All forms of diabetes are characterized by the death of insulin producing ? cells. In Type 1 diabetes (T1D), this leads to the reliance on exogenous insulin for survival. In addition, there is ? cell loss in T2D and in individuals who lose islet or pancreas allografts. New therapies have been developed that can modulate the course of T1D but the effective application of these therapies requires a clearer understanding of the pathologic processes including the timing and precipitants of ? cell death because these therapies have not led to permanent remission of disease. However, death of ? cells is silent - it cannot be detected in vivo until it has progressed to such an extent that metabolic function is impaired. The goal of the proposed work is to address this need by developing an assay that can measure ? cell death in a quantitative manner in vivo. Our proposed assay is based on the understanding that epigenetic modifications control the tissue specific expression of the insulin gene. Since the only significant source of insulin is ? cells, demethylated insulin DNA in the serum or plasma reflects the death of ? cells and release of their DNA. We have developed a novel assay that measures demethylated insulin DNA with a nested PCR reaction in mice and have validated its ability to detect ? cell death in 2 models of T1D. In NOD mice, increased levels of demethylated insulin DNA were detectable in the serum at a time when the glucose level was not elevated. We now propose to develop this approach for use with human samples. In the first aim we will optimize the assay for detection of demethylated insulin-coding DNA in human serum. We will select primers for detection of demethylated insulin DNA as well as the conditions for the nested PCR reactions. We will then characterize the assay including the sensitivity and reproducibility, and test whether serum or plasma is optimal starting material. We will also determine how the total amount of DNA affects the assay results. In the 3rd aim, we will determine baseline responses in healthy control subjects, patients with long standing T1D without detectable C-peptide, and new onset disease. We will test whether the assay results vary in childhood since T1D is most frequently diagnosed in children. Once this assay is optimized, it may have use in clinical settings to study ? cell death such as in individuals at hig risk for development of T1D (in the TrialNet Natural History Study), immune therapy trials, tracking islet and pancreas transplants, and others. Moreover, future application of this assay will include studies of T2D and understanding control of human ? cell growth and regeneration.
All forms of diabetes involve death of ? cells but we do not have a means of measuring ? cell death in vivo. We propose to develop a new assay that will address this need based on identification of ? cell specific insulin DNA. The assay will have broad use for understanding ? death in diabetes and for developing new therapies to treat and prevent diabetes.
|Herold, Kevan C; Usmani-Brown, Sahar; Ghazi, Tara et al. (2015) ? cell death and dysfunction during type 1 diabetes development in at-risk individuals. J Clin Invest 125:1163-73|
|Tooley, James E; Herold, Kevan C (2014) Biomarkers in type 1 diabetes: application to the clinical trial setting. Curr Opin Endocrinol Diabetes Obes 21:287-92|
|Usmani-Brown, Sahar; Lebastchi, Jasmin; Steck, Andrea K et al. (2014) Analysis of ?-cell death in type 1 diabetes by droplet digital PCR. Endocrinology 155:3694-8|
|Hughes, Jing W; Herold, Kevan C (2013) Novel SIRT1 mutation linked to autoimmune diabetes in humans. Cell Metab 17:311-2|