institute of Health and NIH 5K12 DK063704 (to P. Luppi and M. Trucco) and W81XWH-06-1-0317 from the Department of Defense (M. Trucco)
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.