Early metabolic markers that anticipate loss of insulin independence in type 1 diabetic islet allograft recipients

Abstract

The objective of this study was to identify predictors of insulin independence and to establish the best clinical tools to follow patients after pancreatic islet transplantation (PIT). Sequential metabolic responses to intravenous (I.V.) glucose (I.V. glucose tolerance test [IVGTT]), arginine and glucose-potentiated arginine (glucose-potentiated arginine-induced insulin secretion [GPAIS]) were obtained from 30 patients. We determined the correlation between transplanted islet mass and islet engraftment and tested the ability of each assay to predict return to exogenous insulin therapy. We found transplanted islet mass within an average of 16 709 islet equivalents per kg body weight (IEQ/kg BW; range between 6602 and 29 614 IEQ/kg BW) to be a poor predictor of insulin independence at 1 year, having a poor correlation between transplanted islet mass and islet engraftment. Acute insulin response to IVGTT (AIR(GLU) ) and GPAIS (AIR(max) ) were the most accurate methods to determine suboptimal islet mass engraftment. AIR(GLU) performed 3 months after transplant also proved to be a robust early metabolic marker to predict return to insulin therapy and its value was positively correlated with duration of insulin independence. In conclusion, AIR(GLU) is an early metabolic assay capable of anticipating loss of insulin independence at 1 year in T1D patients undergoing PIT and constitutes a valuable, simple and reliable method to follow patients after transplant.

Figure 1. Intravenous glucose tolerance test in islet transplant recipients at 3, 6 and 12 months posttransplant

(A) Glucose kinetics over 20 min after 300 mg/kg dextrose administration over 1 min starting at t = 0. (B) Levels of insulin and (C) C-peptide release were plotted against nondiabetic control subjects. Thirty patients were assessed at 3 months, 19 patients were assessed at 6 months and 27 patients were assessed at 12 months post last infusion. Data are expressed as mean ± SE.

Figure 2. Arginine stimulation test in islet transplant recipients at 3, 6 and 12 months posttransplant

(A) Glucose kinetics over 20 min after 5 g arginine administration over 30 s starting at t = 0. (B) Levels of insulin and (C) C-peptide release were plotted against nondiabetic control subjects. Thirty patients were assessed at 3 months, 19 patients were assessed at 6 months and 27 patients were assessed at 12 months post last infusion. Data are expressed as mean ± SE.

Figure 3. Panel (A): Glucose potentiation of arginine induced insulin secretion (GPAIS) in islet transplant recipients at 3, 6 and 12 months posttransplant

Thirteen patients were assessed at 3 months, 8 patients were assessed at 6 months and 13 patients were assessed at 12 months post last infusion. (A) Glucose kinetics over 20 min after 5 g arginine administration over 30 s starting at t = 0. (B) Levels of insulin and (C) C-peptide release were plotted against nondiabetic control subjects. Data are expressed as mean ± SE. Panel (B): Slope of glucose-potentiation is calculated as the change in insulin release in response to arginine from the normoglycemic to the hyperglycemic condition, divided by the change in plasma glucose. The posttransplant calculated slope in PIT recipients was 1.2 ± 0.4 (p = 0.02) at 3 months, 1.2 ± 0.5 (p = 0.02) at 6 months, 1.6 ± 1.1 (p = 0.07) at 12 months and 1.8 ± 1.0 (p = 0.08) at 24 months, compared to a slope of 5.1 ± 1.4 for controls.

Figure 4. Comparison of total transplanted islet mass and metabolic assessment in the posttransplant period

Panel (A): Bar representation of transplanted islet mass and insulin requirement status at 12 months post last islet infusion. Insulin-dependent group (n = 14) and insulin-independent group (n = 15). Mean and SEM were calculated in each group. Statistical significance was considered at p < 0.05. Panel (B): Relation of total transplanted islet mass post last infusion with insulin secretion AUC at 12 months post last infusion (n = 29). Statistical significance was considered at p < 0.05. Panel (C): Relation of total transplanted islet mass post last infusion with HbA1C level at 3 months post last infusion normalized to pretransplant level (n = 29). Statistical significance was considered at p < 0.05.

Figure 5. Comparison of total transplanted islet mass and insulin and C-peptide levels after IVGTT, arginine stimulation test and glucose-potentiated arginine test

Panel (A): Relation of total transplanted islet mass post last infusion with insulin secretion AUC after IVGTT at 3 months post last infusion (n = 27). Statistical significance was considered at p < 0.05. Panel (B): Relation of total transplanted islet mass post last infusion with insulin secretion AUC after arginine stimulation test at 3 months post last infusion (n = 25). Statistical significance was considered at p < 0.05. Panel (C): Relation of total transplanted islet mass post last infusion with insulin secretion AUC after glucose-potentiated arginine test at 3 months post last infusion (n = 9). Statistical significance was considered at p < 0.05. Panel (D): Relation of total transplanted islet mass post last infusion with c-peptide secretion AUC after IVGTT at 3 months post last infusion (n = 27). Statistical significance was considered at p < 0.05. Panel (E): Relation of total transplanted islet mass post last infusion with c-peptide secretion AUC after arginine stimulation test at 3 months post last infusion (n = 25). Statistical significance was considered at p < 0.05. Panel (F): Relation of total transplanted islet mass post last infusion with c-peptide secretion AUC after glucose-potentiated arginine test at 3 months post last infusion (n = 9). Statistical significance was considered at p < 0.05.

Figure 6. Distribution plots for Insulin secretion using the ROC curve and analysis for IVGTT, arginine stimulation test and GPAIS

Panels (A)–(C): All sequential AIR_GLU, AIR_ARG and AIR_MAX for UW patients during 24-month follow-up. Acute insulin response data were segregated according to exogenous insulin dependence. Mean and SEM were calculated in each group. Data are also stratified according to whether the test was performed 3, 6 or 12 months after the last islet transplantation. Statistically significant differences are expressed as *(p < 0.05), **(p < 0.01) and ***(p < 0.001). Panels (D)–(F) represent the receiver operator characteristic for AIR_GLU, AIR_ARG and AIR_MAX, respectively. It was determined at the time of each assay whether the patient required insulin to achieve normoglycemia and recorded as “On Insulin” or “Off Insulin.” The ROC graph recorded a point for each data pair (quantitative result, clinical outcome) as if it was the critical value for a predictive assay and considering the data set at that point as true positives and false positives. All data from sequential measurements at 3, 6 and 12 months post last islet infusion was included. Area under the ROC curve was then calculated. Tests which cannot discriminate between true and false positives show a sensitivity plot that is not significantly different from the line of identity and a p-value >0.05 when the AUC is calculated. Cutoff values that generate the highest sensitivity and specificity using the best likelihood ratios were chosen for each assay.

Figure 7. Distribution plots for C-peptide secretion using the ROC curve and analysis for IVGTT, arginine stimulation test and GPAIS

Panels (A)–(C): All sequential ACR_GLU, ACR_ARG and ACR_MAX for UW patients during 24-month follow-up. Acute C-peptide response data was segregated according to their exogenous insulin dependency. Mean and SEM were calculated in each group. Data is also stratified according to whether the test was performed 3, 6 or 12 months after the last islet transplantation. Statistically significant differences were expressed as *(p < 0.05), **(p < 0.01) and ***(p < 0.001). Panels (D)–(F) represent the receiver operator characteristics for ACR_GLU, ACR_ARG and ACR_MAX, respectively. Tests, which cannot discriminate between true and false positives, show a sensitivity plot that is not significantly different from the line of identity and a p-value >0.05 when the AUC is calculated. All data from sequential measurements at 3, 6 and 12 months post last islet infusion was used. Area under the ROC curve was then calculated. Cutoff values that generated the highest sensitivity and specificity using the best likelihood ratios were chosen for each assay.

Figure 8. Comparison between acute insulin and peptide response in 32 islet transplant recipients from three institutions

Patients were divided according to their exogenous insulin requirement 12 months posttransplant. Acute insulin and C-peptide response is expressed as mean ± SEM. Cutoff values from Figures 6 and 7 are overlapped with the bars, representative of the data. Statistically significant differences were expressed as *(p < 0.05) and **(p < 0.01). Nondiabetic controls are represented for comparison. Panels (A) and (B): Bar representation of the AIR_GLU and ACR_GLU response at 3, 6 and 12 months after last transplant. Panels (C) and (D): Bar representation of the AIR_ARG and ACR_ARG response at 3, 6 and 12 months after last transplant. Panels (E) and (F): Bar representation of the AIR_MAX and ACR_MAX response at 3, 6 and 12 months after last transplant.

Figure 9. Insulin IVGTT performed at 3 months as a predictor of posttransplant insulin independence and long-term graft function

Panel (A): Correlation between IVGTT acute insulin response at 3 months and posttransplant insulin independence. Panel (B): Comparison between IVGTT insulin AUC performed at 3 months and long-term insulin independence. Data are mean ± SEM. Statistical significant differences expressed as *(p < 0.05), **(p < 0.01) and ***(p < 0.001).