MS1 induces a spontaneous formation of free-floating insulin-positive PIs.

<p><b><i>A.</i></b> Light and phase contrast microscopy showing the formation of βTC3 PIs over 8 d. <b><i>B.</i></b> IF staining of βTC3 monolayers. Blue- DAPI, Red- Insulin and merge. <b><i>C.</i></b> 3-D reconstruction of z-stack confocal images of a representative PI. Blue- DAPI, Red- Insulin and merge.</p

Col-IV and laminin are detected in and around the PI.

<p><b><i>A.</i></b> IF staining of MS1 cells. Blue-DAPI, Green-CD31, Red-BS1 and merge. White/Yellow represents double positive cells. <b><i>B.</i></b> RT-PCR for laminin β1 and col-IV in MS1, whole murine islet preps, and βTC3 cells. Laminin α1 and α2 were not detected (data not shown). <b><i>C.</i></b> IF staining of MS1 cells. Blue- DAPI, Green- col-IV, White-Laminin, and merge. <b><i>D.</i></b> 3-D reconstruction of z-stack imaging of an 8 d old PI. <b><i>E.</i></b> Non-consecutive z-stack confocal images of a PI. Blue- DAPI, Red- Insulin, Green- col-IV, White- laminin.</p

Baseline and glucose stimulated insulin expression and secretion are enhanced in MS1-induced PIs.

<p><b><i>A.</i></b> FACS staining for proinsulin expression in monolayers (dotted line) and PIs (solid line). Grey histogram- isotype control. MFI: mono = 6579, PIs = 7486 <b><i>B.</i></b> Quantitative RT-PCR analysis of insulin 1 expression in βTC3 monolayers (closed bars) or PIs (opened bars). Data represents an average of 3 independent experiments. *<i>p<0.025</i>. Monolayers and PIs were cultured for 8 d, washed with media lacking glucose, and incubated with escalating glucose levels for 6 h. <b><i>C.</i></b> Insulin ELISA analysis of supernatant from βTC3 monolayers and PIs. Experiment represents three independent repeats. N = 3 per group. Two-way ANOVA analysis<b>.</b> <i>^†p<0.001, *p<0.0001.</i></p

Evaluation of β cell loss in MS1-induced PIs.

<p><b><i>A.</i></b> FACS staining for 7-AAD viability dye of monolayers (dotted line) and PIs (solid line). Grey histogram unstained control. Black arrow show 7-AAD positive cells. <b><i>B.</i></b> Non-consecutive z-stack confocal images of a 14 d old PI. Blue- DAPI, Red- Insulin, Green- cleaved caspase-3. White arrows point to caspase-3 positive cells.</p

Circulating Differentially Methylated Amylin DNA as a Biomarker of β-Cell Loss in Type 1 Diabetes

<div><p>In type 1 diabetes (T1D), β-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR) of β-cell-derived DNA in the blood can serve as a biomarker of β-cell death in T1D. Amylin is highly expressed by β-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA) may serve as a biomarker of β-cell death in T1D. β cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human β-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD) mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect β-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of β-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of β-cell loss.</p></div

Methylation specific primers show increased demethylated amylin DNA in the blood of patients with recent onset T1D.

<p><b><i>A</i>.</b> DMI values for healthy control (HC, closed circles) and recent onset T1D patients (RO, closed squares), p<0.015. <b><i>B</i>.</b> ROC analysis of patient data. AUC = 0.866, with 95% confidence interval 0.72 to 1.01, p<0.0017. <b><i>C</i>.</b> Correlation analysis between HbA1c and DMI values in RO patients. <b><i>D</i>.</b> Data presentation of insulin/amylin DMI per RO patient. Pearson’s r = 0.63, p<0.028.</p

Demethylated amylin DNA is increased in the blood of pre-diabetic NOD mice during T1D progression.

<p>8 wk old female NOD mice were housed in SPF conditions and monitored for 12 weeks for the development of diabetes. Blood from each animal was collected on wk 8, 14, 18 and 20. <b><i>A</i>.</b> IPGTT values of pre-diabetic NOD mice at various ages over 120 minutes. <b><i>B</i>.</b> Immunofluorescence staining of representative islets from NOD mice at various ages. Blue- DAPI. Green- Amylin. Red- Insulin. White- GLUT2. Note the appearance of insulin^-amylin⁺GLUT2⁺ β-cells in islets from diabetic NOD mice <b><i>C</i>.</b> Aggregate DMI and glucose values in NOD mice collected over 12 weeks (n = 14). <b><i>D</i>.</b> Representative data from four individual NOD mice. DNA concentration in the serum was measured using picogreen. DMI was calculated on bisulfite treated serum-derived DNA. Variability in disease onset and β-cell DNA is characteristic of the spontaneous nature of T1D in the NOD mouse model.</p

Methylation-specific primers show a high degree of specificity and sensitivity and detect demethylated DNA in primary human islets and enriched human β-cells.

<p><b><i>A</i>.</b> Methylation sensitive DNA digestion was performed on magnetically enriched β-cells and liver DNA using the McrBC enzyme. Digested DNA was subjected to semi-quantitative PCR (cycles 27 and 30) and run on agarose gel. unTx- untreated DNA receiving all reaction components except McrBC enzyme. Pos- positive control using cloned native amylin DNA. NTC- no template control. <b><i>B</i>.</b> Schematic depiction of differentially methylated CpG dinucleotides in the human amylin coding region used for the design of methylation-specific primers. <b><i>C</i>.</b> Human methylation-specific primers were tested over a wide range of copy numbers for DeMeth and Meth DNA (25 to 25x10³) to determine assay sensitivity and specificity using cloned DNA sequences. (R² = 0.9930, p<0.0001). <b><i>D</i>.</b> qPCR reaction using methylation-sensitive primers on bisulfite treated DNA from liver, purified human islets, peripheral blood mononuclear cells (PBMCs) and magnetic beads enriched human β-cells. Islet fractions from three individual donors were used. Data represents DMI values from single donor and consists of two independent repeats.</p

Primer sequences and PCR protocols for human amylin analysis.

<p>Primer sequences and PCR protocols for human amylin analysis.</p

A schematic depiction of amylin-based biomarker assay for the detection of β-cell loss in T1D.

<p>β-cells within the islets of Langerhans die, releasing genomic DNA into circulation. Blood samples are taken from subject and DNA is purified and subjected bisulfite conversion. Bisulfite converted DNA is subjected to first step PCR reaction using methylation unspecific primers, and run on agarose gel. First step PCR product is purified from agarose gel and used as template for qPCR using methylation-specific primers.</p