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Many factors influence the outcome of islet transplantation. As islets in the early posttransplant setting are supplied with oxygen by diffusion only and are in a hypoxic state in the portal system, we tested whether small human islets are superior to large islets both in vitro and in vivo. We assessed insulin secretion of large and small islets and quantified cell death during hypoxic conditions simulating the intraportal transplant environment. In the clinical setting, we analyzed the influence of transplanted islet size on insulin production in patients with type 1 diabetes. Our results provide evidence that small islets are superior to large islets with regard to in vitro insulin secretion and show a higher survival rate during both normoxic and hypoxic culture. Islet volume after 48 h of hypoxic culture decreased to 25% compared with normoxic culture at 24 h due to a preferential loss of large islets. In human islet transplantation, the isolation index (islet volume as expressed in islet equivalents/islet number), or more simply the islet number, proved to be more reliable to predict stimulated C-peptide response compared with islet volume. Thus, islet size seems to be a key factor determining human islet transplantation outcome.

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.

The success of pancreatic islet transplantation depends largely on the capacity of the islet graft to survive the initial phase immediately after transplantation until revascularization is completed. Endothelin-1 (ET-1) is a strong vasoconstrictor which has been involved in solid organ graft failure but is also known to be a potent mitogenic/anti-apoptotic factor which could also potentially enhance the survival of the transplanted islets.Characterization of the endothelin system with regard to a potential endothelin agonist/antagonist treatment.Regulated expression of the endothelin system in human and rat pancreatic islets and beta-cell lines was assessed by means of immunohistochemistry, competition binding studies, western blot, RT-PCR, real-time PCR and transplant studies.ET-1, ETA- and ETB-receptor immunoreactivity was identified in the endocrine cells of human and rat pancreatic islets. The corresponding mRNA was detectable in rat beta-cell lines and isolated rat and human pancreatic islets. Competition binding studies on rat islets revealed binding sites for both receptor types. ET-1 stimulated the phosphorylation of mitogen-activated protein kinase, which was prevented by ETA- and ETB-receptor antagonists. After exposure to hypoxia equal to post-transplant environment oxygen tension, mRNA levels of ET-1 and ETB-receptor of human islets were robustly induced whereas ETA-receptor mRNA did not show significant changes. Immunostaining signals for ET-1 and ETA-receptor of transplanted rat islets were markedly decreased when compared to native pancreatic sections.In pancreatic islets, ET-1 and its receptors are differentially expressed by hypoxia and after transplantation. Our results provide the biological basis for the study of the potential use of endothelin agonists/antagonists to improve islet transplantation outcome.

To become insulin independent, patients with type 1 diabetes mellitus require transplantation of at least two donor pancreata because of massive beta-cell loss in the early post-transplantation period. Many studies describing the introduction of new immunosuppressive protocols have shown that this loss is due to not only immunological events but also nonimmunological factors. To test to what extent hypoxia may contribute to early graft loss, we analyzed the occurrence of apoptotic events and the expression of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-dependent alpha subunit and a constitutive beta subunit. Histological analysis of human and rat islets revealed nuclear pyknosis as early as 6 h after hypoxic exposure (1% O2). Moreover, immunoreactivity to activated caspase-3 was observed in the core region of isolated human islets. Of note, both of these markers of apoptosis topographically overlap with HIF-1alpha immunoreactivity. HIF-1alpha mRNA was detected in islets from human and rat as well as in several murine beta-cell lines. When exposed to hypoxia, mouse insulinoma cells (MIN6) had an increased HIF-1alpha protein level, whereas its mRNA level did not alter. In conclusion, our data provide convincing evidence that reduced oxygenation is an important cause of beta-cell loss and suggest that HIF-1alpha protein level is an indicator for hypoxic regions undergoing apoptotic cell death. These observations suggest that gene expression under the control of HIF-1 represents a potential therapeutic tool for improving engraftment of transplanted islets.