The work in this dissertation presents methods for study of glucose stimulated insulin secretion as well as other hormones
involved in glucose homeostasis. A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of
Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver
sinusoidal glucose waveforms to the islet chamber. Insulin levels in the perfusate were measured using an online competitive
electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration
points ranging from 2–8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3–6 min.
Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations
to this forcing period. Exposing groups of 6–10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent
pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of
oscillatory insulin secretion. A dual detection microscopy system was developed to simultaneously image intracellular messengers and
monitor insulin secretion from islets of Langerhans. Glucose stimulated insulin secretion plays a critical role in glucose homeostasis,
but the underlying mechanism is still unclear. To develop an automated system to simultaneously study the correlation between
intracellular events and insulin secretion, fluorescence imaging and laser induced fluorescence detection for insulin immunoassay were
integrated into a single microscopy system. Intracellular calcium ([Ca2+]i) was used as a representative secondary messenger and studied
with insulin secretion from islets during exposure to constant and oscillatory glucose levels. Both of [Ca2+]i and insulin were
oscillatory during constant glucose levels and entrained to a small amplitude sinusoidal wave of glucose (0.5 – 2 mM). [Ca2+]i and insulin
oscillations were temporally but not necessarily quantitatively correlated. Oscillatory glucose waveforms amplified insulin oscillation
amplitude without further increasing [Ca2+]i, which could be related to amplifying pathway. The developed system was also applied to study
the effect of glucokinase activator 22 on [Ca2+]i and insulin secretion. These results indicated the robustness of the developed method,
which can be potentially expanded to include other intracellular messengers to study the mechanism of glucose stimulated insulin
secretion. A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid
polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled
changes in the perfusion media within 5 s, allowing rapid exchange of constant glucose concentrations delivered to groups of islets. The
perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate
(FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was
injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers
with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged
from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from
groups of 4−10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio
and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple
peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of
glucose homeostasis. The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror image
aptamers, so-called Spiegelmers, is demonstrated. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity
assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM.
Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin
gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and
insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an
important step towards incorporation into an online measurement system for monitoring dynamic secretion from single
islets. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of
the Doctor of Philosophy. / Spring Semester 2016. / April 4, 2016. / affinity assay, capillary electrophoresis, hormone, islet of Langerhans, microfluidics / Includes bibliographical references. / Michael G. Roper, Professor Directing Dissertation; P. Bryant Chase, University Representative;
John G. Dorsey, Committee Member; Kenneth L. Knappenberger, Jr., Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_360490 |
| Contributors | Yi, Lian (authoraut), Roper, Michael Gabriel (professor directing dissertation), Chase, P. Bryant (university representative), Dorsey, John G. (committee member), Knappenberger, Kenneth L. (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting department) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource (119 pages), computer, application/pdf |
| Rights | 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. |
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