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Stimulation of Human and Rat Islet β-Cell Proliferation with Retention of Function by the Homeodomain Transcription Factor Nkx6.1 ^,

Sarah W. Stedman Nutrition and Metabolism Center, Departments of Pharmacology and Cancer Biology, Medicine, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27704,¹ Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Virginia Health System, Charlottesville, Virginia 22903,² Islet Cell Laboratory, Baylor University Medical Center, Dallas, Texas 75226³

Received 29 September 2007/ Returned for modification 5 November 2007/ Accepted 6 March 2008

The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet β-cell development, but its effects on adult β-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-βGAL), 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 β 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 β-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of β-cell mass in treatment of both major forms of diabetes.

Published ahead of print on 17 March 2008.

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