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 125I-labeled protein A. The ratio (mean +/- SEM) of insulins, I/II, 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 less than 0.01). The increase in content of rat insulin I compared to II in the growth hormone-tumor-bearing animals was confirmed by radioimmunoassay of gel slices. To determine whether the difference in contents of rat insulins I and II 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 greater than 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 less than 0.01) at 16.0 mM glucose. Therefore, under conditions that stimulate insulin biosynthesis, there was a marked preferential synthesis of rat insulin I relative to II. 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.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bell G. I., Pictet R. L., Rutter W. J., Cordell B., Tischer E., Goodman H. M. Sequence of the human insulin gene. Nature. 1980 Mar 6;284(5751):26–32. doi: 10.1038/284026a0. [DOI] [PubMed] [Google Scholar]
- Bünzli H. F., Glatthaar B., Kunz P., Mülhaupt E., Humbel R. E. Amino acid sequence of the two insulins from mouse (Maus musculus). Hoppe Seylers Z Physiol Chem. 1972 Mar;353(3):451–458. doi: 10.1515/bchm2.1972.353.1.451. [DOI] [PubMed] [Google Scholar]
- Chan S. J., Keim P., Steiner D. F. Cell-free synthesis of rat preproinsulins: characterization and partial amino acid sequence determination. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1964–1968. doi: 10.1073/pnas.73.6.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clark J. L., Steiner D. F. Insulin biosynthesis in the rat: demonstration of two proinsulins. Proc Natl Acad Sci U S A. 1969 Jan;62(1):278–285. doi: 10.1073/pnas.62.1.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cordell B., Bell G., Tischer E., DeNoto F. M., Ullrich A., Pictet R., Rutter W. J., Goodman H. M. Isolation and characterization of a cloned rat insulin gene. Cell. 1979 Oct;18(2):533–543. doi: 10.1016/0092-8674(79)90070-9. [DOI] [PubMed] [Google Scholar]
- Corden J., Wasylyk B., Buchwalder A., Sassone-Corsi P., Kedinger C., Chambon P. Promoter sequences of eukaryotic protein-coding genes. Science. 1980 Sep 19;209(4463):1406–1414. doi: 10.1126/science.6251548. [DOI] [PubMed] [Google Scholar]
- DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
- Giddings S. J., Chirgwin J., Permutt M. A. The effects of fasting and feeding on preproinsulin messenger RNA in rats. J Clin Invest. 1981 Apr;67(4):952–960. doi: 10.1172/JCI110145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gruss P., Efstratiadis A., Karathanasis S., König M., Khoury G. Synthesis of stable unspliced mRNA from an intronless simian virus 40--rat preproinsulin gene recombinant. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6091–6095. doi: 10.1073/pnas.78.10.6091. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howell S. L., Taylor K. W. The secretion of newly synthesized insulin in vitro. Biochem J. 1967 Mar;102(3):922–927. doi: 10.1042/bj1020922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Itoh N., Nose K., Okamoto H. Purification and characterization of proinsulin mRNA from rat B-cell tumor. Eur J Biochem. 1979 Jun;97(1):1–9. doi: 10.1111/j.1432-1033.1979.tb13079.x. [DOI] [PubMed] [Google Scholar]
- Kaelin D., Renold A. E., Sharp G. W. Glucose stimulated proinsulin biosynthesis rates of turn off after cessation of the stimulus. Diabetologia. 1978 May;14(5):329–335. doi: 10.1007/BF01223025. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lacy P. E., Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 1967 Jan;16(1):35–39. doi: 10.2337/diab.16.1.35. [DOI] [PubMed] [Google Scholar]
- Lin B. J., Haist R. E. Insulin biosynthesis: effects of carbohydrates and related compounds. Can J Physiol Pharmacol. 1969 Sep;47(9):791–801. doi: 10.1139/y69-133. [DOI] [PubMed] [Google Scholar]
- Lomedico P., Rosenthal N., Efstratidadis A., Gilbert W., Kolodner R., Tizard R. The structure and evolution of the two nonallelic rat preproinsulin genes. Cell. 1979 Oct;18(2):545–558. doi: 10.1016/0092-8674(79)90071-0. [DOI] [PubMed] [Google Scholar]
- Markussen J. Mouse insulins--separation and structures. Int J Protein Res. 1971;3(3):149–155. doi: 10.1111/j.1399-3011.1971.tb01705.x. [DOI] [PubMed] [Google Scholar]
- Martin J. M., Akerblom H. K., Garay G. Insulin secretion in rats with elevated levels of circulating growth hormone due to MtT-W15 tumor. Diabetes. 1968 Nov;17(11):661–667. doi: 10.2337/diab.17.11.661. [DOI] [PubMed] [Google Scholar]
- Morris G. E., Korner A. The effect of glucose on insulin biosynthesis by isolated islets of Langerhans of the rat. Biochim Biophys Acta. 1970 Jun;208(3):404–413. doi: 10.1016/0304-4165(70)90213-8. [DOI] [PubMed] [Google Scholar]
- Peake G. T., McKeel D. W., Mariz I. K., Jarett L., Daughaday W. H. Insulin storage and release in rats bearing growth hormone secreting tumors. Diabetes. 1969 Sep;18(9):619–624. doi: 10.2337/diab.18.9.619. [DOI] [PubMed] [Google Scholar]
- Perler F., Efstratiadis A., Lomedico P., Gilbert W., Kolodner R., Dodgson J. The evolution of genes: the chicken preproinsulin gene. Cell. 1980 Jun;20(2):555–566. doi: 10.1016/0092-8674(80)90641-8. [DOI] [PubMed] [Google Scholar]
- Permutt M. A. Effect of glucose on initiation and elongation rates in isolated rat pancreatic islets. J Biol Chem. 1974 May 10;249(9):2738–2742. [PubMed] [Google Scholar]
- Permutt M. A., Kipnis D. M. Insulin biosynthesis. I. On the mechanism of glucose stimulation. J Biol Chem. 1972 Feb 25;247(4):1194–1199. [PubMed] [Google Scholar]
- Rall L. B., Pictet R. L., Rutter W. J. Synthesis and accumulation of proinsulin and insulin during development of the embryonic rat pancreas. Endocrinology. 1979 Sep;105(3):835–841. doi: 10.1210/endo-105-3-835. [DOI] [PubMed] [Google Scholar]
- Reiser J., Wardale J. Immunological detection of specific proteins in total cell extracts by fractionation in gels and transfer to diazophenylthioether paper. Eur J Biochem. 1981 Mar;114(3):569–575. doi: 10.1111/j.1432-1033.1981.tb05182.x. [DOI] [PubMed] [Google Scholar]
- Renart J., Reiser J., Stark G. R. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3116–3120. doi: 10.1073/pnas.76.7.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith L. F. Species variation in the amino acid sequence of insulin. Am J Med. 1966 May;40(5):662–666. doi: 10.1016/0002-9343(66)90145-8. [DOI] [PubMed] [Google Scholar]
- Tanese T., Lazarus N. R., Devrim S., Recant L. Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans. J Clin Invest. 1970 Jul;49(7):1394–1404. doi: 10.1172/JCI106357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
- Villa-Komaroff L., Efstratiadis A., Broome S., Lomedico P., Tizard R., Naber S. P., Chick W. L., Gilbert W. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727–3731. doi: 10.1073/pnas.75.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]