β-Cell Death and Mass in Syngeneically Transplanted Islets Exposed to Short- and Long-Term Hyperglycemia

Abstract

We studied the effects of hyperglycemia on ␤-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient ␤-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted ␤-cell mass (0.13 ؎ 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulintreated groups (group 1: 0.048 ؎ 0.002 mg; group 4: 0.046 ؎ 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased ␤-cell apoptosis (group 1: 0.30 ؎ 0.05%; group 4: 0.42 ؎ 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 ؎ 0.09%; group 5: 0.48 ؎ 0.08%) compared with normal pancreas (0.04 ؎ 0.03%; P < 0.001). In contrast, on day 30, ␤-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 ؎ 0.03%) but not in normoglycemic mice (group 6: 0.12 ؎ 0.02%); ␤-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 ؎ 0.02 mg; group 6: 0.102 ؎ 0.009 mg; P < 0.01). In summary, even in optimal conditions, ϳ60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to ␤-cell death. Increased apoptosis and reduced ␤-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted ␤-cells.

Adenoviral overproduction of interleukin-1 receptor antagonist increases beta cell replication and mass in syngeneically transplanted islets, and improves metabolic outcome

“…Beta cell apoptosis Sections were double-stained by immunoperoxidase for apoptotic nuclei using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) technique (In Situ Cell Death Detection Kit, ApopTag; Intergene, Oxford, UK) and by alkaline phosphatase for the endocrine non-beta cells of the islets [2]. A cocktail of antibodies (DakoCytomation) including rabbit anti-swine glucagon (final dilution 1:1,000), rabbit antihuman somatostatin (final dilution 1:1,000) and rabbit antihuman pancreatic polypeptide (final dilution 1:500) was used [2].…”

“…A cocktail of antibodies (DakoCytomation) including rabbit anti-swine glucagon (final dilution 1:1,000), rabbit antihuman somatostatin (final dilution 1:1,000) and rabbit antihuman pancreatic polypeptide (final dilution 1:500) was used [2]. We stained the endocrine non-beta cells instead of beta cells because the high glucose concentrations used in most of the experimental conditions are associated with beta cell degranulation and may result in weak insulin staining.…”

“…We stained the endocrine non-beta cells instead of beta cells because the high glucose concentrations used in most of the experimental conditions are associated with beta cell degranulation and may result in weak insulin staining. We have previously shown the validity of staining the endocrine non-beta cells to determine beta cell apoptosis [2]. When assessing apoptotic nuclei we excluded necrotic regions.…”

“…Sections were double stained with immunoperoxidase for BrdU using a cell proliferation kit (Amersham, Amersham, UK) with a modified protocol [2]; for endocrine non-beta cells of the islets the cocktail of antibodies described above was used. To validate this measurement, beta cell replication was also determined in sections stained for beta cells with an anti-insulin antibody.…”

“…In the initial days after transplantation islets are particularly vulnerable [1], and more than half of the islet tissue is lost due to increased beta cell apoptosis and necrosis [2]. This initial beta cell death occurs before immunological rejection or recurrence of autoimmunity, is found in syngeneic, allogeneic and xenogeneic transplants, increases the beta cell mass needed to achieve normoglycaemia after transplantation, and may contribute to the long-term failure of the graft [3].…”