Insulin Pulses Less Effective Than Continuous Insulin in Inhibiting PEPCK mRNA Levels Stimulated by cAMP and Dexamethasone in Perifused Hepatoma Cells

Hepatic glucose production is stimulated in vitro twice as effectively by pulsatile as by continuous glucagon, given equivalent time-averaged doses. Efficacy studies of pulsatile insulin have yielded conflicting results. In the rat hepatoma cell line H-4-II-E-C3, insulin rapidly (t½ 15 min) inhibits transcription of the gene and lowers mRNA levels for the gluconeogenic enzyme. PEPCK via a receptor-mediated process. We attached H-4-II-E-C3 cells to Cytodex-3 μcarriers and used a perifusion column system to test whether pulsatile insulin is more or less effective than equivalent time-averaged doses of continuous insulin. PEPCK transcription was induced by inclusion of cAMP analogue 8-(4-chlorophenyl-thio)-cAMP (0.1 mM) and dexamethasone (0.5 μM) in the perifusion medium. Three columns were exposed either to continuous, pulsatile, or no insulin. After 3 h, total nucleic acid was extracted, and mRNA^PEPCK was measured with a sensitive-solution hybridization assay. Continuous insulin inhibited PEPCK expression in a dose-dependent fashion with EC₅₀ 1 × 10⁻¹¹ M. Equivalent time-averaged amounts of insulin delivered as pulses achieved significant inhibition but less effectively than continuous insulin. The apparent EC50 for pulsatile insulin increased from 2 × 10⁻¹¹ M to 5 × 10⁻¹¹ M as the oscillatory period was raised from 5 to 20 min, respectively. These observations suggest that insulin-mediated inhibition of PEPCK gene transcription is diminished by a pulsatile mode of administration in marked contrast to the pulse enhancement demonstrated for glucagon-mediated hepatic glucose production.