Exocytosis Protein DOC2B as a Biomarker of Type 1 Diabetes

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. Type 1 diabetes (T1D) is characterized by autoimmune destruction of β-cell mass, and the preclinical phase of T1D is marked by declining β-cell function (1, 2). Studies of early intervention in T1D have shown limited effectiveness yet have generally shown greater success in subjects that retain greater insulin secretory capacity and in those with the shortest time since clinical onset of disease (3, 4). However, prevention efforts to protect β-cell mass are hindered by the limited availability of early biomarkers to predict accurately β-cell destruction and subsequent progression to clinical disease. In healthy β-cells, insulin secretion requires soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins and associated accessory regulatory proteins to promote the docking, priming, and fusion of insulin vesicles at the plasma membrane. Two target membrane SNARE proteins—syntaxin 1/4 and SNAP25/23—and one vesicle-associated SNARE protein—VAMP2—constitute the SNARE core complex (5). Assembly of the SNARE complex occurs when one vesicle-associated SNARE binds two cognate target membrane SNARE proteins in a heterotrimeric ratio (6). SNARE complex assembly is also facilitated by the accessory regulatory protein, double C2-domain β (DOC2B) (7, 8). Several studies have established that in animal models, deficiencies in DOC2B result in glucose intolerance and insulin-secretion defects (9, 10). Conversely, overexpression of DOC2B, using a global transgenic mouse model, enhances insulin secretion and peripheral glucose uptake (11). Although DOC2B deficiency in rodents has been linked to type 2 diabetes (12), the association between DOC2B protein levels and T1D is still unknown. Deficient first-phase insulin secretion is a hallmark of preclinical T1D (1, 2); thus, the ability to assess early pancreatic β-cell destruction is critically important for the prediction of disease onset. Currently, risk prediction for T1D relies heavily on family history, genetic screening, and the presence of antibodies against β-cell antigens that often appear relatively late in the progression of disease. The use of autoantibodies in evaluating T1D risk is limited, as >50% of autoantibody-positive patients remain disease free, even at 5 years follow-up (13). Risk scores have been established (14) but remain insufficient to provide an accurate prognosis or an accurate measurement of β-cell health, as many autoantibody-positive individuals are slow to progress through the stages (15) of preclinical disease. To improve early prediction of T1D, ongoing studies seek to investigate the levels of circulating factors that reflect declining β-cell health, such as proinsulin (16), heat shock protein 90 (17), and unmethylated insulin DNA (18), as potential biomarkers of T1D. Another potential source of biomarkers is the blood-derived platelet, which is currently being investigated in diseases, such as Alzheimer’s disease (19) and cancer (20), and has been implicated in T1D. Changes in the platelet proteome and morphology have been noted in T1D; for instance, altered intracellular Ca2+ (21), enhanced formation of microparticles (22), and altered morphology (23) have been reported to result in platelet hyper-reactivity and development of vasculopathies. Importantly, platelets harbor many of the same exocytosis proteins as the pancreatic β-cell, including SNARE isoforms and accessory regulatory proteins (24). Biomarkers of β-cell destruction in blood have more clinical potential than those in pancreatic islets, as islet procurement is not feasible for routine diagnosis; therefore, we investigated the correlation between DOC2B protein abundance in blood-derived platelets and pancreatic islets in nonobese diabetic (NOD) mice and T1D humans. We found that protein abundance of DOC2B is reduced in platelets and islets from prediabetic NOD mice compared with control mice. Furthermore, DOC2B protein abundance is reduced in platelets and islets from humans with new-onset T1D compared with matched controls. Notably, we also reveal that DOC2B levels are substantially increased in T1D human platelets after islet transplantation, when C-peptide levels were markedly increased. Materials and Methods Animals Animals were maintained under protocols approved by the Indiana University Institutional Animal Care and Use Committee and following the National Research Council Guidelines for the Care and Use of Laboratory Animals. Female NOD NOD/ShiLtJ (RRID:IMSR_JAX:001976) and major histocompatibility complex-matched control nonobese diabetes-resistant (NOR; RRID:IMSR_JAX:002050) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). We have observed that female NOD mice begin to convert to T1D at 17 to 18 weeks of age, with an average conversion rate of 78% by 20 weeks of age, as previously reported (25). Random blood glucose analysis was performed weekly to monitor conversion to T1D, which is characterized by nonfasting blood glucose levels >250 mg/dL for 3 consecutive days. To assess DOC2B levels before conversion to T1D, pancreatic islets were isolated, using a method as described previously (26), at 7 weeks (earliest time point for sufficient islet cell yield), 13 weeks (intermediate time point), and 16 weeks (latest time point before conversion to T1D) of age. Islet isolation yield decreases in mice <8 weeks of age (27). Islet lysates were then used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Mouse blood was collected (from 13- and 16-week-old mice; blood was insufficient from 7-week-old mice), and platelets were isolated as previously described (24). Platelet lysates were then used for SDS-PAGE and immunoblotting. Human subjects All human studies were conducted in keeping with the principles set out in the Declaration of Helsinki. This protocol was approved by the Indiana University Institutional Review Board. For evaluation of DOC2B levels in human platelets (new-onset T1D study), subjects aged 8 to 14 years (11 boys and six girls) with new-onset T1D were recruited over an 18-month period. Consent was obtained from parents, with assent from the pediatric subjects. Subjects were diagnosed with T1D if they met the criteria of one or more positive autoantibodies with clinical features of T1D—hyperglycemia, weight loss, and normal body mass index (BMI)—or those who were autoantibody negative but <10 years old at diagnosis. Exclusion criteria were as previously described (17). For each visit, subjects received $25. Subjects had blood drawn at diagnosis and at the first follow-up appointment, 7 to 10 weeks after diagnosis. Insulin treatment of T1D subjects was started at the time of diagnosis. Nondiabetic control subjects (eight boys and six girls) were recruited from the community and matched to T1D subjects based on sex, age, and BMI (see Table 1 for demographic data). Samples were de-identified and coded by the clinical team before distribution to the research laboratory for platelet isolation and analyses. Platelets were isolated by centrifugation from blood, as previously described (28), and lysed for SDS-PAGE and immunoblotting. Upon quantification of the data for each sample, the clinical team reidentified samples to permit grouping of data into T1D vs nondiabetic for statistical comparisons. Table 1. Pediatric T1D Study Demographics Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Values displayed are means ± standard deviation unless otherwise noted. Abbreviation: HbA1c, hemoglobin A1c. a For BMI calculations, one T1D subject did not have a diagnosis height, and one non-T1D control did not have a registration height. For these subjects, the heights from clinic follow-up were used to calculate BMI. b The following three diabetes-associated antibodies were tested: glutamic acid decarboxylase, micro insulin autoantibodies, and insulinoma-associated antigen-2. c For C-peptide at diagnosis, n = 13. View Large Table 1. Pediatric T1D Study Demographics Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Values displayed are means ± standard deviation unless otherwise noted. Abbreviation: HbA1c, hemoglobin A1c. a For BMI calculations, one T1D subject did not have a diagnosis height, and one non-T1D control did not have a registration height. For these subjects, the heights from clinic follow-up were used to calculate BMI. b The following three diabetes-associated antibodies were tested: glutamic acid decarboxylase, micro insulin autoantibodies, and insulinoma-associated antigen-2. c For C-peptide at diagnosis, n = 13. View Large For evaluation of DOC2B levels in human islets (T1D islet transplantation study), samples were obtained from T1D islet transplantation recipients, as approved by the City of Hope Institutional Review Board. Two subjects, aged 43 and 52 years, were recruited for human islet transplantation, based on the following criteria: T1D diagnosis with frequent or life-threatening hypoglycemia, with or without unawareness symptoms. Blood was obtained from both subjects before transplantation (Day 0) and on Days 30 and 75 after islet transplantation (see Supplemental Table 1 for demographic data). Platelets were isolated by centrifugation from blood, as previously described (28), and lysed for SDS-PAGE and immunoblotting. Islet cell transplantation For the T1D islet transplant study, human pancreata were procured from ABO-compatible, cross-match-negative cadaveric donors. The islets were isolated under cyclic guanosine monophosphate conditions by the Southern California Islet Cell Resource Center at City of Hope using a modified Ricordi method. Islets were maintained in culture for up to 72 hours before transplantation. Islets were transplanted intraportally with heparinized saline (35 U/kg recipient body weight) using a transhepatic percutaneous approach. Clinical/laboratory assays For the new-onset T1D study, autoantibodies to glutamic acid decarboxylase (GAD)-65, insulin, and islet antigen 2A (IA-2A) were assayed from peripheral blood at diagnosis at Mayo Medical Laboratories (Rochester, MN). Glycated hemoglobin A1c (HbA1c) was also measured at diagnosis and at the first clinic follow-up (7 to 10 weeks after diagnosis), using the A1cNow system or the DCA2000 analyzer (Bayer, Tarrytown, NY). C-Peptide was measured in stored serum samples using the C-peptide enzyme-linked immunosorbent assay kit (Alpco, Salem, NH; detection range 20 to 3000 pM). For the T1D islet transplant study, plasma C-peptide measurements were performed by the Northwest Lipid Metabolism and Diabetes Laboratory (Seattle, WA), using the C-Peptide II Assay (Tosoh Bioscience, San Francisco, CA; detection range 0.02 to 30 ng/mL). A fasting C-peptide <0.2 ng/mL and 6-minute glucagon-stimulated C-peptide <0.3 ng/mL were used to confirm T1D diagnosis before islet transplant. Autoantibodies (GAD-65, IA-2A, micro insulin autoantibodies, and zinc transporter 8) were analyzed using radiobinding assays by the Autoantibody/HLA Service Center at the Barbara Davis Center for Diabetes (Aurora, CA). Ex vivo islet preparations Non-T1D human cadaveric pancreatic islets were obtained through the Integrated Islet Distribution Program at City of Hope. The islets were prepared and treated with a cytokine mixture (10 ng/mL tumor necrosis factor-α, 100 ng/mL interferon-γ, and 5 ng/mL interleukin-1β; ProSpec, East Brunswick, NJ) for 72 hours, as previously described (29). The islets were then used in quantitative real-time polymerase chain reaction (qRT-PCR) analysis or SDS-PAGE, followed by immunoblotting. Immunofluorescence Human paraffin-embedded pancreatic tissue sections were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Five sections from formalin-fixed paraffin-embedded tissue samples were obtained from T1D (n = 3) and age- and BMI-matched nondiabetic (n = 3) donors. Pancreas sections were immunostained with primary and secondary antibodies, listed in Supplemental Table 2. Slides were counterstained to mark the nuclei, using 4′,6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, CA) and viewed using a BZ X-700 fluorescence microscope (Keyence, Itasca, IL). All human T1D samples were prepared and processed at the same time; confocal images were taken with identical acquisition settings. Islet immunofluorescence was assessed by imaging 20 to 30 islets (grouping of four or more insulin-positive cells) per subject. Analysis was performed in a blinded fashion using Image-Pro Software (Media Cybernetics, Rockville, MD) to quantify fluorescence intensities using methods described previously (30). Defined regions of interest were used to delimit islets from adjacent acinar tissue, and average intensity measurements of insulin and DOC2B were quantified by the splitting of the merged image into two color channels with the same region of interest. Immunoblotting Platelet and islet protein lysates for the NOD mouse study were resolved on a 10% SDS-PAGE gel and transferred to standard polyvinylidene difluoride (PVDF; Bio-Rad, Hercules, CA). Platelet proteins from the new-onset T1D study were resolved on a 10% SDS-PAGE gel using an SE400 air-cooled 18 × 16 cm vertical protein electrophoresis unit (Hoefer, Holliston, MA) and transferred to standard PVDF (Bio-Rad). Platelet proteins from the T1D islet transplant study were resolved on a 12% SDS-PAGE gel using a Criterion 13.3 × 8.7-cm vertical electrophoresis unit (Bio-Rad) and transferred to standard PVDF. All blots were probed, as outlined in Supplemental Table 2. qRT-PCR Total RNA was isolated from human islets using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and assessed using the QuantiTect SYBR Green RT-PCR kit (Qiagen). Primers used for the detection of human Doc2b (hDoc2b) are as follows: forward: 5′-CCAGTAAGGCAAATAAGCTC-3′ and reverse: 5′-GGGTTTCAGCTTCTTCA-3′. Standard tubulin primers (Cat: QT00089775; Qiagen) were used for normalization. Statistical analysis Data were evaluated for statistical significance using Student t test for comparison of two groups; analysis of variance and Tukey post hoc test (GraphPad Software, La Jolla, CA) were used for comparison of more than two groups. Data are expressed as the average ± standard error of the mean (SEM). Results Low DOC2B levels in prediabetic NOD mouse platelets and islets To investigate whether DOC2B protein levels are altered in the blood before onset of T1D, we examined platelet DOC2B abundance in young prediabetic NOD mice and major histocompatibility complex-matched NOR mice. Immunoblotting revealed that platelets from 16- and 13-week-old NOD mice exhibited up to a 90% reduction in DOC2B protein levels [Fig. 1(a)] compared with NOR platelets. Furthermore, islets from 16- and 13-week-old NOD mice showed at least a 65% reduction in DOC2B protein levels [Fig. 1(b)] compared with NOR islets. NOD islets from as early as 7 weeks of age showed a 90% reduction in DOC2B protein [Fig. 1(b)]. The average blood glucose levels from random blood testing of NOD and NOR mice were below 250 mg/dL at 7, 13, and 16 weeks (Supplemental Table 3), indicating that the mice had not yet converted to diabetes. These data show that DOC2B protein abundance is reduced in both platelets and islets of prediabetic mice. Figure 1. View largeDownload slide 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 1. View largeDownload slide 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. Low DOC2B levels in new-onset T1D human platelets In the new-onset T1D study, we quantified protein content of DOC2B using platelets from new-onset T1D subjects compared with controls (Table 1). Platelets from new-onset T1D subjects exhibited reduced protein levels of DOC2B for both genders, both at diagnosis and at the first clinic follow-up, 7 to 10 weeks later. When boys and girls were assessed separately, DOC2B levels were reduced in boys by >60% compared with nondiabetic control subjects, persisting even after insulin treatment of the patient and reduction of HbA1c (Fig. 2). The substantial loss of DOC2B at T1D diagnosis was selective for DOC2B compared with another exocytosis protein, syntaxin 4 (Supplemental Fig. 1). These data indicate that DOC2B is decreased in T1D platelets, independent of glycemic control, relative to nondiabetic human platelets, and that platelet DOC2B levels are already diminished at T1D diagnosis. Figure 2. View largeDownload slide 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 2. View largeDownload slide 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. Ex vivo proinflammatory cytokine treatment reduces human islet DOC2B levels T1D is associated with elevated circulating proinflammatory cytokines that damage β-cells (31). As the attainment of pancreatic islets from living T1D subjects is virtually impossible, we evaluated the relationship between T1D and DOC2B levels by treating human cadaveric nondiabetic islets (Supplemental Table 4) ex vivo with proinflammatory cytokines in an effort to simulate the circulating milieu. Cytokine treatment (interleukin-1β, tumor necrosis factor-α, interferon-γ) elevated the levels of islet inducible nitric oxide synthase [iNOS; Fig. 3(a)], consistent with the reported effects of cytokine exposure (32). Correspondingly, DOC2B protein and messenger RNA (mRNA) levels were reduced by 30% and 50%, respectively (Fig. 3). These data suggest that a T1D-like milieu can decrease DOC2B levels in human islets. Figure 3. View largeDownload slide 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 3. View largeDownload slide 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. Reduced DOC2B protein in human early-onset T1D islets To investigate changes in DOC2B levels in T1D human pancreata, we used paraffin-embedded slides (obtained from nPOD) from cadaveric donors for DOC2B immunofluorescence evaluation in early-onset pediatric T1D (5 years or less with T1D; n = 3) vs matched nondiabetic controls [n = 3; Fig. 4(a) and Supplemental Table 5]. With the measurement of relative immunofluorescent intensities, we observed a decrease in DOC2B abundance in T1D islets vs in nondiabetic controls [Fig. 4(b)]. Although the relative number of DOC2B-positive β-cells in nondiabetic and T1D islets was similar [Fig. 4(c)], DOC2B intensity was reduced in T1D β-cells. Figure 4. View largeDownload slide 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 4. View largeDownload slide 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. DOC2B levels are restored after clinical islet transplantation In the T1D islet transplantation study (Supplemental Table 1), we found that the pretransplant platelet DOC2B levels were very low in both subjects relative to a hDOC2B protein standard curve (Fig. 5; Day 0). Notably, within 30 days of transplantation, each T1D islet recipient showed a robust increase in platelet DOC2B protein, which persisted to 75 days after transplantation (Fig. 5; Days 30 and 75). These data coincide with changes in C-peptide levels in these subjects: whereas each subject had low to almost undetectable fasting/glucagon-stimulated C-peptide levels before transplantation, the C-peptide levels were substantially increased by 30 days after transplantation (Supplemental Table 6). As C-peptide levels are indicative of overall islet function, these data suggest that in humans, DOC2B levels in platelets correlate with relative functional β-cell mass. Figure 5. View largeDownload slide 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. Figure 5. View largeDownload slide 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. Discussion The ability to detect β-cell destruction is critical in accurately predicting prognosis during the preclinical phase of T1D; hence, the current need for additional early biomarkers. We show that DOC2B protein levels are substantially reduced in platelets and islets from prediabetic NOD mice vs NOR control mice. Furthermore, we reveal that levels of hDOC2B are significantly lower at the time of diagnosis in platelets of pediatric patients with new-onset T1D than platelets from matched control subjects. Notably, DOC2B levels were reduced at 7 to 10 weeks postdiagnosis, despite therapeutic remediation of hyperglycemia in the human subjects. Consistent with this, islet DOC2B protein levels were reduced in early-onset T1D pancreatic tissue samples compared with matched controls. Loss of DOC2B protein and mRNA could be recapitulated by exposure of nondiabetic human islets to proinflammatory cytokines ex vivo, suggesting that the inflammatory milieu in prediabetic and T1D humans may cause DOC2B loss. Remarkably, clinical islet transplant recipients exhibit a restoration of DOC2B levels in platelets compared with their own nearly undetectable levels of platelet DOC2B before receiving the transplanted islets. These data suggest that the DOC2B protein is a candidate biomarker of prediabetes and T1D, with the levels possibly reporting relative functional β-cell mass. This study establishes an association between T1D and levels of an exocytosis protein in blood-derived platelets and pancreatic islets. Reduced DOC2B in islets is indicative of deficient islet functional health (9). Strikingly, platelet DOC2B levels in islet transplant recipients correlated with the presence of a functional islet mass. This correlative finding supports the possibility that the platelet DOC2B stems not necessarily from the pancreas per se, as islets are grafted into the liver in these human recipients, but that the platelets and/or precursor megakaryocytes may be sampling DOC2B from the islets, irrespective of islet location. It also remains possible that the increased DOC2B content stems from “rested,” native, residual islets of the transplanted patients. However, this is inconsistent with our pediatric platelet data, showing that even after insulin therapy to ameliorate new-onset hyperglycemia, DOC2B levels remained deficient. Mechanistically, questions arise as to if and how platelets and islets communicate to determine DOC2B levels. Supporting the concept of platelet-islet communication, it has been demonstrated that islet transplantation in patients with T1D stabilizes platelet abnormalities, as transplant recipient platelets show normal volume and activation (33). Indeed, β-cells release exosomes as a way of shuttling various microRNAs, mRNAs, and proteins to targeted peripheral cells (34). β-Cell exosomes were also recently shown to carry proteins, such as GAD-65, IA-2, and proinsulin, to dendritic cells, which then become activated (35). Could islets similarly release DOC2B mRNA or protein, which is subsequently taken up by megakaryocytes or by the platelets themselves? Furthermore, platelets can selectively absorb proteins from the blood (36). In fact, platelet sequestration of tumor-specific proteins was detected in animals harboring small tumors (36). Notably, a direct interaction between platelets and pancreatic β-cells has been reported, and protein from platelets was shown to be transferred to β-cells (37). Is it possible that the platelets are sampling the islets to mirror islet DOC2B levels? As the data presented herein are correlative in nature, future studies will be required to determine the detailed molecular mechanism regulating changes in DOC2B protein abundance in human T1D samples. The concept of DOC2B as a biomarker is appealing, as DOC2B levels in platelets and islets are significantly decreased in normoglycemic NOD mice, months before their conversion to T1D. Female NOD mice typically convert to T1D between 18 and 24 weeks of age, but as early as 5 weeks of age, NOD mouse islets show signs of insulitis, resulting from an initial phase of pancreatic inflammation that reduces β-cell function and mass (38). Given that DOC2B content in human islets decreased upon islet exposure to proinflammatory cytokines, which was sufficient to evoke iNOS expression, it is possible that the cytokine-induced drop in islet DOC2B signals reduced islet viability. Although it has been demonstrated by multiple groups that whole-body DOC2B knockout mice show deficient glucose-stimulated insulin secretion (9, 10), β-cell mass was not evaluated. Whereas it is also possible that DOC2B expression is genetically repressed in NOD mice, the genetics of NOD mice have been well studied, and DOC2B was not identified as deviating from control (39). DOC2B mRNA expression was also decreased in response to proinflammatory cytokine exposure in nondiabetic human islets, suggesting that DOC2B might undergo transcriptional repression during T1D development. Indeed, promoter methylation and silencing of the DOC2B gene have been reported to occur in cancer cell types (40). Future studies will be required to determine the detailed molecular mechanism regulating changes in DOC2B protein abundance in human T1D samples. Whereas findings were obtained, there are also some limitations to the conclusions that can be derived from this study. First, this work derives from two pilot studies with relatively small sample sizes of human pediatric subjects (n = 14 controls and n = 17 patients with T1D, new-onset T1D study) and adult T1D islet transplant subjects (n = 2, T1D islet transplantation study). Secondly, the pediatric cohort was evaluated at clinical diagnosis of T1D, so these patients already have full-blown disease. A larger, future study would benefit from a prospective design that includes subjects who are significantly at risk but who are normoglycemic. We are also limited in that only the first clinical follow-up samples were obtained, and we do not have further follow-up data for a longitudinal evaluation of changes in DOC2B. Lastly, the number of adult T1D clinical islet transplantation subjects followed was limited by our initial requirement for collection of fresh blood for platelet isolation to be consistent with our pediatric T1D cohort. Given our focus on DOC2B as an early predictor of T1D, future studies will examine serum and plasma, both of which contain abundant and detectable levels of DOC2B. This will permit studies of larger numbers of T1D transplant recipients and evaluation of stored samples from a variety of repositories to enable more generalized conclusions. In summary, we demonstrate reduced abundance of the exocytosis protein, DOC2B, in prediabetes (NOD mice) or the early onset of T1D (humans). In addition, we show a correlation between the platelet and islet DOC2B levels, suggesting that platelet evaluation may provide prognostic information about T1D risk and progression. Furthermore, transplantation of healthy, functional islets increased the levels of DOC2B in T1D human platelets. Given the need to assess β-cell destruction accurately at an early stage, DOC2B may be a viable biomarker for T1D. Abbreviations: Abbreviations: BMI body mass index DOC2B double C2 domain protein-β GAD glutamic acid decarboxylase HbA1c hemoglobin A1c hDOC2B human double C2 domain protein-β IA-2A islet antigen 2A iNOS inducible nitric oxide synthase mRNA messenger RNA NOD nonobese diabetic NOR nonobese diabetes resistant nPOD Network for Pancreatic Organ Donors with Diabetes PVDF polyvinylidene difluoride qRT-PCR quantitative real-time polymerase chain reaction SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM standard error of the mean SNARE soluble N-ethylmaleimide-sensitive factor-attachment protein receptor T1D type 1 diabetes Acknowledgments We are grateful to Dr. Bart Roep for critical evaluation of this manuscript. The authors thank Dr. Janice Blum for conceptual contributions and assistance with the human platelet sampling, and Gurneet Sangha (Purdue University) for assistance with the NOD and NOR sample collections. We also are indebted to nPOD, the Integrated Islet Distribution Program, and the Southern California Islet Center at City of Hope for providing human pancreata slides and isolated human islets, respectively. Adult patient blood samples were obtained at City of Hope under Institutional Review Board No. 12466 (F.K.). Pediatric patient blood samples were obtained at Indiana University School of Medicine under Institutional Review Board No. 1201007745 (L.A.D.). Research reported in this publication also includes work performed in the Integrative Genomics and Bioinformatics Core, Drug Discovery and Structural Biology Core (in collaboration with Dr. John Williams), and Light Microscopy/Digital Imaging Core, all supported by the National Cancer Institute, Cancer Center Support Grant P30CA33572, to City of Hope. Nancy Linford provided editing assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Portions of this work were presented at the Levine Symposium and the Scientific Sessions of the American Diabetes Association. Financial Support: This work was supported, in part, by a predoctoral fellowship from the Indiana Clinical and Translational Sciences Institute (UL1TR001108) to A.A. and grants from the National Institutes of Health (DK067912 and DK102233) and JDRF (2-SRA-2015-138-S-B and 1-SRA-2016-242-Q-R) to D.C.T. Research reported in this publication also includes work performed with support from the National Institutes of Health P30CA33572. Author Contributions: A.A. performed the majority of the studies, wrote/edited the manuscript, and contributed to the discussion. E.O. assisted in islet and platelet isolation and in procuring/preparing human T1D platelets and pancreata and contributed to the discussion. A.S.M.M. performed the staining and quantification of the human pancreata and contributed to the discussion. M.A. performed the human islet studies and contributed to the discussion. M.C. performed adult human platelet isolation and contributed to the discussion. M.D., J.H.-S., F.K. and M.E.-S. assisted with clinical transplant sample procurement studies and contributed to the discussion. D.C.T. and L.A.D. conceived of the study, contributed to the discussion, and reviewed/edited the manuscript. All authors read and approved the final version of the manuscript. D.C.T. is the guarantor of this work and as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. Ferrannini E , Mari A , Nofrate V , Sosenko JM , Skyler JS ; DPT-1 Study Group . 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Oxford University Press
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Copyright © 2018 Endocrine Society
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0021-972X
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10.1210/jc.2017-02492
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Abstract

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. Type 1 diabetes (T1D) is characterized by autoimmune destruction of β-cell mass, and the preclinical phase of T1D is marked by declining β-cell function (1, 2). Studies of early intervention in T1D have shown limited effectiveness yet have generally shown greater success in subjects that retain greater insulin secretory capacity and in those with the shortest time since clinical onset of disease (3, 4). However, prevention efforts to protect β-cell mass are hindered by the limited availability of early biomarkers to predict accurately β-cell destruction and subsequent progression to clinical disease. In healthy β-cells, insulin secretion requires soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins and associated accessory regulatory proteins to promote the docking, priming, and fusion of insulin vesicles at the plasma membrane. Two target membrane SNARE proteins—syntaxin 1/4 and SNAP25/23—and one vesicle-associated SNARE protein—VAMP2—constitute the SNARE core complex (5). Assembly of the SNARE complex occurs when one vesicle-associated SNARE binds two cognate target membrane SNARE proteins in a heterotrimeric ratio (6). SNARE complex assembly is also facilitated by the accessory regulatory protein, double C2-domain β (DOC2B) (7, 8). Several studies have established that in animal models, deficiencies in DOC2B result in glucose intolerance and insulin-secretion defects (9, 10). Conversely, overexpression of DOC2B, using a global transgenic mouse model, enhances insulin secretion and peripheral glucose uptake (11). Although DOC2B deficiency in rodents has been linked to type 2 diabetes (12), the association between DOC2B protein levels and T1D is still unknown. Deficient first-phase insulin secretion is a hallmark of preclinical T1D (1, 2); thus, the ability to assess early pancreatic β-cell destruction is critically important for the prediction of disease onset. Currently, risk prediction for T1D relies heavily on family history, genetic screening, and the presence of antibodies against β-cell antigens that often appear relatively late in the progression of disease. The use of autoantibodies in evaluating T1D risk is limited, as >50% of autoantibody-positive patients remain disease free, even at 5 years follow-up (13). Risk scores have been established (14) but remain insufficient to provide an accurate prognosis or an accurate measurement of β-cell health, as many autoantibody-positive individuals are slow to progress through the stages (15) of preclinical disease. To improve early prediction of T1D, ongoing studies seek to investigate the levels of circulating factors that reflect declining β-cell health, such as proinsulin (16), heat shock protein 90 (17), and unmethylated insulin DNA (18), as potential biomarkers of T1D. Another potential source of biomarkers is the blood-derived platelet, which is currently being investigated in diseases, such as Alzheimer’s disease (19) and cancer (20), and has been implicated in T1D. Changes in the platelet proteome and morphology have been noted in T1D; for instance, altered intracellular Ca2+ (21), enhanced formation of microparticles (22), and altered morphology (23) have been reported to result in platelet hyper-reactivity and development of vasculopathies. Importantly, platelets harbor many of the same exocytosis proteins as the pancreatic β-cell, including SNARE isoforms and accessory regulatory proteins (24). Biomarkers of β-cell destruction in blood have more clinical potential than those in pancreatic islets, as islet procurement is not feasible for routine diagnosis; therefore, we investigated the correlation between DOC2B protein abundance in blood-derived platelets and pancreatic islets in nonobese diabetic (NOD) mice and T1D humans. We found that protein abundance of DOC2B is reduced in platelets and islets from prediabetic NOD mice compared with control mice. Furthermore, DOC2B protein abundance is reduced in platelets and islets from humans with new-onset T1D compared with matched controls. Notably, we also reveal that DOC2B levels are substantially increased in T1D human platelets after islet transplantation, when C-peptide levels were markedly increased. Materials and Methods Animals Animals were maintained under protocols approved by the Indiana University Institutional Animal Care and Use Committee and following the National Research Council Guidelines for the Care and Use of Laboratory Animals. Female NOD NOD/ShiLtJ (RRID:IMSR_JAX:001976) and major histocompatibility complex-matched control nonobese diabetes-resistant (NOR; RRID:IMSR_JAX:002050) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). We have observed that female NOD mice begin to convert to T1D at 17 to 18 weeks of age, with an average conversion rate of 78% by 20 weeks of age, as previously reported (25). Random blood glucose analysis was performed weekly to monitor conversion to T1D, which is characterized by nonfasting blood glucose levels >250 mg/dL for 3 consecutive days. To assess DOC2B levels before conversion to T1D, pancreatic islets were isolated, using a method as described previously (26), at 7 weeks (earliest time point for sufficient islet cell yield), 13 weeks (intermediate time point), and 16 weeks (latest time point before conversion to T1D) of age. Islet isolation yield decreases in mice <8 weeks of age (27). Islet lysates were then used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Mouse blood was collected (from 13- and 16-week-old mice; blood was insufficient from 7-week-old mice), and platelets were isolated as previously described (24). Platelet lysates were then used for SDS-PAGE and immunoblotting. Human subjects All human studies were conducted in keeping with the principles set out in the Declaration of Helsinki. This protocol was approved by the Indiana University Institutional Review Board. For evaluation of DOC2B levels in human platelets (new-onset T1D study), subjects aged 8 to 14 years (11 boys and six girls) with new-onset T1D were recruited over an 18-month period. Consent was obtained from parents, with assent from the pediatric subjects. Subjects were diagnosed with T1D if they met the criteria of one or more positive autoantibodies with clinical features of T1D—hyperglycemia, weight loss, and normal body mass index (BMI)—or those who were autoantibody negative but <10 years old at diagnosis. Exclusion criteria were as previously described (17). For each visit, subjects received $25. Subjects had blood drawn at diagnosis and at the first follow-up appointment, 7 to 10 weeks after diagnosis. Insulin treatment of T1D subjects was started at the time of diagnosis. Nondiabetic control subjects (eight boys and six girls) were recruited from the community and matched to T1D subjects based on sex, age, and BMI (see Table 1 for demographic data). Samples were de-identified and coded by the clinical team before distribution to the research laboratory for platelet isolation and analyses. Platelets were isolated by centrifugation from blood, as previously described (28), and lysed for SDS-PAGE and immunoblotting. Upon quantification of the data for each sample, the clinical team reidentified samples to permit grouping of data into T1D vs nondiabetic for statistical comparisons. Table 1. Pediatric T1D Study Demographics Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Values displayed are means ± standard deviation unless otherwise noted. Abbreviation: HbA1c, hemoglobin A1c. a For BMI calculations, one T1D subject did not have a diagnosis height, and one non-T1D control did not have a registration height. For these subjects, the heights from clinic follow-up were used to calculate BMI. b The following three diabetes-associated antibodies were tested: glutamic acid decarboxylase, micro insulin autoantibodies, and insulinoma-associated antigen-2. c For C-peptide at diagnosis, n = 13. View Large Table 1. Pediatric T1D Study Demographics Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Characteristic Non-T1D Controls T1D Subjects Number of subjects 14 17 Age in years (range) 11.4 (8.0–14.3) 10.3 (4.3–14.1) Sex (boys), % 55 57 BMI, kg/m2a 20.2 ± 3.0 17.5 ± 2.8 Number of autoantibodies positiveb – 0 AutoAb positive: 1 1 AutoAb positive: 5 2 AutoAb positive: 9 3 AutoAb positive: 2 Basal insulin requirement before hospital discharge, units/kg/day – 0.30 ± 0.09 C-Peptide at diagnosis (pM)c – 110 ± 169 (13–608) HbA1c at diagnosis (range), % – 11.0 ± 1.7 (7.5 ± 14.2) HbA1c at first follow-up (range), % – 7.7 ± 0.8 (6.4–9.0) Values displayed are means ± standard deviation unless otherwise noted. Abbreviation: HbA1c, hemoglobin A1c. a For BMI calculations, one T1D subject did not have a diagnosis height, and one non-T1D control did not have a registration height. For these subjects, the heights from clinic follow-up were used to calculate BMI. b The following three diabetes-associated antibodies were tested: glutamic acid decarboxylase, micro insulin autoantibodies, and insulinoma-associated antigen-2. c For C-peptide at diagnosis, n = 13. View Large For evaluation of DOC2B levels in human islets (T1D islet transplantation study), samples were obtained from T1D islet transplantation recipients, as approved by the City of Hope Institutional Review Board. Two subjects, aged 43 and 52 years, were recruited for human islet transplantation, based on the following criteria: T1D diagnosis with frequent or life-threatening hypoglycemia, with or without unawareness symptoms. Blood was obtained from both subjects before transplantation (Day 0) and on Days 30 and 75 after islet transplantation (see Supplemental Table 1 for demographic data). Platelets were isolated by centrifugation from blood, as previously described (28), and lysed for SDS-PAGE and immunoblotting. Islet cell transplantation For the T1D islet transplant study, human pancreata were procured from ABO-compatible, cross-match-negative cadaveric donors. The islets were isolated under cyclic guanosine monophosphate conditions by the Southern California Islet Cell Resource Center at City of Hope using a modified Ricordi method. Islets were maintained in culture for up to 72 hours before transplantation. Islets were transplanted intraportally with heparinized saline (35 U/kg recipient body weight) using a transhepatic percutaneous approach. Clinical/laboratory assays For the new-onset T1D study, autoantibodies to glutamic acid decarboxylase (GAD)-65, insulin, and islet antigen 2A (IA-2A) were assayed from peripheral blood at diagnosis at Mayo Medical Laboratories (Rochester, MN). Glycated hemoglobin A1c (HbA1c) was also measured at diagnosis and at the first clinic follow-up (7 to 10 weeks after diagnosis), using the A1cNow system or the DCA2000 analyzer (Bayer, Tarrytown, NY). C-Peptide was measured in stored serum samples using the C-peptide enzyme-linked immunosorbent assay kit (Alpco, Salem, NH; detection range 20 to 3000 pM). For the T1D islet transplant study, plasma C-peptide measurements were performed by the Northwest Lipid Metabolism and Diabetes Laboratory (Seattle, WA), using the C-Peptide II Assay (Tosoh Bioscience, San Francisco, CA; detection range 0.02 to 30 ng/mL). A fasting C-peptide <0.2 ng/mL and 6-minute glucagon-stimulated C-peptide <0.3 ng/mL were used to confirm T1D diagnosis before islet transplant. Autoantibodies (GAD-65, IA-2A, micro insulin autoantibodies, and zinc transporter 8) were analyzed using radiobinding assays by the Autoantibody/HLA Service Center at the Barbara Davis Center for Diabetes (Aurora, CA). Ex vivo islet preparations Non-T1D human cadaveric pancreatic islets were obtained through the Integrated Islet Distribution Program at City of Hope. The islets were prepared and treated with a cytokine mixture (10 ng/mL tumor necrosis factor-α, 100 ng/mL interferon-γ, and 5 ng/mL interleukin-1β; ProSpec, East Brunswick, NJ) for 72 hours, as previously described (29). The islets were then used in quantitative real-time polymerase chain reaction (qRT-PCR) analysis or SDS-PAGE, followed by immunoblotting. Immunofluorescence Human paraffin-embedded pancreatic tissue sections were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Five sections from formalin-fixed paraffin-embedded tissue samples were obtained from T1D (n = 3) and age- and BMI-matched nondiabetic (n = 3) donors. Pancreas sections were immunostained with primary and secondary antibodies, listed in Supplemental Table 2. Slides were counterstained to mark the nuclei, using 4′,6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, CA) and viewed using a BZ X-700 fluorescence microscope (Keyence, Itasca, IL). All human T1D samples were prepared and processed at the same time; confocal images were taken with identical acquisition settings. Islet immunofluorescence was assessed by imaging 20 to 30 islets (grouping of four or more insulin-positive cells) per subject. Analysis was performed in a blinded fashion using Image-Pro Software (Media Cybernetics, Rockville, MD) to quantify fluorescence intensities using methods described previously (30). Defined regions of interest were used to delimit islets from adjacent acinar tissue, and average intensity measurements of insulin and DOC2B were quantified by the splitting of the merged image into two color channels with the same region of interest. Immunoblotting Platelet and islet protein lysates for the NOD mouse study were resolved on a 10% SDS-PAGE gel and transferred to standard polyvinylidene difluoride (PVDF; Bio-Rad, Hercules, CA). Platelet proteins from the new-onset T1D study were resolved on a 10% SDS-PAGE gel using an SE400 air-cooled 18 × 16 cm vertical protein electrophoresis unit (Hoefer, Holliston, MA) and transferred to standard PVDF (Bio-Rad). Platelet proteins from the T1D islet transplant study were resolved on a 12% SDS-PAGE gel using a Criterion 13.3 × 8.7-cm vertical electrophoresis unit (Bio-Rad) and transferred to standard PVDF. All blots were probed, as outlined in Supplemental Table 2. qRT-PCR Total RNA was isolated from human islets using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and assessed using the QuantiTect SYBR Green RT-PCR kit (Qiagen). Primers used for the detection of human Doc2b (hDoc2b) are as follows: forward: 5′-CCAGTAAGGCAAATAAGCTC-3′ and reverse: 5′-GGGTTTCAGCTTCTTCA-3′. Standard tubulin primers (Cat: QT00089775; Qiagen) were used for normalization. Statistical analysis Data were evaluated for statistical significance using Student t test for comparison of two groups; analysis of variance and Tukey post hoc test (GraphPad Software, La Jolla, CA) were used for comparison of more than two groups. Data are expressed as the average ± standard error of the mean (SEM). Results Low DOC2B levels in prediabetic NOD mouse platelets and islets To investigate whether DOC2B protein levels are altered in the blood before onset of T1D, we examined platelet DOC2B abundance in young prediabetic NOD mice and major histocompatibility complex-matched NOR mice. Immunoblotting revealed that platelets from 16- and 13-week-old NOD mice exhibited up to a 90% reduction in DOC2B protein levels [Fig. 1(a)] compared with NOR platelets. Furthermore, islets from 16- and 13-week-old NOD mice showed at least a 65% reduction in DOC2B protein levels [Fig. 1(b)] compared with NOR islets. NOD islets from as early as 7 weeks of age showed a 90% reduction in DOC2B protein [Fig. 1(b)]. The average blood glucose levels from random blood testing of NOD and NOR mice were below 250 mg/dL at 7, 13, and 16 weeks (Supplemental Table 3), indicating that the mice had not yet converted to diabetes. These data show that DOC2B protein abundance is reduced in both platelets and islets of prediabetic mice. Figure 1. View largeDownload slide 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 1. View largeDownload slide 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. Low DOC2B levels in new-onset T1D human platelets In the new-onset T1D study, we quantified protein content of DOC2B using platelets from new-onset T1D subjects compared with controls (Table 1). Platelets from new-onset T1D subjects exhibited reduced protein levels of DOC2B for both genders, both at diagnosis and at the first clinic follow-up, 7 to 10 weeks later. When boys and girls were assessed separately, DOC2B levels were reduced in boys by >60% compared with nondiabetic control subjects, persisting even after insulin treatment of the patient and reduction of HbA1c (Fig. 2). The substantial loss of DOC2B at T1D diagnosis was selective for DOC2B compared with another exocytosis protein, syntaxin 4 (Supplemental Fig. 1). These data indicate that DOC2B is decreased in T1D platelets, independent of glycemic control, relative to nondiabetic human platelets, and that platelet DOC2B levels are already diminished at T1D diagnosis. Figure 2. View largeDownload slide 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 2. View largeDownload slide 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. Ex vivo proinflammatory cytokine treatment reduces human islet DOC2B levels T1D is associated with elevated circulating proinflammatory cytokines that damage β-cells (31). As the attainment of pancreatic islets from living T1D subjects is virtually impossible, we evaluated the relationship between T1D and DOC2B levels by treating human cadaveric nondiabetic islets (Supplemental Table 4) ex vivo with proinflammatory cytokines in an effort to simulate the circulating milieu. Cytokine treatment (interleukin-1β, tumor necrosis factor-α, interferon-γ) elevated the levels of islet inducible nitric oxide synthase [iNOS; Fig. 3(a)], consistent with the reported effects of cytokine exposure (32). Correspondingly, DOC2B protein and messenger RNA (mRNA) levels were reduced by 30% and 50%, respectively (Fig. 3). These data suggest that a T1D-like milieu can decrease DOC2B levels in human islets. Figure 3. View largeDownload slide 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 3. View largeDownload slide 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. Reduced DOC2B protein in human early-onset T1D islets To investigate changes in DOC2B levels in T1D human pancreata, we used paraffin-embedded slides (obtained from nPOD) from cadaveric donors for DOC2B immunofluorescence evaluation in early-onset pediatric T1D (5 years or less with T1D; n = 3) vs matched nondiabetic controls [n = 3; Fig. 4(a) and Supplemental Table 5]. With the measurement of relative immunofluorescent intensities, we observed a decrease in DOC2B abundance in T1D islets vs in nondiabetic controls [Fig. 4(b)]. Although the relative number of DOC2B-positive β-cells in nondiabetic and T1D islets was similar [Fig. 4(c)], DOC2B intensity was reduced in T1D β-cells. Figure 4. View largeDownload slide 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 4. View largeDownload slide 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. DOC2B levels are restored after clinical islet transplantation In the T1D islet transplantation study (Supplemental Table 1), we found that the pretransplant platelet DOC2B levels were very low in both subjects relative to a hDOC2B protein standard curve (Fig. 5; Day 0). Notably, within 30 days of transplantation, each T1D islet recipient showed a robust increase in platelet DOC2B protein, which persisted to 75 days after transplantation (Fig. 5; Days 30 and 75). These data coincide with changes in C-peptide levels in these subjects: whereas each subject had low to almost undetectable fasting/glucagon-stimulated C-peptide levels before transplantation, the C-peptide levels were substantially increased by 30 days after transplantation (Supplemental Table 6). As C-peptide levels are indicative of overall islet function, these data suggest that in humans, DOC2B levels in platelets correlate with relative functional β-cell mass. Figure 5. View largeDownload slide 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. Figure 5. View largeDownload slide 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. Discussion The ability to detect β-cell destruction is critical in accurately predicting prognosis during the preclinical phase of T1D; hence, the current need for additional early biomarkers. We show that DOC2B protein levels are substantially reduced in platelets and islets from prediabetic NOD mice vs NOR control mice. Furthermore, we reveal that levels of hDOC2B are significantly lower at the time of diagnosis in platelets of pediatric patients with new-onset T1D than platelets from matched control subjects. Notably, DOC2B levels were reduced at 7 to 10 weeks postdiagnosis, despite therapeutic remediation of hyperglycemia in the human subjects. Consistent with this, islet DOC2B protein levels were reduced in early-onset T1D pancreatic tissue samples compared with matched controls. Loss of DOC2B protein and mRNA could be recapitulated by exposure of nondiabetic human islets to proinflammatory cytokines ex vivo, suggesting that the inflammatory milieu in prediabetic and T1D humans may cause DOC2B loss. Remarkably, clinical islet transplant recipients exhibit a restoration of DOC2B levels in platelets compared with their own nearly undetectable levels of platelet DOC2B before receiving the transplanted islets. These data suggest that the DOC2B protein is a candidate biomarker of prediabetes and T1D, with the levels possibly reporting relative functional β-cell mass. This study establishes an association between T1D and levels of an exocytosis protein in blood-derived platelets and pancreatic islets. Reduced DOC2B in islets is indicative of deficient islet functional health (9). Strikingly, platelet DOC2B levels in islet transplant recipients correlated with the presence of a functional islet mass. This correlative finding supports the possibility that the platelet DOC2B stems not necessarily from the pancreas per se, as islets are grafted into the liver in these human recipients, but that the platelets and/or precursor megakaryocytes may be sampling DOC2B from the islets, irrespective of islet location. It also remains possible that the increased DOC2B content stems from “rested,” native, residual islets of the transplanted patients. However, this is inconsistent with our pediatric platelet data, showing that even after insulin therapy to ameliorate new-onset hyperglycemia, DOC2B levels remained deficient. Mechanistically, questions arise as to if and how platelets and islets communicate to determine DOC2B levels. Supporting the concept of platelet-islet communication, it has been demonstrated that islet transplantation in patients with T1D stabilizes platelet abnormalities, as transplant recipient platelets show normal volume and activation (33). Indeed, β-cells release exosomes as a way of shuttling various microRNAs, mRNAs, and proteins to targeted peripheral cells (34). β-Cell exosomes were also recently shown to carry proteins, such as GAD-65, IA-2, and proinsulin, to dendritic cells, which then become activated (35). Could islets similarly release DOC2B mRNA or protein, which is subsequently taken up by megakaryocytes or by the platelets themselves? Furthermore, platelets can selectively absorb proteins from the blood (36). In fact, platelet sequestration of tumor-specific proteins was detected in animals harboring small tumors (36). Notably, a direct interaction between platelets and pancreatic β-cells has been reported, and protein from platelets was shown to be transferred to β-cells (37). Is it possible that the platelets are sampling the islets to mirror islet DOC2B levels? As the data presented herein are correlative in nature, future studies will be required to determine the detailed molecular mechanism regulating changes in DOC2B protein abundance in human T1D samples. The concept of DOC2B as a biomarker is appealing, as DOC2B levels in platelets and islets are significantly decreased in normoglycemic NOD mice, months before their conversion to T1D. Female NOD mice typically convert to T1D between 18 and 24 weeks of age, but as early as 5 weeks of age, NOD mouse islets show signs of insulitis, resulting from an initial phase of pancreatic inflammation that reduces β-cell function and mass (38). Given that DOC2B content in human islets decreased upon islet exposure to proinflammatory cytokines, which was sufficient to evoke iNOS expression, it is possible that the cytokine-induced drop in islet DOC2B signals reduced islet viability. Although it has been demonstrated by multiple groups that whole-body DOC2B knockout mice show deficient glucose-stimulated insulin secretion (9, 10), β-cell mass was not evaluated. Whereas it is also possible that DOC2B expression is genetically repressed in NOD mice, the genetics of NOD mice have been well studied, and DOC2B was not identified as deviating from control (39). DOC2B mRNA expression was also decreased in response to proinflammatory cytokine exposure in nondiabetic human islets, suggesting that DOC2B might undergo transcriptional repression during T1D development. Indeed, promoter methylation and silencing of the DOC2B gene have been reported to occur in cancer cell types (40). Future studies will be required to determine the detailed molecular mechanism regulating changes in DOC2B protein abundance in human T1D samples. Whereas findings were obtained, there are also some limitations to the conclusions that can be derived from this study. First, this work derives from two pilot studies with relatively small sample sizes of human pediatric subjects (n = 14 controls and n = 17 patients with T1D, new-onset T1D study) and adult T1D islet transplant subjects (n = 2, T1D islet transplantation study). Secondly, the pediatric cohort was evaluated at clinical diagnosis of T1D, so these patients already have full-blown disease. A larger, future study would benefit from a prospective design that includes subjects who are significantly at risk but who are normoglycemic. We are also limited in that only the first clinical follow-up samples were obtained, and we do not have further follow-up data for a longitudinal evaluation of changes in DOC2B. Lastly, the number of adult T1D clinical islet transplantation subjects followed was limited by our initial requirement for collection of fresh blood for platelet isolation to be consistent with our pediatric T1D cohort. Given our focus on DOC2B as an early predictor of T1D, future studies will examine serum and plasma, both of which contain abundant and detectable levels of DOC2B. This will permit studies of larger numbers of T1D transplant recipients and evaluation of stored samples from a variety of repositories to enable more generalized conclusions. In summary, we demonstrate reduced abundance of the exocytosis protein, DOC2B, in prediabetes (NOD mice) or the early onset of T1D (humans). In addition, we show a correlation between the platelet and islet DOC2B levels, suggesting that platelet evaluation may provide prognostic information about T1D risk and progression. Furthermore, transplantation of healthy, functional islets increased the levels of DOC2B in T1D human platelets. Given the need to assess β-cell destruction accurately at an early stage, DOC2B may be a viable biomarker for T1D. Abbreviations: Abbreviations: BMI body mass index DOC2B double C2 domain protein-β GAD glutamic acid decarboxylase HbA1c hemoglobin A1c hDOC2B human double C2 domain protein-β IA-2A islet antigen 2A iNOS inducible nitric oxide synthase mRNA messenger RNA NOD nonobese diabetic NOR nonobese diabetes resistant nPOD Network for Pancreatic Organ Donors with Diabetes PVDF polyvinylidene difluoride qRT-PCR quantitative real-time polymerase chain reaction SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM standard error of the mean SNARE soluble N-ethylmaleimide-sensitive factor-attachment protein receptor T1D type 1 diabetes Acknowledgments We are grateful to Dr. Bart Roep for critical evaluation of this manuscript. The authors thank Dr. Janice Blum for conceptual contributions and assistance with the human platelet sampling, and Gurneet Sangha (Purdue University) for assistance with the NOD and NOR sample collections. We also are indebted to nPOD, the Integrated Islet Distribution Program, and the Southern California Islet Center at City of Hope for providing human pancreata slides and isolated human islets, respectively. Adult patient blood samples were obtained at City of Hope under Institutional Review Board No. 12466 (F.K.). Pediatric patient blood samples were obtained at Indiana University School of Medicine under Institutional Review Board No. 1201007745 (L.A.D.). Research reported in this publication also includes work performed in the Integrative Genomics and Bioinformatics Core, Drug Discovery and Structural Biology Core (in collaboration with Dr. John Williams), and Light Microscopy/Digital Imaging Core, all supported by the National Cancer Institute, Cancer Center Support Grant P30CA33572, to City of Hope. Nancy Linford provided editing assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Portions of this work were presented at the Levine Symposium and the Scientific Sessions of the American Diabetes Association. Financial Support: This work was supported, in part, by a predoctoral fellowship from the Indiana Clinical and Translational Sciences Institute (UL1TR001108) to A.A. and grants from the National Institutes of Health (DK067912 and DK102233) and JDRF (2-SRA-2015-138-S-B and 1-SRA-2016-242-Q-R) to D.C.T. Research reported in this publication also includes work performed with support from the National Institutes of Health P30CA33572. Author Contributions: A.A. performed the majority of the studies, wrote/edited the manuscript, and contributed to the discussion. E.O. assisted in islet and platelet isolation and in procuring/preparing human T1D platelets and pancreata and contributed to the discussion. A.S.M.M. performed the staining and quantification of the human pancreata and contributed to the discussion. M.A. performed the human islet studies and contributed to the discussion. M.C. performed adult human platelet isolation and contributed to the discussion. M.D., J.H.-S., F.K. and M.E.-S. assisted with clinical transplant sample procurement studies and contributed to the discussion. D.C.T. and L.A.D. conceived of the study, contributed to the discussion, and reviewed/edited the manuscript. All authors read and approved the final version of the manuscript. D.C.T. is the guarantor of this work and as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. Ferrannini E , Mari A , Nofrate V , Sosenko JM , Skyler JS ; DPT-1 Study Group . 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Mar 1, 2018

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