title: Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.
abstract: BACKGROUND
  Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function.
  
  
  METHODS
  Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM).
  
  
  RESULTS
  Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% +/- 9% vs 47% +/- 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% +/- 2%) and the A1AT-treated groups (99% +/- 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE).
  
  
  CONCLUSIONS
  Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.
authors.affiliation: Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: G
authors.initials: G
authors.lastname: Loganathan
authors.forename: R K
authors.initials: RK
authors.lastname: Dawra
authors.forename: S
authors.initials: S
authors.lastname: Pugazhenthi
authors.forename: A C
authors.initials: AC
authors.lastname: Wiseman
authors.forename: M A
authors.initials: MA
authors.lastname: Sanders
authors.forename: A K
authors.initials: AK
authors.lastname: Saluja
authors.forename: D E R
authors.initials: DE
authors.lastname: Sutherland
authors.forename: B J
authors.initials: BJ
authors.lastname: Hering
authors.forename: A N
authors.initials: AN
authors.lastname: Balamurugan
mesh_terms: D002102:Cadaver; D018929:Cell Culture Techniques / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism; D006085:Graft Survival; D006801:Humans; D007328:Insulin / Q000302:isolation & purification / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D010196:Pancreatic Elastase / Q000378:metabolism; D014019:Tissue Donors; D014182:Transplantation, Autologous; D014184:Transplantation, Homologous; D014357:Trypsin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D007328:Insulin; D000515:alpha 1-Antitrypsin; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin