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Webster KL, Mirmira RG. Beta cell dedifferentiation in type 1 diabetes: sacrificing function for survival? Front Endocrinol (Lausanne) 2024; 15:1427723. [PMID: 38904049 PMCID: PMC11187278 DOI: 10.3389/fendo.2024.1427723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/04/2024] [Accepted: 05/27/2024] [Indexed: 06/22/2024] Open
Abstract
The pathogeneses of type 1 and type 2 diabetes involve the progressive loss of functional beta cell mass, primarily attributed to cellular demise and/or dedifferentiation. While the scientific community has devoted significant attention to unraveling beta cell dedifferentiation in type 2 diabetes, its significance in type 1 diabetes remains relatively unexplored. This perspective article critically analyzes the existing evidence for beta cell dedifferentiation in type 1 diabetes, emphasizing its potential to reduce beta cell autoimmunity. Drawing from recent advancements in both human studies and animal models, we present beta cell identity as a promising target for managing type 1 diabetes. We posit that a better understanding of the mechanisms of beta cell dedifferentiation in type 1 diabetes is key to pioneering interventions that balance beta cell function and immunogenicity.
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Affiliation(s)
| | - Raghavendra G. Mirmira
- Kovler Diabetes Center and the Department of Medicine, The University of Chicago, Chicago, IL, United States
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2
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Jiang H, Jiang FX. Human pluripotent stem cell-derived β cells: Truly immature islet β cells for type 1 diabetes therapy? World J Stem Cells 2023; 15:182-195. [PMID: 37180999 PMCID: PMC10173812 DOI: 10.4252/wjsc.v15.i4.182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 01/30/2023] [Accepted: 03/20/2023] [Indexed: 04/26/2023] Open
Abstract
A century has passed since the Nobel Prize winning discovery of insulin, which still remains the mainstay treatment for type 1 diabetes mellitus (T1DM) to this day. True to the words of its discoverer Sir Frederick Banting, “insulin is not a cure for diabetes, it is a treatment”, millions of people with T1DM are dependent on daily insulin medications for life. Clinical donor islet transplantation has proven that T1DM is curable, however due to profound shortages of donor islets, it is not a mainstream treatment option for T1DM. Human pluripotent stem cell derived insulin-secreting cells, pervasively known as stem cell-derived β cells (SC-β cells), are a promising alternative source and have the potential to become a T1DM treatment through cell replacement therapy. Here we briefly review how islet β cells develop and mature in vivo and several types of reported SC-β cells produced using different ex vivo protocols in the last decade. Although some markers of maturation were expressed and glucose stimulated insulin secretion was shown, the SC-β cells have not been directly compared to their in vivo counterparts, generally have limited glucose response, and are not yet fully matured. Due to the presence of extra-pancreatic insulin-expressing cells, and ethical and technological issues, further clarification of the true nature of these SC-β cells is required.
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Affiliation(s)
- Helen Jiang
- Sir Charles Gairdner Hospital, University of Western Australia, Perth 6009, Australia
| | - Fang-Xu Jiang
- School of Biomedical Sciences, University of Western Australia, Perth 6009, Australia
- School of Health and Medical Sciences, Edith Cowan University, Perth 6027, Australia
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3
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Zhang Y, Fang X, Wei J, Miao R, Wu H, Ma K, Tian J. PDX-1: A Promising Therapeutic Target to Reverse Diabetes. Biomolecules 2022; 12:1785. [PMID: 36551213 PMCID: PMC9775243 DOI: 10.3390/biom12121785] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Revised: 11/18/2022] [Accepted: 11/19/2022] [Indexed: 12/02/2022] Open
Abstract
The pancreatic duodenum homeobox-1 (PDX-1) is a transcription factor encoded by a Hox-like homeodomain gene that plays a crucial role in pancreatic development, β-cell differentiation, and the maintenance of mature β-cell functions. Research on the relationship between PDX-1 and diabetes has gained much attention because of the increasing prevalence of diabetes melitus (DM). Recent studies have shown that the overexpression of PDX-1 regulates pancreatic development and promotes β-cell differentiation and insulin secretion. It also plays a vital role in cell remodeling, gene editing, and drug development. Conversely, the absence of PDX-1 increases susceptibility to DM. Therefore, in this review, we summarized the role of PDX-1 in pancreatic development and the pathogenesis of DM. A better understanding of PDX-1 will deepen our knowledge of the pathophysiology of DM and provide a scientific basis for exploring PDX-1 as a potential target for treating diabetes.
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Affiliation(s)
- Yanjiao Zhang
- Institute of Metabolic Diseases, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
| | - Xinyi Fang
- Institute of Metabolic Diseases, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
- Graduate College, Beijing University of Chinese Medicine, Beijing 100029, China
| | - Jiahua Wei
- Graduate College, Changchun University of Chinese Medicine, Changchun 130117, China
| | - Runyu Miao
- Institute of Metabolic Diseases, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
- Graduate College, Beijing University of Chinese Medicine, Beijing 100029, China
| | - Haoran Wu
- Graduate College, Beijing University of Chinese Medicine, Beijing 100029, China
| | - Kaile Ma
- Institute of Metabolic Diseases, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
| | - Jiaxing Tian
- Institute of Metabolic Diseases, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China
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Role of the Transcription Factor MAFA in the Maintenance of Pancreatic β-Cells. Int J Mol Sci 2022; 23:ijms23094478. [PMID: 35562869 PMCID: PMC9101179 DOI: 10.3390/ijms23094478] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Revised: 04/16/2022] [Accepted: 04/17/2022] [Indexed: 02/04/2023] Open
Abstract
Pancreatic β-cells are specialized to properly regulate blood glucose. Maintenance of the mature β-cell phenotype is critical for glucose metabolism, and β-cell failure results in diabetes mellitus. Recent studies provide strong evidence that the mature phenotype of β-cells is maintained by several transcription factors. These factors are also required for β-cell differentiation from endocrine precursors or maturation from immature β-cells during pancreatic development. Because the reduction or loss of these factors leads to β-cell failure and diabetes, inducing the upregulation or inhibiting downregulation of these transcription factors would be beneficial for studies in both diabetes and stem cell biology. Here, we discuss one such factor, i.e., the transcription factor MAFA. MAFA is a basic leucine zipper family transcription factor that can activate the expression of insulin in β-cells with PDX1 and NEUROD1. MAFA is indeed indispensable for the maintenance of not only insulin expression but also function of adult β-cells. With loss of MAFA in type 2 diabetes, β-cells cannot maintain their mature phenotype and are dedifferentiated. In this review, we first briefly summarize the functional roles of MAFA in β-cells and then mainly focus on the molecular mechanism of cell fate conversion regulated by MAFA.
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Huang D, Wang R. Exploring the mechanism of pancreatic cell fate decisions via cell-cell communication. MATHEMATICAL BIOSCIENCES AND ENGINEERING : MBE 2021; 18:2401-2424. [PMID: 33892552 DOI: 10.3934/mbe.2021122] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
The endocrine and exocrine cells in pancreas originate initially from a group of apparently identical endoderm cells in the early gut. The endocrine and exocrine tissues are composed of islet/acinar and duct cells respectively. To explore the mechanism of pancreas cell fate decisions, we first construct a minimal mathematical model related to pancreatic regulations. The regulatory mechanism of acinar-to-islet cell conversion is revealed by bifurcation analysis of the model. In addition, Notch signaling is critical in determining the fate of endocrine and exocrine in the developing pancreas and it is a typical mediator of lateral inhibition which instructs adjacent cells to make different fate decisions. Next, we construct a multicellular model of cell-cell communication mediated by Notch signaling with trans-activation and cis-inhibition. The roles of Notch signaling in regulating fate decisions of endocrine and exocrine cells during the differentiation of pancreatic cells are explored. The results indicate that high (or low) level of Notch signaling drive cells to select the fate of exocrine (or endocrine) progenitor cells. The networks and the models presented here might be good candidates for providing qualitative mechanisms of pancreatic cell fate decisions. These results can also provide some insight on choosing perturbation strategies for further experimental analysis.
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Affiliation(s)
- Dasong Huang
- Department of Mathematics, Shanghai University, Shanghai 200444, China
| | - Ruiqi Wang
- Department of Mathematics, Shanghai University, Shanghai 200444, China
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Randolph LN, Bhattacharyya A, Lian XL. Human beta cells generated from pluripotent stem cells or cellular reprogramming for curing diabetes. REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE 2019; 5:42-52. [PMID: 30984818 PMCID: PMC6457681 DOI: 10.1007/s40883-018-0082-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2018] [Accepted: 09/24/2018] [Indexed: 10/28/2022]
Abstract
Diabetes is a group of metabolic diseases characterized by aberrantly high blood glucose levels caused by defects in insulin secretion, its action, or both, which affects approximately 30.3 million people (9.4% of the population) in the United States. This review will focus on using human β cells to treat and cure diabetes because β cells are absent, due to an autoimmune destruction, in Type 1 diabetes or dysfunctional in Type 2 diabetes. In order to generate enough functional β cells for diabetes treatment (0.1 to 1 billion cells to treat one patient), a basic science approach by mimicking what happens in normal pancreatic development must be closely aligned with engineering. Two general approaches are discussed here. The first one uses human pluripotent stem cells (hPSCs) to perform directed differentiation of hPSCs to β cells. This is advantageous because hPSCs grow indefinitely, providing a virtually unlimited source of material. Therefore, if we develop an efficient β cell differentiation protocol, we can essentially generate an unlimited amount of β cells for disease modeling and diabetes treatment. The second approach is cellular reprogramming, with which we may begin with any cell type and covert it directly into a β cell. The success of this cellular reprogramming approach, however, depends on the discovery of a robust and efficient transcription factor cocktail that can ignite this process, similar to what has been achieved in generating induced pluripotent stem cells. This discovery should be possible through identifying the important transcription factors and pioneer factors via recent advances in single-cell RNA sequencing. In short, a new renaissance in pancreas developmental biology, stem cell engineering, and cellular reprogramming for curing diabetes appears to be on the horizon.
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Affiliation(s)
- Lauren N. Randolph
- Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, 16802, USA
- The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA
| | - Agamoni Bhattacharyya
- Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, 16802, USA
- The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA
| | - Xiaojun Lance Lian
- Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, 16802, USA
- The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA
- Department of Biology, Pennsylvania State University, University Park, PA, 16802, USA
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Farack L, Golan M, Egozi A, Dezorella N, Bahar Halpern K, Ben-Moshe S, Garzilli I, Tóth B, Roitman L, Krizhanovsky V, Itzkovitz S. Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas. Dev Cell 2018; 48:115-125.e4. [PMID: 30503750 DOI: 10.1016/j.devcel.2018.11.001] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2018] [Revised: 09/14/2018] [Accepted: 10/31/2018] [Indexed: 12/27/2022]
Abstract
Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. Here, we developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of "extreme" beta cells with elevated mRNA levels of insulin and other secretory genes. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER-enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in db/db diabetic mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales.
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Affiliation(s)
- Lydia Farack
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Matan Golan
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Adi Egozi
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Nili Dezorella
- Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Keren Bahar Halpern
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Shani Ben-Moshe
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Immacolata Garzilli
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Beáta Tóth
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Lior Roitman
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Valery Krizhanovsky
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
| | - Shalev Itzkovitz
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
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Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis. Pancreas 2017; 46:605-613. [PMID: 28099259 DOI: 10.1097/mpa.0000000000000759] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
OBJECTIVES The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. METHODS Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. RESULTS pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. CONCLUSIONS pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.
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Yin C. Molecular mechanisms of Sox transcription factors during the development of liver, bile duct, and pancreas. Semin Cell Dev Biol 2016; 63:68-78. [PMID: 27552918 DOI: 10.1016/j.semcdb.2016.08.015] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2016] [Revised: 08/13/2016] [Accepted: 08/18/2016] [Indexed: 12/15/2022]
Abstract
The liver and pancreas are the prime digestive and metabolic organs in the body. After emerging from the neighboring domains of the foregut endoderm, they turn on distinct differentiation and morphogenesis programs that are regulated by hierarchies of transcription factors. Members of SOX family of transcription factors are expressed in the liver and pancreas throughout development and act upstream of other organ-specific transcription factors. They play key roles in maintaining stem cells and progenitors. They are also master regulators of cell fate determination and tissue morphogenesis. In this review, we summarize the current understanding of SOX transcription factors in mediating liver and pancreas development. We discuss their contribution to adult organ function, homeostasis and injury responses. We also speculate how the knowledge of SOX transcription factors can be applied to improve therapies for liver diseases and diabetes.
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Affiliation(s)
- Chunyue Yin
- Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
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10
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Sunitha MM, Srikanth L, Santhosh Kumar P, Chandrasekhar C, Sarma PVGK. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells. Cell Biol Int 2016; 40:1084-93. [PMID: 27514733 DOI: 10.1002/cbin.10654] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2016] [Accepted: 07/17/2016] [Indexed: 11/06/2022]
Abstract
Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.
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Affiliation(s)
- Manne Mudhu Sunitha
- Stem Cell laboratory, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India
| | - Lokanathan Srikanth
- Stem Cell laboratory, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India
| | - Pasupuleti Santhosh Kumar
- Stem Cell laboratory, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India
| | - Chodimella Chandrasekhar
- Department of Haematology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
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Lee J, Kim K, Yu SW, Kim EK. Wnt3a upregulates brain-derived insulin by increasing NeuroD1 via Wnt/β-catenin signaling in the hypothalamus. Mol Brain 2016; 9:24. [PMID: 26956881 PMCID: PMC4782570 DOI: 10.1186/s13041-016-0207-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2015] [Accepted: 02/26/2016] [Indexed: 12/15/2022] Open
Abstract
Background Insulin plays diverse roles in the brain. Although insulin produced by pancreatic β-cells that crosses the blood–brain barrier is a major source of brain insulin, recent studies suggest that insulin is also produced locally within the brain. However, the mechanisms underlying the production of brain-derived insulin (BDI) are not yet known. Results Here, we examined the effect of Wnt3a on BDI production in a hypothalamic cell line and hypothalamic tissue. In N39 hypothalamic cells, Wnt3a treatment significantly increased the expression of the Ins2 gene, which encodes the insulin isoform predominant in the mouse brain, by activating Wnt/β-catenin signaling. The concentration of insulin was higher in culture medium of Wnt3a-treated cells than in that of untreated cells. Interestingly, neurogenic differentiation 1 (NeuroD1), a target of Wnt/β-catenin signaling and one of transcription factors for insulin, was also induced by Wnt3a treatment in a time- and dose-dependent manner. In addition, the treatment of BIO, a GSK3 inhibitor, also increased the expression of Ins2 and NeuroD1. Knockdown of NeuroD1 by lentiviral shRNAs reduced the basal expression of Ins2 and suppressed Wnt3a-induced Ins2 expression. To confirm the Wnt3a-induced increase in Ins2 expression in vivo, Wnt3a was injected into the hypothalamus of mice. Wnt3a increased the expression of NeuroD1 and Ins2 in the hypothalamus in a manner similar to that observed in vitro. Conclusion Taken together, these results suggest that BDI production is regulated by the Wnt/β-catenin/NeuroD1 pathway in the hypothalamus. Our findings will help to unravel the regulation of BDI production in the hypothalamus. Electronic supplementary material The online version of this article (doi:10.1186/s13041-016-0207-5) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Jaemeun Lee
- Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333 Techno Jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu, 42988, South Korea.
| | - Kyungchan Kim
- Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333 Techno Jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu, 42988, South Korea.
| | - Seong-Woon Yu
- Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333 Techno Jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu, 42988, South Korea.
| | - Eun-Kyoung Kim
- Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333 Techno Jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu, 42988, South Korea. .,Neurometabolomics Research Center, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333 Techno Jungang-daero, Hyeonpung-myeon, Dalseong-gun, Daegu, 42988, South Korea.
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Yu K, Fischbach S, Xiao X. Beta Cell Regeneration in Adult Mice: Controversy Over the Involvement of Stem Cells. Curr Stem Cell Res Ther 2016; 11:542-546. [PMID: 25429702 PMCID: PMC5078597 DOI: 10.2174/1574888x10666141126113110] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2014] [Revised: 10/17/2014] [Accepted: 11/24/2014] [Indexed: 01/06/2023]
Abstract
Islet transplantation is an effective therapy for severe diabetes. Nevertheless, the short supply of donor pancreases constitutes a formidable obstacle to its extensive clinical application. This shortage heightens the need for alternative sources of insulin-producing beta cells. Since mature beta cells have a very slow proliferation rate, which further declines with age, great efforts have been made to identify beta cell progenitors in the adult pancreas. However, the question whether facultative beta cell progenitors indeed exist in the adult pancreas remains largely unresolved. In the current review, we discuss the problems in past studies and review the milestone studies and recent publications.
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Affiliation(s)
- Ke Yu
- Beijing Key Laboratory of Diabetes Prevention and Care, Department of Endocrinology, Lu He Hospital, Capital Medical University, Beijing, China
| | - Shane Fischbach
- Division of Pediatric Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh,USA
- Division of Biology and Medicine, Brown University, Providence,USA
| | - Xiangwei Xiao
- Division of Pediatric Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh,USA
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13
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Lizio M, Ishizu Y, Itoh M, Lassmann T, Hasegawa A, Kubosaki A, Severin J, Kawaji H, Nakamura Y, Suzuki H, Hayashizaki Y, Carninci P, Forrest ARR. Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line. Front Genet 2015; 6:331. [PMID: 26635867 PMCID: PMC4650373 DOI: 10.3389/fgene.2015.00331] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2015] [Accepted: 10/30/2015] [Indexed: 12/22/2022] Open
Abstract
Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment.
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Affiliation(s)
- Marina Lizio
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan
| | - Yuri Ishizu
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan
| | - Masayoshi Itoh
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; RIKEN Preventive Medicine and Diagnosis Innovation Program Yokohama, Japan
| | - Timo Lassmann
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; Telethon Kids Institute, The University of Western Australia Subiaco, WA, Australia
| | - Akira Hasegawa
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan
| | | | - Jessica Severin
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan
| | - Hideya Kawaji
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; RIKEN Preventive Medicine and Diagnosis Innovation Program Yokohama, Japan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center Ibaraki, Japan
| | | | - Harukazu Suzuki
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan
| | - Yoshihide Hayashizaki
- RIKEN Center for Life Science Technologies Yokohama, Japan ; RIKEN Preventive Medicine and Diagnosis Innovation Program Yokohama, Japan
| | - Piero Carninci
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan
| | - Alistair R R Forrest
- RIKEN Center for Life Science Technologies Yokohama, Japan ; Division of Genomic Technologies, RIKEN Center for Life Science Technologies Yokohama, Japan ; QEII Medical Centre and Centre for Medical Research, Harry Perkins Institute of Medical Research, The University of Western Australia Nedlands, WA, Australia
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14
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Lv L, Chen H, Sun J, Lu D, Chen C, Liu D. PRMT1 promotes glucose toxicity-induced β cell dysfunction by regulating the nucleo-cytoplasmic trafficking of PDX-1 in a FOXO1-dependent manner in INS-1 cells. Endocrine 2015; 49:669-82. [PMID: 25874535 DOI: 10.1007/s12020-015-0543-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 01/27/2015] [Indexed: 11/26/2022]
Abstract
Protein N-arginine methyltransferase-1 (PRMT1), the major asymmetric arginine methyltransferase, plays important roles in various cellular processes. Previous reports have demonstrated that levels and activities of PRMT1 can vary in animals with type 2 diabetes mellitus. The aim of this study was to assess the expression and mechanism of action of PRMT1 during glucose toxicity-induced β cell dysfunction. Liposome-mediated gene transfection was used to transfect INS-1 cells with siPRMT1, which inhibits PRMT1 expression, and pALTER-FOXO1, which overexpresses forkhead box protein O1 (FOXO1). The cells were then cultured in media containing 5.6 or 25 mmol/L glucose with or without the small molecule PRMT1 inhibitor AMI-1 for 48 h. The protein levels of PRMT1, the arginine methylated protein α-metR, FOXO1, Phospho-FOXO1, pancreas duodenum homeobox-1 (PDX-1), and the intracellular localization of PDX-1 and FOXO1 were then measured by western blotting. FOXO1 methylation was detected by immunoprecipitated with anti-PRMT1 antibody and were immunoblotted with α-metR. The levels of insulin mRNA were measured by real-time fluorescence quantitative PCR. Glucose-stimulated insulin secretion (GSIS) and intracellular insulin content were measured using radioimmunoassays. Intracellular Ca(2+) ([Ca(2+)]i) was detected using Fura-2 AM. Intracellular cAMP levels were measured using ELISA. Chronic exposure to high glucose impaired insulin secretion, decreased insulin mRNA levels and insulin content, increased intracellular [Ca(2+)]i and cAMP levels, and abolishes their responses to glucose. Inhibiting PRMT1 expression improved insulin secretion, increased mRNA levels and insulin content by regulating the intracellular translocation of PDX-1 and FOXO1, decreasing the methylation of FOXO1, and reducing intracellular [Ca(2+)]i and cAMP concentrations. Transient overexpression of constitutively active FOXO1 in nuclear reversed the AMI-1-induced improvement of β cell function without changing arginine methylation. It is concluded therefore that PRMT1 regulates GSIS in INS-1 cells, through enhanced methylation-induced nuclear localization of FOXO1, which subsequently suppresses the nuclear localization of PDX-1. Our results suggest a novel mechanism that might contribute to the deficient insulin secretion observed under conditions of chronically hyperglycemia.
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Affiliation(s)
- Lixia Lv
- Department of Endocrinology and Metabolism, The Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing, 400010, China
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15
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Peschke E, Bähr I, Mühlbauer E. Experimental and clinical aspects of melatonin and clock genes in diabetes. J Pineal Res 2015; 59:1-23. [PMID: 25904189 DOI: 10.1111/jpi.12240] [Citation(s) in RCA: 81] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Accepted: 04/20/2015] [Indexed: 12/15/2022]
Abstract
The pineal hormone melatonin influences insulin secretion, as well as glucagon and somatostatin secretion, both in vivo and in vitro. These effects are mediated by two specific, high-affinity, seven transmembrane, pertussis toxin-sensitive, Gi-protein-coupled melatonin receptors, MT1 and MT2. Both isoforms are expressed in the β-cells, α-cells as well as δ-cells of the pancreatic islets of Langerhans and are involved in the modulation of insulin secretion, leading to inhibition of the adenylate cyclase-dependent cyclic adenosine monophosphate as well as cyclic guanosine monophosphate formation in pancreatic β-cells by inhibiting the soluble guanylate cyclase, probably via MT2 receptors. In this way, melatonin also likely inhibits insulin secretion, whereas using the inositol triphosphate pathway after previous blocking of Gi-proteins by pertussis toxin, melatonin increases insulin secretion. Desynchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genomewide association studies pinpointing variances of the MT2 receptor as a risk factor for this rapidly spreading metabolic disturbance. As melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. Observations of the circadian expression of clock genes (Clock, Bmal1, Per1,2,3, and Cry1,2) in pancreatic islets, as well as in INS1 rat insulinoma cells, may indicate that circadian rhythms are generated in the β-cells themselves. The circadian secretion of insulin from pancreatic islets is clock-driven. Disruption of circadian rhythms and clock function leads to metabolic disturbances, for example, type 2 diabetes. The study of melatonin-insulin interactions in diabetic rat models has revealed an inverse relationship between these two hormones. Both type 2 diabetic rats and patients exhibit decreased melatonin levels and slightly increased insulin levels, whereas type 1 diabetic rats show extremely reduced levels or the absence of insulin, but statistically significant increases in melatonin levels. Briefly, an increase in melatonin levels leads to a decrease in stimulated insulin secretion and vice versa. Melatonin levels in blood plasma, as well as the activity of the key enzyme of melatonin synthesis, AA-NAT (arylalkylamine-N-acetyltransferase) in pineal, are lower in type 2 diabetic rats compared to controls. In contrast, melatonin and pineal AA-NAT mRNA are increased and insulin receptor mRNA is decreased in type 1 diabetic rats, which also indicates a close relationship between insulin and melatonin. As an explanation, it was hypothesized that catecholamines, which reduce insulin levels and stimulate melatonin synthesis, control insulin-melatonin interactions. This conviction stems from the observation that catecholamines are increased in type 1 but are diminished in type 2 diabetes. In this context, another important line of inquiry involves the fact that melatonin protects β-cells against functional overcharge and, consequently, hinders the development of type 2 diabetes.
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Affiliation(s)
| | - Ina Bähr
- Institute of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Halle, Germany
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16
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Wang Z, You J, Xu S, Hua Z, Zhang W, Deng T, Fang N, Fang Q, Liu H, Peng L, Wang P, Lou J. Colocalization of insulin and glucagon in insulinoma cells and developing pancreatic endocrine cells. Biochem Biophys Res Commun 2015; 461:598-604. [PMID: 25912877 DOI: 10.1016/j.bbrc.2015.04.072] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2015] [Accepted: 04/14/2015] [Indexed: 01/13/2023]
Abstract
A significant portion of human and rat insulinomas coexpress multiple hormones. This character termed as multihormonality is also observed in some early pancreatic endocrine cells which coexpress insulin and glucagon, suggesting an incomplete differentiation status of both cells. Here we demonstrate that insulinoma cells INS-1 and INS-1-derived single cell clone INS-1-15 coexpressed insulin and glucagon in a portion of cells. These two hormones highly colocalized in the intracellular vesicles within a cell. Due to the existence of both PC1/3 and PC2 in INS-1-derived cells, proglucagon could be processed into glucagon, GLP-1 and GLP-2. These glucagon-family peptides and insulin were secreted simultaneously corresponding to the elevating glucose concentrations. The coexpression and partial colocalization of insulin and glucagon was also observed in rat fetal pancreatic endocrine cells, but the colocalization rate was generally lower and more diverse, suggesting that in the developing pancreatic endocrine cells, insulin and glucagon may be stored in nonidentical pools of secreting vesicles and might be secreted discordantly upon stimulus.
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Affiliation(s)
- Zai Wang
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Jia You
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Shiqing Xu
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Zhan Hua
- Department of General Surgery, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Wenjian Zhang
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Tingting Deng
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Ni Fang
- Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, PR China
| | - Qing Fang
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Honglin Liu
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Liang Peng
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China
| | - Peigang Wang
- Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China
| | - Jinning Lou
- Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, PR China.
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17
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Riley KG, Gannon M. Pancreas Development and Regeneration. PRINCIPLES OF DEVELOPMENTAL GENETICS 2015:565-590. [DOI: 10.1016/b978-0-12-405945-0.00031-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2025]
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18
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Cross-fostering and improved lactation ameliorates deficits in endocrine pancreatic morphology in growth-restricted adult male rat offspring. J Dev Orig Health Dis 2014; 1:234-44. [PMID: 25141871 DOI: 10.1017/s2040174410000383] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Uteroplacental insufficiency and poor postnatal nutrition impair adult glucose tolerance and insulin secretion in male rat offspring, which can be partially ameliorated by improving postnatal nutrition. Uteroplacental insufficiency was induced in the WKY rat on day 18 of pregnancy (Restricted) compared to sham-operated Controls. Pups were then cross-fostered onto Control or Restricted mothers one day after birth resulting in: (Pup-on-Mother) Control-on-Control, Control-on-Restricted, Restricted-on-Control and Restricted-on-Restricted. Endocrine pancreatic morphology and markers of intrinsic β-cell function and glucose homeostasis were assessed in male offspring at 6 months. Pancreatic and hepatic gene expression was quantified at postnatal day 7 and 6 months. Restricted pups were born 10-15% lighter than Controls and remained lighter at 6 months. Relative islet and β-cell mass were 51-65% lower in Restricted-on-Restricted compared to Controls at 6 months. Non-fasting plasma C-reactive protein levels were also increased, suggestive of an inflammatory response. Overall, the average number of islets, small islets and proportion of β-cells per islet correlated positively with birth weight. Intrinsic β-cell function, estimated by insulin secretion relative to β-cell mass, was unaffected by Restriction, suggesting that the in vivo functional deficit was attributable to reduced mass, not function. Importantly, these deficits were ameliorated when lactational nutrition was normalized in Restricted-on-Control offspring, who also showed increased pancreatic Igf1r, Pdx1 and Vegf mRNA expression at 7 days compared to Control-on-Control and Restricted-on-Restricted. This highlights lactation as a critical period for intervention following prenatal restraint, whereby deficits in endocrine pancreatic mass and associated impaired in vivo insulin secretion can be ameliorated.
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19
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Silymarin induces expression of pancreatic Nkx6.1 transcription factor and β-cells neogenesis in a pancreatectomy model. Molecules 2014; 19:4654-68. [PMID: 24739928 PMCID: PMC6271357 DOI: 10.3390/molecules19044654] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2013] [Revised: 03/21/2014] [Accepted: 03/31/2014] [Indexed: 12/12/2022] Open
Abstract
A physio-pathological feature of diabetes mellitus is a significant reduction of β-pancreatic cells. The growth, differentiation and function maintenance of these cells is directed by transcription factors. Nkx6.1 is a key transcription factor for the differentiation, neogenesis and maintenance of β-pancreatic cells. We reported that silymarin restores normal morphology and endocrine function of damaged pancreatic tissue after alloxan-induced diabetes mellitus in rats. The aim of this study was to analyze the effect of silymarin on Nkx6.1 transcription factor expression and its consequence in β cells neogenesis. Sixty male Wistar rats were partially pancreatectomized and divided into twelve groups. Six groups were treated with silymarin (200 mg/Kg p.o) for periods of 3, 7, 14, 21, 42 and 63 days. Additionally, an unpancreatectomized control group was used. Nkx6.1 and insulin gene expression were assessed by RT-PCR assay in total pancreatic RNA. β-Cell neogenesis was determined by immunoperoxidase assay. Silymarin treated group showed an increase of Nkx6.1 and insulin genic expression. In this group, there was an increment of β-cell neogenesis in comparison to pancreatectomized untreated group. Silymarin treatment produced a rise in serum insulin and serum glucose normalization. These results suggest that silymarin may improve the reduction of β pancreatic cells observed in diabetes mellitus.
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20
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Takeuchi H, Nakatsuji N, Suemori H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 2014; 4:4488. [PMID: 24671046 PMCID: PMC3967149 DOI: 10.1038/srep04488] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2014] [Accepted: 03/10/2014] [Indexed: 12/11/2022] Open
Abstract
Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs.
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Affiliation(s)
- Hiroki Takeuchi
- Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
| | - Norio Nakatsuji
- 1] Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Ushinomiya-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan [2] Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
| | - Hirofumi Suemori
- Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
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21
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Aziz MTA, El-Asmar MF, Rezq AM, Wassef MAA, Fouad H, Roshdy NK, Ahmed HH, Rashed LA, Sabry D, Taha FM, Hassouna A. Effects of a novel curcumin derivative on insulin synthesis and secretion in streptozotocin-treated rat pancreatic islets in vitro. Chin Med 2014; 9:3. [PMID: 24422903 PMCID: PMC3896850 DOI: 10.1186/1749-8546-9-3] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2013] [Accepted: 01/13/2014] [Indexed: 12/29/2022] Open
Abstract
Background Hyperglycemia induces activation of the c-Jun N-terminal kinase (JNK) pathway, which suppresses insulin gene expression and reduces DNA binding of pancreatic and duodenal homeobox factor (PDX)-1. This study aims to investigate the effects of a novel curcumin derivative (NCD) on JNK signaling pathway on insulin synthesis and secretion in streptozotocin (STZ)-treated rat pancreatic islets in vitro. Methods Isolated rat pancreatic islets were divided into five groups: untreated control group; group treated with NCD (10 μM); group exposed to STZ (5 mM); group treated with NCD (10 μM) and then exposed to STZ (5 mM); and group exposed to STZ (5 mM) and then treated with NCD (10 μM). The pancreatic islets from all groups were used for DNA fragmentation assays and quantitative assessments of the JNK, Pdx1, glucose transporter-2 (GLUT2), heme oxygenase (HO)-1, transcription factor 7-like 2 (TCF7L2), and glucagon-like peptide (GLP)-1 gene expression levels. The intracellular calcium, zinc, and the phosphorylated and total JNK protein levels were assessed. The insulin (secreted/total) and C-peptide levels were examined in islet culture medium. Results NCD protected pancreatic islets against STZ-induced DNA damage, improved total insulin (P = 0.001), secreted insulin (P = 0.001), and C-peptide levels (P = 0.001), normalized mRNA expressions of insulin, Pdx1, and GLUT2 (P = 0.0001), and significantly elevated calcium and zinc levels (P = 0.0001). All effects were significant when islets were treated with NCD before STZ (P = 0.05). JNK gene overexpression and JNK protein levels induced by STZ were significantly inhibited after NCD treatment of islets ( P = 0.0001). NCD-treated islets showed significantly elevated gene expressions of HO-1, TCF7L2, and GLP-1 (P = 0.0001), and these upregulated gene expressions were more significantly elevated with NCD treatment before STZ than after STZ (P = 0.05). Conclusions NCD improved insulin synthesis and secretion in vitro in isolated pancreatic islets treated with STZ through inhibition of the JNK pathway, up-regulation of the gene expressions of HO-1, TCF7L2, and GLP-1 and enhancing effects on calcium and zinc levels.
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Affiliation(s)
| | | | | | | | - Hanan Fouad
- Medical Biochemistry Department, Faculty of Medicine, Cairo University, POB 11562, Cairo, Egypt.
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22
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Bazwinsky-Wutschke I, Bieseke L, Mühlbauer E, Peschke E. Influence of melatonin receptor signalling on parameters involved in blood glucose regulation. J Pineal Res 2014; 56:82-96. [PMID: 24117965 DOI: 10.1111/jpi.12100] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2013] [Accepted: 09/20/2013] [Indexed: 12/18/2022]
Abstract
The pineal hormone melatonin is known to influence insulin secretion via the G-protein-coupled receptor isoforms MT1 and MT2. The present study was aimed to further elucide the impact of melatonin on blood glucose regulation. To this end, mouse lines were used, in which one of the two or both melatonin receptors were deleted. In comparison with wild-type mice of the same age (8-12 months old), increased plasma insulin and melatonin levels and decreased blood glucose levels and body weights were detected in the MT1- and double-knockout lines. The elimination of melatonin receptor signalling also altered blood glucose concentrations, body weight and melatonin and insulin levels when comparing wild-type and receptor knockout mice of different ages (6 wk and 8-12 months old); such changes, however, were dependent on the type of receptor deleted. Furthermore, reverse transcription polymerase chain reaction results provided evidence that melatonin receptor deficiency has an impact on transcript levels of pancreatic islet hormones as well as on pancreatic and hepatic glucose transporters (Glut1 and 2). Under stimulated insulin secretion in the presence of melatonin in the rat insulinoma β-cells INS-1, the Glut1 transcript level was decreased. In conclusion, the present findings demonstrate that melatonin receptor knockout types affect blood glucose levels, body weight, plasma levels of melatonin and insulin, as well as pancreatic hormone and Glut1 expression in significantly different manners.
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MESH Headings
- Analysis of Variance
- Animals
- Blood Glucose/genetics
- Blood Glucose/metabolism
- Body Weight/genetics
- Cell Line, Tumor
- Female
- Glucagon/analysis
- Glucagon/genetics
- Glucagon/metabolism
- Glucose Transporter Type 1/analysis
- Glucose Transporter Type 1/genetics
- Glucose Transporter Type 1/metabolism
- Insulin/blood
- Male
- Melatonin/blood
- Mice
- Mice, Knockout
- Organ Specificity
- RNA, Messenger/analysis
- RNA, Messenger/genetics
- Receptor, Melatonin, MT1/genetics
- Receptor, Melatonin, MT1/metabolism
- Receptor, Melatonin, MT2/genetics
- Receptor, Melatonin, MT2/metabolism
- Somatostatin/analysis
- Somatostatin/genetics
- Somatostatin/metabolism
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23
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Arcidiacono B, Iiritano S, Chiefari E, Brunetti FS, Gu G, Foti DP, Brunetti A. Cooperation between HMGA1, PDX-1, and MafA is Essential for Glucose-Induced Insulin Transcription in Pancreatic Beta Cells. Front Endocrinol (Lausanne) 2014; 5:237. [PMID: 25628604 PMCID: PMC4292585 DOI: 10.3389/fendo.2014.00237] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2014] [Accepted: 12/18/2014] [Indexed: 01/03/2023] Open
Abstract
The high-mobility group AT-hook 1 (HMGA1) protein is a nuclear architectural factor that can organize chromatin structures. It regulates gene expression by controlling the formation of stereospecific multiprotein complexes called "enhanceosomes" on the AT-rich regions of target gene promoters. Previously, we reported that defects in HMGA1 caused decreased insulin receptor expression and increased susceptibility to type 2 diabetes mellitus in humans and mice. Interestingly, mice with disrupted HMGA1 gene had significantly smaller islets and decreased insulin content in their pancreata, suggesting that HMGA1 may have a direct role in insulin transcription and secretion. Herein, we investigate the regulatory roles of HMGA1 in insulin transcription. We provide evidence that HMGA1 physically interacts with PDX-1 and MafA, two critical transcription factors for insulin gene expression and beta-cell function, both in vitro and in vivo. We then show that the overexpression of HMGA1 significantly improves the transactivating activity of PDX-1 and MafA on human and mouse insulin promoters, while HMGA1 knockdown considerably decreased this transactivating activity. Lastly, we demonstrate that high glucose stimulus significantly increases the binding of HMGA1 to the insulin (INS) gene promoter, suggesting that HMGA1 may act as a glucose-sensitive element controlling the transcription of the INS gene. Together, our findings provide evidence that HMGA1, by regulating PDX-1- and MafA-induced transactivation of the INS gene promoter, plays a critical role in pancreatic beta-cell function and insulin production.
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Affiliation(s)
- Biagio Arcidiacono
- Department of Health Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy
| | - Stefania Iiritano
- Department of Health Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy
| | - Eusebio Chiefari
- Department of Health Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy
| | - Francesco S. Brunetti
- Department of Medical and Surgical Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy
| | - Guoqiang Gu
- Department of Cell and Developmental Biology, Center of Stem Cell Biology, Vanderbilt Medical Center, Nashville, TN, USA
| | - Daniela Patrizia Foti
- Department of Health Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy
| | - Antonio Brunetti
- Department of Health Sciences, University “Magna Græcia” of Catanzaro, Catanzaro, Italy
- *Correspondence: Antonio Brunetti, Department of Health Sciences, University “Magna Græcia” of Catanzaro, Viale Europa (Località Germaneto), Catanzaro 88100, Italy e-mail:
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24
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Tsui S, Dai W, Lu L. CCCTC-binding factor mediates effects of glucose on beta cell survival. Cell Prolif 2013; 47:28-37. [PMID: 24354619 DOI: 10.1111/cpr.12085] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2013] [Accepted: 10/07/2013] [Indexed: 12/17/2022] Open
Abstract
OBJECTIVES Pancreatic islet β-cell survival is paramount for regulation of insulin activity and for maintaining glucose homeostasis. Recently, Pax6 has been shown to be essential for many vital functions in β-cells, although many molecular mechanisms of its homeostasis in β-cells remain unclear. The present study investigates novel effects of glucose- and insulin-induced CCCTC-binding factor (CTCF) activity on Pax6 gene expression as well as for subsequent effects of insulin-activated signalling pathways, on β-cell proliferation. MATERIALS AND METHODS Pancreatic β-TC-1-6 cells were cultured in DMEM and stimulated with high concentrations of glucose (5-125 mm); cell viability was assessed by MTT assay. Effects of CTCF on Pax6 were evaluated in the high glucose-induced environment and CTCF/Erk-suppressed cells, by promoter reporter and western blotting analyses. RESULTS Increases in glucose and insulin concentrations upregulated CTCF and consequently downregulated Pax6 in β-cell survival and proliferation. Knocking-down CTCF directly affected Pax6 transcription through CTCF binding and blocked the response to glucose. Altered Erk activity mediated effects of CTCF on controlling Pax6 expression, which partially regulated β-cell proliferation. CONCLUSIONS CTCF functioned as a molecular mediator between insulin-induced upstream Erk signalling and Pax6 expression in these pancreatic β-cells. This pathway may contribute to regulation of β-cell survival and proliferation.
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Affiliation(s)
- S Tsui
- Department of Medicine, David Geffen School of Medicine University of California Los Angeles, Torrance, CA, 90502, USA
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25
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Sane F, Caloone D, Gmyr V, Engelmann I, Belaich S, Kerr-Conte J, Pattou F, Desailloud R, Hober D. Coxsackievirus B4 can infect human pancreas ductal cells and persist in ductal-like cell cultures which results in inhibition of Pdx1 expression and disturbed formation of islet-like cell aggregates. Cell Mol Life Sci 2013; 70:4169-80. [PMID: 23775130 PMCID: PMC11113870 DOI: 10.1007/s00018-013-1383-4] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2013] [Revised: 05/14/2013] [Accepted: 05/23/2013] [Indexed: 12/31/2022]
Abstract
The role of enteroviruses, especially Coxsackievirus B (CVB), in type 1 diabetes is suspected, but the mechanisms of the virus-induced or aggravated pathogenesis of the disease are unknown. The hypothesis of an enterovirus-induced disturbance of pancreatic β-cells regeneration has been investigated in the human system. The infection of human pancreas ductal cells and pancreatic duct cell line, PANC-1, with CVB4E2 has been studied. Primary ductal cells and PANC-1 cells were infectable with CVB4E2 and a RT-PCR assay without extraction displayed that a larger proportion of cells harbored viral RNA than predicted by the detection of the viral capsid protein VP1 by indirect immunofluorescence. The detection of intracellular positive- and negative-strands of enterovirus genomes in cellular extracts by RT-PCR and the presence of infectious particles in supernatant fluids during the 37 weeks of monitoring demonstrated that CVB4E2 could persist in the pancreatic duct cell line. A persistent infection of these cells resulted in an impaired expression of Pdx1, a transcription factor required for the formation of endocrine pancreas, and a disturbed formation of islet-like cell aggregates of which the viability was decreased. These data support the hypothesis of an impact of enteroviruses onto pancreatic ductal cells which are involved in the renewal of pancreatic β-cells.
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Affiliation(s)
- Famara Sane
- Laboratoire de Virologie/ EA3610, Université Lille 2, Faculté de Médecine, CHRU, 59120 Loos-lez-Lille, France
| | - Delphine Caloone
- Laboratoire de Virologie/ EA3610, Université Lille 2, Faculté de Médecine, CHRU, 59120 Loos-lez-Lille, France
| | - Valéry Gmyr
- Laboratoire Biothérapie du diabète, INSERM U859 CHRU de Lille, 59045, Lille, France
| | - Ilka Engelmann
- Laboratoire de Virologie/ EA3610, Université Lille 2, Faculté de Médecine, CHRU, 59120 Loos-lez-Lille, France
| | - Sandrine Belaich
- Laboratoire Biothérapie du diabète, INSERM U859 CHRU de Lille, 59045, Lille, France
| | - Julie Kerr-Conte
- Laboratoire Biothérapie du diabète, INSERM U859 CHRU de Lille, 59045, Lille, France
| | - François Pattou
- Laboratoire Biothérapie du diabète, INSERM U859 CHRU de Lille, 59045, Lille, France
| | - Rachel Desailloud
- Service d’Endocrinologie-Diabétologie-Nutrition, UPJV CHU, 80054 Amiens, France
| | - Didier Hober
- Laboratoire de Virologie/ EA3610, Université Lille 2, Faculté de Médecine, CHRU, 59120 Loos-lez-Lille, France
- Laboratoire de Virologie/EA3610, Institut Hippocrate, CHRU Lille, 152 rue du Dr Yersin, 59120 Loos-Lez-Lille, France
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26
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O'Dowd JF, Stocker CJ. Endocrine pancreatic development: impact of obesity and diet. Front Physiol 2013; 4:170. [PMID: 23882220 PMCID: PMC3714448 DOI: 10.3389/fphys.2013.00170] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2013] [Accepted: 06/18/2013] [Indexed: 12/16/2022] Open
Abstract
During embryonic development, multipotent endodermal cells differentiate to form the pancreas. Islet cell clusters arising from the pancreatic bud form the acini tissue and exocrine ducts whilst pancreatic islets form around the edges of the clusters. The successive steps of islet differentiation are controlled by a complex network of transcription factors and signals that influence cell differentiation, growth and lineage. A Westernized lifestyle has led to an increased consumption of a high saturated fat diet, and an increase in maternal obesity. The developing fetus is highly sensitive to the intrauterine environment, therefore any alteration in maternal nutrition during gestation and lactation which affects the in-utero environment during the key developmental phases of the pancreas may change the factors controlling β-cell development and β-cell mass. Whilst the molecular mechanisms behind the adaptive programming of β-cells are still poorly understood it is established that changes arising from maternal obesity and/or over-nutrition may affect the ability to maintain fetal β-cell mass resulting in an increased risk of type 2 diabetes in adulthood.
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Affiliation(s)
- Jacqueline F O'Dowd
- Metabolic Diseases Group, Clore Laboratory, University of Buckingham Buckingham, UK
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27
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Bastidas M, Showalter SA. Thermodynamic and structural determinants of differential Pdx1 binding to elements from the insulin and IAPP promoters. J Mol Biol 2013; 425:3360-77. [PMID: 23796517 DOI: 10.1016/j.jmb.2013.06.011] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2013] [Revised: 06/11/2013] [Accepted: 06/13/2013] [Indexed: 10/26/2022]
Abstract
In adult mammals, the production of insulin and other peptide hormones, such as the islet amyloid polypeptide (IAPP), is limited to β-cells due to tissue-specific expression of a set of transcription factors, the best known of which is pancreatic duodenal homeobox protein 1 (Pdx1). Like many homeodomain transcription factors, Pdx1 binds to a core DNA recognition sequence containing the tetranucleotide 5'-TAAT-3'; its consensus recognition element is 5'-CTCTAAT(T/G)AG-3'. Currently, a complete thermodynamic profile of Pdx1 binding to near-consensus and native promoter sequences has not been established, obscuring the mechanism of target site selection by this critical transcription factor. Strikingly, while Pdx1 responsive elements in the human insulin promoter conform to the pentanucleotide 5'-CTAAT-3' sequence, the Pdx1 responsive elements in the human iapp promoter all contain a substitution to 5'-TTAAT-3'. The crystal structure of Pdx1 bound to the consensus nucleotide sequence does not explain how Pdx1 identifies this natural variation, if it does at all. Here we report a combination of isothermal calorimetric titrations, NMR spectroscopy, and extensive multi-microsecond molecular dynamics calculations of Pdx1 that define its interactions with a panel of natural promoter elements and consensus-derived sequences. Our results show a small preference of Pdx1 for a C base 5' relative to the core TAAT promoter element. Molecular mechanics calculations, corroborated by experimental NMR data, lead to a rational explanation for sequence discrimination at this position. Taken together, our results suggest a molecular mechanism for differential Pdx1 affinity to elements from the insulin and iapp promoter sequences.
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Affiliation(s)
- Monique Bastidas
- Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802, USA.
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28
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Li B, Zhou X, Wu J, Zhou H. From gut changes to type 2 diabetes remission after gastric bypass surgeries. Front Med 2013; 7:191-200. [PMID: 23553469 DOI: 10.1007/s11684-013-0258-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2012] [Accepted: 01/14/2013] [Indexed: 12/18/2022]
Abstract
Increasing evidence suggests that the gut may influence the host's metabolism and ultimately change the outcomes of type 2 diabetes mellitus (T2DM). We review the evidence on the relationship between the gut and T2DM remission after gastric bypass surgery, and discuss the potential mechanisms underlying the above relationship: gut anatomical rearrangement, microbial composition changes, altered gut cells, and gut hormone modulation. However, the exact changes and their relative importance in the metabolic improvements after gastric bypass surgery remain to be further clarified. Elucidating the precise metabolic mechanisms of T2DM resolution after bypass surgery will help to reveal the molecular mechanisms of pathogenesis, and facilitate the development of novel diagnoses and preventative interventions for this common disease.
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Affiliation(s)
- Bing Li
- Key Laboratory of Systems Biology, SIBS-Novo Nordisk Translational Research Centre for PreDiabetes, Shanghai Institutes for Biological Sciences, CAS, Shanghai, China
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29
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Melatonin and pancreatic islets: interrelationships between melatonin, insulin and glucagon. Int J Mol Sci 2013; 14:6981-7015. [PMID: 23535335 PMCID: PMC3645673 DOI: 10.3390/ijms14046981] [Citation(s) in RCA: 115] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2013] [Revised: 03/07/2013] [Accepted: 03/11/2013] [Indexed: 12/15/2022] Open
Abstract
The pineal hormone melatonin exerts its influence in the periphery through activation of two specific trans-membrane receptors: MT1 and MT2. Both isoforms are expressed in the islet of Langerhans and are involved in the modulation of insulin secretion from β-cells and in glucagon secretion from α-cells. De-synchrony of receptor signaling may lead to the development of type 2 diabetes. This notion has recently been supported by genome-wide association studies identifying particularly the MT2 as a risk factor for this rapidly spreading metabolic disturbance. Since melatonin is secreted in a clearly diurnal fashion, it is safe to assume that it also has a diurnal impact on the blood-glucose-regulating function of the islet. This factor has hitherto been underestimated; the disruption of diurnal signaling within the islet may be one of the most important mechanisms leading to metabolic disturbances. The study of melatonin–insulin interactions in diabetic rat models has revealed an inverse relationship: an increase in melatonin levels leads to a down-regulation of insulin secretion and vice versa. Elucidation of the possible inverse interrelationship in man may open new avenues in the therapy of diabetes.
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30
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Villasenor A, Marty-Santos L, Dravis C, Fletcher P, Henkemeyer M, Cleaver O. EphB3 marks delaminating endocrine progenitor cells in the developing pancreas. Dev Dyn 2012; 241:1008-19. [PMID: 22434763 DOI: 10.1002/dvdy.23781] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
BACKGROUND Understanding the process by which pancreatic beta-cells acquire their "fate" is critical to the development of in vitro directed differentiation protocols for cell replacement therapies for diabetics. To date, these efforts are hampered by a paucity of markers that distinguish pancreatic endocrine cells at different stages of differentiation. RESULTS Here, we identify EphB3 as a novel pro-endocrine marker and use its expression to track delaminating islet lineages. First, we provide a detailed developmental expression profile for EphB3 and other EphB family members in the embryonic pancreas. We demonstrate that EphB3 transiently marks endocrine cells as they delaminate from the pancreatic epithelium, prior to their differentiation. Using a Tet-inducible EphB3(rtTA-lacZ) reporter line, we show that short-term pulse-labeled EphB3(+) cells co-express Pdx1, Nkx6.1, Ngn3, and Synaptophysin, but not insulin, glucagon, or other endocrine hormones. Prolonged labeling tracks EphB3(+) cells from their exit from the epithelium to their differentiation. CONCLUSIONS These studies demonstrate that pro-endocrine cell differentiation during late gestation, from delamination to maturation, takes approximately 2 days. Together, these data introduce EphB3 as a new biomarker to identify beta-cells at a critical step during their step-wise differentiation and define the timeframe of endocrine differentiation.
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Affiliation(s)
- Alethia Villasenor
- Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, USA
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31
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Matsuoka TA. Molecular mechanism of pancreatic β-cell dysfunction under diabetic conditions. Diabetol Int 2012. [DOI: 10.1007/s13340-012-0091-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
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Tsui S, Gao J, Wang C, Lu L. CTCF mediates effect of insulin on glucagon expression. Exp Cell Res 2012; 318:887-95. [PMID: 22426149 DOI: 10.1016/j.yexcr.2012.03.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2011] [Revised: 02/29/2012] [Accepted: 03/01/2012] [Indexed: 11/15/2022]
Abstract
Pancreatic islet α-cell development and glucagon production are mainly regulated by Pax6 in the homeobox gene families. However, the molecular mechanism fine-tuning the regulation of these events in α-cell still remains unclear. In ocular cells, Pax6 transcription is regulated by CTCF through its binding to specific sites in Pax6 promoter. In this study, CTCF-mediated regulations of islet α-cell development and glucagon production were investigated in both CTCF transgenic mice and α-TC-1-6 cells. Over-expression of CTCF in transgenic mice affected development of pancreatic islets by significantly suppressing α-cell population in both embryonic and adult pancreases. The effect of CTCF on Pax6 gene expression and subsequently, on pro-glucagon production was however, examined in pancreatic islet α-cells. Over-expression and knock-down of CTCF directly affected Pax6 expression. More importantly, the CTCF binding sites upstream from Pax6 p0 promoter were required for regulating p0 promoter activity in islet α-cells. Stimulation of α-cells with insulin resulted in a significant increase in CTCF expression and a decrease in Pax6 expression, and consequently suppressed pro-glucagon expression. In contrast, these insulin-induced effects were blocked by knockdown of CTCF mRNA with specific siRNA in α-cells. Altogether, our results demonstrated for the first time that CTCF functions as a switch-like molecule between the insulin signaling and the regulations of Pax6 and glucagon expression in pancreatic islet α-cells.
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Affiliation(s)
- Shanli Tsui
- Department of Medicine, David Geffen School of Medicine University of California Los Angeles, Torrance, CA 90502, USA
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33
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Endoplasmic reticulum stress and insulin biosynthesis: a review. EXPERIMENTAL DIABETES RESEARCH 2012; 2012:509437. [PMID: 22474424 PMCID: PMC3303544 DOI: 10.1155/2012/509437] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/14/2011] [Accepted: 12/06/2011] [Indexed: 12/21/2022]
Abstract
Insulin resistance and pancreatic beta cell dysfunction are major contributors to the pathogenesis of diabetes. Various conditions play a role in the pathogenesis of pancreatic beta cell dysfunction and are correlated with endoplasmic reticulum (ER) stress. Pancreatic beta cells are susceptible to ER stress. Many studies have shown that increased ER stress induces pancreatic beta cell dysfunction and diabetes mellitus using genetic models of ER stress and by various stimuli. There are many reports indicating that ER stress plays an important role in the impairment of insulin biosynthesis, suggesting that reduction of ER stress could be a therapeutic target for diabetes. In this paper, we reviewed the relationship between ER stress and diabetes and how ER stress controls insulin biosynthesis.
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Riedel MJ, Asadi A, Wang R, Ao Z, Warnock GL, Kieffer TJ. Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas. Diabetologia 2012; 55:372-81. [PMID: 22038519 DOI: 10.1007/s00125-011-2344-9] [Citation(s) in RCA: 119] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2011] [Accepted: 08/30/2011] [Indexed: 12/18/2022]
Abstract
AIMS/HYPOTHESIS In adult human islets, insulin and glucagon production is largely restricted to individual cell populations. The production of these hormones is less segregated during development and during the differentiation of human pluripotent stem cells towards pancreatic lineages. We therefore sought to characterise the transcription factor profile of these cells that co-produce insulin and glucagon in the developing human pancreas, and thus to gain insight into their potential fate during normal pancreas development. METHODS An immunohistochemical analysis was performed on human pancreas sections from fetal donors aged 9 to 21 weeks and from adult donors between the ages of 17 and 55 years. RESULTS Endocrine cells were observed within the pancreas at all ages examined, with cells co-producing insulin and glucagon observed as early as 9 weeks of fetal age. The population of cells that co-produce insulin and glucagon generally decreased in prevalence with age, with negligible numbers in adult pancreas. From 9 to 16 weeks, the population of glucagon-only cells increased, while the insulin-only cells decreased in abundance. Cells that co-produced insulin and glucagon also produced the alpha cell transcription factor, aristaless related homeobox (ARX), and lacked the beta cell transcription factors pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1) and v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA). CONCLUSIONS/INTERPRETATION Our results indicate that cells co-producing insulin and glucagon in the developing human pancreas share a transcription factor profile that is similar to that of mature alpha cells and suggest that some maturing alpha cells briefly exhibit ectopic insulin expression. Thus cells that co-produce insulin and glucagon may represent a transient cell population, which gives rise to mature alpha cells.
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Affiliation(s)
- M J Riedel
- Laboratory of Molecular and Cellular Medicine, Department of Cellular and Physiological Sciences, Life Sciences Institute, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z3
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35
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Kuwabara T, Kagalwala MN, Onuma Y, Ito Y, Warashina M, Terashima K, Sanosaka T, Nakashima K, Gage FH, Asashima M. Insulin biosynthesis in neuronal progenitors derived from adult hippocampus and the olfactory bulb. EMBO Mol Med 2011; 3:742-54. [PMID: 21984534 PMCID: PMC3377118 DOI: 10.1002/emmm.201100177] [Citation(s) in RCA: 101] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2011] [Revised: 08/04/2011] [Accepted: 08/08/2011] [Indexed: 12/15/2022] Open
Abstract
In the present study, we demonstrated that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from the hippocampus and olfactory bulb (OB). Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampal and OB-derived neural stem cells. Our analysis indicated that the balance between Wnt3, which triggers the expression of insulin via NeuroD1, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on diabetic (DB) status. We also show that adult neural progenitors derived from DB animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of DB animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes.
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Affiliation(s)
- Tomoko Kuwabara
- Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City, Japan.
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36
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Lombardo MF, De Angelis F, Bova L, Bartolini B, Bertuzzi F, Nano R, Capuani B, Lauro R, Federici M, Lauro D, Donadel G. Human placental lactogen (hPL-A) activates signaling pathways linked to cell survival and improves insulin secretion in human pancreatic islets. Islets 2011; 3:250-8. [PMID: 21765243 PMCID: PMC3219159 DOI: 10.4161/isl.3.5.16900] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The search for factors either promoting islets proliferation or survival during adult life is a major issue for both type 1 and 2 diabetes mellitus. Among factors with mitogenic activity on pancreatic β-cells, human placental lactogen (hPL) showed stronger activity when compared to the other lactogen hormones: growth hormone (GH) and prolactin (PRL). The aim of the present work is to elucidate the biological and molecular events of hPL isoform A (hPL-A) activity on human cultured islets. We used pure human pancreatic islets and insulinoma cell lines (βTC-1 and RIN, murine and rat respectively) stimulated with hPL-A recombinant protein and we compared hPL-A activity with that of hGH. We showed that hPL-A inhibits apoptosis, both in insulinoma and human islets, by the phosphorylation of AKT protein. Indeed, the antiapoptotic role of hPL-A was mediated by PI3K, p38 and it was independent by PKA, Erk1/2. Compared with hGH, hPL-A modulated at different intervals and/or intensity by the phosphorylation of JAKs/STATs and MAPKinases. Moreover, hPL-A induced PDX-1 intracellular expression, improving beta cell activity and ameliorating insulin secretion in response to high glucose stimulation. Our data support the idea that hPL-A is involved in the regulation of beta cells activity. Importantly, we found that hPL-A can preserve and improve the ability of purified human pancreatic islets cultured to secrete insulin in vitro.
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Affiliation(s)
- Marco F Lombardo
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | | | - Luca Bova
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | - Barbara Bartolini
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | - Federico Bertuzzi
- Cell Therapy for Type 1 Diabetes Unit; San Raffaele Scientific Institute; Milan, Italy
| | - Rita Nano
- Cell Therapy for Type 1 Diabetes Unit; San Raffaele Scientific Institute; Milan, Italy
| | - Barbara Capuani
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | - Renato Lauro
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | - Massimo Federici
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | - Davide Lauro
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
| | - Giulia Donadel
- Department of Internal Medicine; University of Rome Tor Vergata; Rome
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Oxidative stress and redox modulation potential in type 1 diabetes. Clin Dev Immunol 2011; 2011:593863. [PMID: 21647409 PMCID: PMC3102468 DOI: 10.1155/2011/593863] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2011] [Accepted: 03/09/2011] [Indexed: 12/21/2022]
Abstract
Redox reactions are imperative to preserving cellular metabolism yet must be strictly regulated. Imbalances between reactive oxygen species (ROS) and antioxidants can initiate oxidative stress, which without proper resolve, can manifest into disease. In type 1 diabetes (T1D), T-cell-mediated autoimmune destruction of pancreatic β-cells is secondary to the primary invasion of macrophages and dendritic cells (DCs) into the islets. Macrophages/DCs, however, are activated by intercellular ROS from resident pancreatic phagocytes and intracellular ROS formed after receptor-ligand interactions via redox-dependent transcription factors such as NF-κB. Activated macrophages/DCs ferry β-cell antigens specifically to pancreatic lymph nodes, where they trigger reactive T cells through synapse formation and secretion of proinflammatory cytokines and more ROS. ROS generation, therefore, is pivotal in formulating both innate and adaptive immune responses accountable for islet cell autoimmunity. The importance of ROS/oxidative stress as well as potential for redox modulation in the context of T1D will be discussed.
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Salbaum JM, Kappen C. Diabetic embryopathy: a role for the epigenome? ACTA ACUST UNITED AC 2011; 91:770-80. [PMID: 21538816 DOI: 10.1002/bdra.20807] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2010] [Revised: 02/04/2011] [Accepted: 02/11/2011] [Indexed: 12/28/2022]
Abstract
Embryonic development under adverse conditions, such as maternal diabetes or obesity during pregnancy, constitutes a major risk factor for birth defects, as well as for long-term health consequences and disease susceptibility in the offspring. While contributions from epigenetic changes have been invoked previously to explain the long-term changes in terms of developmental programming, we here review how maternal metabolism may directly affect the embryonic epigenome in relationship to teratogenic processes. We consider four epigenetic modalities--DNA methylation, non-coding RNA, transcription factors, and histone modifications--and their contribution to epigenetic memory, and discuss how epigenomic changes may mediate the altered control of embryonic gene expression brought about by maternal diabetes. In combination, the epigenomic modalities serve to define transcription-permissive domains of the genome, resulting in distinct epigenomic landscapes in different developmental cell types. We evaluate experimental approaches to characterize the epigenome in adverse pregnancy conditions, highlighting the role of next-generation sequencing on the technological side, while emphasizing the necessity to study defined cell populations in terms of biologic impact. Finally, we outline the challenges in moving from findings that correlate epigenomics to developmental phenotypes to scenarios that establish teratogenic causality.
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Affiliation(s)
- J Michael Salbaum
- Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, USA.
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Gefen-Halevi S, Rachmut IH, Molakandov K, Berneman D, Mor E, Meivar-Levy I, Ferber S. NKX6.1 promotes PDX-1-induced liver to pancreatic β-cells reprogramming. Cell Reprogram 2011; 12:655-64. [PMID: 21108535 DOI: 10.1089/cell.2010.0030] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Reprogramming adult mammalian cells is an attractive approach for generating cell-based therapies for degenerative diseases, such as diabetes. Adult human liver cells exhibit a high level of developmental plasticity and have been suggested as a potential source of pancreatic progenitor tissue. An instructive role for dominant pancreatic transcription factors in altering the hepatic developmental fate along the pancreatic lineage and function has been demonstrated. Here we analyze whether transcription factors expressed in mature pancreatic β-cells preferentially activate β-cell lineage differentiation in liver. NKX6.1 is a transcription factor uniquely expressed in β-cells of the adult pancreas, its potential role in reprogramming liver cells to pancreatic lineages has never been analyzed. Our results suggest that NKX6.1 activates immature pancreatic markers such as NGN-3 and ISL-1 but not pancreatic hormones gene expression in human liver cells. We hypothesized that its restricted capacity to activate a wide pancreatic repertoire in liver could be related to its incapacity to activate endogenous PDX-1 expression in liver cells. Indeed, the complementation of NKX6.1 by ectopic PDX-1 expression substantially and specifically promoted insulin expression and glucose regulated processed hormone secretion to a higher extent than that of PDX-1 alone, without increasing the reprogrammed cells. This may suggest a potential role for NKX6.1 in promoting PDX-1 reprogrammed cells maturation along the β-cell-like lineage. By contrast, NKX6.1 repressed PDX-1 induced proglucagon gene expression. The individual and concerted effects of pancreatic transcription factors in adult extra-pancreatic cells, is expected to facilitate developing regenerative medicine approaches for cell replacement therapy in diabetics.
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Affiliation(s)
- Shiraz Gefen-Halevi
- Sheba Regenerative Medicine, Stem cells and Tissue engineering Center , Sheba Medical Center, Tel-Hashomer, Israel
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Balentine CJ, Berger DH, Liu SH, Chen C, Nemunaitis J, Brunicardi FC. Defining the cancer master switch. World J Surg 2011; 35:1738-45. [PMID: 21286716 DOI: 10.1007/s00268-010-0941-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BACKGROUND Recent research has focused on signaling cascades and their interactions yielding considerable insight into which genetic pathways are targeted and how they tend to be altered in tumors. Therapeutic interventions now can be designed based on the knowledge of pathways vital to tumor growth and survival. These critical targets for intervention, master switches for cancer, are termed so because the tumor attempts to "flip the switch" in a way that promotes its survival, whereas molecular therapy aims to "switch off" signals important for tumor-related processes. METHODS Literature review. CONCLUSIONS Defining useful targets for therapy depends on identifying pathways that are crucial for tumor growth, survival, and metastasis. Because not all signaling cascades are created equal, selecting master switches or targets for intervention needs to be done in a systematic fashion. This discussion proposes a set of criteria to define what it means to be a cancer master switch and provides examples to illustrate their application.
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41
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Stein R. Insulin Gene Transcription: Factors Involved in Cell Type–Specific and Glucose‐Regulated Expression in Islet β Cells are Also Essential During Pancreatic Development. Compr Physiol 2011. [DOI: 10.1002/cphy.cp070202] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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Rapamycin suppresses the expansion and differentiation of porcine neonatal pancreas cell clusters. Transplantation 2010; 90:717-24. [PMID: 20622751 DOI: 10.1097/tp.0b013e3181eceaaf] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND The role of rapamycin in pancreas stem cells remains to be clearly elucidated. Herein, we evaluated the effects of rapamycin on porcine neonatal pancreas cell clusters (NPCCs), which primarily comprised pancreatic precursors, and attempted to find an intracellular mechanism about the harmful effects of rapamycin. METHODS Porcine NPCCs were treated with rapamycin in a monolayer, and the apoptosis and proliferation were determined via caspase-3 assay and H-thymidine uptake analysis. The expression of transcription factors was assessed via reverse-transcriptase polymerase chain reaction and Western blotting. For the in vivo study, the porcine NPCCs were transplanted into the kidney subcapsules of normal nude mice and treated with rapamycin. RESULTS Rapamycin treatment significantly reduced the number of β cells, glucose-stimulated insulin secretion, and the insulin contents in the monolayer-cultured porcine NPCCs. Furthermore, rapamycin treatment increased the apoptosis and inhibited the proliferation of β cells in the culture dishes. The expressions of the insulin, pancreatic and duodenal homeobox-1, and NeuroD/Beta2 genes were down-regulated via rapamycin treatment. The expression of insulin-like growth factor-II was significantly down-regulated, but the expression of Foxo1 was simultaneously inversely increased, and the translocation of Foxo1 from the cytoplasm to the nucleus was induced by rapamycin treatment. Moreover, rapamycin treatment induced a marked reduction in the relative volume and absolute mass of β cells in the porcine NPCCs grafts at 8 weeks after transplantation in the normal nude mice. CONCLUSIONS Here, we demonstrate that rapamycin treatment suppresses the expansion and differentiation of porcine NPCCs, and the alteration of Foxo1 and insulin-like growth factor-II gene expression might be the crucial factors.
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Wolf G, Hessabi B, Karkour A, Henrion U, Dahlhaus M, Ostmann A, Giese B, Fraunholz M, Grabarczyk P, Jack R, Walther R. The activation of the rat insulin gene II by BETA2 and PDX-1 in rat insulinoma cells is repressed by Pax6. Mol Endocrinol 2010; 24:2331-42. [PMID: 20943817 DOI: 10.1210/me.2009-0220] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The transcriptional transactivator Pax6 binds the pancreatic islet cell-specific enhancer sequence (PISCES) of the rat insulin I gene. However the human, mouse, and rat insulin gene II promoters do not contain a PISCES element. To analyze the role of Pax6 in those PISCES-less promoters, we investigated its influence on rat insulin gene II expression and included in our studies the main activators: pancreatic and duodenal homeobox protein-1 (PDX-1) and BETA2/E47. Luciferase assays, Northern blots, and RIA were used to study effects of Pax6 overexpression, gel shift and chromatin precipitation assays to study its binding to the DNA, and yeast two-hybrid assays and glutathione S transferase capture assays to investigate its interactions with PDX-1 and BETA2. Finally, glucose-dependent intracellular transport of Pax6 was demonstrated by fluorescence microscopy. Overexpression of Pax6 prevents activation of the rat insulin II gene by BETA2 and PDX-1 and hence suppresses insulin synthesis and secretion. In vitro, Pax6 binds to the A-boxes, thereby blocking binding of PDX-1, and at the same time, its paired domain interacts with BETA2. Fluorescence microscopy demonstrated that the nuclear-cytoplasmic localization of Pax6 and PDX-1 are oppositely regulated by glucose. From the results, it is suggested that at low concentrations of glucose, Pax6 is localized in the nucleus and prevents the activation of the insulin gene by occupying the PDX-1 binding site and by interacting with BETA2.
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Affiliation(s)
- Gabriele Wolf
- Department of Medical Biochemistry and Molecular Biology, University of Greifswald, Klinikum, Greifswald, Germany
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Banerjee I, Sharma N, Yarmush M. Impact of co-culture on pancreatic differentiation of embryonic stem cells. J Tissue Eng Regen Med 2010; 5:313-23. [PMID: 20717889 DOI: 10.1002/term.317] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Accepted: 04/16/2010] [Indexed: 02/06/2023]
Abstract
Promise of cellular therapy for type 1 diabetes has inspired the search for transplantable cell sources, and embryonic stem cells (ESCs) have emerged as strong candidates. We have developed a directed differentiation protocol to obtain insulin-producing cells from ESCs. The ESCs are first induced towards a homogeneous monolayer of definitive endoderm-like cells by co-culture with primary hepatocytes. Pancreatic commitment is induced by plating the ESC-derived endoderms on Matrigel, along with Sonic hedgehog inhibition and retinoid induction. More than 70% of differentiated cells positively upregulated Pdx-1, along with pro-endocrine transcription factors Ngn3, β2/neroD1, Nkx2.2 and Nkx6.1. Final maturation to islet-specific cells is achieved by co-culturing the ESC-derived pancreatic endocrine cells with endothelial cells, which resulted in Insulin 1 upregulation in 60% of the cell population, along with high levels of IAPP and Glut2. The differentiated cell population also secreted high levels of insulin. Our findings illustrate the significant effect of co-culture in different stages of differentiation and maturation of ESCs in vitro. Such a high yield of pancreatic islet cells has not yet been reported. Our findings establish a robust protocol for islet differentiation.
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Affiliation(s)
- Ipsita Banerjee
- Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA.
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Abstract
Low birth weight is an important risk factor for impaired glucose tolerance and diabetes later in life. One hypothesis is that fetal beta-cells inherit a persistent defect as a developmental response to fetal malnutrition, a primary cause of intrauterine growth restriction (IUGR). Our understanding of fetal programing events in the human endocrine pancreas is limited, but several animal models of IUGR extend our knowledge of developmental programing in beta-cells. Pathological outcomes such as beta-cell dysfunction, impaired glucose tolerance, and diabetes are often observed in adult offspring from these animal models, similar to the associations of low birth weight and metabolic diseases in humans. However, the identified mechanisms underlying beta-cell dysfunction across models and species are varied, likely resulting from the different methodologies used to induce experimental IUGR, as well as from intraspecies differences in pancreas development. In this review, we first present the evidence for human beta-cell dysfunction being associated with low birth weight or IUGR. We then evaluate relevant animal models of IUGR, focusing on the strengths of each, in order to define critical periods and types of nutrient deficiencies that can lead to impaired beta-cell function. These findings frame our current knowledge of beta-cell developmental programing and highlight future research directions to clarify the mechanisms of beta-cell dysfunction for human IUGR.
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Affiliation(s)
- Alice S. Green
- Department of Animal Sciences, University of Arizona, Tucson, AZ
| | - Paul J. Rozance
- Department of Pediatrics, University of Colorado, Denver, CO
| | - Sean W. Limesand
- Department of Animal Sciences, University of Arizona, Tucson, AZ
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McDonald E, Krishnamurthy M, Goodyer CG, Wang R. The Emerging Role of SOX Transcription Factors in Pancreatic Endocrine Cell Development and Function. Stem Cells Dev 2009; 18:1379-88. [DOI: 10.1089/scd.2009.0240] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022] Open
Affiliation(s)
- Erin McDonald
- Children’s Health Research Institute, University of Western Ontario, London, Ontario, Canada
| | - Mansa Krishnamurthy
- Children’s Health Research Institute, University of Western Ontario, London, Ontario, Canada
| | - Cynthia G. Goodyer
- Department of Pediatrics, McGill University Health Centre, Children’s Hospital Research Institute, Montreal, Quebec, Canada
| | - Rennian Wang
- Children’s Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
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47
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Vincent RK, Odorico JS. Reduced serum concentration is permissive for increased in vitro endocrine differentiation from murine embryonic stem cells. Differentiation 2009; 78:24-34. [PMID: 19446949 DOI: 10.1016/j.diff.2009.03.006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2009] [Accepted: 03/15/2009] [Indexed: 10/20/2022]
Abstract
Embryonic stem cells (ESCs) have been shown to be capable of differentiating into pancreatic progenitors and insulin-producing cells in vitro. However, before ESC derivatives can be used in clinical settings, efficient selective differentiation needs to be achieved. Essential to improving ESC differentiation to islet endocrine cells is an understanding of the influences of extrinsic signals and transcription factors on cell specification. Herein, we investigate the influence of serum-supplemented growth conditions on the differentiation of murine ESCs to endocrine lineages in the context of over-expression of two pancreatic transcription factors, Pdx1 and Ngn3. To study the effect of different serum formulations and concentrations on the ability of murine ESCs to differentiate into endocrine cells in vitro, cells were grown into embryoid bodies and then differentiated in various serum replacement (SR), fetal calf serum (FCS) and serum-free conditions. Using immunohistochemistry and quantitative real-time RT-PCR (QPCR), we found that, of the conditions tested, 1% SR differentiation medium resulted in the highest levels of insulin-1 mRNA and significantly increased the total number of insulin-expressing cells. Applying this knowledge to cell lines in which Pdx1 or Ngn3 transgene expression could be induced by exposure to doxycycline we differentiated TetPDX1 and TetNgn3 ESCs under conditions of either 10% FCS or 1% SR medium. In the presence of 10% serum, induced expression of either Pdx1 or Ngn3 in differentiating ESCs resulted in modest increases in hormone transcripts and cell counts. However, changing the serum formulation from 10% FCS to 1% SR significantly enhanced the number of insulin+/C-peptide+ cells in parallel with increased insulin-1 transcript levels in both inducible cell lines. In summary, these data demonstrate that induced expression of key pancreatic transcription factors in combination with low serum/SR concentrations increases endocrine cell differentiation from murine ESCs.
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Affiliation(s)
- Robert K Vincent
- Division of Transplantation, Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792, USA
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Wang H, Wang S, Hu J, Kong Y, Chen S, Li L, Li L. Oct4 is expressed in Nestin-positive cells as a marker for pancreatic endocrine progenitor. Histochem Cell Biol 2009; 131:553-63. [PMID: 19224238 DOI: 10.1007/s00418-009-0560-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/22/2009] [Indexed: 01/18/2023]
Abstract
There are abundant progenitor cells in the developing pancreas, but molecular markers for these cells are lacking. Octamer-binding transcription factor-4 (Oct4) is an important transcription factor for keeping the features of self-renewal and pluripotency of embryonic stem cells. It's well known that Oct4, as a totipotent stem cells marker, just is expressed in totipotent stem cells. In the present study, we collected ten human fetal pancreases, and found that Oct4 mRNA and protein were expressed in human fetal pancreas samples by RT-PCR, western blot and immunohistochemistry assays. Using double-staining, we demonstrated that Oct4 was not co-expressed with Chromogranin A (a peptide expressed in endocrine cells), but partially co-expressed with Ngn3 (a transcription factor expressed in pancreatic endocrine precursor cells) and Nestin (a intermediate filament, Nestin-positive cells isolated from islets can be induced to express insulin) in human fetal pancreases. Indeed, we prepared Nestin-positive cells from human fetal pancreas by cell selection, and found that these cells expressed Oct4 and Ngn3. The Nestin-positive cells displayed a rapid duplication and could differentiate into osteoblasts, fat and endocrine cells in vitro. These results indicated that the Nestin-positive cells in the fetal age should be pancreatic progenitor cells. Overall, our study suggested that Oct4 was a marker for pancreatic endocrine progenitor.
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Affiliation(s)
- Hong Wang
- Stem Cell Research Center, Health Science Center, Peking University, No. 38 Xueyuan Road, Haidian District, Beijing, China.
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Francini F, Del Zotto H, Massa ML, Gagliardino JJ. Selective effect of INGAP-PP upon mouse embryonic stem cell differentiation toward islet cells. ACTA ACUST UNITED AC 2009; 153:43-8. [DOI: 10.1016/j.regpep.2008.12.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2008] [Revised: 11/07/2008] [Accepted: 12/15/2008] [Indexed: 12/28/2022]
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Martin CC, Flemming BP, Wang Y, Oeser JK, O’Brien RM. Foxa2 and MafA regulate islet-specific glucose-6-phosphatase catalytic subunit-related protein gene expression. J Mol Endocrinol 2008; 41:315-28. [PMID: 18753309 PMCID: PMC2614309 DOI: 10.1677/jme-08-0062] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP/G6PC2) is a major autoantigen in both mouse and human type 1 diabetes. IGRP is selectively expressed in islet beta cells and polymorphisms in the IGRP gene have recently been associated with variations in fasting blood glucose levels and cardiovascular-associated mortality in humans. Chromatin immunoprecipitation (ChIP) assays have shown that the IGRP promoter binds the islet-enriched transcription factors Pax-6 and BETA2. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2. Single binding sites for these factors were identified in the proximal IGRP promoter, mutation of which resulted in decreased IGRP fusion gene expression in betaTC-3, Hamster insulinoma tumor (HIT), and Min6 cells. ChiP assays have shown that the islet-enriched transcription factor Pdx-1 also binds the IGRP promoter, but mutational analysis of four Pdx-1 binding sites in the proximal IGRP promoter revealed surprisingly little effect of Pdx-1 binding on IGRP fusion gene expression in betaTC-3 cells. In contrast, in both HIT and Min6 cells mutation of these four Pdx-1 binding sites resulted in a approximately 50% reduction in fusion gene expression. These data suggest that the same group of islet-enriched transcription factors, namely Pdx-1, Pax-6, MafA, BETA2, and Foxa2, directly or indirectly regulate expression of the two major autoantigens in type 1 diabetes.
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Affiliation(s)
| | | | | | | | - Richard M. O’Brien
- To whom correspondence should be addressed: Department of Molecular Physiology and Biophysics, 8415 MRB IV, 2213 Garland Ave, Vanderbilt University Medical School, Nashville, TN 37232-0615, Telephone (615) 936-1503; Facsimile (615) 322-7236, E-mail:
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