Abstract
Insulin-degrading enzyme (IDE) functions in the catabolism of bioactive peptides. Established roles include degrading insulin and the amyloid beta peptide (Aβ), linking it to diabetes and Alzheimer’s disease. IDE is primarily located in the cytosol, and a longstanding question is how it gains access to its peptide substrates. Reports suggest that IDE secreted by an unconventional pathway participates in extracellular hydrolysis of insulin and Aβ. We find that IDE release from cultured HEK-293 or BV-2 cells represents only ~1% of total cellular IDE, far less than has been reported previously. Importantly, lactate dehydrogenase (LDH) and other cytosolic enzymes are released at the same relative level, indicating that extracellular IDE results from a loss of cell integrity, not secretion. Lovastatin increases IDE release from BV-2 cells as reported, but this release is mirrored by LDH release. Cell viability assays indicate lovastatin causes a loss of cell integrity, explaining its effect on IDE release. IDE is present in an exosome-enriched fraction from BV-2 cell conditioned media, however it represents only ~0.01% of the total cellular enzyme and is unlikely to be a significant source of IDE. These results call into question the secretion of IDE and its importance in extracellular peptide degradation.
Highlights
Insulin-degrading enzyme (IDE) is a zinc metallopeptidase that degrades a number of physiological peptides, the best documented in vivo substrates being insulin and amyloid β-peptide (Aβ)
Since lactate dehydrogenase (LDH) in the media of cultured cells is considered a marker for cell lysis, our results suggest that IDE found in conditioned media results from loss of cell integrity rather than specific secretion, at least in the cell lines studied
The result of this analysis showed a small amount of IDE detectable in the media over a 8 h incubation period, and this amount was essentially the same as the percentage of LDH found in the media (Fig. 1 and Supplementary Fig. S1)
Summary
Insulin-degrading enzyme (IDE) is a zinc metallopeptidase that degrades a number of physiological peptides, the best documented in vivo substrates being insulin and amyloid β-peptide (Aβ). There have been a number of studies reporting the secretion of IDE from cells[6,8,9,10,11,12,13] and this secreted form of IDE has been suggested to play an important role in degrading insulin and Aβ. The lack of inhibition of IDE secretion by brefeldin A and monensin was confirmed by Bulloj et al.[6] This led to the suggestion that IDE was secreted from cells via an unconventional mechanism, similar to that proposed for a number of other proteins[14]. To our surprise we could not confirm any specific secretion of IDE, finding that lactate dehydrogenase (LDH) and other cytosolic enzymes were released at the same relative levels as IDE. The lines show linear regression fits to the IDE values (solid line) or LDH values (dashed line)
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