Alterations of a Putative Phospholipid Translocase, Atp10c

150 Pages·2016·1.62 MB·English

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Doctoral Dissertations Graduate School
5-2012
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Sarah Elizabeth Hurst
[email protected]
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Hurst, Sarah Elizabeth, "In Vitro Alterations of a Putative Phospholipid Translocase, Atp10c, and Its Role in
Glucose Metabolism. " PhD diss., University of Tennessee, 2012.
https://trace.tennessee.edu/utk_graddiss/1308
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To the Graduate Council:
I am submitting herewith a dissertation written by Sarah Elizabeth Hurst entitled "In Vitro
Alterations of a Putative Phospholipid Translocase, Atp10c, and Its Role in Glucose
Metabolism." I have examined the final electronic copy of this dissertation for form and content
and recommend that it be accepted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy, with a major in Comparative and Experimental Medicine.
Madhu Dhar, Major Professor
We have read this dissertation and recommend its acceptance:
John Biggerstaff, Guoxun Chen, Carla Sommardahl
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
In Vitro Alterations of a Putative Phospholipid Translocase,
Atp10c, and Its Role in Glucose Metabolism

A Thesis Presented for the
Doctor of Philosophy
Degree
The University of Tennessee, Knoxville

Sarah Elizabeth Hurst
May 2012
DEDICATION

Once upon a time, chemist and physicist Marie Curie said,
“I am among those who think that science has great beauty.
A scientist in his laboratory is not only a technician:
He is also a child placed before natural phenomena
which impress him like a fairy tale.”
This dissertation is dedicated to the child in all of us
who are still fascinated by the fairy tale that is science.
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ACKNOWLEDGEMENTS

There are many people that deserve acknowledgement and thanks for their help in
the completion of this thesis dissertation. First and foremost, I must thank my Heavenly
Father for all the wondrous blessings He bestows on me from day to day. I am truly
thankful for His grace and mercy, which is sufficient for a sinner like me. Secondly, I’d
like to acknowledge my parents, W. Dale and Judy Hurst, and my grandparents, Gerald
and Virginia Hurst and John and Lelia Woodruff. Their pride in my accomplishments
motivates me and pushes me onward to future goals. I also must thank my friends for
their unfailing love and support. I wouldn’t have made it through the past few years
without it!
Without hesitation, I’d like to thank my advisor and mentor, Dr. Madhu Dhar.
During my time here at the University of Tennessee-Knoxville, I came to respect her as
both a mentor and friend, and as a skilled scientist and researcher. She is definitely one of
the most dedicated and generous people that I have ever had the privilege to work with,
and to know. She believed in me in times when I needed it the most and took a chance on
me when no one else would. For that I dedicate the work in this dissertation to her. I am
truly grateful for all you have done in my life.
I would also like to acknowledge the assistance and guidance from the remaining
members of my dissertation committee, Drs. John Biggerstaff, Guoxun Chen, and Carla
Sommardahl. Their willingness and dedication to my graduate research experience was
greatly appreciated. I am very thankful for all their help and encouragement over the last
few years. I would also like to give a special thanks to several generous collaborators,
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Drs. Sujata Agarwal, Steven Minkin, John Dunlap, Nolan Hoffmann, Jeffrey Elmendorf,
and Ling Zhao, and Mr. Keith Prater for their help in learning and running real-time PCR
experiments, in immunofluorescence techniques and imaging, and lastly, in procuring the
L6-G4myc samples and the mouse tissue samples from the DIO and ob/ob mice. Without
these individuals, I truly would have been lost and this project would not have ended as it
has done.
Last, but not least, I would like to give a heartfelt thanks to all the faculty and
staff in the Department of Large Animal Clinical Sciences, especially Ms. Catheryn
Hance and Ms. Linda Beets. Their help was immediate and much appreciated! I also need
to thank the Comparative and Experimental Medicine Program and the University of
Tennessee Veterinary Medical Center, and its associated faculty and staff. Ms. Kim
Rutherford and Ms. Misty Bailey proved invaluable assistance when asked and always
provided a smiling face and a kind word when it was most needed. Moreover, the work
in this dissertation would not have been possible without the funding agencies, The
American Diabetes Association, The University of Tennessee Center of Excellence in
Livestock Diseases and Human Health, and the University of Tennessee Obesity
Research Center. Additional thanks to the unsung heroes, Ms. Nancy Neilson, Ms. Sarah
Elliot, the soon-to-be Dr. Erin Bartley, and Ms. Lisa Amelse, who have helped me so
much along the way!
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ABSTRACT

Atp10c/ATP10C is a putative aminophospholipid translocase or “flippase” that
encodes for a type 4 P-type ATPase. Based on previous research, we hypothesize that
Atp10c/ATP10C due to its flippase nature plays a role in diet-induced obesity and type 2
diabetes as Atp10c heterozygous mice display this phenotype.
For purposes of this dissertation, Atp10c/ATP10C was characterized both
generally and biologically to gleam information about its molecular weight, cellular
location and possible biological roles and/or functions. Multiple experiments, both in
vitro and in vivo, were performed in order to accomplish these characterizations and are
discussed at length.
Essential results from this dissertation work include the validation of ATP10C’s
expected molecular weight (~165 kD), the localization of ATP10C to the plasma
membrane, its possible co-localization with GLUT4, and the high expression of Atp10c in
key tissues (skeletal muscle and adipose tissue) of two mice models, one a model of diet-
induced obesity mice and another a genetic model (ob/ob).
To identify molecular and cellular targets of ATP10C, Atp10c expression was
altered in vitro in C2C12 and L6-G4myc skeletal muscle myotubes. Functional outcomes
of GLUT4 translocation and glucose uptake assays were performed resulting in the
significant alteration of normal GLUT4 regulation and a significant decrease in glucose
uptake. Additionally, when the expression of Atp10c was altered both experimentally and
genetically, significant up-regulation of native and activated mitogen-activated protein
kinases, p38, and ERK1/2 were observed. These results demonstrate that Atp10c-
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silencing does affect these main mitogen-activated protein kinase proteins and in turn,
glucose metabolism via both insulin-independent and insulin-dependent manners.
Taken together, all these results presented in this dissertation indicate that
Atp10c/ATP10C has an important role in the regulation of glucose metabolism, at least in
part via the mitogen-activated protein kinase pathway, and as such, plays a significant
role in the development of insulin resistance and type 2 diabetes.

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TABLE OF CONTENTS
CHAPTER I………………………………………………………………….....................1
Introduction………………………………………………………..........................1
Disease Epidemics………………………………………….......................1
Genetics, Environment, or Factors of Both………………….....................2
CHARTER II.......................................................................................................................4
Literature Review.....................................................................................................4
Insulin Resistance and Type 2 Diabetes......................................................4
Cell Culture Models to Study Insulin Resistance and Type 2
Diabetes........................................................................................................8
P-type ATPases and Atp10c/ATP10C.......................................................10
Glucose Metabolism Pathways and Insulin Resistance.............................20
Phosphatidylinositol-3-Kinase (PI3K) Pathway............................25
Mitogen-Activated Protein Kinase (MAPK) Pathway..................27
Gene/Protein Modification Techniques.....................................................31
RNAi..............................................................................................31
Overexpression..............................................................................32
Research Project Overview.......................................................................33
CHARTER III...................................................................................................................35
Experimental Investigations...................................................................................35
General Characterization...........................................................................35
Abstract..........................................................................................35
Introduction...................................................................................36
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Materials and Methods...................................................................37
Cell Culture....................................................................................38
Mouse Treatment and Sample Collection......................................39
RNA, cDNA synthesis, and qPCR.................................................39
Transfection of ATP10C plasmids.................................................40
Preparation of Cellular, Extracts, Immunoblotting, and
Immunofluorescence......................................................................41
Immunofluorescence quantitation..................................................42
Results and Discussion..................................................................43
Conclusions....................................................................................54
Biological Characterization.......................................................................56
Abstract..........................................................................................56
Introduction....................................................................................57
Materials and Methods...................................................................60
Cell Culture and Treatments..........................................................61
Mouse Treatments and Sample Collection....................................62
siRNA Transfection.......................................................................62
RNA, cDNA, and qPCR................................................................63
Preparation of Cellular, Extracts and Immunoblotting..................64
Immunofluorescence and Immunocytochemistry..........................65
Glucose Uptake..............................................................................66
Densitometry Analysis...................................................................67
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