Regulation Of Insulin Gene Expression Research Articles

Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin.

Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.

Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures.

To study the regulation of insulin gene expression by physiological regulators, primary cultures of rat islet cells were transfected with portions of the rat insulin I gene 5'-flanking sequence linked to the reporter gene chloramphenicol acetyltransferase (CAT). Incubation of the cells in increasing glucose concentrations led to a parallel increase in both CAT activity and CAT mRNA levels. Pretreatment of the cells with the beta-cell-specific toxin streptozotocin reduced CAT activity 97%. Beta-Cell-specific expression of CAT was also demonstrated by co-staining the transfected cells with antisera to both CAT and insulin. Experiments showing a reduction in the response to glucose in the presence of the calcium channel blocker verapamil suggest that calcium plays a role in the glucose response, possibly via regulation of factors interacting with this limited portion of the insulin gene.

Rat Insulin II Gene Expression by Extraplacental Membranes: A Non-Pancreatic Source for Fetal Insulin

Abstract Using cDNA cloning, ribonuclease protection, and Northern hybridization analysis, we showed that insulin gene expression occurs in yolk sac-derived fetal extraplacental membranes throughout the last half of rat fetal development. The mRNA product of the ancestral rat insulin II but not the duplicated rat insulin I gene was present in high copy number, and its abundance was regulated during development. Insulin mRNA was present in extraplacental membranes before pancreatic differentiation; membrane insulin mRNA content greatly exceeded that in pancreas until the last 2 days of gestation when content in each tissue became similar. Polyadenylation and intron splicing occurred at the same sites used in pancreas, but initiation of transcription occurred at multiple sites in membranes. Minces of membranes maintained in culture produced approximately 10 ng of radioimmunoassayable insulin/mg membrane protein/day. Over a 4-day period, approximately 50 times more insulin accumulated in medium than that present in membranes at the time of isolation. These studies indicate that yolk sac is a source for insulin during fetal development and that the mechanisms regulating insulin gene expression in this tissue differ from those in pancreatic beta cells.

Hyperinsulinemia in transgenic mice carrying multiple copies of the human insulin gene.

We are investigating human insulin gene expression in transgenic mice. An 8.8 kilobase (kb) human genomic DNA fragment, including the insulin gene (1.4 kb) and 2 kb of 5' human flanking sequences, was introduced into mouse embryos by pronuclear microinjection. Two lines of transgenic mice have been established, both of which carry the intact human gene in multiple copies. Animals from both lines have significantly higher insulin levels than control mice, and the degree of hyperinsulinemia shows a positive correlation with human gene copy number in the two lines. Expression of the human gene is confirmed by the detection of human C-peptide in plasma. Tissue specificity of expression is maintained, with human insulin mRNA detectable only in the pancreas. The transgenics maintain normal fasting blood glucose in spite of their high insulin levels, but preliminary studies show them to be glucose intolerant when given a glucose load. These mice provide a model system for further studies on the regulation of insulin gene expression and on the effects of chronic hyperinsulinemia on glucose homeostasis.

Insulin-glycerolipid mediators and gene expression.

Insulin is an anabolic polypeptide hormone with pleiotrophic effects. During the decades since the initial description by Banting and Best, the acute effects of insulin have been widely studied with particular focus on the mechanism or mechanisms of insulin activation of hexose transport and regulation of metabolic enzyme activity. However, recently there has been a major expansion of investigation to include insulin regulation of gene expression with multiple insulin-sensitive specific mRNAs now reported. In this review, we explore the involvement of insulin-induced changes in plasma membrane glycerolipid metabolism in the transmembrane signaling process required for insulin regulation of mRNA levels. Insulin increases diacylglycerol levels in insulin-responsive cells, and synthetic diacylglycerols or their phorbol ester diacylglycerol analogs, such as 4 beta,9 alpha,12 beta,13 alpha, 20-pentahydroxytiglia-1,6-dien-3-one 12 beta-myristate 13-acetate (TPA), mimic insulin regulation of ornithine decarboxylase mRNA, c-fos mRNA, and phosphoenolpyruvate carboxykinase mRNA levels. This suggests that insulin regulation of specific mRNA levels may be mediated by insulin-induced changes in phospholipid metabolism and that diacylglycerol may play a pivotal role in insulin regulation of gene expression.

Regulation of insulin-gene expression. Implications for gene therapy.

DURING the past decade, the focus of mammalian molecular biology has evolved from the characterization of gene structure to the regulation of gene expression. In parallel, the ability to manipulate...

Tissue-specific binding of a nuclear factor to the insulin gene promoter

Using a DNA-binding gel mobility shift assay, a protein-DNA complex was detected using the rat II insulin promoter and a nuclear extract from rat insulinoma cells. This complex was detected using extracts from other insulin-producing cell lines but was not found using nuclear extracts from non-insulin-producing pancreatic cell lines or other cell types. Binding of the tissue-specific protein to the labelled promoter fragment was effectively competed by both rat and human insulin genes but not by the SV40 TATA box. This protein-DNA interaction may be involved in tissue-specific regulation of insulin gene expression.

Glucose regulates preproinsulin messenger RNA levels in a clonal cell line of simian virus 40-transformed B cells

In HIT-T15 cells grown in the absence of glucose, Northern blot analysis of total RNA revealed a major 0.5 kb preproinsulin (ppI) mRNA transcript which co-migrated with the mature transcript from a human insulinoma. In 4 h tissue cultures, glucose (2–20 mM) stimulated HIT cell ppI mRNA levels in a markedly dose-dependent manner. Glucose-stimulated ppI mRNA was (i) inhibited by actinomycin D, suggesting that regulation may be in part transcriptional, and (ii) potentiated by agents known to activate B cell protein kinases. HIT cells represent a unique model for investigating long term regulation of insulin gene expression and biosynthesis.