Abstract
The half-life of insulin mRNA at various glucose concentrations was determined by filter hybridization techniques in isolated rat islets incubated with 3H-labeled uridine followed by a chase incubation at 3.3 or 17 mM glucose. High glucose induced a greater stabilization of insulin mRNA than of other poly(A) + RNAs or total cellular RNA. In RIN-5F insulinoma cells, an insulin-producing cell line, cholera toxin, but not glucose, induced a stabilization of insulin mRNA. After 24 h of culture of islets with actinomycin D or alpha-amanitin at several glucose concentrations, insulin mRNA content was decreased in comparison to controls only at higher glucose concentrations. The biosynthesis of islet proteins other than insulin was strongly decreased by actinomycin D at all glucose concentrations. Insulin biosynthesis was inhibited proportionately to the observed decreases in insulin mRNA content. We conclude that inhibition of insulin mRNA degradation is an important component in increasing the insulin mRNA content in response to glucose, thereby augmenting the effects of glucose stimulation on insulin gene transcription (5). This stabilization may be partly mediated by cAMP as evidenced by the similar responses to cholera toxin in the RIN-5F cells. Furthermore, the results of experiments with actinomycin D suggest that the degradation of insulin mRNA may require the continuous production of a factor(s) which could be either RNA or protein in nature.
Highlights
The half-life of insulin mRNA at variousglucose periods, modulation of islet insulin mRNA content occurs concentrationswas determined byfilter hybridization techniquesinisolatedrat islets incubated with ‘H
(4).In the accompanying report ( 5 ) we show that glucose stimulates insulin gene transcription in a specific manner, and that thiseffect may be mediated in part via increases in islet cAMP levels induced by glucose[6]
The effect of glucose on the loss of [3H]uridine-labeled insulin mRNA in islets precultured at 11mM glucose is shown in Fig. 1.The amount of labeled insulin mRNA remaining at various time points is expressed as theper centof the initial value at zero time in each experiment
Summary
We have studied the effects of cholera creasing the insulinmRNA content in response to glucose, thereby augmenting theeffects of glucose stimulation on insulin gene transcription [5]. This stabilization may be partly mediated bycAMP as evidenced toxin on insulin mRNA half-life inthe insulin-producing RIN-5F insulinoma cell line [11]. The increase in insulin mRNA content in P-cells in response to glucose and cholera toxin is an additive effect resulting from both stabilization of insulin mRNA and stimulation of transcription of the insulin gene ( 5 ). Over short periods of 1-2 h glucose regulates insulin biosynthesis in isolated islets of Langerhans mainly at the level of translation [2, 3],l while over longer
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