Continuous measurement of oxygen consumption by pancreatic islets.
The rate of oxygen consumption is an important measure of mitochondrial function in all aerobic cells. In pancreatic beta cells, it is linked to the transduction mechanism that mediates glucose-stimulated insulin secretion. However, measurement of oxygen consumption over long periods of time is technically difficult owing to the error resulting from baseline drift and the challenge of measuring small changes in oxygen tension. We have adapted an ultrastable oxygen sensor based on the detection of the decay of the phosphorescent emission from an oxygen-sensitive dye to a previously developed islet flow culture system. The drift of the sensor is approximately 0.3%/24 h, allowing for the continuous measurement of oxygen consumption by 300 islets (or about 6 x 10(5) cells) for hours or days. Rat islets placed in the perifusion chamber for 24 h were well maintained as reflected by membrane integrity, insulin secretion, and oxygen consumption. Both acute changes in oxygen consumption as induced by glucose and chronic changes as induced by sequential pulses of azide were resolved. The features of the flow culture system--aseptic conditions, fine temporal control of the composition of the media, and the collection of outflow fractions for measurement of insulin, and other products--facilitate a systematic approach to assessing metabolic and functional viability in responses to a variety of stimuli. Applications to the measurement of effects of hypoxia on insulin secretion, membrane integrity, and the redox state of cytochromes are demonstrated. The system has particular application to the field of human islet transplantation, where assessment and the study of islet viability have been hampered by a lack of experimental methods.
Sweet
Ian R
IR
Department of Medicine, University of Washington, Seattle, Washington 98195, USA. isweet@u.washington.edu
Khalil
Gamal
G
Wallen
Angela R
AR
Steedman
Mark
M
Schenkman
Kenneth A
KA
Reems
Jo Anna
JA
Kahn
Steven E
SE
Callis
James B
JB
D000818:Animals; D015374:Biosensing Techniques; D002138:Calibration; D002462:Cell Membrane / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism; D004566:Electrodes; D066298:In Vitro Techniques; D007328:Insulin / Q000032:analysis; D007515:Islets of Langerhans / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D010084:Oxidation-Reduction; D010100:Oxygen / Q000097:blood; D010101:Oxygen Consumption / Q000502:physiology*; D010477:Perfusion; D051381:Rats; D011913:Rats, Inbred BB
D003580:Cytochromes; D007328:Insulin; D010100:Oxygen