ATP and ADP levels are critical regulators of glucosestimulated 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. Diabetes 53: [401][402][403][404][405][406][407][408][409] 2004 F ree ATP and ADP levels are understood to couple glucose metabolism with the closing of ATPsensitive K ϩ (K ATP ) channels and the ionic events leading to the exocytosis of insulin (1,2). Furthermore, the phosphorylation potential (ATP/ADP/P i ) may augment insulin secretion beyond that mediated by K ATP channels (3). Although it is well established that the critical ATP and ADP levels depend on islet substrate metabolism and mitochondrial electron transport (4 -6), it is less broadly appreciated that ADP stimulates electron transport and ATP production (7,8) as a feedback regulator of ATP/ADP/P i (9,10). Thus, mitochondrial ATP production, the ATP/ADP/P i , and ultimately the insulin secretory rate depend dually on both substrate supply as well as energy demand.Although the mechanisms by which mitochondria sense ADP levels and control ATP/ADP/P i are not fully established, a likely candidate is cytochrome c oxidase, the rate-limiting step in the electron transport chain (11). Wilson et al. (11,12) validated a mathematical model correlating substrate supply and energy demand (i.e., as phosphorylation potential) to the redox state and oxygen cons...

Platelets play an important role in hemostasis by forming a thrombotic plug that seals the vessel wall and promotes vascular healing. After platelets adhere and aggregate at the wound site, their next step is to generate contractile forces through the coordination of physicochemical interactions between actin, myosin, and αIIbβ3 integrin receptors that retract the thrombus' size and strengthen its adhesion to the exposed matrix. Although platelet contractile forces (PCF) are a definitive feature of hemostasis and thrombosis, there are few approaches that can directly measure them. In this study, we describe the development of an approach to measure PCF in microthrombi using a microscopic flexible post force sensor array. Quasi-static measurements and live microscopic imaging of thrombin-activated platelets on the posts were conducted to assay the development of PCF to various hemostatic conditions. Microthrombi were observed to produce forces that monotonically increased with thrombin concentration and activation time, but forces subsided when thrombin was removed. PCF results were statistically similar on arrays of posts printed with fibronectin or fibrinogen. PCF measurements were combined with clot volume measurements to determine that the average force per platelet was 2.1 ± 0.1 nN after 60 min, which is significantly higher than what has been measured with previous approaches. Overall, the flexible post arrays for PCF measurements is a promising approach for evaluating platelet functionality, platelet physiology and pathology, the impacts of different matrices or agonists on hemostatic responses, and in providing critical information regarding platelet activity that can guide new hemostatic or thrombotic strategies.

The detection of glucose-stimulated cytochrome c reduction and oxygen consumption may have utility as criteria for the assessment of human islet quality.

High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38 lo cells and culture derived megakaryocytes (MKs). As previously published, 3 replicate microarray data sets from 3 different sources of organ donor marrow were analyzed using the software program Rosetta Resolver® [1]. After setting a stringent p-value of ≤0.001 with a fold change cut-off of ≥3 in expression level, dynamin 3 (DNM3) was identified to be differentially expressed during the course of MK development with a mean fold-change of 8.2±2.1 (mean±S.D.). DNM3 is a member of a family of mechanochemical enzymes (DNM1, DNM2 & DNM3) known for their participation in membrane dynamics by hydrolyzing nucleotides to link cellular membranes to the actin cytoskeleton. Real-time qRT-PCR confirmed that DNM3 increased by 20.7±3.4-fold (n=4, p=0.09) during megakaryocytopoiesis and Western blot analysis showed that DNM3 protein was expressed in human MKs. Confocal microscopy revealed that DNM3 was distributed diffusely throughout the cytoplasm of MKs with a punctate appearance in pro-platelet processes. Immunogold electron microscopy also showed that DNM3 is widely distributed in the cytoplasm of MKs with no apparent localization to specific organelles. The open reading frame of DNM3 was cloned from cultured derived human MKs and determined to be 100% identical to the protein encoded by the DNM3 transcript variant ENST00000367731 published in the Ensemble database. Over expression of DNM3 in umbilical cord blood CD34+ cells resulted in an increase in total nucleated cells, an amplification of total colony forming cells (CFCs) and colony forming unitmegakaryoyctes (CFU-MKs), and a concomitant increase in the expression of NFE-2 and β1 tubulin. Together these findings provide the first evidence that a member of the dynamin family of mechanochemical enzymes is present in human MKs and indicate that DNM3 is an excellent candidate for playing an important role in mediating cytoskeleton and membrane changes that occur during MK/platelet development.