Abstract
Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.
Highlights
Starvation and diabetes both caused a marked in- studied [1].This enzyme is generally considered one of the crease in theconcentration of hepatic phosphoenolpy- regulatorystepsin the gluconeogenic pathway [2] andits ruvate carboxykinase mRNA while the administration synthesis rate is acutely regulated by a number of hormones of insulin to diabetic rats or refeeding glucose to [1].P-enolpyruvate carboxykinaseinliver and kidney are starved animals caused a markedreduction in thelevels of enzyme mRNA as measured by hybridization using a cDNA probe
In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with therelative levels of translatable mRNA for theenzyme
Translatable mRNA levels for hepatic but not renal P-enolpyruvate carboxykinase aremarkedly increasedby administration of N',O'-dibutyryladenosine 3':5'-monophosphoric acid [4], whereas acidosis induces the concentrationof enzyme mRNA in kidney but not liver [4].Translatable P-enolpyruvate carboxykinase mRNA in both tissues can be induced by glucocorticoids ( 5, 6 ), but the extent of this induction in liver is lower than noted after Bt2cAMP' administration [6]
Summary
From the Department of Biochemistry, Case Western Reserve University, School of Medicine, Cleveland, Ohio 444106 Starvation and diabetes both caused a marked in- studied [1].This enzyme is generally considered one of the crease in theconcentration of hepatic phosphoenolpy- regulatorystepsin the gluconeogenic pathway [2] andits ruvate carboxykinase mRNA while the administration synthesis rate is acutely regulated by a number of hormones of insulin to diabetic rats or refeeding glucose to [1].P-enolpyruvate carboxykinaseinliver and kidney are starved animals caused a markedreduction in thelevels of enzyme mRNA as measured by hybridization using a cDNA probe. Two hours after refeeding glucose to a starved rat, these nuclear RNA species could nolonger be detected by hybridization to of mature P-enolpyruvate carboxykinase mRNA present in the nuclei (but not cytosol)from rat liver. The nitrocellulose filters were prehybridized for at least 1h at 50 "C in a medium containing 50%formamide, 10%dextran sulfate,5X SSC, 1.5mg/ml of heat-denatured salmon testis DNA, 0.556 sodium dodecyl
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