Access Type

Open Access Dissertation

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Bhanu P. Jena

Abstract

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.

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