Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1

Abstract

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.

FIG. 1.

Manipulation of Nkx6.1 expression in rat islets regulates [³H]thymidine incorporation and GSIS. (A) Rat islets were treated with Ad-siNkx6.1 or Ad-siRNAcontrol for 18 h and then cultured for an additional 80 h. Suppression of Nkx6.1 mRNA levels was confirmed by real-time PCR (black bars). Uptake of [methyl-³H]thymidine into genomic DNA was also measured (white bars). Data represent the means ± standard errors of the means from three independent experiments, each performed in triplicate. Nkx6.1 mRNA levels (*) and [³H]thymidine incorporation (#) in Ad-siNkx6.1-treated islets were lower than those in control islets, with a P value of <0.001. (B to D) Rat islets were treated with AdCMV-Nkx6.1 or AdCMV-βGAL for 18 h and maintained in culture for an additional 80 h. (B) RT-PCR analysis with primers specific for total Nkx6.1 (rat and hamster), hamster Nkx6.1 (AdCMV-Nkx6.1 contains the hamster Nkx6.1 cDNA), and, as a loading control, rat glucose-6-phosphate dehydrogenase (G6PDH). (C) [³H]thymidine incorporation into genomic DNA. Data represent the means ± standard errors of the means for three independent experiments, each involving triplicate pools of 30 islets per condition. *, a P value of <0.0001 in comparison to AdCMV-βGAL-treated islets. (D) GSIS in rat islets. Data are the means ± standard errors of means from three independent experiments, each performed in triplicate. *, a P value of <0.005 in comparison to AdCMV-βGAL-treated islets.

FIG. 2.

Nkx6.1 stimulates rat islet β-cell replication. (A) Histochemical staining of islet sections. Islet sections were stained with antibodies that detect BrdU incorporation (top panels) or BrdU (brown nuclear staining) and insulin (pink cytosolic staining) (bottom panels). (B) Immunofluorescence analysis of BrdU and insulin expression (top panels) or BrdU and glucagon expression (bottom panels) in sections of AdCMV-Nkx6.1-treated rat islets. The far-right panels are the overlay of insulin and BrdU staining (top) or glucagon and BrdU staining (bottom). (C) Serial sections of AdCMV-Nkx6.1-treated islets stained for BrdU (top left), Nkx6.1 (top right), or DAPI (4′,6′-diamidino-2-phenylin-dole) nuclear stain (bottom left). The bottom right panel shows the overlay of all three signals. The yellow arrows identify the BrdU-labeled nuclei, which in all cases costain with Nkx6.1 and DAPI. (D) Effects of Nkx6.1 overexpression on islet cell number. Data represent the means ± standard errors of the means for two independent experiments, each involving six independent groups of 10 islets per condition. *, a P value of <0.005 in comparison to AdCMV-βGAL-treated islets.

FIG. 3.

Effects of Nkx6.1 on gene expression in adult rat pancreatic islets. Five independent groups of rat pancreatic islets were treated with AdCMV-Nkx6.1 or AdCMV-βGAL, and duplicate RNA samples were utilized for microarray analysis. (A) Heat map demonstrating consistent upregulation (green) or downregulation (red) of Nkx6.1-regulated genes. (B) Confirmation of microarray results for key cell cycle-regulatory and functional genes by quantitative real-time PCR. Data represent the means ± standard errors of the means for five independent experiments. Rb1, retinoblastoma protein 1; GK, glucokinase. (C) ChIP analysis of interactions of Nkx6.1 with the cyclin A2, B1, and E1 promoters. Data are represented as the relative levels of association of Nkx6.1 with genomic fragments following immunoprecipitation with anti-Nkx6.1 serum, normalized to the amount of DNA recovered with normal serum, and represent the means ± standard errors of the means from three independent experiments, each performed in triplicate. *, a P value of <0.005 for the increase of Nkx6.1 association with the cyclin A2 and B1 genes in AdCMV-Nkx6.1-treated islets compared to that in AdCMV-βGAL-treated islets.

FIG. 4.

Time course studies and sufficiency of cyclin E for activation of islet cell replication. (A) Time course of induction of cyclins D1, A1, B1, and E1 in response to Nkx6.1 overexpression in rat islets. Relative expression is normalized to the level in untreated islets (no virus) cultured for 24 h. (B) Time course of stimulation of [³H]thymidine incorporation into rat islets by AdCMV-Nkx6.1 treatment relative to AdCMV-βGAL or no treatment, expressed relative to incorporation into no-virus control islets at each time point. For panels A and B, data are means ± standard errors of the means for three independent experiments, and the asterisk indicates significant differences between Nkx6.1-overexpressing cells and controls (P < 0.05). (C) Overexpression of human cyclin E1 is sufficient to increase [³H]thymidine incorporation in rat islets. Rat islets were treated with recombinant adenoviruses containing the human cyclin E cDNA (AdCVM-CycE) or the GFP cDNA (AdCMV-GFP). For these studies, [³H]thymidine was added in the 80- to 96-h time period after viral treatment. Data are means ± standard errors of the means from three independent experiments. *, a P value of <0.05 in comparison to the control.

FIG. 5.

Nkx6.1 overexpression in rat islets increases the levels of multiple cyclin proteins. AdCMV-Nkx6.1- or AdCMV-βGAL-treated rat islets were harvested for immunoblot analysis with antibodies specific for Nkx6.1, cyclin A, B1, D1, D2, or E, or γ-tubulin as a loading control. (A) Representative immunoblot. (B) Quantitative gel scan data from three independent experiments. *, a P value of <0.05 in comparison with AdCMV-βGAL-treated islets; ns, not significant.

FIG. 6.

Cyclin B1 overexpression partially rescues the effect of Nkx6.1 suppression on rat islet replication. Primary rat islets were treated with Ad-tA or Ad-tA plus Ad-t-cyclin B1 and cotreated with Ad-siNkx6.1 or Ad-siRNAcontrol. (A) Representative RT-PCR analysis using primer pairs that amplify human cyclin B1, total cyclin B1 (rat and human), or glucose-6-phosphate dehydrogenase (G6PDH) as a loading control. (B) [³H]thymidine incorporation into genomic DNA. Data represent the means ± standard errors of the means from three experiments, each involving triplicate pools of 30 islets per condition. *, significant decrease in thymidine incorporation in Ad-tA-treated/Ad-siNkx6.1-treated islets compared to the Ad-siRNA control-treated islets (P < 0.0005). #, increase in Ad-siRNAcontrol-treated, cyclin B-expressing (Ad-t-cyclin B1-treated) islets compared to control (Ad-tA-treated) islets (P < 0.0001).

FIG. 7.

Effects of Nkx6.1 overexpression on human islets. Human islets were treated with AdCMV-Nkx6.1 or AdCMV-βGAL. (A) Immunoblot analysis showing representative data from two of four separate human islet aliquots. (B) [³H]thymidine incorporation. Data are the means from four independent experiments, each performed in triplicate. *, a P value of <0.0005 in comparison to AdCMV-βGAL-treated islets. (C) GSIS in human islets, expressed as the ratio of insulin secreted at a stimulatory glucose concentration (16.7 mM) to insulin secreted at a basal glucose concentration (2.5 mM). Data represent means ± standard errors of the means from four independent experiments performed on the same islet aliquots used in the experiments whose results are shown in panels A and B.

FIG. 8.

Schematic summary of the effects of overexpressed Nkx6.1 on proteins involved in all phases of the cell cycle. Nkx6.1 exerts these effects via an array of direct and indirect actions as indicated in the key at the lower left and as elaborated in the text.