Inside the Insulin Secretory Granule

Abstract

The pancreatic β-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy β-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing β-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.

Nutrient Regulation of Pancreatic Islet β-Cell Secretory Capacity and Insulin Production

“…Following oxidative protein folding in the ER, proinsulin is trafficked into the Golgi, where it is packaged with other regulated secretory cargo, such as the processing hormones, PC1/3, PC2, and CPE, into the budding secretory granule [ 7 , 83 ]. Although the process of protein cargo selection and sorting into the budding granule is not well understood, aggregation of the granule cargo in the late Golgi and/or trans -Golgi network (TGN) is considered a rate-limiting step in the sorting process [ 139 , 140 ].…”

“…Insulin molecules are assembled into a hexameric arrangement stabilized by two central Zn 2+ ions through interactions with histidine (B10) side chains ( Figure 1 C,D) [ 3 , 4 , 5 ]. While insulin comprises approximately 90% of the granule content, additional granule proteins are necessary to promote granule formation, maturation, and exocytosis [ 6 , 7 ]. These include the granin proteins, chromogranin A (CgA), chromogranin B (CgB), and VGF; the prohormone processing enzymes, prohormone convertases 1/3 and 2 (PC1/3 and PC2) and carboxypeptidase E (CPE); regulators of vesicle pH, including the vacuolar-(v)-ATPase pump and chloride channels; the zinc transporter, ZnT8; and soluble N-ethyl maleimide sensitive factor attachment protein receptor protein (SNARE) complexes containing vesicle-associated membrane protein-2 (VAMP2) and synaptotagmins 7 and 9.…”

Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking

“…In regards to the case of molecular aggregation, we propose here a study on two FP-tagged peptide markers of the insulin secretory granule (ISG) with similar MW transiently expressed in living INS-1E cells (an immortalized model of β-like cells), namely: proinsulin-GFP and proIAPP (Islet Amyloid Polypeptide)-Emerald ( Figure 4 ). These two peptides, according to the known structural organization of the ISG [ 32 ], are supposed to be localized in the same subgranule compartment, i.e., the luminal ‘halo’ which surrounds a dense core of crystallized insulin ( Figure 4 A). Proinsulin-GFP is processed by intragranular enzymes with the release of a GFP-tagged C-peptide (MW: 32.4 kDa); similarly, proIAPP-Emerald undergoes endo-proteolytic processing leading to the generation of Emerald-tagged propetide-2 (MW: 29 kDa) [ 32 ].…”

“…These two peptides, according to the known structural organization of the ISG [ 32 ], are supposed to be localized in the same subgranule compartment, i.e., the luminal ‘halo’ which surrounds a dense core of crystallized insulin ( Figure 4 A). Proinsulin-GFP is processed by intragranular enzymes with the release of a GFP-tagged C-peptide (MW: 32.4 kDa); similarly, proIAPP-Emerald undergoes endo-proteolytic processing leading to the generation of Emerald-tagged propetide-2 (MW: 29 kDa) [ 32 ]. INS-1E cells were transfected and imaged at 2 µs/pixel scan-speed to capture molecular diffusion ( Figure 4 B).…”

Islet Biology and Metabolism

“…Zooming in deeper within the beta-cell, we approach the subcellular compartments inside which insulin is synthesised, processed, and stored. These vesicles, termed insulin secretory granules, and the proteins involved in their formation, maturation, and secretion, are comprehensively discussed by Germanos et al [6] within this Special Issue. This article is further complemented by a review on the technical advances and limitations in the isolation of insulin secretory granules for analysis by Norri et al [7], which reflects particularly on knowledge gaps in the field with respect to insulin granule proteomics.…”