PC2 and PC1/3 are Ca2+-dependent enzyme activities with an acidic pH 5 – 5.5 optimum . Fortunately, the β granule contains an intraorganellar environment of 1 – 10 mM free Ca2+ and acidic pH 5.5, which ideally suits the requirements for optimal PC2, PC1/3 and CP-H activities within this organelle. This also ensures that insulin is produced mainly in the intracellular β-granule compartment in which it is stored . To render PC2 and PC1/3 fully active for proinsulin processing in a newly formed β granule, it follows that activation of the proton-pumping ATPase and Ca2+-translocation proteins  are key regulatory events to control proinsulin conversion.
Both PC2 and PC1/3 are initially synthesized as preproprotein precursor molecules themselves, with the “pre” signal peptide region enabling translocation into the RER lumen during translation as with the signal peptide region of proinsulin (see earlier). ProPC2 and proPC1/3 are transported to β granules along with their proinsulin substrate in the β cell’s secretory pathway and undergo maturation beginning in the TGN . However, unlike proinsulin, proPC2 and proPC1/3 are thought to be accompanied by individual chaperon molecules, 7B2 and proSAAS, respectively, that specifically inhibit these endopeptidases’ activity . Proteolytic cleavage of 7B2 by PC2 in the TGN/immature β-granule compartment alleviates the inhibition on proPC2 promoting its maturation and activation to mature PC2. Indeed, 7B2 has an important role in controlling PC2 activity in vivo. The 7B2 knockout mouse has multiple neuroendocrine disorders, similar yet more severe than the PC2 null mouse . In contrast, the role of proSAAS in regulating proinsulin processing is doubtful, since proSAAS null mice have normal insulin production and proSAAS is not highly expressed in β cells [96,97].
As previously indicated, the biosynthesis of proPC2 and proPC1/3 is stimulated predominately at a translational level coordinately with that of proinsulin [88,91,92]. In the long term (>12h), glucose also regulates PC2 and PC1/3 gene transcription in parallel with the preproinsulin gene . Thus, it seems that proinsulin conversion is adaptable to changes in glucose by coordinate regulation of the endopeptidases that catalyze processing [91,92].
The mature β-granule storage pool
A mature β granule is retained from anywhere between a few hours to several days, awaiting transport to the β cell’s plasma membrane and exocytosis under stimulatory conditions, characteristic of a regulated secretory pathway [88,92] (Figure 6.4). It should be noted that under normal conditions, the storage compartment of insulin in mature β granules far exceeds the compartment undergoing transport/exocytosis, so that during a 1-h stimulation by glucose only ∼1 – 2% of the insulin content of a primary islet β cell is secreted . The insulin content of a β cell is kept at a relatively constant level under normal physiologic conditions where secreted insulin is rapidly replaced at the biosynthetic level. However, in the long term there is also an additional regulatory component that maintains insulin stores at optimal levels, via insulin degradation . The half-life of a β granule is several days, but if it is not used for exocytosis it is eventually degraded by fusion with lysosomal compartments by autophagy (also known previously as crinophagy) .
Dysfunctional proinsulin processing in diabetes
In type 2 diabetes where there is hyperinsulinemia to compensate for peripheral insulin resistance, an increased proportion of the secreted insulin is actually proinsulin or split proinsulin conversion intermediates (mostly des 31,32 proinsulin) so that it is also a hyperproinsulinemic state . It is possible that genetic defects in the proinsulin conversion enzyme genes or the insulin gene itself hamper proinsulin conversion, resulting in an increased proportion of proinsulin secreted. However, such genetic mutations are very rare, yet hyperproinsulinemia is a common trait of type 2 diabetes . As such, an increased proportion of secreted proinsulin likely occurs as a consequence of β-cell secretory dysfunction in type 2 diabetes .
In common obesity-linked type 2 diabetes there is chronic hyperglycemia and dyslipidemia [88,91,92]. As a consequence, the β cell is working very hard, with both proinsulin synthesis and insulin secretion are upregulated in an attempt to compensate for peripheral insulin resistance. Normally in β cells there is preferential exocytosis of newly formed β granules, but under such chronic stimulation from hyperglycemia/ hyperlipidemia newly synthesized proinsulin is not retained long enough to be fully converted to insulin and C-peptide, and as a consequence a greater proportion of proinsulin (as well as des 31,32 proinsulin) is secreted [88,91]. It should also be noted that chronic dyslipidemia adversely affects secretory capacity of β cells. Elevated fatty acid levels increase the amount of insulin secreted from the β cell, but in contrast, fatty acids modestly inhibit glucose-induced proinsulin biosynthesis, which in turn markedly decreases insulin content of islet β cells in vivo . A similar situation might also be envisaged with the prolonged use of sulfonylureas, which though potent inducers of insulin secretion, do not stimulate proinsulin synthesis and decrease insulin content , thus also reducing the insulin secretory capacity of the β cell. In general, the chronic hyperglycemia and dyslipidemia in obesity-linked type 2 diabetes are constantly making the β cell work harder to produce sufficient insulin to compensate for increased metabolic load and peripheral insulin resistance [91,92]. But in the long run this eventually leads to β-cell dysfunction of which the hyperproinsulinemia is symptomatic. Interestingly, if the β cell in type 2 diabetes patients is allowed to rest, the β-cell secretory dysfunction in vivo is reduced. This emphasizes the importance of protecting β-cell mass and function in the treatment of obesity-linked type 2 diabetes [88,91,92].
Work from our laboratories cited in this chapter was supported by the following grants: R01 DK-050610, R01 DK-055267, and the JDRF and Brehm Coalition (C.J. Rhodes); R01 DK-058096 and the Canada Research Chair in Diabetes and Pancreatic Beta-cell Function (V. Poitout), R01 DK 50203, R01 DK-055091 and R01 DK-042502 (R. Stein).