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. 2020 Jun 5;10(16):7351-7368.
doi: 10.7150/thno.44459. eCollection 2020.

Protein acetylation derepresses Serotonin Synthesis to potentiate Pancreatic Beta-Cell Function through HDAC1-PKA-Tph1 signaling

Yuqing Zhang  1   2 , Shushu Wang  1 , Linlin Zhang  1 , Feiye Zhou  1 , Kecheng Zhu  1 , Qin Zhu  1 , Qianqian Liu  1 , Yun Liu  1 , Lei Jiang  1 , Guang Ning  1 , Yufang Bi  1 , Libin Zhou  1 , Xiao Wang  1
Affiliations

Protein acetylation derepresses Serotonin Synthesis to potentiate Pancreatic Beta-Cell Function through HDAC1-PKA-Tph1 signaling

Yuqing Zhang et al. Theranostics. .

Abstract

Rationale: Protein acetylation is tightly linked to transcriptional control and energy metabolism. However, the role of protein acetylation in islet function remains enigmatic. This study aims to determine how protein acetylation controls β-cell function and explore the underlying mechanism. Methods: The gene-expression profiles were analyzed for rat islets in response to two histone deacetylase (HDAC) inhibitors. Insulin secretion, tryptophan hydroxylase 1 (Tph1) expression, and serotonin synthesis of rat islets were detected after HDAC inhibitor treatment both in vivo and ex vivo. β-cell-specific Tph1-overexpressing transgenic rats and β-cell-specific Tph1 knockout mice were constructed to evaluate the role of Tph1 in β-cell function. The deacetylation of PKA in β-cells by HDAC1 was investigated by adenoviral infection, immunoprecipitation, and western blot. Results: Inhibition of HDACs greatly potentiated pancreatic β-cell function and reprogrammed transcriptional landscape of islets. Among the commonly up-regulated genes by two pan-HDAC inhibitors, Tph1 displayed the most prominent change. Specifically, inhibition of HDAC1 and HDAC3 by MS-275 strongly promoted Tph1 expression and endogenous serotonin synthesis in rat islets, concomitantly with enhanced insulin secretory capacity in vivo and ex vivo. β-cell-specific Tph1-overexpressing transgenic rats exhibited improved glucose tolerance and amplified glucose-stimulated insulin secretion. On the contrary, β-cell-specific Tph1 knockout mice displayed glucose intolerance and impaired insulin secretion with aging. Moreover, depletion of Tph1 in β-cells abrogated MS-275-induced insulin hypersecretion. Overexpression of HDAC1, not HDAC3, inhibited Tph1 transcriptional activity and decreased MS-275-stimulated Tph1 expression. Mechanistically, HDAC1 deacetylated PKA catalytic subunit and decreased its activity, resulting in Tph1 transcriptional repression. The acetylation mimetic K62Q mutant of PKA increased its catalytic activity. HDAC1 inhibition exerted a synergistic effect with cAMP/PKA signal on Tph1 expression. Conclusions: The present findings highlight a novel role of HDAC1-PKA-Tph1 signaling in governing β-cell functional compensation by derepressing serotonin synthesis.

Keywords: Beta-cell function; HDAC1; PKA; Protein acetylation; Serotonin; Tph1.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Effects of HDAC inhibitors on insulin secretion and transcriptional landscape of rat islets. (A) Rat islets were treated with 200 nM TSA at 3.3 mM glucose for the indicated time, and the cumulative insulin secretion was measured. (B) Rat islets were pretreated with 200 nM TSA at 3.3 mM glucose for 24 h, and then stimulated with 3.3, 8.3 or 16.7 mM glucose for 1 h. The supernatant was taken for insulin secretion assay. (C) Rat islets were stimulated with 3.3, 8.3 or 16.7 mM glucose in the presence or absence of 5 mM SB for 1 h, and insulin secretion was measured. (D) Rat islets were pretreated with 5 mM SB at 3.3 mM glucose for 24 h, and then stimulated with 3.3, 8.3 or 16.7 mM glucose for 1 h. The supernatant was taken for insulin secretion assay. (E) Comparison of the differentially up-regulated genes in TSA- and SB-treated rat islet transcriptomes. (F) KEGG pathway analysis of the commonly upregulated genes by both TSA and SB. (G) Top 10 commonly upregulated-genes in TSA-treated islet transcriptome. (H) Top 10 commonly upregulated-genes in SB-treated islet transcriptome. (I) qRT-PCR analysis of Tph1, Tph2, and Ddc mRNA expressions in rat islets incubated with 200 nM TSA and 5 mM SB at 3.3 mM glucose for 24 h. (J) Western blot analysis of Tph1 protein expression in rat islets incubated with 200 nM TSA and 5 mM SB at 3.3 mM glucose for 24 h. Data are expressed as mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control (CON).
Figure 2
Figure 2
HDAC1 inhibition increases serotonin synthesis and β-cell function of rat islets. After rat islets were incubated with 200 nM TSA at 3.3 mM glucose for 24 h, islet serotonin content (A) and serotonin secretion (B) were measured by ELISA. (C) Rat islets were incubated with 500 μM 5-hydroxytryptamine (5-HT) and 500 μM 5-hydroxytryptophan (5-HTP) for the indicated time, and then insulin secretion was measured. Tph1 mRNA expressions in rat islets (D) and INS-1 cells (E) treated with 200 nM TSA, 5 mM SB, 3 μM MS-275, 10 μM CI-994, 5 μM PCI-34051, and 10 μM Tubacin at 3.3 mM glucose for 24 h. (F) Rat islets were pretreated with 3 μM MS-275 and 10 μM CI-994 at 3.3 mM glucose for 24 h, then stimulated with 3.3 and 16.7 mM glucose (3.3G and 16.7G) for 1 h, and insulin secretion was measured. After mice were injected with either saline vehicle or MS-275 (20 mg/kg body weight) for consecutive 7 days, (G) Immunofluorescent staining was performed for serotonin (red), insulin (green) and DAPI (blue) in the pancreatic sections from mice injected with MS-275 or saline (scale bars, 20 μm). Body weight (H), fasting blood glucose (I), random blood glucose (J) and random serum insulin levels (K) were measured (n=6). (L) Blood glucose levels during IPGTT of mice injected with either saline or MS-275 for 5 days (n=6-7). (M) Serum insulin levels were measured before and after glucose injection from vehicle- or MS-275-treated mice (n=6). (N) Islets were isolated from mice injected with either saline or MS-275 for 7 days, then stimulated with 3.3 and 16.7 mM glucose for 1 h, and insulin secretion were measured. Data are expressed as mean ± SEM of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control (CON or vehicle).
Figure 3
Figure 3
Enhanced β-cell function in β-cell-specific Tph1-overexpressing transgenic rats. (A and B) Tph1 mRNA (n=6) and protein levels in islets from wild-type (WT) and Tph1 transgenic male rat line #10 (Tg-10). (C and D) Flag protein level in islets and other tissues from WT and Tph1 transgenic rats. (E) Immunofluorescence staining for serotonin (green), insulin (red), and glucagon (red) in the pancreatic sections from WT and Tph1 transgenic rats (scale bars, 20 μm). Body weight (F) and food intake (G) of WT and Tph1 transgenic rats (n=10). (H) Fasted and fed blood glucose levels of WT and Tph1 transgenic rats (n=8). (I) Fasted and fed serum insulin levels of WT and Tph1 transgenic rats (n=6-7). (J and K) Blood glucose (n=8) and serum insulin (n=6-7) levels were measured during IPGTT from WT and Tph1 transgenic rats. (L) Blood glucose levels were measured at the indicated time after insulin injection (n=10). (M) Islets from WT and Tph1 transgenic rats were stimulated with various concentrations of glucose for 1 h, and then insulin secretion was assayed. All the experiments were performed on 10-week-old WT and Tph1 transgenic rats. *P<0.05, **P<0.01, ***P<0.001 vs. WT mice.
Figure 4
Figure 4
β-cell-specific Tph1 knockout mice exhibit impaired insulin secretion. (A) Detection of Tph1 mRNA expression in islets from WT and βTph1KO mice by RT-PCR. (B) Islets isolated from WT and βTph1KO mice were incubated in 16.7 mM glucose overnight, and then Tph1 protein expression was detected. (C) Body weight of WT and βTph1KO mice at the age of 8, 24, and 36 weeks (n=8-14). (D and E) Fasting and random blood glucose levels in 8-week-old WT and βTph1KO mice (n=10-12). (F) Blood glucose levels during IPGTT of 8-week-old WT and βTph1KO mice (n=6). (G) Islets isolated from 8-week-old WT and βTph1KO mice were pretreated with 10 μM CI-994 and 3 μM MS-275 at 3.3 mM glucose for 24 h, then stimulated with 3.3 and 16.7 mM glucose (3.3G and 16. 7G) for 1 h, and insulin secretion was assayed. (H and I) Fasting and random blood glucose levels in 24-week-old WT and βTph1KO mice (n=10-11). (J) Blood glucose levels during IPGTT of 24-week-old WT and βTph1KO mice (n=8-10). (K) Introperitoneal insulin tolerance tests (ITT) of 24-week-old WT and βTph1KO mice (n=6). (L) Insulin levels before and after glucose loading in 24-week-old WT and βTph1KO mice (n=7-9). (M) Islets isolated from 24-week-old WT and βTph1KO mice were stimulated with 3.3 and 16.7 mM glucose for 1 h and insulin secretion was assayed. *P<0.05, **P<0.01, ***P<0.001, vs. WT or CON. #P<0.05 vs. WT-16.7G.
Figure 5
Figure 5
Tph1 transcription is regulated by HDAC1 in β-cells. (A) INS-1 cells were transfected with rat Tph1 promoter, and then treated with 3 μM MS-275 and 200 nM TSA at 3.3 mM glucose for the indicated time. Dual-luciferase assay was performed. (B) Tph1 promoter activities in INS-1 cells transfected with empty vector (EV), HDAC1 or HDAC3 plasmid. (C) Rat islets were transfected with vector or flag-tagged HDAC1-overexpressing adenovirus, and then treated with 3 μM MS-275 and 10 μM CI-994 for 24 h. Tph1 mRNA expression was determined. (D) After INS-1 cells were treated with 5 mM SB and 3 μM MS-275 for 24 h, total H3Kac, H3K9ac, H3K18ac and H3K27ac were determined by western blot of extracted histones. (E) INS-1 cells were transfected with HDAC1-overexpressing adenovirus and then treated with 3 μM MS-275 for 24 h. H3K9ac, H3K18ac and H3K27ac were determined. (F) Top 10 upregulated KEGG pathways from ChIP-seq analysis of genes whose promoter regions were upregulated in SB-treated rat islets. (G) Overlap of the up-regulated genes in SB-treated islet ChIP-seq (yellow circle), TSA-treated islet transcriptome (red circle) and SB-treated islet transcriptome (blue circle). (H) H3K27ac patterns of Tph1, Tph2 and Ddc promoters from ChIP-seq. Data are expressed as mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control (CON, EV, or Ad-Vector).
Figure 6
Figure 6
HDAC1 deacetylates and regulates the catalytic activity of Prkaca. (A) CREB Ser133 phosphorylation levels in INS-1 cells treated with 3 μM MS-275 for the indicated time. (B) INS-1 cells were transfected with control vector (CON) or HDAC1-overexpressing adenovirus (HDAC1), and then stimulated with or without 5 μM foskolin (FSK) for 1 h. CREB phosphorylation level was assayed by western blot. (C) His-tagged HDAC1 and flag-tagged Prkar1a, Prkar1b, Prkaca or Prkacb were co-expressed in HEK-293T cells. The co-immunoprecipitated His-tagged HDAC1 was detected in Flag beads purified proteins. (D) Western blot detection of endogenous PKA-catalytic subunit (PKA-C) level of immunoprecipitated HDAC1 from INS-1 cells. (E) Western blot detection of endogenous HDAC1 level of immunoprecipitated PKA-C from INS-1 cells. Acetylation levels of ectopically expressed Prkaca (F) and Prkacb (G) in HDAC1-overexpressing HEK-293T cells. (H) Acetylation level of endogenous PKA-C in INS-1 cells transfected with vector or HDAC1-overexpressing adenovirus. (I) Acetylation level of endogenous PKA-C in INS-1 cells transfected with vector or HDAC1 silencing adenovirus. (J) Flag-tagged wild-type, K62Q or K62R mutant of Prkaca was overexpressed in HEK-293T cells and the levels of Prkaca acetylation and CREB phosphorylation were determined. (K) INS-1 cells were transfected with wild-type or K62Q of Prkaca adenovirus and then treated with 5 μM FSK at 5.6 mM glucose for 1 h. CREB Ser133 phosphorylation was determined. (L) Relative Tph1 mRNA expression levels in rat islets transfected with control vector, Prkaca wild-type (WT) or K62Q mutant adenovirus in the presence of 3.3 mM glucose, 3.3 mM glucose plus 5 μM FSK or 16.7 mM glucose for 24 h. Data are expressed as mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control (CON). #P<0.05, ##P<0.01.
Figure 7
Figure 7
Synergistic effect of HDAC1 inhibition and cAMP/PKA signal on Tph1 expression. (A) Relative Tph1 mRNA levels in rat islets transfected with control vector or HDAC1-overexpressing adenovirus in the presence or absence of 5 μM forskolin (FSK) at 5.6 mM glucose for 24 h. (B) Tph1 mRNA expression in rat islets incubated with 3 μM MS-275 (MS) in the presence or absence of 10 μM H89 for 24 h. (C) Rat islets were treated with 3.3 and 16.7 mM glucose (3.3G and 16. 7G) for 24 h, and then Hdac1 mRNA expression was detected. (D) Rat islets transfected with control vector or HDAC1-overexpressing adenovirus were further treated with 3.3 and 16.7 mM glucose for 24 h, and Tph1 mRNA expression was detected. (E) Tph1 mRNA expression in rat islets incubated with 3 μM MS-275 in the presence of 3.3 or 16.7 mM glucose for 24 h. Tph1 mRNA (F) and protein expressions (G) in rat islets incubated with 10 μM CI-994 (CI) and 5 μM FSK for 24 h. (H) Tph1 mRNA expression in rat islets incubated with 10 nM Exendin-4 and 3 μM MS-275 for 24 h. (I) The schematic illustration summarizes the effect of protein acetylation on insulin secretion via HDAC1-PKA-Tph1 signaling. In the basal state, HDAC1 binds to and deacetylates Prkaca, thus disrupting CREB phosphorylation to repress Tph1 transcription. Under the condition of increased insulin demand, HDAC1 inhibition increases acetylation level of Prkaca, leading to CREB phosphorylation and subsequent Tph1 transcriptional derepression. This could further augment glucose- or GLP-1 analogue-elicited Tph1 expression. Tph1-mediated serotonin production triggers the adaptive insulin hypersecretion from β-cells. Data are expressed as mean ± SEM of three independent experiments. *P<0.05, **P<0.01 vs. control (CON) or 3.3G. #P<0.05.
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