title: Insulin pulses less effective than continuous insulin in inhibiting PEPCK mRNA levels stimulated by cAMP and dexamethasone in perifused hepatoma cells.
abstract: 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 (t1/2 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 microcarriers 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 microM) 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 x 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 x 10(-11) M to 5 x 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.
authors.affiliation: Department of Medicine, University of Washington, Seattle.
authors.forename: H C
authors.initials: HC
authors.lastname: Harrison
authors.forename: C J
authors.initials: CJ
authors.lastname: Goodner
authors.forename: M A
authors.initials: MA
authors.lastname: Berrie
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D010477:Perfusion / Q000379:methods; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D013873:Thionucleotides / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D007328:Insulin; D012333:RNA, Messenger; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D003907:Dexamethasone; D000242:Cyclic AMP; D010729:Phosphoenolpyruvate Carboxykinase (GTP)