Real-time RT-PCR measurements of glucose-induced changes in HIF subunits and HIF-target gene mRNA levels in cultured rat islets.

After 1 week preculture in serum-free RPMI medium containing 5 g/l BSA and 10 mmol/l glucose (G10), rat islets were cultured 18 h in the presence of 2, 5, 10 or 30 mmol/l glucose (insulin secretion during culture was (mean ± SEM ng.islet−1.h−1) 0.06±0.01 in G2, 0.09±0.01 in G5, 0.94±0.22 in G10 and 3.17±0.47 in G30). Glucose-induced changes in the mRNA levels of selected genes were measured by real time RT-PCR. Tbp mRNA levels and Gene/Tbp mRNA ratios were expressed relative to the level in G2. Data are means ± SEM for 4 experiments. The threshold cycle measured in G2 (real time PCR performed with islet cDNA equivalent to 2 ng total RNA) are shown as rough indicators of gene mRNA levels.apbpvs. islets cultured in G2;cpvs. islets cultured in G10 (one-way ANOVA+test of Newman-Keuls).Adm: Adrenomedullin; Aldoa: Aldolase A; Arnt (Hif1b): Aryl hydrocarbon receptor nuclear translocator (Hypoxia-inducible factor 1, beta subunit); Car12: Carbonic anhydrase 12; Eno1: Enolase 1, (alpha); Gapdh: Glyceraldehyde-3-phosphate dehydrogenase; Hif1a: Hypoxia-inducible factor 1, alpha subunit; Hif2a: Hypoxia-Inducible Factor 2, alpha subunit; Ldha: Lactate dehydrogenase A; Mct4 (Slc16a3): Monocarboxylate transporter 4 (solute carrier family 16, member 3); Pdk1: Pyruvate dehydrogenase kinase, isozyme 1; Pkm2: Pyruvate kinase, muscle; Tbp: TATA box binding protein; Tpi1: Triosephosphate isomerase 1.