Transplantation of pancreatic islets to adrenal gland is promoted by agonists of growth-hormone-releasing hormone

Abstract

Here, we evaluate an alternative approach of preconditioning pancreatic islets before transplantation using a potent agonist of growth-hormone-releasing hormone (GHRH) to promote islet viability and function, and we explore the adrenal gland as an alternative transplantation site for islet engraftment. The endocrine microenvironment of the adrenal represents a promising niche with the unique advantages of exceptional high oxygen tension and local anti-inflammatory and immunosuppressive properties. GHRH agonists have been shown to promote islet graft survival and function, which may help to reduce the islet mass necessary to reverse diabetes. In the present study, the most potent GHRH agonist MR403 was tested on insulinoma cells, isolated rat islets, and adrenal β-cell cocultures in vitro. GHRH receptor is expressed on both adrenal cells and islets. MR403 caused a significant increase in cell viability and proliferation and revealed an antiapoptotic effect on insulinoma cells. Viability of rat islets was increased after treatment with the agonist and in coculture with adrenal cells. Rat islets were transplanted into diabetic mice to the intraadrenal transplant site and compared with the classical transplants underneath the kidney capsule. Graft function and integration were tested by metabolic follow-up and immunohistochemical staining of intraadrenal grafts. A rapid decrease occurred in blood glucose levels in both models, and all animals reached normoglycemia within the first days after transplantation. Our studies demonstrated that the adrenal may be an attractive site for islet transplantation and that GHRH analogs might allow reduction of the islet mass needed to reverse a diabetic status.

Fig. 1.

Expression of GHRH receptor (GHRH-R) in isolated rat adrenal cells. RT-PCR analysis of GHRH-R in isolated rat adrenal cells. Rat pituitary was used as positive control, water was used as negative control, and β-actin was used as loading control.

Fig. 2.

In vitro effects of the GHRH agonist MR403 on INS-1 cells. (A) MR403 (10⁻⁸ M) significantly improved viability (15% compared with control) after 24 h in culture (n = 3). (B) Apoptosis as indicated by activity of caspases 3/7 was significantly reduced by 14% (10⁻⁸ M) or 18% (10⁻⁹ M) after treatment with MR403 for 24 h (n = 4). (C) MR403 significantly stimulated cell proliferation (16% versus control) after 24 h in culture (n = 3). Altogether, MR403 pretreatment showed maximum effect on all tested parameters at a concentration of 10⁻⁸ M. **P < 0.01; *P < 0.05.

Fig. 3.

In vitro effect of the GHRH agonist MR403 on islet viability on islets alone and on islets within the islet–adrenal coculture system (n = 4). Islets were cultured either alone or in coculture with adrenal cells and treated with MR403 (10⁻⁸ M) for 24 h. Addition of GHRH agonist alone to islets resulted in an increase of viability, and a significant improvement (56% compared with control) was seen in coculture condition and addition of the GHRH agonist. **P < 0.01 compared with islets alone.

Fig. 4.

Islet transplantation into the adrenal gland. Serial cryosections of retrieved islet-bearing adrenals were stained for insulin (green) to detect pancreatic islets within the adrenal tissue. Sections were costained with DAPI for visualization of cell nuclei (blue). Representative images are shown by bright field (Left) and fluorescent microscopy (Right).

Fig. 5.

Islet transplantation (300 islet equivalents) beneath the kidney capsule (standard model) or in the adrenal of streptozotocin-induced diabetic NOD-SCID mice. (A) Intraadrenal transplantation model: the left adrenal was carefully exposed (Left and Center), and islets concentrated to a minimal volume were injected through the upper pole of the gland (Right). (B) All animals of both groups showed a rapid and persistent recovery from diabetes upon transplantation. Retrieval of the graft-bearing kidney or adrenal gland, respectively, induced immediate recurrence of diabetes. (C) On day 10 following islet transplantation, animals were subjected to an i.p. glucose tolerance test. All animals, irrespective of the transplantation site, were able to revert initial hyperglycemia to normal ranges within 2 h.