Ramzy_Asadi_Kieffer_-_Online_appendix_-_09Mar2020.pdf (2.2 MB)

Revisiting proinsulin processing: Evidence that human β-cells process proinsulin with prohormone convertase (PC) 1/3 but not PC2

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posted on 14.04.2020 by Ada Admin, Adam Ramzy, Ali Asadi, Timothy J Kieffer
Insulin is first produced in pancreatic β-cells as the precursor prohormone proinsulin. Defective proinsulin processing has been implicated in the pathogenesis of both type 1 and type 2 diabetes. Though there is substantial evidence that mouse β-cells process proinsulin using prohormone convertase 1/3 (PC1/3) then prohormone convertase 2 (PC2), this finding has not been verified in human β-cells. Immunofluorescence with validated antibodies reveals that there was no detectable PC2 immunoreactivity in human β-cells and little PCSK2 mRNA by in situ hybridization. Similarly, rat β-cells were not immunoreactive for PC2. In all histological experiments, PC2 immunoreactivity in neighbouring α-cells acts as a positive control. In donors with type 2 diabetes, β-cells had elevated PC2 immunoreactivity, suggesting that aberrant PC2 expression may contribute to impaired proinsulin processing in β-cells of patients with diabetes. To support histological findings using a biochemical approach, human islets were used for pulse-chase experiments. Despite inhibition of PC2 function by temperature blockade, brefeldin-A, chloroquine, and multiple inhibitors that blocked production of mature glucagon from proglucagon, β-cells retained the ability to produce mature insulin. Conversely, suppression of PC1/3 blocked processing of proinsulin but not proglucagon. By demonstrating that healthy human β-cells process proinsulin by PC1/3 but not PC2 we suggest that there is a need to revise the longstanding theory of proinsulin processing.

Funding

This work was supported by grants from the Canadian Diabetes Association and the Canadian Institutes of Health Research (CIHR Foundation Scheme). A.R. gratefully acknowledges studentship support from the Canadian Institutes of Health Research (Vanier Canada Graduate Scholarship) and Vancouver Coastal Health (CIHR-UBC MD/PhD Studentship).

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