MP25: High Throughput Hypoxic Modulation of Islet Calcium Response and Preconditioning
Joe F. Lo, Ph.D.(1), Yong Wang, M.D.(2), David T. Eddington, Ph.D.(1)
(1) Bioengineering Department, (2) Department of Surgery, School of Medicine,
University of Illinois at Chicago
Type I diabetes can be treated with the transplant of islets of Langerhans in the hepatic portal vein. However, this environment represents a decrease in available oxygen (hypoxia), and the islets must adapt and later recruit microvasculature post-transplantation. Moreover, the insulin response, correlated with intracellular calcium concentration, is unknown for islets under hypoxia (~8% volumetric O2 for hepatic portal). While there are large number of studies on the success and the complications of islet transplants, there is no demonstration of how islets can operate or even recover under hypoxic transplant conditions. Quantifying islet response to oxygen levels can elucidate the role respiration has on glucose metabolism, and may provide critical understanding of diabetes beyond transplant research.
Islet trapping and perfusion were achieved using microfluidic structures previously published by our group. Furthermore, novel diffusion-based oxygen delivery was integrated into the microfluidic device. Highly controlled oxygen concentrations of 0%, 5%, 10%, 21% and 100% volumetric concentrations (balance air) were delivered to islets across a 100 µm gas-permeable polydimethylsiloxane, PDMS membrane. Islets were monitored for their intracellular calcium fluxes via ratiometric FURA-2 AM intensities on an Olympus IX71 inverted microscope. Using this system, oxygen can be delivered and modulated in timescale of several minutes.
Islet exposure to hypoxia predictably showed impairment in calcium response to 14mM glucose, and thus insulin secretion. Furthermore, oxygen concentration exhibits a dose response with 10% oxygen exposure leading to a 50% depressed calcium response but retaining the phasic overshoot and oscillation seen in normal glucose response. 5% oxygen was taken as the “hypoxic standard” and islet response under this hypoxia showed no phasic response under 7, 14, and 25mM glucose exposure, showing hypoxic response is neither improved nor worsened by short-term elevated glucose exposure.
Furthermore, preconditioning of islets was tested to recover islet response under 5% hypoxia. Preconditioning of islets to hypoxia and 100% hyperoxia for 20 minutes does not improve calcium response. Also hyperoxic exposure might be related to poor antioxidant capacity of islet cells. However, cycling of 5% and 21% oxygen with 5 minute intervals for 4 cycles, totally 20 minutes of hypoxia precondition, recovers both maginitude (~70%) and phasic response including oscillations related to membrane calcium ion channels.
The response of islet glucose stimulation in low oxygen shows that hypoxic impairment critically affects islet performance. Preconditioning using constant hypoxia or hyperoxia cannot prepare islets for hypoxic operations. Intermittent hypoxia at 5 minute intervals showed promise to recover calcium responses for islet operation under 5% hypoxia. These results show both insights to islet oxygen/glucose metabolism, as well as provide a possible enhancement to islet transplant therapy using high-throughput microfluidic devices.
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