Acinar cell proteases reduce insulin granules of human islets in vitro and degrade insulin after transplantation: Preventive effect of Alpha-1 antitrypsin

G. Loganathan¹, R. Dawra², S. Pugazhenthi³, Z. Guo⁴, S. Soltani¹, A. Wiseman³, M. Sanders⁵, K. Papas¹, A. Saluja², D. Sutherland¹, B. Hering¹, A.N. Balamurugan^1
1 Schulze Diabetes Institute, Dept. of Surgery, University of Minnesota, Minneapolis, USA; ² Dept. of Surgery, Basic and Translational Research, Minneapolis, USA; ³ University of Colorado Denver, Aurora, USA; ⁴ Sanford Project, Sanford Health/University of South Dakota, Sioux Falls, USA; ⁵ University Imaging Centers, St.Paul, MN, USA

Objective: We previously reported the dysfunctional environment created by acinar cell proteases within impure human islet cultures which effectively degrade insulin in the culture medium. In this study, the effects of proteases on beta cell insulin granules were examined at the sub-cellular level by transmission electron microscopy (TEM) and the effect of transplanting acinar cells adjacent to islet graft.

Methods: Immunogold particles were used to detect insulin in the granules after exposure of human islets to various protease treatments (Fig H). The number of insulin granules and insulin-labeled particles were counted in 30 beta cells using TEM. For in vivo studies (n=15), pure hand-picked islets (2000 IEQ) were transplanted under the renal capsule of diabetic mice in the controls (Fig E) while experimental groups received equal amounts of acinar cells transplanted either with pure islets (Fig F), or separate but near to the pure islet grafts (Fig G).

Results: Figures A and B depict normal beta cell ultrastructure with normal amounts of insulin granules. Broken beta cell architecture and reduced numbers of insulin granules after protease cocktail treatment (Fig C and D). The average insulin granule count per beta cell after trypsin treatment was 39 compared to 112 insulin granules / beta cell in control islets (Fig H).Whereas immediate reversal of diabetes was achieved in all mice receiving pure islets and hyperglycemia was sustained in all mice receiving impure islets, a delayed (7-10 days) diabetic reversal was observed in all mice receiving separately transplanted islets and acinar cells indicating insulin degradation.

Conclusions: Our previous report indicated that A1AT supplementation to islet culture inhibits the protease effect of insulin degradation and reduced islet cell loss. A1AT treatment may inhibit the cellular degradation of beta cells and intracellular insulin granules.