Glucagon-like peptide-1 (GLP-1) is an
incretin hormone derived from the
proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic beta-cell-specific phenotype:
insulin, GLUT-2, and
glucokinase. The aim of this study was to investigate the potential role of
GLP-1 for the gene therapy of
glucose-insensitive pancreatic beta-cells. We transfected mouse
insulinoma cells with
a DNA fragment of the human
proglucagon gene containing the nucleotide sequence encoding for human
GLP-1 but lacking the coding region for
glucagon. Two constructs were generated: In one, the expression of
GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/
GLP-1), and the second was regulated by the rat
insulin II promoter (RIP)/
GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the
protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/
GLP-1 cells did not gain a greater
glucose sensitivity as a result of the transfection with
GLP-1, compared with cells transfected with the plasmid alone, RIP/
GLP-1 was capable of regulating the gene expression of
insulin and
GLP-1 based on the concentration of
glucose in the culture medium. Detection of the counterpart
proteins (
insulin and
GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis.
GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous
insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the
glucose-dependent binding of IDX-1 to the
insulin promoter in RIP/
GLP-1 cells but not in CMV/
GLP-1 cells or control cells. Finally, we observed that although the
GLP-1-dependent secretion of
insulin was mediated by an increase in cAMP levels, the transcription of the
insulin gene, in response to
GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of
GLP-1 for the gene or
cell therapy of diabetes.