Robert Jason Walker - ETD Index Page

Robert Jason Walker
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Doctoral Dissertation
Robert Jason Walker
rjwalke@gmail.com
Beta-2-Adrenergic Receptor Regulates Insulin
Signaling to Reduce Cell Death in Müller Cells
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Doctor of Philosophy
NA
Integrated Program in Biomedical Sciences
Neuroscience
Jena Steinle Ph.D.
Jsteinl1@uthsc.edu
Suleiman Bahouth, Ph.D.
Edward Chaum, M.D. Ph.D.
Monica Jablonski, Ph.D.
Tonia Rex, Ph.D.
Graduation Date: May 2012
beta-adrenergic receptors, diabetes, Muller
cells, retina
World-Wide Web Access

Abstract
Changes in the diabetic retina likely occur in response to a variety of insults, including high
glucose, oxidative stress, and increased levels of inflammatory markers. Müller cells become
activated and express increased glial fibrillary acidic protein levels in diabetes. This suggests that
these cells are in a reactive state that may alter their regulation of inflammatory markers, glucose
transport, oxidative stress, growth factors, and cell survival. During the onset of diabetes, there is
a loss of sympathetic nerve activity that takes place in different regions of the body. Previous
studies from our lab suggested that loss of sympathetic neurotransmission induces diabetic-like
changes to the retina, specifically in Müller cells and endothelial cells.
In previous studies, we found that retinal Müller cells possess beta-1 and beta-2 adrenergic
receptors, and with those findings we used a non-specific beta agonist, isoproterenol as a
treatment to stimulate both receptors. In addition we used a selective beta-2-adrenergic receptor
agonist, salmeterol, to selectively stimulate the beta-2-adrenergic receptor in Müller cells.
Salmeterol significantly increased tyrosine phosphorylation of the insulin receptor, Akt, and
significantly reduced apoptosis in retinal Müller cells. Our data showed that salmeterol reduced
TNF-α levels. TNF-α phosphorylates IRS-1 on Ser307 and inhibits insulin signaling.
Salmeterol inhibited , TNF-α and thereby increased insulin signal transduction.
Based upon these findings, we generated shRNA against TNF-α and IRS-1. Knockdown of IRS-
1 with shRNA significantly increased in cell death compared to retinal Müller cell samples
cultured in 5mM glucose alone. However stimulation of beta-2-adrenergic receptor prevented
cell death through IRS-1 in a hyperglycemic environment. We demonstrated that knockdown of
TNF-α by shRNA significantly reduced the phosphorylation of serine 307 on IRS-1, leading to
increased Akt activity. Our results are in agreement with other studies in adipose cells, that
found TNF-α to be a negative regulator of the insulin receptor signaling. We believe that these
results provide a putative mechanism for the effects of beta-adrenergic receptors in reducing
retinal damage in diabetes. These studies will help to further characterize the function of beta-2-
adrenergic receptors in the retina and also in a disease state, such as diabetic retinopathy.
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Revised 02 May 2012