Open Access Dissertation
Date of Award
Bhanu P. Jena
The insulin secreting porosome is a supramolecular lipo-protein complex that
measures roughly 100 – 120 nm in diameter. Porosomes allow transient fusion of insulin
secretory granules to the cell plasma membrane and mediates partial release of secretory
contents. Post secretion, the secretory granule reseals and re-enters to the cell interior.
This is in contrast to the ‘total fusion’ phenomenon, where secretory vesicles completely
fuse at the cell plasma membrane and release all of the contents to the cell exterior. This
study involved a deeper understanding of the transient or ‘kiss-and-run’ mechanism of
cell secretion that involves the insulin secreting porosome complex. In addition to the
porosome, two other components of transient cell secretion, namely the t/v SNARE
complex and the insulin secreting granules (ISGs) were also studied. We demonstrated
for the very first time in the history of porosomes, its functional and stable reconstitution
into live insulin secreting mouse insulinoma cells leading to improved glucose stimulated
insulin releasing from the reconstituted cells. Further, we demonstrated a drop in
intracellular pH once a cell has been stimulated for secretion. This lowering of pH is critical
for locking in place, the t/v SNARE complex that are present at the base of the porosome.
We also demonstrated a loss in glucose stimulated insulin secretion upon prevention of
intracellular acidification utilizing Bafilomycin A, a pharmacological inhibitor of the
vacuolar proton pump (vH+ ATPase). The vH+ ATPase is also present on the insulin
secretory granule membrane, which led to our fourth aim of this study. Valproate, is an
FDA approved anticonvulsant that is widely used in the treatment of various neurological
disorders such as epilepsy and mood disorders by disturbing vH+ ATPase activity in the
neurons. Since, vH+ ATPase is also present on ISG membrane we wanted to understand
effects of valproate on insulin secretion. We demonstrated that valproate treatment
significantly reduces glucose stimulated insulin secretion. Additionally, we also
demonstrated that valproate leads to de-localization of one of the cytosolic subunits of
vH+ ATPase from the ISG membrane, preventing complete assembly of the proton pump.
These results coherently suggest the importance of porosomes in transient cell secretion
and its critical regulation via interaction with various proteins namely SNARE complex
and ISG membrane proteins that allows for cell secretion.
Naik, Akshata Ramesh, "Molecular Machinery For The ‘kiss And Run’ Mechanism Of Insulin Secretion" (2019). Wayne State University Dissertations. 2287.