A transgene coding for a human insulin analog has a mitogenic effect on murine embryonic beta cells.

Abstract

We have investigated the mitogenic effect of three mutant forms of human insulin on insulin-producing f3 cells of the developing pancreas. We examined transgenic embryonic and adult mice expressing (i) Insulin is a peptide hormone that plays a crucial role in the control of blood glucose. It contains two separate polypeptide chains, the A chain containing 21 amino acid residues and the B chain containing 30 residues, which are linked to each other by a pair of disulfide bonds (1). Insulin is synthesized by ,3 cells of the pancreatic islets in the form of a precursor called preproinsulin (1), which is processed into proinsulin in the rough endoplasmic reticulum and transported via a regulated secretory pathway to storage granules, where it is cleaved to yield insulin and a 31-residue fragment called the C peptide. A small amount of proinsulin (0.5-2%) escapes from this route and is released through a constitutive, unregulated pathway (1-4). The expression of insulin solely by ,B cells has been controversial (5, 6). However, since we previously documented the expression of a chimeric gene containing the insulin I promoter in neurons of transgenic mice (7), the possibility was raised that, in addition to ,3 cells, insulin may be expressed by other cell types but at concentrations that are below the level of detection of the immunohistochemical techniques. To test this possibility, we initiated an analysis of transgenic mice overexpressing a human insulin analog. The transgene codes for the expression of a proinsulin molecule in which the amino acid histidine in position 10 of the B chain was replaced by aspartic acid. The substitution at B10 produced an aberrant intracellular sorting of the mutant hormone ([AspBlO]-proinsulin) resulting in secretion of up to 15% of the humanThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6239[AspBlO]proinsulin via an unregulated pathway (8). However, processing of mouse and human proinsulin within secretory granules in the islets of transgenic mice was not impaired, and the residual mouse and most of the human proinsulin were converted to insulin and secreted normally (1). Surprisingly, this mutant form of insulin has a 4-to 5-fold higher biological activity than normal insulin (9, 10). The appearance of the vaginal plug was considered to be day 0.5 of gestation. Pregnant females were killed by cervical dislocation, the embryos were dissected, the trunk was processed for Southern blotting analysis, and the head and abdomen were fixed in 4% paraformaldehyde buffered to pH 7.4 with 0.1 M sodium phosphate buffered saline (PBS) for 1 hr. Embryos were examined daily from embryonic day (E) 7.5, corresponding to four or five pairs of somites (15) to E17.5. Adult mice were perfused through the heart with fixative solution; the pancreas was removed and post-fixed for 1 hr in

Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy

“…It is possible that the use of B10-insulin may have been another factor. This insulin is superior to normal insulin in biological activity [29] and has some mitogenic properties [30–32]. However, there was no evidence of excessive mitogenic activity or tumour development, while liver function tests remained normal throughout, with no lymphocytic infiltrates.…”

Gene therapy of streptozotocin‐induced diabetes by intramuscular delivery of modified preproinsulin genes

“…B10-insulin has a single amino acid substitution (histidine to aspartic acid) at [28][29][30] and chemical storage stability of this protein [30,31], as well as superior biological activity in terms of insulin receptor binding as tested in vitro [32][33][34][35]. A caveat is that B10-insulin has some mitogenic activity for several cell lines [36], primary cultures of smooth muscle cells [37], hepatocytes [38] and embryonic beta-islet cells [39]. At suprapharmacological doses B10-insulin induced adenocarcinomas in female rats [40], but establishing the relevance of this finding will require further studies.…”

Diabetes therapy by lentiviral hepatic insulin gene expression without transformation of liver

“…In this insulin analogue the histidine in position 10 of the B chain of the insulin molecule is replaced by aspartic acid [4]. The mutation of this insulin analogue has been shown to induce mitogenic effects [5][6][7], and according to the authors' [1] conclusion, could also be responsible for the observed liver transdifferentiation. Using the furin-cleavable insulin for the lentiviral transductions we detected a decrease in blood glucose concentration of STZ-diabetic rats from 22.3±3.4 to 15.6±2.1 mmol/l after 3 days and to 15.0±2.8 mmol/l after 30 days.…”