Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming.
Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets.
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California; komori@coh.org.
Keiko
K
Omori
Center for Development of Advanced Medical Technology and Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan.
Eiji
E
Kobayashi
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Hirotake
H
Komatsu
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Jeffrey
J
Rawson
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Garima
G
Agrawal
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Mounika
M
Parimi
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Alina R
AR
Oancea
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Luis
L
Valiente
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Kevin
K
Ferreri
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Ismail H
IH
Al-Abdullah
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Fouad
F
Kandeel
Division of Inflammation Research, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan; and.
Masafumi
M
Takahashi
Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope, Duarte, California;
Yoko
Y
Mullen
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D006801:Humans; D007515:Islets of Langerhans / Q000293:injuries* / Q000503:physiopathology*; D018384:Oxidative Stress / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D017694:Rewarming / Q000009:adverse effects*
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D011518:Proto-Oncogene Proteins
Bbc3/PUMA; cold preservation; islet death; islet transplantation; type 1 diabetes