Inhibitory Effect of the α₁-Antitrypsin Pittsburgh Type-Mutant (α₁-PI_M/R) on Proinsulin Processing in the Regulated Secretory Pathway of the Pancreatic β-Cell Line MIN6

Abstract

To elucidate its effect on proinsulin processing, we introduced the expression of a Pittsburgh type-mutant, α₁-protease inhibitor _M/R (α₁-PI_M/R) and its chimera protein with growth hormone (GH) (GHα₁-PI_M/R) into MIN6 cells. In metabolic labeling and chasing experiments with [³H]-Leu and [³⁵S]-Met, proinsulin appeared in the medium during stimulatory secretion only from MIN6 clones expressing GHα₁-PI_M/R and, surprisingly, α₁-PI_M/R, but not from the clones of either the control or α₁-PI. The major part of α₁-PI_M/R was secreted through the constitutive pathway and about 10% of total secreted α₁-PI_M/R in the chase periods entered the regulated pathway. On the other hand, GHα₁-PI_M/R was mainly transported to the secretory granules and about 80% of the total secreted GHα₁-PI_M/R in the chase periods was secreted during stimulatory secretion. In the first 3 h chase periods without stimulation, only α₁-PI_M/R and no GHα₁-PI_M/R appeared in the medium, thus suggesting that α₁-PI_M/R might be transported through a constitutive-like pathway for those periods. The α₁-PI, which had no inhibitory effect on proinsulin processing, showed similar secretion pathways to those of α₁-PI_M/R. This implies that some part of α₁-PI_M/R and α₁-PI entered the regulated pathway, not due to any specific interaction between the processing endoproteases and serine protease inhibitors, but due to some type of passive transport in a nonselective manner. The inhibitory effect of α₁-PI_M/R in the regulated secretory pathway was slightly but clearly evident when it was expressed in MIN6 β-cells.