Dataset Information

Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function.

ABSTRACT: Type 1 and type 2 diabetes result from a deficit in insulin production and beta-cell mass. Methods to expand beta-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [(3)H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates beta-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating beta-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [(3)H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human beta-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [(3)H]thymidine incorporation. CCK is a beta-cell proliferative factor that is effective in both mouse and human islets. CCK triggers beta-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.

SUBMITTER: Lavine JA

PROVIDER: S-EPMC2626203 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function.

Lavine Jeremy A JA Raess Philipp W PW Davis Dawn Belt DB Rabaglia Mary E ME Presley Brent K BK Keller Mark P MP Beinfeld Margery C MC Kopin Alan S AS Newgard Christopher B CB Attie Alan D AD

Molecular endocrinology (Baltimore, Md.) 20081009 12

Type 1 and type 2 diabetes result from a deficit in insulin production and beta-cell mass. Methods to expand beta-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [(3)H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry an ...[more]

PMID: 18845673

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Project description:The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet Î²-cell development, but its effects on adult Î²-cell function, survival, and proliferation are not well understood. In the present study, we demonstrated that treatment of primary rat pancreatic islets with a cytomegalovirus promoter-driven recombinant adenovirus containing the Nkx6.1 cDNA (AdCMV-Nkx6.1) causes dramatic increases in [methyl-3H] thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation and in the number of cells per islet relative to islets treated with a control adenovirus (AdCMV-Î²GAL), whereas suppression of Nkx6.1 expression reduces thymidine incorporation. Immunocytochemical studies reveal that >80% of BrdU-positive cells in AdCMV-Nkx6.1-treated islets are Î² cells. Microarray, real-time PCR, and immunoblot analyses reveal that overexpression of Nkx6.1 in rat islets causes concerted upregulation of a cadre of cell cycle control genes, including those encoding cyclins A, B, and E, and several regulatory kinases. Cyclin E is upregulated earlier than the other cyclins, and adenovirus-mediated overexpression of cyclin E is shown to be sufficient to activate islet cell proliferation. Moreover, chromatin immunoprecipitation assays demonstrate direct interaction of Nkx6.1 with the cyclin A2 and B1 genes. Overexpression of Nkx6.1 in rat islets caused a clear enhancement of glucose-stimulated insulin secretion (GSIS), whereas overexpression of Nkx6.1 in human islets caused an increase in the level of [3H]thymidine incorporation that was twice the control level, along with complete retention of GSIS. We conclude that Nkx6.1 is among the very rare factors capable of stimulating Î²-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of Î²-cell mass in treatment of both major forms of diabetes. Keywords: Insulin secretion, islet biology, transcription factor, cell cycle regulation, diabetes We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from rat pancreatic islets was hybridized to the microarray slide in the presence of labeled rat reference RNA (RRR, Stratagene, LaJolla, CA). Cultures were treated with adenoviruses expressing either the hamster form of Nkx6.1 or the beta-galactosidase enzyme. 5 biological replicates each representing independent islet isolations were used for microarray analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5. Labeled subject cRNA was co-hybridized to Operon rat 27K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled RRR. Slides were hybridized, washed, and scanned on a Gene Pix 5000 microarray scanner.

Dataset Information

Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function.

Publications

Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function.

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