ß-Cell death is decreased in women with gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) affects approximately 7-17 % of all pregnancies and has been recognized as a significant risk factor to neonatal and maternal health. Postpartum, GDM significantly increases the likelihood of developing type 2 diabetes (T2D). While it is well established that insulin resistance and impaired ß-cell function contribute to GDM development, the role of active ß-cell loss remains unknown. Differentially methylated circulating free DNA (cfDNA) is a minimally invasive biomarker of ß-cell loss in type 1 diabetes mellitus. Here we use cfDNA to examine the levels of ß-cell death in women with GDM. METHODS: Second to third-trimester pregnant women with GDM were compared with women with normal pregnancy (PRG), women at postpartum (PP), and non-pregnant (NP) women. Fasting glucose levels, insulin, and C-peptide levels were measured. Serum samples were collected and cfDNA purified and bisulfite treated. Methylation-sensitive probes capable of differentiating between ß-cell-derived DNA (demethylated) and non-ß-cell-derived DNA (methylated) were used to measure the presence of ß-cell loss in the blood. RESULTS: GDM was associated with elevated fasting glucose levels (GDM = 185.9 ± 5.0 mg/dL) and reduced fasting insulin and c-peptide levels when compared with NP group. Interestingly, ß-cell derived insulin DNA levels were significantly lower in women with GDM when compared with PRG, NP, and PP groups (demethylation index: PRG = 7.74 × 10(-3) ± 3.09 × 10(-3), GDM = 1.01 × 10(-3) ± 5.86 × 10(-4), p < 0.04; NP = 4.53 × 10(-3) ± 1.62 × 10(-3), PP = 3.24 × 10(-3) ± 1.78 × 10(-3)). CONCLUSIONS: These results demonstrate that ß-cell death is reduced in women with GDM. This reduction is associated with impaired insulin production and hyperglycemia, suggesting that ß-cell death does not contribute to GDM during the 2nd and 3rd trimester of pregnancy.

A Minimally-invasive Blood-derived Biomarker of Oligodendrocyte Cell-loss in Multiple Sclerosis.

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). Minimally invasive biomarkers of MS are required for disease diagnosis and treatment. Differentially methylated circulating-free DNA (cfDNA) is a useful biomarker for disease diagnosis and prognosis, and may offer to be a viable approach for understanding MS. Here, methylation-specific primers and quantitative real-time PCR were used to study methylation patterns of the myelin oligodendrocyte glycoprotein (MOG) gene, which is expressed primarily in myelin-producing oligodendrocytes (ODCs). MOG-DNA was demethylated in O4(+) ODCs in mice and in DNA from human oligodendrocyte precursor cells (OPCs) when compared with other cell types. In the cuprizone-fed mouse model of demyelination, ODC derived demethylated MOG cfDNA was increased in serum and was associated with tissue-wide demyelination, demonstrating the utility of demethylated MOG cfDNA as a biomarker of ODC death. Collected sera from patients with active (symptomatic) relapsing-remitting MS (RRMS) demonstrated a higher signature of demethylated MOG cfDNA when compared with patients with inactive disease and healthy controls. Taken together, these results offer a minimally invasive approach to measuring ODC death in the blood of MS patients that may be used to monitor disease progression.

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

In type 1 diabetes (T1D), ß-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR) of ß-cell-derived DNA in the blood can serve as a biomarker of ß-cell death in T1D. Amylin is highly expressed by ß-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA) may serve as a biomarker of ß-cell death in T1D. ß 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 ß-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 ß-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 ß-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of ß-cell loss.

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin ß1 in ß Cells.

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin ß1. A wide array of integrin ß subunits was detected in ßTC3 and NIT-1 insulinomas as well as in primary islets, with integrin ß1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin ß1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer ß-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin ß1 in PIs. Antibody-mediated blocking of integrin ß1 led to alterations in ß-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin ß1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in ß cells.

In vitro formation of ß cell pseudoislets using islet-derived endothelial cells.

ß cell pseudoislets (PIs) are used for the in vitro study of ß-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage ß-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of ß-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to ß-cells cultured as monolayers. Key extracellular matrix proteins that were absent in ß-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining ß cell function in a native 3-D configuration and can be used for examining ß-cell/iEC interactions in vitro.