Reagents were obtained from the following sources: rat aortic smooth muscle cells (SMCs) (A10 cells) from the American Type Culture Collection (Manassas, VA, USA); DMEM, penicillin–streptomycin, and FBS from Gibco (Grand Island, NY, USA). Human C-peptide was kindly provided by Eli Lilly (Indianapolis, IN, USA). Scrambled human C-peptide (the same amino acid residues as in C-peptide, but assembled in random order) was from Sigma Genosys (Cambridge, UK). Whatman GF/C filters were from Whatman International (Maidstone, UK). Rabbit anti-human PDGF-beta receptor antibody, and rabbit anti-human p42/p44 MAP kinase antibody came from Upstate Biotechnology (Lake Placid, NY, USA). Rabbit anti-phospho-human p42/p44 MAP kinase was supplied by Cell Signaling Technology (Beverly, MA, USA). Chemiluminescence detection kits and [3H]-thymidine came from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Rat aortic SMCs (A10 cells) were grown in DMEM containing 5.5 mmol/l glucose and 10% FBS, pH 7.4, at 37°C in a humidified 5% CO2/95% air atmosphere. Third or fourth passage cells from the purchase were grown for 3 weeks in DMEM containing 5.5 or 20 mmol/l glucose with or without C-peptide or scrambled C-peptide (1 to 100 nmol/l), and were used in subsequent experiments. For experiments with acute stimulation with C-peptide, cells were serum-starved overnight and stimulated with C-peptide or scrambled C-peptide for 10 min.
Assay of proliferation activities in SMCs
Cells were plated on six-well plates at a density of 1×104 cells/cm2 and grown in each experimental medium as described above. The proliferation activity in SMCs was assessed by determining [3H]-thymidine incorporation into DNA as previously described .
After incubation with each experimental medium for the indicated periods, cells were washed three times with ice-cold PBS and lysed in a buffer containing 50 mmol/l Tris–HCl, pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EGTA, 1 mmol/l phenylmethylsulphonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mmol/l Na3VO4, and 1 mmol/l NaF at 4°C. Samples containing the same amount of protein were electrophoresed on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with the first antibody, followed by incubation with an HRP-conjugated anti-rabbit polyclonal IgG antibody. The proteins were visualised using ECL chemiluminescence detection kits. Protein expression was quantified by densitometry.
Assay of glucose transport activities in SMCs
After 3 weeks of culture in normal or high-glucose conditions with or without C-peptide, unlabelled and labelled 2-deoxyglucose (0.1 mmol/l, 0.74 kBq/well) were added to the cells in the PBS buffer (138 mmol/l NaCl, 8.1 mmol/l Na2HPO4, 2.6 mmol/l KCl, 0.5 mmol/l MgSO4, 0.1 mmol/l CaCl2, 1.5 mmol/l KH2PO4, at pH 7.4) with 1% BSA. The cells were then incubated for 6 min at 37°C. The reaction was stopped by washing the cells three times with ice-cold PBS. The cells were solubilised in 1 ml of solution. Radioactivity was quantified using a liquid scintillation counter (LSC-5100; Aloka, Tokyo, Japan).
Results were expressed as means±SEM. Statistical analyses were made by one-way ANOVA with the Bonferroni correction for multiple comparisons. A p value of p<0.05 was considered statistically significant.