Regulation of ATP/ADP in pancreatic islets.

ATP and ADP levels are critical regulators of glucose-stimulated insulin secretion. In many aerobic cell types, the phosphorylation potential (ATP/ADP/P(i)) is controlled by sensing mechanisms inherent in mitochondrial metabolism that feed back and induce compensatory changes in electron transport. To determine whether such regulation may contribute to stimulus-secretion coupling in islet cells, we used a recently developed flow culture system to continuously and noninvasively measure cytochrome c redox state and oxygen consumption as indexes of electron transport in perifused isolated rat islets. Increasing substrate availability by increasing glucose increased cytochrome c reduction and oxygen consumption, whereas increasing metabolic demand with glibenclamide increased oxygen consumption but not cytochrome c reduction. The data were analyzed using a kinetic model of the dual control of electron transport and oxygen consumption by substrate availability and energy demand, and ATP/ADP/P(i) was estimated as a function of time. ATP/ADP/P(i) increased in response to glucose and decreased in response to glibenclamide, consistent with what is known about the effects of these agents on energy state. Therefore, a simple model representing the hypothesized role of mitochondrial coupling in governing phosphorylation potential correctly predicted the directional changes in ATP/ADP/P(i). Thus, the data support the notion that mitochondrial-coupling mechanisms, by virtue of their role in establishing ATP and ADP levels, may play a role in mediating nutrient-stimulated insulin secretion. Our results also offer a new method for continuous noninvasive measures of islet cell phosphorylation potential, a critical metabolic variable that controls insulin secretion by ATP-sensitive K(+)-dependent and -independent mechanisms.

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.

Cre recombinase-dependent expression of a constitutively active mutant allele of the catalytic subunit of protein kinase A.

Using the cre-loxP recombination system, we generated a line of mice expressing a constitutively active catalytic subunit of Protein Kinase A (PKA) in a temporally and spatially regulated fashion. In the absence of cre recombinase the modified catalytic subunit allele is functionally silent, but after recombination the mutant allele is expressed, resulting in enhanced PKA effects at basal cAMP levels. Mice expressing the modified protein in hepatocytes using albumin-cre transgenics show defects in glucose homeostasis, glycogen storage, fructose 2,6-bisphosphate levels, and induction of glucokinase mRNA during feeding. Similar to animals lacking glucokinase in the liver (Postic et al.: J Biol Chem 274:305-315, 1999), these mice also have defects in glucose-stimulated insulin secretion, a hallmark of Type II diabetes. The widespread expression of PKA and the involvement of this kinase in a myriad of signaling pathways suggest that these animals will provide critical tools for the study of PKA function in vivo.

Systematic screening of potential beta-cell imaging agents.

The beta-cell loss seen in diabetes mellitus could be monitored clinically by positron emission tomography (PET) if imaging agents were sufficiently specific for beta-cells to overcome the high ratio of non-beta-cell to beta-cell tissue in pancreas. In this report, we present a screening assay for identifying beta-cell-specific compounds that is based on the relative accumulation and retention by islet, INS-1, and exocrine (PANC-1) cells of candidate molecules. Molecules thought to have a high affinity for beta-cells were tested and included glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone were the most specific, but the specificity ratios fell well below those needed to attain robust signal to background ratio as a PET imaging agent for quantifying beta-cell mass. In vivo tests of the biodistribution of glibenclamide and fluorodithizone in rats indicated that the compounds were not specifically associated with pancreas, bearing out the predictions of the in vitro screen.