i

INVESTIGATING THE ROLE OF CRABP1 IN ADIPOSE BIOLOGY

by

JOSHUA E. MILLER

Submitted in partial fulfillment of the requirements For the degree of Master of Science

Thesis Adviser: Dr. Noa Noy

Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY

May, 2017

ii

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis of

Joshua E. Miller

candidate for the degree of Master of Science*.

Committee Chair: Ruth Keri, Ph.D.

Committee Member: Hua Lou, Ph.D.

Committee Member: Noa Noy, Ph.D.

Committee Member: Monica Montano, Ph.D.

Committee Member: David Danielpour

Date of Defense: January 13th, 2017

*We also certify that written approval has been obtained for any proprietary material contained therein. iii

Table of Contents

TABLE OF CONTENTS______iii

LIST OF FIGURES______iv-v

ACKNOWLEDGEMENTS______vi-vii

ABSTRACT______1

CHAPTER 1: Background and Statement of Purpose______2-8

CHAPTER 2: Materials and Methods______9-14

CHAPTER 3: Examination of the role of CRABP1 in Adipocytes______15-30

CHAPTER 4: Conclusions, Discussion and Future Directions______31-37

Bibliography______38-41

iv

LIST OF FIGURES

Figure 1.1 functions through two distinct nuclear hormone receptors______3

Figure 3.1. CRABP transcript expression declines upon adipocyte differentiation______19

Figure 3.2. CRABP1 expression is diminished upon adipocyte differentiation______20

Figure 3.3. CRABP1 transcript expression in WAT greatly exceeds that of D0 3T3-

L1 cells______21

Figure 3.4. Severe ablation of CRABP1 transcript upon high fat feeding and vitamin

A deficient diet______22

Figure 3.5. Dietary changes alter the mouse white adipose tissue______23-24

Figure 3.6. Generation of stable 3T3-

L1s______25

Figure 3.7. Overexpression of CRABP1 does not significantly alter RAR target in

WAT______26 v

Figure 3.8. CRABP1 overexpression does not significantly reduce FABP4 transcript______27

Figure 3.9. Retinoic Acid inhibits adipocyte differentiation while CRABP1 overexpression induces less lipid droplet formation______28

Figure 3.10. CRABP1 overexpression reduces CEBPα protein expression______29

Figure 3.11. CRABP1 overexpression does not affect phosphorylation of HSL at Ser

563______30

vi

ACKNOWLEDGEMENTS I would like to thank my graduate school mentor, Dr. Noa Noy. Noa contributed greatly to my training as a scientist, both in that she challenged me to convey my ideas concisely and confidently and she trained me to really reflect on the meaning of the data before jumping to conclusions. Her passion and enthusiasm for research drove me to persevere in the face of innumerable failures, and I am a stronger person for it. She provided me with opportunities to interact with leaders in the field and to develop a diverse depth of knowledge. She will be deeply missed. I would like to express my gratitude to the members of the Noy lab, both past and present, who have guided my development as a scientist. I thank you for your patience in teaching me lab techniques, providing me with useful tips and protocols and overseeing the honing of my lab skills.

Thank you for your advice when planning experiments and for being generous with lending samples and reagents when I needed them. Finally, thank you for being more than a colleague when I needed a friend. You became like second family to me.

To my thesis committee, Drs. Keri, Montano, Lou and Danielpour, I am so grateful for your participation in my development as a scientist. Thank you for your help and guidance throughout my project and your patience when I was following a hypothesis that you may not have believed in. You challenged me to think critically and for that I thank you.

I would like to thank my friends and fellow pharmacology graduate students for all of their help and support in the good times and bad. You created a community and I am so grateful that I could be a part of it. To Leslie and Sahil, you have become two of vii my closest friends, and I want you to know that I could not have survived graduate school without you.

Finally I would like to thank my family, who have loved and supported me throughout my time in this program.

1

Investigating The Role of CRABP1 In Adipose Biology

Abstract

by

JOSHUA E. MILLER

Dysfunctional regulation of adipose tissue is a key risk factor for a number of diseases such as type 2 diabetes(1). Consequently, understanding the role of specific involved in adipose biology is critical to treatment of these serious illnesses.

Administration of the vitamin A metabolite, retinoic acid has been shown to improve insulin responses and protect against diet-induced obesity in several mouse studies(2,3).

One of its carrier proteins, CRABP2 has been shown to participate in adipose tissue biology by enhancing retinoic acid-induced transcriptional modulation of target genes(4,5). Due to its role in adipose tissue biology, we hypothesized that CRABP1 may also be involved. Our studies show that CRABP1 is down regulated upon induction of adipocyte differentiation. However, high fat diet feeding depletes CRABP1 from mouse white adipose tissue, suggesting that there may be an interesting link between CRABP1 and fat accumulation in the adipose tissue.

2

Chapter 1: Background and Statement of Purpose 1.1 Background

Vitamin A Vitamin A (all-trans retinol) is a key nutrient involved in numerous biological

processes, ranging from development and vision to maintenance of the immune

system(6). Many of its functions are carried out by a key metabolite, known as all-

trans retinoic acid (RA), which is synthesized from all-trans retinol by a series of

dehydrogenases(7). Retinoic acid is then able to travel to the nucleus, where it binds

directly to specific nuclear hormone receptors, including PPARβ/δ and the three

isotypes of RAR, known as RARα, RARβ and RARγ(6,8). Binding of RA to these

nuclear hormone receptors alters the transcription of specific target genes. Although

retinoic acid is largely hydrophobic and is able to traverse the nuclear membrane by

itself, its delivery to specific nuclear hormone receptors is facilitated by a group of

proteins from the intracellular lipid binding protein family(9).

It has been well established that the effect of retinoic acid on a given cell is

dependent upon the expression of specific lipid binding proteins and nuclear hormone

receptors(10-13). For example, fatty acid binding protein 5 (FABP5) shuttles retinoic

acid to peroxisome proliferator activated receptor β/δ, PPARβ/δ, which is a nuclear

hormone receptor that regulates genes involved in promoting cell proliferation(8,14-

16). However, when cellular retinoic acid binding protein 2 (CRABP2) is the

predominant intracellular lipid binding protein in the cell, it will deliver retinoic acid 3

to the (RAR), which regulates a different set of genes that

induce differentiation, growth arrest and cellular (17).

Figure 1.1 Retinoic acid functions through two distinct nuclear hormone

receptors

All-trans retinoic acid is capable of binding to either CRABP2 or FABP5

in the cytosol, after which it is shuttled to either RAR or PPARβ/δ respectively. In

cells which highly express CRABP2, delivery of retinoic acid to RAR will induce

transcription of genes involved in apoptosis and cell cycle arrest. When FABP5 is

highly expressed, retinoic acid will be delivered to PPARβ/δ, which will modulate 4

transcription of genes that induce pro-survival pathways. Figure adapted from

Vreeland, A. (2015). Cellular Retinoic Acid-Binding Protein 2 Cooperates with HuR

to Stabilize RNA and Inhibit Tumor Growth . (Electronic Thesis or Dissertation).

Retrieved from https://etd.ohiolink.edu, with contributions from Dr. Liraz Levi and

Dr. Noa Noy.

CRABPs

Among the lipid binding proteins, two homologs, the cellular retinoic acid

binding proteins 1 and 2 (CRABPs) share ~75% homology and bind to all-trans

retinoic acid with sub-nanomolar affinity(12,18). CRABP2 has been shown to have

two distinct functions. In the presence of retinoic acid, CRABP2 enhances delivery of

retinoic acid to RAR to modulate transcription of target genes. However, in the

absence of retinoic acid, CRABP2 has been shown to directly cooperate with an RNA

binding protein known as HuR(19,20). When CRABP2 binds to HuR it enhances the

affinity of HuR for target mRNAs that bear an AU-rich region in their 3’UTR(21-24).

This interaction with HuR allows CRABP2 to enhance the stability of these target

mRNAs. Despite its homology with CRABP2, CRABP1 is unable to deliver retinoic

acid to RAR to activate transcription(10,25). In fact, some studies suggest that

CRABP1 may enhance the delivery of retinoic acid to downstream metabolic

enzymes. Several studies have shown that overexpression of CRABP1 decreases

RAR transcriptional activity(26-28) It has also been reported that CRABP1

overexpression reduces the expression of retinoic acid target genes such as RARβ,

while decreasing RA half-life and increasing production of polar metabolites(26,29). 5

However, the scope of current literature on the function of CRABP1 remains limited,

warranting further studies to better characterize its role in the cell.

Adipose Tissue

Adipose tissue, more commonly known as fat, has long been regarded as a hub for

lipid storage and a potential source of energy under fasting conditions(30). Adipose

tissue can be divided into two main categories based on its function: white adipose

tissue (WAT) where stored lipids are available to be used as an energy source and

brown adipose tissue (BAT) where utilization of fat is diverted to production of heat

instead of energy(31). Aside from its role in lipid storage, WAT has been shown to

play a critical role as a major endocrine organ, secreting cytokines that alter the

storage of lipids and the insulin sensitivity of other organs(32,33). Consequently,

alterations in adipose tissue functionally impact the whole body. In disease states

such as obesity where there is aberrant accumulation of adipose tissue, the effects

range from insulin resistance and type 2 diabetes mellitus, to an increased risk for

heart disease and stroke(30).

Adipogenesis

In order for WAT to effectively develop the capacity to regulate lipid synthesis and degradation as well as its endocrine capabilities, it must undergo a series of remodeling steps as mature white adipocytes are formed from preadipocyte precursor cells(30,34,35). This process is known as adipogenesis or adipocyte differentiation.

Through the use of well-established preadipocyte cell lines, such as the mouse preadipocyte cell line, 3T3-L1s, the major drivers of this process have been thoroughly 6 characterized. As a preadipocyte undergoes adipogenesis, there is a transcriptional cascade that is induced, which requires the down regulation of many genes required to maintain a preadipocyte phenotype(36,37). These genes are referred to as preadipocyte markers because they function to inhibit the expression of key transcription factors needed for the development of the mature adipocyte. Examples of these genes include

Pref-1, Sox9 and KLF2(4). In the absence of inhibitors of differentiation, adipogenesis initiates with the increase in two transcription factors known as CEBPβ and CEBPδ. Loss of both of these transcription factors has been shown to inhibit adipogenesis in vitro(37).

Up regulation of these transcription factors down regulates preadipocyte markers while simultaneously inducing expression of CEBPα and PPARγ, which are the two most important transcription factors for adipogenesis to proceed to completion. While there is some overlap between the target genes of these two transcription factors, in vitro studies have shown that PPARγ is both necessary and sufficient for adipogenesis to occur, while

CEBPα is not essential. However, in the absence of CEBPα, cells have been shown to exhibit poor insulin sensitivity(34,35). Along with their critical roles in adipogenesis, these factors are also critical for the maintenance and development of mature adipocyte function, which can be severely impaired in disease states such as obesity(30).

Vitamin A in Diabetes and Obesity

Vitamin A has been reported to exert many beneficial effects in the context of

diabetes and obesity(2,4). Supplementing mice with retinoic acid has been shown to

protect mice from diet-induced obesity and enhance their insulin sensitivity. Several 7

of the effects of vitamin A in the context of adipose biology are due to CRABP2

delivery of RA to RAR. Berry et. al. demonstrated that in comparison to wild type

C57BL/6Ntac, CRABP2 heterozygous mice exhibited increased weight gain specific

to the white adipose tissue, along with a decrease in markers such as Pref-1 that

maintain preadipocytes and prevent adipogenesis(4). It has been concluded that this

CRABP2-RAR axis effectively suppresses dietary-induced obesity both by inhibiting

adipogenesis in preadipocytes and by enhancing lipid oxidation and energy utilization

in the mature adipocyte. Clearly retinoic acid and CRABP2 play essential roles in the

context of adipose biology. Given the homology with CRABP1, it begs the question

of whether it may have some function in the context of this tissue.

Statement of Purpose

This project has two key specific aims. 1) We sought to identify whether

CRABP1 would be a relevant protein to study in the context of adipose biology and

then to determine 2) what effect, if any that CRABP1 would have on the process of

differentiation. If these aims were successful, I would then further examine specific

physiological consequences of CRABP1 in adipocytes, specifically whether it would

affect processes such as insulin sensitivity and lipolysis that are critical to healthy

adipose tissue function and become aberrantly regulated in many diseases. Given a

single report by Park et. al., that alleges CRABP1 is regulated by thyroid hormone

through the protein RIP140, I aimed to confirm the regulation of CRABP1 over the 8

course of adipocyte differentiation and I was able to more strongly hypothesize that

CRABP1 would play a role in adipose biology(38).

9

Chapter 2: Materials and Methods

2.1 Chemicals and Reagents. All-trans retinoic acid (RA) was purchased from

Calbiochem. Oil red o powder, Insulin, Dexamethasone and IBMX were provided from

Sigma.

2.2 Buffers. 10X SDS running buffer was prepared with 120 g Tris (Fisher), 576 g

Glycine (Fisher) and 40 g SDS (Fisher) dissolved in 4 L of ddH2O. It was diluted in ddH2O 10-fold to make 1X solution used to run polyacrylamide gels.

1X Transfer Buffer was made by adding 30.3 g Tris, 144 g Glycine and 20% Methanol to ddH2O and making up a 1 L solution.

10X PBS was made by adding 80 g NaCl (Fisher), 2 g KCL (Fisher), 14.4 g Na2HPO4

(Fisher), and 2.4 g KH2PO4 (Fisher) to 1 L ddH2O. This was subsequently diluted 10- fold to make 1X PBS. The solution was autoclaved before use.

RIPA Buffer was prepared using 0.88g NaCl, 1 ml of 1M Tris (pH 6.8), 2.78 ml EDTA

(Fisher), 1 ml of 10% SDS, 1 ml Triton X-100 (Fisher), and 1g Deoxycholate (Fisher) in

100 ml ddH2O. Upon usage, a 1:100 dilution of protease inhibitor (Thermo) was added.

5X SDS Loading Buffer was prepared by making up a 20 ml solution of 50 mg

Bromophenol blue (Fisher), 1.54 g DTT (Fisher), 10 ml Glycerol (Fisher), 1 g SDS

(Fisher), and 0.79 g Tris-HCl (Fisher) in ddH2O. 950 ul aliquots were made and stored at

-20 degrees Celsius. Prior to use, aliquots were thawed, and 50 ul of beta mercaptoethanol was added. 10

TBS-T Consisted of 50 mM Tris, 150 mM NaCl and 0.1% Tween 20 (Fisher) dissolved in ddH2O.

2.3 Cell Lines. NIH3T3-L1 mouse preadipocytes were the primary cell line used in this study. 293T human embryonic kidney cells were used to generate lentivirus to create stable 3T3-L1 cells harboring either EGFP or EGFP-CRABP1.

2.4 Antibodies. The antibody against CRABP1 was purchased from Sigma (mouse;

C1608). The antibodies against PPARγ (rabbit; #24435), CEBPα (rabbit; #22955), and p-

HSL (Ser563) (mouse; #41395) were from Cell Signaling. The antibody directed at

FABP4 was purchased from R&D Systems (goat; AF1443). The antibody for GAPDH was purchased from Santa Cruz (mouse; sc-32233). The antibody against CRABP2

(mouse) was generously provided by Cecile Rochette-Egly (Institut Génétique Biologie

Moléculaire Cellulaire). Secondary antibodies were purchased from Biorad, including goat anti-mouse (#1721011), goat anti-rabbit (#1706515) and donkey anti-goat

(STAR206P).

2.5 Quantitative real-time PCR. QPCR was carried out using TaqMan chemistry and

Assays-on-Demand probes (Applied Biosystems) for CRABP2, Mm00801691_m1; Pref-

1, Mm00494477_m1; FABP4, Mm00445880_m1; Cyp26a1 Mm00514484_g1;

ADAM17 Mm00456428_m118s; and CRABP1, Mm00442777_m1. Eukaryotic 18S

4352930 (Applied Biosystems) ribosomal RNA was used for normalization.

2.6 Mice. Male C57BL/6Ntac mice (4-6 weeks old) were fed either regular chow or a high fat/high sucrose diet for 17 weeks. The high fat/high sucrose diet consisted of 35%

(w/w) fat (D12331 from Research Diets). Within each group, some mice were fed a 11 regular diet devoid of Vitamin A (Research Diet #BV577) while others were fed the high fat/high sucrose diet without Vitamin A (D12053104)(39). Weight gain was monitored across the 17 weeks. Epididymal white adipose tissue was collected and retinoid content was measured as previously described(40,41). All experiments were done in accordance of IACUC protocol.

2.7 Statistics. Statistics from experiments in Figure 3.5 were analyzed by ANOVA and complemented with Tukey’s test (p<0.05). All other statistical analysis was determined by unpaired t-test.

2.8 QPCR. Media was removed from confluent cells and RNA was extracted by incubation for 5 minutes in 1 mL TRI Reagent (MRC)/plate at room temperature (RT).

200 ul of Chloroform (Fisher) was added to the lysate and mixed vigorously for 15 seconds, followed by a 2-minute incubation at RT. The lysate was clarified by centrifugation for 15 minutes at 12,000 x G at 4 degrees Celsius. After centrifugation, the top layer was extracted and mixed with equal volume of Isopropanol (Fisher). After vigorous shaking, the samples were incubated for 10 minutes at RT and then spun down for 10 minutes at 12,000 x G at 4 degrees Celsius. The supernatant was removed and the pellets dried and then resuspended in 1 mL of 70% ethanol. Following a final spin for 5 minutes at 7,500 x G, the pellet was dried and resuspended in 30 ul ddH2O. RNA concentration was determined using the NanoDrop 2000. 1 ug of RNA was used to reverse transcribe cDNA by the RNA to cDNA EcoDry Kit (Clontech). The cDNA was diluted 5-fold in ddH2O and utilized for QPCR. Four technical replicates were analyzed for each sample. Eukaryotic 18S was used as a housekeeping gene to which all data was analyzed. 12

2.9 Western Blotting. Media was removed from confluent cells and plates were washed once with 1X PBS. Cells were lysed using 150-300 ul of RIPA buffer. Upon addition of

RIPA to each plate, the plates were incubated on a rocker at 4 degrees Celsius for 10 minutes. After scraping the lysate, it was incubated on ice for 5 minutes, after which the lysates were clarified by centrifugation for 15 minutes at 4 degrees Celsius and 15,000 rpm. Following clarification, the pellet was removed by pipet. In the case of mature adipocytes, fat was removed by gentle pipetting. Protein concentration of the lysate was determined by performing a Bradford assay. Lysate was mixed in 5X SDS Loading

Buffer and boiled for 5 minutes at 100 degrees Celsius. All samples were spun down and the requisite amount of each sample was loaded onto 10-15% polyacrylamide gels. All

Western Blots were run alongside Dual Color Precision Plus Protein Standards (Biorad).

Proteins were transferred onto Nitrocellulose membranes (Millipore) at 4 degrees Celsius and then blocked for at least one hour in either 10% milk of 10% bovine serum albumin

(BSA) dissolved in TBS-T. Membranes were then briefly washed with TBS-T and incubated in primary antibody at 1-1000 dilution overnight, shaking at 4 degrees Celsius.

The following day, the primary antibody solution was removed and four ten-minute washes were performed using TBS-T. After washing, one of three secondary antibodies

(Biorad) were incubated with the membrane at either a 1-2500 or 1-5000 dilution for 1 hour at RT. Four ten-minute washes were performed with TBS-T and the membranes were incubated in Pierce ECL Western Blotting Substrate for 5 minutes. Membranes were incubated with film between a few seconds to 30 minutes and then developed.

2.10 Generation of Stable 3T3-L1s. 293T cells were grown to near confluence and then transfected in suspension using Polyfect (Qiagen). Along with Polyfect, the cells were 13 transfected with pCMV, pM2DG and a lentiviral vector containing either EGFP or

EGFP-CRABP1 in serum free media. After 16 hours, the media was replaced. The following day, the media was decanted into a 50 mL conical tube and spun for 5 minutes.

The supernatant was collected and added to 3T3-L1 cells with 8 ug/mL of polybrene.

Meanwhile, the 293T cells were given fresh media for 24 hours. The next day, the media was collected into a conical tube, spun down, and the virus containing media was again added to the 3T3-L1 cells. After 48 hours, some 3T3-L1 plates were lysed with RIPA, and a western blot was performed to confirm overexpression. Meanwhile, the remaining plates were selected with puromycin and overexpression was verified by Western Blot.

2.11 Differentiation of 3T3-L1s. 3T3-L1 mouse preadipocytes were cultured to confluence in DMEM (Corning) with 10% FBS. At confluence, the media was replenished and the cells were incubated for 48 hours. After 48 hours, the media was replaced with DMEM containing 10% FBS, 250 nM Dexamethasone, 10 ug/mL Insulin and 0.5 mM IBMX. Insulin, Dexamethasone and IBMX were sterile filtered through a

0.2 mm filter (Millipore) before being added to the media. The cells were cultured in this media for 72 hours, after which the media was replaced with DMEM with 10%FBS. The cells were then cultured for another 72 hours after which the differentiation process was verified by assessing the status of markers of differentiation such as FABP4 and by staining for lipid droplets using Oil Red O. Cells treated with retinoic acid were given 0.2 uM upon differentiation and were supplemented with an equivalent dose every two days until Day 6 of differentiation.

2.12 Oil Red O Staining. Media was removed from plates and they were rinsed with 2 mL of 1X PBS. The wash was removed and the plates were fixed with 2 ml of 10% 14 formalin, which was incubated on the plates between 30 minutes to 1 hour. After the incubation period, the formalin was removed and the plates were washed with 2 ml of ddH2O. The water was removed and the plates were incubated for 5 minutes in 60%

Isopropanol.

Stock solution of oil red o was prepared by dissolving 300 mg of oil red o powder

(Sigma) in 100 ml of 99% Isopropanol. 30 ml of stock oil red o solution was mixed with

20 ml of ddH2O and incubated at RT for 10 minutes. This solution was then filtered through a funnel and 2 ml of this working solution was added to each plate after removing the 60% isopropanol. The working solution was rotated along the dish and allowed to stand for 5 minutes before removal. Excess stain was removed by washing the plates with ddH2O. The plates were then counter stained with hematoxylin (Harris

Sigma) for 1 minute. The hematoxylin was then rinsed off with ddH2O. Lipids were stained red, whereas undifferentiated cells remained unstained(42,43).

15

Chapter 3: Examination of the role of CRABP1 in adipocytes

In order to confirm whether CRABP1 would be relevant in the context of adipose biology, I first needed an established system to study the process of adipogenesis and mature adipose function. Given the previous studies that elucidated the role of CRABP2 in this context, I chose to utilize the established 3T3-L1 mouse preadipocyte cell line for my studies. Using an established differentiation protocol, I would be able to examine the effect of CRABP1 at critical junctures in the adipogenesis process, including the molecular characteristics of adipocytes from the beginning of differentiation until mature adipocytes had been formed. Work from Berry et. al. (2010) has previously used this model to demonstrate that CRABP2 expression is rapidly depleted in response to induction of adipogenesis both at the transcript and protein level(5). Given this information, I set out to examine CRABP1 expression throughout the differentiation process and ensure that it would be a logical choice to study in this system.

As shown in Figure 3.1, I was able to confirm that CRABP2 transcript was diminished upon induction of adipogenesis. Using FABP4 transcript as a surrogate marker of successful adipocyte differentiation (Figure 3.1B), the data clearly show that along with CRABP2, CRABP1 transcript was strikingly reduced as differentiation proceeded. Further work was done to examine the effect of adipogenesis on the protein expression of both CRABPs. Both CRABP2 and CRABP1 protein were detectable prior to induction of differentiation, but in line with the effects at the transcript level, both proteins’ expression was ablated as the adipocytes were cultured to day 6 (Figure 3.2). In the case of CRABP2 there is an explanation for this phenomenon, as loss of CRABP2 is necessary to blunt RAR-mediated upregulation of preadipocyte , allowing 16 adipogenesis to continue(5). However, CRABP1 has not been shown to exert effects on

RAR target genes in these cells, while data regarding the role of CRABP1 on RAR targets in other cell lines remains controversial. Consequently, these data raise the question of why CRABP1 expression is being down regulated as preadipocytes transition to mature adipocytes.

In order to verify that CRABP1 remains relevant to adipose biology in vivo, I assessed the expression of CRABP1 transcript in mouse WAT. Strikingly, the CRABP1 transcript was more highly expressed in mouse WAT than in the 3T3-L1 cells (Figure

3.3). In an attempt to understand the impact of different diets on CRABP1 expression, I also assessed the CRABP1 transcript levels from WAT of mice fed either regular chow or a high fat diet in which there was or was not any dietary vitamin A. As observed in

Figure 3.4, feeding the mice a high fat diet severely depleted CRABP1 transcript from

WAT, whereas depletion of Vitamin A on a regular diet regime reduced CRABP1 levels though not with statistical significance. While mice fed regular chow with or without vitamin A showed no significant changes in weight, absence of vitamin A resulted in significantly leaner mice compared to mice fed a high fat diet with sufficient vitamin A levels (Figure 3.5A). Interestingly, when mice were fed a high fat diet with sufficient levels of Vitamin A, the expression level of PPARγ was significantly up regulated

(Figure 3.5B). PPARγ is a master regulator of adipogenesis, hence these data suggest that the high fat diet induced an increase in adipogenesis(44). With the increase in adipogenesis, CRABP1 levels are depleted, which may indicate a potential connection between CRABP1 and high fat feeding in WAT. However it remains unclear whether this connection is merely correlation or if there is some cause and effect relationship between 17 the two characteristics. What is clear is that the high fat diet strongly reduced the retinoid content in the WAT (Figure 3.5C). This may mean that the loss of CRABP1 is directly tied to the retinoids in WAT, which are depleted by high fat feeding.

In order to assess whether CRABP1 exerts a direct effect on adipocytes, I generated 3T3-L1 cells stably expressing either EGFP or CRABP1 (Figure 3.6). Since I had observed low CRABP1 expression as the adipocytes became more differentiated, I hypothesized that it was likely that CRABP1 would be more important early in differentiation before the endogenous protein is depleted. First, I assessed whether ectopic expression of CRABP1 would affect the expression of genes like Pref-1 and

ADAM17, which are markers of preadipocytes and play a role in deterring adipogenesis from occurring(45-49). I hypothesized that overexpression of CRABP1 would enhance the expression of these transcripts, which would slow adipogenesis. The data showed slight increases at the transcript level, with the increase in Pref-1 transcript being statistically significant (Figure 3.7). Both transcripts had previously been reported to be targets of RAR. In order to dissect between RAR targets and preadipocyte markers, I examined the effect of CRABP1 overexpression on Cyp26a1, which is an RAR target but not a marker of preadipocytes. The results demonstrated that CRABP1 overexpression had no effect on Cyp26a1 transcript(50). It appeared from these experiments that

CRABP1 did not significantly up regulate these target genes and that prior to adipogenesis CRABP1 did not enhance expression of the RA metabolizing enzyme,

Cyp26a1.

Due to the lack of significant effects early in adipogenesis, I chose to determine whether stable overexpression of CRABP1 would affect the differentiation status of the 18 adipocytes after six days of differentiation. As shown in Figure 3.8, the differentiation marker, FABP4 was strongly enhanced after six days of differentiation(51). Though the

CRABP1 overexpressing adipocytes show a slightly lower FABP4 level, there was no statistically significant change. I attempted to analyze the effect of CRABP1 overexpression on storage of lipids in the adipocytes by Oil Red O staining. As shown in

Figure 3.9, lipid-storing mature adipocytes are reduced by treatment with exogenous retinoic acid (0.2µM every two days). In the absence of exogenous retinoic acid, the staining is stronger in EGFP expressing adipocytes than in adipocytes stably overexpressing CRABP1. Along with the FABP4 transcript levels, this suggests that

CRABP1 may reduce adipogenesis, though the exact mechanism remains unclear. To better understand the effects that may occur at the protein level, I performed a western blot on lysates collected after six days of differentiation in the presence or absence of retinoic acid. As expected, retinoic acid treatment decreased the expression of these markers. While overexpression of CRABP1 did not alter the expression of PPARγ or

FABP4, it clearly reduced expression of CEBPα both in the presence and absence of exogenous retinoic acid (Figure 3.10).

Along with understanding the potential role that CRABP1 could play in the context of adipogenesis, I wanted to determine whether CRABP1 would have any effect on mature adipocyte function. In order to determine whether CRABP1 overexpression had any effect on lipolysis, I performed a Western Blot for phosphorylated hormone sensitive lipase, which is a rate-limiting enzyme that is activated by phosphorylation.

Phosphorylated HSL is involved in the breakdown of triacylglycerol to diacylglycerol 19 and free fatty acids(52). As observed in Figure 3.11, overexpression of CRABP1 did not alter the phosphorylation of hormone sensitive lipase at serine 563.

A 1.2 1 CRABP1 0.8

0.6 CRABP2

0.4

0.2 fold mRNA/18S RNA mRNA/18S fold RNA 0 DAY: 0 3 6 8 B 3500 3000 2500 2000 /18S RNA /18SRNA 1500 1000 Fabp4 500 0 fold fold DAY: 0 3 6 8

Figure 3.1 CRABP transcript expression declines upon adipocyte differentiation A) 3T3-L1 cells were grown to confluence and were cultured for 48 hours prior to

addition of differentiation mix (Day 0). The cells were grown in differentiation

media for 3 days (Day 3), after which the media was replaced and the cells were 20

grown five more days (Day 8). In agreement with previously reported data,

CRABP2 transcript levels decline upon adipocyte differentiation. Similarly,

CRABP1 transcript levels are greatly diminished as soon as three days after

initiating adipogenesis. B) FABP4 transcript, a marker of adipogenesis, is poorly

expressed before differentiation and even after three days. However, upon Day 6

of differentiation, FABP4 expression is markedly induced and remains high

through Day 8.

CRABP1 CRABP2

GAPDH GAPDH

DAY: 0 3 6 0 3 6

Figure 3.2 CRABP1 protein expression is diminished upon adipocyte differentiation Both CRABP1 and CRABP2 protein expression are detectable prior to induction of differentiation. After three days of differentiation, the protein levels of both CRABPs are greatly reduced and are hardly detectable after 6 days.

21

1.2

1

0.8

0.6

0.4

CRABP1 mRNA/ 18S RNA CRABP1mRNA/ 18S RNA 0.2

0 WAT RD 3T3L1 D0

Figure 3.3 CRABP1 transcript expression in WAT greatly exceeds that of D0 3T3- L1 cells QPCR data from mouse white adipose tissue exhibits markedly higher CRABP1 transcript in vivo than the mouse preadipocyte cell model just prior to adipogenesis.

22

Figure 3.4 Severe ablation of CRABP1 transcript upon high fat feeding and vitamin A deficient diet QPCR analysis of CRABP1 transcript levels from WAT of mice fed a regular diet

(RD), a high fat/high sucrose diet (HFD), a regular diet without vitamin A (RD-A) or

a high fat diet without vitamin A (HFD-A) showed that high fat feeding significantly

depleted mouse WAT of CRABP1 transcript. Feeding the mice a diet without vitamin

A alone also significantly reduces CRABP1 transcript levels. Three mice were

analyzed for each group, and significance is denoted by p<0.05. Asterisks indicate

significance compared to RD.

23

24

Figure 3.5 Dietary changes alter the mouse white adipose tissue A) Mouse weight was monitored across the course of the feeding regimen. Within

the study, mice were fed diets that either contained vitamin A (VAS) or had no

vitamin A content (VAD). While mice fed normal chow did not show significant

weight gain regardless of vitamin a status, high fat/ high sucrose feeding

significantly increased the weight of the mice. However, mice fed a high fat/high

sucrose diet without vitamin A were significantly leaner than the mice fed a high

fat/high sucrose diet with sufficient levels of vitamin a (VAS HFD). B) PPARγ

transcript levels were measured across the different diets. While mice fed the

normal chow diet showed no significant changes regardless of vitamin a status,

high fat/high sucrose feeding significantly up regulated PPARγ transcript levels.

However, mice fed a high fat diet with sufficient vitamin a levels exhibit 25

markedly higher PPARγ transcript levels. C) Retinoids were extracted from

mouse WAT of mice fed the different feeding regimes and measured by HPLC.

As expected, mice fed chow deficient in vitamin a (VAD) have significantly

lower retinoid levels. Likewise, mice fed a high fat/high sucrose diet with

deficient vitamin a also exhibit significant decreases in retinoids. However, even

feeding a mouse a diet with sufficient vitamin a levels that is also high in fat also

significantly reduces retinoids, though not to the same extent as removing them

from the diet. Data were analyzed with ANOVA complemented with Tukey’s test

to compare each group to the other statistically. Different letters indicate

statistical differences between different diets. Different letters also signify p<0.05.

Studies conducted in this figure were performed by Dr. Liraz Levi.

Figure 3.6 Generation of stable 3T3-L1s Stable 3T3-L1s were generated as described previously in Chapter 2. After six days of differentiation, lysates were collected and Western blotting was performed to verify overexpression of CRABP1. 26

1.8 * 1.6 1.4 1.2 1 EGFP 0.8 CRABP1 0.6 Fold mRNA/18S RNA mRNA/18S RNA Fold 0.4 0.2 0 Pref1 Adam17 Cyp26a1

Figure 3.7 Overexpression of CRABP1 does not significantly alter RAR target genes in 3T3-L1 cells QPCR analysis from Day 0 adipocytes was performed to assess potential effects on the preadipocyte markers, Pref-1 and Adam17. While Pref-1 transcript increased, the effect on ADAM17 transcript expression was not statistically significant. Analysis of a third gene, Cyp26a1, which is also an RAR target, exhibited no significant changes either.

Data are the result of three independent experiments. An asterisk indicates a p value<0.05.

27

1800 1600 1400 1200 1000 800 600

fold Fabp4/ 18S RNA 18S RNA Fabp4/ fold 400 200 0 EGFP CRABP1 EGFP CRABP1

Figure 3.8 CRABP1 overexpression does not significantly reduce FABP4 transcript FABP4 transcript was measured before differentiation and at Day 6 of differentiation in

3T3-L1s stably over expressing EGFP or EGFP-CRABP1. As expected, at Day 0 neither condition yielded high FABP4 transcript. Differentiation of EGFP expressing L1s induced high transcript expression, which was slightly less in CRABP1 expressing L1s. 28

RD HFD-A

RD-A HFD

Figure 3.9 Retinoic Acid inhibits adipocyte differentiation while CRABP1 overexpression induces less lipid droplet formation Stable 3T3-L1s were differentiated and cultured until Day 6 in the presence (+RA) or absence (-RA) of excess 0.2 uM retinoic acid. As expected retinoic acid reduces staining of lipid droplets as an indicator of decreased adipogenesis. Staining of L1s differentiated without exogenous retinoic acid display increased staining in the EGFP expressing L1s compared to those expressing CRABP1. 29

0.2 0.4

0.6 0.000

0.001

0.003

0.004

0.002

Figure 3.10 CRABP1 overexpression reduces CEBPα protein expression Stable 3T3-L1s were differentiated and cultured to Day 6 in the presence or absence of exogenous retinoic acid. Upon blotting for three proteins that are highly expressed in mature adipocytes, PPARγ and FABP4 levels were not significantly changed when

CRABP1 was over expressed, though they did decline upon treatment with RA.

However, regardless of whether ectopic RA was added, CEBPα levels were reduced when CRABP1 was over expressed.

30

1.2

1.0

0.8

Figure 3.11 CRABP1 overexpression does not affect phosphorylation of HSL at Ser 563 Stable 3T3-L1s were differentiated and cultured to Day 6. However, CRABP1 overexpression did not significantly affect phosphorylation of hormone sensitive lipase at

Ser 563. The experiment was performed with three biological replicates for each condition.

31

Chapter 4: Conclusions, Discussion and Future Directions

4.1 General Conclusions and Discussion

Despite attempts to uncover a function for CRABP1 and a possible link to adipose biology, its exact role remains unclear. I hypothesized that CRABP1 would be a relevant protein to study in adipose tissue, and that it would be highly expressed in 3T3-L1 adipocytes. Interestingly, the former hypothesis was correct because the latter was wrong.

CRABP1 expression was detectable both by transcript and protein at Day 0 in 3T3-L1 cells, but its expression decreased as differentiation proceeded. This was in line with what had been previously reported about CRABP2 as well as what I observed when I repeated the experiment. The notion that CRABP1 and CRABP2 are both down regulated during adipogenesis is interesting for many reasons. Historically it has been reported that

CRABP1 and CRABP2 display different temporal and spatial expression patterns. This would suggest that perhaps the two proteins do not need to coexist because they perform similar functions, which would not be an unreasonable hypothesis given that the two proteins share near 75% homology and display no significant region of difference in their protein sequence. The co-expression of both CRABPs and similar down regulation of both their transcript and protein levels during adipogenesis does little to discourage this notion that there may be functional redundancy within the context of adipose biology.

However, what was more striking was the data obtained from mouse white adipose tissue. Not only was CRABP1 mRNA expression much higher in mouse white adipose tissue than it was in Day 0 3T3-L1s, it was also severely depleted upon high fat/high sucrose feeding. Given the connection between a high fat diet and many of the diseases of interest related to adipose tissue, I believe this observation warrants further 32 study. These data raise many interesting possibilities. Is there a causal relationship between a high fat diet and depletion of CRABP1, or is the data merely correlative?

Feeding a high fat diet feeding greatly depleted retinoid levels in the WAT, suggesting that there could be retinoid regulation of CRABP1 at play instead of there being a direct link between high fat diet and CRABP1 expression. Also, a high fat diet did not have nearly as profound an impact on PPARγ expression in WAT when the diet lacked vitamin

A. This casts doubt on my hypothesis that increased adipogenesis and increased PPARγ may be responsible for down regulation of CRABP1 transcript, though as the data currently stand, the question remains to be resolved.

My second hypothesis was that CRABP1 may play an important role in adipogenesis. This hypothesis was based on the idea that CRABP1 may need to be down regulated in order for adipogenesis to occur, as is true with CRABP2. The current data are insufficient to provide a complete answer to this question. While overexpression of

CRABP1 induced a significant increase in Pref-1 transcript levels, the observed increase in ADAM17 transcript was not statistically significant. However, there are caveats to the approach we took to address this question. I examined the expression of the preadipocyte markers at a time point where I observed that endogenous CRABP1 was high, thus it is likely that further overexpression would not have a significant impact. What may have been more insightful would be to compare the stable L1s at early stages in differentiation as endogenous CRABP1 levels are depleted. I could observe the rate at which the preadipocyte markers’ expression decreased and early differentiation transcription factors such as CEBPβ and CEBPδ are induced. This would provide a more accurate assessment of whether CRABP1 was directly impacting the initiation of adipogenesis. 33

One pitfall of the existing data that I have generated is that the overexpression studies were carried out using a CRABP1-EGFP fusion protein. Given that EGFP is a large protein tag (27kDa), it is possible that its fusion with CRABP1 might alter

CRABP1 function. In fact, it has been reported previously that expression of CRABP2 fused to GFP altered the native localization of CRABP2(9). To ensure that the EGFP tag is not altering CRABP1 function, I would repeat the overexpression studies using a lentivirus without a tag to determine whether an untagged construct would significantly impact adipogenesis. Due to the lack of a quality control for CRABP1 function, I would repeat the experiments with the untagged CRABP1 and use previously reported data on untagged CRABP2 in adipocytes as a control for the experiment.

Attempts to study the effect of CRABP1 in mature adipocytes have provided preliminary data that CRABP1 only has a modest effect on adipogenesis in the context of the 3T3-L1 differentiation model. At Day 6 of differentiation, FABP4 transcript was slightly less when CRABP1 was over expressed, but the protein expression does not appear to be effected. However, oil red o staining was slightly decreased when CRABP1 was over expressed compared to EGFP. CRABP1 expression also appears to have decreased CEBPα protein expression, both with and without retinoic acid, though the magnitude of the effect is not particularly strong. From these data is seems that CRABP1 may not directly affect adipocyte differentiation, or if there is an effect, it is very mild.

Key Findings

1) CRABP1 was expressed in Day 0 adipocytes, but was strongly reduced upon induction of adipocyte differentiation. This pattern resembles what has been previously reported about CRABP2 and confirms the results shown by Park et. al. (38). 34

2) CRABP1 is depleted in mouse WAT when mice are fed a high fat diet.

3) CRABP1 overexpression modestly decreases CEBPα protein expression in differentiated adipocytes.

4.2 Future Directions

Although the function of CRABP1 still remains unclear in the context of adipocyte differentiation, these studies reveal that exploring its potential role in adipose biology is a valid endeavor. Moving forward, there are a number of critical questions that merit examination. The majority of the data suggests that CRABP1 may not exert profound effects in the context of the 3T3-L1-cell line and that perhaps it is not the best model for study. However, generating a knockdown of CRABP1 might provide useful insight related to the expression of preadipocyte markers at day 0 when CRABP1 expression was highest. Given the lack of conclusive data regarding what role CRABP1 may play in adipogenesis, it would be important to assess whether silencing of CRABP1 expression accelerates the differentiation process, which is my current hypothesis. It is also worth noting that since CEBPα protein expression was decreased upon CRABP1 expression, it is possible that the effect of CRABP1 may be less related to adipogenesis and more directly associated with CEBPα expression. Given that CEBPα is induced later in adipogenesis largely through other family members such as CEBPβ and CEBPδ, there is a possibility that CRABP1’s effects could originate early in differentiation when

CRABP1 expression is still high. Coupling CRABP1 overexpression with CRABP1 35 knockdown could provide insights regarding CRABP1 function if its effect occurs in the middle of differentiation instead of before or after it occurs.

The expression of CRABP1 transcript was significantly greater in vivo, which is encouraging given functional outcomes in vivo will likely be more translatable than outcomes from cells grown on plastic. If I were to continue to with cell studies, I would want to culture adipocytes from mouse white adipose tissue first since the expression of

CRABP1 transcript is much higher, and this may provide an improved model to assess

CRABP1 function. However, it is worth noting that having protein expression data from the mouse WAT would be more useful and there is no guarantee that the higher transcript expression will result in higher rates of translation. An attempt to complete western blots for CRABP1 using adipose tissue lysates was unsuccessful (data not shown) and this raises the question of whether there is sufficient CRABP1 protein expression for detection. Upon generating the primary cell line I would want to verify CRABP1 protein expression before repeating any of the experiments done with the 3T3-L1 cells. Another weakness of the data I have generated is that it focuses too narrowly on transcriptional outcomes and largely ignores questions that would address potential translational effects.

The bulk of my data shows the effect of CRABP1 overexpression on transcript levels of targets, but when I examined CEBPα protein, I observed that CRABP1 did impact its expression. Though the focus on transcript expression was largely made out of convenience, it is important that such a preliminary study not rule out alternative hypotheses. Our work focuses on previous reports that CRABP1 may affect RA catabolism and a separate hypothesis from work in our lab that CRABP1 may be interacting with CRABP2 to block its functions. Regardless of the validity of either 36 hypothesis, our approaches led us to the same conclusion. If either hypothesis is correct in adipose tissue, CRABP1 should blunt RAR effects on the cell, and enhance differentiation. Our current data conflict with this postulate. In the future, it will be critical to identify whether CRABP1 is playing some role in the context of retinoic acid signaling considering its similarities with its homolog are considerable.

To determine whether CRABP1 exerts RAR related effects, I would treat day 0 adipocytes with retinoic acid for thirty minutes and compare the expression of preadipocyte markers to cells that received no exogenous retinoic acid. In studies from

Berry et. al., the effect was always contingent upon the presence of retinoic acid and

RAR, and so it would be important for me to verify that CRABP1 was not in fact operating through a similar mechanism. I would want to verify the results of this experiment with transactivation assays in these cells, looking at the effect of either

CRABP1 overexpression or knockdown on RAR activity. If CRABP1 functions through a retinoic acid related mechanism, I would evaluate the retinoic acid levels in the cell to determine potential effects on metabolism.

The most compelling future direction of this project should come in the form of in vivo studies that address whether CRABP1 plays a direct role in the function of adipose tissue. I would feed CRABP1 knockout mice with either regular chow or a high fat diet and compare them to wild type mice with the same diets to assess whether there are specific phenotypic changes that have not been previously identified. I would perform a glucose tolerance test to determine whether loss of CRABP1 expression affected the ability of the cells to take up glucose. I would complement this by looking at the effect of loss of CRABP1 on the expression of adipose specific genes such as GLUT4 to 37 determine whether genes involved in glucose uptake are affected. I would also examine insulin sensitivity of the adipose tissue by performing an insulin tolerance test. I would weigh the adipose tissue from wild type and knockout mice on both diets to determine whether CRABP1 affects accumulation of adipose tissue and I would couple this with

H&E staining to assess whether CRABP1 causes any adipocyte hypertrophy or changes in the types of adipocytes present. All of these phenotypic characteristics could address whether CRABP1 is relevant to study in the context of obesity and type 2 diabetes, two common, chronic conditions that adversely affect millions of people worldwide. In light of the many unanswered questions regarding CRABP1 function within any system, studying it in the adipose tissue has the potential to yield many exciting avenues that may lead to treatment, but regardless will enrich our understanding of vitamin a biology.

38

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