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. 2015 Mar 20;10(3):e0121204.
doi: 10.1371/journal.pone.0121204. eCollection 2015.

The effects of exendin-4 treatment on graft failure: an animal study using a novel re-vascularized minimal human islet transplant model

Afaf Sahraoui  1 , Maria Sörhede Winzell  2 , Tracy Gorman  3 , Dave M Smith  2 , Stanko Skrtic  2 , Merete Hoeyem  1 , Shadab Abadpour  1 , Lars Johansson  4 , Olle Korsgren  5 , Aksel Foss  6 , Hanne Scholz  1
Affiliations

The effects of exendin-4 treatment on graft failure: an animal study using a novel re-vascularized minimal human islet transplant model

Afaf Sahraoui et al. PLoS One. .

Abstract

Islet transplantation has become a viable clinical treatment, but is still compromised by long-term graft failure. Exendin-4, a glucagon-like peptide 1 receptor agonist, has in clinical studies been shown to improve insulin secretion in islet transplanted patients. However, little is known about the effect of exendin-4 on other metabolic parameters. We therefore aimed to determine what influence exendin-4 would have on revascularized minimal human islet grafts in a state of graft failure in terms of glucose metabolism, body weight, lipid levels and graft survival. Introducing the bilateral, subcapsular islet transplantation model, we first transplanted diabetic mice with a murine graft under the left kidney capsule sufficient to restore normoglycemia. After a convalescent period, we performed a second transplantation under the right kidney capsule with a minimal human islet graft and allowed for a second recovery. We then performed a left-sided nephrectomy, and immediately started treatment with exendin-4 with a low (20μg/kg/day) or high (200μg/kg/day) dose, or saline subcutaneously twice daily for 15 days. Blood was sampled, blood glucose and body weight monitored. The transplanted human islet grafts were collected at study end point and analyzed. We found that exendin-4 exerts its effect on failing human islet grafts in a bell-shaped dose-response curve. Both doses of exendin-4 equally and significantly reduced blood glucose. Glucagon-like peptide 1 (GLP-1), C-peptide and pro-insulin were conversely increased. In the course of the treatment, body weight and cholesterol levels were not affected. However, immunohistochemistry revealed an increase in beta cell nuclei count and reduced TUNEL staining only in the group treated with a low dose of exendin-4 compared to the high dose and control. Collectively, these results suggest that exendin-4 has a potential rescue effect on failing, revascularized human islets in terms of lowering blood glucose, maintaining beta cell numbers, and improving metabolic parameters during hyperglycemic stress.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests to declare: Afaf Sahraoui, Merete Hoeyem, Shadab Abadpour, Aksel Foss, and Hanne Scholz have no financial interests in the funding organization. Neither do the authors hold any pending patents, nor are the authors involved in product development. The authors also do not promote any product distributed by AstraZeneca. Co-author Lars Johansson has an academic position with Uppsala University, Sweden, and is a paid employee of AstraZeneca. Co-authors Maria Sörhede Winzell, Tracy Gorman, Dave M. Smith, and Stanko Skrtic are also paid employees by AstraZeneca. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials

Figures

Fig 1
Fig 1. Time frame of experimental design.
In the double islet transplantation model, the first mouse islet graft is used to achieve normoglycemia in diabetic mice. The second minimal human islet graft is allowed a convalescent period before the first mouse graft is removed to induce hyperglycemia (red arrow), and treatment -/+ exendin-4 with a low (20μg/kg/day) or high (200μg/kg/day) dose was initialized.
Fig 2
Fig 2. Exendin-4 treatment improves non-fasting blood glucose regardless of dose.
Mice were transplanted with a minimal human islet graft (250 islets) and treated with or without a low (20μg/kg/day) or high (200μg/kg/day) dose of exendin-4 for 15 days while monitoring glycemic levels and body weight for saline treated controls (black line), and exendin-4 treated groups (20μg/kg/day (orange line) and 200μg/kg/day (green line)). (a) Random blood glucose levels over the first 12 days of treatment (b) with corresponding AUC representing the different groups. (c) Body weight during the first 12 days of treatment (d) with corresponding AUC calculated for each group. **p<0.01 by one-way ANOVA followed by Bonferroni correction vs. corresponding saline treated control, and values are expressed as mean ±SD.
Fig 3
Fig 3. Acute administration of exendin-4 improves the oral glucose tolerance test.
Mice transplanted with a minimal human islet graft (250 islets) were treated with or without exendin-4 (20 or 200μg/kg/day) for 15 days. At day 13 day of +/-exendin-4 treatment, an oral glucose tolerance test was performed as indicated in methods. The control group (black line) was compared to non-diabetic controls (pink line), and exendin-4 treated groups (20μg/kg/day (orange line), 200μg/kg/day (green line)). (a) Blood glucose levels during the oral glucose tolerance test with (b) AUC calculated per mice in the different groups. Results are expressed as mean ±SD for each column. **** p< 0.0001 by one-way ANOVA followed by Bonferroni correction vs. saline treated control.
Fig 4
Fig 4. Exendin-4 treatment increases C-peptide and pro-insulin secretion in mice transplanted with minimal human islet grafts.
Circulating levels of fasting blood glucose (a) and C-peptide (b) after 15 days of treatment -/+ exendin-4 (20 or 200μg/kg/day). (c) Ratio of fasting C-peptide to fasting blood glucose at day 15 of treatment was calculated for each mouse. (d) Pro-insulin was measured as indicated in methods. Values for each column are expressed as mean ±SD. * p<0, 05, ** p <0, 01, *** p< 0, 001 by one-way ANOVA followed by Bonferroni correction vs. saline treated control.
Fig 5
Fig 5. Exendin-4 treatment improves endogenous GLP-1 production, reduces glucagon release and improves triglyceride profile.
Blood sampling was performed on day 7 and 15 of treatment -/+ exendin-4 with a low (20μg/kg/day) or high (200μg/kg/day) dose. Analysis of selected metabolic parameters was performed and the difference (∆) between day 7 and 15 was calculated for each mouse as indicated in methods for (a) GLP-1, (b) glucagon, (c) triglycerides and (d) total cholesterol. Results are expressed as mean ±SD. * p<0, 05, ** p<0, 01, *** p<0, 001 by one-way ANOVA followed by Bonferroni correction vs. saline treated control.
Fig 6
Fig 6. Exendin-4 treatment increases transplanted human beta cell mass, and reduces apoptosis.
After 15 days of treatment -/+ exendin-4 treatment with a low (20μg/kg/day) or high (200μg/kg/day) dose, the cell nuclei in the grafts were counted (a). The ratio of TUNEL positive cells to insulin was calculated in the transplanted grafts after 15 days of exendin-4 treatment. Immunofluorescence staining was performed as indicated in methods, and representative images (c) show DAPI (blue), insulin (red) and TUNEL (green) staining in transplanted human islets treated with a low (20μg/kg/day) or high (200μg/kg/day) dose of exendin-4, or saline (control). The right-hand panel (I-III) shows TUNEL staining indicated by the arrows. The results for each column are expressed as mean ±SD. **** p<0, 0001 by one-way ANOVA followed by Bonferroni correction vs. saline treated control.
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Grants and funding

This study was funded through a research program established at Oslo University Hospital (OUS) supported by AstraZeneca in a scientific collaboration (OUS project number 37813). AS and HS received financial support from this program funding. MH has no relevant disclosures. The funder provided support in the form of salaries for authors MSW, TG, DMS, SS and LJ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section.