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Skarmaliorakis I, Vasilopoulou A, Gutierrez de Piñeres V, Yannakoulia M, Anastasiou CA, Mantzoros CS. Regulation of proglucagon derived peptides by carbohydrate and protein ingestion in young healthy males-A randomized, double-blind, cross-over trial. Clin Nutr 2025; 44:33-40. [PMID: 39612864 DOI: 10.1016/j.clnu.2024.11.025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Revised: 10/09/2024] [Accepted: 11/11/2024] [Indexed: 12/01/2024]
Abstract
BACKGROUND & AIMS The ingestion of macronutrients triggers the release of several incretin peptides from the gastrointestinal system, which have both insulinotropic and satiety-inducing properties. The effect of the meal's macronutrient content on the secretion of these peptides has not been adequately studied, particularly concerning the secretion of the newly characterized proglucagon-derived peptides (PGDPs). We aimed to examine the effect of a meal's macronutrient content, specifically its protein versus carbohydrate content, on postprandial PGDPs responses in healthy men. METHODS Ten apparently healthy, normal-weight males completed a trial consisting of two interventions in a randomized, double-blind, crossover design. In one intervention, participants consumed an isocaloric high-protein breakfast (65 g of glucose, 60 g of protein), while in the other, participants consumed a carbohydrate-rich breakfast (125 g of glucose). Levels of all seven PGDPs, namely glucagon-like peptide-1 and -2 (GLP-1 and GLP-2), oxyntomodulin, glicentin, major pro-glucagon fragment (MPGF), glucagon and proglucagon, as well as glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide total and total plus (GIP total and GIP total plus) levels were measured at baseline, every 15 minutes for the first hour and every 30 minutes for the second and third hours after each meal. RESULTS The two interventions produced similar glycemic and insulinemic responses, while total amino acids increased more over time in response to protein administration. Levels of proglucagon (F(8) = 4.114, p = 0.001) and the primarily pancreas-secreted glucagon and MPGF (F(8) = 3.088, p = 0.005) rose significantly during the protein intervention. GIP total and GIP total plus increased in response to carbohydrate ingestion. No major overall differences were observed for the primarily intestinally secreted GLP-1, oxyntomodulin and glicentin between the two arms of the trial, although their levels tended to increase earlier in response to carbohydrates and later in response to protein administration, especially in the case of GLP-2 levels. CONCLUSIONS The carbohydrate vs. protein content of a meal differentially increases the levels of GIP and PGDPs during the postprandial period. Dose-response studies and comparisons with lipid intake may further advance our knowledge of the physiology of these clinically important molecules and their implications in energy homeostasis.
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Affiliation(s)
- Ioannis Skarmaliorakis
- Department of Nutrition and Dietetics, School of Health Sciences & Education, Harokopio University, GR 17671 Athens, Greece
| | - Antonia Vasilopoulou
- Department of Nutrition and Dietetics, School of Health Sciences & Education, Harokopio University, GR 17671 Athens, Greece
| | - Valeria Gutierrez de Piñeres
- Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Mary Yannakoulia
- Department of Nutrition and Dietetics, School of Health Sciences & Education, Harokopio University, GR 17671 Athens, Greece
| | - Costas A Anastasiou
- Department of Nutrition and Dietetics, School of Health Sciences & Education, Harokopio University, GR 17671 Athens, Greece
| | - Christos S Mantzoros
- Department of Internal Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
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Gong K, Xue C, Feng Z, Pan R, Wang M, Chen S, Chen Y, Guan Y, Dai L, Zhang S, Jiang L, Li L, Wang B, Yin Z, Ma L, Iwakiri Y, Tang J, Liao C, Chen H, Duan Y. Intestinal Nogo-B reduces GLP1 levels by binding to proglucagon on the endoplasmic reticulum to inhibit PCSK1 cleavage. Nat Commun 2024; 15:6845. [PMID: 39122737 PMCID: PMC11315690 DOI: 10.1038/s41467-024-51352-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2023] [Accepted: 08/02/2024] [Indexed: 08/12/2024] Open
Abstract
Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.
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Affiliation(s)
- Ke Gong
- Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
- Department of Physiology, Faculty of Basic Medical Sciences, Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Shiyan, Hubei, China
- Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, Hefei University of Technology, Hefei, Anhui, China
| | - Chao Xue
- College of Life Sciences, Key Laboratory of Bioactive Materials of Ministry of Education, Nankai University, Tianjin, China
| | - Zian Feng
- Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Ruru Pan
- Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Mengyao Wang
- Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, Hefei University of Technology, Hefei, Anhui, China
| | - Shasha Chen
- Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, Hefei University of Technology, Hefei, Anhui, China
| | - Yuanli Chen
- Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, Hefei University of Technology, Hefei, Anhui, China
| | - Yudong Guan
- Department of Geriatrics, and Shenzhen Clinical Research Centre for Geriatrics, Shenzhen People's Hospital, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Lingyun Dai
- Department of Geriatrics, and Shenzhen Clinical Research Centre for Geriatrics, Shenzhen People's Hospital, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong, China
| | - Shuang Zhang
- Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, Hefei University of Technology, Hefei, Anhui, China
| | - Liwei Jiang
- Laboratory of Immunoengineering, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
| | - Ling Li
- Laboratory of Immunoengineering, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
| | - Bei Wang
- Department of Pathology, China-Japan Friendship Hospital, Beijing, China
| | - Zequn Yin
- Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Likun Ma
- Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Yasuko Iwakiri
- Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA
| | - Junming Tang
- Department of Physiology, Faculty of Basic Medical Sciences, Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Shiyan, Hubei, China
| | - Chenzhong Liao
- Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher Education Institutes, Hefei University of Technology, Hefei, Anhui, China
| | - Houzao Chen
- Department of Biochemistry & Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
| | - Yajun Duan
- Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
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Marafie SK, Al-Mulla F. An Overview of the Role of Furin in Type 2 Diabetes. Cells 2023; 12:2407. [PMID: 37830621 PMCID: PMC10571965 DOI: 10.3390/cells12192407] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2023] [Accepted: 09/27/2023] [Indexed: 10/14/2023] Open
Abstract
Post-translational modifications (PTMs) play important roles in regulating several human diseases, like cancer, neurodegenerative disorders, and metabolic disorders. Investigating PTMs' contribution to protein functions is critical for modern biology and medicine. Proprotein convertases (PCs) are irreversible post-translational modifiers that have been extensively studied and are considered as key targets for novel therapeutics. They cleave proteins at specific sites causing conformational changes affecting their functions. Furin is considered as a PC model in regulating growth factors and is involved in regulating many pro-proteins. The mammalian target of the rapamycin (mTOR) signaling pathway is another key player in regulating cellular processes and its dysregulation is linked to several diseases including type 2 diabetes (T2D). The role of furin in the context of diabetes has been rarely explored and is currently lacking. Moreover, furin variants have altered activity that could have implications on overall health. In this review, we aim to highlight the role of furin in T2D in relation to mTOR signaling. We will also address furin genetic variants and their potential effect on T2D and β-cell functions. Understanding the role of furin in prediabetes and dissecting it from other confounding factors like obesity is crucial for future therapeutic interventions in metabolic disorders.
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Affiliation(s)
- Sulaiman K. Marafie
- Biochemistry and Molecular Biology Department, Dasman Diabetes Institute, P.O. Box 1180, Dasman 15462, Kuwait
| | - Fahd Al-Mulla
- Genetics and Bioinformatics Department, Dasman Diabetes Institute, P.O. Box 1180, Dasman 15462, Kuwait
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Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice. Mol Metab 2022; 66:101627. [PMID: 36374777 PMCID: PMC9664468 DOI: 10.1016/j.molmet.2022.101627] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2022] [Revised: 10/23/2022] [Accepted: 10/28/2022] [Indexed: 11/06/2022] Open
Abstract
OBJECTIVE The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.
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5
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Asadi F, Dhanvantari S. Pathways of Glucagon Secretion and Trafficking in the Pancreatic Alpha Cell: Novel Pathways, Proteins, and Targets for Hyperglucagonemia. Front Endocrinol (Lausanne) 2021; 12:726368. [PMID: 34659118 PMCID: PMC8511682 DOI: 10.3389/fendo.2021.726368] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Accepted: 09/13/2021] [Indexed: 12/15/2022] Open
Abstract
Patients with diabetes mellitus exhibit hyperglucagonemia, or excess glucagon secretion, which may be the underlying cause of the hyperglycemia of diabetes. Defective alpha cell secretory responses to glucose and paracrine effectors in both Type 1 and Type 2 diabetes may drive the development of hyperglucagonemia. Therefore, uncovering the mechanisms that regulate glucagon secretion from the pancreatic alpha cell is critical for developing improved treatments for diabetes. In this review, we focus on aspects of alpha cell biology for possible mechanisms for alpha cell dysfunction in diabetes: proglucagon processing, intrinsic and paracrine control of glucagon secretion, secretory granule dynamics, and alterations in intracellular trafficking. We explore possible clues gleaned from these studies in how inhibition of glucagon secretion can be targeted as a treatment for diabetes mellitus.
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Affiliation(s)
- Farzad Asadi
- Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada
- Program in Metabolism and Diabetes, Lawson Health Research Institute, London, ON, Canada
| | - Savita Dhanvantari
- Department of Pathology and Laboratory Medicine, Western University, London, ON, Canada
- Program in Metabolism and Diabetes, Lawson Health Research Institute, London, ON, Canada
- Imaging Research Program, Lawson Health Research Institute, London, ON, Canada
- Department of Medical Biophysics, Western University, London, ON, Canada
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6
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Abstract
The kexin-like proprotein convertases perform the initial proteolytic cleavages that ultimately generate a variety of different mature peptide and proteins, ranging from brain neuropeptides to endocrine peptide hormones, to structural proteins, among others. In this review, we present a general introduction to proprotein convertase structure and biochemistry, followed by a comprehensive discussion of each member of the kexin-like subfamily of proprotein convertases. We summarize current knowledge of human proprotein convertase insufficiency syndromes, including genome-wide analyses of convertase polymorphisms, and compare these to convertase null and mutant mouse models. These mouse models have illuminated our understanding of the roles specific convertases play in human disease and have led to the identification of convertase-specific substrates; for example, the identification of procorin as a specific PACE4 substrate in the heart. We also discuss the limitations of mouse null models in interpreting human disease, such as differential precursor cleavage due to species-specific sequence differences, and the challenges presented by functional redundancy among convertases in attempting to assign specific cleavages and/or physiological roles. However, in most cases, knockout mouse models have added substantively both to our knowledge of diseases caused by human proprotein convertase insufficiency and to our appreciation of their normal physiological roles, as clearly seen in the case of the furin, proprotein convertase 1/3, and proprotein convertase 5/6 mouse models. The creation of more sophisticated mouse models with tissue- or temporally-restricted expression of specific convertases will improve our understanding of human proprotein convertase insufficiency and potentially provide support for the emerging concept of therapeutic inhibition of convertases.
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Affiliation(s)
- Manita Shakya
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, USA
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, USA
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7
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Novel Homozygous Inactivating Mutation in the PCSK1 Gene in an Infant with Congenital Malabsorptive Diarrhea. Genes (Basel) 2021; 12:genes12050710. [PMID: 34068683 PMCID: PMC8151971 DOI: 10.3390/genes12050710] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 05/02/2021] [Accepted: 05/07/2021] [Indexed: 01/03/2023] Open
Abstract
Proprotein convertase 1/3 (PC1/3), encoded by the PCSK1 gene, is expressed in neuronal and (entero)endocrine cell types, where it cleaves and hence activates a number of protein precursors that play a key role in energy homeostasis. Loss-of-function mutations in PCSK1 cause a recessive complex endocrinopathy characterized by malabsorptive diarrhea and early-onset obesity. Despite the fact that neonatal malabsorptive diarrhea is observed in all patients, it has remained understudied. The aim of this study was to investigate the enteroendocrine pathologies in a male patient with congenital PCSK1 deficiency carrying the novel homozygous c.1034A>C (p.E345A) mutation. This patient developed malabsorptive diarrhea and metabolic acidosis within the first week of life, but rapid weight gain was observed after total parenteral nutrition, and he displayed high proinsulin levels and low adrenocorticotropin. In vitro analysis showed that the p.E345A mutation in PC1/3 resulted in a (near) normal autocatalytic proPC1/3 processing and only partially impaired PC1/3 secretion, but the processing of a substrate in trans was completely blocked. Immunohistochemical staining did not reveal changes in the proGIP/GIP and proglucagon/GLP-1 ratio in colonic tissue. Hence, we report a novel PCSK1 deficient patient who, despite neonatal malabsorptive diarrhea, showed a normal morphology in the small intestine.
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8
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Saikia M, Holter MM, Donahue LR, Lee IS, Zheng QC, Wise JL, Todero JE, Phuong DJ, Garibay D, Coch R, Sloop KW, Garcia-Ocana A, Danko CG, Cummings BP. GLP-1 receptor signaling increases PCSK1 and β cell features in human α cells. JCI Insight 2021; 6:141851. [PMID: 33554958 PMCID: PMC7934853 DOI: 10.1172/jci.insight.141851] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 12/29/2020] [Indexed: 02/06/2023] Open
Abstract
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates glucose-stimulated insulin secretion. GLP-1 is classically produced by gut L cells; however, under certain circumstances α cells can express the prohormone convertase required for proglucagon processing to GLP-1, prohormone convertase 1/3 (PC1/3), and can produce GLP-1. However, the mechanisms through which this occurs are poorly defined. Understanding the mechanisms by which α cell PC1/3 expression can be activated may reveal new targets for diabetes treatment. Here, we demonstrate that the GLP-1 receptor (GLP-1R) agonist, liraglutide, increased α cell GLP-1 expression in a β cell GLP-1R-dependent manner. We demonstrate that this effect of liraglutide was translationally relevant in human islets through application of a new scRNA-seq technology, DART-Seq. We found that the effect of liraglutide to increase α cell PC1/3 mRNA expression occurred in a subcluster of α cells and was associated with increased expression of other β cell-like genes, which we confirmed by IHC. Finally, we found that the effect of liraglutide to increase bihormonal insulin+ glucagon+ cells was mediated by the β cell GLP-1R in mice. Together, our data validate a high-sensitivity method for scRNA-seq in human islets and identify a potentially novel GLP-1-mediated pathway regulating human α cell function.
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Affiliation(s)
- Mridusmita Saikia
- Department of Biomedical Sciences and
- Baker Institute for Animal Health, Cornell University College of Veterinary Medicine, Ithaca, New York, USA
| | | | | | | | | | | | | | | | | | - Reilly Coch
- Cayuga Medical Center, Ithaca, New York, USA
| | - Kyle W Sloop
- Diabetes and Complications, Lilly Research Laboratories, Lilly, Indianapolis, Indiana, USA
| | | | - Charles G Danko
- Department of Biomedical Sciences and
- Baker Institute for Animal Health, Cornell University College of Veterinary Medicine, Ithaca, New York, USA
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Southey BR, Zhang P, Keever MR, Rymut HE, Johnson RW, Sweedler JV, Rodriguez-Zas SL. Effects of maternal immune activation in porcine transcript isoforms of neuropeptide and receptor genes. J Integr Neurosci 2021; 20:21-31. [PMID: 33834688 PMCID: PMC8103820 DOI: 10.31083/j.jin.2021.01.332] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Revised: 12/11/2020] [Accepted: 02/09/2021] [Indexed: 12/17/2022] Open
Abstract
The prolonged effects of maternal immune activation in response stressors during gestation on the offspring's molecular pathways after birth are beginning to be understood. An association between maternal immune activation and neurodevelopmental and behavior disorders such as autism and schizophrenia spectrum disorders has been detected in long-term gene dysregulation. The incidence of alternative splicing among neuropeptides and neuropeptide receptor genes, critical cell-cell signaling molecules, associated with behavior may compromise the replicability of reported maternal immune activation effects at the gene level. This study aims to advance the understanding of the effect of maternal immune activation on transcript isoforms of the neuropeptide system (including neuropeptide, receptor and connecting pathway genes) underlying behavior disorders later in life. Recognizing the wide range of bioactive peptides and functional receptors stemming from alternative splicing, we studied the effects of maternal immune activation at the transcript isoform level on the hippocampus and amygdala of three-week-old pigs exposed to maternal immune activation due to viral infection during gestation. In the hippocampus and amygdala, 29 and 9 transcript isoforms, respectively, had maternal immune activation effects (P-value < 0.01). We demonstrated that the study of the effect of maternal immune activation on neuropeptide systems at the isoform level is necessary to expose opposite effects among transcript isoforms from the same gene. Genes were maternal immune activation effects have also been associated with neurodevelopmental and behavior disorders. The characterization of maternal immune activation effects at the transcript isoform level advances the understanding of neurodevelopmental disorders and identifies precise therapeutic targets.
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Affiliation(s)
- Bruce R Southey
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
| | - Pan Zhang
- Illinois Informatics Institute, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
| | - Marissa R Keever
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
| | - Haley E Rymut
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
| | - Rodney W Johnson
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA.,Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
| | - Jonathan V Sweedler
- Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA.,Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
| | - Sandra L Rodriguez-Zas
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA.,Illinois Informatics Institute, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA.,Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA.,Department of Statistics, University of Illinois at Urbana-Champaign, Urbana, 61801 IL, USA
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10
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Acosta-Montalvo A, Saponaro C, Kerr-Conte J, Prehn JHM, Pattou F, Bonner C. Proglucagon-Derived Peptides Expression and Secretion in Rat Insulinoma INS-1 Cells. Front Cell Dev Biol 2020; 8:590763. [PMID: 33240888 PMCID: PMC7683504 DOI: 10.3389/fcell.2020.590763] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2020] [Accepted: 10/20/2020] [Indexed: 11/26/2022] Open
Abstract
Rat insulinoma INS-1 cells are widely used to study insulin secretory mechanisms. Studies have shown that a population of INS-1 cells are bi-hormonal, co-expressing insulin, and proglucagon proteins. They coined this population as immature cells since they co-secrete proglucagon-derived peptides from the same secretory vesicles similar to that of insulin. Since proglucagon encodes multiple peptides including glucagon, glucagon-like-peptide-1 (GLP-1), GLP-2, oxyntomodulin, and glicentin, their specific expression and secretion are technically challenging. In this study, we aimed to focus on glucagon expression which shares the same amino acid sequence with glicentin and proglucagon. Validation of the anti-glucagon antibody (Abcam) by Western blotting techniques revealed that the antibody detects proglucagon (≈ 20 kDa), glicentin (≈ 9 kDa), and glucagon (≈ 3 kDa) in INS-1 cells and primary islets, all of which were absent in the kidney cell line (HEK293). Using the validated anti-glucagon antibody, we showed by immunofluorescence imaging that a population of INS-1 cells co-express insulin and proglucagon-derived proteins. Furthermore, we found that chronic treatment of INS-1 cells with high-glucose decreases insulin and glucagon content, and also reduces the percentage of bi-hormonal cells. In line with insulin secretion, we found glucagon and glicentin secretion to be induced in a glucose-dependent manner. We conclude that INS-1 cells are a useful model to study glucose-stimulated insulin secretion, but not that of glucagon or glicentin. Our study suggests Western blotting technique as an important tool for researchers to study proglucagon-derived peptides expression and regulation in primary islets in response to various metabolic stimuli.
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Affiliation(s)
- Ana Acosta-Montalvo
- INSERM, U1190, Lille, France.,European Genomic Institute for Diabetes, Lille, France.,University of Lille, Lille, France
| | - Chiara Saponaro
- INSERM, U1190, Lille, France.,European Genomic Institute for Diabetes, Lille, France.,University of Lille, Lille, France
| | - Julie Kerr-Conte
- INSERM, U1190, Lille, France.,European Genomic Institute for Diabetes, Lille, France.,University of Lille, Lille, France
| | - Jochen H M Prehn
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland
| | - François Pattou
- INSERM, U1190, Lille, France.,European Genomic Institute for Diabetes, Lille, France.,University of Lille, Lille, France.,Chirurgie Endocrinienne et Métabolique, CHU Lille, Lille, France
| | - Caroline Bonner
- INSERM, U1190, Lille, France.,European Genomic Institute for Diabetes, Lille, France.,University of Lille, Lille, France.,Institut Pasteur de Lille, Lille, France
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11
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Winters A, Ramos-Molina B, Jarvela TS, Yerges-Armstrong L, Pollin TI, Lindberg I. Functional analysis of PCSK2 coding variants: A founder effect in the Old Order Amish population. Diabetes Res Clin Pract 2017; 131:82-90. [PMID: 28719828 PMCID: PMC5572827 DOI: 10.1016/j.diabres.2017.06.023] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Revised: 06/27/2017] [Accepted: 06/27/2017] [Indexed: 02/06/2023]
Abstract
AIMS In humans, noncoding variants of PCSK2, the gene encoding prohormone convertase 2 (PC2), have been previously associated with risk for and age of onset of type 2 diabetes (T2D). The aims of this study were to identify coding variants in PCSK2; to determine their possible association with glucose handling; and to determine functional outcomes for coding variants in biochemical studies. METHODS Exome-wide genotyping was performed on 1725 Old Order Amish (OOA) subjects. PCSK2 coding variants were tested for association with diabetes-related phenotypes. In vitro analyses using transfected human PC2-encoding constructs were performed to determine the impact of each mutation on PC2 activity. RESULTS We identified 10 rare missense coding variants in PCSK2 in various genomic databases. R430W (rs200711626) is greatly enriched in the OOA population (MAF 4.3%). This variant is almost twice as common (MAF 7.4%) in OOA individuals with T2D as in OOA individuals with normal or with normal/impaired glucose tolerance (MAF 3.9% and 2.9%, respectively; p=0.25 and p=0.10). In vitro experiments revealed a broadening of the pH optimum for the R430W variant, which may result in increased activity against PCSK2 substrates. CONCLUSIONS Although the association of the R430W variation with T2D in the OOA population did not reach significance, based upon the broadened pH profile of R430W PC2, we speculate that the presence of this substitution may result in altered processing of PCSK2 substrates, ultimately leading to increased conversion to diabetes.
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Affiliation(s)
- Alexandra Winters
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, United States; Division of Endocrinology, Diabetes, and Nutrition, University of Maryland School of Medicine, Baltimore, MD 21201, United States
| | - Bruno Ramos-Molina
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, United States
| | - Timothy S Jarvela
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, United States
| | - Laura Yerges-Armstrong
- Division of Endocrinology, Diabetes, and Nutrition, University of Maryland School of Medicine, Baltimore, MD 21201, United States
| | - Toni I Pollin
- Division of Endocrinology, Diabetes, and Nutrition, University of Maryland School of Medicine, Baltimore, MD 21201, United States
| | - Iris Lindberg
- Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
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12
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Guizzetti L, McGirr R, Dhanvantari S. Two dipolar α-helices within hormone-encoding regions of proglucagon are sorting signals to the regulated secretory pathway. J Biol Chem 2014; 289:14968-80. [PMID: 24727476 DOI: 10.1074/jbc.m114.563684] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229-240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting "signature."
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Affiliation(s)
| | - Rebecca McGirr
- the Metabolism/Diabetes and Imaging Programs, Lawson Health Research Institute, London, Ontario N6A 4V2, Canada
| | - Savita Dhanvantari
- From the Departments of Medical Biophysics, the Metabolism/Diabetes and Imaging Programs, Lawson Health Research Institute, London, Ontario N6A 4V2, Canada Pathology, and Medicine, University of Western Ontario, London, Ontario N6A 3K7 and
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13
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Abstract
Glucose homeostasis is precisely regulated by glucagon and insulin, which are released by pancreatic α- and β-cells, respectively. While β-cells have been the focus of intense research, less is known about α-cell function and the actions of glucagon. In recent years, the study of this endocrine cell type has experienced a renewed drive. The present review contains a summary of established concepts as well as new information about the regulation of α-cells by glucose, amino acids, fatty acids and other nutrients, focusing especially on glucagon release, glucagon synthesis and α-cell survival. We have also discussed the role of glucagon in glucose homeostasis and in energy and lipid metabolism as well as its potential as a modulator of food intake and body weight. In addition to the well-established action on the liver, we discuss the effects of glucagon in other organs, where the glucagon receptor is expressed. These tissues include the heart, kidneys, adipose tissue, brain, small intestine and the gustatory epithelium. Alterations in α-cell function and abnormal glucagon concentrations are present in diabetes and are thought to aggravate the hyperglycaemic state of diabetic patients. In this respect, several experimental approaches in diabetic models have shown important beneficial results in improving hyperglycaemia after the modulation of glucagon secretion or action. Moreover, glucagon receptor agonism has also been used as a therapeutic strategy to treat obesity.
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14
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Jones HB, Reens J, Brocklehurst SR, Betts CJ, Bickerton S, Bigley AL, Jenkins RP, Whalley NM, Morgan D, Smith DM. Islets of Langerhans from prohormone convertase-2 knockout mice show α-cell hyperplasia and tumorigenesis with elevated α-cell neogenesis. Int J Exp Pathol 2014; 95:29-48. [PMID: 24456331 DOI: 10.1111/iep.12066] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2013] [Accepted: 11/14/2013] [Indexed: 01/24/2023] Open
Abstract
Antagonism of the effects of glucagon as an adjunct therapy with other glucose-lowering drugs in the chronic treatment of diabetes has been suggested to aggressively control blood glucose levels. Antagonism of glucagon effects, by targeting glucagon secretion or disabling the glucagon receptor, is associated with α-cell hyperplasia. We evaluated the influence of total glucagon withdrawal on islets of Langerhans using prohormone convertase-2 knockout mice (PC2-ko), in which α-cell hyperplasia is present from a young age and persists throughout life, in order to understand whether or not sustained glucagon deficit would lead to islet tumorigenesis. PC2-ko and wild-type (WT) mice were maintained drug-free, and cohorts of these groups sampled at 3, 12 and 18 months for plasma biochemical and morphological (histological, immunohistochemical, electron microscopical and image analytical) assessments. WT mice showed no islet tumours up to termination of the study, but PC2-ko animals displayed marked changes in islet morphology from α-cell hypertrophy/hyperplasia/atypical hyperplasia, to adenomas and carcinomas, these latter being first encountered at 6-8 months. Islet hyperplasias and tumours primarily consisted of α-cells associated to varying degrees with other islet endocrine cell types. In addition to substantial increases in islet neoplasia, increased α-cell neogenesis associated primarily with pancreatic duct(ule)s was present. We conclude that absolute blockade of the glucagon signal results in tumorigenesis and that the PC2-ko mouse represents a valuable model for investigation of islet tumours and pancreatic ductal neogenesis.
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Affiliation(s)
- Huw B Jones
- Department of Pathological Sciences, AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK
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Phenotypic and functional characterization of glucagon-positive cells derived from spontaneous differentiation of D3-mouse embryonic stem cells. Cytotherapy 2013; 15:122-31. [PMID: 23260092 DOI: 10.1016/j.jcyt.2012.08.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2012] [Accepted: 08/14/2012] [Indexed: 01/06/2023]
Abstract
BACKGROUND Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established. METHODS D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon(+) cells. RESULTS Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon(+) cells co-expressed neurofilament middle and some glucagon-like peptide-1(+) cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1(+) cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon(+) cells did not display Ca(2+) oscillations typical of pancreatic α-cells. DISCUSSION These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins.
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McGirr R, Guizzetti L, Dhanvantari S. The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals. J Endocrinol 2013; 217:229-40. [PMID: 23418362 DOI: 10.1530/joe-12-0468] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.
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Affiliation(s)
- Rebecca McGirr
- Metabolism and Diabetes and Imaging Programs, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, Canada
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17
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Sugiyama C, Yamamoto M, Kotani T, Kikkawa F, Murata Y, Hayashi Y. Fertility and pregnancy-associated ß-cell proliferation in mice deficient in proglucagon-derived peptides. PLoS One 2012; 7:e43745. [PMID: 22928026 PMCID: PMC3426535 DOI: 10.1371/journal.pone.0043745] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2012] [Accepted: 07/23/2012] [Indexed: 12/15/2022] Open
Abstract
Proglucagon, which is encoded by the glucagon gene (Gcg), is the precursor of several peptide hormones, including glucagon and glucagon-like peptide 1 (GLP-1). Whereas glucagon stimulates hepatic glycogenolysis and gluconeogenesis, GLP-1 stimulates insulin secretion to lower blood glucose and also supports ß-cell proliferation and protection from apoptotic stimuli. Pregnancy is a strong inducer of change in islet function, however the roles of proglucagon-derived peptides in pregnancy are only partially understood. In the present study, we analyzed fertility and pregnancy-associated changes in homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcggfp/gfp), which lack all the peptides derived from proglucagon. Female Gcggfp/gfp mice could deliver and raise Gcggfp/gfp pups to weaning and Gcggfp/gfp pups from Gcggfp/gfp dams were viable and fertile. Pregnancy induced ß-cell proliferation in Gcggfp/gfp mice as well as in control mice. However, serum insulin levels in pregnant Gcggfp/gfp females were lower than those in control pregnant females under ad libitum feeding, and blood glucose levels in pregnant Gcggfp/gfp females were higher after gestational day 12. Gcggfp/gfp females showed a decreased pregnancy rate and smaller litter size. The rate of successful breeding was significantly lower in Gcggfp/gfp females and was not improved by experience of breeding. Taken together, proglucagon-derived peptides are not required for pregnancy-associated ß-cell proliferation, however, are required for regulation of blood glucose levels and normal reproductive capacity. Gcggfp/gfp mice may serve as a novel model to analyze the effect of mild hyperglycemia during late gestational periods.
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Affiliation(s)
- Chisato Sugiyama
- Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Michiyo Yamamoto
- Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
| | - Tomomi Kotani
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Fumitaka Kikkawa
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yoshiharu Murata
- Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
| | - Yoshitaka Hayashi
- Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan
- * E-mail:
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18
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Cyr NE, Stuart RC, Zhu X, Steiner DF, Nillni EA. Biosynthesis of proTRH-derived peptides in prohormone convertase 1 and 2 knockout mice. Peptides 2012; 35:42-8. [PMID: 22421509 DOI: 10.1016/j.peptides.2012.02.024] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2012] [Revised: 02/27/2012] [Accepted: 02/27/2012] [Indexed: 10/28/2022]
Abstract
Prohormone convertases (PCs) 1 and 2 are the primary endoproteases involved in the post-translational processing of proThyrotropin Releasing Hormone (proTRH) to give rise to TRH and other proposed biologically active non-TRH peptides. Previous evidence suggests that PC1 is responsible for most proTRH cleavage events. Here, we used the PC1 and PC2 knockout (KO) mouse models to examine the effects of PC1 or PC2 loss on proTRH processing. The PC1KO mouse presented a decrease in five proTRH-derived peptides, whereas the PC2KO mouse showed only lesser reduction in three TRH (Gln-His-Pro), TRH-Gly (Gln-His-Pro-Gly), and the short forms preproTRH(178-184) (pFQ(7)) and preproTRH(186-199) (pSE(14)) of pFE(22) (preproTRH(178-199)). Also, PC1KO and not PC2KO showed a decrease in pEH(24) indicating that PC1 is more important in generating this peptide in the mouse, which differs from previous studies using rat proTRH. Furthermore, downstream effects on thyroid hormone levels were evident in PC1KO mice, but not PC2KO mice suggesting that PC1 plays the more critical role in producing bioactive hypophysiotropic TRH. Yet loss of PC1 did not abolish TRH entirely indicating a complementary action for both enzymes in the normal processing of proTRH. We also show that PC2 alone is responsible for catalyzing the conversion of pFE(22) to pFQ(7) and pSE(14), all peptides implicated in regulation of suckling-induced prolactin release. Collectively, results characterize the specific roles of PC1 and PC2 in proTRH processing in vivo.
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Affiliation(s)
- Nicole E Cyr
- Division of Endocrinology, Department of Medicine, The Warren Alpert Medical School of Brown University/Rhode Island Hospital, Providence, RI 02903, USA
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19
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Hayashi Y, Yamamoto M, Mizoguchi H, Watanabe C, Ito R, Yamamoto S, Sun XY, Murata Y. Mice deficient for glucagon gene-derived peptides display normoglycemia and hyperplasia of islet {alpha}-cells but not of intestinal L-cells. Mol Endocrinol 2009; 23:1990-9. [PMID: 19819987 DOI: 10.1210/me.2009-0296] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcg(gfp/gfp) mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcg(gfp/gfp) mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcg(gfp/gfp) mice were not significantly different from those in Gcg(+/+) and Gcg(gfp/+) mice, even after starvation for 16 h. Serum insulin levels in Gcg(gfp/gfp) mice were lower than in Gcg(+/+) and Gcg(gfp/+) on ad libitum feeding, but no significant differences were observed on starvation. Islet alpha-cells and intestinal L-cells were readily visualized in Gcg(gfp/gfp) and Gcg(gfp/+) mice under fluorescence. The Gcg(gfp/gfp) postnatally developed hyperplasia of islet alpha-cells, whereas the population of intestinal L-cells was not increased. In the Gcg(gfp/gfp), expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of alpha-cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model.
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Affiliation(s)
- Yoshitaka Hayashi
- Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Japan.
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20
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Richards MP, McMurtry JP. The avian proglucagon system. Gen Comp Endocrinol 2009; 163:39-46. [PMID: 18938167 DOI: 10.1016/j.ygcen.2008.09.010] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2008] [Revised: 09/04/2008] [Accepted: 09/25/2008] [Indexed: 10/21/2022]
Abstract
Understanding how the proglucagon system functions in maintaining glycemic control and energy balance in birds, as well as defining its specific roles in regulating metabolism, gastrointestinal tract function and food intake requires detailed knowledge of the components that comprise this system. These include proglucagon, a precursor protein from which glucagon and two glucagon-like peptide hormones (GLP-1 and GLP-2) are derived, and the membrane bound G-protein-coupled receptors that specifically bind glucagon, GLP-1 and GLP-2 to mediate their individual physiological actions. Another key feature of the proglucagon system that is important for regulating its activity in different tissues involves post-translational processing of the proglucagon precursor protein and the individual peptide hormones derived from it. Currently, there is limited information about the proglucagon system in birds with the majority of that coming from studies involving chickens. By summarizing what is currently known about the proglucagon system in birds, this review aims to provide useful background information for future investigations that will explore the nature and actions of this important hormonal system in different avian species.
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Affiliation(s)
- Mark P Richards
- United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, Beltsville, MD 20705-2350, USA.
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Richards MP, McMurtry JP. Expression of proglucagon and proglucagon-derived peptide hormone receptor genes in the chicken. Gen Comp Endocrinol 2008; 156:323-38. [PMID: 18299131 DOI: 10.1016/j.ygcen.2008.01.014] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/09/2007] [Revised: 12/13/2007] [Accepted: 01/16/2008] [Indexed: 11/24/2022]
Abstract
To better understand how the proglucagon system functions in birds, we utilized a molecular cloning strategy to sequence and characterize the chicken proglucagon gene that encodes glucagon, glucagon-like peptide (GLP)-1 and GLP-2. This gene has seven exons and six introns with evidence for an additional (alternate) first exon and two promoter regions. We identified two distinct classes of proglucagon mRNA transcripts (PGA and PGB) produced by alternative splicing at their 3'-ends. These were co-expressed in all tissues examined with pancreas and proventriculus showing the highest levels of each. Although both mRNA classes contained coding sequence for glucagon and GLP-1, class A mRNA lacked that portion of the coding region (CDS) containing GLP-2; whereas, class B mRNA had a larger CDS that included GLP-2. Both classes of mRNA transcripts exhibited two variants, each with a different 5'-end arising from alternate promoter and alternate first exon usage. Fasting and refeeding had no effect on proglucagon mRNA expression despite significant changes in plasma glucagon levels. To investigate potential differences in proglucagon precursor processing among tissues, mRNA expression for two prohormone convertase (PC) genes was analyzed. PC2 mRNA was predominantly expressed in pancreas and proventriculus, whereas PC1/3 mRNA was more highly expressed in duodenum and brain. We also determined mRNA expression of the specific receptor genes for glucagon, GLP-1 and GLP-2 to help define major sites of hormone action. Glucagon receptor mRNA was most highly expressed in liver and abdominal fat, whereas GLP-1 and GLP-2 receptor genes were highly expressed in the gastrointestinal tract, brain, pancreas and abdominal fat. These results offer new insights into structure and function of the chicken proglucagon gene, processing of the precursor proteins produced from it and potential activity sites for proglucagon-derived peptide hormones mediated by their cognate receptors.
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Affiliation(s)
- Mark P Richards
- Animal Biosciences and Biotechnology Laboratory, USDA, ARS, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, BARC-East, Beltsville, MD 20705-2350, USA.
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Tegge AN, Southey BR, Sweedler JV, Rodriguez-Zas SL. Comparative analysis of neuropeptide cleavage sites in human, mouse, rat, and cattle. Mamm Genome 2008; 19:106-20. [PMID: 18213482 DOI: 10.1007/s00335-007-9090-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2007] [Accepted: 12/11/2007] [Indexed: 01/02/2023]
Abstract
Neuropeptides are an important class of signaling molecules that result from complex and variable posttranslational processing of precursor proteins and thus are difficult to identify based solely on genomic information. Bioinformatics prediction of precursor cleavage sites can support effective biochemical characterization of neuropeptides. Neuropeptide cleavage models were developed using comprehensive human, mouse, rat, and cattle precursor data sets and used to compare predicted neuropeptide processing across these species. Logistic regression and artificial neural network models were used to predict cleavages based on amino acid and physiochemical properties of amino acids at precursor sequence locations proximal to cleavage. Correct cleavage classification rates across species and models ranged from 85% to 100%, suggesting that amino acid and amino acid properties have major impact on the probability of cleavage and that these factors have comparable effects in human, mouse, rat, and cattle. The variable accuracy of each species-specific model to predict cleavage sites indicated that there are species- and precursor-specific processing patterns. Prediction of mouse cleavages using rat models was highly accurate, yet the reverse was not observed. Sensitivity and specificity revealed that logistic models are well suited to maximize the rate of true noncleavage predictions with moderate rates of true cleavage predictions; meanwhile, artificial neural networks maximize the rate of true cleavage predictions with moderate to low true noncleavage predictions. Logistic models also provided insights into the strength of the amino acid associations with cleavage. Prediction of neuropeptide cleavage sites using human, mouse, rat, and cattle models are available at http://www.neuroproteomics.scs.uiuc.edu/neuropred.html .
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Affiliation(s)
- Allison N Tegge
- Department of Animal Sciences, University of Illinois, Urbana, Illinois, 61801, USA
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Stein J, Shah R, Steiner D, Dey A. RNAi-mediated silencing of prohormone convertase (PC) 5/6 expression leads to impairment in processing of cocaine- and amphetamine-regulated transcript (CART) precursor. Biochem J 2006; 400:209-15. [PMID: 16800814 PMCID: PMC1635452 DOI: 10.1042/bj20060506] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Understanding the functions of the widely expressed PCs (prohormone/proprotein convertases), including PC5/6, furin and PACE4 (paired basic amino acid cleaving enzyme 4), in animal models is difficult since individual knockouts of these PCs in mice exhibit early embryonic lethality. To investigate the roles of PC5/6 in processing pro-CART (pro-cocaine- and amphetamine-regulated transcript), an important anorexigenic peptide precursor, we have generated GH3 cells silenced for PC5/6 expression by RNAi (RNA interference). We show, following transient knockdown of PC5/6 in these neuroendocrine cells, that generation of the two bioactive forms, CART I (amino acids 42-89/55-102) and CART II (amino acids 49-89/62-102), from pro-CART is impaired due to a lack particularly of the A isoform of PC5/6. The results indicate that PC5/6A shares specificities primarily with PC2 (PC5/6A<PC2) in cleaving the pairs of basic residues, KR (40, 41 //53, 54/) and KK (47, 48//60, 61/), within the pro-CART isoforms [see Dey, Zhu, Carroll, Turck, Stein and Steiner (2003) J. Biol. Chem. 278, 15007-15014]. We do not find any significant role of PC5/6A in processing the RXXR (29-32/) site for production of intermediate CART (amino acids 33-102) from long pro-CART. The findings taken altogether indicate that PC5/6 participates in normal processing of pro-CART.
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Affiliation(s)
- Jeffrey Stein
- *Department of Biochemistry and Molecular Biology, University of Chicago, 5841 South Maryland Avenue, MC-1027, Chicago, IL 60637, U.S.A
| | - Rohan Shah
- *Department of Biochemistry and Molecular Biology, University of Chicago, 5841 South Maryland Avenue, MC-1027, Chicago, IL 60637, U.S.A
| | - Donald F. Steiner
- *Department of Biochemistry and Molecular Biology, University of Chicago, 5841 South Maryland Avenue, MC-1027, Chicago, IL 60637, U.S.A
- †Howard Hughes Medical Institute, University of Chicago, 5841 South Maryland Avenue, MC-1028, Chicago, IL 60637, U.S.A
| | - Arunangsu Dey
- *Department of Biochemistry and Molecular Biology, University of Chicago, 5841 South Maryland Avenue, MC-1027, Chicago, IL 60637, U.S.A
- To whom correspondence should be addressed (email )
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Stein J, Steiner DF, Dey A. Processing of cocaine- and amphetamine-regulated transcript (CART) precursor proteins by prohormone convertases (PCs) and its implications. Peptides 2006; 27:1919-25. [PMID: 16784796 DOI: 10.1016/j.peptides.2005.10.028] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/15/2005] [Accepted: 10/04/2005] [Indexed: 11/20/2022]
Abstract
Cocaine- and amphetamine-regulated transcript (CART) peptides are expressed in several neuroendocrine tissues, including hypothalamus, pituitary, gut, adrenal and pancreas, and are involved in regulating important biological processes including feeding/appetite, drug reward and stress. CART is synthesized as larger, inactive peptide precursors (pro-CART) that require endoproteolytic processing to generate smaller, active forms. Prohormone/proprotein convertases (PCs), a family of calcium-dependent, serine endoproteases, have been shown to cleave many protein precursors in the regulated/constitutive secretory pathway to generate smaller fragments. In our previous studies, we have demonstrated the important roles of the two neuroendocrine-specific PCs, PC2 and PC1/3, in processing the two pro-CART isoforms, long (102aa) and short (89aa), to generate the bioactive CART peptides, I (55-102/42-89) and II (62-102/49-89) as well as the intermediate fragments, 10-89 and 33-102. Our subsequent studies have revealed the participation of another PC family member, PC5/6A (the soluble isoform of a widely expressed PC, PC5/6), in cleaving both precursor isoforms. We conclude that PC5/6A contributes to the normal efficient processing of pro-CART and is functionally more redundant with PC2 than PC1/3 in generating both CART I and II.
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Affiliation(s)
- Jeffrey Stein
- Department of Biochemistry and Molecular Biology, The University of Chicago, IL 60637, USA
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25
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Martin GR, Beck PL, Sigalet DL. Gut hormones, and short bowel syndrome: The enigmatic role of glucagon-like peptide-2 in the regulation of intestinal adaptation. World J Gastroenterol 2006; 12:4117-29. [PMID: 16830359 PMCID: PMC4087358 DOI: 10.3748/wjg.v12.i26.4117] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Short bowel syndrome (SBS) refers to the malabsorption of nutrients, water, and essential vitamins as a result of disease or surgical removal of parts of the small intestine. The most common reasons for removing part of the small intestine are due to surgical intervention for the treatment of either Crohn's disease or necrotizing enterocolitis. Intestinal adaptation following resection may take weeks to months to be achieved, thus nutritional support requires a variety of therapeutic measures, which include parenteral nutrition. Improper nutrition management can leave the SBS patient malnourished and/or dehydrated, which can be life threatening. The development of therapeutic strategies that reduce both the complications and medical costs associated with SBS/long-term parenteral nutrition while enhancing the intestinal adaptive response would be valuable.
Currently, therapeutic options available for the treatment of SBS are limited. There are many potential stimulators of intestinal adaptation including peptide hormones, growth factors, and neuronally-derived components. Glucagon-like peptide-2 (GLP-2) is one potential treatment for gastrointestinal disorders associated with insufficient mucosal function. A significant body of evidence demonstrates that GLP-2 is a trophic hormone that plays an important role in controlling intestinal adaptation. Recent data from clinical trials demonstrate that GLP-2 is safe, well-tolerated, and promotes intestinal growth in SBS patients. However, the mechanism of action and the localization of the glucagon-like peptide-2 receptor (GLP-2R) remains an enigma. This review summarizes the role of a number of mucosal-derived factors that might be involved with intestinal adaptation processes; however, this discussion primarily examines the physiology, mechanism of action, and utility of GLP-2 in the regulation of intestinal mucosal growth.
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Affiliation(s)
- G-R Martin
- Department of Gastrointestinal Sciences, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW., Calgary, Alberta T2N 4N1, Canada.
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Southey BR, Rodriguez-Zas SL, Sweedler JV. Prediction of neuropeptide prohormone cleavages with application to RFamides. Peptides 2006; 27:1087-98. [PMID: 16494967 DOI: 10.1016/j.peptides.2005.07.026] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2005] [Accepted: 07/13/2005] [Indexed: 11/24/2022]
Abstract
Genomic information is becoming available for an ever-wider range of animals with the genes for several well-characterized peptide families, such as the RFamides, detected in a surprisingly diverse set of these animals. While bioinformatic tools allow the prediction of the RFamide-related prohormones from genetic information, it is more difficult to accurately predict the final processed peptides because of the large number of processing steps required to convert a prohormone into mature bioactive peptides. Several statistical-based methods for predicting basic site cleavages in prohormones are described, and their ability to predict the basic site cleavages in a variety of RFamide-related peptides from vertebrates and invertebrates is reported. Specifically, the cleavages in the invertebrate FMRFamides, and the vertebrate NPFFa, RFRPa, and PrRPa peptide families are modeled. The three models compared here are based on known cleavage motifs, a logistic regression, and artificial neural networks. Improvements in the accuracy and precision of the cleavage estimates will lead to increased utilization of these models for predicting bioactive neuropeptides before experimental verification is available.
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Affiliation(s)
- Bruce R Southey
- Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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27
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Ugleholdt R, Poulsen MLH, Holst PJ, Irminger JC, Orskov C, Pedersen J, Rosenkilde MM, Zhu X, Steiner DF, Holst JJ. Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor. J Biol Chem 2006; 281:11050-7. [PMID: 16476726 DOI: 10.1074/jbc.m601203200] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.
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Affiliation(s)
- Randi Ugleholdt
- Department of Medical Physiology, the Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
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Kacprzak MM, Than ME, Juliano L, Juliano MA, Bode W, Lindberg I. Mutations of the PC2 substrate binding pocket alter enzyme specificity. J Biol Chem 2005; 280:31850-8. [PMID: 16002408 DOI: 10.1074/jbc.m505567200] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.
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Affiliation(s)
- Magdalena M Kacprzak
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA
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