Diabetes Volume 69, July 2020 1451 Revisiting Proinsulin Processing: Evidence That Human b-Cells Process Proinsulin With Prohormone Convertase (PC) 1/3 but Not PC2 Adam Ramzy,1 Ali Asadi,1 and Timothy J. Kieffer1,2 Diabetes 2020;69:1451–1462 | https://doi.org/10.2337/db19-0276 Insulin is first produced in pancreatic b-cells as the B-chain and COOH-terminal A-chain with a connecting precursor prohormone proinsulin. Defective proinsulin C-peptide, many studies have attempted to clarify how processing has been implicated in the pathogenesis of b-cells excise the C-peptide to liberate mature insulin. both type 1 and type 2 diabetes. Though there is sub- Current theory posits that the B-chain–C-peptide junction stantial evidence that mouse b-cells process proinsulin is cleaved by prohormone convertase 1/3 (PC1/3; gene using prohormone convertase 1/3 (PC1/3) and then pro- PCSK1) before cleavage at the C-peptide–A-chain junction fi hormone convertase 2 (PC2), this nding has not been by prohormone convertase 2 (PC2; gene PCSK2). PC2 fi b fl veri ed in human -cells. Immuno uorescence with val- knockout mice have impaired processing at the C-A ISLET STUDIES idated antibodies revealed that there was no detectable junction, resulting in a buildup of des-31,32 proinsulin (2), PC2 immunoreactivity in human b-cells and little PCSK2 and PC1/3 knockout mice have severely impaired process- mRNA by in situ hybridization. Similarly, rat b-cells were ing at the B-C junction, resulting in a buildup of des-64,65 not immunoreactive for PC2. In all histological experi- proinsulin (3). Based on relative processing rates of intact ments, PC2 immunoreactivity in neighboring a-cells acted as a positive control. In donors with type 2 diabe- human proinsulin versus des-31,32 proinsulin or des-64,65 tes, b-cells had elevated PC2 immunoreactivity, sug- proinsulin by rat PC1/3 and PC2, processing at the B-C gesting that aberrant PC2 expression may contribute junction by PC1/3 likely occurs before processing by PC2 at to impaired proinsulin processing in b-cells of patients the C-A junction (4), but some data have countered this with diabetes. To support histological findings using hypothesis by showing more buildup of des-64,65 proinsu- a biochemical approach, human islets were used lin during the processing of rat insulin 2 in islets (5). for pulse-chase experiments. Despite inhibition of PC2 While past work supports the theory that primary function by temperature blockade, brefeldin A, chloro- mouse b-cells process proinsulin sequentially by PC1/3 quine, and multiple inhibitors that blocked production of and then PC2, there is some indication that PC2 may not mature glucagon from proglucagon, b-cells retained the be as important in human b-cells as it is in mouse b-cells. ability to produce mature insulin. Conversely, suppression Humans with mutant PCSK1 have circulating hyperproin- of PC1/3 blocked processing of proinsulin but not proglu- sulinemia (6), but there are no associations between PCSK2 cagon. By demonstrating that healthy human b-cells pro- polymorphisms and circulating proinsulin (7). Though cess proinsulin by PC1/3 but not PC2, we suggest that a human b-cell line (EndoC-bH2) has abundant PCSK1 and there is a need to revise the long-standing theory of pro- PCSK2 (8), RNA-sequencing experiments on sorted pri- insulin processing. mary a-cells and b-cells indicate higher expression of PCSK2 than PCSK1 in mouse b-cells (9), whereas human In 1967, Steiner et al. (1) demonstrated with pulse-chase b-cells expressed ;20 times more PCSK1 than PCSK2 (10) experiments that insulin is generated from a larger pre- (Supplementary Fig. 1). Additionally, Davalli et al. (11) cursor they named “proinsulin.” After the general structure reported a deficiency of immunoreactive PC2 in human islet fi b of insulin was identi ed as containing an NH2-terminal -cells transplanted into mice and some human insulinomas 1Laboratory of Molecular and Cellular Medicine, Department of Cellular and This article contains supplementary material online at https://doi.org/10.2337/ Physiological Sciences, Life Sciences Institute, The University of British Columbia, db20-4567/suppl.12074721. Vancouver, British Columbia, Canada © 2020 by the American Diabetes Association. Readers may use this article as 2 Department of Surgery, The University of British Columbia, Vancouver, British long as the work is properly cited, the use is educational and not for profit, and the Columbia, Canada work is not altered. More information is available at https://www.diabetesjournals Corresponding author: Timothy J. Kieffer, [email protected] .org/content/license. Received 13 March 2019 and accepted 3 April 2020 1452 Human b-Cells Process Proinsulin With PC1/3 Diabetes Volume 69, July 2020 are not immunoreactive for PC2 (12). Collectively, there is no Immunohistofluorescence definitive evidence that proinsulin is processed by both PC1/3 Immunofluorescent staining was performed as previously and PC2 in human b-cells,andthereissomeindicationthat described (21). Briefly, sections were deparaffinized in PC2, while critical for proglucagon processing in a-cells xylene (three times for 5 min) and rehydrated in graded (13), is not abundantly expressed in human b-cells. ethanol (100%, two times for 5 min, 95% for 5 min, 70% In this study, we use validated antibodies and oligonu- for 5 min, and PBS for 10 min) before heat-induced epitope cleotide probes to determine that primary human b-cells retrieval in an EZ-Retriever microwave oven (BioGenex, Fre- have no detectable PC2 and little detectable PCSK2.We mont, CA) for 15 min at 95°C in 10 mmol/L citrate buffer also performed pulse-chase experiments and suppressed (0.5% Tween 20, pH 6.0) (Thermo Fisher Scientific, Waltham, the function of PC2 by temperature blockade (14), bre- MA). Samples were blocked in Dako Protein Block, Serum Free feldin A (15,16), the weak base chloroquine (14), and (Dako Canada, Burlington, Ontario Canada), and incubated multiple inhibitors (17,18). In the absence of full PC2 overnight in primary antibody diluted in Dako Antibody function, human b-cells can produce mature insulin, but Diluent. The following day, slides were washed and incubated neighboring a-cells produce little mature glucagon. in secondary antibody (Alexa Fluor–conjugated secondary Moreover, we provide evidence that PC1/3 is responsible antibodies; Life Technologies) for 1 h at room temperature for processing human proinsulin by inhibiting the function before mounting and counterstaining with VECTASHIELD of PC1/3 using two inhibitors (18,19) to impair formation HardSet Mounting Medium with nuclear stain DAPI (Vector of mature insulin with no significant effect on proglucagon Laboratories, Burlingame, CA). All images were captured processing. These findings provide a more advanced un- and analyzed with an ImageXpress Micro XLS System derstanding of the processing of proinsulin and suggest the (Molecular Devices, LLC, San Jose, CA) with a scientific need to reconsider the widely accepted thought that human CMOS camera, a Nikon 20X Plan Apo objective (numer- proinsulin is processed sequentially by PC1/3 and then PC2. ical aperture of 0.75, 1-6300-0196; Nikon, Tokyo, Japan), Our findings suggest that in human b-cells, PC1/3 is re- and DAPI (DAPI-5060B), FITC (FITC-3540B), Cy3 (Cy3– sponsible for processing human proinsulin without PC2. 4040B), Texas Red (TXRED-4040B), and Cy5 (Cy5– 4040A) filter cubes. Image analysis was performed on RESEARCH DESIGN AND METHODS MetaXpress software (version 6.2.3.733; Molecular Devi- Experimental Models and Subject Details ces, LLC). HumanpancreastissuebiopsieswerecollectedbytheIke Barber Human Islet Transplant Laboratory (Vancouver, British Cell Sorting Columbia, Canada). Human pancreas tissue biopsies and A total of 10,000–20,000 human islet equivalents were cadaveric human islets were provided by the Alberta Diabetes dispersed in 0.05% trypsin before magnetic bead purifica- Institute IsletCore (Edmonton, Alberta, Canada) after isolation tion according to a published protocol (22). by standardized protocol (20). Sample collection and islet Western Blotting isolation were approved by the Human Research Ethics Board Groups of 250 mouse islets, 1,000 human islet equivalents, at the University of Alberta (Pro00013094). All donors’ fam- or ;1 million EndoC-bH1 cells were lysed in 200 mL lysis ilies gave informed consent for the use of pancreatic tissue in buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.02% research, and all work with human tissues was approved by the Na azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium Research Ethics Board (H14-02949), The University of British deoxycholate, 1 mmol/L phenylmethyl sulfonyl fluoride, Columbia (Vancouver, British Columbia, Canada). Basic donor and protease inhibitor cocktail [Sigma-Aldrich, St. Louis, demographics are detailed in Supplementary Tables 2 and 3. MO]) as per a published protocol (15). Levels of proteins of All experiments with animals were approved by the University interest were assessed by fluorescent Western blotting of British Columbia Animal Care Committee and carried out methods using two PC2 antibodies (1:1,000, MAB6018, in accordance with the Canadian Council on Animal Care R&D Systems; and 1:1,000, PA5-14594, Thermo Fisher Guidelines. Scientific), anti-PC1/3 antibody (1:2,500; gift from Lakshmi Devi), and an anti–a-tubulin antibody (1:1,000; Sigma- Paraffin-Embedded Samples Aldrich) and detected on a LI-COR Odyssey 9120 Imaging C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) system (LI-COR Biosciences, Lincoln, NE). were sacrificed at 12 weeks of age. Following euthanasia, the pancreas was quickly dissected out of mice, washed in PBS, and In Situ Hybridization fixed in 4% paraformaldehyde overnight before being trans- For all in situ hybridization experiments, we used tissue ferred to 70% ethanol for storage prior to paraffin embedding samples collected and sectioned in all RNase-free solu- and sectioning (5-mm thickness) (Wax-it Histology Services tions (including paraformaldehyde, 70% ethanol, and Inc., Vancouver, British Columbia, Canada).