A Pre-Existent Hypoxic Gene Signature Predicts Impaired Islet Graft Function and Glucose Homeostasis

Abstract

A major obstacle to clinical islet transplantation is the poor function of islets after engraftment. Hypoxia has been suggested to be one critical insult contributing to the poor performance of islet grafts. However, in the context of islet transplantation it remains to be established if hypoxic exposure prior to the event of transplantation will result in impaired graft function. Genome transcript profiling analysis of eight independent human islet isolates with seven preparations of lazer dissected human beta cells from human pancreata revealed increased expression of characteristic hypoxia-response genes in isolated islets. Increased genes included LDHA, CITED2 and DDIT4 as well as PDK1, MAFF and SLC7A1. Therefore, isolated human islets exhibit a gene expression profile characteristic for that of cells responding to hypoxic stress and this gene signature is present prior to the event of transplantation. Next we tested how a pre-existent hypoxic gene signature would effect islet transplant outcomes. Hypoxia-pre-exposed (1% O2 for 16h) murine islet grafts were transplanted under the renal capsule of diabetic syngeneic recipients. Hypoxia-pre-exposed grafts provided poor glucose homeostasis after transplantation; mean blood glucose was >15mM for the majority of the post operative observation period for hypoxiapre-exposed grafts versus normoxic (21% O2) grafts (P < 0.05; n = 6). Mice receiving hypoxia-pre-exposed islet grafts also showed impaired insulin release in the fed state and a defect in glucose stimulated insulin secretion. Histological analysis of the islet grafts revealed evident islet structures and definitive insulin labeling with equivalent levels of PDX-1 mRNA expression; suggesting a defect in beta cell function rather than an outright loss of beta cells as the cause of poor glycaemic control. Some studies show hypoxia activates HIF-1α which induces high LDHA expression; in islets LDHA would impair insulin secretion by favoring glycolysis and lactate production. Western blot analysis of murine hypoxia-pre-exposed islets demonstrated increased HIF-1α and a concomitant increase in LDHA. Further, static incubation studies showed hypoxia-exposed islets exhibited dysregulated glucose responsiveness with elevated basal insulin secretion. This study demonstrates that hypoxic exposure of islets prior to transplantation has an overall negative impact on graft function and glucose homeostasis. Given isolated human islets, like hypoxic exposed murine islets, show high LDHA levels this may explain by extrapolation, the usual requirement to transplant large numbers of islets to reverse diabetes in clinical trials. Early intervention in reversing the hypoxic changes may be critical to improve graft function and transplant outcome.