Evaluation of rat insulin messenger RNA in pancreatic and extrapancreatic tissues

Abstract

Summary. The purpose of these studies was to determine whether insulin detected immunochemically in extrapancreatic tissues of the adult rat is synthesized in situ by quantitating mRNA in these tissues. A blot hybridization assay was utilized with cloned 32p-proinsulin cDNA. The lower limit of detection was estimated to be 3pg. Proinsulin mRNA concentration was found to be 1000-1500 p~g in isolated pancreatic islets and was easily detected in total pancreatic RNA at 10-15 pg/ ~tg. Proinsulin mRNA was quantitated in rat insulinoma cells adapted to culture at levels 1 : 50 those in normal islets. Samples of RNA (20-50 p~g) enriched about 50-fold for mRNA sequences by repeated oligo-deoxythymidylate chromatography were assayed. No insulin mRNA was detected in 50 ~tg samples of RNA from brain or in 20 ~tg samples from subsections of brain or other extrapancreatic tissues. RNA samples were undegraded as assessed by ability to stimulate protein synthesis in a cell-free system. Proinsulin mRNA from pancreas was added to brain homogenates and recovered intact. Brain RNA samples with insulin mRNA levels 1:1000 that of pancreas would be predicted to have 50-75 pg proinsulin mRNA/50 ~tg sample assayed if present. Because none was found, brain must have a concentration < 1 : 6,000 that of pancreas. These findings suggest that immunoassayable insulin detected in extrapancreatic tissues of the adult rat is synthesized by the pancreas.Keywords: RNA blot hybridization, rat, proinsulin mRNA, brain mRNA, pancreatic islets.A number of peptides originally described as hormones from gut have been identified in brain [1,2]. Some are present in approximately equal concentrations in hypothalamus and gut (e. g. somatostatin) [3]. Others, such as insulin, are present at levels 1000-fold less in brain than in pancreas [4,5]. The identification of pg quantities of less abundant peptides per g of brain has been facilitated by radioimmunoassays and by localization with immunocytochemical techniques [1][2][3][4][5].The immunochemical detection of hormones in brain leaves unanswered the important question of their origin. Demonstration of insulin synthesis in brain or elsewhere would strongly imply as yet undescribed functions for this peptide. There are no data to answer this question, except reports of increased levels of insulin in brain compared with plasma under various conditions [4][5][6]. The low abundance of insulin in brain precludes the use of radiolahelled amino-acid incorporation to document synthesis directly [7].Using an RNA blot hybridization technique, we have assayed for proinsulin I and II mRNA's (which are 93% homologous) in brain and several other tissues of the adult rat, using a cloned 32p-labelled probe complementary to rat insulin I mRNA [8]. 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. The present limit of sensitivity of this assay is about 5 p...

Control of Insulin mRNA Stability in Rat Pancreatic Islets

“…in the presence of serum, the ␤-cell content of insulin mRNAs was at least 40,000 -100,000 molecules, depending upon the glucose concentration of the culture medium. This finding is in agreement with the early and indirect assessment (6), which indicated a ␤-cell insulin mRNA content of 50,000 -150,000 molecules. A typical mammalian cell contains ϳ360,000 mRNAs in its cytoplasm (26).…”

Regulation of insulin preRNA splicing by glucose

“…The estimate of 1 ϫ 10 5 insulin II mRNA molecules per ␤ cell in cultured islets is similar to the previous estimate of 50,000-150,000 insulin mRNAs per ␤ cell by Northern blot hybridization with RNA from freshly isolated rat islets (24).…”

Insulin, leptin, and food reward: update 2008

“…The first issue is the need for circulating signals to have access to CNS circuitry. The presence of insulin in the CNS was reported in 1979 (88), and many studies established that the predominant amount of insulin in the CNS can be accounted for by receptor-mediated transport into the CNS (46, 52, 53, 79,106,115,164,165). Although intermittent reports have suggested that insulin can be synthesized locally in the developed CNS, quantities appear to be negligible, particularly compared with the affinity of the receptor for insulin.…”