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.

Keywords: granin; granule; insulin; islet amyloid polypeptide (IAPP); pancreatic β-cell; secretory pathway; trans-Golgi network (TGN).

Figure 1

Overview of the β-cell secretory pathway. Following synthesis in the ER, proteins transit the Golgi apparatus to the TGN, and those destined for the regulated secretory pathway are sorted by entry into ISGs. This event relies on soluble protein aggregation which is under the control of Ca²⁺ and H⁺, and several proteins may also interact with membrane components of the TGN that are enriched in β-cell SGs. Some non-SG proteins can also slip into ISGs but are removed as a byproduct of the sorting by exit mechanism, which specifically escorts proteins from the maturing SG via receptor mediated recognition and vesicle budding. Concurrently, Zn²⁺, Ca²⁺, and H⁺ taken up by the maturing SG will bind to certain proteins to enhance their condensation and prevent their exit, in a process termed sorting by retention. While the free concentration of Ca²⁺ is in the micromolar range and decreases proximal to distal along the secretory pathway, the β-cell SG holds 50–100 mM Ca²⁺ bound to luminal proteins. Similarly, the total amount of Zn²⁺ bound to luminal proteins in the SG is in the range of 20–30 mM, although its free concentration is elevated in the distal secretory pathway relative to the proximal secretory pathway. Finally, the pH of the newly formed ISG can be estimated as similar to that of a constitutive vesicle (~5.7), but this will drop to 5.2 in the MSG. Notably, these values represent the free H⁺ concentration, but there exists no indication of the amount of H⁺ that is bound to luminal proteins.

Figure 2

Prohormone Processing in the β-cell. (A) Sequence of proinsulin processing. After entry into the ISG, proinsulin is converted to insulin and C-peptide via cleavage at two sites of dibasic amino acid residues. The 31–32 Arg-Arg site is located at the C-peptide/B-chain junction and the 64–65 Lys-Arg site is located between the C-peptide/A-chain junction. Cleavage at one dibasic site by endoprotease PC1/3 or PC2 produces the split proinsulin molecules, which precedes C-terminal trimming of exposed residues by exoprotease CPE to produce the des proinsulin molecules. One round of endo/exoprotease activity is followed by the same action at the other dibasic site. (B) Sequence of proIAPP processing. The C-terminal proregion of proIAPP is cleaved in the TGN prior to ISG entry. Next, in no particular order, within the maturing ISG the N-terminal proregion of proIAPP is removed and the exposed C-terminal glycine residue is amidated to produce IAPP. IAPP may then be further processed into smaller fragments by β-secretase 2. (C) Processing events and products during secretory granule maturation in the human β-cell SG. des 31,32 proinsulin is the major proinsulin intermediate in human β-cells and is elevated in the circulation of those with T2D along with proIAPP_1–48.

Figure 3

IAPP precursor/product amino acid sequence in human (H), mouse (M), and rat (R). The green glycine residue is amidated after the C-terminal cleavage site is processed. Red residues denote dibasic sites of endoproteolytic processing. Blue residues indicate a modified sequence between species at cleavage sites that could account for their differential specificity to PC enzymes.

Figure 4

Trafficking of the granins. Upon exposure to mild acidity (pH < 6.4) within the Golgi apparatus, the granins will bind Ca²⁺, which triggers their aggregation and interaction with other granin members. In the TGN, several of these members will interact with target molecules in the membrane to drive the formation of SGs at distinct sites from within the lumen.

Figure 5

Enzyme suppression and activation. The major processing enzymes of the insulin SG are synthesized as inactive precursors in the ER. Through mechanisms unique to each member, their activity (indicated in red) is suppressed during transit. Activation is principally driven by ionic changes; several enzymes require certain conditions for trafficking into the ISG and conformational activation, and all members require a low pH for optimal enzymatic activity. This will naturally play out through the TGN and the maturing SG as the luminal composition is modified.

Figure 6

Channels and transporters of the insulin SG. An array of transmembrane components controls the luminal composition of the insulin SG and transform the SG into a responsive store that is utilized by the beta cell for cytosolic signaling.

Figure 7

Granule refinement. The cation dependent mannose 6 phosphate receptor (M6PR) binds to certain proteins modified with a mannose 6 phosphate group (namely, procathepsin B) in the TGN, and this complex enters the ISG. During maturation, this complex will exit the SG via small transport vesicles and traffic to the endolysosomal system. A byproduct of this process may be the removal of soluble components in the general vicinity of the budding vesicle.