The KINGS Ins2+/G32S mouse: a novel model of beta cell endoplasmic reticulum stress and human diabetes

Animal models are important tools in diabetes research as ethical and logistical constraints limit access to human tissue. Beta cell dysfunction is a common contributor to the pathogenesis of most types of diabetes. Spontaneous hyperglycaemia developed in a colony of C57BL/6J mice at King’s College London (KCL). Sequencing identified a mutation in the Ins2 gene, causing a glycine to serine substitution at position 32 on the B chain of the preproinsulin2 molecule. Mice with the Ins2+/G32S mutation were named KCL Ins2 G32S (KINGS) mice. The same mutation in humans (rs80356664) causes dominantly inherited neonatal diabetes. Mice were characterised and beta cell function was investigated. Male mice became overtly diabetic at around 5 weeks of age whereas female mice had only slightly elevated non-fasting glycaemia. Islets showed decreased insulin content and impaired glucose-induced insulin secretion, which was more severe in males. Transmission electron microscopy and studies of gene and protein expression showed beta cell endoplasmic reticulum (ER) stress in both sexes. Despite this, beta cell numbers were only slightly reduced in older animals. In conclusion, the KINGS mouse is a novel model of a human form of diabetes that may be useful to study beta cell responses to ER stress.

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Loss of MANF Causes Childhood Onset Syndromic Diabetes due to Increased Endoplasmic Reticulum Stress

10.2337/figshare.13622993 ◽

2021 ◽

Author(s):

Hossam Montaser ◽

Kashyap A Patel ◽

Diego Balboa ◽

Hazem Ibrahim ◽

Väinö Lithovius ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Beta Cell ◽

Er Stress ◽

Beta Cells ◽

Beta Cell Function ◽

Cell Function ◽

Beta Cell Development ◽

Human Beta Cell ◽

And Function

MANF is an endoplasmic reticulum resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse beta cells, its precise role in human beta cell development and function is unknown. Herein, we show that lack of MANF in humans results in diabetes due to increased ER stress leading to impaired beta cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the MANF gene. To study the role of MANF in human beta cell development and function, we knocked out the MANF gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of MANF induced mild ER stress and impaired insulin processing capacity of beta cells in vitro. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in diabetic recipients. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human beta cell function and demonstrate the crucial role of MANF in this process.

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Loss of MANF Causes Childhood Onset Syndromic Diabetes due to Increased Endoplasmic Reticulum Stress

10.2337/figshare.13622993.v1 ◽

2021 ◽

Author(s):

Hossam Montaser ◽

Kashyap A Patel ◽

Diego Balboa ◽

Hazem Ibrahim ◽

Väinö Lithovius ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Beta Cell ◽

Er Stress ◽

Beta Cells ◽

Beta Cell Function ◽

Cell Function ◽

Beta Cell Development ◽

Human Beta Cell ◽

And Function

MANF is an endoplasmic reticulum resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse beta cells, its precise role in human beta cell development and function is unknown. Herein, we show that lack of MANF in humans results in diabetes due to increased ER stress leading to impaired beta cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the MANF gene. To study the role of MANF in human beta cell development and function, we knocked out the MANF gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of MANF induced mild ER stress and impaired insulin processing capacity of beta cells in vitro. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in diabetic recipients. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human beta cell function and demonstrate the crucial role of MANF in this process.

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Featured Article: Deterioration of visual function mediated by senescence-associated endoplasmic reticulum stress in inflammatory tie2-TNF mice

Experimental Biology and Medicine ◽

10.1177/1535370218794915 ◽

2018 ◽

Vol 243 (12) ◽

pp. 976-984 ◽

Cited By ~ 2

Author(s):

Raji Lenin ◽

Peter G Nagy ◽

Jordy Gentry ◽

Rajashekhar Gangaraju

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Endothelial Activation ◽

Premature Senescence ◽

Retinal Diseases ◽

Old Patients ◽

Mature Adult ◽

Visual Deficits ◽

Gene And Protein Expression ◽

Age Dependent

Stress-associated premature senescence plays a major role in retinal diseases. In this study, we investigated the relationship between endothelial dysfunction, endoplasmic reticulum (ER) stress, and cellular senescence in the development of retinal dysfunction. We tested the hypothesis that constant endothelial activation by transmembrane tumor necrosis factor-α (tmTNF-α) exacerbates age-induced visual deficits via senescence-mediated ER stress in this model. To address this, we employed a mouse model of chronic vascular activation using endothelial-specific TNF-α-expressing (tie2-TNF) mice at 5 and 10 months of age. Visual deficits were exhibited by tie2-TNF mice at both 5 months and 10 months of age, with the older mice showing statistically significant loss of visual acuity compared with tie2-TNF mice at age 5 months. The neural defects, as measured by electroretinogram (ERG), also followed a similar trend in an age-dependent fashion, with 10-month-old tie2-TNF mice showing the greatest decrease in “b” wave amplitude at 25 cd.s.m2 compared with age-matched wildtype (WT) mice and five-month-old tie2-TNF mice. While gene and protein expression from the whole retinal extracts demonstrated increased inflammatory (Icam1, Ccl2), stress-associated premature senescence (p16, p21, p53), and ER stress (Grp78, p-Ire1α, Chop) markers in five-month-old tie2-TNF mice compared with five-month-old WT mice, a further increase was seen in 10-month-old tie2-TNF mice. Our data demonstrate that tie2-TNF mice exhibit age-associated increases in visual deficits, and these data suggest that inflammatory endothelial activation is at least partly at play. Given the correlation of increased premature senescence and ER stress in an age-dependent fashion, with the loss of visual functions and increased endothelial activation, our data suggest a possible self-enhanced loop of unfolded protein response pathways and senescence in propagating neurovascular defects in this model. Impact statement Vision loss in most retinal diseases affects the quality of life of working age adults. Using a novel animal model that displays constant endothelial activation by tmTNF-α, our results demonstrate exacerbated age-induced visual deficits via premature senescence-mediated ER stress. We have compared mice of 5 and 10 months of age, with highly relevant human equivalencies of approximately 35- and 50-year-old patients, representing mature adult and middle-aged subjects, respectively. Our studies suggest a possible role for a self-enhanced loop of ER stress pathways and senescence in the propagation of retinal neurovascular defects, under conditions of constant endothelial activation induced by tmTNF-α signaling.

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Diazoxide-induced β-cell rest reduces endoplasmic reticulum stress in lipotoxic β-cells

Journal of Endocrinology ◽

10.1677/joe-08-0251 ◽

2008 ◽

Vol 199 (1) ◽

pp. 41-50 ◽

Cited By ~ 35

Author(s):

Ernest Sargsyan ◽

Henrik Ortsäter ◽

Kristofer Thorn ◽

Peter Bergsten

Keyword(s):

Endoplasmic Reticulum ◽

Insulin Secretion ◽

Er Stress ◽

Cell Function ◽

Β Cells ◽

Β Cell ◽

Eukaryotic Translation ◽

Β Cell Function ◽

Activating Transcription Factor ◽

The Unfolded Protein Response

Elevated levels of glucose and lipids are characteristics of individuals with type 2 diabetes mellitus (T2DM). The enhanced nutrient levels have been connected with deterioration of β-cell function and impaired insulin secretion observed in these individuals. A strategy to improve β-cell function in individuals with T2DM has been intermittent administration of KATP channel openers. After such treatment, both the magnitude and kinetics of insulin secretion are markedly improved. In an attempt to further delineate mechanisms of how openers of KATP channels improve β-cell function, the effects of diazoxide on markers of endoplasmic reticulum (ER) stress was determined in β-cells exposed to the fatty acid palmitate. The eukaryotic translation factor 2-alpha kinase 3 (EIF2AK3; also known as PERK) and endoplasmic reticulum to nucleus signaling 1 (ERN1; also known as IRE1) pathways, but not the activating transcription factor (ATF6) pathway of the unfolded protein response, are activated in such lipotoxic β-cells. Inclusion of diazoxide during culture attenuated activation of the EIF2AK3 pathway but not the ERN1 pathway. This attenuation was associated with reduced levels of DNA-damage inducible transcript 3 (DDIT3; also known as CHOP) and β-cell apoptosis was decreased. It is concluded that reduction of ER stress may be a mechanism by which diazoxide improves β-cell function.

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Inheritance of Mildly Activating ABCC8 Mutation From a Mother With MODY Causes Permanent Neonatal Diabetes Mellitus (NDM) in Two Siblings Who Also Carry a Second Inactivating Mutation: Genetic Testing Allows for Improved Treatment With Sulfonylureas (SU)

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.923 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A452-A452

Author(s):

Maria Victoria Salguero Bermonth ◽

Lisa Letourneau-Freiberg ◽

Nancy Devine ◽

Siri Atma W Greeley

Keyword(s):

Genetic Testing ◽

Beta Cell ◽

Beta Cell Function ◽

Cell Function ◽

Family Members ◽

Neonatal Diabetes ◽

Cell Surface Expression ◽

High Dose ◽

C Peptide ◽

Significant Difference

Abstract Background: Heterozygous activating mutations in KCNJ11 or ABCC8 are the most common cause of neonatal diabetes (NDM). ABCC8 (SUR1) mutations more often cause transient NDM. Inactivating ABCC8 mutations can cause congenital hyperinsulinism (HI), but very rarely will such mutations be inherited together. Mildly activating KATP mutations can also be a cause of MODY, but even if genetic testing is considered, many commercial testing panels do not include these genes, despite the significant difference in treatment that can result due to sulfonylurea (SU) responsiveness. Clinical Case: The proband was diagnosed with DM at 11 months old and fortuitously treated with SU for 3 years. He was switched to insulin and had poor DM control thereafter. Sister was diagnosed at 3.5 months old and had poor DM control on insulin. Mother was diagnosed with DM at 27 years old and treated with various medications including insulin. Genetic testing revealed that mother carried ABCC8 mutation R1380C previously described to cause transient NDM and/or later-onset DM consistent with her phenotype. Both children inherited this mutation from her and inherited a variant (L1148R) from their father without diabetes that has been reported in association with HI. The L1148R allele may reduce cell surface expression thereby increasing the relative expression and pathogenic effect of the R1380C allele that has not previously been described to cause permanent NDM. We assessed SU responsiveness by measuring maximal beta-cell function through combined mixed meal and arginine testing. Mother exhibited easily detectable C-peptide levels at baseline that improved by SU treatment. In contrast, the children displayed almost undetectable baseline beta-cell function with variable response to SU: the sister who had been chronically poorly controlled on insulin therapy displayed barely improved C-peptide production, while her brother who had previously been treated with SU as an infant had markedly improved beta-cell function on SU. Within two months of continued treatment with high doses of SU only, he was able to start lowering his SU dose with improved glycemia. His sister was started on high-dose SU in addition to insulin, but continued to have difficulty adhering to her treatment regimen. Her blood sugar improved after the addition of long-acting GLP-1 agonist (liraglutide) but she later became pregnant and returned to insulin only. Her glycemic control improved when re-started on SU after pregnancy. The mother exhibited excellent DM on a lower dose of exclusive SU therapy. Clinical Lesson: Genetic testing can dramatically alter management and must be pursued in both NDM and family members with diabetes later in life. Careful assessment of clinical characteristics along with genetic testing for segregation patterns in family members can greatly improve understanding of the causality of previous uncharacterized variants.

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The Current State of Beta-Cell-Mass PET Imaging for Diabetes Research and Therapies

Biomedicines ◽

10.3390/biomedicines9121824 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1824

Author(s):

Pierre Cheung ◽

Olof Eriksson

Keyword(s):

Positron Emission Tomography ◽

Beta Cell ◽

Disease Progression ◽

Beta Cell Function ◽

Cell Function ◽

Cell Mass ◽

Beta Cell Mass ◽

Emission Tomography ◽

Diabetes Research ◽

Positron Emission

Diabetes is a chronic metabolic disease affecting over 400 million people worldwide and one of the leading causes of death, especially in developing nations. The disease is characterized by chronic hyperglycemia, caused by defects in the insulin secretion or action pathway. Current diagnostic methods measure metabolic byproducts of the disease such as glucose level, glycated hemoglobin (HbA1c), insulin or C-peptide levels, which are indicators of the beta-cell function. However, they inaccurately reflect the disease progression and provide poor longitudinal information. Beta-cell mass has been suggested as an alternative approach to study disease progression in correlation to beta-cell function, as it behaves differently in the diabetes physiopathology. Study of the beta-cell mass, however, requires highly invasive and potentially harmful procedures such as pancreatic biopsies, making diagnosis and monitoring of the disease tedious. Nuclear medical imaging techniques using radiation emitting tracers have been suggested as strong non-invasive tools for beta-cell mass. A highly sensitive and high-resolution technique, such as positron emission tomography, provides an ideal solution for the visualization of beta-cell mass, which is particularly essential for better characterization of a disease such as diabetes, and for estimating treatment effects towards regeneration of the beta-cell mass. Development of novel, validated biomarkers that are aimed at beta-cell mass imaging are thus highly necessary and would contribute to invaluable breakthroughs in the field of diabetes research and therapies. This review aims to describe the various biomarkers and radioactive probes currently available for positron emission tomography imaging of beta-cell mass, as well as highlight the need for precise quantification and visualization of the beta-cell mass for designing new therapy strategies and monitoring changes in the beta-cell mass during the progression of diabetes.

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Imeglimin Ameliorates β-cell Apoptosis by Modulating the Endoplasmic Reticulum Homeostasis Pathway

10.2337/figshare.16654780.v1 ◽

2021 ◽

Author(s):

Jinghe Li ◽

Ryota Inoue ◽

Yu Togashi ◽

Tomoko Okuyama ◽

Aoi Satoh ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Cell Survival ◽

Cell Apoptosis ◽

Cell Function ◽

Cell Mass ◽

Β Cells ◽

Β Cell ◽

Akita Mice ◽

The Impact

The effects of imeglimin, a novel anti-diabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation, after treatment with thapsigargin, and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell (<a>hPSC)-derived β-like cells</a>. <a>Taken together, imeglimin modulates ER homeostasis pathway, which results in the prevention of β-cell apoptosis both in vitro and in vivo.</a>

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Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes

eLife ◽

10.7554/elife.38519 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 37

Author(s):

Diego Balboa ◽

Jonna Saarimäki-Vire ◽

Daniel Borshagovski ◽

Mantas Survila ◽

Päivi Lindholm ◽

...

Keyword(s):

Beta Cell ◽

Er Stress ◽

Cell Mass ◽

Gene Mutations ◽

Beta Cell Mass ◽

Neonatal Diabetes ◽

Beta Cell Apoptosis ◽

Beta Cell Proliferation ◽

Sequencing Analysis ◽

Mtorc1 Signaling

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 induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits 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.

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Characterization of 5-(2-18F-fluoroethoxy)-L-tryptophan for PET imaging of the pancreas

F1000Research ◽

10.12688/f1000research.9129.1 ◽

2016 ◽

Vol 5 ◽

pp. 1851 ◽

Cited By ~ 5

Author(s):

Ahmed Abbas ◽

Christine Beamish ◽

Rebecca McGirr ◽

John Demarco ◽

Neil Cockburn ◽

...

Keyword(s):

Beta Cell ◽

Er Stress ◽

Beta Cell Function ◽

Cell Function ◽

Cell Mass ◽

Beta Cell Mass ◽

Wild Type ◽

Time Activity ◽

System L ◽

Akita Mice

Purpose: In diabetes, pancreatic beta cell mass declines significantly prior to onset of fasting hyperglycemia. This decline may be due to endoplasmic reticulum (ER) stress, and the system L amino acid transporter LAT1 may be a biomarker of this process. In this study, we used 5-(2-18F-fluoroethoxy)-L-tryptophan (18F-L-FEHTP) to target LAT1 as a potential biomarker of beta cell function in diabetes. Procedures: Uptake of 18F-L-FEHTP was determined in wild-type C57BL/6 mice by ex vivo biodistribution. Both dynamic and static positron emission tomography (PET) images were acquired in wild-type and Akita mice, a model of ER stress-induced diabetes, as well as in mice treated with streptozotocin (STZ). LAT1 expression in both groups of mice was evaluated by immunofluorescence microscopy. Results: Uptake of 18F-L-FEHTP was highest in the pancreas, and static PET images showed highly specific pancreatic signal. Time-activity curves showed significantly reduced 18F-L-FEHTP uptake in Akita mice, and LAT1 expression was also reduced. However, mice treated with STZ, in which beta cell mass was reduced by 62%, showed no differences in 18F-L-FEHTP uptake in the pancreas, and there was no significant correlation of 18F-L-FEHTP uptake with beta cell mass. Conclusions: 18F-L-FEHTP is highly specific for the pancreas with little background uptake in kidney or liver. We were able to detect changes in LAT1 in a mouse model of diabetes, but these changes did not correlate with beta cell function or mass. Therefore, 18F-L-FEHTP PET is not a suitable method for the noninvasive imaging of changes in beta cell function during the progression of diabetes.

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