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熊本大

Beneficial Effect of Insulin Treatment on Islet Transplantation Outcomes in Akita Mice

<div><p>Islet transplantation is a promising potential therapy for patients with type 1 diabetes. The outcome of islet transplantation depends on the transplantation of a sufficient amount of β-cell mass. However, the initial loss of islets after transplantation is problematic. We hypothesized the hyperglycemic status of the recipient may negatively affect graft survival. Therefore, in the present study, we evaluated the effect of insulin treatment on islet transplantation involving a suboptimal amount of islets in Akita mice, which is a diabetes model mouse with an <i>Insulin 2</i> gene missense mutation. Fifty islets were transplanted under the left kidney capsule of the recipient mouse with or without insulin treatment. For insulin treatment, sustained-release insulin implants were implanted subcutaneously into recipient mice 2 weeks before transplantation and maintained for 4 weeks. Islet transplantation without insulin treatment did not reverse hyperglycemia. In contrast, the group that received transplants in combination with insulin treatment exhibited improved fasting blood glucose levels until 18 weeks after transplantation, even after insulin treatment was discontinued. The group that underwent islet transplantation in combination with insulin treatment had better glucose tolerance than the group that did not undergo insulin treatment. Insulin treatment improved graft survival from the acute phase (i.e., 1 day after transplantation) to the chronic phase (i.e., 18 weeks after transplantation). Islet apoptosis increased with increasing glucose concentration in the medium or blood in both the <i>in vitro</i> culture and <i>in vivo</i> transplantation experiments. Expression profile analysis of grafts indicated that genes related to immune response, chemotaxis, and inflammatory response were specifically upregulated when islets were transplanted into mice with hyperglycemia compared to those with normoglycemia. Thus, the results demonstrate that insulin treatment protects islets from the initial rapid loss that is usually observed after transplantation and positively affects the outcome of islet transplantation in Akita mice.</p></div

Upregulated genes (>5-fold) classified as ‘inflammatory response genes’.

<p>Upregulated genes (>5-fold) classified as ‘inflammatory response genes’.</p

Upregulated genes (>5-fold) classified as ‘chemotaxis genes’.

<p>Upregulated genes (>5-fold) classified as ‘chemotaxis genes’.</p

Upregulated genes (>5-fold) classified as ‘immune response genes’.

<p>Upregulated genes (>5-fold) classified as ‘immune response genes’.</p

Groups and protocol.

<p>(A) Schematic drawing of the experiment for evaluating the effect of insulin treatment on islet transplantation. Heterozygous male Akita mice (<i>Ins2</i>^WT/C96Y) were used as recipients. Small insulin implants were used for insulin treatment. Glucose monitoring was started from 5 weeks of age. In mice transplanted with 50 islets without insulin treatment (50-islet TP), islet transplantation was performed at 10–12 weeks of age. In mice transplanted with 50 islets in combination with insulin treatment (50-islet TP+INS Tx), insulin implants were inserted when the mice reached 8–10 weeks of age. After 2 weeks, islet transplantation was performed (10–12 weeks of age). Implants were removed 2 weeks after islet transplantation (12–14 weeks of age). Glucose monitoring was continued until 18 weeks after islet transplantation (28–30 weeks of age). (B) Numbers of mice in each group. (C) Images of 50 (left) or 200 (right) islets transplanted under the left kidney capsule of Akita mice. Images were obtained immediately after transplantation. Arrows indicate catheter insertion sites, and arrowheads indicate the outer boundary of the transplanted islets.</p

GO term analysis of genes upregulated >5-fold in hyperglycemic Akita mice compared to normoglycemic mice.

<p>GO term analysis of genes upregulated >5-fold in hyperglycemic Akita mice compared to normoglycemic mice.</p

Insulin treatment improved islet transplantation outcomes.

<p>(A–C) Fasting blood glucose levels of Akita (<i>Ins2</i>^WT/C96Y, untreated) mice (broken line with open triangles), the INS Tx group (solid line with crosses), and C57BL/6 WT mice (solid line with closed triangles). (A) Insulin implants were inserted at week −2 and removed at week 2. There were significant differences between the INS-treated and INS-untreated groups from 2 to 14 weeks (*<i>p</i><0.05, ANOVA). (B) Fasting blood glucose levels in the 200-islet TP group (broken line with open squares). Two hundred islets were transplanted at week 0, and removed at week 13. (C) Fasting blood glucose levels of the 50-islet group (broken line with open circles) and 50-islet TP+INS Tx group (solid line with closed circles). Insulin treatment significantly lowered the blood glucose levels (**<i>p</i><0.01, ANOVA). (D) Average fasting blood glucose levels of individual mice in each group from the time of insulin implant removal (week 2) until graft removal. Each dot represents the mean values in 1 mouse. The 50-islet TP+INS Tx group had significantly lower average fasting blood glucose levels than the 50-islet TP group (*<i>p</i><0.05, two-tailed unpaired Student’s <i>t-</i>test).</p

Islet apoptosis increased with increasing glucose concentration.

<p>(A) Islets were cultured in low-glucose (100 mg/dL, left) and high-glucose (900 mg/dL, right) media for 24 hours. Lower panels show high-magnification images. (B) The caspase-3/7–positive area increased after a 24-hour exposure to various glucose concentrations from 0 to 600 mg/dL. (C) Quantitative analysis of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095451#pone-0095451-g005" target="_blank">Figure 5B:</a> media with glucose concentrations of 0 mg/dL (dotted line), 50 mg/dL (small broken line), 100 mg/dL (dot-broken line), 200 mg/dL (large broken line), 300 mg/dL (middle broken line), and 600 mg/dL (solid line) were used.</p