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. 2023 Jul 10;26(8):107311.
doi: 10.1016/j.isci.2023.107311. eCollection 2023 Aug 18.

Type 2 diabetes susceptibility gene GRK5 regulates physiological pancreatic β-cell proliferation via phosphorylation of HDAC5

Shugo Sasaki  1   2 , Cuilan Nian  1   2   3 , Eric E Xu  1   3 , Daniel J Pasula  1   2 , Helena Winata  1   2   3 , Sanya Grover  1 , Dan S Luciani  1   2 , Francis C Lynn  1   2   3   4
Affiliations

Type 2 diabetes susceptibility gene GRK5 regulates physiological pancreatic β-cell proliferation via phosphorylation of HDAC5

Shugo Sasaki et al. iScience. .

Abstract

Restoring functional β cell mass is a potential therapy for those with diabetes. However, the pathways regulating β cell mass are not fully understood. Previously, we demonstrated that Sox4 is required for β cell proliferation during prediabetes. Here, we report that Sox4 regulates β cell mass through modulating expression of the type 2 diabetes (T2D) susceptibility gene GRK5. β cell-specific Grk5 knockout mice showed impaired glucose tolerance with reduced β cell mass, which was accompanied by upregulation of cell cycle inhibitor gene Cdkn1a. Furthermore, we found that Grk5 may drive β cell proliferation through a pathway that includes phosphorylation of HDAC5 and subsequent transcription of immediate-early genes (IEGs) such as Nr4a1, Fosb, Junb, Arc, Egr1, and Srf. Together, these studies suggest GRK5 is linked to T2D through regulation of β cell growth and that it may be a target to preserve β cells during the development of T2D.

Keywords: Cell biology; Human metabolism.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
β cell specific GRK5 knockout mice displayed impaired glucose tolerance (A) Scheme for generation of β cell specific GRK5 knockout mice (Pdx1-CreER; GRK5fl/fl). (B) GRK5 mRNA expression levels in 8- and 18-week-old mice; tamoxifen was injected at 6-week. CT: GRK5fl/fl, KO: Pdx1-CreER; GRK5fl/fl. n = 4 for 8-week, n = 5 for 18-week. ∗p < 0.05. (C) Western blotting for GRK5 and GAPDH. CT: GRK5fl/fl, KO: Pdx1-CreER; GRK5fl/fl. n = 4. (D) Body weight, fasting blood glucose, and non-fasted blood glucose levels over time. Average blood glucose levels across 30 weeks. n = 6 for each group. ∗p < 0.05. (E) OGTT profiles for 6-, 10-, 14-, and 18- week-old mice. n = 6 for each group. ∗p < 0.05. (F) ITT profiles at 16-week. n = 6 for each group. (G) Serum insulin levels during OGTT. CT: GRK5fl/fl, KO: Pdx1-CreER; GRK5fl/fl. n = 5–6. ∗p < 0.05.
Figure 2
Figure 2
β cell specific GRK5 knockout mice displayed reduced β cell mass with less proliferation and more apoptosis (A) Immunohistochemical analysis for insulin in the pancreas at 18-week. Scale bar, 1 mm. (B) Quantification for β cell number. n = 7. ∗p < 0.05. (C) Immunohistochemical analysis for insulin and EdU at 8-week. Arrows indicate EdU-positive cells. Scale bar, 50 μm. (D) Quantification for EdU-positive β cell number. n = 4. ∗p < 0.05. (E) Immunohistochemical analysis for insulin and TUNEL at 8-week. Arrows indicate TUNEL-positive cells. Scale bar, 50 μm. (F) Quantification for TUNEL-positive β cell number. n = 4. ∗p < 0.05. (G) Cdkn1a gene expression levels in the islet. CT: GRK5fl/fl, KO: Pdx1-CreER; GRK5fl/fl. n = 5–6. ∗p < 0.05.
Figure 3
Figure 3
RNA-sequencing revealed alteration of mass regulation-related gene expressions in β cell specific GRK5 knockout mice (A) The number of differentially expressed genes (DEGs) in GRK5 and SOX4 knockout islets. Overlapped DEGs are shown. (B) Panther pathway analysis in β cell specific GRK5 KO islets at 18-week. Pathways with p < 0.01 are shown. (C) FPKM for DEGs related to AKT signaling, ERK signaling, cell cycle/proliferation, and IEGs are shown. CT: GRK5fl/fl, KO: Pdx1-CreER; GRK5fl/fl. n = 5. ∗p < 0.05.
Figure 4
Figure 4
Phosphorylation of HDAC5 is altered in β cell specific GRK5 knockout mice and human islets treated with amlexanox (A) Western blotting for HDAC5 phosphorylation. 80 mouse islets (18-week) were applied to each lane. CT: GRK5fl/fl, KO: Pdx1-CreER; GRK5fl/fl. p/tHDAC5 ratio: phospho HDAC5/total HDAC5 ratio. n = 6. ∗p < 0.05. (B) Western blotting for HDAC5 phosphorylation status after stimulation. 80 mouse islets (18-week) were applied to each lane. The islets were treated by LG (2.8 mM low glucose) and HG + KCl (16 mM high glucose +30 mM KCl) for 1 h. n = 5. ∗p < 0.05. (C) Western blotting for HDAC5 phosphorylation status after stimulation. 100 human islets were applied to each lane. The islets were treated by LG, HG + KCl and Amx (50 μM amlexanox) for 1 h. n = 3. ∗p < 0.05. (D) Scheme for β cell mass regulation by GRK5. HDAC5 is suppressing activity of MEF2 that transcribes IEGs and regulates cell mass. GRK5 inactivates HDAC5 by phosphorylation, and de-suppresses MEF2 activity, resulting in β cell mass expansion.
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