Transfection of pancreatic-derived beta-cells with a minigene encoding for human glucagon-like peptide-1 regulates glucose-dependent insulin synthesis and secretion.
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.
Division of Diabetes, Endocrinology, and Metabolism, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.
Hongxiang
H
Hui
Run
R
Yu
Corinne
C
Bousquet
Riccardo
R
Perfetti
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D000242:Cyclic AMP / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005934:Glucagon / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D052216:Glucagon-Like Peptide 1; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D010190:Pancreatic Neoplasms; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D052336:Proglucagon; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured
D007328:Insulin; D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; D052336:Proglucagon; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000242:Cyclic AMP; D005947:Glucose