We recently described the persistence of detectable serum proinsulin in a large majority
of individuals with longstanding type 1 diabetes (T1D), including individuals with
undetectable serum C-peptide. Here, we sought to further explore the mechanistic etiologies
of persistent proinsulin secretion in T1D at the level of the islet, using tissues
obtained from human donors. Immunostaining for proinsulin and insulin was performed
on human pancreatic sections from the Network for Pancreatic Organ Donors with Diabetes
(nPOD) collection (n = 24). Differential proinsulin processing enzyme expression was
analyzed using mass spectrometry analysis of human islets isolated from pancreatic
sections with laser capture microdissection (n = 6). Proinsulin processing enzyme
mRNA levels were assessed using quantitative real-time PCR in isolated human islets
(n = 10) treated with or without inflammatory cytokines. Compared to nondiabetic controls,
immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed
increased numbers of cells with proinsulin-enriched, insulin-poor staining. T1D donor
islets also exhibited increased proinsulin fluorescence intensity relative to insulin
fluorescence intensity. Laser capture microdissection followed by mass spectrometry
revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3
(PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of
human islets with inflammatory cytokines reduced mRNA expression of the processing
enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight
into altered proinsulin processing in long-duration T1D and suggest that reduced β
cell prohormone processing is associated with proinflammatory cytokine-induced reductions
in proinsulin processing enzyme expression.
Abbreviations
- CPE = carboxypeptidase E
- ER = endoplasmic reticulum
- IR = infrared
- LCM = laser capture microdissection
- LFQ = label-free quantification
- nPOD = network for pancreatic organ donors with type 1 diabetes
- PC1/3 = prohormone convertase 1/3
- PC2 = prohormone convertase 2
- T1D = type 1 diabetes
- UV = ultraviolet
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Article info
Publication history
Published online: August 09, 2019
Accepted:
August 5,
2019
Received in revised form:
July 12,
2019
Received:
March 19,
2019
Footnotes
Emily K. Sims, MD is an Assistant Professor of Pediatrics at the Indiana University School of Medicine. Dr. Sims presented part of this work at the 2018 annual meeting of the Central Society for Clinical and Translational Research. Dr. Sims is physician scientist with a long-term career goal of addressing clinically relevant problems in diabetes utilizing molecular discovery. Her research focus revolves around the investigation of molecular mechanisms contributing to β cell dysfunction and the development of diabetes, as well as the identification and verification of circulating biomarkers of β cell dysfunction. Dr. Sims's goal is to ultimately utilize this knowledge for development of biomarkers and β cell-targeted therapeutics that allow for a more tailored approach to treatment of patients with, or at-risk for, diabetes.
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