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. 2019 Aug 14:2019:8915818.
doi: 10.1155/2019/8915818. eCollection 2019.

FXR Mediates Adenylyl Cyclase 8 Expression in Pancreatic β-Cells

Xiangchen Kong  1 , Bingfeng Li  1 , Yushen Deng  2 , Xiaosong Ma  1
Affiliations

FXR Mediates Adenylyl Cyclase 8 Expression in Pancreatic β-Cells

Xiangchen Kong et al. J Diabetes Res. .

Abstract

Adenylyl cyclase 8 (ADCY8) and Farnesoid X Receptor (FXR) have been identified in pancreatic β-cells and play important roles in insulin secretion. But the mechanisms underlying with respect to the regulation of ADCY8 expression in β-cells, particularly whether FXR is involved, remain unexplored. We now show that ADCY8 expression is decreased in Goto-Kakizaki (GK) rat islets compared with healthy Wistar controls. We also found that reduced ADCY8 is associated with decreased expression of FXR. Consistently, ADCY8 expression was suppressed by the knockdown of FXR in INS-1 832/13 cells, as well as the islets from FXR knockout mice. On the contrary, ADCY8 expression was increased in FXR-overexpressed INS-1 832/13 cells or in the case of FXR activation. Mechanistically, FXR directly binds to Adcy8 promoter and recruits the histone acetyltransferase Steroid Receptor Coactivator 1 (SRC1), thereby resulting in the increased acetylation of histone H3 in Adcy8 locus, promoting Adcy8 gene transcription in β-cells. Thus, this study indicates that FXR is a critical transcription factor that mediates ADCY8 expression in pancreatic β-cells and has characterized the chromatin modification associated with Adcy8 transcription.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
ADCY8 expression is decreased in GK rat islets. (a) mRNA level of ADCY8 in Wistar and GK islets. Values represent as means ± SEM. n = 5; ∗∗ P < 0.01. (b) Representative blots of ADCY8 protein expression of three independent experiments in Wistar and GK islets. Data are means ± SEM. ∗ P < 0.05.
Figure 2
Figure 2
Expression of FXR in islets from Wistar and GK rats. (a, b) Cytosol and nuclear proteins extracted from Wistar and GK rat pancreases were subjected to western blot analysis. Histone3 and β-actin were used as nuclear and cytosol internal control, respectively. Data are means ± SEM. n = 3; ∗ P < 0.05. (c) Representative immunostaining images for FXR (green), insulin (red), DAPI (blue), and merge of the three in single β-cells isolated from Wistar and GK rats; n = 4. Scale bars represent 5 μm. (d) Statistical analysis of FXR expression in β-cells isolated from Wistar and GK rats. Data are means ± SEM. n = 4; ∗ P < 0.05. (e) FXR protein expression in Wistar and GK rat islets. Data represent means ± SEM. n = 3; ∗ P < 0.05. (f) Insulin secretion in islets isolated from Wistar and GK rats. The islets were treated with 16.8 mM glucose in the presence of DMSO or 5 μM GW4064 for 30 min. Data are means ± SEM. n = 4; ∗ P < 0.05.
Figure 3
Figure 3
FXR regulates ADCY8 expression in pancreatic β-cells. mRNA (a) and protein (b) level of ADCY8 in FXR knockdown INS-1 832/13 cells. Data are means ± SEM. n = 4‐5; ∗ P < 0.05. mRNA (c) and protein (d) level of ADCY8 in FXR knockout mouse islets. 100-150 islets were used in each experiment. Data represent means ± SEM. n = 3‐5; ∗ P < 0.05. mRNA (e) and protein (f) expression of ADCY8 in FXR-overexpressed INS-1 832/13 cells. Data are means ± SEM. n = 4; ∗ P < 0.05. mRNA (g) and protein (h) expression of ADCY8 in INS-1 832/13 cells after 24 h treatment with 5 μM GW4064. Data are means ± SEM. n = 4‐7; ∗∗ P < 0.01.
Figure 4
Figure 4
FXR directly binds to the Adcy8 promoter. (a) A putative FXR binding site in the Adcy8 promoter of different species. (b) 293T cells were transfected with a luciferase reporter driven by the Adcy8 promoter, followed by treatment with 5 μM GW4064 for 24 h prior to luciferase activity analysis for the reporter expression. Data are means ± SEM. n = 3; ∗∗ P < 0.01. ChIP analysis for FXR occupancy in the promoter of Adcy8 in INS-1 832/13 cells treated with 5 μM GW4064 for 24 h (c) or in FXR knockdown INS-1 832/13 cells (d). Data are means ± SEM of three independent experiments. ∗ P < 0.05.
Figure 5
Figure 5
FXR mediates SRC1 binding and histone H3 acetylation at the promoter of Adcy8. ChIP assay was performed to detect the binding of SRC1 (a) and histone H3 acetylation (b) at the Adcy8 promoter in INS-1 832/13 cells treated with 5 μM GW4064 for 24 h. Data represent means ± SEM. n = 3; ∗ P < 0.05. FXR knockdown INS-1 832/13 cells were subjected ChIP assay to test SRC1 binding (c) and histone H3 acetylation (d) at the Adcy8 promoter. Data are means ± SEM. n = 3; ∗ P < 0.05, ∗∗ P < 0.01. mRNA (e) and protein (f) expression of ADCY8 in scramble or SRC1 knockdown INS-1 832/13 cells treated with 5 μM GW4064 for 24 h. Data are means ± SEM. n = 3; ∗ P < 0.05.
Figure 6
Figure 6
Effect of forskolin on insulin secretion and cAMP production in pancreatic β-cells. (a) Insulin secretion of islets isolated from FXR+/+ and FXR−/− mice. The islets were stimulated with 2.8 mM glucose or 16.8 mM glucose in the presence or absence of 10 μM forskolin for 30 min. (b) Fold increase of forskolin-potentiated insulin secretion in FXR+/+ and FXR−/− mouse islets. Data are means ± SEM. n = 4; ∗ P < 0.05, ∗∗ P < 0.01. (c) Insulin secretion of scramble and shFXR INS-1 832/13 cells treated with the condition same with (a). (d) Fold increase of forskolin-potentiated insulin secretion in scramble and shFXR INS-1 832/13 cells. Data represent means ± SEM. n = 4‐10; ∗ P < 0.05, ∗∗ P < 0.01. cAMP production in islets from FXR+/+ and FXR−/− mice (e) and scramble and shFXR INS-1 832/13 cells (f) incubated with 16.8 mM glucose in the presence or absence of 10 μM forskolin for 30 min. Data are means ± SEM. n = 3 of each group. ∗ P < 0.05, ∗∗ P < 0.01.
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