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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...

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.

Five healthy adult men were given metoclopramide (10 and 20 mg) iv, and in repeated tests almost always developed transient restlessness lasting from 10-30 min. The effects of L-dopa and dexamethasone on metoclopramide-induced increases in cortisol concentration were determined. These response values were compared with those of a control. After an injection of 10 mg metoclopramide, the cortisol level increased significantly only at 40 min; the ACTH level did not change. The cortisol rise was suppressed by dexamethasone pretreatment. Pretreatment with 0.5 g L-dopa resulted in a decrease in the PRL level from -20 min to 20 min, and the increase in cortisol seen at 40 min was cancelled. The ACTH level did not change. After injecting 20 mg metoclopramide, the ACTH level increased significantly from 20 min to 60 min and the cortisol level showed a significant increase from 20 min to 120 min. Pretreatment with dexamethasone resulted in a decrease in these hormones. The L-dopa pretreatment did not reduce even the rise in the PRL level which resulted from the administration of 20 mg metoclopramide. These findings suggest that the ACTH and cortisol response to metoclopramide is a stress-mediated effect. Plasma cortisol responses to 20 mg metoclopramide and insulin-induced hypoglycemia were studied and compared in seven volunteers and found to be similar.

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