Plasma GAD65, a Marker for Early β-Cell Loss After Intraportal Islet Cell Transplantation in Diabetic Patients

Abstract

Context and objective: Intraportal islet transplantation can restore insulin production in type 1 diabetes patients, but its effect is subject to several interfering processes. To assess the influence of β-cell loss before and during engraftment, we searched for a real-time marker of β-cell destruction. Previous studies showed that 65-kDa isoform of glutamate decarboxylase (GAD65) is discharged by chemically damaged rat β-cells. We therefore examined the utility of the GAD65 assay to detect and quantify destruction of human β-cells in vitro and in vivo.

Design and participants: A time-resolved fluorescence immunoassay was used to measure GAD65 discharge from β-cells after administration of toxins or after intraportal transplantation. The study in patients involved type 1 diabetes recipients of 56 implants.

Results: GAD65 was discharged from cultured human β-cells between 4 and 24 hours after acute insult and proportional to the number of dying cells. It was also detected in plasma during the first 24 hours after intraportal transplantation of human islet cell grafts. Diabetic nude rat recipients without hyperglycemic correction exhibited higher plasma GAD65 levels than those with normalization. In type 1 diabetes recipients of grafts with 2-5 × 10(6) β-cells per kilogram of body weight, five of six with plasma GAD65 greater than 1 ng/mL failed to increase plasma C-peptide by greater than 0.5 ng/mL at posttransplant month 2, whereas five of six with undetectable plasma GAD 65 and 15 of 19 with intermediate levels did result in such increase.

Conclusion: Plasma GAD65 qualifies as a marker for early β-cell loss after intraportal transplantation. Further studies are needed to extend its clinical utility.

Trial registration: ClinicalTrials.gov NCT00623610.

Figure 1.. Flow diagram of subgroups analyzed for GAD65 levels and implant function.

ND, undetectable.

Figure 2.. A, Plasma glucose and GAD65 concentrations in rats injected at time 0 with STZ (60 mg/kg body weight, full circles) or with solvent (open circles). Data represent mean ± SEM of three (control) or four (STZ) animals. Statistical significance of difference between control and STZ: *, P < .05, **, P < .01. B, Immunofluorescent double staining for GAD65 (red) and insulin (green) or immunostaining for macrophages (ED1, brown) on pancreatic sections of STZ-treated and control rats. Nuclei are stained with 4′,6′-diamino-2-phenylindole (blue) in fluorescence or hematoxylin.

Figure 3.. Plasma glucose, C-peptide, and GAD65 levels in STZ-diabetic nude rats after intraportal transplantation of human islet cell grafts.

A, Animals were divided into two subgroups according to their 2-hour fasting glycemia at PT week 1: the normoglycemic group (open circles) consisted of four animals receiving grafts from three different preparations, and the hyperglycemic group (filled circles) consisted of three animals receiving grafts from two different preparations. B, Plasma GAD65 levels in the two subgroups in function of time after the transplant. Statistical significance of difference between subgroups: *, P < .05; †, P < .01.

Figure 4.. A, Correlation between plasma GAD65 levels at 60 minutes PT in type 1 diabetic patients with β-cell mass in graft as expressed by β-cell number and C-peptide content. Open circles represent grafts with plasma GAD65 1 ng/mL or less; filled circles represent grafts with plasma GAD65 greater than 1 ng/ mL, which were identified as outliers by ROUT (Robust regression and Outlier removal) test (set Q = 1%); filled square represents a graft with plasma GAD65 greater than 1 ng/ mL, which was not identified as an outlier; the full lines represent regression lines without omission of outliers and the dashed lines their 95% confidence interval (CI). Statistical significance of the correlation: left panel, P = .001; right panel, P < .001. After omission of outliers: left panel, r² = 0.179; right panel, r² = 0.401; P < .001 for both. B, Correlation between plasma GAD65 levels at PT minute 60 and plasma C-peptide increment (differences between before the transplant and PT month 2). Open circles represent grafts with low plasma GAD65 levels (≤1 ng/mL or < 0.04 ng/mL when GAD65 levels were normalized to transplanted β-cell mass) and C-peptide increment of 0.5 ng/mL or less; open squares represent grafts with low plasma GAD65 and C-peptide increment greater than 0.5 ng/mL; filled circles represent grafts with disproportionally plasma GAD65 levels (outliers in panel A); filled square represents a graft with plasma GAD65 greater than 1 ng/ mL, which was not identified as an outlier in panel A. Statistical significance of the correlation: left panel, P = .0050; right panel, P = .0035.