Nur Shabrina Amirruddin1,2, Wei Xuan Tan1,2, Yaw Sing Tan3, Daphne Su-Lyn Gardner4, Yong Mong Bee4, Chandra Shekhar Verma3,5,6, Shawn Hoon7, Kok Onn Lee2, Adrian Kee Keong Teo8,9,10. 1. Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore. 2. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore. 3. Bioinformatics Institute, A*STAR, Singapore, Republic of Singapore. 4. Department of Endocrinology, Singapore General Hospital, Singapore, Republic of Singapore. 5. Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore. 6. School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore. 7. Molecular Engineering Laboratory, IMCB, A*STAR, Singapore, Republic of Singapore. 8. Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore. ateo@imcb.a-star.edu.sg. 9. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore. ateo@imcb.a-star.edu.sg. 10. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore. ateo@imcb.a-star.edu.sg.
Abstract
AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. METHODS: We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. RESULTS: The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. CONCLUSIONS/ INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.
AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. METHODS: We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. RESULTS: The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. CONCLUSIONS/ INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.
Authors: Ming Liu; Israel Hodish; Leena Haataja; Roberto Lara-Lemus; Gautam Rajpal; Jordan Wright; Peter Arvan Journal: Trends Endocrinol Metab Date: 2010-08-18 Impact factor: 12.015
Authors: Felicia W Pagliuca; Jeffrey R Millman; Mads Gürtler; Michael Segel; Alana Van Dervort; Jennifer Hyoje Ryu; Quinn P Peterson; Dale Greiner; Douglas A Melton Journal: Cell Date: 2014-10-09 Impact factor: 41.582