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Metab-Immune analysis of the non-obese diabetic mouse
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. (Kristina Lejon)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1A diabetes mellitus or T1D is a chronic disease characterized by T cell mediated destruction of the insulin producing β cells in the islets of Langerhans. The classical symptoms include high glucose levels in urine and blood, polyuria, and polydipsia. Complications associated with T1D include blindness, amputations, and end-stage renal disease, and premature death. The non-obese diabetic (NOD) mouse, first described in 1980, is widely used as a model organism for T1D. T1D disease in the NOD mouse shares a number of similarities to human T1D including dependence on genetic and environmental factors. More than 30 disease associated gene regions or loci (termed insulin dependent diabetes, or Idd, loci) have been associated with T1D development in NOD. For some of these Idds, the corresponding region in human has been linked to the development of T1D in human.

T1D, both in humans and mice, is recognized as a T cell mediated disease. However, many studies have shown the importance of both the metabolome and the immune system in the pathogenesis of the disease. Appearance of autoantibodies in the serum of patients is the first sign of pathogenesis. However, molecular and cellular events precede the immune attack on the β-cell immunity. It has been shown that patients who developed T1D have an altered metabolome prior to the appearance of autoantibodies. Although much is known about the pathogenesis of T1D, the contribution of the environment/immune factors triggering the disease is still to be revealed. 

In the present study both metabolic and immune deviations observed in the NOD mouse was analyzed. Serum metabolome analysis of the NOD mouse revealed striking resemblance to the human metabolic profile, with many metabolites in the TCA cycle significantly different from the non-diabetic control B6 mice. In addition, an increased level of glutamic acid was of the most distinguishing metabolite. A detailed bioinformatics analysis revealed various genes/enzymes to be present in the Idd regions. Compared to B6 mice, many of the genes correlated to the metabolic pathways, showed single nucleotide polymorphism (SNP), which can eventually affect the functionality of the protein. A genetic analysis of the increased glutamic acid revealed several Idd regions to be involved in this phenotype. The regions mapped in the genetic analysis harbor important enzymes and transporters related to glutamic acid. In-vitro islet culture with glutamic acid led to increased beta cell death indicating a toxic role of glutamic acid specifically towards insulin producing beta cells.

In the analysis of the immune system, B cells from NOD mice, which are known to express high levels of TACI, were stimulated with APRIL, a TACI ligand. This resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. NOD mice have previously been shown to react vigorously to T-dependent antigens upon immunization. In this study we confirmed this as NOD mice showed an enhanced and prolonged immune response to hen egg lysozyme. Thus, serum IgG levels were significantly increased in the NOD mice and were predominantly of the IgG1 subtype. Immunofluorescence analysis revealed increased number of germinal centers in the NOD mice. Transfer of purified B and T cells from NOD to an immune deficient mouse could reproduce the original phenotype as seen in the NOD mice.    

Collectively, this thesis has analyzed the metabolomics and immune deviations observed in the NOD mice.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2016. , 62 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1777
Keyword [en]
NOD mouse, Type 1 diabetes, B cells, glutamic acid, metabolomics
National Category
Immunology
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-119444ISBN: 978-91-7601-404-2 (print)OAI: oai:DiVA.org:umu-119444DiVA: diva2:920957
Public defence
2016-05-13, A5_R0, Byg 6A, NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2016-04-21 Created: 2016-04-19 Last updated: 2016-04-20Bibliographically approved
List of papers
1. Altered metabolic signature in Pre-Diabetic NOD Mice
Open this publication in new window or tab >>Altered metabolic signature in Pre-Diabetic NOD Mice
Show others...
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, e35445- p.Article in journal (Refereed) Published
Abstract [en]

Altered metabolism proceeding seroconversion in children progressing to Type 1 diabetes has previously been demonstrated. We tested the hypothesis that non-obese diabetic (NOD) mice show a similarly altered metabolic profile compared to C57BL/6 mice. Blood samples from NOD and C57BL/6 female mice was collected at 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13 and 15 weeks and the metabolite content was analyzed using GC-MS. Based on the data of 89 identified metabolites OPLS-DA analysis was employed to determine the most discriminative metabolites. In silico analysis of potential involved metabolic enzymes was performed using the dbSNP data base. Already at 0 weeks NOD mice displayed a unique metabolic signature compared to C57BL/6. A shift in the metabolism was observed for both strains the first weeks of life, a pattern that stabilized after 5 weeks of age. Multivariate analysis revealed the most discriminative metabolites, which included inosine and glutamic acid. In silico analysis of the genes in the involved metabolic pathways revealed several SNPs in either regulatory or coding regions, some in previously defined insulin dependent diabetes (Idd) regions. Our result shows that NOD mice display an altered metabolic profile that is partly resembling the previously observation made in children progressing to Type 1 diabetes. The level of glutamic acid was one of the most discriminative metabolites in addition to several metabolites in the TCA cycle and nucleic acid components. The in silico analysis indicated that the genes responsible for this reside within previously defined Idd regions.

Place, publisher, year, edition, pages
Public Library of Science, 2012
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-54276 (URN)10.1371/journal.pone.0035445 (DOI)000305341600149 ()22514744 (PubMedID)
Note

This work was supported by the Kempe Foundation, the Medical Faculty at Umeå University, Insamlingsstiftelsen at Umeå University, Magnus Bergvalls stiftelse, JDRF (1-2008-1011), and the Children Diabetes Foundation in Sweden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Available from: 2012-04-23 Created: 2012-04-23 Last updated: 2017-12-07Bibliographically approved
2. Elevated Systemic Glutamic Acid Level in the Non-Obese Diabetic Mouse is Idd Linked and Induces Beta Cell Apoptosis
Open this publication in new window or tab >>Elevated Systemic Glutamic Acid Level in the Non-Obese Diabetic Mouse is Idd Linked and Induces Beta Cell Apoptosis
2017 (English)In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 150, no 2, 162-171 p.Article in journal (Refereed) Published
Abstract [en]

Although type 1 diabetes (T1D) is a T-cell-mediated disease in the effector stage, the mechanism behind the initial beta cell assault is less understood. Metabolomic differences, including elevated levels of glutamic acid, have been observed in patients with T1D before disease onset, as well as in pre-diabetic non-obese diabetic (NOD) mice. Increased levels of glutamic acid damage both neurons and beta cells, implying that this could contribute to the initial events of T1D pathogenesis. We investigated the underlying genetic factors and consequences of the increased levels of glutamic acid in NOD mice. Serum glutamic acid levels from a (NODxB6) F-2 cohort (n = 182) were measured. By genome-wide and Idd region targeted microsatellite mapping, genetic association was detected for six regions including Idd2, Idd4 and Idd22. In silico analysis of potential enzymes and transporters located in and around the mapped regions that are involved in glutamic acid metabolism consisted of alanine aminotransferase, glutamic-oxaloacetic transaminase, aldehyde dehydrogenase 18 family, alutamyl-prolyl-tRNA synthetase, glutamic acid transporters GLAST and EAAC1. Increased EAAC1 protein expression was observed in lysates from livers of NOD mice compared with B6 mice. Functional consequence of the elevated glutamic acid level in NOD mice was tested by culturing NOD. Rag2(-/-) Langerhans' islets with glutamic acid. Induction of apoptosis of the islets was detected upon glutamic acid challenge using TUNEL assay. Our results support the notion that a dysregulated metabolome could contribute to the initiation of T1D. We suggest that targeting of the increased glutamic acid in pre-diabetic patients could be used as a potential therapy.

Keyword
beta cell apoptosis, genetics, glutamic acid, Idd, non-obese diabetic mice
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-119451 (URN)10.1111/imm.12674 (DOI)000394790400005 ()27649685 (PubMedID)
Available from: 2016-04-19 Created: 2016-04-19 Last updated: 2017-11-30Bibliographically approved
3. Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies, enhanced plasma cell differentiation and immunoglobulin production
Open this publication in new window or tab >>Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies, enhanced plasma cell differentiation and immunoglobulin production
2016 (English)In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 149, no 3, 297-305 p.Article in journal (Refereed) Published
Abstract [en]

B cells have an important pathogenic role in the development of type 1 diabetes in the non-obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACIhigh-expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation-inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up-regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down-regulate TACI. Availability of the TACI ligand B-cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4-hydroxy-3-nitrophenylacetyl hapten-conjugated hen egg lysozyme, NOD mice produced higher titres of low-affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse.

Keyword
affinity maturation, B cells, B-cell activating factor, non-obese diabetic mouse, transmembrane activat or, calcium modulator and cyclophilin ligand interactor
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-119452 (URN)10.1111/imm.12651 (DOI)000387360300005 ()27444337 (PubMedID)
Note

Originally published in thesis in manuscript form with the title: Increased expression of TACI in the NOD mouse results in enhanced plasma cell differentiation and immunoglobulin production

Available from: 2016-04-19 Created: 2016-04-19 Last updated: 2017-11-30Bibliographically approved
4. B cell intrinsic defects lead to enhanced immune response in the NOD mice
Open this publication in new window or tab >>B cell intrinsic defects lead to enhanced immune response in the NOD mice
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-119453 (URN)
External cooperation:
Available from: 2016-04-19 Created: 2016-04-19 Last updated: 2016-09-08

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