Cultured pancreatic ductal cells undergo cell cycle re-distribution and beta-cell-like differentiation in response to glucagon-like peptide-1.
The intestinal hormone glucagon-like peptide-1 (GLP-1) has been shown to promote an increase in pancreatic beta-cell mass via proliferation of islet cells and differentiation of non-insulin-secreting cells. In this study, we have characterized some of the events that lead to the differentiation of pancreatic ductal cells in response to treatment with human GLP-1. Rat pancreatic ductal (ARIP) cells were cultured in the presence of GLP-1 and analyzed for cell counting, cell cycle distribution, expression of cyclin-dependent-kinase (Cdk) inhibitors, transcription of beta-cell-specific genes, loss of ductal-like phenotype and acquisition of beta-cell-like gene expression profile. Exposure of ARIP cells to 10 nM GLP-1 induced a significant reduction in the cell replication rate and a significant decrease in the percentage of cells in S phase of the cell cycle. This was associated with an increase in the number of cells in G0-G1 phase and a reduction of cells in G2-M phase. Western blot analysis for the Cdk inhibitors, kinase inhibitor protein 1 (p27(Kip1)) and Cdk-interacting protein 1 (p21(Cip1)), demonstrated a significant increase in p27(Kip1) and p21(Cip1) levels within the first 24 h from the beginning of GLP-1 treatment. As cells slowed down their proliferation rate, GLP-1 also induced a time-dependent expression of various beta-cell-specific mRNAs. The glucose transporter GLUT-2 was the first of those factors to be expressed (24 h treatment), followed by insulin (44 h) and finally by the enzyme glucokinase (56 h). In addition, immunocytochemistry analysis showed that GLP-1 induced a time-dependent down-regulation of the ductal marker cytokeratin-20 (CK-20) and a time-dependent induction of insulin expression. Finally, GLP-1 promoted a glucose-dependent secretion of insulin, as demonstrated by HPLC and RIA analyses of the cell culture medium. The present study has demonstrated that GLP-1 induces a cell cycle re-distribution with a decrease in cell proliferation rate prior to promoting the differentiation of cells towards an endocrine-like phenotype.
Bulotta
A
A
Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.
Hui
H
H
Anastasi
E
E
Bertolotto
C
C
Boros
L G
LG
Di Mario
U
U
Perfetti
R
R
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D005934:Glucagon / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1; D005941:Glucokinase / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D051273:Glucose Transporter Type 2; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010183:Pancreatic Ducts / Q000166:cytology* / Q000187:drug effects*; D010446:Peptide Fragments / Q000494:pharmacology*; D011498:Protein Precursors / Q000494:pharmacology*; D051381:Rats; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C493956:CDKN1A protein, human; C493958:Cdkn1a protein, rat; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D005941:Glucokinase; D005947:Glucose