Misfolded proinsulin accumulates in expanded pre-Golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of Akita mice.

A missense mutation of the insulin 2 gene (Cys96Tyr) in Akita mice disrupting one of the two interchain disulfide bonds results in intracellular accumulation of misfolded proinsulin. We analyzed the secretory pathway of pancreatic beta cells by electron microscopy and morphometry and identified sites of proinsulin accumulation by quantitative immunogold electron microscopy in this protein-folding disease. In Akita mice beta cells, the volume density of dilated endoplasmic reticulum subdomains was increased by 2.9-fold, resulting in a 1.7-fold increased volume density of the entire rough endoplasmic reticulum. The volume density of pre-Golgi intermediates was increased by 4.9-fold, and that of the Golgi apparatus was increase by 3.4-fold. The relative labeling intensity for proinsulin was 2.1-fold higher in dilated endoplasmic reticulum subdomains and 2.9-fold higher in pre-Golgi intermediates as compared with narrow endoplasmic reticulum, resulting in a significantly different distribution pattern between Akita and control mice beta cells (Chi2= 29.97, P<0.001). The numerical density of insulin secretory granules was equal in Akita and control mice beta cells. However, their volume density and average volume were reduced to 20% and their average diameter to 58% in Akita mice. Together, these data demonstrate that misfolded proinsulin accumulates mainly in pre-Golgi intermediates and to a lesser extent in dilated endoplasmic reticulum subdomains, providing evidence for the importance of pre-Golgi intermediates in a protein folding disease.