Transplantation of betacellulin-transduced islets improves glucose intolerance in diabetic mice

Abstract

Type 1 diabetes is an autoimmune disease caused by permanent destruction of insulin-producing pancreatic β cells and requires lifelong exogenous insulin therapy. Recently, islet transplantation has been developed, and although there have been significant advances, this approach is not widely used clinically due to the poor survival rate of the engrafted islets. We hypothesized that improving survival of engrafted islets through ex vivo genetic engineering could be a novel strategy for successful islet transplantation. We transduced islets with adenoviruses expressing betacellulin, an epidermal growth factor receptor ligand, which promotes β-cell growth and differentiation, and transplanted these islets under the renal capsule of streptozotocin-induced diabetic mice. Transplantation with betacellulin-transduced islets resulted in prolonged normoglycemia and improved glucose tolerance compared with those of control virus-transduced islets. In addition, increased microvascular density was evident in the implanted islets, concomitant with increased endothelial von Willebrand factor immunoreactivity. Finally, cultured islets transduced with betacellulin displayed increased proliferation, reduced apoptosis and enhanced glucose-stimulated insulin secretion in the presence of cytokines. These experiments suggest that transplantation with betacellulin-transduced islets extends islet survival and preserves functional islet mass, leading to a therapeutic benefit in type 1 diabetes.

Figure 1

Betacellulin prevents cytokine-induced apoptosis in isolated islets. (a) Mouse islets were transduced with adenoviruses expressing betacellulin (Ad-BTC) or β-galactosidase (Ad-LacZ), and then treated with interleukin (IL)-1β (1 U ml⁻¹) and interferon-γ (100 U ml⁻¹) in the presence or absence of anti-BTC antibody (500 ng ml⁻¹). Following a 48-h incubation, the extent of apoptosis was determined by the amount of ApoPercentage dye accumulated in islets. (b) Islet extracts were prepared 24 h after cytokine treatment and the expression levels of apoptosis-related proteins were examined by western blot. Relative intensity showed the ratio of the expression compared with that of β-actin. (c) After 48 h of cytokine treatment, β-cell proliferation was measured by 5-bromo-2-deoxyuridine (BrdU) incorporation in islets transduced with either Ad-BTC or Ad-LacZ. (d) Islets were treated with recombinant human betacellulin (10 ng ml⁻¹) until the indicated time points and subjected to western blot analysis with anti-p-Akt or anti-Akt antibody. Each value represents the mean±s.e.m. (n=9). **P<0.01 vs untreated control; ^##P<0.01 vs Ad-LacZ.

Figure 2

Betacellulin preserves glucose-stimulated insulin secretion (GSIS) in isolated islets. (A) Betacellulin or β-galactosidase-transduced islets were treated with cytokines and then stained with hematoxylin–eosin (H&E) or immunostained with anti-insulin antibody. Representative stainings from four separate experiments are shown. (B) GSIS was quantified by enzyme-linked immunosorbent assay (ELISA) and normalized to total protein content. Each value represents the mean±s.e.m. (n=9). **P<0.01 vs untreated control; ^##P<0.01 vs Ad-LacZ.

Figure 3

Betacellulin transduction improves islet engraftment. Blood glucose levels were monitored in streptozotocin (STZ)-induced diabetic mice transplanted with betacellulin or β-galactosidase-transduced islets for 65 days. Each value represents the mean±s.e.m. (n=9). **P<0.01 vs STZ; ^#P<0.05, ^##P<0.01 vs adenoviruses expressing β-galactosidase (Ad-LacZ).

Figure 4

Betacellulin increases glucose-stimulated insulin secretion from transplanted islets. Thirty-five days after transplantation, diabetic mice were fasted overnight and injected intraperitoneally with 2 g kg⁻¹ glucose solution. (a) Blood glucose profiles and the corresponding area under the curve (AUC) values were measured during the glucose tolerance test. (b) Plasma insulin levels were determined under basal conditions. Each value represents the mean±s.e.m. (n=9–12). **P<0.01 vs untreated control; ^#P<0.05 vs adenoviruses expressing β-galactosidase (Ad-LacZ).

Figure 5

Betacellulin increases new blood vessel formation in transplanted islets. Fourteen (for betacellulin (BTC) staining) or 35 (for hematoxylin–eosin (H&E), insulin, and von Willebrand factor (vWF) staining) days after transplantation, diabetic mice were terminated and islet grafts were retrieved. (a) Paraffin-embedded islet grafts were sectioned and stained with H&E or immunostained with anti-insulin or anti-BTC antibody. (b) Paraffin-embedded islet grafts were immunostained with anti-vWF antibody. The black arrowheads indicate internal-positive blood vessels, the red arrowheads indicate newly formed blood vessels and the blue arrowheads indicate transplanted islets. Photomicrographs are representative of three independent experiments.