Menin‐regulated Pbk controls high fat diet‐induced compensatory beta cell proliferation

Article6 April 2021Open Access Source DataTransparent process Menin-regulated Pbk controls high fat diet-induced compensatory beta cell proliferation Jian Ma orcid.org/0000-0001-8223-8327 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Bowen Xing Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Yan Cao Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Xin He orcid.org/0000-0002-2131-2092 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Kate E Bennett Division of Gastroenterology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Chao Tong Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Chiying An Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Taylor Hojnacki Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Zijie Feng Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Sunbin Deng Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Sunbin Ling Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Gengchen Xie Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Yuan Wu Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Yue Ren Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Ming Yu Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Bryson W Katona Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Division of Gastroenterology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Hongzhe Li Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Ali Naji Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Xianxin Hua Corresponding Author [email protected] orcid.org/0000-0003-4862-4691 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Jian Ma orcid.org/0000-0001-8223-8327 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Bowen Xing Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Yan Cao Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Xin He orcid.org/0000-0002-2131-2092 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Kate E Bennett Division of Gastroenterology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Chao Tong Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Chiying An Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Taylor Hojnacki Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Zijie Feng Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Sunbin Deng Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Sunbin Ling Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Gengchen Xie Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Yuan Wu Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Yue Ren Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Ming Yu Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Bryson W Katona Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Division of Gastroenterology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Hongzhe Li Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Ali Naji Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Xianxin Hua Corresponding Author [email protected] orcid.org/0000-0003-4862-4691 Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Search for more papers by this author Author Information Jian Ma1,2, Bowen Xing1, Yan Cao1,2, Xin He1, Kate E Bennett3, Chao Tong4, Chiying An1,2, Taylor Hojnacki1, Zijie Feng1, Sunbin Deng5, Sunbin Ling1, Gengchen Xie1, Yuan Wu1, Yue Ren6, Ming Yu2, Bryson W Katona1,3, Hongzhe Li6, Ali Naji2 and Xianxin Hua *,1,2 1Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 2Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 3Division of Gastroenterology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 4Department of Biology, University of Pennsylvania, Philadelphia, PA, USA 5Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA 6Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA *Corresponding author. Tel: +1 215 746 5565; E-mail: [email protected] EMBO Mol Med (2021)13:e13524https://doi.org/10.15252/emmm.202013524 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pancreatic beta cells undergo compensatory proliferation in the early phase of type 2 diabetes. While pathways such as FoxM1 are involved in regulating compensatory beta cell proliferation, given the lack of therapeutics effectively targeting beta cell proliferation, other targetable pathways need to be identified. Herein, we show that Pbk, a serine/threonine protein kinase, is essential for high fat diet (HFD)-induced beta cell proliferation in vivo using a Pbk kinase deficiency knock-in mouse model. Mechanistically, JunD recruits menin and HDAC3 complex to the Pbk promoter to reduce histone H3 acetylation, leading to epigenetic repression of Pbk expression. Moreover, menin inhibitor (MI) disrupts the menin–JunD interaction and augments Pbk transcription. Importantly, MI administration increases beta cell proliferation, ameliorating hyperglycemia, and impaired glucose tolerance (IGT) in HFD-induced diabetic mice. Notably, Pbk is required for the MI-induced beta cell proliferation and improvement of IGT. Together, these results demonstrate the repressive role of the menin/JunD/Pbk axis in regulating HFD-induced compensatory beta cell proliferation and pharmacologically regulating this axis may serve as a novel strategy for type 2 diabetes therapy. Synopsis Pancreatic beta cells undergo compensatory proliferation in the early phase of type 2 diabetes. Understanding the mechanism and regulation of compensatory beta cell proliferation may allow for improved treatment options for diabetes. Herein we elucidated that the menin/JunD/Pbk axis is important in compensatory beta-cell proliferation. Pbk is crucial for regulating compensatory pancreatic beta cell proliferation of high fat diet (HFD) fed mice. Menin and HDAC3 complex were recruited by JunD to epigenetically repress Pbk expression. Menin-JunD interaction was interrupted by small molecule menin inhibitors (MIs), leading to upregulating of Pbk gene expression, beta cell proliferation, and improved glucose tolerance in diet-induced obese and diabetic mice. Pbk is required for MI-induced beta cell proliferation and improved glucose tolerance in HFD-induced diabetic mice. The paper explained Problem An insufficient number of functional pancreatic beta cells is a central feature of both type 1 and advanced type 2 diabetes. As such, there has been considerable interest in exploring strategies for inducing beta cell regeneration as a novel mechanism for diabetes treatment. Pancreatic beta cells undergo compensatory proliferation in the early phase of obesity or high fat diet (HFD)-induced type 2 diabetes (T2D), but is underexplored. Understanding the underlying mechanism of compensatory beta cell proliferation as well as the regulation of this proliferation is crucial and may lead to improved treatments for diabetes. Results Pbk was identified as a key protein for mediating HFD-induced compensatory beta cell proliferation using a Pbk kinase dysfunctional mouse model. Mechanistically, the transcription factor JunD recruits menin and HDAC3 complex to the Pbk promoter to reduce histone H3 acetylation, leading to epigenetic repression of Pbk expression. Pharmacologically blocking the menin–JunD interaction menin inhibitors (MIs) increased compensatory beta cell proliferation, resulting in both improved hyperglycemia and glucose tolerance in HFD-induced diabetic mice, demonstrating the key impact of MIs on influencing expression of Pbk and beta cell proliferation in mouse models. Impacts The current studies illustrate the novel role of the menin/JunD/Pbk axis as a key regulator of compensatory beta cell proliferation, unraveling a novel approach to enhancing beta cell proliferation by regulating the menin pathway through administering MIs. These novel findings highlight the potential to further develop MIs to increase Pbk expression and beta cell regeneration, which is a novel mechanism of action for developing future islet function-enhancing drugs for improving treatment of diabetes. Introduction Beta cells in pancreatic islets undergo compensatory proliferation in the early phase of obesity and high fat diet (HFD)-induced type 2 diabetes (T2D); however, long-term stress in beta cells induced by these conditions eventually results in beta cell death and a reduced number of functional beta cells (Finegood et al, 2001; Hanley et al, 2010; Saisho et al, 2010; Linnemann et al, 2014; Wang et al, 2015). As such, there has been considerable interest in understanding how compensatory beta cell proliferation is regulated, and how the underlying mechanism can be further explored for improving diabetes treatment (Kulkarni et al, 2004; Linnemann et al, 2014; El Ouaamari et al, 2016). A number of factors such as transcription factor FoxM1, incretin hormone GLP-1, liver-derived protease inhibitor serpinB1, RNA splicing processing protein Argonaute2, and others are involved in the compensatory beta cell proliferation (Baggio & Drucker, 2007; Dai et al, 2017; El Ouaamari et al, 2016; Kawamori et al, 2017; Ning et al, 2006; Tattikota et al, 2014; Yamamoto et al, 2017), but other unknown pathways that regulate this process remain to be identified. Pbk, a serine/threonine protein kinase, facilitates cell cycle control and mitotic progression (Abe et al, 2000; Gaudet et al, 2000; Fujibuchi et al, 2005). Pbk mRNA is detected in limited human tissue types and is most abundant in testis, placenta, thymus, and activated lymphoid cells (Abe et al, 2000; Zhu et al, 2007). Normally, Pbk expression is suppressed in non-transformed cells from differentiated tissues including pancreatic beta cells (Ayllon & O'Connor, 2007; Joel et al, 2015; Herbert et al, 2018). Furthermore, it remains unclear whether Pbk plays any role in beta cell proliferation. Menin, which is encoded by the MEN1 gene, is an established key regulator of beta cell mass, as ablation of the Men1 gene leads to increased beta cell proliferation in mice (Crabtree et al, 2001; Bertolino et al, 2003; Schnepp et al, 2006; Karnik et al, 2007). Men1 gene deletion also reverses pre-existing hyperglycemia in high fat diet (HFD)-induced diabetic mice as well as in obese (db/db) mice (Schnepp et al, 2006; Yang et al, 2010a; Yang et al, 2010b) and ameliorates gestational diabetes in pregnant mice (Karnik et al, 2007). Menin interacts with various protein partners and regulates beta cell homeostasis via multiple pathways including regulation of gene transcription and cell proliferation (Agarwal et al, 1999; Jin et al, 2003; Hughes et al, 2004; Milne et al, 2005; Grembecka et al, 2012; Gurung et al, 2013; Matkar et al, 2013). Among the different interacting partners, the menin–MLL interaction plays a suppressing role in islet cells by driving expression of cell cycle inhibitors p27 and p18 (Karnik et al, 2005; Milne et al, 2005). In addition, menin also binds JunD, an AP-1 family transcription factor (Agarwal et al, 1999; Kim et al, 2003). X-ray crystallographic studies showed that menin's structure harbors a deep central pocket that binds MLL or JunD (Huang et al, 2012; Shi et al, 2012). Small molecule menin inhibitors (MIs), including MI-463 and MI-503, were developed to target this pocket and block the menin–MLL interaction, and MIs are effective at suppressing MLL-fusion protein-induced leukemia in vivo (Borkin et al, 2015). Pbk is upregulated in Men1-excised islets in mouse models (Yang et al, 2010a) and in human pancreatic neuroendocrine tumor (PNET) tissues with MEN1 loss-of-function mutations (Jiang et al, 2014). These findings prompted us to investigate whether menin regulates Pbk expression as well as the mechanism of this regulation and also whether Pbk is crucial for the beta cell compensatory proliferation induced by stressors such as HFD. Herein, for the first time we demonstrated the crucial role of Pbk in regulating HFD-induced beta cell compensatory proliferation and uncovered the repressive function of the menin/JunD/Pbk axis in regulating compensatory beta cell proliferation. We showed that administration of MI, which can interrupt the menin/JunD/Pbk axis, upregulates Pbk expression, improves compensatory beta cell proliferation, and potently improves hyperglycemia in HFD-induced diabetic mice. These studies may accelerate the development of new types of diabetes therapies directed at promoting Pbk-dependent beta cell proliferation. Results Pbk is upregulated in islets of mice with high fat diet (HFD)-induced obesity and crucial for beta cell proliferation Recent gene expression profile assays in pancreatic beta cells or islets from HFD rodent models show increased Pbk expression, opening a question about the possible role of enhanced Pbk expression in regulating HFD-induced pancreatic beta cell compensatory proliferation. Analysis of Pbk expression profile in normal human and mouse tissues shows that Pbk protein/mRNA expression is mainly restricted to bone marrow, testis, and the gastrointestinal tract (Appendix Fig S1A and B). Moreover, further analysis of the mRNA expression profile of mice on HFD vs control mice (Malpique et al, 2014; Dusaulcy et al, 2019) indicates that Pbk is upregulated in the islets of mice on HFD (Fig 1A and Appendix Fig S1C). To confirm these findings, we performed qRT–PCR with the isolated islets from the C57BL/6 mice on HFD or chow diet, showing that Pbk mRNA level was higher in islets from HFD-induced obese mice than that from the control lean mice. In contrast, Pbk mRNA was not significantly increased in other metabolically active tissues such as fat and liver, and was even downregulated in muscle (Fig 1B). Figure 1. Pbk kinase expression is upregulated in pancreatic beta cells of HFD-induced diabetic mice with beta cell compensatory proliferation Volcano plot showing the fold change (y-axis) versus adjusted (adj.) P value (x-axis) of the beta cell transcriptomes between chow and HFD-fed mice (16 weeks). Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj. P value < 0.01 (two-tailed paired Student's t-test). Genes with > 10-fold upregulated expression are labeled with their name. Pbk gene expression is indicated by the arrow. RNA-seq data were available from Dusaulcy et al 2019 (https://doi.org/10.1371/journal.pone.0213299.t004). qPCR for detecting Pbk mRNA level in different metabolically active organs from DIO and control mice (n = 3 per group). **P = 0.0029 (islets), ***P = 0.0007 (muscle) (two-tailed unpaired Student's t-test). ns, not statistically significant difference. PBK mRNA level comparison in human islets from obese (BMI > 30) or lean donors (BMI < 25) (n = 3 per group). *P = 0.0261 (two-tailed unpaired Student's t-test). Representative images of insulin (green) and Pbk (red, indicated by white arrow) double immuno-staining in pancreas of DIO mice with 12 weeks HFD feeding (60% fat diet, n = 4) or age-matched control mice (10% fat diet, n = 4). Nuclei were labeled by DAPI (blue). Islet area was circled with white dashed line. Scale bar: 50 μm. Quantification of the percentage of Pbk-positive beta cells. Four mice for each group, and 10 islet images per mouse were analyzed. *P = 0.0150 (two-tailed unpaired Student's t-test). WB data showed an upregulation of Pbk expression level in islets of mice on HFD compared with that of control lean mice. Beta cell mass comparison between DIO and control mice (n = 4 per group). *P = 0.0419 (two-tailed unpaired Student's t-test). Representative images of insulin (green) and BrdU (red, indicated by white arrow) double immuno-staining in pancreas of DIO mice with 12 weeks HFD feeding (60% fat diet, n = 4) or age-matched control mice (10% fat diet, n = 4). Nuclei were labeled by DAPI (blue). Islet area was circled with white dashed line. Scale bar: 50 μm. Quantification of the percentage of BrdU positive-β-cells. Four mice for each group, and 5–10 islet images per mouse were analyzed. *P = 0.0230 (two-tailed unpaired Student's t-test). The representative image of co-staining of Pbk and BrdU in pancreatic sections. Islet area was circled with white dashed line. Pbk and BrdU co-staining are denoted by white arrows. Scale bar: 50 μm. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean value of four mice from 5 sections for each mouse were presented. Pbk KD in INS-1 cells suppresses cell growth. Western blot data showed the decreased Pbk expression level in INS-1 cells with Pbk-targeted shRNA transduction. Cell growth curve was from three independent experiments (n = 3). ***P < 0.0001 (Vector:shRNA-#1), *P = 0.0181 (Vector:shRNA-#5), (Two-way ANOVA). The role of ectopic expression of Pbk-WT and Pbk-Mut on INS-1 cell growth. Western blot data showed the overexpression of V5-flaged wild-type and mutant Pbk in INS-1 cells. Cell growth curve was from three independent experiments (n = 3). **P = 0.0069 (Two-way ANOVA). ns, not statistically significant difference. Cell cycle analysis via PI staining followed by flow cytometry for the cells with Pbk overexpression. PIME cells steadily transduced with vector, Pbk-WT, and Pbk-Mut expression plasmids. Purified recombinant His-Erk2 protein incubated with the purified PBK in the presence or absence of ATP (100 μM) or menin inhibitor (1 μM or 0.1 μM), as indicated. After SDS–PAGE, the phosphorylation of Erk2 was detected with the pERK1/2 antibody. Anti-ERK1/2 antibody showed that equal amount of Erk2 was used for the kinase assay. Purified recombinant His-Erk2 proteins were incubated with IP-ed Pbk from PIME cells (WT or menin-/-) for the kinase activity assay. The phosphorylation of Erk2 was detected with the anti-pERK1/2 antibody. The Western blot incubated with the anti-ERK1/2 antibody showed that an equal amount of Erk2 was loaded for the kinase assay. Data information: Data are represented as mean ± SEM. Download figure Download PowerPoint Moreover, increased PBK expression was also identified in human islets from obese donors (BMI > 30) as compared to the lean donors (BMI < 25) (Fig 1C, Appendix Table S1). Upregulated Pbk protein was confirmed via detecting an increased number of Pbk/insulin co-stained beta cells from the HFD-induced obese mice (Fig 1D and E) and an increased Pbk protein expression in isolated islet samples of HFD-induced obese mice relative to the control lean mice (Fig 1F). Consistent with increased Pbk expression, beta cell mass and BrdU uptake in beta cells of HFD-induced obese mice were also increased (Fig 1G–I). Further, Pbk and BrdU co-localized, with approximately 70% of Pbk-positive cells also showing BrdU positivity, indicating Pbk-mediated cell proliferation is cell-autonomous (Fig 1J and K). Together, these results support that HFD upregulates Pbk expression selectively in islets of mice, correlating with enhanced beta cell proliferation. Next, to elucidate the relationship between upregulated Pbk expression and beta cell proliferation, we showed that shRNA-mediated Pbk knockdown (KD) suppressed growth of INS-1 cells, a rat insulinoma-derived cell line (Fig 1L), whereas ectopic expression of Pbk promoted INS-1 cell growth (Fig 1M). Notably, a K64K65 to AA mutation in Pbk, which deactivates catalytic activity of Pbk (Gaudet et al, 2000), does not promote INS-1 cell growth as seen with wild-type Pbk (Fig 1M). To further determine whether Pbk expression is crucial for cell proliferation, we treated Pbk overexpressing and control PIME cells, a mouse islet-derived cell line with an inducible floxed Men1 gene (Appendix Fig S2), with Pbk-specific inhibitor OTS514 (Ikeda et al, 2016). The results showed that ectopic expression of Pbk increased the sensitivity of PIME cells to the Pbk inhibitor (Fig EV1A). Furthermore, upregulated Pbk expression drove more cells into G2/M phase (Fig 1N), increased phosphorylation of Erk1/2 and JunD, and upregulated CcnB1 expression (Fig EV1B and C). Consistently, the Pbk-mutant cells had fewer cells in G2/M phase (Fig 1N). Taken together, these results indicate that Pbk kinase activity plays a crucial role in promoting beta cell proliferation. Click here to expand this figure. Figure EV1. Ectopic expression of Pbk activates cell growth-related intracellular signaling pathways A. Pbk overexpression PIME cells and control cells were treated by OTS 514 with different concentrations for 72 h. The cell growth was assessed by MTS assay. Five independent replicates (n = 5). Data are represented as mean ± SEM. ***P = 0.0004 (Two-way ANOVA). B, C. The effect of Pbk overexpression on phosphorylation levels of JNK, P38, ERK1/2, and JunD, as well as total protein level of CcnB1 in INS-1 cells (B) and PIME cells (C). Download figure Download PowerPoint To explore the substrate of Pbk kinase, we performed the in vitro kinases assay with purified Pbk and Erk2, both with or without ATP. The results show that incubation of Pbk with Erk2 in the presence of ATP indeed induced Erk2 phosphorylation (Fig 1O, lane 3). In contrast and as expected, removal of either Pbk or ATP from the assay abolished Erk2 phosphorylation (Fig 1O, lane 1 and 2, respectively). On the other hand, addition of various concentrations of menin inhibitor (MI) (Borkin et al, 2015), which was used to induce menin-dependent Pbk upregulation in the subsequent in vitro or in vivo experiments, did not affect the Pbk-mediated Erk2 phosphorylation (Fig 1O, lanes 4–5), indicating MI does not physically impact Pbk kinase activity. As a control, the amount of purified Erk2 was comparable among each of the reactions (Fig 1O, lanes 1–5, lower panel). In addition, we further examined in cells whether upregulated Pbk could also lead to improved kinase activity. To this end, we immunoprecipitated (IP-ed) Pbk from wild-type PIME cells and menin KO PIME cells. First, we confirmed the upregulation of Pbk expression in menin KO PIME cells compared with the control PIME cells (Fig 1P, Input Pbk). Then, we IP-ed Pbk from the same amount of PIME cell lysates (with or without menin) and performed in vitro kinase assays showing that larger amounts of Pbk induced higher level of Erk2 phosphorylation (Fig 1P). Together, these results indicate that Pbk can phosphorylate Erk2. Generation of Pbk kinase-inactivated mice and the essential role of Pbk in maintaining normal glucose tolerance To investigate the in vivo role of Pbk in regulating HFD-induced beta cell proliferation, we generated Pbk kinase inactivation mutant Knockin (KI) mice by introducing KI of Pbk with a K64K65→AA mutation (referred to as PbkKI/KI hereafter) (Fig 2A and B). Homozygous PbkKI/KI mice were identified using restriction fragment length polymorphism (RFLP) analysis as well as DNA sequencing (Fig 2B–D). Figure 2. Pbk kinase activity is crucial for HFD-induced beta cell compensatory proliferation A. A diagram for generation of Pbk K64K65 → AA KI mice using CRISPR/Cas9 system. B. Target region in exon5 of murine Pbk locus. Mutant nucleotides for inducing KK → AA mutation were highlighted with blue. Two mismatched bases with silence mutations that generate a novel Mwol site are labeled in yellow. The amplicon and enzyme cutting size are indicated. C. PCR genotyping of tail genomic DNA for PbkKI/KI mice using RFLP. Genomic fragments from exon 5 were amplified by PCR and digested with Mwol. D. DNA sequence confirming the mutation from K64K65 to AA in PbkKI/KI mice from the tail genomic DNA compared with PbkWT/WT mice. E. IPGTT (glucose at 2 g/kg of body weight, i.p.) on 11-week-old male PbkKI/KI mice and PbkWT/WT mice (n = 4 for each group). All mice were starved overnight (16 h) before testing. F. Islet number in each pancreas section on 11-week-old male PbkKI/KI and PbkWT/WT mice (n = 4 for each group). *P = 0.0443 (two-tailed unpaired S