Age-Dependent Sensitivity to Streptozotocin of Pancreatic Islets Isolated from Female NOD Mice

AbstractThe notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4+ and CD8+ T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing β cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4+ and CD8+ T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4+ and CD8+ T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies.

Download Full-text

Fas and Fas Ligand Immunoexpression in Pancreatic Islets of NOD Mice during Spontaneous and Cyclophosphamide-Accelerated Diabetes

Annals of the New York Academy of Sciences ◽

10.1196/annals.1288.019 ◽

2003 ◽

Vol 1005 (1) ◽

pp. 166-169 ◽

Cited By ~ 4

Author(s):

SHIVA REDDY ◽

JACQUELINE M. ROSS

Keyword(s):

Pancreatic Islets ◽

Fas Ligand ◽

Nod Mice

Download Full-text

Feasibility of Localized Metabolomics in the Study of Pancreatic Islets and Diabetes

Metabolites ◽

10.3390/metabo9100207 ◽

2019 ◽

Vol 9 (10) ◽

pp. 207 ◽

Cited By ~ 2

Author(s):

Oscar Alcazar ◽

Luis F. Hernandez ◽

Ashley Tschiggfrie ◽

Michael J. Muehlbauer ◽

James R. Bain ◽

...

Keyword(s):

Pancreatic Islets ◽

Aqueous Humor ◽

General Circulation ◽

Biomarker Discovery ◽

Nod Mice ◽

Health Condition ◽

Plasma Metabolites ◽

Islet Transplant ◽

Metabolic Biomarkers ◽

Longitudinal Dataset

(1) Background: Disruption of insulin production by native or transplanted pancreatic islets caused by auto/allo-immunity leads to hyperglycemia, a serious health condition and important therapeutic challenge due to the lifelong need for exogeneous insulin administration. Early metabolic biomarkers can prompt timely interventions to preserve islet function, but reliable biomarkers are currently lacking. We explored the feasibility of “localized metabolomics” where initial biomarker discovery is made in aqueous humor samples for further validation in the circulation. (2) Methods: We conducted non-targeted metabolomic studies in parallel aqueous humor and plasma samples from diabetic and nondiabetic mice. Metabolite levels and associated pathways were compared in both compartments as well as to an earlier longitudinal dataset in hyperglycemia-progressor versus non-progressor non-obese diabetic (NOD) mice. (3) Results: We confirmed that aqueous humor samples can be used to assess metabolite levels. About half of the identified metabolites had well-correlated levels in the aqueous humor and plasma. Several plasma metabolites were significantly different between diabetic and nondiabetic animals and between males and females, and many of them were correlated with the aqueous humor. (4) Conclusions: This study provides proof-of-concept evidence that aqueous humor samples enriched with islet-related metabolites and representative of the immediate islet microenvironment following intraocular islet transplant can be used to assess metabolic changes that could otherwise be overlooked in the general circulation. The findings support localized metabolomics, with and without intraocular islet transplant, to identify biomarkers associated with diabetes and islet allograft rejection.

Download Full-text

Natural Protection From Type 1 Diabetes in Non Obese Diabetic (Nod) Mice is Characterized by a Unique Pancreatic Islet Phenotype

10.2337/figshare.13643393 ◽

2021 ◽

Author(s):

Joanne Boldison ◽

Terri C. Thayer ◽

Joanne Davies ◽

F. Susan Wong

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Pancreatic Islets ◽

Pancreatic Islet ◽

Nod Mice ◽

Significant Shift ◽

Immune Infiltrate ◽

Cell Compartments ◽

Natural Protection

The non-obese diabetic (NOD) mouse develops spontaneous type 1 diabetes, with some features of disease that are very similar to the human disease. However, a proportion of NOD mice are naturally-protected from developing diabetes, and currently studies characterising this cohort are very limited. Here, using both immunofluorescence and multi-parameter flow cytometry we focus on the pancreatic islet morphology and immune infiltrate observed in naturally-protected NOD mice. We show that naturally-protected NOD mice are characterised by an increased frequency of insulin-containing, smaller sized, pancreatic islets. Although mice remain diabetes free, florid immune infiltrate remains. However, this immune infiltrate is skewed towards a regulatory phenotype in both T and B-cell compartments. Pancreatic islets have an increased frequency of IL-10 producing B cells and associated cell surface markers. Resident memory CD69+CD8+ T cells show a significant shift towards reduced CD103 expression, while CD4+ T cells have increased FoxP3+CTLA4+ expression. These data indicate that naturally-protected NOD mice have a unique islet signature and provide new insight into regulatory mechanisms within pancreatic islets.

Download Full-text

SUN-658 CHL1 Increases Insulin Secretion&Negatively Regulates the Poliferation of Pancreatic β Cell

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1269 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Tao Yang ◽

Qi Fu ◽

Hemin Jiang

Keyword(s):

Cell Cycle ◽

Insulin Secretion ◽

Pancreatic Islets ◽

High Fat Diet ◽

Nod Mice ◽

Pancreatic Β Cell ◽

High Fat ◽

Β Cells ◽

Β Cell ◽

Min6 Cells

Abstract CHL1 Increases Insulin Secretion & Negatively Regulates The Poliferation Of Pancreatic β Cell Objective: CHL1 belongs to neural recognition molecules of the immunoglobulin superfamily, is mainly expressed in the nervous system. CHL1 is involved in neuronal migration, axonal growth, and dendritic projection. RNA sequencing of single human islet cells confirmed that CHL1 had an expression difference in β cells of type 2 diabetes and healthy controls. However, whether CHL1 gene regulates islet function remained to be explored. Methods: PCR and Western Blot were applied to investigate the tissue distribution of CHL1 in wild-type C57BL/6J mice. The islet expression of CHL1 gene was observed in pancreatic islets of NOD mice and high-fat-diet C57BL/6J mice of different ages. MIN6 cells with siRNA to silence CHL1 or with lentivirus to overexpress CHL1 were constructed. Effects of the gene on proliferation, apoptosis, cell cycle and insulin secretion were determined by using CCK8, EdU, TUNEL, AV/PI, GSIS, electron microscopy and flow cytometry. Results: CHL1 was localized on the cell membrane and expressed in the nervous system, islet of pancreas and gastrointestinal tract. CHL1 was hypoexpressed in the pancreatic islets of obese mice, hyperexpressed in the pancreatic islets of NOD mice and in vitro after treated with cytokines. After silencing CHL1 in MIN6 cells, insulin secretion decreased in 20 mM glucose with down-regulation of INS1, SLC2A2 gene, and transmission electron microscope showed the number of insulin secretary granules <50nm from the cell membrane was significantly reduced. Silencing of CHL1 in MIN6 cells induced cell proliferation, reduced apoptosis rate, prolonged the S phase of cell cycle and shortened the G1 phase with downregulated expression of p21, p53 and up-regulated expression of cyclin D1, opposite results were found in CHL1 over-expressing MIN6 cells. Proliferation induced by silencing of CHL1 was inhibited by ERK inhibitor (PD98059), which indicates that ERK pathway is essential for signaling by these molecules in pancreatic β cell. Conclusion: The expression of CHL1 gene was significantly decreased in the pancreatic islets of obese mice induced by high-fat diet. The low expression of CHL1 gene promotes the proliferation of MIN6 cells through the ERK pathway and affect cell cycle through the p53 pathway. This may be one of the mechanisms that pancreatic β cells compensatory hyperplasia in the stage of obesity-induced pre-diabetes.

Download Full-text

Faculty Opinions recommendation of Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718167483.793498156 ◽

2014 ◽

Author(s):

Lucienne Chatenoud ◽

Sylvaine You

Keyword(s):

T Cells ◽

Nod Mice ◽

Dependent Manner ◽

Autoreactive T Cells ◽

Thymic Development ◽

Age Dependent

Download Full-text

Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713543114 ◽

2017 ◽

Vol 114 (48) ◽

pp. E10418-E10427 ◽

Cited By ~ 44

Author(s):

Javier A. Carrero ◽

Derrick P. McCarthy ◽

Stephen T. Ferris ◽

Xiaoxiao Wan ◽

Hao Hu ◽

...

Keyword(s):

T Cells ◽

Pancreatic Islets ◽

Islet Cells ◽

Autoimmune Diabetes ◽

Nod Mice ◽

Diabetes Incidence ◽

Glucose Response ◽

Blocking Antibody ◽

Multiple Parameters ◽

Pancreatic Islets Of Langerhans

Treatment of C57BL/6 or NOD mice with a monoclonal antibody to the CSF-1 receptor resulted in depletion of the resident macrophages of pancreatic islets of Langerhans that lasted for several weeks. Depletion of macrophages in C57BL/6 mice did not affect multiple parameters of islet function, including glucose response, insulin content, and transcriptional profile. In NOD mice depleted of islet-resident macrophages starting at 3 wk of age, several changes occurred: (i) the early entrance of CD4 T cells and dendritic cells into pancreatic islets was reduced, (ii) presentation of insulin epitopes by dispersed islet cells to T cells was impaired, and (iii) the development of autoimmune diabetes was significantly reduced. Treatment of NOD mice starting at 10 wk of age, when the autoimmune process has progressed, also significantly reduced the incidence of diabetes. Despite the absence of diabetes, NOD mice treated with anti–CSF-1 receptor starting at 3 or 10 wk of age still contained variably elevated leukocytic infiltrates in their islets when examined at 20–40 wk of age. Diabetes occurred in the anti–CSF-1 receptor protected mice after treatment with a blocking antibody directed against PD-1. We conclude that treatment of NOD mice with an antibody against CSF-1 receptor reduced diabetes incidence and led to the development of a regulatory pathway that controlled autoimmune progression.

Download Full-text