| description |
Insulin shows postprandial transient secretion with high-dose, and fasting sustained secretion with low-dose, selectively controling multiple functions via its temporal patterns and doses. However, how specific temporal patterns and doses of insulin control gene expression remains unknown. We have previously shown that the additional secretion-like pulse stimulation induced a transient activation of S6K, and productions of fructose-1,6-bisphosphate and glycogen, and that the basal secretion-like ramp stimulation induced sustained suppressions of G6Pase and PEPCK at low-dose of insulin. Here we performed RNA sequencing to comprehensively identify insulin-dependently changed genes in rat hepatoma FAO cells, prior to analysis of the temporal patterns of them. The cells were seeded at a density of 3 × 10 ^ 6 cells/dish on 6-cm dishes (Corning) and cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum at 37°C under 5% CO2 for 2 days prior to starvation. The cells were washed twice with ice-cold phosphate-buffered saline and starved in serum-free media containing 0.01 nM insulin (Sigma) and 10 nM dexamethasone (Wako) for 16 hours. To mimic the in vivo basal secretion during fasting, insulin was added to the media to reach the final concentration at 0.01 nM. The media were changed at 2 and 4 hours before the stimulation. Total RNA was extracted from Fao cells stimulated by 0.01, 1, and 100nM insulin for 0, 15, 30, 60, 90, 120, and 240 min with DNase I digestion to remove the mixed genome DNA. Messenger RNA was enriched from total RNA using poly(A) selection, and the mRNA samples were validated using the 2100 BioAnalyzer (Agilent). Standard Illumina (San Diego, CA, USA) protocols were used to generate 101 bp paired-end read libraries that were sequenced on the Illumina HiSeq 2000 platform. |