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Progressive ER stress over time due to human insulin gene mutation contributes to pancreatic β-cell dysfunction, islet inflammation and compensatory responses

ABSTRACT: Heterozygous human INS gene mutations are known to promote ER stress, leading to β-cell dysfunction and neonatal diabetes. Recent literature challenged the long-standing notion that neonatal diabetes occurs due to ER stress-induced β-cell apoptosis. Importantly, mechanisms of β-cell failure during the disease progression and why the other wild-type (WT) INS allele is unable to function still remain unclear. Here, our computational modelling studies, short-term and long-term expression studies in β-cells revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in decreased insulin secretion but not insulin secretory capacity. By nine weeks of expression of mutant INS, dominant negative effects of mutant INS were evident and β-cell insulin secretory capacity declined. INS+/C109Y patient-derived β-like cells and single cell RNA-Sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. Our results provide deeper insights into the mechanisms of β-cell failure during INS mutation-mediated diabetes disease progression. Decreasing CHOP-10, sXBP1 or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141319 | GEO | 2021/10/03

REPOSITORIES: GEO

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Single-cell RNA sequencing enables transcriptomic analysis of iPSC-derived beta-cells in a model of neonatal diabetes caused by insulin mutations.

Project description:Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated iPSCs from diabetics carrying insulin mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells using a 3D-suspension differentiation protocol. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation to mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunit expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.

2018-12-01 | GSE115257 | GEO

Coordinate control of endoplasmic reticulum chaperone biosynthesis and peptide hormone secretion revealed by a β-cell model of Prader-Willi syndrome

Project description:Prader-Willi syndrome (PWS) is a multisystem disorder caused by loss of expression of a cluster of paternally-expressed, imprinted genes. Neonatal failure to thrive is followed by childhood-onset hyperphagia, obesity, neurobehavioral abnormalities, and hormonal deficits. Prior evidence from a mouse model with a deletion of the orthologous PWS-domain identified abnormal pancreatic islet development with deficient insulin secretion, hypoglucagonemia, and postnatal onset of progressive, lethal hypoglycemia. To investigate PWS-genes in β-cell secretory function, we used CRISPR/Cas9 genome-editing to generate isogenic, clonal INS-1 insulinoma lines with 3.16 Mb deletions of the silent, maternal (control) or active, paternal (PWS) alleles. A significant reduction in basal and glucose-stimulated insulin secretion signifies a cell autonomous insulin secretion deficit in PWS β-cells. Parallel proteome and transcriptome studies revealed reduced levels of secreted peptides and for eleven endoplasmic reticulum (ER) chaperones, including HSPA5 and HSP90B1. In contrast to dosage compensation previously seen for ER chaperones in Hspa5 or Hsp90b1 gene knockouts, compensation is precluded by the widespread deficiency of ER chaperones in PWS cells. Remarkably, but consistent with reduced ER chaperone levels, PWS β-cells are more sensitive to ER stress activation of all three regulatory pathways (XBP1, eIF2α-P, ATF6-N). Therefore, a coordinated, chronic deficit of ER chaperones in PWS β-cells is hypothesized to lead to a delay in ER transit and/or folding of insulin and other cargo along the secretory pathway. These findings provide insight into the pathophysiological basis of hormone deficits in PWS and indicate key roles for PWS-imprinted genes in β-cell secretory function.

2023-04-11 | PXD037874 | Pride

Coordinate control of endoplasmic reticulum chaperone biosynthesis and peptide hormone secretion revealed by a β-cell model of Prader-Willi syndrome

2023-04-11 | PXD034471 | Pride

Coordinate control of endoplasmic reticulum chaperone biosynthesis and peptide hormone secretion revealed by a β-cell model of Prader-Willi syndrome

2023-04-11 | PXD037875 | Pride

Rfx6 target genes in the Min6b1 cell line, a pancreatic beta cell model

Project description:Increasing evidence suggests that loss of beta cell characteristics may cause insulin secretory deficiency in diabetes but the underlying mechanisms remain unclear. Here we show that Rfx6, whose mutation leads to neonatal diabetes in man, is essential to maintain key features of functionally mature beta cells in mice. Rfx6 loss in adult beta cells leads to glucose intolerance, impaired beta cell glucose sensing and to defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway including GLucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca2 channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of M-bM-^@M-^\disallowedM-bM-^@M-^] genes, a group usually specifically repressed in adult beta cells, and thus to the maintenance of beta cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to beta cell failure in man. Two ChIP-Seq experiments have been performed : 1) anti-HA ChIP-Seq on 3HA-Rfx6 transfected Min6b1 cells and 2) anti-HA ChIP-Seq on Min6b1 cells

2014-12-11 | E-GEOD-62844 | biostudies-arrayexpress

Microarray comparing gene expression changes between control, normoglycaemic mice, and mice expressing the human neonatal diabetes mutation Kir6.2-V59M in pancreatic beta-cells left diabetic for 2 weeks

Project description:Activating mutations in the KATP channel cause a rare genetic form of diabetes called neonatal diabetes. These mutations render the channel permanently open results in membrane hyperpolarisation of the pancreatic beta-cell. This prevents calcium influx and impairs insulin secretion. Mice expressing the human neonatal diabetes mutation Kir6.2-V59M specifically in pancreatic beta-cells are diabetic but do not display dyslipidaemia or insulin resistance. In this experiment, gene expression changes were analysed to explore the effect of high blood glucose per se on isolated pancreatic islets

2017-07-05 | E-MTAB-5086 | biostudies-arrayexpress

Saturated fatty acids promotes β-cell toxicity by disabling intracellular lipid droplet formation through S-acylation of FIT2

Project description:Palm and coconut oils are linked to cardiovascular disease and diabetes because of their high saturated fatty acid (SFA) content but exactly how exogenous SFAs, but not unsaturated fatty acids (UFA), are toxic to cells remains unknown. In insulin-producing, β-cells of the Islets of Langerhans, loss of which exacerbates diabetes, we found that SFAs but not UFAs were toxic because they disable a highly conserved lipid droplet biogenesis machinery. We show that palmitate (a major SFA of these oils), but not palmitoleic or oleic, S-acylates the highly conserved ER-resident FITM2 protein, required for lipid coalescence and droplet budding from the ER. The S-acylation marks FITM2 for ubiquitination and proteosomal degradation, leaving SFAs within the ER instead safe sequestration within lipid droplets. ER-stress ensues with rapid induction of ER stress leading to β-cell apoptosis. Specific deletion of FITM2 in β-cells disrupts calcium signaling and key β-cell TFs and exacerbates high fat diet-induced ER stress and diabetes. Rescue by overexpression ameliorates ER-stress and β-cell apoptosis thus demonstrating an important link between lipid species and cell ability to sequester them away from the ER in the form of lipid droplets.

2021-07-08 | GSE133939 | GEO

Rfx6 target genes in the Min6b1 cell line, a pancreatic beta cell model

Project description:Increasing evidence suggests that loss of beta cell characteristics may cause insulin secretory deficiency in diabetes but the underlying mechanisms remain unclear. Here we show that Rfx6, whose mutation leads to neonatal diabetes in man, is essential to maintain key features of functionally mature beta cells in mice. Rfx6 loss in adult beta cells leads to glucose intolerance, impaired beta cell glucose sensing and to defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway including GLucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca2 channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of “disallowed” genes, a group usually specifically repressed in adult beta cells, and thus to the maintenance of beta cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to beta cell failure in man.

2014-12-11 | GSE62844 | GEO

Vitreous proteome of transgenic INSC94Y pigs compared to wild type

Project description:INSC94Y transgenic pigs serve as a valuable model for permanent neonatal diabetes mellitus. Through genetically engineered modifications in the insulin (INS) gene, this model presents with impaired insulin secretion, resulting elevated fasting blood-glucose levels (hyperglycemia) [1].One of the severe complications of diabetes mellitus are hypoglycemia-mediated morphological abnormalities in the retina, also described as diabetic retinopathy (DR). Adjacent to the retina lies the vitreous, a gelatinous, highly hydrated matrix, which is important for ocular function and retinal physiology. It contains variety of proteins and signaling molecules, which are useful for the characterization of the biological activity of the vitreous itself, and may help to elucidate molecular processes occurring in the retina, especially under pathophysiological conditions and in disease. To gain insight into the proteomic profile of porcine vitreous and detect possible differences relevant to DR pathogenesis, we used discovery proteomics for analysis of INSC94Y vitreous compared to wild-type controls. Reference: [1] Renner, S.; Braun-Reichhart, C.; Blutke, A.; Herbach, N.; Emrich, D.; Streckel, E.; Wunsch, A.; Kessler, B.; Kurome, M.; Bahr, A.; et al. Permanent neonatal diabetes in INS(C94Y) transgenic pigs. Diabetes 2013, 62, 1505-1511, doi:10.2337/db12-1065.

2024-08-27 | PXD038198 | Pride

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion

Project description:Pancreatic beta-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair beta-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on beta-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during beta-cell glucose toxicity that impairs expression of proteins with critical roles in beta-cell function.

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