Negative regulation of human insulin gene expression by the 5'-flanking region in non-pancreatic cells

We have examined the effect of the 5'-flanking region of the human insulin gene on its expression in non-pancreatic cells. The presence of the region containing the insulin gene enhancer (-339 to -169 bp) markedly repressed the promoter activity of the insulin gene. This suppressive phenomenon was restored by the addition of forskolin or dibutyryl cAMP, suggesting that this region alone is not sufficient to repress completely insulin gene expression in the presence of extracellular stimuli which increase the intracellular cAMP level. The hypervariable region (HVR) located at -365 bp also repressed the promoter activity. These results show negative regulation of human insulin gene expression in non-pancreatic cells by these regions.     

Evaluation of insulin resistance linkage to rat chromosome 4 in SHR of a Japanese colony

The spontaneously hypertensive rat (SHR) is a model of human insulin resistance syndrome. Quantitative trait loci for cellular defects in glucose and fatty acid metabolism have been mapped to an overlapping region of rat chromosome (RNO) RNO4 in SHR of the National Institute of Health colony, where a deletion in the Cd36 gene has been implicated as the causative mutation of insulin resistance. The present study has examined the potential presence of RNO4 linkage to a series of metabolic phenotypes in F(2) progeny derived from SHR of a Japanese colony (SHR/Izm) without the Cd36 mutation. Our data demonstrate that 'major' insulin resistance gene(s) are unlikely to exist on RNO4 in SHR/Izm and in vitro phenotypes measured in isolated adipocytes do not cosegregate in the F(2) population studied. Thus, it seems to be difficult to explain the underlying genetic mechanisms of insulin resistance by a single major gene on RNO4.     

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.     

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.     

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.     

CCAAT/enhancer-binding protein-beta participates in insulin-responsive expression of the factor VII gene

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.     

Divergent effects of peroxisome proliferator-activated receptor gamma agonists and tumor necrosis factor alpha on adipocyte ApoE expression

ApoE is expressed in multiple mammalian cell types in which it supports cellular differentiated function. In this report we demonstrate that apoE expression in adipocytes is regulated by factors involved in modulating systemic insulin sensitivity. Systemic treatment with pioglitazone increased systemic insulin sensitivity and increased apoE mRNA levels in adipose tissue by 2-3-fold. Treatment of cultured 3T3-L1 adipocytes with ciglitazone increased apoE mRNA levels by 2-4-fold in a dose-dependent manner and increased apoE secretion from cells. Conversely, treatment of adipocytes with tumor necrosis factor (TNF) alpha reduced apoE mRNA levels and apoE secretion by 60%. Neither insulin nor a peroxisome proliferator-activated receptor (PPAR) alpha agonist regulated adipocyte apoE gene expression. In addition, treatment of human monocyte-derived macrophages with ciglitazone did not regulate expression of apoE. Additional analyses using reporter genes indicated that the effect of TNFalpha and PPARgamma agonists on the apoE gene was mediated via distinct gene control elements. The TNFalpha effect was mediated by elements within the proximal promoter, whereas the PPARgamma effect was mediated by elements within a downstream enhancer. However, the addition of TNFalpha substantially reduced the absolute levels of apoE reporter gene response even in the presence of ciglitazone. These results indicate for the first time that adipose tissue expression of apoE is modulated by physiologic regulators of insulin sensitivity.     

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.     

Expression of the human insulin gene in the gastric G cells of transgenic mice

The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.