β cell death and dysfunction during type 1 diabetes development in at-risk individuals

Abstract

Role of the funding source: Funding from the NIH was used for support of the participating clinical centers and the coordinating center. The funding source did not participate in the collection or the analysis of the data.

Background: The β cell killing that characterizes type 1 diabetes (T1D) is thought to begin years before patients present clinically with metabolic decompensation; however, this primary pathologic process of the disease has not been measured.

Methods: Here, we measured β cell death with an assay that detects β cell-derived unmethylated insulin (INS) DNA. Using this assay, we performed an observational study of 50 participants from 2 cohorts at risk for developing T1D from the TrialNet Pathway to Prevention study and of 4 subjects who received islet autotransplants.

Results: In at-risk subjects, those who progressed to T1D had average levels of unmethylated INS DNA that were elevated modestly compared with those of healthy control subjects. In at-risk individuals that progressed to T1D, the observed increases in unmethylated INS DNA were associated with decreases in insulin secretion, indicating that the changes in unmethylated INS DNA are indicative of β cell killing. Subjects at high risk for T1D had levels of unmethylated INS DNA that were higher than those of healthy controls and higher than the levels of unmethylated INS DNA in the at-risk progressor and at-risk nonprogressor groups followed for 4 years. Evaluation of insulin secretory kinetics also distinguished high-risk subjects who progressed to overt disease from those who did not.

Conclusion: We conclude that a blood test that measures unmethylated INS DNA serves as a marker of active β cell killing as the result of T1D-associated autoimmunity. Together, the data support the concept that β cell killing occurs sporadically during the years prior to diagnosis of T1D and is more intense in the peridiagnosis period.

Trial registration: Clinicaltrials.gov NCT00097292.

Funding: Funding was from the NIH, the Juvenile Diabetes Research Foundation, and the American Diabetes Association.

Figure 6. Changes in insulin secretory pattern during progression to T1D in PTP participants followed for up to 4 years.

The proportion of insulin secreted in the first hour of the OGTTs shown in Figure 1C is shown. Day 0 was designated the date of diagnosis of T1D in the progressors and the last visit of the nonprogressors. There was a significant difference in the dynamics of insulin secretion in the progressors versus nonprogressors (n = 10 each group, mean ± SEM from the mixed model are shown; P = 0.04, repeated-measures ANOVA). *P = 0.03.

Figure 5. Insulin secretion during the repeat OGTTs in individuals at high risk for T1D.

(A) The insulin secretory AUC (n = 10 each, mean ± SEM) and (B) change in the AUC (second visit to first visit) are shown for each subgroup. There was not a significant change in the ISR AUC between the 2 visits. (C) The time of the peak rate of insulin secretion (ISR) during the 2-hour OGTT is shown for the first and second visits for the 3 subgroups of high-risk subjects. The time of peak insulin secretion was not significantly different at the first visit, at a time when all participants had an abnormal OGTT. Those who returned with normal glucose tolerance had an earlier time of peak insulin secretion when they returned (the lines represent median values, P = 0.0004, Kruskal-Wallis, overall group comparison; *P < 0.05, ***P < 0.001, Dunn’s multiple comparison test). (D) The percentage of total insulin secreted within the first hour of the second OGTT is shown. The high-risk subjects who returned with normal OGTT secreted a significantly greater proportion of insulin in the first hour compared with those who returned with an abnormal OGTT or diabetic OGTT (P = 0.0005, ANOVA; **P < 0.01, Bonferroni multiple comparisons test) (mean ± SEM).

Figure 4. β Cell death and glucose tolerance in high-risk individuals.

(A) The level of unmethylated INS DNA to methylated INS DNA measured by ddPCR in serum samples from the first visit (n = 27; results from 3 samples were unavailable for technical reasons) was compared with age-matched nondiabetic control subjects (HC) (****P < 0.0001, Student’s t test). The dashed line represents the mean + 2 SD of the nondiabetic control subjects (n = 32) is shown. The dashed line represents the mean + 2 SD of the nondiabetic control subjects. (B) Comparison of ratios in high-risk subjects, progressors, and nonprogressors from the PTP study and healthy control subjects. Data shown are the least squares (mean ± SEM) from the mixed model (using ID as a class variable). Ratios were higher in the high-risk group compared with the others (P < 0.0001, ANOVA; ****P < 0.0001). (C) Change in glucose AUC during OGTTs performed on 2 visits in high-risk participants. The second test was performed 94 ± 15 days after the first. Groups were designated by the outcome of the OGTT at the second visit. One-third of subjects had a normal glucose tolerance test when they returned (Nl), one-third repeated the finding of dysglycemia (AbnGT), and one-third showed a diabetic glucose tolerance test (DM). Change in glucose AUC during the 2-hour OGTT is shown (n = 10 each, P = 0.0002, ANOVA; **P < 0.01, ****P < 0.0001, Bonferroni multiple comparison test). (D) Ratios at the time of the first visit are shown for those who returned with a normal (n = 10), an abnormal (n = 8), and a diabetic (n = 9) OGTT and compared with the ratios in controls (mean + SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. controls, Bonferroni multiple comparison test and ANOVA). Ratios were elevated in each subgroup compared with controls but were not significantly different between groups.

Figure 3. Levels of unmethylated INS DNA in recipients of autologous islet transplants.

The levels of unmethylated INS DNA were measured in 4 individuals with pancreatitis who underwent a pancreatectomy, followed by intraportal transplantation of the autologous islets. The number of islet equivalents (IEQ) that were transplanted is shown.

Figure 2. β Cell death in progressors and nonprogressors to T1D in the PTP study followed for long periods of time.

(A) Comparison of the average levels (mean ± SEM) of unmethylated INS DNA (i.e., ratio of unmethylated/methylated INS DNA) in the at-risk progressors, nonprogressors, and healthy control subjects (HC) (P = 0.048, ANOVA; *P < 0.05, Bonferroni multiple comparison test). (B) All of the individual ratio measurements are shown for the at-risk progressors (n = 10, 49 measurements) and nonprogressors (n = 10, 56 measurements) and were compared with the average levels (n = 32, 62 measurements). Six measurements (of a total of 105) were unobtainable for technical reasons. The dashed line represents the mean + 2 SD of the nondiabetic control subjects. (C) The ISR AUC for the progressors and nonprogressors at the study visits over the observation period prior to the diagnosis of T1D in the progressors or the last study visit in the nonprogressors. The least-square mean ± SEM from the repeated-measures ANOVA is shown. Neither the 2 curves nor the individual time points differ significantly. (D) The relationship between the change in the ratio and the change in the ISR AUC (from the first study visit) is shown for the progressors and nonprogressors (progressors: Pearson correlation coefficient, r = –0.475, P = 0.0026; nonprogressors: P = NS). All of the data points are shown.

Figure 1. CONSORT diagram showing allocation of the study subjects.

The TrialNet Natural History study commenced in 2004. The number of subjects screened reflects the total number of subjects since enrollment. The first at-risk subject was enrolled in 2005 and the last was enrolled in 2008. The high-risk subjects were identified between 2010 and 2013. The diagram shows the allocation of subjects to the “at-risk” group that was prospectively followed and the “high-risk” group that was studied on 2 occasions.