Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1.
The homeodomain transcription factor Nkx6.1 plays an important role in pancreatic islet beta-cell development, but its effects on adult beta-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-(3)H] 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-betaGAL), 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 beta 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 [(3)H]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 beta-cell replication with retention or enhancement of function, properties that may be exploitable for expansion of beta-cell mass in treatment of both major forms of diabetes.
Schisler
Jonathan C
JC
Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Duke Independence Park Facility, 4321 Medical Park Drive, Suite 200, Durham, NC 27704, USA.
Fueger
Patrick T
PT
Babu
Daniella A
DA
Hohmeier
Hans E
HE
Tessem
Jeffery S
JS
Lu
Danhong
D
Becker
Thomas C
TC
Naziruddin
Bashoo
B
Levy
Marlon
M
Mirmira
Raghavendra G
RG
Newgard
Christopher B
CB
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D019925:Cyclin A / Q000235:genetics; D056751:Cyclin A2; D019926:Cyclin B / Q000235:genetics; D056744:Cyclin B1; D016213:Cyclins / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005947:Glucose / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D046509:Tissue Culture Techniques
C534699:CCNB1 protein, human; C503722:Ccna2 protein, rat; C534701:Ccnb1 protein, rat; D019925:Cyclin A; D056751:Cyclin A2; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D017931:DNA Primers; D018398:Homeodomain Proteins; D007328:Insulin; C105201:NKX6-1 protein, human; C491327:Nkx6-1 protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D005947:Glucose