I hypothesize that maternal hyperglycemia induces changes in DNA
methylation in the developing heart thus increasing the risk of
congenital heart defects due to abnormal mRNA expression of cardiac
important genes. I have three specific aims to test this
hypothesis. Aim 1: To analyze the cardiac phenotypes of embryos
born to dams with hyperglycemia compared with pups of euglycemic
pregnancies. Aim 2: To identify methylationsensitive DNA loci
within developing fetal hearts that are more vulnerable to maternal
hyperglycemia using a
high-resolution genome-wide cytosine methylation profiling assay.
Aim 3: To juxtapose differential mRNA expression in developing
fetal hearts with changes in genome wide DNA methylation.
Research strategy:
Diabetes will be induced in standard 8 week old CD-1 WT female mice
with a one time intraperitoneal injection of 150 mg/kg of
streptozotocin (STZ). Histological analysis of fetal cardiac
morphology will be performed on the hearts of the hyperglycemic and
the euglycemic pupps at day E16.5. We will extract the hearts at
different timepoints and use genome wide cytosine methylation
profiling to delineate the difference between pupps of
hyperglycemic mothers versus controls. We will then juxtapose
differential mRNA expression. With this information, we can then in
the future obtain gene candidates that may be the targets for
intervention.
Expected Results:
We anticipate that maternal diabetes will alter DNA methylation of
specific genes related to cardiac development. We believe that by
sequencing the entire genome of the extracted DNA after creating
our library following the modified HELP-tagging assay and sequence,
we will identify candidate genes that are implicated in
dysregulation of the methylation patterns that ultimately
predispose these embryos to congenitally acquired cardiac
lesions.
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The overall objective of this study is to identify selective
activators and inhibitors of the two-pore-domain potassium channel,
TALK-1. TALK-1 channels are key regulators of pancreatic -cell
electrical excitability, Ca2+ homeostasis and insulin secretion.
KCNK16 the gene that codes for TALK-1 is the most abundant K+
channel transcript of the islet and TALK-1 is the most
islet-restricted ion channel. Moreover, a mutation in KCNK16
results in neonatal diabetes and a nonsynonymous polymorphism in
KCNK16 causes an increased predisposition for type-2 diabetes.
However, our understanding of the role(s) of TALK-1 channel in
human islets remains obscure due to a lack of specific and potent
pharmacology. Therefore, this project will utilize a robust
thallium (Tl+) based fluorescent assay in a high throughput screen
(HTS) to identify pharmacological probes of the human TALK-1
channel. The assay will be performed on a tetracycline inducible
TALK-1 cell line, which was selected for based on its performance
in the Tl+ assay. The TALK-1 Tl+ assay was validated with primary
screens of two small molecule libraries including the Spectrum
Collection (~2320 molecules) and a bioactive lipid library (~928
molecules), which identified a small cohort of activators and
inhibitors of TALK-1. The primary screens were utilized to optimize
the Tl+ assay for use with the TALK-1 cell line in a large HTS.
Building on these preliminary studies, this proposal plans to
perform a HTS on the human TALK-1 channel with 35,000 structurally
diverse small molecules from the Vanderbilt HTS small molecule
library. This will be accomplished using 1. A Tl+ flux based HTS,
which will be followed by 2. Secondary assays utilizing Tl+ flux,
electrophysiology and Ca2+ imaging to support rapid hit-to-lead
progression. This will be followed by assays to determine the
mechanism of action of TALK-1 regulation including TALK-1 in
single
channel inside out membrane patches as well as inhibition of
G-protein-coupled receptor signaling, and finally 3.Molecular
regulators of TALK-1 activity identified in this HTS will be
utilized to test the influence of TALK-1 channels on human and
mouse islet cell electrical activity, Ca2+ homeostasis and insulin
secretion.
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Macroautophagy (hereafter referred to as autophagy) is an intracellular catabolic pathway involved in recycling of cellular components. The normal functioning of a variety of cell types important in both diabetes and obesity, including pancreatic beta cells and adipocytes, depends heavily on autophagy. Thus, it is critically important to understand the mechanisms that underlie this process. MAP1LC3B (LC3) is one of the central proteins involved in autophagy, playing essential roles in both the formation of autophagosomes as well as the capturing cargo for degradation by autophagy. Emerging evidence suggests nuclear forms of LC3 are also important in autophagy. However, the functions of nuclear LC3 are still largely unexplored and little information is currently available regarding LC3’s interacting partners in the nucleus. Work from our group has shown that in the nucleus, LC3 is contained within ~1000 kDa complexes. We also identified an initial list of candidate LC3-interacting proteins using mass spectrometry. These studies have revealed a number of intriguing candidate interacting partners of nuclear LC3, including ribosomal subunits and tubulin. These findings lead us to hypothesize that additional functions of nuclear LC3 remain to be discovered, and that the identification of bona fide nuclear LC3 interacting proteins could provide new insights into how autophagy maintains cellular and organismal homeostasis. Here, we propose to leverage and expand on our initial findings by i) determining how the nuclear LC3 interactome is physiologically regulated and ii) validating key interacting partners of nuclear LC3 and exploring the functional significance of their interactions. These studies will thus set the stage for obtaining external funding for future work focused on identifying new functions of nuclear LC3. Ultimately, these studies should provide a framework that will inform our understanding of how autophagy functions as a general regulator of metabolism and how this pathway can be exploited to help prevent and treat diabetes and obesity.