Postpartum Circulating Cell-Free Insulin DNA Levels Are Higher in Women with Previous Gestational Diabetes Mellitus Who Develop Type 2 Diabetes in Later Life.

BACKGROUND: Women with previous gestational diabetes mellitus (GDM) have evidence of postpartum ß-cell dysfunction, which increases their risk of developing type 2 diabetes (T2DM) later in life. Elevated levels of circulating cell-free preproinsulin (INS) DNA correlate with dying ß-cells in both mice and humans. The aim of this study was to determine if cell-free circulating INS DNA levels are higher in women with previous GDM who develop T2DM. METHODS: We used droplet digital (dd) PCR to measure the levels of cell-free circulating methylated and unmethylated INS DNA in plasma from 97 women with normal glucose tolerance (NGT), 12 weeks following an index GDM pregnancy. Women were assessed for up to 10 years for the development of T2DM. RESULTS: In the follow-up period, 22% of women developed T2DM. Compared with NGT women, total cell-free INS DNA levels were significantly higher in women who developed T2DM (P = 0.02). There was no difference in cell-free circulating unmethylated and methylated INS DNA levels between NGT women and women who developed T2DM (P = 0.09 and P = 0.07, respectively). CONCLUSIONS: In women with a previous index GDM pregnancy, postpartum levels of cell-free circulating INS DNA are significantly higher in those women who later developed T2DM.

Comparative analysis of diagnostic platforms for measurement of differentially methylated insulin DNA.

Circulating cell-free DNA (cfDNA) has been intensively investigated as a diagnostic and prognostic marker for various cancers. In recent years, presence of unmethylated insulin cfDNA in the circulation has been correlated with pancreatic ß-cell death and risk of developing type 1 diabetes. Digital (d)PCR is an increasingly popular method of quantifying insulin cfDNA due to its ability to determine absolute copy numbers, and its increased sensitivity when compared to the more routinely used quantitative PCR. Multiple platforms have been developed to carry out dPCR. However, not all technologies perform comparably, thereby necessitating evaluation of each platform. Here, we compare two dPCR platforms: the QuantStudio 3D (QS3D, Applied Biosystems) and the QX200 (Bio-Rad), to measure copies of unmethylated/methylated insulin plasmids. The QS3D detected greater copy numbers of the plasmids than the QX200 (manual mode), whereas QX200 demonstrated minimal replicate variability, increased throughput, ease of use and the potential for automation. Overall, the performance of QX200, in our hands, was better suited to measure differentially methylated insulin cfDNA.

Droplet Digital PCR for Measuring Absolute Copies of Gene Transcripts in Human Islet-Derived Progenitor Cells.

Transcript analysis is a routinely used method to assess the expression profile of progenitor cells at different stages starting from their isolation to differentiation into specific lineages. It is a powerful way to understand similarities and differences between different cell types as well to estimate successful differentiation process. Transcript measurement is most commonly done using polymerase chain reaction (PCR) but other methods such as in situ hybridization, RNA sequencing are available. The quantitative PCR using TaqMan chemistry is a highly sensitive and reproducible method that measures gene transcripts as a relative abundance. With recent advances in technology, absolute quantitation of genes to single copy level is possible using digital PCR platforms.Digital PCR is an improved method of PCR in which a single reaction is partitioned into multiple mini reactions. Gene transcripts are measured in each of these mini reactions thereby improving assay sensitivity and making absolute quantitation possible. Here we describe the generation of human islet-derived progenitor cells and measuring gene transcripts in these cells at different passages using digital droplet PCR.

Expression of miR-206 in human islets and its role in glucokinase regulation.

Inappropriate insulin secretion from ß-cells is considered as an early sign of impaired glucose tolerance and type 2 diabetes (T2D). Glucokinase (GCK) is an important enzyme that regulates glucose metabolism and ensures that the normal circulating glucose concentrations are maintained. GCK expression is induced by glucose and regulated via transcription factors and regulatory proteins. Recently, microRNA-206 (miR-206) was reported to regulate GCK and alter glucose tolerance in normal and high-fat diet-fed mice. Although the study findings have implications for human diabetes, studies in human islets are lacking. Here, we analyze human islets from individuals without or with T2D, using TaqMan-based real-time qPCR at the tissue (isolated islet) level as well as at single cell resolution, to assess the relationship between miR-206 and GCK expression in normal and T2D human islets. Our data suggest that, unlike mouse islets, human islets do not exhibit any correlation between miR-206 and GCK transcripts. These data implicate the need for further studies aimed toward exploring its potential role(s) in human islets.