Insulin Gene Expression in the Developing Rat Pancreas

Abstract

In the developing rat pancreas from about day 15 until birth there is a marked increase in the concentration of insulin. The present experiments were designed to determine whether enhanced insulin biosynthesis is accompanied by a coordinate increment in proinsulin mRNA. Using a sensitive RNA filter hybridization technique and a cloned rat proinsulin 32P-cDNA as a hybridizing probe, the concentration of proinsulin mRNA was measured. A greater than 900-fold accumulation of proinsulin mRNA was found, which closely paralleled that of immunoreactive insulin. Proinsulin mRNA and immunoreactive insulin were highest 1–2 days after birth, more than eightfold higher than that in adult pancreas. The patterns of digestion by a number of restriction endonucleases of the two nonallelic insulin genes were identical in adult and fetal DNA and in DNA from an insulinoma. We found no evidence of major gene rearrangement during embryogenesis as a prerequisite to insulin gene expression. Glucose stimulated insulin biosynthesis at the earliest time examined (day 15 of gestation). Insulin biosynthesis was enhanced almost fourfold while proinsulin mRNA differed by less than 20% during a 2-h incubation. This suggested that the short-term effect of glucose on insulin biosynthesis in the developing rat pancreas occurs at the level of translation of existing mRNA, similar to that noted in adult pancreatic islets. These data taken together suggest that the same controls for insulin biosynthesis in the adult exist at these early times in pancreatic development and that the controls for insulin biosynthesis appear coordinately with the appearance of increased insulin during endocrine pancreatic differentiation.

Modulation of proinsulin messenger RNA after partial pancreatectomy in rats. Relationships to glucose homeostasis.

“…During fetal pancreatic development there is a specific increase in insulin content relative to total pancreatic protein (39), which is preceded by a comparable increase in proinsulin mRNA (18,19). Reduction of proinsulin mRNA has been established as a result of fasting (20), and ability to increase proinsulin mRNA concentration has been shown in fasted rats after glucose injection (21).…”

“…Modulation of proinsulin mRNA concentration has been shown in studies assessing the physiologic regulation of insulin production (17)(18)(19)(20)(21)(22). A correlation has been shown in rats between pancreatic proinsulin mRNA concentration and rates of in vivo incorporation of [3Hjleucine into proinsulin, showing comparable ability to assess rates of insulin biosynthesis (16,17).…”

Evaluation of rat insulin messenger RNA in pancreatic and extrapancreatic tissues

“…Blot hybridization provides a sensitive and specific method for identification and quantitation of messenger RNA's [9,13,26,30]. Cloned cDNA probes can be labelled to specific activities similar to those obtained when iodinating peptides for use in radioimmunoassays.…”

“…In each RNA blot analysis a known amount of rat proinsulin eDNA was electrophoresed to provide a standard and to control tbr variations in efficiency of transfer and hybridization. The amount of eDNA bound to specific bands on filters was determined by liquid scintillation counting (Fig.l) or by densitometric analysis (Table 1) of autoradiograms using the gel scanning accessory of a recording spectrophotometer (Beckman DU-8, Palo Alto, CA) as described previously [9,26,32]. Fig, 2.…”

“…ProinsulinI cDNA hybridizes readily to both genes under the conditions of hybridization employed [9]. The presence of proinsulin mRNA in extrapancreatic tissues would provide an independent line of evidence for synthesis.…”