In VSMC, both physiologic concentrations of C-peptide and NF-κB inhibitors reduce high glucose-induced proliferation. VSMC were incubated with 25 mmol/l glucose in the presence or absence of C-peptide (C-P) for 48 hours, and assayed for proliferation by measuring the nuclear incorporation of BrdU (DNA synthesis). In UASMC, BrdU incorporation showed cellular proliferation in high glucose (vs. 5.6 mmol/l, p = 0.002). C-peptide reduced high glucose-induced UASMC proliferation (p < 0.01), while addition of scrambled C-peptide (Scr C-P) did not have a significant effect. Addition of the NF-κB inhibitors PDTC (20 μM) and Bay-11-7082 (1 μM) also showed a decrease in proliferation (vs. 25mmol/l glucose, p < 0.01). In AoSMC, BrdU incorporation showed cellular proliferation in high glucose (vs. 5.6 mmol/l glucose, p = 0.048). C-peptide reduced high glucose-induced AoSMC proliferation (p < 0.01), while addition of scrambled C-peptide (Scr C-P) did not have a significant effect. Addition of the NF-κB inhibitors PDTC (20 μM) and Bay-11-7082 (1 μM) also showed a decrease in proliferation (vs. 25 mmol/l glucose, p < 0.01). Values are mean ± SD of 10 different experiments run in triplicate. Modified with kind permission from Elsevier, Figure 1 [9].

In HAEC and UASMC, C-peptide internalizes as punctuate structures. HAEC (A) and UASMC (B) were incubated for 30 minutes with 1 mmol/l Alexa Fluor 488-labeled C-peptide at 37°C, washed with medium, and imaged by confocal microscopy. The C-peptide probe localized at the periphery of the cell and in the cytoplasm, represented by the green punctuate staining. The figure shows a representative z-section across one cell. Modified with kind permission from Springer Science+Business Media, Figure 1 [100].

Physiologic concentrations of C-peptide reduce adherence of U-937 to HAEC under high-glucose conditions. HAEC were cultured in 25 mmol/l glucose in the presence or absence of 0.5 nmol/l C-peptide (C-P) for 4 hours. U-937 were added for 1 hour then counted. Boxplot graphs show the median values (limits of the lines are 5th and 95th centiles) of number of adherent U-937 per well. High glucose (25 mmol/l) increased the number of adherent U-937 compared to low glucose (5.6 mmol/l) (p = 0.004). Addition of C-peptide reduced the number of adherent U-937 compared to 25 mmol/l glucose alone (p < 0.01). Heat-inactivated (HI) C-peptide did not significantly alter adherence of U-937. TNF-α, used as a positive control, produced more than a five-fold increase in adherent U- 937 in comparison to normal glucose. Modified with kind permission from Springer Science+Business Media, Figure 4A [10].

Representative immunoblot depicting the 65-kDa band of the p65 subunit in AoSMC cultured in 5.6 mmol/l or 25 mmol/l glucose in the presence or absence of C-peptide (C-P) for 48 hours. In high glucose, AoSMC in the presence of C-peptide showed a decreased NF-κB nuclear translocation as compared to high glucose alone. Scrambled C-peptide (Scr C-P) did not have significant effect. To show equal loading of the gel, staining for β-actin is also shown. Modified with kind permission from Elsevier, Figure 5 [9].