Exocytosis Protein DOC2B as a Biomarker of Type 1 Diabetes

Abstract

Context: Efforts to preserve β-cell mass in the preclinical stages of type 1 diabetes (T1D) are limited by few blood-derived biomarkers of β-cell destruction.

Objective: Platelets are proposed sources of blood-derived biomarkers for a variety of diseases, and they show distinct proteomic changes in T1D. Thus, we investigated changes in the exocytosis protein, double C2 domain protein-β (DOC2B) in platelets and islets from T1D humans, and prediabetic nonobese diabetic (NOD) mice.

Design, patients, and main outcome measure: Protein levels of DOC2B were assessed in platelets and islets from prediabetic NOD mice and humans, with and without T1D. Seventeen new-onset T1D human subjects (10.3 ± 3.8 years) were recruited immediately following diagnosis, and platelet DOC2B levels were compared with 14 matched nondiabetic subjects (11.4 ± 2.9 years). Furthermore, DOC2B levels were assessed in T1D human pancreatic tissue samples, cytokine-stimulated human islets ex vivo, and platelets from T1D subjects before and after islet transplantation.

Results: DOC2B protein abundance was substantially reduced in prediabetic NOD mouse platelets, and these changes were mirrored in the pancreatic islets from the same mice. Likewise, human DOC2B levels were reduced over twofold in platelets from new-onset T1D human subjects, and this reduction was mirrored in T1D human islets. Cytokine stimulation of normal islets reduced DOC2B expression ex vivo. Remarkably, platelet DOC2B levels increased after islet transplantation in patients with T1D.

Conclusions: Reduction of DOC2B is an early feature of T1D, and DOC2B abundance may serve as a valuable in vivo indicator of β-cell mass and an early biomarker of T1D.

Figure 1.

DOC2B protein abundance is reduced in platelets and islets of prediabetic NOD mice. (a) Platelets were isolated from 16- or 13-week-old group-housed female NOD and age-matched NOR mice, and proteins were resolved on SDS-PAGE for immunoblotting. DOC2B levels were quantified relative to tubulin immunoblotting (IB) in the same lane. Dashed, vertical lines indicate splicing of lanes from within the same gel exposure. Data are shown as means ± SEM (n = 3 to 6 mice per group); *P < 0.05. (b) Islets were isolated from 16-, 13-, or 7-week-old group-housed female NOD and age-matched NOR mice, and proteins were resolved on SDS-PAGE for immunoblotting. DOC2B levels were quantified relative to tubulin loading in the same lane. Dashed, vertical lines indicate splicing of lanes from within the same gel exposure. Data are shown as means ± SEM for DOC2B (n = 3 to 7 mice per group); *P < 0.05.

Figure 2.

DOC2B protein abundance is reduced in platelets from new-onset pediatric T1D human subjects. Platelets were isolated from patients with new-onset T1D at the time of diagnosis (“Diagnosis”) and 7 to 10 weeks later (“First Follow-up”) and from matched controls (“Control”). Platelet proteins were resolved on SDS-PAGE for immunoblotting. Standard curves were generated using recombinantly expressed and purified hDOC2B protein on each gel to confirm that the band intensities of DOC2B in human platelets fell within the dynamic range of the curve on the same gel. DOC2B was quantified relative to protein loading, determined by Ponceau S staining in the same lane (37 to 68 kDa segment). Data are shown as means ± SEM for DOC2B [n = 11 to 14 per group (sex-combined group, eight boys per group, three to six girls per group)]; *P < 0.05, Diagnosis vs Control; #P < 0.05 Follow-up vs Control.

Figure 3.

DOC2B protein and messenger RNA (mRNA) abundance are reduced in adult human islets subjected to treatment with proinflammatory cytokines. Human adult cadaveric islets were incubated under control conditions or with proinflammatory cytokines for 72 hour at 37°C. Islet protein lysates were resolved by SDS-PAGE for (a) immunoblotting or for (b) RNA extraction and qRT-PCR analysis. In addition to hDOC2B, tubulin and iNOS levels were evaluated by immunoblotting. Bars represent means ± SEM for four or five independent sets of human islets evaluated for protein and mRNA analyses, respectively; ****P < 0.0001; **P < 0.002.

Figure 4.

DOC2B protein levels are reduced in islets from pediatric T1D humans. Slides obtained from nPOD, comprised of early-onset T1D and age-matched nondiabetic (ND) human pancreata, were immunostained for the presence of DOC2B or insulin in 4′,6-diamidino-2-phenylindole (DAPI)-positive cells. (a) Representative images: top six panels, 100 μm; bottom six panels, 25 μm. Boxed areas indicate the precise regions of top panel images used to create the higher magnification images seen in bottom panels. White arrows indicate insulin and DOC2B-positive regions to demonstrate co-localization. (b) Tabulated relative intensities; n = 3 donors; *P < 0.05. (c) Number of DOC2B-positive β-cells; P = not significant.

Figure 5.

DOC2B levels in adult T1D human platelets are increased after clinical islet transplantation. Platelets obtained from two clinical islet transplant recipients before (Day 0) islet infusion or on Days 30 and 75 postinfusion were evaluated by quantitative immunoblotting for DOC2B protein content: (a) subject COH-027, (b) subject COH-028. Ponceau S staining and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) show the relative protein loading of the membranes used for immunoblotting. COH, City of Hope.