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. Author manuscript; available in PMC: 2018 Sep 5.
Published in final edited form as: Cell Metab. 2017 Sep 5;26(3):568–575.e3. doi: 10.1016/j.cmet.2017.08.013

Persistence of Pancreatic Insulin mRNA Expression and Proinsulin Protein in Type 1 Diabetes Pancreata

Clive Wasserfall 1, Harry S Nick 2, Martha Campbell-Thompson 1, Dawn Beachy 2, Leena Haataja 3, Irina Kusmartseva 1, Amanda Posgai 1, Maria Beery 1, Christopher Rhodes 4, Ezio Bonifacio 5, Peter Arvan 3, Mark Atkinson 1,6
PMCID: PMC5679224  NIHMSID: NIHMS901211  PMID: 28877460

SUMMARY

The canonical notion that type 1 diabetes (T1D) results following a complete destruction of β-cells has recently been questioned as small amounts of C-peptide are detectable in patients with longstanding disease. We analyzed protein and gene expression levels for proinsulin, insulin, C-peptide and islet amyloid polypeptide within pancreatic tissues from T1D, autoantibody positive (Ab+) and control organs. Insulin and C-peptide levels were low to undetectable in extracts from the T1D cohort; however, proinsulin and INS mRNA were detected in the majority of T1D pancreata. Interestingly, heterogeneous nuclear RNA (hnRNA) for insulin and INS-IGF2, both originating from the INS promoter, were essentially undetectable in T1D pancreata, arguing for a silent INS promoter. Expression of PCSK1, a convertase responsible for proinsulin processing, was reduced in T1D pancreata, supportive of persistent proinsulin. These data implicate the existence of β-cells enriched for inefficient insulin/C-peptide production in T1D patients, potentially less susceptible to autoimmune destruction.

In Brief

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Wasserfall et al. analyze rare pancreatic samples from T1D patients. They find proinsulin protein and persistence of insulin mRNA, but decreased proconvertase PCSK1 and undetectable INS hnRNA. These findings indicate the presence, in long-term T1D subjects, of a population of β-cells with impaired insulin gene transcription and inefficient proinsulin processing.

INTRODUCTION

Type 1 diabetes (T1D) develops as a result of endogenous insulin insufficiency due to autoimmune destruction of the insulin producing pancreatic β-cells (Atkinson et al., 2014). Two long-standing dogmas in T1D maintain that symptomatic onset occurs when 90–95% of β-cells are destroyed, and that within months to a few years after recent onset, all β-cells are lost (Atkinson et al., 2014; Eisenbarth, 1986). However, recent data collected from pancreata with disease of varying durations suggest that β-cell mass at diagnosis is more variable than once appreciated. Indeed β-cells, while rare in number, can exist for decades after disease onset (Campbell-Thompson et al., 2016; Keenan et al., 2010; Meier et al., 2005). Not surprisingly, this emerging concept has further fueled an interest in identifying factors that either promote the formation of new β-cells or promote enhanced survival and function of existing β-cells, in combination with an immunotherapy capable of blocking the destruction of such cells (Ludvigsson, 2014).

Contemporaneous with this pathology-based literature, emerging metabolic data from living T1D patients supports reconsideration of the notion that complete β-cell loss occurs in T1D (both quantitatively as well as functionally). Indeed, a citation classic involving analysis of NIH Diabetes Control and Complications Trial (DCCT) study participants using the conventional assay noted that after 5–15 years of disease, only 22 percent had detectable C-peptide (DCCT Research Group, 1987). However, a vast majority of subjects with established T1D, upwards of 80%, have been posited as being so called “microsecretors” of insulin, made possible by newly developed ultrasensitive C-peptide assays (Oram et al., 2014; Wang et al., 2012).

With this, we sought to better characterize the molecular mechanisms of endocrine hormone production and processing in the pancreas throughout the natural history of T1D. These studies have only recently become possible due to the availability of high-quality tissues through organ donation programs, such as the JDRF Network for Pancreatic Organ donors with Diabetes (nPOD) (Campbell-Thompson, 2015). We hypothesized that in T1D, a population of endocrine cells selected for resistance to autoimmune destruction may exist, possibly representing a depot for unprocessed proinsulin given extremely low levels of insulin and C-peptide in T1D. To address this concept, we determined the expression (both protein and gene) of a series of hormones in human pancreatic tissues obtained from relevant study groups, including those with longstanding T1D. These analyses included immunohistochemistry (IHC), acid-ethanol extractions of pancreatic tissue blocks followed by quantification of the various peptide hormones by ELISA, gene expression studies of tissue blocks by real time qPCR, and in situ hybridization (ISH) of tissue sections for mRNA.

RESULTS AND DISCUSSION

Pancreatic Hormone Production by Immunoflourescence Staining

All pancreatic tissue blocks were obtained from 106 study organs (Table 1), with a summary of all assays per donor summarized in Table S1. The study groups include those that either had no history of diabetes and were T1D autoantibody (Ab) negative (controls), no diabetes but were Ab positive (Ab+, Table S2) and those diagnosed with T1D.

Table 1.

Network for Pancreatic Organ Donors with Diabetes (nPOD) tissue donors are categorized by disease state (left column), and for each donor type, the number of donors (N), the number of male (M) and female donors (F), age (years), disease duration (years), and age at disease onset (years) are listed. NA indicates not applicable.

Donor Type N Gender (N) Age Median (Range) Disease Duration Median (Range) Age at disease onset Median (Range)
M F
No Diabetes 50 32 18 26.9 (0.3–75) NA NA
Autoantibody Positive 16 9 7 33.2 (2.2–69) NA NA
Type 1 Diabetes 40 24 16 27.5 (10.7–61) 13.0 (1–52) 12.4 (2–33.8)
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As a histological illustration of our patient groups, we performed immunolocalization for proinsulin (green), insulin (red) and glucagon (yellow) on an islet from a representative control (no history of diabetes, Figure 1A,F,K, and P), along with random proinsulin and insulin staining of individual cells in the exocrine pancreas (Figure 1A, inset). Representative images from a patient with short duration T1D (Figure 1B,C,G,H,L,M,Q, and R) illustrate the heterogeneity and progression of disease with islets displaying significant proinsulin and insulin staining as well as islets where only glucagon is detected (Figure 1C, inset). Here, we note a patient with longer duration T1D, demonstrating the continued expression of both proinsulin and insulin despite seven years of disease (Figure 1D,I,N and S). Analogous to the cells staining positive for insulin in the exocrine tissue of the control donor, a representative patient with two year duration of T1D similarly illustrates the presence of cells in the exocrine pancreas that harbor proinsulin, insulin as well as glucagon (Figure 1E,J,O, and T, inset).

Figure 1. Proinsulin, insulin, and glucagon expression in type 1 diabetes (T1D).

Figure 1

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(A–E) Composite images of pancreas sections stained for proinsulin (green), insulin (red), glucagon (yellow), and DAPI (nuclei, blue) are shown for sections containing islets from (A) a control 24-year old male Caucasian (nPOD 6131), (B–C) a 12-year old male African American with T1D for 1 year (nPOD 6052), (D) a 23-year old female Caucasian with T1D for 7 years (nPOD 6070), and (E) a section containing pancreas exocrine tissue from a 12.5 year old female Caucasian with T1D for 2 years (nPOD 6371).

(F–T) The individual channels for (F–J) proinsulin (green), (K–O) insulin (red), and (P–T) glucagon (yellow) are also shown.

Insets display: (A) proinsulin and insulin positive cells in the exocrine pancreas, (C) an insulin negative, proinsulin negative, glucagon positive islet and (E) proinsulin, insulin and glucagon positive cells in the exocrine pancreas.

Scale bars represent 50 μm. See also Figure S1

Persistence of Proinsulin in T1D Pancreata Identified by Protein Extraction

Following acid-ethanol extraction of protein from randomly localized pancreatic blocks, levels of proinsulin, insulin, C-peptide, islet amyloid polypeptide, and glucagon were determined and normalized against total protein. Extracted insulin (p=0.60), proinsulin (p=0.95), and C-peptide levels (p=0.71) were comparable for single Ab+ subjects (n=12) versus the four subjects with multiple Ab; hence, all Ab+ donors were analyzed together as one group. Insulin protein was detected in amounts similar to those determined from control pancreas tissues in only five of the T1D pancreata, while the majority of samples (17/24) displayed insulin levels at the lower end of the assay detection limits with two additional samples that were completely undectable (Figure 2A and Supplemental Figure 1A, p<0.0001). However, proinsulin protein levels in T1D pancreata were surprisingly similar to the Ab+ cohort, albeit slightly lower than controls (Figure 2B, p<0.01). C-peptide levels were very low to undetectable in T1D pancreata relative to the control and Ab+ tissues (Figure 2C, p<0.0001, not detected (ND)=8/24). Consequently, proinsulin/insulin and proinsulin/C-peptide ratios were significantly elevated in T1D pancreatic extracts versus control and Ab+ pancreata (Supplemental Figure 1B,C, p<0.0001). In line with the C-peptide data, levels of islet amyloid polypeptide, a β-cell co-secretory molecule, were essentially undetectable in the majority of T1D pancreata relative to controls (Figure 2D, ND=13/18). Due to limited sample availability, glucagon levels were only determined in a subset of controls and T1D donors, yet no significant differences in the quantity of this analyte were observed between these groups (Figure 2E).

Figure 2. Total protein extracts and gene expression from human pancreas sections.

Figure 2

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(A–H) ELISAs of acid-ethanol extracts from human pancreas tissue sections for (A) Insulin (μInternational Units (μIU)), (B) Proinsulin, (C) C-Peptide, (D) Islet Amyloid Polypeptide, and (E) Glucagon (values were normalized against total protein). ND = not detected/examined are noted on each graph. Data are presented as median with interquartile range.

(F–H) Correlation between extracted proinsulin and extracted insulin in (F) Controls, (G) autoantibody positive (Ab+), and (H) T1D is shown. (H) For T1D subjects, correlation analysis is reported for all data as well as a separate analysis of the five subjects with insulin detected in the normal range (Detectable INS Only) with the latter represented by the trend line.

(I–N) Real time qPCR Cq values for control and T1D pancreata for (I) INS, (J) unspliced INS heterogeneous nuclear RNA (hnRNA), (K) INS-IGF2 readthrough mRNA, (L) IAPP, (M) GCG, and (N) SST expression were compared. For INS hnRNA, INS-IGF2, and IAPP, statistical analyses were not performed due to the number of T1D samples with undetectable RNA (ND = not detected/examined).

p-values are indicated on the figure.

When analyzing the relationship between proinsulin and insulin levels within the pancreatic extracts, there was a significant correlation between these analytes in control (Figure 2F, p<0.0001), but not in the T1D or Ab+ pancreata (Figure 2G,H). We then performed a correlation analysis comparing the percent insulin positive area from IHC stained tissue sections against the extracted insulin for each respective patient. For both T1D and control pancreata, the r values were above 0.4, with the correlation for T1D reaching significance (Supplemental Figure 1D,E).

Gene Expression Analysis Identifies Persistant INS mRNA Expression in T1D Pancreata

To further investigate the persistence of proinsulin protein in T1D pancreata (Figure 2B), we next assayed insulin (INS) mRNA, INS heterogeneous nuclear RNA (hnRNA), and the mRNAs encoding INS-IGF2, islet amyloid polypeptide (IAPP), glucagon (GCG), and somatostatin (SST) expression by real time qPCR. It should be noted that we provide Cq values for all genes since we felt it invalid to normalize when, in so many instances, we observed undetectable expression. Contrary to our expectations but consistent with the detection of proinsulin protein (Figure 2B), we observed low but reproducible levels of INS mRNA in T1D pancreata (Figure 2I). Although the T1D INS mRNA was much lower than control, we observed consistent expression across pancreata, even in those with long duration T1D. We next studied de novo transcription from the INS gene promoter, using primers that would only detect unspliced hnRNA for INS (Table S3). Surprisingly, compared with control pancreata, we detected very little if any de novo synthesis of INS hnRNA indicating that the INS promoter was essentially silent in T1D pancreata (Figure 2J, ND=17/30 in T1D samples versus 0/34 controls). Levels of INS-IGF2 read through mRNA, which also originates from the human INS promoter, supports an inactive INS promoter since 28/31 T1D pancreata demonstrated no detectable mRNA (Figure 2K).

To assess the notion of whether these INS hnRNA results were potentially influence by sample degradation afforded by the action of digestive enzymes or variability in pancreatic recovery procedures influenced these results (e.g., warm and cold ischemia time), we measured hnRNA levels for calmodulin 1 (CALM1), serine protease inhibitor Kazal-type 1 (SPINK1), cyclophilin A (PPIA) and chymotrypsin C (CTRC) by real time qPCR. We observed that hnRNA levels for these pancreatic genes were comparable across T1D and control samples (Supplemental Figure 2). Collectively, these data support an interpretation that the INS promoter displays significantly reduced activity in the vast majority of T1D subjects.

In agreement with the the islet amyloid polypeptide protein data (Figure 2D), IAPP mRNA levels were substantially lower in T1D, with 17/31 undetectable, while control pancreata contained detectable message in all cases (Figure 2L). In contrast, we observed no significant differences between T1D and control pancreata for GCG and SST (Figures 2M,N). These data suggest that T1D pancreata have cells, either in islets or scattered in the exocrine regions of the pancreas, that contain INS mRNA and proinsulin protein, but with limited INS promoter activity and little to no production of the co-secretory molecule, IAPP.

Distribution of Insulin or Glucagon Single Positive Cells in the Exocrine Tissue

To further explore the intrapancreatic location of these cells, we performed IHC using insulin (red) and glucagon (blue) antibodies (Figures 3A,B,C,D, and E), as well as ISH for INS mRNA (Figures 3F,G,H,I, and J) in a representative control and longer duration (7 and 35 years) T1D pancreata. These efforts demonstrated the presence of both insulin positive and negative islets in the seven year duration patient (Figure 3B) whereas no insulin positive islets were detected in the long duration pancreas (Figure 3D,E insets). Interestingly, we observed small clusters or single insulin positive cells scattered through the exocrine pancreas in both the control (Figure 3A) and the seven year duration T1D pancreas (Figure 3C). These scattered insulin postive cells were also present in the long duration T1D pancreas (35 years) and were either detected in the exocrine pancreas (Figure 3D) or localized adjacent to ducts (Figure 3E).

Figure 3. Evaluation of insulin/glucagon IHC, INS ISH, prohormone convertase and protease gene expression from human pancreas.

Figure 3

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(A–E) Insulin (red) and glucagon (blue) protein was detected by IHC, and (F–J) insulin mRNA (pink) was detected by ISH in pancreas sections from donors without diabetes (A,F; Control; nPOD 6172) and T1D pancreata with 7 (B,C,G,H; nPOD 6070) and 35 year duration (D,E,I,J; nPOD 6031). Insets in (D) and (E) illustrate glucagon only positive islets. Red arrows indicate single insulin positive cells located in the exocrine tissue and peri-ductal. Scale bars represent 50μm.

(K–O) For control (n=5) and T1D subjects (n=11), IHC analysis of glucagon and insulin within pancreas sections from control donors as well as short (0–7 years; n=5) or long (8–35 years; n=6) duration T1D were analyzed for (K) insulin positive cells per islet; cell counts within the (L–M) islets and (N–O) exocrine tissue.

(P–R) qPCR for control versus T1D pancreata (P) PCSK1, (Q) PCSK2, and (R) CPE.

p-values indicated on the figure.

We performed a semiquantitative assessment of insulin positive cells within islets, noting both small clusters (2–5 insulin positive cells) and single cells, in a subset of control (n=5), short duration (0–7 years; n=5) and longer duration (8–35 years; n=6) T1D pancreata, involving co-staining for insulin and glucagon (Figure 3K,L,M,N, and O). Whereas control pancreata contained significant numbers of insulin positive cells in islets, reduced numbers of such cells were identified in the short duration T1D cases while in longer duration pancreata, they were essentially absent (Figure 3K). Additionally, pseudo-atrophic (glucagon+insulin-) islets were only detected in the T1D groups (Figure 3L). Consistent with the number of insulin positive cells per islet (Figure 3K), the number of glucagon+insulin+ islets showed a similar distribution (Figure 3M, p<0.01). Interestingly, control pancreata contained a significant population of both insulin positive single cells (Figure 3N, p<0.01) and small clusters (Figure 3O, p<0.01), while short and long duration T1D pancreata harbor both populations, albeit in reduced numbers relative to controls. Similar to these single insulin positive cells in longer duration T1D pancreata, we also observed glucagon positive cells in both the exocrine pancreas and peri-ductal regions (Supplemental Figure 3).

Consistent with these IHC findings (Figure 3A–E and K-O), studies involving acid-ethanol extraction (Figure 2A–B, Supplemental Figure 1), RNA expression of proinsulin/insulin (Figure 2I), and ISH for INS mRNA clearly demonstrate that T1D pancreata harbor INS mRNA in islets (Figure 3G) as well as in many cells scattered through the exocrine pancreas (Figure 3H). Here, we would highlight, in particular, those in the exocrine region of longer duration T1D pancreata (Figure 3I,J). Our observations not only corroborate previous findings of residual β-cells in long-duration T1D pancreas (Keenan et al., 2010), but extend them by providing semiquatitative data on insulin positive cell numbers. Both efforts support the need to better understand the production of endocrine hormones, both at the protein and RNA level, throughout the natural history of T1D.

Proconvertase PCSK1 is Diminished in T1D Pancreata

Proinsulin is processed to insulin and C-peptide by the prohormone convertases (PCSK1 and PCSK2) and carboxypetidase E (CPE) (Goodge and Hutton, 2000). Glucagon is similarly processed by PCSK2 and CPE in α-cells, which do not express PCSK1 (Friis-Hansen et al., 2001; Orskov et al., 1987) and which are spared destruction in T1D. To address a mechanistic explanation for the presence of INS mRNA and proinsulin protein in contrast to low levels of mature insulin and essentially undetectable C-peptide in T1D pancreata (Figure 2A–C and I), we addressed whether inflammation might contribute to defective proinsulin processing in T1D. This action was taken given previous in vitro reports that proinflammatory cytokines inhibit the protein levels of PCSK1 and PCSK2 (Hostens et al., 1999). Specifically, we measured IFN-γ, IL-1β, and TNFα expression in the pancreas by real time qPCR (Supplemental Figure 4). IFN-γ was undetectable in both groups whereas IL-1β levels were similar (Supplemental Figure 4A). mRNA levels for TNFα were undetectable in approximately 30% of T1D and 50% of control samples (Supplemental Figure 4B), precluding comprehensive statistical analysis for this cytokine. Hence, while reducing the potential that proinflammatory cytokines underscore our mRNA based observations, we believe additional studies addressing this issue are warrented moving forward.

We also evaluated the expression levels of three prohormone convertases/proteases in control and T1D pancreata. PCSK1 mRNA expression was reduced among T1D pancreata compared to controls (Figure 3P, p<0.01). In contrast, PCSK2 and CPE were expressed at similar levels in controls and T1D tissues (Figure 3Q,R). Indeed, the lower levels of PCSK1 in T1D pancreata are supportive of the reduction/absence of insulin and C-peptide. Furthermore, the detection of low levels of insulin in some pancreata may reflect the gradual but slow maturation from any residual PCSK1 activity in T1D pancreata.

Conclusions

Herein, in aggregate, we report the persistence of proinsulin protein and INS mRNA within the pancreata of individuals with varying durations of T1D, including those with long duration disease. Evidence for this includes direct staining of proinsulin by immunolocalization and IHC (Figures 1 and 3A-E), measurable extracts of proinsulin by ELISA (Figure 2B), ISH staining (Figure 3F–J) and qPCR analysis of INS mRNA (Figure 2I) in β-cells within islets as well as in scattered single cells within the exocrine regions of T1D pancreata. Furthermore, cell counts demonstrate the remarkable heterogeneity and persisitence of single insulin positive cells and small clusters in T1D pancreata. While insulin levels were significantly lower and C-peptide as well as islet amyloid polypeptide essentially undetectable in T1D pancreata compared with no-diabetes controls, proinsulin and INS mRNA were detectable in virtually every T1D subject. The novel finding regarding complete absence of INS hnRNA and the INS-IGF2 mRNA in many T1D pancreata, given that both messages originate from the insulin promoter, demonstrates that the insulin gene is most likely silent in T1D pancreata, whilst the presence of INS mRNA is suggestive of the persistence of long-lived message (Evans-Molina et al., 2007; Lee and Gorospe, 2010; Welsh et al., 1985).

The significant correlation between proinsulin and insulin concentrations in extracts from the control pancreata (Figure 2F) further implies that these assays are truly measuring the cognate analytes. While proinsulin is synthesized, it is likely not undergoing approapriate processing into mature insulin and C-peptide in the majority of T1D pancreata (Figure 2H). The presence of proinsulin (Figure 2B) coupled with the low levels of insulin along with the near absence of C-peptide and islet amyloid polypeptide (Figure 2A,C, and D) further supports the notion that the molecular machinery for processing the full-length proinsulin protein may be disrupted. In addition, our data demonstrating a significant reduction in the levels of PCSK1 mRNA in T1D pancreata also provide a strong argument for the observed persistence of proinsulin. Alternatively, our findings could also be interpreted as supporting the notion that proinsulin (along with islet amyloid polypeptide) is so rapidly secreted that there is insufficient time for it to be retained in residual β-cells for full processing to insulin. This concept has been previously associated with type 2 diabetes (Alarcon et al., 2016).

In sum, these data indicate that proinsulin is present in the pancreas in individuals with longstanding T1D; whether this is occurring in dedifferentiated β-cells (Talchai et al., 2012) or in cells attempting to become β-cells cannot be concluded from this data. The absence of insulin hnRNA and INS-IGF2 mRNA is suggestive of the insulin gene not being actively transcribed (Evans-Molina et al., 2007; Lee and Gorospe, 2010; Welsh et al., 1985), potentially as a means to avoid autoimmune killing (Rui et al., 2017). Therefore, there is a clear need for further studies of the mechanisms underlying long-lasting INS mRNA and proinsulin protein expression despite low to no INS promoter activity. Future analysis of the lineage of the cells expressing proinsulin/insulin in longstanding T1D pancreata and the molecular machinery involved in processing the full-length protein into insulin and C-peptide is warranted. Indeed, it is our belief that these observations are important for expanding our understanding of the pancreas and β-cell biology in the context of T1D.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author Mark A Atkinson ( atkinson@ufl.edu)

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Donors and Sample Processing

The JDRF nPOD program (www.jdrfnpod.com) recovered transplant-quality pancreata from organ donors with T1D as previously described (Campbell-Thompson et al., 2012b). All procedures were approved by the University of Florida Institutional Review Board (201400486) and the United Network for Organ Sharing (UNOS) according to federal guidelines, with informed consent obtained from each donor’s legal representative. For each donor, a medical chart review was performed in addition to assays for T1D associated Ab (Wasserfall et al., 2016) (Table S2) and C-peptide (Campbell-Thompson et al., 2012a), with T1D diagnosed according to the guidelines established by the ADA. Demographic data, hospitalization duration, and organ transport time were obtained from hospital records or UNOS. Pancreata were recovered, placed in transport media on ice, and shipped via organ courier to the University of Florida where tissues were processed by a licensed Pathology Assistant as previously described (Campbell-Thompson et al., 2012b).

METHOD DETAILS

Immunoflourescence

Sections (4μm) from formalin fixed paraffin embedded samples were evaluated for immunolocalization (Table S1). Immunolocalization was performed after deparaffinization and rehydration, blocking, and incubation with primary antibodies to proinsulin (GS-9A8, Developmental Studies Hybridoma Bank, Iowa City, IA), insulin (A056401, Dako, Carpinteria CA), and glucagon (A056501, Dako). Detection of primary antibody binding was performed by multiplex staining using polymer horseradish peroxidase (HRP, ARH1001EA, PerkinElmer, Waltham MA) followed by tyramine amplification with fluorophores 520, 570, and 670 with DAPI counterstain (OPAL 4-color, NEL94001KT, PerkinElmer). Slides were mounted and sections visualized using a Zeiss AxioskopPlus microscope.

Acid-Ethanol Extraction of Pancreatic Proteins

This method was adapted from a method developed for extraction of whole rodent pancreas (Andersson and Sandler, 2001). It is not feasible to perform such a procedure on whole human pancreas, but from initial experimentation with control tissue, we determined 150μm cryosections of pancreas (3×50μm) collected into cryotubes to be suitable. 1mL of acid-ethanol (1.5% HCl in 70% ethanol) was added to each cryotube, and this was incubated overnight at −20°C. The following day the tissue was homogenized using a dounce and again incubated overnight at −20°C. The following day the tubes were centrifuged at 800 × g and the resulting supernatant neutralized 1:1 with 1M Tris pH 7.5 for use in subsequent Bradford, ELISA, and Luminex assays (described below).

Proinsulin, Insulin, C-peptide, Islet Amyloid Polypeptide and Glucagon by ELISA and Luminex

Commercially available kits from ALPCO (Salem, NH) were utilized to measure total proinsulin, insulin and C-peptide from pancreas protein extracts as indicated in Table S1. The proinsulin, insulin and C-peptide assays are specific and do not cross react with each other. Glucagon was measured with a commercial ELISA assay provided by Mercodia (Winston Salem, NC). Islet amyloid polypeptide was measured by an assay from Millipore (Billerica, MA) using magnetic bead technology (Luminex). A total protein Bradford assay from Thermo-Fisher (Waltham, MA) was performed on each supernatant and used to normalize mass and extraction efficiency.

Immunohistochemistry

Sections (4μm) from formalin fixed paraffin embedded pancreas tissues were deparaffinized and rehydrated with serial passage through changes of xylene and graded ethanol. All slides were subjected to heat induced antigen retrieval in Target Retrieval Solution (Dako). The tissue sections were stained for insulin as a part of routine collection protocol for nPOD tissues (previously described (Campbell-Thompson et al., 2016; Campbell-Thompson et al., 2012b)) or double stained for insulin (polyclonal guinea pig anti-insulin,1:1000 dilution, Dako (Santa Clara, CA)) and glucagon (monoclonal mouse anti-glucagon, 1:5000 dilution, Abcam, Cambridge, MA) by immunohistochemistry (IHC), scanned using an Aperio CS Scanscope (Leica/Aperio, Vista, CA), and stored in the nPOD online digital pathology database (eSLIDE version 12, Leica/Aperio). For donors indicated in Table S1, scanned images of insulin-stained slides available from the block(s) nearest to the tissues used for total protein extraction (described above) were evaluated for fractional insulin area using Indica Labs, Inc image analysis software (Corrales, NM). For insulin and glucagon double stained slides, antigen-antibody binding was visualized using the EnVision G/2 Doublestain System (Dako). For control (n=5) and T1D subjects (n=11), glucagon + insulin positive islets, glucagon positive islets, insulin positive single cells, and clusters of two to five insulin positive cells were annotated by hand using the Aperio viewing platform and analyzed using Image Scope, Leica Biosystems analysis software (Version 12.1.0.5029, Buffalo Grove, IL).

RNA Extraction

Tissue from pancreas was flash frozen in RNAlater (Qiagen, Valencia, CA) an average of 16h from cross clamp. Total RNA was isolated following homogenization in Qiagen RNeasy Plus Mini Kit isolation buffer as per the manufacturer’s instructions including treatment with DNase 1. RNA concentrations were determined using a Nanodrop 2000C (Thermo Scientific, Waltham, MA) and when necessary, integrity was verified by visualization of ribosomal RNA by gel electrophoresis and ethidium bromide staining.

Primer Design

We utilized the public Primer-Blast software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (Ye et al., 2012)) which incorporates the Primer3 program (Rozen and Skaletsky, 2000) for primer design, genome-wide BLAST analysis along with the Needleman-Wunsch (NW) global alignment algorithm (Needleman and Wunsch, 1970) to identify internal homology between primers and any unwanted targets in the human genome and to satisfy the requirements for primer specificity compared to both the human transcriptome and genome. Primers were designed as exonic primers spanning an intron when possible, with an optimal Tm of 60 °C. For hnRNA studies, RNA (free of genomic DNA) was isolated from frozen human pancreatic tissue and evaluated by real time qPCR using an exon derived primer and a primer in the adjacent intron to evaluate hnRNA levels, thus evaluating promoter activity. Human gene symbols, based on HUGO were used throughout this study with primer sequences and the NCBI accession numbers provided in Table S3.

RT-qPCR

All samples were confirmed to be free of DNA contamination in no reverse transcriptase controls with an exon/intron primer pair. cDNA was produced with SuperScript II (Invitrogen, Carlsbad, CA) using oligo dT priming and subsequently utilized for real time quantitative PCR (RT-qPCR) using Thermo Luminaris Color HiGreen Fluorescein qPCR Master Mix (Thermo Scientific). 0.5–1 μg of total RNA was used for each 20 μl cDNA reaction which was then diluted to 200 μl and 2 μl of diluted cDNA employed for each 25 μl RT-qPCR reaction containing 600 nM of each primer pair. Individual RT-qPCR reactions were carried out in duplicate in a Biorad MyiQ. Un-normalized Cq values were displayed in all data since many of the genes displayed no detectable amplification (ND) from the T1D pancreata, thus precluding the use of normalization factors and ultimately statistical analysis. We have however extensive data on multiple reference genes from these same patient samples verifying total RNA integrity and lack of genomic DNA contamination. Regarding each gene primer set, we obtain proper amplification from the no diabetes controls with total reproducibility. For each donor, the specific genes examined by RT-qPCR are indicated in Table S1.

In Situ Hybridization

Sections (4μm) from fresh frozen pancreas samples were evaluated for in situ hybridization (ISH) as indicated in Table S1. ISH was performed using the RNAscope 2.0 High Definition kit (Advanced Cell Diagnostics (ACD), Hayward, California, USA) according to manufacturer’s instructions. A human insulin probe (ACD catalogue number 313571, NCBI reference sequence NM_000207.2) was hybridized to sections followed by alkaline phosphatase detection with Fast Red chromogen. Slides were mounted and scanned using an Aperio CS Scanscope (Leica/Aperio, Vista, CA) with image files stored in an online pathology database (eSLIDE version 12, Leica/Aperio).

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical parameters including exact n, the definition of center, dispersion and precision measures (mean and 95% confidence intervals or median and interquartile range) are reported in the Figures and Figure Legends. Data were judged to be statistically significant when p < 0.05 by either Kruskal-Wallis with Dunn’s post-test for for multiple comparisons or unpaired, two-tailed Mann-Whitney test for comparison of two groups, and the Spearman correlation with linear regression as indicated in Figures and Figure Legends. Data were analyzed and graphed using GraphPad Prism software version 6.02.

Supplementary Material

1
2

Highlights.

  • Proinsulin is detectable whereas insulin and C-peptide are reduced in T1D pancreas

  • Insulin mRNA is evident in T1D pancreas while insulin hnRNA is undetectable

  • PCSK1 is reduced in T1D pancreas, suggesting incomplete processing of proinsulin

  • Intra-islet insulin+ cells are lost with T1D evolution; single insulin+ cells remain

Acknowledgments

We would like to thank the organ donors and their families for the gift of tissues, without which these studies could not have been performed. Additionally, we are greatly appreciative of the immunolocalization staining performed by Dr. Ann Fu and technical assistance of Sean McGrail (University of Florida). The proinsulin monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. These efforts were supported by NIH AI42288 and UC4 DK108132 (M.A.), JDRF (M.A.), The Leona M. and Harry B. Helmsley Charitable Trust (M.A.), and the Brehm Coalition (M.A., P.A., C.R.). We thank Dr. Edward Phelps for his illustrative services. The graphical abstract was generated, in part, using an image derived from Servier Medical Art at smart.servier.com, available via a Creative Commons Attribution 3.0 Unported License.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes 4 figures and 3 tables and can be found with this article at xxxxx.

AUTHOR CONTRIBUTIONS

C.W. conceived of the study, researched the data, and wrote the manuscript, H.S.N. conceived of the study, researched the data, and reviewed/edited the manuscript, M.C.T. researched the data and reviewed/edited the manuscript, D.B. and M.B. researched the data and reviewed/edited the manuscript, L.H., I.K., and, A.P., C.R., E.B. and P.A. contributed to discussion and reviewed/edited the manuscript, M.A. conceived of the study and reviewed/edited the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS901211-supplement-1.pdf (6.2MB, pdf)
2
NIHMS901211-supplement-2.xlsx (46KB, xlsx)

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