1. Abstract
Objective
Methods
Results
Summary and Conclusions•Demonstrate diabetes progression in NOD model
•Investigate DNMT gene expression: RT-PCR
•Investigate DNMT protein level using immunofluorescence
•IPGTT on NOD Mice with Insulin ELISA
•Collection of mRNA and Gene Expression Assay, RT-PCR on DNMT1,
DNMT3A, Ins2 and B-Actin
•Frozen tissue sections of NOD pancreas on slides
•Immunofluorescence and histomorphic analysis using ImageJ of
confocal images: staining for Insulin and DNMT1
•NOD mice exhibit diabetes progression
•Insulin secretion decreased
•DNMT1 and DNMT3A levels increased in the
diabetic group
•DNMT1/3A are elevated and likely contribute to
gene silencing in diabetes; beta cell islet and
immune cells
Methyl
Group Added
by DNMT
DAPI
Insulin
DNMT1
Merge
NOD
Wk8
NOD
Wk12
NOD
Wk16
C57Bl6
Control
Insulin secretion decreased in week 16 (diabetic) group; confirmed by
insulin ELISA and Gene Expression Assay and IF. DNMT1 and DNMT3A
increased in expression, confirmed by gene expression and IF (DNMT1 only)
Healthy
Islets
Diseased
Islets
Immune
Infiltrate
Wk 8-10
Islet
Wk 8-10
Infiltrate
Wk 12-14
Islet
Wk 12-14
Infiltrate
Wk 16
Islet
Wk 16
Infiltrate
Examination of DNA Methyltransferase (DNMT) Levels During the Progression of Type 1
Diabetes
1,2Lauren A. Kenna, 1,2Eitan Akirav, 1Tandra Chakraborty
Image from: Miescher F. 2011.
Controlling Patterns of DNA
Methylation. Medicalpress, Genetics
Type 1 Diabetes (T1D) is defined by an autoimmune destruction of
pancreatic β-cells over time resulting in complete loss of insulin production
and blood glucose regulation. Studies show that many autoimmune
diseases, including diabetes, can be induced by epigenetic changes leading
to insulin deficiency, resistance and glucose intolerance. Epigenetics is the
process by which genes are regulated for expression due to environmental
factors and can directly impact phenotype. These changes are often due to
methylation of DNA, where an enzyme, DNMT, adds a methyl group to a
CpG in gene promoter region to physically block transcription. To examine
the levels; prediabetic and diabetic non-obese diabetic mice (NOD), a
model of human type 1 diabetes, were monitored over a period of 8 weeks;
pancreatic sections and blood samples were obtained to establish the role
of DNMT in islet inflammation. Intraperitoneal Glucose Tolerance Test
(IPGTT) was performed on the day of animal sacrifice to monitor disease
progression with age. DNMT1 expression measured by RT-PCR on whole
pancreatic lysates showed a 3.5 fold increase over time, while insulin gene
expression decreased by ~5 folds in 16 week mice when compared to 8-10
week old animals. DNMT3A increased overall by ~2 fold in expression in
the week 16-diabetic group when compared to the week 8-10 group.
Histomorphic analysis of frozen pancreatic sections stained for DNMT1,
insulin and DAPI showed an increase of 45% in DNMT1 positive area
relative to insulin. This data shows that DNMT1 and DNMT3A is expressed
in higher levels in diabetic pancreatic islets of NOD mice than healthy islets,
suggesting that DNMT1/3A may contribute to gene silencing the islet.
1Adelphi University, Garden City, NY USA, 2Winthrop University Hospital Biomedical Research Institute, Mineola, NY USA

2. DAPI
Insulin
DNMT1
Merge
NOD
Wk8
NOD
Wk12
NOD
Wk16
C57Bl6
Control
Outline insulin
positive area and
calculate
Transfer outline to
DNMT channel,
calculate positive
nuclei area
Trace infiltrate
area, calculate
positive nuclei
area
Analysis of Immunofluorescence on frozen sections

3. References
Miescher F. 2011. Controlling Patterns of DNA Methylation. Medicalpress, Genetics. [cited
2016 March 29]. Available from:http://medicalxpress.com/news/2011-10-patterns-dna-
methylation.html
Lienert, F., Wirbelauer, C., Som, I., Dean, A., Mohn, F., & Schübeler, D. (2011).
Identification of genetic elements that autonomously determine DNA methylation
states. Nature genetics, 43(11), 1091-1097.