Keith E. Dionne

The effect of pO2s reduced below physiological levels on GSIR by isolated islets of Langerhans was investigated with a microperifusion apparatus that provided control of pO2 and rapid dynamic response. Second-phase insulin secretion was reduced substantially by hypoxia. The response to lower pO2 was rapid and reversible. Although the steady, normoxic (pO2 = 142 mmHg) second-phase secretion rate varied widely from one islet preparation to another, the ratio of Sx to S142 for each preparation could be represented by a single curve that exhibited a continuous reduction with decreasing pO2. For rat islets perifused 1 day after isolation, the secretion rate was nearly 100% of the normoxic value at a pO2 of 60 mmHg, 50% at 27 mmHg (P50, the pO2 at which the S142 is reduced by 50%), and approximately 2% at 5 mmHg. Oxygen sensitivity of second-phase secretion rate declined after 1 wk of in vitro culture: P50 was 13 mmHg after 1 wk and remained at 10 mmHg after 2-5 wk of culture. Canine islets exhibited a P50 of 16 mmHg after 1 wk of culture. The reduction in insulin secretion is thought to be associated with the existence of pO2 gradients outside and inside the isolated islets, resulting in exposure of islet cells to low pO2 levels that decrease radially from the periphery to the core. We hypothesize that the effect of low pO2 on S is manifested through depletion of the energy stores of the beta-cells. The effect of hypoxia on S may be an important factor in some in vitro secretion studies and may play a critical role in the effectiveness of transplanted islets before their revascularization and of immunoisolated islet implantation devices.

The goal of islet transplantation in human diabetes is to maintain the islet grafts in the recipients without the use of immunosuppression. One approach is to encapsulate the donor islets in permselective membranes. Hollow fibers fabricated from an acrylic copolymer were used to encapsulate small numbers of rat islets that were immobilized in an alginate hydrogel for transplantation in diabetic mice. The fibers were biocompatible, prevented rejection, and maintained normoglycemia when transplanted intraperitoneally; hyperglycemia returned when the fibers were removed at 60 days. Normoglycemia was also maintained by subcutaneous implants that had an appropriately constructed outer surface on the fibers.

One approach to insulin replacement therapy is transplantation of islets of Langerhans immunoisolated from host tissue by a semipermeable membrane. In this state, islets depend on diffusion of nutrients and wastes to and from the 0-cell to provide a suitable environment for survival and secretion. A perifusion system was constructed to test glucose-stimulated (100–300 mg/100 ml) insulin secretion from whole islets, or small (5–10 cell) aggregates, under controlled pO2. First phase insulin secretion from adult rat islets was unaffected by hypoxic levels of pO2, but second phase secretion was rapidly reduced at pO2 levels below 60 mmHg in the bulk media. Secretion from single-cell aggregates was unaffected until pO2 levels dropped to 12 mmHg, at which point secretion progressively decreased with falling pO2. A theoretical reaction/diffusion model was developed to correlate intraislet pO2 with reduced insulin secretion. Oxygen limited secretion was reversible, and not a result of decreased cell viability, as ascertained by both long-term static culture and trypan blue staining. Insulin secretion is more sensitive to hypoxia than is cell viability, in part because O2 uptake increases with glucose stimulation. These results indicate that O2 may be the limiting factor in the ability of immunoisolated islets to respond to blood glucose changes. We conclude that maintenance of a sufficiently high islet pO2 for maximal insulin secretion may be an important issue for graft design and implant site selection.

The effective diffusion coefficient of oxygen in pancreatic islets is an important parameter that is needed for the development of models of viability and function of pancreatic islets that are transplanted to cure diabetes, especially when encapsulated in devices. We have developed a method for estimating the maximum oxygen consumption rate (Vmax) and the oxygen permeability in tissue (αtDt) in a suspension of spherical aggregates of different sizes from measurements of the bulk oxygen partial pressure in batch oxygen consumption experiments. The method made use of a model of oxygen consumption and diffusion in the spheres and a parameter estimation scheme. Measurements made with three different batches of rat islets yielded batch averages of Vmax = 3.40 ± 0.77 × 10-8 mol/(cm3 s) and αtDt = 1.34 ± 0.36 × 10-14 mol/(cm mm Hg s). Using a literature estimate for the solubility (αt) yields Dt = 1.31 ± 0.36 × 10-5 cm2/s at 37 °C. These oxygen permeability and diffusion coefficient values are ∼38% and ∼47% of their values in water, respectively.