Measurement of Small Molecules in Islets of Langerhans
The work in this dissertation presents methods developed for the measurement of small molecules in islets of Langerhans, including reactive oxygen species and amino acids. Reactive oxygen species were measured using fluorescence imaging. The conditions of dye loading, incubat...
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Florida State University
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Online Access: | http://purl.flvc.org/fsu/fd/FSU_2016SP_Wang_fsu_0071E_13074 |
Summary: | The work in this dissertation presents methods developed for the measurement of small molecules in islets of Langerhans,
including reactive oxygen species and amino acids. Reactive oxygen species were measured using fluorescence imaging. The conditions of dye
loading, incubation, and imaging were optimized to develop a robust and reproducible protocol to measure these species using the
fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). The protocol that was developed minimized photobleaching and
leakage of H2DCF from murine islets and utilized a normalization procedure to further reduce experimental variability. The method allowed
for ~25 min of DCF measurement in living islets. We used the developed protocol to compare DCF fluorescence from batches of islets
incubated in varying glucose concentrations and observed ~1.5-fold higher fluorescence signals in 3 vs. 20 mM glucose. The presence of
diazoxide increased DCF fluorescence at all glucose concentrations tested while addition of 30 mM K+, to increase [Ca2+]i, reduced the
fluorescence by ~15%. With the developed protocol, all experimental methods tested to increase [Ca2+]i resulted in a decrease in DCF
fluorescence, potentially indicating involvement of ROS in intracellular signaling cascades. To characterize the potential roles that
amino acids may play in islet physiology, derivatization of amino acids in high-salt buffers commonly used in islet experiments with
naphthalene-2,3-dicarboxaldehyde and micellar electrokinetic chromatography separation conditions were optimized. The optimized conditions
used D-norvaline as the internal standard and allowed quantification of 14 amino acids with limit of detections ranging from 0.2 nM to 7
nM. The relative standard deviations (RSDs) of the migration times were 0.04% – 0.54% and the RSDs of the peak areas were 0.2% – 5.8% for
the various amino acids. The effects of glucose and 2,4-dinitrophenol on amino acid secretions from islets were tested and a suppressive
effect of glucose on gamma-aminobutyric acid release was observed, likely acting through adenosine triphosphate inactivation of glutamate
decarboxylase. To understand the secretion dynamics of these amino acids, a microfluidic system was developed to perform online monitoring
of the secretion profiles of amino acids from 2 – 5 islets. The device contained an islet chamber with the ability to perfuse stimulants,
and an amino acid measurement system with derivatization and electrophoretic separation integrated on a single microchip. The setup was
optimized to allow -15 kV to be applied to the device for high efficiency and rapid separations of derivatized amino acids. The
compositions of the derivatization and separation buffers were optimized to prevent precipitations in the channels, which allowed
continuous monitoring of secretion for over 2 hours. With this method, 10 amino acids were resolved with limits of detection ranging from
1 – 20 nM. When murine islets were perfused with 3 mM glucose, the secretion rates of 9 amino acids were measured and ranged from 30 to
400 fmol islet-1 min-1. As the glucose concentration was increased to 20 mM, the dynamic changes of amino acids were monitored. The
biological relevance of the amino acid secretions was verified using 2,4-dinitrophenol as an inhibitor of the proton motive force. The
microfluidic system was also used to measure dynamic changes of amino acid release from human islets, which showed different release
profiles compared to their murine counterparts. === A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of
the requirements for the degree of Doctor of Philosophy. === Spring Semester 2016. === March 29, 2016. === AMINO ACIDS, FLUORESCENCE, ISLETS OF LANGERHANS, MICROFLUIDICS, REACTIVE OXYGEN SPECIES === Includes bibliographical references. === Michael G. Roper, Professor Directing Dissertation; Wu-Min Deng, University Representative;
Joseph B. Schlenoff, Committee Member; Oliver Steinbock, Committee Member. |
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