Biochemical and Biophysical Research Communications 524 (2020) 936e942

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Biochemical and Biophysical Research Communications

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GSTO1 regulates insulin biosynthesis in pancreatic b cells

** * Linlin Wang a, b, 1, Lei Lei c, 1,TaoXua, b, , You Wang a, a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China b College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China c The State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100050, China article info abstract

Article history: Insulin biosynthesis and secretion by pancreatic b cells are critical for the maintenance of blood glucose Received 10 January 2020 homeostasis. Here, we show that the expression of glutathione S-transferase omega-1 (GSTO1) is Accepted 28 January 2020 upregulated in the primary islet cells of diabetic Goto-Kakizaki (GK) rats. Knocking out GSTO1 upregu- Available online 11 February 2020 lated insulin transcripts and increased the insulin content in both INS-1 cells and primary islet cells. In contrast, overexpression of GSTO1 reduced the insulin content. Furthermore, knocking out GSTO1 Keywords: increased the expression of pancreatic duodenal homeobox-1 (PDX1) at both the transcription and GSTO1 levels. These results indicate that GSTO1 may be involved in the regulation of insulin biosynthesis Pancreatic b cell Insulin biosynthesis by modulating the transcriptional expression of PDX1. © PDX1 2020 Elsevier Inc. All rights reserved. Diabetes

1. Introduction and plays a pro-inflammatory role in models of inflammation, co- litis and obesity [10e13]. The omega class of glutathione transferases (GSTs) is an ancient Pancreatic b cells are unique cells in the islets of Langerhans that branch of the GST superfamily. In contrast to other GST families, synthesize, store and secrete the hypoglycemic hormone insulin omega class GSTs contain a cysteine residue in the active site and and play a vital role in the maintenance of blood glucose homeo- exhibit distinct catalytic properties [1]. Glutathione S-transferase stasis [14]. Elevated extracellular glucose concentrations trigger omega-1 (GSTO1), a member of the omega class of glutathione insulin release from pancreatic b cells by increasing ATP produc- þ transferases, was first found to be upregulated in a multidrug- tion, membrane depolarization and Ca2 influx [15]. Although resistant lymphoma cell line and was identified as a stress pancreatic b cells depend on oxidative metabolism for ATP syn- response protein [2]. GSTO1 is widely distributed in many tissues, thesis, the expression of is relatively low in such as the heart, liver, prostate, colon and pancreas [3]. Poly- these cells. This imbalance may reflect a special role for redox morphisms in GSTO1 have been associated with a range of disor- signaling in the regulation of pancreatic b cell function; however, ders, including Alzheimer and Parkinson disease, chronic the biological mechanism is not fully understood. GSTO1 has the obstructive pulmonary disease, vascular dementia, stroke, and ability to catalyze multiple enzymatic reactions, such as those of amyotrophic lateral sclerosis [1,3e6]. Recent studies have shown thiol transferase and dehydroascorbate reductase and is involved in þ that GSTO1 regulates intracellular Ca2 concentration by modu- the glutathionylation cycle [5,6,16]. These results suggest that lating cardiac ryanodine receptor calcium release channels [7,8]. In GSTO1 may play an important role in the regulation of redox addition, GSTO1 has been identified as a key regulator of NLRP3 signaling. In this study, we aimed to investigate the role of GSTO1 in inflammasome activation upon deglutathionylation of NEK7 [9] the regulation of pancreatic b cell function.

2. Materials and methods * Corresponding author. ** Corresponding author. National Laboratory of Biomacromolecules, CAS Center 2.1. Cell culture for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. E-mail addresses: [email protected] (T. Xu), [email protected] (Y. Wang). INS-1 cells were cultured in RPMI 1640 medium (Gibco) sup- 1 Contributed equally to this work. plemented with 10% fetal bovine serum (FBS, Gibco), 1 mM sodium https://doi.org/10.1016/j.bbrc.2020.01.151 0006-291X/© 2020 Elsevier Inc. All rights reserved. L. Wang et al. / Biochemical and Biophysical Research Communications 524 (2020) 936e942 937

Fig. 1. GSTO1 expression is increased in the primary islet cells of diabetic GK rats. A: The mRNA and protein expression levels of GSTO1 in the islet cells of Wistar rats and diabetic GK rats, as determined by RNA-seq and proteomics analysis at the indicated time points. Cells from the GK rats and Wistar rats are shown in red and green, respectively. expression line chart data represent the means ± SEM of repeated experiments (n ¼ 3). *P < 0.01, **P < 0.001, and ***P < 0.0001 as determined by adjusted ANOVAs. B: GSTO1 levels in the pancreas sections from the GK rats and WST rats at 8 weeks of age. C: The mean fluorescence intensity of the GSTO1 expression in rat pancreas sections. Data were compared by one-way ANOVAs, *p < 0.05, **p < 0.01 and ***p < 0.005. Three independent experiments were completed. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) pyruvate (Sigma), 50 mM 2-mercaptoethanol (Sigma), 100 U/mL program was 35 cycles at 95 C for 30 s, 58 C for 30 s, 72 C for penicillin (HyClone), and 100 mg/mL streptomycin (HyClone) in a 2.5 min, and an extension at 72 C for 10 min. The PCR products humidified atmosphere of 5% CO2 at 37 C. were visualized on a 1% agarose gel.

2.2. Generation of GSTO1-knockout (KO) INS-1 cells 2.4. Preparation of pancreatic islets

The GSTO1-KO INS-1 cell line was constructed by CRISPR/Cas9 Pancreatic islets from male paired WT and GSTO1-KO mice were technology. Briefly, INS-1 cells were transfected with a sgRNA isolated through 0.5 mg/mL collagenase digestion. After the plasmid targeting GSTO1 using Lipofectamine 2000 (Invitrogen), digestion was terminated in 10% FBS, the digested pancreas was and GFP-positive cells were sorted with flow cytometry to generate rinsed with HBSS, and the islets were separated on a Ficoll density monoclonal stable cell lines. The GSTO1 KO effect was confirmed by gradient. After three washes with HBSS, the islets were eventually western blot analysis. placed in Hanks’ buffer and handpicked while visualized through a dissection microscope. 2.3. Generation of GSTO1-KO mice 2.5. Immunohistochemistry GSTO1-KO mice were generated with C57/B6J mice. For gene editing, sgRNAs targeted from exon 1 to exon 4 were injected into The pancreases from male paired WT and GSTO1-KO mice were the cytoplasm of fertilized eggs. The genotypes of the knockout dissected and fixed with 4% paraformaldehyde in PBS for 24 h. After mice were determined by PCR of genomic DNA extracted from dehydration, the samples were embedded in paraffin. Continuous mouse tails. Taq mix was activated at 95 C for 3 min, and the PCR paraffin sections from each sample were sliced at 5 mm intervals 938 L. Wang et al. / Biochemical and Biophysical Research Communications 524 (2020) 936e942

Fig. 2. The effect of GSTO1 on glucose-stimulated insulin release from INS-1 cells and primary mouse islet cells. AeB: Verification of GSTO1-knockout in INS-1 cells (A) and primary islet cells in the C57/B6J mice (B) by western blot analysis. b-actin was used as a reference protein in the western blot analysis to determine protein levels. CeD: The amount of insulin secreted from the INS-1 cells (C) and the islet cells isolated from the GSTO1-knockout mice (D) treated with one of two different glucose concentrations (2.8 mM and 16.8 mM). Data were compared by one-way ANOVAs, *p < 0.05, **p < 0.01 and ***p < 0.005. and then stained according to a standard IHC protocol. 1 cells were preincubated for 2 h in 2.8 mM glucose in Krebs-Ringer bicarbonate HEPES buffer (KRBB) (114 mM NaCl, 4.7 mM KCl, 2.6. Western blotting 1.2 mM KH2PO4, 1.16 mM MgSO4, 0.5 mM MgCl2, 2.5 mM CaCl2, and 20 mM HEPES with 0.2% BSA at pH 7.4). The cells were then stim- The cells and islet samples were lysed in standard RIPA buffer ulated with 2.8 mM and 16.7 mM glucose KRBB for 1 h. The amount (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and of insulin secreted in the KRBB was measured with an insulin ELISA 50 mM Tris at pH 8.0) with proteinase inhibitors (Sigma). Then, kit (Millipore). The protein content of the cells was used for samples of the centrifuged supernatant were adjusted to have the normalization. For the intracellular insulin content, the cells and same total protein concentration and were separated by SDS-PAGE, islet samples were lysed in RIPA lysis buffer and quantified through transferred onto PVDF membranes (Millipore), and incubated with the use of an insulin ELISA kit. primary antibodies against GSTO1 (Proteintech), PDX1 (CST), b- actin (Sigma) and a-tubulin (Sigma). The membranes were then 2.8. Quantitative real-time PCR incubated with the appropriate HRP-conjugated secondary anti- bodies (Sungene Biotech), and bands were detected with enhanced Total RNA was prepared directly from the cells using TRIzol luminescence (GE Healthcare). A minimum of three independent (Invitrogen) according to the manufacturer’s instructions. Reverse western blots were obtained, and a typical example is presented. transcription (RT) was performed using SuperQuick RT MasterMix (CWBio). Quantitative PCR was performed with cDNA (1:20 dilu- 2.7. Detection of insulin by ELISA tion), 2 SYBR Green PCR Mix, and specific primers (10 mM). The 18S primer was used for normalization, and DCt was calculated to To measure the secreted insulin, WT and GSTO1-knockout INS- determine the relative expression levels. L. Wang et al. / Biochemical and Biophysical Research Communications 524 (2020) 936e942 939

Fig. 3. The insulin content was increased in GSTO1-knockout INS-1 cells and primary islet cells. AeB: Total insulin content of the GSTO1-KO INS-1 cells (A) and islet cells isolated from the GSTO1-KO mice (B) as measured by ELISAs. (CeD) The mRNA level of insulin-1 was detected by qRT-PCR with 18S used as a reference gene. (E) The insulin content upon GSTO1 overexpression was detected by ELISA. Data represent the means ± SEM (n ¼ 3). Data were compared by one-way ANOVAs, *p < 0.05, **p < 0.01 and ***p < 0.005.

3. Results indicated that GSTO1 may be involved in the progressive deterio- ration of pancreatic b cells in GK rats. 3.1. Expression of GSTO1 is upregulated in the pancreatic b cells of diabetic GK rats 3.2. GSTO1 regulates insulin biosynthesis in vitro and in vivo

In our previous study, both the transcriptome and proteome To investigate the function of GSTO1 in pancreatic b cells, a data showed that the expression of GSTO1 was upregulated in the GSTO1-knockout INS-1 cell line was constructed by CRISPR/Cas9 islets of diabetic GK rats compared with that in the islets of Wistar technology. The elimination of GSTO1 protein expression in the rats (Fig. 1A). To confirm these previously obtained omics data, GSTO1-knockout cell line was confirmed by the western blot immunohistochemistry was performed to determine the protein analysis results (Fig. 2A). Insulin release from pancreatic b cells þ level of GSTO1 in the pancreas of GK rats and age-matched Wistar depends on an increase in intracellular Ca2 concentration upon rats. The results showed that the expression level of the GSTO1 stimulation with high-glucose levels. It has been reported that þ protein was significantly upregulated in the pancreatic b cells of the GSTO1 plays a role in regulating intracellular Ca2 concentration by GK rats compared with that of the control rats (Fig. 1B and C), which modulating ryanodine receptor 2 (RyR2) calcium release channels 940 L. Wang et al. / Biochemical and Biophysical Research Communications 524 (2020) 936e942

Fig. 4. Knocking out GSTO1 significantly increased the expression of Pdx1. AeB: The mRNA expression level of Pdx1 in GSTO1-KO INS-1 cells was analyzed by RT-PCR (A) and primary islet cells isolated from the GSTO1-KO mice (B). CeD: The expression of Pdx1 was analyzed by western blot in GSTO1 KO cells (C) and primary islets isolated from GSTO1 KO mice (D). (E) The mean fluorescence intensity of Pdx1 expression in GSTO1-KO mice. Data represent the means ± SEM (n ¼ 3). Data were compared by one-way ANOVAs, *p < 0.05, **p < 0.01 and ***p < 0.005. L. Wang et al. / Biochemical and Biophysical Research Communications 524 (2020) 936e942 941

INS-1 cells. The results showed that the PDX1 expression was significantly increased at both the mRNA and protein levels in the GSTO1-knockout INS-1 cells (Fig. 4A and C). Consistently, the upregulation of PDX1 mRNA and protein levels was observed in primary islet cells isolated from GSTO1-knockout mice (Fig. 4B and D). The upregulation of PDX1 in the pancreatic b cells was confirmed in GSTO1-knockout primary islet cells by immunofluo- rescence staining (Fig. 4E). These results suggested that PDX1 may be involved in GSTO1 regulation of insulin biosynthesis.

4. Discussion

Reactive oxygen species (ROS) are continuously produced by cells to facilitate their adaption to the immediate redox environ- ment. The generation and elimination of ROS need to be constantly balanced by glutathione and redox-sensitive enzymes such as glutathione transferases (GSTs), glutathione peroxidases (GPxs) and catalases [22]. ROS are normally produced by glucose meta- bolism, including glycolysis and mitochondrial oxidative phos- phorylation [23,24]. In macrophages, ROS can be induced by LPS and have been shown to induce posttranslational modification of [25]. To maintain high ROS levels during an inflammatory Fig. 5. The GSTO1 model showing the regulation of insulin biosynthesis in pancreatic b response, LPS stimulation causes the suppression of enzymes, cells. including the catalases and Gpxs that protect against oxidative stress. However, the expression of antioxidant enzymes such as catalases and GPxs in pancreatic islets is lower than it is in other [7]. Therefore, the effect of GSTO1 on glucose-stimulated insulin tissues, leaving b cells vulnerable to oxidative stress [26,27]. Glu- secretion was investigated. However, the amount of insulin tathionylation is defined as a posttranslational modification of secreted upon stimulation with high-glucose treatment was not protein thiol groups in which disulfides form in combination with significantly different in the GSTO1-knockout INS-1 cells than it glutathione. Modification of glutathionylation results in the alter- was in the control INS-1 cells. However, the amount of insulin ation of the protein structure and activity in response to changes in secreted upon low-glucose treatment from GSTO1-knockout INS- the oxidation state [28]. GSTO1, as a member of the GST super- 1 cells was significantly increased compared with that secreted family, has dehydroascorbate reductase and thiol transferase ac- from the control INS-1 cells (Fig. 2B). To confirm these results, tivities and plays a significant role in the glutathionylation cycle glucose-stimulated insulin secretion from the primary islets iso- [5,6,16]. In this study, we found that GSTO1 expression was upre- lated from global-GSTO1-knockout mice and age-matched control gulated in the primary islet cells of diabetic GK rats, which suggests mice was also measured. The deficiency of GSTO1 expression in the that GSTO1 may play a role in the progressive deterioration of primary islets from the global-GSTO1-knockout mice was verified pancreatic b cell function in diabetic GK rats. by the western blot analysis results (Fig. 2C). Consistently, we found GSTO1 reportedly inhibits ryanodine receptor 2 (RyR2) and ac- that, upon high-glucose stimulation, the amount of insulin secreted tivates RyR1, which binds transiently to RyRs through its active site from the GSTO1-knockout islet cells was not significantly different cysteine residue (Cys 32) in a concentration-dependent manner [7]. than that secreted from the control islet cells, while low-glucose The RyR protein is a member of a family of ryanodine receptors that treatment significantly increased the amount of insulin secreted form channels to transport positively charged calcium ions within from the GSTO-knockout islet cells (Fig. 2D). cells. These channels are embedded in the outer membrane and act Because of the increase in the amount of insulin secreted upon as a sarcoplasmic reticulum, a storage center for calcium ions low-glucose treatment, the insulin content was measured in [8,29,30]. In excitable pancreatic b-cells, an increase in intracellular þ GSTO1-knockout INS-1 cells and primary islet cells. The results Ca2 concentration is the primary trigger for glucose-stimulated showed a significant increase in the insulin content in both the insulin secretion (GSIS) [31]. We speculated that GSTO1 might in- GSTO1-knockout INS-1 cells and the primary islet cells (Fig. 3A and fluence insulin secretion through ROS-stimulated RyR [32]. How- B). Furthermore, the mRNA expression levels of insulin were also ever, knocking out GSTO1 had no effect on 16.8 mM glucose- significantly upregulated in both the GSTO1-knockout INS-1 cells stimulated insulin secretion. Instead, both the insulin content and and primary islet cells (Fig. 3C and D). In contrast, GSTO1 over- insulin transcripts were increased in the GSTO1-knockout INS- expression significantly reduced the insulin content in the INS- 1 cells and primary mouse islet cells. PDX1 is a key insulin tran- 1 cells (Fig. 3E). These results indicate that GSTO1 is involved in the scriptional factor and plays an important role in the development regulation of insulin biosynthesis. and maturation of pancreatic b cells [33,34]. We found that knocking out GSTO1 increased PDX1 expression at both the mRNA 3.3. PDX1 expression is upregulated in pancreatic b cells in the and protein levels. These results suggest that GSTO1 may partici- absence of GSTO1 pate in the regulation of insulin biosynthesis by modulating PDX1 transcription. Pancreas and duodenum homeo-box1 (PDX1) is a well-known On the basis of these findings, we propose that GSTO1 is transcription factor of insulin and plays a critical role in the expressed at relatively low levels to maintain pancreatic b cell maturation and maintenance of pancreatic b cell identity [17e21]. function under physiological conditions. Oxidative stress induced To investigate whether the upregulation of insulin mRNA levels in by chronic hyperglycemia or free fatty acids upregulates GSTO1 the absence of GSTO1 is mediated by PDX1, the mRNA and protein expression, which in turn inhibits the biosynthesis of insulin expression levels of PDX1 were measured in the GSTO1-knockout through the downregulation of Pdx1 transcription and reduced ROS 942 L. Wang et al. / Biochemical and Biophysical Research Communications 524 (2020) 936e942 generation (Fig. 5). Our results suggest that GSTO1 may play an [12] D. Menon, et al., GSTO1-1 modulates metabolism in macrophages activated e important role in mediating redox signaling in pancreatic b cells. through the LPS and TLR4 pathway, J. Cell Sci. 128 (10) (2015) 1982 1990. [13] D. Menon, et al., GSTO1-1 plays a pro-inflammatory role in models of inflammation, colitis and obesity, Sci. Rep. 7 (1) (2017) 17832. Author contributions [14] Y.J. Park, M. 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