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Comparative Study
. 2005 May 17;102(20):7297-302.
doi: 10.1073/pnas.0502168102. Epub 2005 May 9.

The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells

Jonathan C Schisler  1 , Per Bo Jensen, David G Taylor, Thomas C Becker, Filip Krag Knop, Shiro Takekawa, Michael German, Gordon C Weir, Danhong Lu, Raghavendra G Mirmira, Christopher B Newgard
Affiliations
Comparative Study

The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells

Jonathan C Schisler et al. Proc Natl Acad Sci U S A. .

Abstract

We have previously described rat insulinoma INS-1-derived cell lines with robust or poor glucose-stimulated insulin secretion (GSIS). In the current study, we have further resolved these lines into three classes: class 1, glucose-unresponsive/glucagon-expressing; class 2, glucose-unresponsive/glucagon-negative; and class 3, glucose-responsive/glucagon-negative. The transcription factor Nkx2.2 was expressed with relative abundance of 3.3, 1.0, and 1.0 in class 1, class 2, and class 3 cells, respectively, whereas Nkx6.1 expression had the opposite trend: 1.0, 2.6, and 6.4 in class 1, class 2, and class 3 cells, respectively. In class 1 cells, overexpressed Nkx6.1 suppressed glucagon expression but did not affect the levels of several other prominent beta cell transcription factors. RNA interference (RNAi)-mediated suppression of Nkx6.1 in class 3 cells resulted in a doubling of glucagon mRNA, with no effect on Pdx1 levels, whereas suppression of Pdx1 in class 3 cells caused a 12-fold increase in glucagon transcript levels, demonstrating independent effects of Nkx6.1 and Pdx1 on glucagon expression in beta cell lines. RNAi-mediated suppression of Nkx6.1 expression in class 3 cells also caused a decrease in GSIS from 13.9- to 3.7-fold, whereas suppression of Pdx1 reduced absolute amounts of insulin secretion without affecting fold response. Finally, RNAi-mediated suppression of Nkx6.1 mRNA in primary rat islets was accompanied by a significant decrease in GSIS relative to control cells. In sum, our studies have revealed roles for Nkx6.1 in suppression of glucagon expression and control of GSIS in islet beta cells.

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Figures

Fig. 1.
Fig. 1.
GSIS from INS-1-derived cell lines. Insulin secretion was measured at 1 or 20 mM glucose in a 2-h static incubation assay in the indicated INS-1-derived cell lines. Data represent the mean ± SEM for three to four independent assays, each performed in triplicate. *, Cell lines 832/13 and 833/15 had larger fold responses to stimulatory glucose than the other four cell lines, with P < 0.0025.
Fig. 2.
Fig. 2.
Differential expression of glucagon (A), Nkx 2.2 (B), and Nkx 6.1 (C) in INS-1-derived cell lines. Shown are representative gels and bar graph summaries, with the latter representing the mean ± SEM for three independent experiments per cell line, each performed in duplicate. #, Class 1 cells have more glucagon and Nkx2.2 mRNA, respectively, than the other two classes of cells, with P < 0.008. *, Class 2 cells have more Nkx6.1 mRNA than class 1 cells, with P < 0.05; **, class 3 cells have more Nkx6.1 mRNA than the other two classes, with P < 0.001.
Fig. 3.
Fig. 3.
Overexpression of Nkx6.1 in class 1 cells suppresses glucagon expression. Class 1 cells were treated with AdCMV-Nkx6.1 or AdCMV-βGAL. (A) Semiquantitative multiplex PCR analysis of hamster Nkx6.1 and glucagon mRNAs, with α-tubulin as an internal control. (B) Immunoblot analysis of Nkx6.1 protein levels for the same range of viral titers as used in A. (C) The dose-dependent effects of Nkx6.1 overexpression on glucagon mRNA levels quantified by real-time PCR. Data represent the mean ± SEM for four independent experiments per viral dose, with each measurement in duplicate.
Fig. 4.
Fig. 4.
EMSA analysis of Nkx6.1 binding to the G1 element of the glucagon promoter. (A) Nuclear extracts were prepared from 832/13 cells treated with AdCMV-Nkx6.1 (lanes 1 and 2) or no virus (lanes 3 and 4) and mixed with a radiolabeled oligonucleotide corresponding to the G1 element of the glucagon promoter. (B) Effects of Ad-siNkx6.1 (Left) and Ad-siPdx1 (Right) viruses on their target proteins in 832/13 cells. Lanes 1, 2, 5, and 6 are from Ad-siLuc-treated cells, lanes 3 and 4 are from Ad-siNkx6.1-treated cells, and lanes 7 and 8 are from Ad-siPdx1-treated cells. (C) Effects of Ad-siPdx1 on Nkx6.1 protein level (Left) and of Ad-siNkx6.1 on Pdx1 protein level (Right) in 832/13 cells. Lanes 1, 2, 5, and 6 are from Ad-siRNA control-treated cells, lanes 3 and 4 are from Ad-siPdx1-treated cells, and lanes 7 and 8 are from Ad-siNkx6.1-treated cells. Before gel electrophoresis, samples were untreated (-) or treated (+) with anti-Nkx6.1 or anti-Pdx1 antibodies as indicated to cause supershift of the specific Nkx6.1/G1 (SS Nkx6.1) or Pdx1/G1 (SS Pdx1) complexes. Data are representative of two or three independent experiments.
Fig. 5.
Fig. 5.
Suppression of glucagon promoter function by Nkx6.1. (A) αTC1.6 cells were cotransfected with the pBAT12.Nkx6.1 plasmid expressing Nkx6.1 and plasmids containing the luciferase reporter under control of the glucagon promoter, CMV promoter, TK promoter, or no promoter (promoterless). Luciferase activity is expressed as fold repression relative to activity in cells cotransfected with the glucagon/luciferase reporter construct and the pBAT12 plasmid lacking a cDNA insert (empty vector). Data represent the mean ± SEM for three independent cotransfections. (B) Suppression of endogenous glucagon expression in αTC1.6 cells in response to adenovirus-mediated overexpression of Nkx6.1. Data represent the mean ± SEM for two independent experiments, each performed in triplicate. (C) ChIP assay of Nkx6.1 binding to the endogenous glucagon promoter in a class 3 cell line (832/3). Immunoprecipitation reactions were performed with nonspecific rabbit serum or anti-Nkx6.1 antibody, followed by real-time PCR analysis of associated DNA with primers specific for the glucagon or myoD genes. Data are expressed as fold increase in recovery of input glucagon or myoD DNA by immunoprecipitation with the respective antibodies and represent the mean ± SEM for five independent experiments, each performed in triplicate. Primary data for these experiments (real-time PCR amplification curves) are available upon request from the authors. *, P < 0.01.
Fig. 6.
Fig. 6.
Effects of RNA interference-mediated suppression of Nkx6.1 or Pdx1 expression in 832/13 cells. 832/13 cells were treated with Ad-siNkx6.1 (left group of bars), Ad-siPdx1 (center group of bars), or Ad-siRNAcontrol (right group of bars), and glucagon, Nkx6.1, Pdx1, and GLUT2 mRNA levels were analyzed by real-time PCR. Data are normalized to the levels in Ad-siRNAcontrol-treated cells and represent the mean ± SEM for three to five independent experiments, each performed in triplicate. Numbers below the graph indicate the average fold changes in expression of the indicated mRNAs in response to Nkx6.1 or Pdx1 suppression. Symbols denote statistically significant increases or decreases in mRNA levels: *, P < 0.05; **, P < 0.01; #, P < 0.002.
Fig. 7.
Fig. 7.
Effects of Nkx6.1 and Pdx1 suppression on GSIS in class 3 cells. 832/13 cells were treated with the indicated siRNA-containing recombinant adenoviruses, followed by assay of insulin secretion in response to 3 or 15 mM glucose. (A) Insulin secretion expressed as microunits per ml per every 2 h. (B) Insulin secretion expressed as fold response. Data represent the mean ± SEM for three to five independent experiments, each performed in triplicate. *, Fold response was decreased in Ad-siNkx6.1-treated cells, with P < 0.001.
Fig. 8.
Fig. 8.
Suppression of Nkx6.1 expression impairs GSIS in rat islets. Rat islets were treated with Ad-siNkx6.1 or Ad-siRNAcontrol viruses, resulting in a 46% decrease in Nkx6.1 mRNA in the Ad-siNkx6.1-treated cells. Data represent the mean ± SEM for three independent experiments, with data normalized to insulin content. *, Fold response in Ad-siNkx6.1-treated cells was significantly reduced (P = 0.012), and insulin secretion at stimulatory glucose was decreased by 56% (P = 0.001).
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