FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1975347125> ?p ?o ?g. }

• W1975347125 endingPage "6650" @default.
• W1975347125 startingPage "6642" @default.
• W1975347125 abstract "Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mm glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 ± 0.2 min and amplitude of 0.8 ± 0.1 mm glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca2+ and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca2+ ([Ca2+]i) revealed that decreases in [Ca2+]i preceded increases in glucose consumption by 7.4 ± 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca2+]i preceded increases in oxygen consumption by 1.5 ± 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca2+ has stopped entering the cell. Glycolysis stimulates further Ca2+ entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca2+]i. A model to explain these results is described. Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mm glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 ± 0.2 min and amplitude of 0.8 ± 0.1 mm glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca2+ and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca2+ ([Ca2+]i) revealed that decreases in [Ca2+]i preceded increases in glucose consumption by 7.4 ± 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca2+]i preceded increases in oxygen consumption by 1.5 ± 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca2+ has stopped entering the cell. Glycolysis stimulates further Ca2+ entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca2+]i. A model to explain these results is described. intracellular free Ca2+concentration ATP-sensitive K+ channel phosphofructokinase muscle isoform of PFK glucose oxidase flow injection analysis Glucose-stimulated insulin secretion is of paramount importance in maintenance of glucose homeostasis. Defects in this process are a critical component of type 2 diabetes and further understanding of stimulus-secretion coupling is required to enable better management of this prevalent disease. Unlike other secretory pathways that rely on receptor binding to initiate secretion, glucose-stimulated insulin secretion requires metabolism of the sugar to generate appropriate intracellular signaling events. The intertwining of metabolism with secretion results in a complex control process, which has yet to be completely elucidated. The present model stipulates that glucose is rapidly transported into the β-cell through glucose transporter-2. Glycolysis ultimately produces ATP, which activatesK ATP channels causing the cell to depolarize. Cellular depolarization opens L-type Ca2+ channels, which allow entry of Ca2+ and subsequent triggering of exocytosis (see Ref. 1.Newgard C.B. McGarry J.D. Annu. Rev. Biochem. 1995; 64: 689-719Crossref PubMed Scopus (494) Google Scholar for review). Recent reports point to NADH produced during glycolysis and its activation of the NADH shuttle in mitochondria as a key step in initiating secretion (2.Dukes I.D. McIntyre M.S. Mertz R.J. Philipson L.H. Roe M.W. Spencer B. Worley III, J.F. J. Biol. Chem. 1994; 269: 10979-10982Abstract Full Text PDF PubMed Google Scholar, 3.Eto K. Tsubamoto Y. Terauchi Y. Sugiyama T. Kishimoto T. Takahashi N. Yamauchi N. Kubota N. Murayama S. Aizawa T. Akanuma Y. Aizawa S. Kasai H. Yazaki Y. Kadowaki T. Science. 1999; 283: 981-985Crossref PubMed Scopus (394) Google Scholar). According to this model, metabolism should be activated prior to Ca2+ entry into the cell and several lines of evidence support this view. Glucose-stimulated pancreatic β-cells exhibit rises in ATP/ADP ratio and/or oxygen consumption, which precede increases in the Ca2+ levels (4.Civelek V.N. Deeney J.T. Kubik K. Schultz V. Tornheim K. Corkey B.E. Biochem. J. 1996; 315: 1015-1019Crossref PubMed Scopus (58) Google Scholar, 5.Nilsson T. Schultz V. Berggren P.O. Corkey B.E. Tornheim K. Biochem. J. 1996; 314: 91-94Crossref PubMed Scopus (112) Google Scholar). In addition, pyridine and flavin nucleotide fluorescence increases precede rises in intracellular Ca2+([Ca2+]i)1(6.Pralong W.F. Bartley C. Wollheim C.B. EMBO J. 1990; 9: 53-60Crossref PubMed Scopus (233) Google Scholar, 7.Gilon P. Henquin J.C. J. Biol. Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar). At the same time, however, for many cells Ca2+ has been shown to be a primary activator of mitochondrial respiration by activation of Ca2+-dependent dehydrogenases (8.Denton R.M. McCormack J.G. Am. J. Physiol. 1985; 249: E543-E554Crossref PubMed Google Scholar). Indeed, evidence supports the possibility of activation of key dehydrogenases by Ca2+ in the β-cell (9.Malaisse W.J. Sener A. Mol. Cell. Biochem. 1991; 107: 95-102Crossref PubMed Scopus (12) Google Scholar, 10.Sener A. Rasschaert J. Malaisse W.J. Biochim. Biophys. Acta. 1990; 1019: 42-50Crossref PubMed Scopus (51) Google Scholar, 11.McCormack J.G. Longo E.A. Corkey B.E. Biochem. J. 1990; 267: 527-530Crossref PubMed Scopus (71) Google Scholar). In addition, simultaneous measurements of [Ca2+]iand NAD(P)H fluorescence have illustrated that increasing [Ca2+]i by K+-induced depolarization will pace metabolism (12.Pralong W.F. Spat A. Wollheim C.B. J. Biol. Chem. 1994; 269: 27310-27314Abstract Full Text PDF PubMed Google Scholar). Recent measurements of intracellular free ATP by a luciferase assay in single β-cells have revealed that ATP levels increase without Ca2+, but increase further with Ca2+ indicative of both mechanisms occurring in β-cells (13.Kennedy H.J. Pouli A.E. Ainscow E.K. Jouaville L.S. Rizzuto R. Rutter G.A. J. Biol. Chem. 1999; 274: 13281-13291Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). An important feature of glucose stimulation that is not readily explained is the oscillatory nature of insulin secretion. Since oscillatory secretion is lost in type II diabetes (14.O'Rahilly S. Turner R.C. Matthews D.R. N. Engl. J. Med. 1988; 318: 1225-1230Crossref PubMed Scopus (503) Google Scholar, 15.Polonsky K.S. Given B.D. Hirsch L.J. Tillil H. Shapiro E.T. Beebe C. Frank B.H. Galloway J.A. Van Cauter E. N. Engl. J. Med. 1988; 318: 1231-1239Crossref PubMed Scopus (525) Google Scholar), considerable attention has been focused on the cause and regulation of the oscillation. Such oscillations can be observed in vivo(14.O'Rahilly S. Turner R.C. Matthews D.R. N. Engl. J. Med. 1988; 318: 1225-1230Crossref PubMed Scopus (503) Google Scholar, 15.Polonsky K.S. Given B.D. Hirsch L.J. Tillil H. Shapiro E.T. Beebe C. Frank B.H. Galloway J.A. Van Cauter E. N. Engl. J. Med. 1988; 318: 1231-1239Crossref PubMed Scopus (525) Google Scholar), in groups of islets in vitro (16.Cunningham B.A. Deeney J.T. Bliss C.R. Corkey B.E. Tornheim K. Am. J. Physiol. 1996; 271: E702-E710Crossref PubMed Google Scholar, 17.Chou H.F. Ipp E. Diabetes. 1990; 39: 112-117Crossref PubMed Scopus (88) Google Scholar, 18.Bersten P. Hellman B. Diabetes. 1993; 42: 670-674Crossref PubMed Scopus (125) Google Scholar, 19.Longo E.A. Tornheim K. Deeney J.T. Varnum B.A. Tillotson D. Prentki M. Corkey B.E. J. Biol. Chem. 1991; 266: 9314-9319Abstract Full Text PDF PubMed Google Scholar), and at single islets (18.Bersten P. Hellman B. Diabetes. 1993; 42: 670-674Crossref PubMed Scopus (125) Google Scholar, 20.Gilon P. Shepherd R.M. Henquin J.C. J. Biol. Chem. 1993; 268: 22265-22268Abstract Full Text PDF PubMed Google Scholar, 21.Martin F. Reig J.A. Soria B. J. Mol. Endocrinol. 1995; 15: 177-185Crossref PubMed Scopus (33) Google Scholar). In addition to oscillations in insulin secretion, oscillations in oxygen consumption (19.Longo E.A. Tornheim K. Deeney J.T. Varnum B.A. Tillotson D. Prentki M. Corkey B.E. J. Biol. Chem. 1991; 266: 9314-9319Abstract Full Text PDF PubMed Google Scholar, 22.Jung S.-K. Aspinwall C.A. Kennedy R.T. Biochem. Biophys. Res. Commun. 1999; 259: 331-335Crossref PubMed Scopus (40) Google Scholar, 23.Jung S.-K. Gorski W. Aspinwall C.A. Kauri L.M. Kennedy R.T. Anal. Chem. 1999; 71: 3642-3649Crossref PubMed Scopus (81) Google Scholar), [Ca2+]i (19.Longo E.A. Tornheim K. Deeney J.T. Varnum B.A. Tillotson D. Prentki M. Corkey B.E. J. Biol. Chem. 1991; 266: 9314-9319Abstract Full Text PDF PubMed Google Scholar, 20.Gilon P. Shepherd R.M. Henquin J.C. J. Biol. Chem. 1993; 268: 22265-22268Abstract Full Text PDF PubMed Google Scholar, 21.Martin F. Reig J.A. Soria B. J. Mol. Endocrinol. 1995; 15: 177-185Crossref PubMed Scopus (33) Google Scholar, 24.Valdeolmillos M. Nadal A. Soria B. Garcia-Sancho J. Diabetes. 1993; 42: 1210-1214Crossref PubMed Google Scholar, 25.Hellman B. Gylfe E. Grapengiesser E. Lund P.E. Berts A. Biochim. Biophys. Acta. 1992; 1113: 295-305Crossref PubMed Scopus (142) Google Scholar), membrane potential (26.Santos R.M. Rosario L.M. Nadal A. Garcia-Sancho J. Soria B. Valdeolmillos M. Pflugers Arch. 1991; 418: 417-422Crossref PubMed Scopus (316) Google Scholar), and K ATP channel activity (27.Larsson O. Kindmark H. Branstrom R. Fredholm B. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5161-5165Crossref PubMed Scopus (91) Google Scholar) have all been observed. One attractive hypothesis explaining these data is that oscillatory insulin secretion is driven by oscillatory glycolysis (28.Tornheim K. Diabetes. 1997; 46: 1375-1380Crossref PubMed Google Scholar). This hypothesis has been supported by several lines of evidence including the observation that oxygen and [Ca2+]ioscillate with similar frequency in similar preparation (19.Longo E.A. Tornheim K. Deeney J.T. Varnum B.A. Tillotson D. Prentki M. Corkey B.E. J. Biol. Chem. 1991; 266: 9314-9319Abstract Full Text PDF PubMed Google Scholar); however, to date no simultaneous measurements of metabolic oscillations and [Ca2+]i oscillations have been made with appropriate temporal resolution to verify the expected temporal relationships. A stipulation of this model, as currently formulated, is that Ca2+ entry into the cell, while possibly amplifying secretory oscillations, does not affect metabolic rates (28.Tornheim K. Diabetes. 1997; 46: 1375-1380Crossref PubMed Google Scholar). Alternative models have also been proposed (29.Detimary P. Gilon P. Henquin J.C. Biochem. J. 1998; 333: 269-274Crossref PubMed Scopus (150) Google Scholar). In this work, we have correlated the temporal changes of glucose level, oxygen level, and [Ca2+]i in single islets. While techniques for oxygen (22.Jung S.-K. Aspinwall C.A. Kennedy R.T. Biochem. Biophys. Res. Commun. 1999; 259: 331-335Crossref PubMed Scopus (40) Google Scholar, 23.Jung S.-K. Gorski W. Aspinwall C.A. Kauri L.M. Kennedy R.T. Anal. Chem. 1999; 71: 3642-3649Crossref PubMed Scopus (81) Google Scholar) and [Ca2+]imeasurements (19.Longo E.A. Tornheim K. Deeney J.T. Varnum B.A. Tillotson D. Prentki M. Corkey B.E. J. Biol. Chem. 1991; 266: 9314-9319Abstract Full Text PDF PubMed Google Scholar, 20.Gilon P. Shepherd R.M. Henquin J.C. J. Biol. Chem. 1993; 268: 22265-22268Abstract Full Text PDF PubMed Google Scholar, 21.Martin F. Reig J.A. Soria B. J. Mol. Endocrinol. 1995; 15: 177-185Crossref PubMed Scopus (33) Google Scholar, 24.Valdeolmillos M. Nadal A. Soria B. Garcia-Sancho J. Diabetes. 1993; 42: 1210-1214Crossref PubMed Google Scholar, 25.Hellman B. Gylfe E. Grapengiesser E. Lund P.E. Berts A. Biochim. Biophys. Acta. 1992; 1113: 295-305Crossref PubMed Scopus (142) Google Scholar) in single islets have been described, this is the first report of measuring glucose in single islets. The glucose sensor used here is based on immobilizing glucose oxidase (GOx) on the surface of a Pt microelectrode. GOx-based sensors have been described before (30.Kamin R.A. Wilson G.S. Anal. Chem. 1980; 52: 1198-1205Crossref Scopus (1154) Google Scholar, 31.Pantano P. Kuhr W.G. Electroanalysis. 1995; 7: 405-416Crossref Scopus (86) Google Scholar), but we have miniaturized the sensor to a total tip diameter of <5 μm, which allows implantation into a single islet with minimal damage. The sensor derives its selectivity and signal generation from GOx, a flavoprotein that is specific for oxidation of glucose (32.Gibson Q.H. Swoboda B.E.P. Massey V. J. Biol. Chem. 1964; 239: 3927-3934Abstract Full Text PDF PubMed Google Scholar, 33.Swoboda B.E.P. Massey V. J. Biol. Chem. 1965; 240: 2209-2215Abstract Full Text PDF PubMed Google Scholar). The following reactions occur in the presence of glucose and enzyme, β­D­glucose+GOx/FAD→glucono­δ­lactone+GOx/FADH2Equation 1 GOx/FADH2+O2→GOx/FAD+H2O2Equation 2 With the Pt electrode poised at a sufficiently positive potential, hydrogen peroxide is electrochemically oxidized at the electrode, H2O2→O2+2H++2e−Equation 3 resulting in an anodic current that is directly proportional to glucose concentration. Sensor design for use in real systems must account for nonlinear response, potential effects of low oxygen level (low oxygen levels can slow the formation of H2O2 and lead to artificially low signals), and possible fouling of the electrode surface by the tissue. In this report, these issues are addressed by recessing the electrode tip in a glass case, coating the electrode with a polymer, and ensuring a sufficient oxygen level in the system. Pt wire (diameter 25 μm) was from Johnson Matthey (Ward Hill, MA). Silver epoxy was from Epoxy Technology, Inc. (Billerica, MA). Epoxy was from Miller-Stephenson Chemical Co. (Danbury, CT). Glucose oxidase (type X-S fromAspergillus niger) was from Sigma. Deionized water as obtained from a Milli-Q Plus system water purifier (Millipore Co., Bedford, MA) was used to make all solutions. All cell culture media and solutions were from Life Technologies, Inc. (Grand Island, NY). All other chemicals were obtained from Aldrich or Sigma. Islets of Langerhans were isolated from mice weighing 20–30 g using controlled collagenase perfusion via the duct as described previously (34.Warnock G.L. Ao Z. Lakey J.R.T. Rajotte R.V. Lanza R.P. Chick W.L. Pancreatic Islet Transplantation: Procurement of Pancreatic Islets. 1. R. G. Landes, Austin, TX1994: 81-95Google Scholar). The isolated islets were put into RPMI 1640 solution allowed to recover overnight in an incubator at 37 °C in a humidified 95% air and 5% CO2. Oxygen microsensors were prepared as described previously (23.Jung S.-K. Gorski W. Aspinwall C.A. Kauri L.M. Kennedy R.T. Anal. Chem. 1999; 71: 3642-3649Crossref PubMed Scopus (81) Google Scholar). The final product consists of a Pt wire recessed inside a glass pipette tip, sealed with epoxy, and coated with cellulose acetate as illustrated in Fig. 1 B. Oxygen is detected at these electrodes by reduction at platinum. The characteristics and performance of the oxygen microsensors were reported previously (23.Jung S.-K. Gorski W. Aspinwall C.A. Kauri L.M. Kennedy R.T. Anal. Chem. 1999; 71: 3642-3649Crossref PubMed Scopus (81) Google Scholar). Glucose sensors were prepared as recessed Pt electrodes using the same initial steps as for preparation of oxygen sensors; however, before coating with cellulose acetate, the Pt surface was electrochemically platinized at −0.2 V versus Ag/AgCl in an aqueous solution of 10 mm hexachloroplatinate in lead acetate (1.6 mm) for 1 min to form Pt particles inside the recess (see Fig. 1 A). GOx was immobilized onto the Pt particles by immersing the electrodes in 5% (w/v) aqueous GOx solution for 10 min and 2% glutaraldehyde for 1 min. Once the enzyme was immobilized, the electrodes were dip-coated with 10% (w/v) cellulose acetate (30 kDa) in dimethylformamide for ∼5 s and then allowed to dry for 10 min. Dipping could be repeated to achieve a thicker coat of polymer. The total tip diameter of the resulting sensors was 3–5 μm, which is much smaller than the islets as illustrated in Fig. 1 C. Calibration and measurement of the response times and flow sensitivities of the sensors were measured in an electrolyte consisting of 50 mm HEPES buffer with 0.15m NaCl at pH 7.4 using flow injection analysis (FIA). The FIA system consisted of a reservoir for electrolyte connected to a pneumatically actuated two-position, six-port valve (Valco AC6UHC) equipped with a 1-ml sample loop. The outlet of the valve was connected to a glass cell via tubing of 0.25-mm inner diameter. The sensors were positioned in the outlet of the tubing using a micromanipulator. The electrolyte solution was fed by gravity at 1 ml/min. The entire FIA system was housed in a Faraday cage. Amperometric data were collected using an EI-400 bipotentiostat (Ensman Instrumentation, Bloomington, IN) which allowed measurements to be made with two sensors simultaneously. In experiments involving single sensors, a Keithley 428 Current Amplifier was used because it generated lower noise. Amperometric data were collected using a National Instruments (Austin, TX) multifunction board and an IBM compatible personal computer at 300 Hz and low-pass filtered with a cutoff frequency of 20 Hz using filters on the potentiostat or current amplifier. For all measurements, glucose sensors and oxygen sensors were poised at +0.60 V and −0.60 V versus Ag/AgCl reference electrode, respectively. Experiments were performed 1 to 3 days after islet isolation, when the islets had adhered to the surface of culture dishes. To perform measurements, islets in Petri dishes were rinsed three times with a modified Krebs-Ringer buffer (KRB) consisting of 118 mm NaCl, 5.4 mm KCl, 2.4 mmCaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, and 20 mmHEPES at pH 7.4. The dish was filled with 4 ml of KRB and placed in a microincubator (Medical Systems, Co., Greenvale, NY) on the stage of a Zeiss Axiovert 35 microscope at 37 °C. For measurements using the glucose sensor inside an islet, a stream of oxygen was continuously flowed above the solution containing the islet to raise oxygen levels in the KRB. As discussed under “Results,” this was necessary to prevent low oxygen levels from interfering with the glucose measurement. For most measurements, the islets were maintained in a static solution on the microscope stage to minimize noise in the electrochemical measurements. Drugs or other additions were made by injecting drug dissolved in KRB into the incubation chamber to generate the desired final concentration. For some experiments, the islets were constantly perfused at 32 μl/s and changes were made by switching solutions that flowed over the islet. In this case, the total volume of the incubation chamber was 400 μl. The response time of the perfusion system, defined as the time to completely change the buffer content of the incubation chamber on the microscope stage, was 38.5 ± 1.2 s (mean ± S.D., n = 5). For all measurements, sensors were positioned normal to the islet surface and advanced to the islet with the use of a micromanipulator (Burleigh, PC-1000). Data collection and electrode potentials were the same as for sensor testing described above. Calcium indicator was loaded by incubating islets in RPMI 1640 containing 2 μm Fura-2/AM (Molecular Probes, Eugene, OR) at 37 °C in a humidified 95% air and 5% CO2 for 30 min. After loading, an islet was attached to the central part of circular 25-mm coverslip coated with poly-l-lysine and incubated in dye-free KRB at 37 °C. The complex of [Ca2+-(Fura-2)] was alternately excited at 340 and 380 nm and the resulting fluorescence from individual islets was collected at 1 Hz through a Fluar 40× oil immersion objective (Zeiss), band pass filter (510 ± 10 nm), and 20-μm pinhole aperture onto a photomultiplier tube using a SPEX CMX cation measurement system and DM3000M data acquisition software (Instruments SA) (35.Aspinwall C.A. Lakey J.R.T. Kennedy R.T. J. Biol. Chem. 1999; 274: 6360-6365Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The ratio of the emission intensities from 340 and 380 nm (F 340/F 380) was used as a measure of [Ca2+]i in the islet. In some experiments, the signal was calibrated to allow measurement of actual [Ca2+]i (36.Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). All values are reported as mean ± S.E. unless stated otherwise. Two-tailed Student'st tests are used to evaluate significant differences observed. The response time (time for signal to change from 10% of the maximum to 90% of the maximum) for glucose sensors was 0.8 ± 0.1 s (n = 8) as determined by FIA. A FIA trace for injection of 20 mm glucose is shown in Fig.2 A for a sensor that had a response time of 0.7 s. The sensors typically yielded a linear response up to 10 mm glucose while some deviation was observed with the sensors of high sensitivity (see Fig. 2 B). As illustrated by Fig. 2 B, the sensitivity was variable for different sensors yielding an average sensitivity of 4.8 ± 0.7 pA/mm (n = 38). The relatively wide linear dynamic range of the sensors was achieved by slowing transport of glucose to the electrode through the use of a recessed electrode and cellulose acetate coating (37.Jung S.K. Wilson G.S. Anal. Chem. 1996; 68: 591-596Crossref PubMed Scopus (101) Google Scholar). An important consideration in using oxidase-based enzyme sensors in biological systems is their oxygen dependence. At a low glucose to oxygen ratio, the signal at the sensor is only dependent on glucose because sufficient oxygen is available to rapidly form H2O2; however, at a higher glucose to oxygen ratio, the oxygen level may not be high enough to rapidly form H2O2 (reaction 2) resulting in a lower signal for the glucose level (37.Jung S.K. Wilson G.S. Anal. Chem. 1996; 68: 591-596Crossref PubMed Scopus (101) Google Scholar, 38.Zhang Y. Wilson G.S. Anal. Chim. Acta. 1993; 281: 513-520Crossref Scopus (93) Google Scholar). The oxygen dependence of the microsensors used here is shown in Fig. 2 C. For a glucose concentration of 10 mm, the sensor signal is independent of the oxygen level as long as the oxygen level is above 150 mm Hg; however, below this level the glucose signal is decreased. We have previously shown that under typical incubation conditions, the oxygen level in the islet interior can be as low as 67 mm Hg (23.Jung S.-K. Gorski W. Aspinwall C.A. Kauri L.M. Kennedy R.T. Anal. Chem. 1999; 71: 3642-3649Crossref PubMed Scopus (81) Google Scholar). Therefore, to avoid this artifact in islet measurements, the oxygen level in islet media was increased by flowing oxygen over the islet chamber. This precaution maintained oxygen in the medium at 405 ± 12 mm Hg (n = 7) and within islets at 214 ± 9 mm Hg (n = 7) even at 20 mm glucose. These levels are sufficiently high to prevent any effect of oxygen on the glucose measurement. Oxygen was added only for experiments involving glucose measurements. Fig.3 A shows a typical time-dependent glucose recording inside a single islet upon changing the perfusion medium from 3 to 10 mm glucose. As expected, the signal increases up to nearly 10 mm glucose. The increase is not instantaneous, requiring 7.4 ± 0.6 min (n = 9) to achieve a steady state level, as the glucose must diffuse into the islet and is constantly being taken up by islet cells and consumed. This delay to a steady level has been predicted based on the effects of mass transport and metabolism of glucose inside islets (39.Bertram R. Pernarowski M. Biophys. J. 1998; 74: 1722-1731Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). To demonstrate that the signals observed are due exclusively to glucose and not an interferent, an electrode identical to the sensor but without the GOx enzyme was also implanted in the islet. As shown in Fig. 3 A, the current at this “dummy” electrode was unaffected by the glucose addition indicating that the signal is due exclusively to glucose. An interesting feature of the glucose signal is that upon reaching its maximum level, it appears to oscillate with a period of 2.4 min. Such oscillations were not seen at the control electrode indicating that the oscillation is not an artifact from the system but rather a real fluctuation in the glucose level (see Fig. 3 A). To determine if this oscillation was metabolic in origin, the effect of 10 mm mannoheptulose, an inhibitor of glucokinase (40.Coore H.G. Randle P.J. Simon E. Kraicer P.F. Shelesnyak M.C. Nature. 1963; 197: 1264-1266Crossref PubMed Scopus (40) Google Scholar, 41.Sweet I.R. Li G. Najafi H. Berner D. Matschinsky F.M. Am. J. Physiol. 1996; 271: E606-E625Crossref PubMed Google Scholar), was tested as illustrated in Fig. 3 B. In Fig. 3 B, as in all trials (n = 5), mannoheptulose caused an abrupt halt to oscillations and small increase in glucose level. Mannoheptulose was verified to not interfere with the sensor operation in separate experiments. Regular fluctuations in glucose level were observed in 82 of 104 islets exposed to 10 mm glucose. A variety of patterns were observed as shown in Fig. 4. In 74 of the islets, we observed a “slow” oscillation with a period of 3.1 ± 0.2 min and an amplitude of 0.8 ± 0.1 mm(n = 21). Frequently (68 of 74 islets), the slow oscillation was accompanied by faster fluctuations during the times of relatively high glucose level (i.e. low glucose consumption). In some cases, the fast oscillations frequently started small with a short period and then increased in amplitude and period until a longer burst of glucose consumption occurred (see Fig. 4,A and C, for examples). For cases with a regular fast oscillation, the period was 15 ± 2 s (n= 15). In 8 islets, only a single pattern of oscillations was observed which had a period of 30 ± 5 s and amplitude of 0.5 ± 0.2 mm (see Fig. 4). The periods and amplitudes of the slow oscillations are significantly longer (p < 0.001) and larger (p < 0.01) than those of the fast oscillations. Oscillations in intraislet oxygen concentration have previously been observed at elevated glucose level (22.Jung S.-K. Aspinwall C.A. Kennedy R.T. Biochem. Biophys. Res. Commun. 1999; 259: 331-335Crossref PubMed Scopus (40) Google Scholar, 23.Jung S.-K. Gorski W. Aspinwall C.A. Kauri L.M. Kennedy R.T. Anal. Chem. 1999; 71: 3642-3649Crossref PubMed Scopus (81) Google Scholar). Fig.5 A illustrates the characteristic oxygen oscillation in the presence of ambient oxygen levels. As shown, much like glucose, both slow and fast oscillations can occur. Slow oscillations had a period of 3.3 ± 0.6 min and an amplitude of 9.7 ± 2.1 mm Hg (n = 6). Fast oscillations had a period of 12.1 ± 1.7 s and an amplitude of 5.7 ± 1.3 mm Hg (n = 6). The periods and amplitudes of the slow oscillations are significantly longer (p < 0.001) and larger (p < 0.01) than those of the fast oscillations. In contrast to the glucose fluctuations, the fast oscillations of oxygen occur at low oxygen levels, i.e. during high oxygen consumption. When two oxygen electrodes were placed at different positions in the same islet, we observed that the oxygen levels fluctuated synchronously (Fig.5 B). This synchronous relationship was observed in all trials (n = 9) and was independent of the relative electrode position. Such synchrony has previously been observed for [Ca2+]i oscillations in islets by Ca2+ imaging techniques (24.Valdeolmillos M. Nadal A. Soria B. Garcia-Sancho J. Diabetes. 1993; 42: 1210-1214Crossref PubMed Google Scholar). These results indicate that small differences in the placement of the electrodes should not affect the observed phase relationship between different measurements. In order to determine the temporal relationship between glucose and oxygen fluctuations, both glucose and oxygen electrodes were simultaneously implanted into single islets during exposure to 10 mm glucose. Typical traces are illustrated in Fig.6. During simultaneous measurements, oxygen oscillations were observed; however, they were not as distinct because of the elevated oxygen level required to obtain reliable glucose measurements (see above). The elevated oxygen level tends to damp out the oxygen oscillation because of both the higher level and increased diffusive flux. Nevertheless, it was consistently observed that both glucose and oxygen oscillated with the same period but out of phase. That is, high glucose consumption correlated with low oxygen consumption and vice versa. This result is consistent with the observation made with independent measurements of oxygen and glucose, which showed no significant difference in the period of glucose and oxygen oscillations. (The out of phase correlation of glucose and oxygen confirms that the glucose fluctuation is not an artifact of the oxygen level change. This" @default.
• W1975347125 created "2016-06-24" @default.
• W1975347125 creator A5019171763 @default.
• W1975347125 creator A5056086091 @default.
• W1975347125 creator A5067730125 @default.
• W1975347125 creator A5075235007 @default.
• W1975347125 date "2000-03-01" @default.
• W1975347125 modified "2023-10-16" @default.
• W1975347125 title "Correlated Oscillations in Glucose Consumption, Oxygen Consumption, and Intracellular Free Ca2+ in Single Islets of Langerhans" @default.
• W1975347125 cites W130501137 @default.
• W1975347125 cites W1485720498 @default.
• W1975347125 cites W1494239480 @default.
• W1975347125 cites W1501608258 @default.
• W1975347125 cites W1510217511 @default.
• W1975347125 cites W1515119897 @default.
• W1975347125 cites W1543532453 @default.
• W1975347125 cites W1573201350 @default.
• W1975347125 cites W1737157464 @default.
• W1975347125 cites W1743153984 @default.
• W1975347125 cites W1759896623 @default.
• W1975347125 cites W1796459452 @default.
• W1975347125 cites W1901140894 @default.
• W1975347125 cites W1933247863 @default.
• W1975347125 cites W1965084066 @default.
• W1975347125 cites W1969914617 @default.
• W1975347125 cites W1970312446 @default.
• W1975347125 cites W1973102979 @default.
• W1975347125 cites W1973533455 @default.
• W1975347125 cites W1974287069 @default.
• W1975347125 cites W1975742198 @default.
• W1975347125 cites W1975943401 @default.
• W1975347125 cites W2014321636 @default.
• W1975347125 cites W2017783616 @default.
• W1975347125 cites W2042381753 @default.
• W1975347125 cites W2043988282 @default.
• W1975347125 cites W2053670472 @default.
• W1975347125 cites W2054243156 @default.
• W1975347125 cites W2056929654 @default.
• W1975347125 cites W2060631487 @default.
• W1975347125 cites W2073436363 @default.
• W1975347125 cites W2075786190 @default.
• W1975347125 cites W2078509049 @default.
• W1975347125 cites W2139132103 @default.
• W1975347125 cites W2141260882 @default.
• W1975347125 cites W2142089485 @default.
• W1975347125 cites W2151706465 @default.
• W1975347125 cites W2161641259 @default.
• W1975347125 cites W2164217251 @default.
• W1975347125 cites W2178375292 @default.
• W1975347125 cites W2336304679 @default.
• W1975347125 cites W2414896080 @default.
• W1975347125 cites W2879608796 @default.
• W1975347125 cites W58251843 @default.
• W1975347125 doi "https://doi.org/10.1074/jbc.275.9.6642" @default.
• W1975347125 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10692473" @default.
• W1975347125 hasPublicationYear "2000" @default.
• W1975347125 type Work @default.
• W1975347125 sameAs 1975347125 @default.
• W1975347125 citedByCount "115" @default.
• W1975347125 countsByYear W19753471252012 @default.
• W1975347125 countsByYear W19753471252013 @default.
• W1975347125 countsByYear W19753471252014 @default.
• W1975347125 countsByYear W19753471252015 @default.
• W1975347125 countsByYear W19753471252016 @default.
• W1975347125 countsByYear W19753471252017 @default.
• W1975347125 countsByYear W19753471252020 @default.
• W1975347125 countsByYear W19753471252021 @default.
• W1975347125 countsByYear W19753471252022 @default.
• W1975347125 countsByYear W19753471252023 @default.
• W1975347125 crossrefType "journal-article" @default.
• W1975347125 hasAuthorship W1975347125A5019171763 @default.
• W1975347125 hasAuthorship W1975347125A5056086091 @default.
• W1975347125 hasAuthorship W1975347125A5067730125 @default.
• W1975347125 hasAuthorship W1975347125A5075235007 @default.
• W1975347125 hasBestOaLocation W19753471251 @default.
• W1975347125 hasConcept C12554922 @default.
• W1975347125 hasConcept C126322002 @default.
• W1975347125 hasConcept C134018914 @default.
• W1975347125 hasConcept C144024400 @default.
• W1975347125 hasConcept C165220095 @default.
• W1975347125 hasConcept C178790620 @default.
• W1975347125 hasConcept C185592680 @default.
• W1975347125 hasConcept C30772137 @default.
• W1975347125 hasConcept C36289849 @default.
• W1975347125 hasConcept C540031477 @default.
• W1975347125 hasConcept C55493867 @default.
• W1975347125 hasConcept C555293320 @default.
• W1975347125 hasConcept C71924100 @default.
• W1975347125 hasConcept C79879829 @default.
• W1975347125 hasConcept C86803240 @default.
• W1975347125 hasConcept C95444343 @default.
• W1975347125 hasConceptScore W1975347125C12554922 @default.
• W1975347125 hasConceptScore W1975347125C126322002 @default.
• W1975347125 hasConceptScore W1975347125C134018914 @default.
• W1975347125 hasConceptScore W1975347125C144024400 @default.
• W1975347125 hasConceptScore W1975347125C165220095 @default.
• W1975347125 hasConceptScore W1975347125C178790620 @default.
• W1975347125 hasConceptScore W1975347125C185592680 @default.

• next