Biosynthesis of rat insulins I and II: evidence for differential expression of the two genes.

Abstract

The purpose of these experiments was to determine the relative content and biosynthetic rate of insulins I and II under various experimental conditions. The two insulins were quantitated by polyacrylamide gel electrophoresis, electrotransfer to nitrocellulose paper, photoaffinity crosslinking, and immunodetection with anti-insulin antibody and '5I-labeled protein A. The ratio (mean ± SEM) of insulins, I/HI, was 1.2 ± 0.2 in Wistar-Furth rats fasted for 4 days, 1.6 ± 0.2 in normal rats, and 5.5 ± 0.8 in growth hormone-tumor-bearing hyperinsulinemic rats (P < 0.01). The increase in content of rat insulin I compared to II in the growth hormone-tumor-bearing animals was confirmed by radioimmunoassay ofgel slices. To determine whether the difference in contents of rat insulins Iand El in the hyperinsulinemic rats was due to increased biosynthesis or a different turnover rate, isolated rat islets were incubated in [3H]leucine for 4 hr with 5.5 mM or 16.0 mM glucose in the incubation medium. Glucose stimulated insulin biosynthesis >8-fold. The ratio of synthesis of rat insulin I relative to II was 0.9 ± 0.1 at 5.5 mM glucose and 9.8 ± 3.3 (P < 0.01) at 16.0 mM glucose. Therefore, under conditions that stimulate insulin biosynthesis, there was a marked preferential synthesis ofrat insulin I relative to H. These studies suggest that the two rat insulin genes are expressed independently and that, under stimulatory conditions, there is preferential expression of the rat insulin I gene.In rats (1, 2), mice (3, 4), and certain fish (5, 6), two different insulins have been found. The rat insulins have been shown to be encoded by two nonallelic genes separated by at least 9,000 base pairs of DNA (7,8). The mRNAs are quite similar, approximately 93% homologous in the coding regions with only 34 of 439 nucleotides different (9, 10). Preproinsulins I and II, the initial translation products, differ by three amino acids in the preregion (7-11), two in the B chain, and two in the C peptide (see Fig. 1).Although the structures of the two genes, RNAs, and preproinsulins have been completely determined, little is known about the regulation of expression of the individual genes. In insulin isolated from Sprague-Dawley rat pancreas by Clark and Steiner (2), analysis by polyacrylamide gel electrophoresis indicated 58% insulin I and 42% insulin II. Biosynthesis of rat insulins I and II has been shown to occur in a ratio of 60:40 in several studies (2,(12)(13)(14).The present experiments were initiated in the course of the development ofan immunoelectrophoretic method for studying pancreatic extracts of insulin. We noted, in a hyperinsulinemic rat, that the ratio of rat insulins, I/IL, appeared to be greater than 5:1, a value very different from that previously reported. We therefore decided to do a more complete analysis of the proportion of rat insulins I and II under various experimental conditions and to examine their relative rates of synthesis. Our analysis confirmed that both the steady-state content and synthesis ofrat ins...

Mouse MafA, homologue of zebrafish somite Maf 1, contributes to the specific transcriptional activity through the insulin promoter

“…We have revealed that MafA activates the rat insulin promoter II, but not promoter I. It has been reported that the responses of rat insulin genes to stimulation are different [33,34]. Our results imply that, under conditions of stimulation, the difference between rat insulin promoters I and II depends on Maf regulation.…”

“…In this study, we indicated that MafA and MafB activate the RIPII and bind to the core site of RIPE3b. Mouse and rat possess two insulin promoters each (insulin promoters I and II), whereas human possesses only one promoter; these insulin promoters have unique transcriptional regulations [33][34][35][36]. Two significant differences appeared when the RIPE3b sequences of these insulin promoters were aligned with the MARE sequence (Fig.…”

“…It is well known that mouse and rat have two insulin genes and that each gene has a specific expression profile [33][34][35][36]. To assess the contribution of large Mafs to the transactivation activity of the insulin gene through the rat insulin promoters I and II (RIPI and II), we first The tree was generated by the UPGMA method using the whole amino acid sequences of the known large Mafs.…”

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

“…Rat I insulin contains no methionine, but there is a substitution of a lysine for a methionine at residue 29 of the rat insulin II B-chain [2,15]. The use of [35S]methionine instead of [3H]leucine in the pulse-labelling period therefore allows for monitoring of only rat insulin II biosynthesis.…”

“…However, the issue of Correspondence address: P.A. Halban, Joslin Diabetes Center, 1 Joslin Place, Boston, MA 02215, USA whether glucose regulates the synthesis of both rat insulins I and II, although addressed, has remained unresolved due to inadequate resolution of rat insulin I from proinsulins and the non-quantitative transfer to nitrocellulose of rat insulin II with the immunoelectrophoretic system used [15][16][17]. To avoid such analytical problems, we have used highperformance liquid chromatography (HPLC) to monitor glucose stimulation of rat insulin I and II biosynthesis in isolated islets.…”