Measurement of Small Molecules in Islets of Langerhans
Wang, Xue (author)
Roper, Michael Gabriel (professor directing dissertation)
Deng, Wu-Min (university representative)
Schlenoff, Joseph B. (committee member)
Steinbock, Oliver (committee member)
Florida State University (degree granting institution)
College of Arts and Sciences (degree granting college)
Department of Chemistry and Biochemistry (degree granting department)
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.
AMINO ACIDS, FLUORESCENCE, ISLETS OF LANGERHANS, MICROFLUIDICS, REACTIVE OXYGEN SPECIES
March 29, 2016.
A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Michael G. Roper, Professor Directing Dissertation; Wu-Min Deng, University Representative; Joseph B. Schlenoff, Committee Member; Oliver Steinbock, Committee Member.
Florida State University
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them.