Pdia4 regulates β‐cell pathogenesis in diabetes: molecular mechanism and targeted therapy
2021; Springer Nature; Volume: 13; Issue: 10 Linguagem: Inglês
10.15252/emmm.201911668
ISSN1757-4684
AutoresTien‐Fen Kuo, Shuo‐Wen Hsu, Shou‐Hsien Huang, Cicero Lee‐Tian Chang, Ching‐Shan Feng, Mingguang Huang, Tzung‐Yan Chen, Meng‐Ting Yang, Si‐Tse Jiang, Tuan‐Nan Wen, C.Y. Yang, Chung‐Yu Huang, Shu‐Huei Kao, Keng‐Chang Tsai, Greta Luyuan Yang, Wen‐Chin Yang,
Tópico(s)Transgenic Plants and Applications
ResumoArticle20 September 2021Open Access Source DataTransparent process Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy Tien-Fen Kuo Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shuo-Wen Hsu Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shou-Hsien Huang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Search for more papers by this author Cicero Lee-Tian Chang orcid.org/0000-0001-5108-6594 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan Search for more papers by this author Ching-Shan Feng Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Search for more papers by this author Ming-Guang Huang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Search for more papers by this author Tzung-Yan Chen Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Search for more papers by this author Meng-Ting Yang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan Search for more papers by this author Si-Tse Jiang orcid.org/0000-0002-5145-258X National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan Search for more papers by this author Tuan-Nan Wen Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chun-Yen Yang orcid.org/0000-0002-8672-2981 Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chung-Yu Huang orcid.org/0000-0003-2909-3805 PhD Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Search for more papers by this author Shu-Huei Kao orcid.org/0000-0003-4618-0898 PhD Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Search for more papers by this author Keng-Chang Tsai orcid.org/0000-0001-8277-9174 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan Search for more papers by this author Greta Yang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Wen-Chin Yang Corresponding Author [email protected] orcid.org/0000-0001-6410-2581 Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan Search for more papers by this author Tien-Fen Kuo Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shuo-Wen Hsu Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shou-Hsien Huang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Search for more papers by this author Cicero Lee-Tian Chang orcid.org/0000-0001-5108-6594 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan Search for more papers by this author Ching-Shan Feng Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Search for more papers by this author Ming-Guang Huang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Search for more papers by this author Tzung-Yan Chen Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Search for more papers by this author Meng-Ting Yang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan Search for more papers by this author Si-Tse Jiang orcid.org/0000-0002-5145-258X National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan Search for more papers by this author Tuan-Nan Wen Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chun-Yen Yang orcid.org/0000-0002-8672-2981 Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Chung-Yu Huang orcid.org/0000-0003-2909-3805 PhD Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Search for more papers by this author Shu-Huei Kao orcid.org/0000-0003-4618-0898 PhD Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Search for more papers by this author Keng-Chang Tsai orcid.org/0000-0001-8277-9174 National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan Search for more papers by this author Greta Yang Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Search for more papers by this author Wen-Chin Yang Corresponding Author [email protected] orcid.org/0000-0001-6410-2581 Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan Search for more papers by this author Author Information Tien-Fen Kuo1, Shuo-Wen Hsu1, Shou-Hsien Huang1,2, Cicero Lee-Tian Chang3, Ching-Shan Feng4, Ming-Guang Huang1,5, Tzung-Yan Chen1,5, Meng-Ting Yang1,6,7,8, Si-Tse Jiang9, Tuan-Nan Wen10, Chun-Yen Yang1, Chung-Yu Huang11, Shu-Huei Kao11, Keng-Chang Tsai12, Greta Yang1 and Wen-Chin Yang *,1,2,4,5,7,8,13,14 1Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan 2Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan 3Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan 4Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan 5Institute of Biotechnology, National Taiwan University, Taipei, Taiwan 6Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan 7Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan 8Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan 9National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan 10Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 11PhD Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 12National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan 13Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan 14Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan *Corresponding author. Tel: +886-2-27872076, E-mail: [email protected] EMBO Mol Med (2021)13:e11668https://doi.org/10.15252/emmm.201911668 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 Loss of β-cell number and function is a hallmark of diabetes. β-cell preservation is emerging as a promising strategy to treat and reverse diabetes. Here, we first found that Pdia4 was primarily expressed in β-cells. This expression was up-regulated in β-cells and blood of mice in response to excess nutrients. Ablation of Pdia4 alleviated diabetes as shown by reduced islet destruction, blood glucose and HbA1c, reactive oxygen species (ROS), and increased insulin secretion in diabetic mice. Strikingly, this ablation alone or in combination with food reduction could fully reverse diabetes. Conversely, overexpression of Pdia4 had the opposite pathophysiological outcomes in the mice. In addition, Pdia4 positively regulated β-cell death, dysfunction, and ROS production. Mechanistic studies demonstrated that Pdia4 increased ROS content in β-cells via its action on the pathway of Ndufs3 and p22phox. Finally, we found that 2-β-D-glucopyranosyloxy1-hydroxytrideca 5,7,9,11-tetrayne (GHTT), a Pdia4 inhibitor, suppressed diabetic development in diabetic mice. These findings characterize Pdia4 as a crucial regulator of β-cell pathogenesis and diabetes, suggesting Pdia4 is a novel therapeutic and diagnostic target of diabetes. SYNOPSIS Pancreatic β-cell failure is associated with diabetes. Pdia4, a protein disulfide isomerase, is identified as a crucial regulator of β-cell pathogenesis and diabetes. Pdia4 interacts with Ndufs3 and p22phox and engages them in mitochondrial and cytosolic ROS production in β-cells. Pharmacological inhibition of Pdia4 disrupts the interaction of Pdia4 and its downstream partners, decreases ROS production, and ameliorates β-cell failure and diabetes. Paper explained Problem A gradual loss of functional pancreatic β-cells is a cardinal feature of type 2 diabetes. Understanding the cellular and molecular mechanisms of β-cell failure is important and may lead to improved diabetes treatment. Results Pdia4 was identified as a key protein for promoting ROS production and pathogenesis in β-cells during diabetes in Leprdb/db mice and HFD-fed B6 mice. Mechanistically, Pdia4 recruited its partners, Ndufs3 and p22phox, to increase ROS generation in the mitochondria and cytosol of β-cells, leading to β-cell failure and the development of diabetes. This recruitment involved interaction and stabilization of Ndufs3 and p22phox by interaction with Pdia4. Furthermore, the Pdia4 inhibitor abolished the interaction of Pdia4 with its partners and, consequently, reduced ROS production in β-cells and improved β-cell failure and diabetes symptoms in Leprdb/db mice. Both the genetics and pharmacological approaches demonstrated that targeting Pdia4 can preserve functional β-cells and ameliorate diabetes in mouse models. Impacts This work illustrates the novel role of the Pdia4/Ndufs3/p22phox cascade as a central regulator of ROS generation in β-cells and further establishes the new link between the Pdia4/Ndufs3/p22phox cascade, which orchestrates oxidative stress, β-cell failure and diabetes. Administration of a first-in-class Pdia4 inhibitor represents a feasible approach for treating β-cell failure during diabetes. The overall findings also highlight the potential of targeting Pdia4 to prevent β-cell loss and treat diabetes. Introduction Globally, 425 million people live with diabetes, which causes about 5 million deaths annually. Diabetes is characterized by a failure of functional β-cells to adapt insulin secretion to compensate for increasing insulin resistance, driving diabetes development (Cerf, 2013). Thus, pancreatic β-cell failure is central to diabetes development (Matthews et al, 1998; Donath & Halban, 2004; Harrity et al, 2006). Accordingly, accumulating data suggest that preserving a portion of functional β-cells can change the clinical outcome of diabetes (Defronzo, 2009; Leahy et al, 2010). However, none of the current anti-diabetic drugs is clinically effective for this preservation. Therefore, identification of the key players in β-cell dysfunction and death help us gain insight and understanding into β-cell pathogenesis and diabetes development and thus aids the development of new strategies for diabetes treatment (Ardestani et al, 2014; Ardestani & Maedler, 2016). The mechanism underlying the maintenance of β-cell number and function is extremely complex and is still poorly understood. During diabetes, endoplasmic reticulum (ER) stress, inflammation, and excess nutrients can induce aberrant reactive oxygen species (ROS) in β-cells and other cell types (Robertson et al, 2004; Robertson, 2004). Under normal physiological conditions, ROS are considered to be essential signaling molecules in β-cells and other cells (Trachootham et al, 2009). Nevertheless, during diabetes, exuberant ROS accumulation leads to β-cell dysfunction and death (Newsholme et al, 2012; Weaver et al, 2015) and peripheral insulin resistance (Evans et al, 2005) in animals and humans with diabetes. Autophagy, apoptosis, and necrosis are implicated in β-cell death (Nakamura et al, 2006; Quan et al, 2012). The mitochondrial electron transport chain (ETC) is thought to be a major machinery for ROS production in β-cells though NADPH oxidase (Nox), ER oxidoreductin 1 (Ero1), and certain pathways may also be implicated (Bindokas et al, 2003; Harrity et al, 2006; Leung & Leung, 2008). In contrast, ROS can be eliminated by antioxidant proteins such as glutathione peroxidase (Gpx), catalase, and superoxide dismutases (Sod). In particular, β-cells are more vulnerable to aberrant ROS than other cell types due to the low expression level of antioxidant proteins (Lenzen et al, 1996; Tiedge et al, 1997). Protein disulfide isomerases (Pdis) in mammals, including eight typical Pdis with CGHC motifs and 13 atypical Pdis with C/SXXC/S motifs, represent a family of multifunctional enzymes with oxidoreductase and chaperone activities (Maattanen et al, 2006). Most of the Pdis have an ER retention motif (Ni & Lee, 2007; Galligan & Petersen, 2012). However, more and more data show that in addition to being present in the cytosol (ER and other organelles), Pdis reside in the nuclei and membrane (Turano et al, 2002) and plasma of different cell types (https://www.proteinatlas.org/ENSG00000155660-Pdia4/cell). Thus, Pdis are thought to possess ER-relevant and ER-irrelevant localizations and functions such as other ER chaperones (Schultz-Norton et al, 2006; Xiong et al, 2012). This family is presumed to implement their functions via multiple mechanisms, e.g., the catalysis of disulfide bonds and conformational maintenance and regulation of their specific interaction partners and substrates (Maattanen et al, 2006; Schultz-Norton et al, 2006). More recently, Pdia1 has been characterized as a molecular chaperone to activate estrogen receptor via stabilizing the receptor (Schultz-Norton et al, 2006; Xiong et al, 2012). The role of Pdis in health and disease is poorly studied (Ni & Lee, 2007; Galligan & Petersen, 2012) though they might be implicated in infection (Naguleswaran et al, 2005; Ou & Silver, 2006), fertilization (Ellerman et al, 2006), coagulation (Manukyan et al, 2008), immunity (Garbi et al, 2006), tumors (Goplen et al, 2006), or cell viability/growth (Li & Lee, 1991; Severino et al, 2007). Emerging evidence obtained from yeast and worms suggests that the function of Pdis is not always redundant (Norgaard et al, 2001; Winter et al, 2007). Pdia4 is structurally unique because it is the largest member with three GCHC motifs in the family. Unlike Pdia3 (Garbi et al, 2006), Pdia4 is not an essential gene since its knockout mice were shown to survive without any noticeable phenotype (Almeida et al, 2011; Kuo et al, 2017). Its expression could be further induced by calcium flux (Li & Lee, 1991), ER stress (Li & Lee, 1991; Parker et al, 2001) and hypoxia (Pawar et al, 2011) in tumors. However, like other Pdis, nothing is known about the role of Pdia4 in diabetes, and its therapeutic potential and molecular basis in diabetes has not been deciphered. In this study, we first evaluated the expression level of Pdia4 in the pancreatic islets and sera in mice and humans. Next, mice with Pdia4 knockout and overexpression were generated to evaluate the impact of Pdia4 on β-cell pathogenesis and diabetes. In parallel, we elucidated the molecular mechanism of Pdia4 in ROS generation and β-cell pathogenesis. Finally, we identified 2-β-D-glucopyranosyloxy1-hydroxytrideca 5,7,9,11-tetrayne (GHTT) as a Pdia4 inhibitor, which was used to assess its anti-diabetic potential in diabetic mice. Results Up-regulation of Pdia4 protein in pancreatic islets and sera of mouse and human origin in response to excess nutrients To explore the likely role of Pdis in β-cells, we first compared the mRNA level of 8 typical Pdis (a1, a2, a3, a4, a5, a6, a13, and a15) and 2 atypical Pdis (a16 and a19) in Min6 cells, a mouse β-cell line, and mouse islets. The real-time polymerase chain reaction (RT–PCR) data showed that 4 Pdis (a1, a3, a4, and a6) had a higher transcriptional level than the rest in those cells (Appendix Fig S1A). In marked contrast, the protein level of Pdia4, but not Pdia1, Pdia3, or Pdia6, was up-regulated by glucose in Min6 cells (Appendix Fig S1B). This up-regulation was consistent with the presence of a putative ER stress responsive element (ERSE) in the Pdia4 promoter (Appendix Fig S1C). Further, high glucose increased the activity of Pdia4 promoter in Min6 cells (Appendix Fig S1D). Next, we investigated the expression pattern of Pdia4 in mouse tissues. We discovered that Pdia4 was expressed in mouse pancreata and islets to a greater extent than in liver, kidney, testis, and fat tissue (Fig 1A). Further, Pdia4 was expressed in β-cells but not α-cells of mouse islets (Fig 1B). However, we could not rule out its expression in other pancreatic cell types. Of note, this expression was up-regulated in response to a high dose of glucose (left, Fig 1C) and palmitate (right, Fig 1C) in Min6 cells. Likewise, Pdia4 was expressed in human islets and this expression was further up-regulated by excess nutrients (Fig 1D). Accordingly, the in vivo studies revealed that Pdia4 was expressed in pancreatic islets of wild-type (WT) control mice and this expression level was further elevated in pancreatic islets of diabetic Leprdb/db mice (Fig 1E). The up-regulation of Pdia4 in pancreatic islets correlated well with diabetes development in Leprdb/db mice and Lepob/ob mice (Fig 1F), two spontaneous mouse models of diabetes. Equally importantly, serum Pdia4 also went up with diabetes development in Leprdb/db mice, high-fat diet (HFD)-fed B6 mice, and diabetic patients (Fig 1G). Since Pdia4 was initially documented as an ER-resident protein with an ER retention motif, KEEL642–645, at its C-terminus (Ni & Lee, 2007; Galligan & Petersen, 2012), we thus examined the subcellular distribution of Pdia4 in Min6 β-cells. Surprisingly, the immunoblotting data indicated that Pdia4 was distributed in the nuclei, cytosol, membrane, mitochondria, and ER of Min6 cells (Fig 1H). Consistently, mass spectroscopy (MS) data also confirmed that despite its KEEL motif, Pdia4 resided in the aforesaid compartments of Min6 β-cells and mouse serum (Appendix Fig S1E). Figure 1. Expression and distribution of Pdia4 in pancreatic islets and/or other tissues A. Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. B. Confocal analysis of Pdia4 and insulin (Ins) or glucagon (Gcg) in mouse islets. The tissues were stained with the indicated antibodies and analyzed using a confocal microscope. Scale bar = 100 μm. C. Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. D. Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis. E. The islets of WT BKS and diabetic Leprdb/db mice underwent immunoblotting analysis. F. IHC analysis of the islets from Leprdb/db and Lepob/ob mice at the indicated ages using anti-Pdia4 antibody and hematoxylin. Scale bar = 100 μm. The dash circles indicate islet regions. G. Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high-fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit. H. Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies. Data information: Data from 3 experiments (A–E, F and H) and more (G) are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. The number of mice (n) is indicated in parentheses. Source data are available online for this figure. Source Data for Figure 1 [emmm201911668-sup-0002-SDataFig1.zip] Download figure Download PowerPoint Overall, the data showed that Pdia4 was expressed in the pancreas and was distributed in different cell compartments. The remainder of the study concentrated on the investigation of Pdia4 in β-cell pathogenesis and diabetes. Reduction of ROS, HbA1c, islet atrophy, and islet cell death, and increase in β-cell function and longevity in Pdia4-deficient mice The fact that the Pdia4 expression increased with diabetes development in mice and humans prompted us to examine whether it could trigger β-cell pathogenesis and diabetes. First, conventional Pdia4 knockout mice (Pdia4−/− B6) were bred as published (Almeida et al, 2011; Kuo et al, 2017). The mice were bred into BKS (Pdia4−/− BKS) and, subsequently, Leprdb/db backgrounds (Pdia4−/− Leprdb/db) in Appendix Fig S2A. WT and Pdia4−/− mice on B6 (Almeida et al, 2011; Kuo et al, 2017) and BKS backgrounds (Appendix Fig S2) were diabetes-free. As expected, Leprdb/db mice spontaneously developed diabetes by 8 weeks of age and this diabetes became more severe with age as evidenced by fasting blood glucose (FBG, Appendix Fig S2A), postprandial blood glucose (PBG, Appendix Fig S2A), the percentage of glycosylated hemoglobin A1c (HbA1c, Appendix Fig S2B), glucose tolerance (Appendix Fig S2C), homeostatic model assessment (HOMA) indices (Appendix Fig S2D), and diabetic incidence (Appendix Fig S2E). In sharp contrast, Pdia4−/−Leprdb/db mice developed borderline diabetes with average FBG and PBG of around 109 and 289 mg/dl at 24 weeks of age, respectively (Appendix Fig S2A). Closer analysis revealed that 58% of the Pdia4−/−Leprdb/db mice whose PBG was 137 mg/dl by 24 weeks of age, were diabetes-free (Pdia4−/−Leprdb/db, Appendix Fig S2E) compared to the rest (42%) which exhibited slight diabetes with PBG around 340 mg/dl (Pdia4−/−Leprdb/db, Appendix Fig S2E). The diabetes-free and diabetic mice had daily food intake of 6.8 and 7.9 g, respectively. Regression analysis indicated a strong correlation between PBG and food intake in Pdia4−/−Leprdb/db mice (R2 = 0.98, Appendix Fig S2F). Consistently, Pdia4−/−Leprdb/db mice, given 6.8 g feed/day/mouse from 4 to 24 weeks of age, showed completely arrested diabetes development (Pdia4−/−Leprdb/db + FR, Appendix Fig S2E). In contrast, a ROS scavenger, vitamin C, at 375 mg/kg failed to affect diabetes development in Leprdb/db mice (Leprdb/db + VitC, Appendix Fig S2E). As published (Brem et al, 2007), Leprdb/db mice showed decreased food intake and PBG with age over a period of 90 weeks (Appendix Fig S3A and B). However, Pdia4−/−Leprdb/db mice, aged 55 weeks and over, had much lower PBG and HbA1c than the age-matched Leprdb/db mice (Appendix Fig S3B and C). Of note, Pdia4−/−Leprdb/db mice showed more effective amelioration of diabetes than the age-matched Leprdb/db mice as shown by water consumption (right, Appendix Fig S3A), PBG (Appendix Fig S3B), glucose tolerance (Appendix Fig S3D), islet preservation (Appendix Fig S3E), survival rate (top, Fig 2A), and life span (bottom, Fig 2A). No significant difference in food intake was observed between Leprdb/db and Pdia4−/−Leprdb/db mice, from 8 to 90 weeks (left, Appendix Fig S3A). Strikingly, all the Pdia4−/−Leprdb/db mice became diabetes-free by the age of 55 weeks and beyond, which correlated with decreased food intake in aged mice (Appendix Fig S3). Figure 2. Pdia4 ablation increases survival rate and longevity and reduces islet atrophy, ROS production, and islet cell death, elevates serum insulin, and potentiates β-cell GSIS in Pdia4−/−Leprdb/db mice A. Survival rate and life span of WT, Pdia4−/− (KO), and Pdia4tg/tg (TG) mice on Leprdb/db background from birth to 120 weeks (Appendix Figs S2A and S6A). B. Pancreata of 18-week-old WT and Pdia4−/− (KO) mice on BKS or Leprdb/db background were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. C. Serum ROS of the 18-week-old mice (B) were determined. D. The same batch mice as in (B) were given water containing BrdU. The BrdU+ cells of the islets were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the islets of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per islet area (0.01 mm2) and TUNEL+ cells per islet area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate islet regions, and the black arrowheads indicate TUNEL+ cells. E, F. ELISA kits were used to quantify serum insulin (E) of the same mice as in (B) and their supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3) was used in GSIS assays. Data information: The number of mice (n) is indicated in parentheses. Data from over three experiments are presented as the mean ± SD. Log rank (A) and one-way ANOVA test (B–F) were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. Source data are available online for this figure. Source Data for Figure 2 [emmm201911668-sup-0003-SDataFig2.zip] Download figure Download PowerPoint We also examined the structure and function of pancreatic islets in WT and Pdia4−/−mice. Islet size in the four mouse lines aged 18 weeks was in the following ascending order: Leprdb/db mice < WT BKS mice = Pdia4−/− BKS mice < Pdia4−/−Leprdb/db mice (αIns, Fig 2B). Also of note, the degree of islet atrophy in both mouse backgrounds correlated with ROS accumulation in islets (dihydroethidium (DHE), Fig 2B) and blood (Fig 2C). To investigate the role of Pdia4 in β-cell proliferation and demise, we checked cell proliferation and death of mouse islets using 5-bromo-2-deoxyuridine (BrdU) labeling assays and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), respectively. BrdU data showed no significant difference in cell proliferation of pancreatic islets between WT and Pdia4−/− mice on BKS and Leprdb/db backgrounds irrespective of Pdia4 content (left, Fig 2D). In sharp contrast, Leprdb/db mice had more TUNEL-positive islet cells, dead cells, and living cells undergoing proliferation and repair, than Pdia4−/−Leprdb/db mice, WT, and Pdia4−/− BKS mice (right, Fig 2D). Both types of assays suggest that Pdia4 is inversely correlated to cell death in mouse islets. Furthermore, we generated islet-specific Pdia4 knockout mice (Pdia4f/fLeprdb/dbCretg/0) to verify the islet-specific function of Pdia4 in islets and diabetes (Appendix Fig S4). The conditional Pdia4 knockout mice (Appendix Fig S4) had similar pre-clinical parameters to the conventional Pdia4 knockout mice (Fig 2 and Appendix Fig S2) in terms of FBG, PBG, HbA1c, serum insulin, islet atrophy, ROS, HOMA indices, and glucose tolerance. Moreover, Pdia4−/− mice had better β-cell function than WT mice on BKS and Leprdb/db backgrounds as evidenced by HOMA-β (Appendix Fig S2D), serum insulin (Fig 2E), and glucose-stimulated insulin secretion (GSIS) (Fig 2F). At 8 weeks of age, Leprdb/db and Pdia4−/−Leprdb/db mice had a high but similar
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