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

<p>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. <i>Tbp</i> mRNA levels and <i>Gene/Tbp</i> 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.</p>a<p><i>p</i><0.05,</p>b<p><i>p</i><0.01 <i>vs.</i> islets cultured in G2;</p>c<p><i>p</i><0.01 <i>vs.</i> islets cultured in G10 (one-way ANOVA+test of Newman-Keuls).</p><p><i>Adm</i>: Adrenomedullin; <i>Aldoa</i>: Aldolase A; <i>Arnt (Hif1b)</i>: Aryl hydrocarbon receptor nuclear translocator (Hypoxia-inducible factor 1, beta subunit); <i>Car12</i>: Carbonic anhydrase 12; <i>Eno1</i>: Enolase 1, (alpha); <i>Gapdh</i>: Glyceraldehyde-3-phosphate dehydrogenase; <i>Hif1a</i>: Hypoxia-inducible factor 1, alpha subunit; <i>Hif2a</i>: Hypoxia-Inducible Factor 2, alpha subunit; <i>Ldha</i>: Lactate dehydrogenase A; <i>Mct4 (Slc16a3)</i>: Monocarboxylate transporter 4 (solute carrier family 16, member 3); <i>Pdk1</i>: Pyruvate dehydrogenase kinase, isozyme 1; <i>Pkm2</i>: Pyruvate kinase, muscle; <i>Tbp</i>: TATA box binding protein; <i>Tpi1</i>: Triosephosphate isomerase 1.