A Thesis

entitled

Induction of the Lipid Regulator PPAR-Delta in FoxO1 Overexpressed Skeletal Muscle

by

Vesna Markovic

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Masters of Science Degree in

Exercise Science

______Dr. Thomas J. McLoughlin, Committee Chair

______Dr. Francis X. Pizza, Committee Member

______Dr. Abraham D. Lee, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo

May 2018

Copyright 2018, Vesna Markovic

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Induction of the Lipid Regulator PPAR-Delta in FoxO1 Overexpressed Skeletal Muscle

by

Vesna Markovic

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Masters of Science Degree in Exercise Science

The University of Toledo

May 2018

The Forkhead box O1 (FoxO1) molecule plays a vital role in the production of metabolism.1, 2 However, the precise mechanisms for its upregulation are yet to be fully understood. 1, 2 Preliminary data has shown that overexpression of FoxO1 in skeletal muscle results in a dramatic decrease in intramuscular triglyceride storage.3, 4 A potent stimulator of fatty acid metabolism in skeletal muscle is peroxisome proliferator activated delta (PPAR-Delta). As such, the purpose of the present study was to investigate the interplay between FoxO1 and PPAR-Delta in the promotion of fatty acid metabolism in skeletal muscle. Our hypothesis is that PPAR-Delta will have a greater protein expression in skeletal muscle overexpressing FoxO1 versus normal skeletal muscle. In this study, western blotting was used to detect PPAR-Delta expression while densitometry was used to quantify expression levels. Our findings show that PPAR-Delta expressed higher levels of protein expression in the overexpressed FoxO1 skeletal muscle versus the wildtype skeletal muscle. This suggests that PPAR-Delta may contribute to adaptive changes in fatty acid metabolism in response to FoxO1 overexpressed skeletal muscle. Further studies need to

v be conducted to investigate the more in-depth relationship between the interplay of

PPAR-Delta and FoxO1 in fatty acid metabolism in skeletal muscle.

vi

This is dedicated to those who have had nothing but endless support for me through this chapter in my life and never have given up on me. To my parents, who have been there with me every step of the way and never letting me falter. Without their help I wouldn’t have gotten where I am at today. To my grandparents, who have done nothing but help me every step of the way and do everything in their power to make sure I succeed and accomplish my dreams. To my best friends, you were always there when I needed you the most.

vii

Acknowledgements

Committee Members: Dr. McLoughlin, you have been my advisor for the past two years of my life and have had endless amounts of patience through my learning process and helping me achieve my “master’s plus” degree. I appreciate the opportunity you bestowed on me to further my education. Furthermore, I value your advice, vital insight on science, and have the upmost respect for you. Dr. Pizza, thank you for all your help and irreplaceable knowledge of science that you have given to me in the vast realm we call science. It has been greatly appreciated. Dr. Lee, thank you for your support and help throughout the program. You always gave me a new perspective on the research and in turn you helped expound my knowledge. Friends: During my time as a graduate student and assistant, you were all there for me to support me in your own ways. If I ever needed anything I knew I could count on you ladies to be there for me no matter what. I greatly appreciate your friendship and your support when I have needed it the most.

Thank you to Laura Magni, Christina Hart, Laurin Jeffers, and Alexia Shrout. To many more wonderful years of friendship to come. Family: I cannot thank my family enough for the support they have given me throughout my life in general. I would like to thank my parents, Tomislav and Suzanna Markovic, for always being there to support me and always believing me in me. You both push me to be the best version of myself. Finally, to my grandparents, Branko and Nada Tasic and Mika and Mica Markovic, you guys are my foundation. Without you I would not be here or be the person I am today. I would not have made it through without any of their constant support, guidance, and love. I do not know how lucky I got to have each and every one of you in my life.

viii

Table of Contents

Abstract ...... v

Acknowledgements ...... viii

Table of Contents ...... x

List of Figures ...... xi

1 Introduction… ...... 1

1.1 Background and Significance ...... 1

1.2 Specific aim...... 3

2 Literature Review...... 4

2.1 FoxO Factors ...... 4

2.1.1 Structure of FoxO’s...... 5

2.2 FoxO1 in Skeletal Muscle...... 6

2.2.1 Role of FoxO1 in energy metabolism ...... 6

2.3 Peroxisome Proliferator-Activated Receptor ...... 8

2.3.1 Structure ...... 9

2.3.2 PPAR-Delta...... 10

2.4 PPAR-Delta and FoxO1...... 11

3 Research Design and Methods ...... 13

3.1 Protein Homogenization ...... 13

3.2 Protein Quantification ...... 14

3.3 Protein Preparation...... 15

3.4 10% Tricine Gel Preparation...... 15

ix

3.5 Western Blotting ...... 16

3.6 Statistical Analysis ...... 17

4 Results...... 18

4.1 The Efficacy of the Model ...... 18

4.2 PPAR-Delta in Overexpressed FoxO1 Skeletal Muscle Tissue ...... 21

5 Discussion...... 23

References...... 26

x

List of Figures

4-1 Total FoxO1 and GAPDH protein expression...... 20

4-2 Total PPAR-Delta and GAPDH protein expression...... 22

xi

Chapter 1

Introduction

1.1 Background and Significance

In every living organism it is essential for survival to adjust macronutrient metabolism to physiological conditions and nutrient availability. In mammals, excess energy is stored primarily as triglycerides, which are mobilized when demand for energy arises.5 It has become increasingly clear that all macronutrients play an important role in the regulation of energy metabolism. However, in muscle, it appears that fatty acids supply a major fraction of the energy required for muscle function and contribute to the intricate regulation of muscle metabolism.1, 6-8

Forkhead box O1 protein (FoxO1) is a key regulator of muscle growth, metabolism, cell proliferation and differentiation predominantly expressed in most muscle types.9-12 A crucial mechanism of FoxO1 is fatty acid metabolism which is a complex process in skeletal muscle. Fatty acids are mobilized from stored triacylglycerols through the activity of hormone sensitive lipases to yield free fatty acids.13 The free fatty acids are activated by coupling to Coenzyme A.13 This step is

1 catalyzed by the acyl-CoA synthetase family of enzymes followed by conversion of acyl-

CoA into Acylcarnitine where the free fatty acids are imported into mitochondria.13

Finally, in mitochondria, the fatty acids are oxidized.13 Some of the steps in this metabolic pathway are known to be regulated by signaling.13 For instance, insulin signaling inhibits expression and activity of lipases such as adipose triglyceride lipase and hormones sensitive lipase.13 Insulin signaling also decreases the rate of fatty acid entry into mitochondria in part by a FoxO-dependent process.13

The FoxO1 appears to play an important role in regulating skeletal muscle homeostasis through upregulation of certain .1, 14 One of these upregulated protein expressions are PPAR-Delta. In skeletal muscle, PPAR-Delta acts as a key regulator of fuel metabolism, promoting a shift from to lipids as the main energy substrate involving FoxO1.1-3,21, 26, 28, 15 It promotes cellular lipid uptake, activation of fatty acids by fatty acyl CoA synthetase, and their mitochondrial uptake and beta-oxidation with decreased glucose oxidation as a consequence.1-3, 14, 16-19 In addition,

PPAR-Delta in skeletal muscle results in enhanced lipid metabolism as an adaptive response to external stimuli such as food availability and prolonged physical activity.1, 11,

12, 21, 26 Given that activation of PPAR-Delta in skeletal muscle enhances lipid use for energy expenditure, which is preferred to glucose and allows glucose to become more available for peripheral organs, this protein could potentially be used as a therapeutic target for some metabolic disorders.1-7,11,21,26,16, 20-23

A protein that is implicated in being upregulated when FoxO1 is activated is

PPAR-Delta.1, 24 We are interested in examining if there is a difference in the expression of PPAR-Delta protein between the wild type skeletal muscle and the transgenic skeletal

2 muscle tissue that overexpresses FoxO1 because compared to our wildtype mice, the transgenic mice showed a reduction in triglyceride levels, reduced fat storage in skeletal muscle, and a constant state of lethargy.3, 4 Therefore, the increased FoxO1 levels in skeletal muscle and associated reduced fat storage may be due to increased PPAR-Delta expression.14 The objective of this study was to examine the protein expression of PPAR-

Delta in FoxO1 overexpressed skeletal muscle versus normal skeletal muscle tissue in mice. The central hypothesis is that PPAR-Delta has an increased expression in the

FoxO1 overexpressed skeletal muscle in transgenic mice versus the skeletal muscle in wildtype mice.

1.2 Specific Aim

To assess the protein expression of PPAR-Delta in FoxO1 overexpressed skeletal muscle tissue versus normal skeletal muscle tissue in wildtype mice.

Hypothesis: PPAR-Delta protein expression in the FoxO1 overexpressed skeletal muscle tissue of the transgenic mice will be greater than that of skeletal muscle tissue in wildtype mice.

3

Chapter 2

Literature Review

Skeletal muscle is the most abundant tissue in the body. 25, 26 It is an active metabolic organ with high plasticity for adaptive responses to varying conditions such as fasting or physical exercise.1 In addition to its primary role in posture and movement, skeletal muscle performs a number of critical functions, such as the regulation of energy and glucose metabolisms. 11, 25 FoxO1, a member of the forkhead transcription factor forkhead box protein O family, is predominantly expressed in most muscle types.10, 11

FoxO1 is a key regulator of muscle growth, metabolism, cell proliferation and differentiation.9 The transcription factor, FoxO1, is implicated in several of these pathways that are linked to muscle metabolism.

2.1 FoxO Transcription Factors

The forkhead transcription factor family is characterized by a winged-helix DNA binding motif and the forkhead domain. 27 The mammalian forkhead transcription factors

4 of the O class have four members: FoxO1, FoxO3, FoxO4, and FoxO6. 28 FoxO1 and

FoxO3 are expressed in nearly all tissues. 28 Multiple signals converge on FoxO factors including , mono- or polyubiquitination, , glycosylation and arginine or methylation. 11, 12, 29 A major mechanism of FoxO1 is phosphorylation.

Phosphorylation of FoxO1 prevents nuclear localization and DNA binding to the regions of its protein targets, thereby inhibiting transcription of its protein targets. 11, 12, 28 AMPK and Akt have opposite effects on FoxO3 localization and activity.

11, 12, 29 While Akt inhibits FoxO1, 3 and 4 by phosphorylation and 14-3-3 binding under mitogenic activation, AMPK phosphorylates FoxO3 at two regulatory sites in skeletal muscle, leading to its activation under stress conditions. 12, 29 Moreover, AMPK activation is associated with increasing levels of FoxO1 and FoxO3 mRNAs and protein content. 12, 29

2.1.1 Structure of FoxO’s

Molecules of FoxO consist of four domains. These domains are a highly conserved forkhead DNA binding domain, a nuclear localization signal, a nuclear export sequence, and a C-terminal transactivation domain. 11, 28 FoxO1 and FoxO3 proteins have similar length of approximately 650 amino-acid residues, whereas FoxO4 and FoxO6 sequence is shorter and are about 500 amino-acid residues. 11, 27 Lastly, some of the regions of the FoxO proteins are highly conserved. The highly conserved regions of the

FoxO proteins include the N-terminal region surrounding first AKT/ phosphorylation site, the forkhead DNA binding domain, the region containing the

5 nuclear localization signal and the part of the C-terminal transactivation domain. 11, 12, 27

FoxOs recognize two response elements: the Daf-16 family member binding element (5′-

GTAAA(T/C)AA) and insulin-responsive element (5′-(C/A)(A/C)AAA(C/T)AA). 11, 28

The core DNA sequence 5′-(A/C)AA(C/T)A-3′ is recognized by all Fox-family members.

11, 30 According to Wang et al. 31 the research showed the following: The transport of

FoxO proteins through the nuclear pore is dependent on active-transport mechanisms. 31

The presence of a nuclear localization sequence is a prerequisite for maintaining proteins in the nucleus, whereas a nuclear export sequence maintains proteins in the .31

FoxO proteins have both a nuclear localization sequence and a nuclear export sequence within the C-terminal DNA binding domain. 31 Kinases and interactions with other proteins modulate the effectiveness of these nuclear localization sequences and nuclear export sequences, which forms the basis of FoxO shuttling in and out of the nuclear compartment. 31 The cytoplasmic sequestration of FoxO proteins is mediated by a combination of binding partners and changes in the properties of FoxO. 31 The chaperone protein 14-3-3 binds to FoxO factors in the nucleus and allows their active export. 31 It also blocks the nuclear localization signal to prevent FoxO re-entry into the nucleus. 31

2.2 FoxO1 in Skeletal Muscle

2.2.1 Role of FoxO1 in Energy Metabolism

FoxO1 plays a significant role in the regulation of skeletal muscle energy metabolism. It is an important regulator of glucose metabolism in skeletal muscle.6, 10, 11,

6

32 FoxO1 can bind directly to the promoter region of the pyruvate dehydrogenase kinase 4

(PDK4), a key factor in maintaining the blood glucose level, to promote its expression during energy deprivation in skeletal muscle. 6, 33 High levels of PDK4 induce a decrease in the activity of pyruvate dehydrogenase, which catalyzes the reaction from pyruvate to acetyl-CoA and leads to lower use of carbohydrates as an energy substrate. 6, 12, 29

Therefore, the increase in PDK4 expression mediated by FoxO1 results in the conservation of glucose and gluconeogenic substrates and a decrease in glycolytic flux by inactivating the pyruvate dehydrogenase complex. 6, 12, 34

In addition to its significant role in glucose metabolism, FoxO1 also has an important role in lipid metabolism. Starvation appears to promote the expression of

FoxO1.35 A critical component of muscle metabolism during fasting is a switch from oxidation of carbohydrates as the major energy source to fatty acids which FoxO1 is involved in.13, 35 Fasting and exercise increase the expression of lipoprotein lipase, the enzyme that hydrolyses plasma triglycerides into fatty acids and glycerol for uptake by the muscle. 21,6, 35 Furthermore, FoxO1 enhances metabolism through additional mechanisms, including cluster differentiation 36 (CD36) recruitment to the plasma membrane and the enhancement of fatty acid oxidation.7, 27 In addition to increasing fatty acid uptake by membrane recruitment of CD36, FoxO1 also suppresses expression of acyl-CoA carboxylase, which reduces levels of the fatty acid oxidation inhibitor malonyl-

CoA. 7, 27, 36 Thus, FoxO1 contributes to regulating muscle glucose and fatty acid preference during periods of fasting or feeding. 7, 27, 36

7

2.3 Peroxisome Proliferator-Activated Receptor

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of nuclear superfamily comprising of three subtypes PPAR-Alpha, PPAR-Gamma, and PPAR-Beta/Delta. Activation of PPAR-

Alpha reduces triglyceride level and is involved in regulation of energy homeostasis. 26, 37

Activation of PPAR-Gamma causes insulin sensitization and enhances glucose metabolism, whereas activation of PPAR-Beta/Delta enhances fatty acids metabolism. 37

Even though all three PPAR isotype genes are located on different , they are often co-expressed at variable levels in different tissues with possible compensatory or complementary roles, including skeletal muscle where they exert specialized or pleiotropic responses.1, 26, 37 PPAR-Alpha is highly expressed in tissues with active fatty acid catabolism, such as in liver, heart, kidney, brown fat, intestine, macrophages, and muscle.1, 26, 37 It modulates all three fatty acid oxidation systems including peroxisomal and mitochondrial beta-oxidation and microsomal ω-oxidation, and plays a key role in lipid metabolism and energy expenditure.1, 26 PPAR-Gamma, expressed as the two isoforms PPAR-Gamma1 and PPAR-Gamma2, is predominantly present in and plays a crucial role in .1, 37 Furthermore, PPAR-Gamma1 is found in other tissues such as in breast, placenta, colon, liver, brain, macrophages and vascular cells.1, 26, 37 PPAR-Beta/Delta has multifaceted roles and is expressed in all organs at various levels, where it can have a function in development, lipid metabolism, energy

8 expenditure, tissue repair and regeneration, and .1, 26, 37 Therefore, the PPAR family plays a major regulatory role in energy and metabolic function.

2.3.1 Structure

All of the PPARs conform to a similar structure. It is a three-module structure, with a binder group involved in a series of hydrogen bonds in front of the ligand- dependent Activation Function, a linker mostly arranged around a phenoxyethyl and an effector end occupying the large cavity of the binding site. 16, 38 Furthermore, PPAR’s have four domains. The domains are activation function 1, DNA binding domain, ligand binding domain and activation function 2. 5 Activation function 1 is considered to mediate ligand independent activation, whereas Activation function 2 mediates ligand dependent activation. 17 The ligand binding domain consists of a ligand binding pocket, a dimerization domain and an activation domain.23 Activation function 2 is a short alpha- helix near the C-terminal of PPARs. 5 All three PPAR isotypes, PPAR-Alpha, PPAR-

Beta/Delta, and PPAR-Gamma, are activated by a variety of molecules, including fatty acids, eicosanoids and phospholipids, and regulate a spectrum of genes involved in development, lipid and carbohydrate metabolism, inflammation, and proliferation and differentiation of many cell types in different tissues.1, 16, 26, 37

9

2.3.2 PPAR-Delta

PPAR-Delta is found predominantly in skeletal muscle.18, 19, 37 In mouse skeletal muscle, PPAR-Delta is several folds more abundant than either PPAR-Alpha or PPAR-

Gamma.18, 37, 39 PPAR- Delta is ubiquitously expressed in whole body regulation energy expenditure.40 This ligand-activated transcription factor is cellularly localized in the nucleus. PPAR-Delta is directly implicated in fatty acid-induced cell proliferation and has a preference to bind to polyunsaturated acids.5, 10, 18, 26, 37, 40 Once it is activated by a ligand, the receptor binds to promoter elements of target proteins and regulates the peroxisomal beta-oxidation pathway of fatty acids and it functions as a transcription activator for the acyl-CoA oxidase.5, 10, 18, 26, 37, 40 In addition, it assists with glucose metabolism, insulin sensitivity, and regulates mitochondrial biogenesis.18, 37

The PGC-la promoter contains a highly conserved PPAR-responsive element that is conserved among human, mouse, and rat, where PPAR-Delta can directly bind suggesting that PPAR-Delta directly stimulates the PGC-la expression.18, 26 PPAR-Delta stimulates the PGC1α expression through a PPRE in its promoter.1 PPAR-Delta directly interacts with PGC-la, and activates the CPT1 together with PGC-la.18, 26 PGC1α co-activates the PPAR-Delta:RXR heterodimers to enhance the expression of proteins involved in fatty acid beta-oxidation.1, 16, 26 PPAR-Delta also stimulates the PGC1b independently of PGC1a through binding of MyoD and RelB and regulates mitochondrial biogenesis.1, 26

10

Its primary role is to enhance reliance of muscle cells on long chain fatty acids during fasting and prolonged exercise and diminish glucose utilization during fasting.18

During starvation, glucose uptake and oxidation are reduced rapidly in muscle, which shifts to use free fatty acids.18 Once activated by fatty acids PPAR-Delta drives the expression of a limited set of proteins thereby inducing adipose differentiation.26, 40

Overexpression of PPAR-Delta enhances fatty acid induction of the adipose-related proteins for fatty acid translocase and lipid binding protein. 40, 41

Mitochondrial oxidative and phosphorylation activity is strongly correlated with insulin sensitivity.18 Exercise, starvation, and cold increase the expression of PGC-la increasing the capacity for mitochondrial energy production through fatty acid- oxidation.18, 37 PPAR-Delta agonists increase the phosphorylation and expression of

AMPK. 1, 6, 26, 37Activated PPAR-Delta stimulates the expression of lipoprotein lipase and

CD36, increasing the flux of fatty acids into the muscle cells.1, 6, 26, 37 It also stimulates the expression of pyruvate dehydrogenase kinase 4, which reduces glucose oxidation.1, 6, 26, 37

2.4 PPAR-Delta and FoxO1

FOXO1 and PPAR-Delta are crucial transcription factors that regulate glucose metabolism, mitochondrial biogenesis, and fatty acid oxidation.1, 6, 18, 26, 40, 41 It is shown that PPAR-Delta increases the protein expression of FoxO1.6 PPAR-delta regulates glucose metabolism, mitochondrial biogenesis, and fatty acid metabolism by inducing

FoxO1 and PGC1-alpha.5, 10 Among the proteins induced by PPAR-Delta in fasting, a role of FoxO1 is particularly important for skeletal muscle. FoxO1 is activated during

11 fasting because of decreased insulin.5-7, 10 PDK4 phosphorylates and inactivates pyruvate dehydrogenase (PDH), inhibiting utilization of pyruvate for acetyl-CoA synthesis, and blocking glucose oxidation and favoring fatty acid oxidation to generate energy. 5-7, 10

FoxO1, through direct binding to a promoter sequence, increases PDK4 therefore being induced by activation of PPAR-Delta which is in large part likely mediated by FoxO1. 5-7,

10

These adaptations induced by PPAR-Delta activation in skeletal muscle appear to be critical for optimal functioning of the muscle to adjust fuel preference by suppressing glucose utilization and by relying more on fats during periods of nutrient shortage or exercise.5, 10 These changes are reversed rapidly upon feeding, when nutrient supply is abundant.5, 10

12

Chapter 3

Research Design and Methods

3.1 Homogenization of Skeletal Muscle

The mice that were used were female and male 12 to 18-week-old FoxO1 transgenic mice or female and male C57BL/6 12 to 18-week-old wildtype mice. The skeletal muscle extracted from these mice were quadriceps. The reagents needed for protein homogenization is NP-40 buffer and the protease inhibitors (Sodium orthovanadate and

HALT). One muscle sample was worked on at a time. The muscle was cut in half to approximately 50 milligrams and the appropriate amount of NP-40 and protease inhibitors (Sodium orthovanadate and HALT) were added to microtubes. Muscles were homogenized using bead homogenization and were lysed twice for two minutes at 30 hertz waiting 15 seconds in between. Once the bead homogenization was completed, the samples were put in a centrifuge and centrifuged for 10 minutes at 4 degrees Celsius. The new tubes are relabeled, and the homogenizing beads are removed. The supernatant is

13 carefully extracted and pipetted into the new labeled tubes and stored in the -80 Celsius freezer.

3.2 Protein Quantification

The materials needed for protein quantification was the DC protein assay kit (Bio-

Rad Laboratories, Inc., Hercules, CA). The components needed are reagent A, reagent B, reagent S, Bovine Serum Albumin (BSA), NP-40 buffer, and the unknown proteins. After determining the appropriate amounts of reagent A and reagent S, the standard curve was made. The BSA used was 1.42 micrograms per milliliter and the total volume of each standard cuvette needs to equate 50 microliters total. The cuvettes with the unknown proteins is 3 microliters of protein and 47 microliters of NP-40 buffer. In the standard curve the appropriate amount of BSA was added first then the appropriate amount of NP-

40 buffer was added second. 250 microliters of A prime was added to each cuvette then 2 milliliters of reagent B was added to each cuvette. The standard curve and unknown proteins are then left to incubated in the dark for 20 minutes. The samples were then vortexed and spectrophotometry is ran on the samples at 680nm. The samples were ran through the spectrophotometer twice, and the average was found for each sample of the standard curve and the unknowns.

14

3.3 Protein Preparation

Protein preparation is prepared in microtubes in quadruplicates containing 50 micrograms of protein for PPAR-Delta or 30 micrograms of protein for FoxO1. The components needed for protein preparation is the protein, homogenization buffer (NP-40) plus protease/phosphatase inhibitors (HALT, Sodium Orthovanadate), and loading buffer

(4x Sodium Dodecyl Sulfate (4x SDS), Dithiothreitol (DTT)). The protease/phosphatase inhibitors are then added to the NP-40 to make NP-40 + i. The amount of loading buffer is a 4:1 ratio of 4 parts 4x SDS to 1 part 2 M DTT. This is made fresh every time protein preparation is done. The order of the solutions in which they are added to the microtubes is homogenization buffer plus protease/phosphatase inhibitors, the protein, and the 4x loading buffer solution. The samples were then vortexed and returned to the -80 degree

Celsius freezer for storage.

3.4 10% Tricine Gel Preparation

The components for the gel are AB-3, 3x gel buffer, glycerol, distilled water

(dH20), 10% Ammonium persulphate (APS), and TEMED. First, the 10% APS solution is made with .02 grams of APS in 180 microliters of dH20 and then vortexed. 10 milliliters of 3x gel buffer, 6 milliliters of AB-3, 3 grams of glycerol, and 11 milliliters of dH20 was added. 150 microliters of the 10% APS solution was added first and 15 microliters of the

TEMED was added second with trituration after each. The gel mixture was pipetted into

15 each casting plate then water saturated butanol was added to the tops of the gel. Plastic wrap was placed on top of the two-casting plates holding the two separate gels and the gel was left to polymerize for an hour. Once the hour is over, the water saturated butanol was removed, and the gels were rinsed with dH20 three times. Finally, each casting plate was filled with dH20 to the top and covered with plastic wrap to stand overnight.

3.5 Western Blotting

The protein samples were heated at 37 degrees Celsius for 5 minutes and cooled.

The protein standard is loaded in the first lane at 10 microliters and the other lanes are loaded with 48 microliters of the proteins for FoxO1 (30 micrograms of protein) and 48 microliters for PPAR-Delta (50 micrograms of protein). The samples were resolved by

SDS-PAGE on 10% tricine gels at 80 milliamps(mA) for 6 hours and transferred to nitrocellulose membrane via semi dry blotting transfer (TransBlot Transfer Cell, Bio-Rad

Laboratories, Inc., Hercules, CA) for 45 minutes at 20 volts (V) constant. Following the transfer, the membranes were blocked in 5% non-fat dry milk in Tris-buffered Saline

(TBS) for 1 hour at room temperature than washed 4 times 5 minutes each in TBS. Once the membranes have completed their washes they are than spliced at 50 kDa to probe for

FoxO1 or not cut for the probing of PPAR-Delta. The membranes are placed in separate appropriate labeled incubation containers with the primary antibody and immunoblotted overnight at 4° C. The membranes are immunoblotted with PPAR-Delta polyclonal antibody (1:500; abcam, Cambridge, MA) or FoxO1 monoclonal antibody (1: 2,000; Cell

Signaling Technology, Beverly, MA) which were added to separate containers from

16

GAPDH monoclonal antibody (1:10,000; Cell Signaling Technology). Equal protein loading was verified using the GAPDH antibody (1:10,000; Cell Signaling Technology).

After the overnight incubation at 4 degrees Celsius, the membranes are then washed 4 times for 5 minutes with tris-buffered saline-tween 20 (TBS-T). The membranes were then incubated for an hour at room temperature in goat-anti rabbit

Alexa Flour 680 secondary antibody. The secondary antibody consists of 8 microliters of

Alexa Flour 680 secondary antibody (1:5000; Molecular Probes, Carlsbad, CA) and 40 milliliters 1% BSA in TBS-T. The proteins were analyzed via infrared detection

(Odyssey 2.1) and bands quantified through densitometry.

3.6 Statistical Analysis

Statistical Analyses were performed using SPSS. An independent t-test was utilized to determine differences in total FoxO1 expression and total PPAR-Delta expression. All the analyses used independent t-test (P ≤0.05) to determine differences between conditions (PPAR-Delta [wildtype vs. transgenic]) (FoxO1[wildtype vs.

Transgenic]) for all dependent variables. Data are reported as mean ± SEM.

17

Chapter 4

Results

4.1 The Efficacy of the Model

To establish the efficacy of the FoxO1 overexpression model, a western blot was performed on the wildtype and transgenic samples, and the samples probed for FoxO1 protein expression with GAPDH used as a loading control. As shown in Figure 1, total

FoxO1 protein expression in the transgenic samples compared to wildtype samples was significantly elevated. There were 8 transgenic samples and 8 wildtype samples. The mean in the transgenic samples probing for FoxO1 is 90.2638 arbitrary units with a SEM of ±35.38438. The mean in the wildtype samples probing for FoxO1 is 0.4838 arbitrary units with a SEM of ±0.25528. After running an independent t-test the significance of the two-tailed test was p<0.001.

As shown in Figure 1, the mean in the transgenic samples probing for GAPDH is

29.4388 arbitrary units with a SEM of ±3.35232. The mean in the wildtype samples probing for GAPDH is 27.3963 arbitrary units with a SEM of ±4.01567. The two-tailed test was .288 which shows that there were no statistical significant differences between

18 the transgenic and wildtype mice, which provides evidence that the samples were loaded equally across the groups.

19

A B

Transgenic FoxO1 Expression. (~75 kDa)

Wildtype FoxO1 Expression. (~75 kDa)

Transgenic GAPDH Expression. (~37 kDa)

Wildtype GAPDH Expression. (~37 kDa)

Figure 1: Total FoxO1 protein expression. Panel A: Representative western blots of total FoxO1 protein expression in wildtype and transgenic samples. GAPDH is representative of total protein loading. Panel B: Quantification of Total FoxO1 and GAPDH protein expression. *, FoxO1 expression is significantly higher in the transgenic than the wildtype samples. P<0.05 for all significant differences. n=8 for all groups.

20

4.2 PPAR-Delta in Overexpressed FoxO1 Skeletal Muscle Tissue

To establish if the protein of interest was present, PPAR-Delta, a western blot was performed on the wildtype and transgenic samples. These samples were probed for

PPAR-Delta using a polyclonal antibody.

As shown in Figure 2, there were 8 transgenic samples and 8 wildtype samples.

The mean in the transgenic samples probing for PPAR-Delta is 57.2813 arbitrary units with an SEM of ±26.03400. The mean in the wildtype samples probing for PPAR-Delta is 31.6463 arbitrary units with an SEM of ±6.07096. The significance of the two-tailed test was .027 which shows that PPAR-Delta protein expression was greater in transgenic and wildtype mice.

As shown in Figure 2, the mean in the transgenic samples probing for GAPDH is

86.1463 arbitrary units with an SEM of ±10.03495. The mean in the wildtype samples probing for GAPDH is 86.3900 arbitrary units with an SEM of ±4.78491. The significance of the two-tailed test was .952 which shows that there was no statistical significance between the mean of FoxO1 expression in transgenic and wildtype mice which provides evidence that the samples were loaded equally across the groups.

21

* Transgenic PPAR-Delta Expression. (~40 kDa)

Wildtype PPAR-Delta Expression. (~40 kDa)

Transgenic GAPDH Expression. (~37 kDa)

Wildtype GAPDH Expression. (~37 kDa)

Figure 2: Total PPAR-Delta protein expression. Panel A: Representative western blot of total PPAR-Delta protein expression in wild type and transgenic samples. Panel B: Quantification of Total PPAR-Delta protein expression. *, PPAR-Delta expression is significantly higher in the transgenic than the wildtype samples. P<0.05 for all significant differences. n=8 for all groups.

22

Chapter 5

Discussion

The purpose of this study was an attempt to garner insight into some metabolic targets involved in fatty acid metabolism that may explain the apparent increased fatty acid metabolism and/or diminished storage as a result of FoxO1 overexpression in skeletal muscle. The findings from this study demonstrate that PPAR-Delta has a greater protein expression in FoxO1 overexpressed skeletal muscle tissue versus wildtype skeletal muscle tissue.

Preliminary data from our lab showed that within the skeletal muscle of the transgenic FoxO1 mice there is a significantly reduced amount of stored triglycerides compared to the wildtype mice.3 Therefore, the increased FoxO1 levels in skeletal muscle and associated reduced fat storage may perhaps be due in part to increased PPAR-Delta expression.14 This may serve to enhance insulin sensitivity in skeletal muscle and, in turn, improve glucose disposal rates of metabolic disorders.14

In addition, these mice were semi-fasted. PPAR-Delta and FoxO1 seem to increase expression under fasted conditions.5, 18In conjecture that FoxO1 is increased,

PDK4 is also increased, which is known to inactivate the pyruvate dehydrogenase

23 complex, which is rate limiting in muscle carbohydrate oxidation, resulting in up- regulation of fatty acid metabolism as a result of FoxO1 activation. 5-7, 10, 18 It is known that during fasting, glucose uptake and oxidation are reduced rapidly in muscle, which shifts to use free fatty acids which is largely mediated by FoxO1. 5-7, 10, 18 This could possibly be another speculation why the results exhibited PPAR-Delta having an increased expression in FoxO1 overexpressed skeletal muscle.

Previous research that was conducted, observed overexpressed PPAR-Delta in mice which presented decreased adiposity. 1, 24, 42 When PPAR-Delta is overexpressed in the muscle in this form it expressed in the adipose tissue, specifically, increases fatty acid oxidation and energy dissipation, resulting in reduced fat mass, better lipid profiles, reduced adiposity, and resistance to a high-fat diet. 1, 24, 42 The observations appeared very similar in our FoxO1 overexpressed transgenic mice which show energy dissipation, reduced mass, and the amount of adipose stored in the muscles.

In addition, preliminary data from a previous study by Kamei et al.,4 observed that

FoxO1 transgenic mice weighed less than the wild-type control mice, had a reduced skeletal muscle mass, and the muscle was paler in color.4 Moreover, running wheel activity was significantly reduced in FOXO1 mice compared with control mice.4 This appears congruent with our lab observation. This could also be another conjecture of

PPAR-Delta increased expression in FoxO1 overexpressed skeletal muscle.

Considering the findings in this study, it is imperative to explicate the role of

PPAR-Delta in fatty acid metabolism in an overexpressed FoxO1 transgenic mouse model in relation to metabolic disorders. This can possibly be accomplished by looking at another agonist of PPAR-Delta such as exercise, to see if PPAR-Delta still has increased

24 expression in FoxO1 overexpressed skeletal. It is known that PPAR-Delta will have an increased expression while FoxO1 will have a decreased expression.1, 33, 8 Further research into the fatty acid metabolism mechanism and the interplay between PPAR-

Delta and FoxO1 will be needed to provide a breakthrough for metabolic and muscle disorders involving skeletal muscle.

.

25

References

1. Manickam R, Wahli W. Roles of Peroxisome Proliferator-Activated Receptor

beta/delta in skeletal muscle physiology. Biochimie. May 2017;136:42-48.

2. Li GQ, Liu XY, Xu SY. [Research advance on signaling pathways and protein

metabolism for skeletal muscle disuse atrophy]. Zhongguo Gu Shang. Nov

2013;26(11):969-972.

3. Schick EE. The effect of FoxO1 on glycemic control and skeletal muscle glucose

uptake and lipid metabolism. The University of Toledo Digital Repository. 2014.

4. Kamei Y, Miura S, Suzuki M, et al. Skeletal muscle FOXO1 (FKHR) transgenic

mice have less skeletal muscle mass, down-regulated Type I (slow twitch/red

muscle) fiber genes, and impaired glycemic control. J Biol Chem. Sep 24

2004;279(39):41114-41123.

5. Nakamura MT, Yudell BE, Loor JJ. Regulation of energy metabolism by long-

chain fatty acids. Prog Lipid Res. Jan 2014;53:124-144.

6. Nahle Z, Hsieh M, Pietka T, et al. CD36-dependent regulation of muscle FoxO1

and PDK4 in the PPAR delta/beta-mediated adaptation to metabolic stress. J Biol

Chem. May 23 2008;283(21):14317-14326.

26

7. Bastie CC, Nahle Z, McLoughlin T, et al. FoxO1 stimulates fatty acid uptake and

oxidation in muscle cells through CD36-dependent and -independent

mechanisms. J Biol Chem. Apr 08 2005;280(14):14222-14229.

8. Sanchez AM. FoxO transcription factors and endurance training: a role for FoxO1

and FoxO3 in exercise-induced angiogenesis. J Physiol. Jan 15 2015;593(2):363-

364.

9. Rudrappa SS, Wilkinson DJ, Greenhaff PL, Smith K, Idris I, Atherton PJ. Human

Skeletal Muscle Disuse Atrophy: Effects on Muscle Protein Synthesis,

Breakdown, and Insulin Resistance-A Qualitative Review. Front Physiol.

2016;7:361.

10. Yan Y, Wang ZB, Tang CK. [PPARs Mediate the Regulation of Energy

Metabolism By Long-Chain Fatty Acids]. Sheng Li Ke Xue Jin Zhan. Feb

2016;47(1):1-6.

11. Tsuchiya K, Ogawa Y. Forkhead box class O family member proteins: The

biology and pathophysiological roles in diabetes. J Diabetes Investig. Nov

2017;8(6):726-734.

12. Nakae J, Kitamura T, Kitamura Y, Biggs WH, 3rd, Arden KC, Accili D. The

forkhead transcription factor Foxo1 regulates adipocyte differentiation. Dev Cell.

Jan 2003;4(1):119-129.

13. Xu X, Gopalacharyulu P, Seppanen-Laakso T, et al. Insulin signaling regulates

fatty acid catabolism at the level of CoA activation. PLoS Genet. Jan

2012;8(1):e1002478.

27

14. Rieck M, Meissner W, Ries S, Muller-Brusselbach S, Muller R. Ligand-mediated

regulation of peroxisome proliferator-activated receptor (PPAR) beta/delta: a

comparative analysis of PPAR-selective agonists and all-trans retinoic acid. Mol

Pharmacol. Nov 2008;74(5):1269-1277.

15. Hegarty BD, Furler SM, Oakes ND, Kraegen EW, Cooney GJ. Peroxisome

proliferator-activated receptor (PPAR) activation induces tissue-specific effects

on fatty acid uptake and metabolism in vivo--a study using the novel

PPARalpha/gamma agonist tesaglitazar. Endocrinology. Jul 2004;145(7):3158-

3164.

16. Ehrenborg E, Krook A. Regulation of skeletal muscle physiology and metabolism

by peroxisome proliferator-activated receptor delta. Pharmacol Rev. Sep

2009;61(3):373-393.

17. Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear

control of metabolism. Endocr Rev. Oct 1999;20(5):649-688.

18. Tanaka T, Yamamoto J, Iwasaki S, et al. Activation of peroxisome proliferator-

activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and

attenuates metabolic syndrome. Proc Natl Acad Sci U S A. Dec 23

2003;100(26):15924-15929.

19. Loviscach M, Rehman N, Carter L, et al. Distribution of peroxisome proliferator-

activated receptors (PPARs) in human skeletal muscle and adipose tissue: relation

to insulin action. Diabetologia. Mar 2000;43(3):304-311.

20. Djouadi F, Aubey F, Schlemmer D, et al. Bezafibrate increases very-long-chain

acyl-CoA dehydrogenase protein and mRNA expression in deficient fibroblasts

28

and is a potential therapy for fatty acid oxidation disorders. Hum Mol Genet. Sep

15 2005;14(18):2695-2703.

21. Djouadi F, Bonnefont JP, Thuillier L, et al. Correction of fatty acid oxidation in

carnitine palmitoyl transferase 2-deficient cultured skin fibroblasts by bezafibrate.

Pediatr Res. Oct 2003;54(4):446-451.

22. Djouadi F, Aubey F, Schlemmer D, Bastin J. Peroxisome proliferator activated

receptor delta (PPARdelta) agonist but not PPARalpha corrects carnitine

palmitoyl transferase 2 deficiency in human muscle cells. J Clin Endocrinol

Metab. Mar 2005;90(3):1791-1797.

23. Lahiri S, Wahli W. Peroxisome proliferator-activated receptor beta/delta: a master

regulator of metabolic pathways in skeletal muscle. Horm Mol Biol Clin Investig.

Dec 1 2010;4(2):565-573.

24. Wang YX, Lee CH, Tiep S, et al. Peroxisome-proliferator-activated receptor delta

activates fat metabolism to prevent obesity. Cell. Apr 18 2003;113(2):159-170.

25. Eijkelenboom A, Burgering BM. FOXOs: signalling integrators for homeostasis

maintenance. Nat Rev Mol Cell Biol. Feb 2013;14(2):83-97.

26. de Lange P, Lombardi A, Silvestri E, Goglia F, Lanni A, Moreno M. Peroxisome

Proliferator-Activated Receptor Delta: A Conserved Director of Lipid

Homeostasis through Regulation of the Oxidative Capacity of Muscle. PPAR Res.

2008;2008:172676.

27. Obsil T, Obsilova V. Structure/function relationships underlying regulation of

FOXO transcription factors. Oncogene. Apr 07 2008;27(16):2263-2275.

29

28. Wang Y, Zhou Y, Graves DT. FOXO transcription factors: their clinical

significance and regulation. Biomed Res Int. 2014;2014:925350.

29. Sanchez AM, Candau RB, Bernardi H. FoxO transcription factors: their roles in

the maintenance of skeletal muscle homeostasis. Cell Mol Life Sci. May

2014;71(9):1657-1671.

30. Brent MM, Anand R, Marmorstein R. Structural basis for DNA recognition by

FoxO1 and its regulation by posttranslational modification. Structure. Sep 10

2008;16(9):1407-1416.

31. Wang Y, Pessin JE. Mechanisms for fiber-type specificity of skeletal muscle

atrophy. Curr Opin Clin Nutr Metab Care. May 2013;16(3):243-250.

32. McLoughlin TJ, Smith SM, DeLong AD, Wang H, Unterman TG, Esser KA.

FoxO1 induces in skeletal myotubes in a DNA-binding-dependent

manner. Am J Physiol Cell Physiol. Sep 2009;297(3):C548-555.

33. Furuyama T, Kitayama K, Yamashita H, Mori N. Forkhead transcription factor

FOXO1 (FKHR)-dependent induction of PDK4 in skeletal

muscle during energy deprivation. Biochem J. Oct 15 2003;375(Pt 2):365-371.

34. Sin TK, Yung BY, Siu PM. Modulation of SIRT1-Foxo1 signaling axis by

resveratrol: implications in skeletal muscle aging and insulin resistance. Cell

Physiol Biochem. 2015;35(2):541-552.

35. Gross DN, van den Heuvel AP, Birnbaum MJ. The role of FoxO in the regulation

of metabolism. Oncogene. Apr 07 2008;27(16):2320-2336.

30

36. Vries RG, Flynn A, Patel JC, Wang X, Denton RM, Proud CG. Heat shock

increases the association of binding protein-1 with initiation factor 4E. J Biol

Chem. Dec 26 1997;272(52):32779-32784.

37. Tyagi S, Gupta P, Saini AS, Kaushal C, Sharma S. The peroxisome proliferator-

activated receptor: A family of nuclear receptors role in various diseases. J Adv

Pharm Technol Res. Oct 2011;2(4):236-240.

38. Farce A, Renault N, Chavatte P. Structural insight into PPARgamma ligands

binding. Curr Med Chem. 2009;16(14):1768-1789.

39. Muoio DM, MacLean PS, Lang DB, et al. Fatty acid homeostasis and induction of

lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated

receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by

PPAR delta. J Biol Chem. Jul 19 2002;277(29):26089-26097.

40. Jehl-Pietri C, Bastie C, Gillot I, Luquet S, Grimaldi PA. Peroxisome-proliferator-

activated receptor delta mediates the effects of long-chain fatty acids on post-

confluent cell proliferation. Biochem J. Aug 15 2000;350 Pt 1:93-98.

41. Bastie C, Luquet S, Holst D, Jehl-Pietri C, Grimaldi PA. Alterations of

peroxisome proliferator-activated receptor delta activity affect fatty acid-

controlled adipose differentiation. J Biol Chem. Dec 8 2000;275(49):38768-

38773.

42. Wang YX, Zhang CL, Yu RT, et al. Regulation of muscle fiber type and running

endurance by PPARdelta. PLoS Biol. Oct 2004;2(10):e294.

31