Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

TLDR

Proinsulin is a single polypeptide chain of insulin subunits linked by a C‑peptide, and its conversion to insulin during secretory vesicle maturation requires two proteases and is inhibited by ionophores that disrupt intracellular H⁺ gradients. The study aimed to test whether proinsulin conversion to insulin takes place in acidic secretory vesicles by incubating cultured rat islet cells with DAMP, a basic compound that accumulates in acidic compartments. The authors used indirect protein A‑gold colocalization with antibodies against DAMP and proinsulin to visualize the distribution of the basic compound and the prohormone within vesicles. DAMP labeling revealed that proinsulin‑containing immature vesicles are acidic, and that mature insulin‑rich vesicles are even more acidic, indicating that proinsulin conversion occurs in an acidic compartment that becomes progressively more acidic as vesicles mature and reaches a critical pH threshold.

Abstract

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.