Isolation and Proteomics of the Insulin Secretory Granule

Abstract

Insulin, a vital hormone for glucose homeostasis is produced by pancreatic beta-cells and when secreted, stimulates the uptake and storage of glucose from the blood. In the pancreas, insulin is stored in vesicles termed insulin secretory granules (ISGs). In Type 2 diabetes (T2D), defects in insulin action results in peripheral insulin resistance and beta-cell compensation, ultimately leading to dysfunctional ISG production and secretion. ISGs are functionally dynamic and many proteins present either on the membrane or in the lumen of the ISG may modulate and affect different stages of ISG trafficking and secretion. Previously, studies have identified few ISG proteins and more recently, proteomics analyses of purified ISGs have uncovered potential novel ISG proteins. This review summarizes the proteins identified in the current ISG proteomes from rat insulinoma INS-1 and INS-1E cell lines. Here, we also discuss techniques of ISG isolation and purification, its challenges and potential future directions.

Keywords: beta-cells; granule protein purification; insulin secretory granule.

Figure 1

The insulin secretory pathway. (A) Proinsulin is synthesised and folded at the endoplasmic reticulum, trafficked to the trans-Golgi network and sorted into budding immature insulin secretory granules. (B) Immature insulin secretory granules undergo maturation where proinsulin is cleaved into mature insulin and condenses with zinc to form the dense core within the mature insulin secretory granule. (C) Aged mature insulin secretory granules that do not undergo secretion are trafficked for degradation within lysosomes or autophagosomes.

Figure 2

ISG membrane and cargo proteins of the immature and mature ISG. ISG proteins associated with the ‘sorting by entry’, ‘sorting by exit’ and ‘sorting by retention’ steps of ISG biogenesis and maturation.

Figure 3

Schematic of insulin granule isolation and purification techniques across the 4 ISG proteomes. Rat insulinoma cells in all ISG proteomic studies are lysed before ISG enrichment through various density gradients or immunoprecipitations. ISG are then purified prior to proteomics analysis by LC-MS/MS to obtain list of ISG proteins. Data collated from Brunner et al., 2007, Hickey et al., 2009, Schvartz et al., 2012 and Li et al., 2018.

Figure 4

Venn diagram of overlapping proteins identified across the 4 ISG proteomes. Total of 5 proteins identified (box) in all proteomic analyses. Data collated from Brunner et al., 2007, Hickey et al., 2009, Schvartz et al., 2012 and Li et al., 2018.