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FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences

Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism

Submitted by: Manya Warrier

In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences

Examination Committee

Major Advisor: Edwin Sanchez, Ph.D.

Academic Sonia Najjar, Ph.D. Advisory Committee: Linda Dokas, Ph.D.

Zi-Jian Xie, Ph.D.

Cynthia Smas, Ph.D.

Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D.

Date of Defense: July 23, 2008

Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism

Manya Warrier

The University of Toledo 2008

DEDICATION

I dedicate this work to my husband who has given me the courage, support and freedom to reach my dreams, my parents and grandparents who have always given their unconditional love and to Dr. Sonia M. Najjar who helped me immensely during the course of this work.

ii ACKNOWLEDGEMENTS

I wish to express my deep sense of gratitude to Dr. Edwin R. Sanchez, for giving me an

opportunity to do this project, his timely advice and support. Our discussions and his

constructive criticisms have greatly helped to improve my scientific thinking. I am

grateful to Dr. Weinian Shou and members of his laboratory for providing us with the animals to initiate this project.

I am greatly obliged to Dr. Sonia M. Najjar, for her keen interest and valuable guidance throughout this work. I would also like to thank other members of my committee,

Drs. Linda A. Dokas, Cynthia M. Smas and Zijian Xie for their insightful suggestions.

Special thanks to Dr. Sumudra Periyasamy, Dr. Dapei Li and other members of my lab for their help throughout my doctoral program. I am also deeply indebted to the members of Dr. Najjar’s lab, especially Ms. Payal Patel. It would have been extremely difficult to complete this study without their help. I truly enjoyed working with them and am always grateful for their co-operation.

Last but not least, my sincere gratitude to all members of the Pharmacology Department who have made this arduous journey truly enjoyable.

iii TABLE OF CONTENTS

Dedication …………………………………………...... ii

Acknowledgements …………………………………………….. iii

Table of Contents ……………………………………………….. iv

Introduction …………………………………………...... 1

Literature ………………………………………………………….7

Materials and Methods …………………………………………..44

Results……………………………………………………………49

Discussion………………………………………………………..69

Conclusions………………………………………………………85

Summary…………………………………………………………87

Bibliography……………………………………………………..91

Abstract…………………………………………………………124

iv INTRODUCTION

Steroid receptors (SR) are hormone-activated transcription factors that belong to the family of nuclear receptors (NR) (Hollenberg et al., 1985). Hormone-free receptors reside either in the cytoplasm or nucleus in association with several proteins, each of which assists the receptor to optimize its functions. In its steady state, the receptor complex contains one molecule of receptor, heat shock protein 90 (HSP90) dimer, p23 and a tetratricopeptide repeat (TPR) protein. Several TPR proteins may exist, but to date

only four such proteins have been shown to be incorporated into the steroid receptor

complexes. They are FK506-binding proteins (FKBP51 and FKBP52), cyclophilin-40

(Cyp40) and protein phosphatase 5 (PP5). The first three are also recognized as larger

members of the immunophilin family because of their ability to bind immunosuppressive drugs, such as FK506 and cyclosporine A (CsA). Nevertheless, all four proteins possess a common TPR domain, through which they bind to the single acceptor site (TPR binding site) created by the HSP90 dimer. Besides the TPR site, immunophilins harbor a PPIase domain that has intrinsic peptidyl-prolyl isomerase (PPIase) enzymatic activity (Galat,

1993). Immunosuppressive drugs may bind to this region and inhibit its activity

(Schreiber and Crabtree, 1992). Protein phosphatase 5 lacks PPIase activity but is a serine/threonine phosphatase. Three of these proteins, except Cyp40, also possess a

PPIase-like region of unknown function.

During protein synthesis, peptide bonds on the amino side of the proline residues are formed in cis-conformation. PPIase enzymes catalyze the conversion of cis-bonds to their energy favorable trans-form, thereby altering protein structure (Galat, 1993). Due to this special feature, proteins with PPIase activity, including immunophilins, are known to

1 regulate transcriptional activities by changing protein-protein interactions (Wu et al.,

2000). However, such reports for TPR-containing immunophilins are rather limited

(Mamane et al., 2000). The major role identified so far for these proteins is in mediating hormone responsiveness and localization of steroid receptors. Evidence to support the latter is plentiful with many reports showing an ability of FKBP52, Cyp40 and PP5 to interact with the motor protein, dynein, to facilitate transport of receptor along cytoskeletal tracts (Galigniana et al., 2002; Galigniana et al., 2004b; Galigniana et al.,

2001)

In regulating receptor hormone-binding affinity and, in turn, transcriptional activity, FKBP51 and FKBP52 are perhaps better researched over other TPR proteins.

The first such evidence came from studies that identified FKBP51 as the cause of glucocorticoid (GC) resistance in squirrel monkeys. Squirrel monkey lymphocytes (SML) express elevated amount of FKBP51 that is incorporated into glucocorticoid receptor

(GR) complexes and lowers its hormone-binding affinity (Reynolds et al., 1999). Further, using African green monkey COS-7 cells, Denny et al (2005) demonstrated a critical requirement of FKBP51 interaction with HSP90 but not PPIase activity for this inhibition. The fact that FKBP51 gene expression itself is induced by glucocorticoids provides a potential but excellent feedback system to attenuate and tightly control GR functions. The inhibitory effect of FKBP51 is also established for progesterone receptor

(PR) (Hubler et al., 2003). In contrast, elevated levels of this protein have been shown to increase androgen receptor (AR) transcriptional activity (Febbo et al., 2005).

Over-expression of human FKBP52 in yeast enhanced hormone-mediated GR signaling by altering hormone potency. Similar to FKBP51, HSP90-binding ability of

2 FKBP52 is important for potentiation (Riggs et al., 2003). Mutating several residues vital for PPIase activity, Riggs et al (2007) discovered that enzymatic activity itself is dispensable, but the domain does play a crucial role in potentiating receptor activity. This mutational analysis further revealed an important role for the amino acid proline in position 119 (P119). FKBP51 has a leucine in this position and mutating it to proline confers a potentiation effect in both yeast and murine cells. Regions outside this residue may also contribute towards the potentiation effect. Enhancement of transcriptional activity by FKBP52 however, is not specific for GR, but also includes AR and PR.

Estrogen (ER) and mineralocorticoid (MR) receptors, however, are not affected by

FKBP52 in this way (Gallo et al., 2007; Riggs et al., 2007).

The mechanisms by which FKBP51 inhibits and FKBP52 stimulates steroid receptor activity are still far from clear. But it was speculated that the notch formed by

P119 makes a specific contact with the ligand-binding domain (LBD) of the receptor-

HSP90 complex and stabilizes a conformational state that is otherwise transient for hormone-binding. FKBP51 with leucine at residue 119 is not able to do so (Riggs et al.,

2007). Even though the exact molecular mechanisms are not yet understood, it is now evident that distinct interactions of two different TPR proteins to steroid receptors via

HSP90 give diversity to receptor functions.

In order to translate these cell-based findings to physiology, mice lacking

FKBP51 and FKBP52 have been created (Tranguch et al., 2005; Yang et al., 2006; Yong et al., 2007b). While FKBP51 deficiency initially showed no apparent phenotypes (Yong et al., 2007b), FKBP52 mutants were defective in select reproductive organs. Male mutant mice were infertile due to penile hypospadias and prostate dysgenesis. Further

3 analysis of affected organs and mouse embryonic fibroblasts (MEFs) derived from

knockout mice revealed compromised AR transcriptional activity as the cause for the above mentioned developmental defects. Even though FKBP52 is present in other reproductive organs, interestingly, loss of this protein only seems to affect certain organs

(Yong et al., 2007b). FKBP52 -/- females were sterile due to selective reduction in PR transcriptional activity especially in the uterus. Uteri of FKBP52 mutant females were unable to support implantation. Molecular analyses of uterine lysates and MEFs from knockout animals were able to show that FKBP52 specifically regulates PR in this organ.

Estrogen responses in the uterus were not down-regulated and were normal or nearly normal (Tranguch et al., 2005; Yang et al., 2006). Thus, these data provide evidence for the selective targeting of steroid receptor signaling through TPR proteins. It is noteworthy that in contrast to previous in vitro findings, ablation of FKBP52 did not change hormone-binding affinity or translocation of steroid receptors (AR or PR), revealing a novel mechanism by which this protein controls SR functions. Taken together, these studies suggest that the TPR proteins are integral part of steroid receptor complexes and their fundamental role is to provide receptors with specificity and selectivity by creating heterogeneous complexes within a cell or tissue.

Despite their well-established roles in glucocorticoid receptor functions, no overt

GR abnormalities were found in FKBP51 or FKBP52 knockout animals. Glucocorticoids are important for a variety of physiological processes, such as development, immune responses and metabolism. It is generally accepted that the major contribution of GCs to physiology occur in response to stress. An example for this is the stimulation of gluconeogenic genes in response to fasting to maintain euglycemia. It is for this reason

4 they are called ‘stress hormones’ and are produced from adrenals when different types of

stress activate the hypothalamic-pituitary-adrenal axis (HPA) (Sapolsky et al., 2000). We,

therefore, concluded that to detect any GR defects, TPR mutants have to be challenged

with a stress event, such that GR functions become essential to maintain homeostasis.

Since our objective was to study how GR controls metabolic pathways in mutant animals,

as a form of stress we subjected them to four weeks of high fat feeding (HF).

Glucocorticoids control a range of metabolic responses and over-stimulation of

these hormones by prolonged stress or by pathological means can lead to disorders that are commonly observed in metabolic syndrome (MS). Indeed, it is now well established that chronic stress can lead to MS (Kyrou and Tsigos, 2007). This also happens to be true in reverse. Patients with type II diabetes or insulin resistance have elevated GC levels and

HPA activity (Walker, 2006). Although these facts support the idea of a global GR antagonist to alleviate, at least, some of the symptoms of metabolic disorders, potent side effects have precluded their usage. Tissue-selective regulation of receptor function is, therefore, a critical issue. In this regard, TPR proteins are known to selectively regulate other steroid receptor functions such as AR and PR (Tranguch et al., 2005; Yang et al.,

2006; Yong et al., 2007b). However, most of the work conducted to understand the role of FKBP51 and FKBP52 in GR actions have been using in vitro systems (Davies et al.,

2005; Riggs et al., 2007; Riggs et al., 2003) . Extrapolating from the above studies, we hypothesized that these proteins may also tissue-specifically regulate metabolic processes controlled by GCs. This is further corroborated by our observation that these mutant animals had no obvious GR defects under normal conditions. If ablation of TPR proteins had a global effect on GR actions, these animals would have behaved like complete GR

5 knockout mice, which show peri-natal lethality (Cole et al., 1995a). Mice with liver- specific inactivation of GR are viable and only show metabolic abnormalities when

subject to severe metabolic stress, such as starvation (Opherk et al., 2004). Treatment

with potent GC agonists, such as dexamethasone (Dex) and starvation are considered

standard procedures to study GR actions. We decided to take a different approach to

understand receptor functions in FKBP mutant mice. The combination of poor diet (diet

rich in fats and sugar) and lack of physical activity are known to increase the risk of

diabetes and insulin resistance in humans. Because our ultimate goal is to design drugs

that target the TPR proteins to selectively regulate GR actions in metabolic disorders, we

wanted to study their functions in an environment analogous to which that leads to MS in

humans. Also, high fat feeding itself is considered as a stress. Rats fed with high fat diet

show increased HPA axis activity and basal corticosterone production (Brindley et al.,

1981; Hulsmann, 1978; Tannenbaum et al., 1997). Thus, we chose high fat feeding as our

primary stressor.

Our results show that FKBP52 and FKBP51 differentially regulate high fat diet

response in mice. High fat fed FKBP52 mutants had elevated glucose and insulin levels

than wild-type (WT) mice and greater hepatic lipid buildup. This phenotype is consistent

with an animal model of insulin resistance due to liver fat causing reduced insulin

clearance. In contrast, FKBP51-/- animals were resistant to diet-induced obesity and

visceral adiposity. Their serum glucose and insulin levels were not affected by the loss of

this protein but they had lower triglyceride (TG) and free fatty acid (FFA) levels. Thus,

this work, for the first time provides in vivo evidence for the role of FKBPs in regulating

intermediary metabolism.

6 LITERATURE

Nuclear receptors are ligand-inducible, DNA-binding transcription factors that control gene expression via extracellular and intracellular signals. The effects of nuclear receptors on transcription are mediated mostly through the recruitment of cell specific coregulators and protein-protein interactions with other transcription factors. The nuclear hormone receptor superfamily includes several receptors that are further divided into subfamilies based on similarities of amino acid sequences and other functional properties.

However, their ability to interact with heat shock proteins (HSPs) and TPR proteins divide NRs broadly into two groups. The first group, formed by thyroid hormone receptors, retinoid acid receptors and various orphan receptors of unknown ligands, do not associate with HSPs or their co-chaperones. The other is comprised of steroid receptors that bind to HSPs and TPR proteins, such as FKBP51 and FKBP52 (Smirnov,

2002). Each of these proteins present in the receptor complex plays a pivotal role in modulating receptor functions. Since the regulation of SR functions by TPR proteins, especially the function of GR in metabolism is the crux of this project, the following sections will provide a brief overview on TPR proteins and SRs with special reference given to GR and its role in metabolism.

Steroid receptors are activated by small lipohilic molecules called steroids and control a variety of physiological processes, such as development, metabolism and reproduction. The classical steroid receptors are GR, MR, AR, PR and ER. These structurally related receptors share similar modes of activation but differ extensively in their physiological functions (Tsai and O'Malley, 1994). In vivo studies using knockout and transgenic animal models revealed an essential role of AR, PR and ER in

7 reproductive functions. GR is ubiquitously expressed and is crucial for glucose

homeostasis and immunity. MR is important for fluid and electrolyte homeostasis

(Edwards, 2005; Heemers and Tindall, 2007; Schoneveld et al., 2004; Viengchareun et

al., 2007).

Nuclear Receptor Structure:

Nuclear receptors exhibit a modular structure (Fig. 1) with independent but

functionally related structural domains (Smirnov, 2002).

FIGURE 1. FUNCTIONAL DOMAINS OF NUCLEAR RECEPTORS

The N terminal region of the receptor (A/B domain) is most variable in size and amino acid sequences and contains transcription activation function-1 (AF-1). This

region appears to act independently of ligand binding and confers specificity to receptor activity by interacting with tissue-specific coregulators. In many cases, this region also synergistically acts with ligand-dependent AF-2 located in the LBD of the receptor (Ma et al., 1999; Metivier et al., 2001). This domain harbors several phosphorylation sites and is the target of various signaling kinases, such as mitogen-activated protein kinases

8 (MAPK) and cyclin-dependent kinases (Benecke et al., 2000). Phosphorylation of these

sites can significantly affect transcriptional activity of the receptor (Shao and Lazar,

1999).

DNA-binding domain (DBD) follows the N-terminal domain and is the most

conserved domain. It is comprised of two zinc fingers and a C-terminal extension (CTE)

of 25 residues and recognizes specific target sequences (hormone response elements;

HRE) in the DNA to activate or repress genes (Benecke et al., 2000). Amino acid

residues of the first zinc finger region (P-box) are required for the recognition of HRE sites. The second zinc finger makes non-specific contacts with the sugar back bone of

DNA and contains a D-box that is involved in the dimerization of the receptor. C-

terminal extension interacts with the bases flanking HRE for a specific and tight binding of HRE with monomeric receptor. Steroid receptors bind as homodimers to inverted repeats of two consensus hexameric HREs separated by a three base pair spacer. The

hinge region links DBD to LBD and varies by length and sequence. It allows free rotation

of DBD relative to LBD by 1800. It harbors nuclear localization signal 1 (NLS1 or NL1)

and contains residues important for interaction with certain coregulators (Aranda and

Pascual, 2001).

Ligand-binding domain is a moderately conserved, multifunctional domain. In

addition to binding ligand, this region facilitates homo- and hetero-dimerization,

interaction with HSPs and is responsible for ligand-dependent transcriptional activity. It

has nuclear localization signal 2 (NLS2 or NL2) and activation function domain 2

(AF-2). This domain is formed by 12 conserved -helical regions numbered from H1 to

H12. A central core layer of three helices create a hydrophobic cavity – the ligand-

9 binding pocket that accommodates ligand. Ligand-binding induces a conformational

change in the receptor altering the orientation of the AF-2 motif which is located in helix

12. This ‘locks’ the ligand in the pocket and also forms a surface for the interaction with

specific coactivators and corepressors which, in turn, modulate receptor actions (Aranda

and Pascual, 2001). Coactivators such as CBP and p300 often possess histone acetyl

transferase activity (HAT) that modifies histones and opens chromatin structure to allow

the recruitment of RNA polymerase to initiate transcription. Corepressors on the other

hand, have histone deacetylases activity (HDAC) that condenses the chromatin to prevent

transcription (Kinyamu and Archer, 2004).

Glucocorticoid Receptor:

Glucocorticoid receptor, a member of steroid receptor family, controls a variety of physiological processes. Its signaling events are initiated by binding to natural (cortisol in humans and corticosterone in rodents) or synthetic ligands called glucocorticoids.

Glucocorticoid receptor sensitivity to its ligands and physiological responses greatly vary among different species, tissues and cell types (Hsu and DeFranco, 1995). The receptor was first identified in rat thymic cytosols by Allan Munck in 1966 (Munck and Brinck-

Johnsen, 1968). Subsequently, human GR was cloned by Ron Evans and his colleagues in

1985 (Heitzer et al., 2007).

In humans, only a single gene exists that codes for GR (hGR). It is located on chromosome 5q31-32 and is composed of nine exons. The protein coding region is formed by exons two-nine and exon one represents the 5’untranslated region. Three transcription-initiation sites are located in exon one (promoters 1A, 1B and 1C) each of

10 which produces a different first exon that is fused to a common exon two (Encio and

Detera-Wadleigh, 1991). Promoter regions of the hGR gene lack a TATA box or CCAAT motif but contain several CpG islands (Zong et al., 1990) and binding sites for transcription factors AP1, AP2, Sp1, YY1 (Ying Yang), NF-kB and CREB (Lu and

Cidlowski, 2004). Exon two contains the coding sequence for AF1 at the N-terminal; exons three and four code for first and the second zinc-finger motif in the DBD respectively; exons five-eight code for AF-2 and a portion of LBD. Exon nine harbors sequences for two alternative carboxy termini of LBD ( and ) and 3’untranslated

regions (Lu and Cidlowski, 2004).

Although only one gene has been identified, multiple GR isoforms exist due to alternative splicing and translation initiation sites. Alternative splicing of exon nine gives rise to two hGR isoforms. Human GR-the classical glucocorticoid receptor consists of

777 amino acids (97kDa) and, in its unliganded state is localized in the cytoplasm.

Human GR has 742 amino acid residues and differs greatly in its C-terminal sequence

(Duma et al., 2006). It is constitutively localized in the nucleus and acts as a dominant negative inhibitor on hGR-mediated transactivation (Schaaf and Cidlowski, 2002). Due to alternative translation initiation sites on hGR, multiple N-terminal isoforms are also produced. These isoforms are termed GR A to D (A, B, C1, C2, C3, D1, D2 and D3) and occur as a result of ribosomal leaky scanning or ribosomal shunting from alternative translation initiation sites located in exon two. It has been suggested that similar isoforms

may also exists for hGR due to their N-terminal structural similarities (Duma et al.,

2006).

11 The mode of action of GR is well defined and like other NRs it regulates transcription by binding to specific response elements in the promoters of target genes

(Grad and Picard, 2007). In its untransformed state, GR primarily resides in the cytoplasm as a part of a large heteromeric complex. The composition of mature GR heterocomplex was determined by Pratt and coworkers and consists of one molecule of the receptor, an HSP90 dimer, one molecule of p23 and one of the four HSP90-binding co-chaperones. Heat shock protein 90 plays a central role in the complex connecting the receptor with other proteins. Its interaction with the receptor occurs via a conserved region on the LBD called Signal Transduction Domain (STD). This association keeps the receptor in a conformation that is ready to receive ligand (Pratt and Toft, 1997). Heat shock protein 90 binding-chaperones, also called TPR proteins due to the presence of

TPR domains, compete for the single binding site generated by the dimer. The TPR is composed of highly degenerate 34 amino acid sequences found in tandem repeats of varying number (Blatch and Lassle, 1999). To date, four major TPR proteins are found to enter the GR complex (Fig. 2). They include FKBP51, FKBP52, Cyp40 and PP5 (Peattie et al., 1992; Pratt and Toft, 1997; Ratajczak et al., 1993; Smith et al., 1993).

FIGURE 2. MATURE GR HETEROCOMPLEXES BOUND TO FOUR DIFFERENT TPR PROTEINS

12 A new model, which deviates from the classical model in its initial stages of ligand-induced GR signaling, has been proposed based on recent findings from our laboratory (Davies et al., 2002). In the classical model, upon ligand-binding, the receptor dissociates from the complex and enters the nucleus where it dimerizes and binds to specific sequences in the DNA. Based on the new model, an early TPR swapping occurs within the receptor complex as a consequence of hormone-binding, prior to its translocation to nucleus (Fig. 3). According to this model, hormone-binding causes

FKBP51, the predominantly bound TPR in the GR complex, to be replaced by FKBP52 followed by concomitant recruitment of the motor protein, dynein (Davies et al., 2002).

The complex then moves along the microtubule network to the nucleus followed by its dissociation and conversion to a transcriptionally active form.

FIGURE 3. NEW MODEL OF GR SIGNALING

13 GR action in the nucleus involves orchestrated cooperation of several factors,

such as coregulators and other transcription factors. GR can be recruited to the DNA

either directly or through other transcription factors. Ligand-bound GR is capable to either transactivate or transrepress its target genes. Transactivation is carried out with the help of several coactivators that possess intrinsic histone acetylase transferase (HAT) activity. Some cofactors that do not possess HAT activity act as adaptor proteins to bridge the receptor with other coregulators and chromatin remodeling complexes.

Transrepression of GR occurs by different mechanisms and has been studied extensively.

The anti-inflammatory action of GR is mostly attributed to repression of inflammatory genes. Transrepression by GR commonly involves an indirect binding (tethering) to other

DNA-bound transcription factors such as AP-1 and NF-kB (Barnes, 1998).

Glucocorticoid Receptor Knockout and Transgenic Models:

In order to translate in vitro findings to GR physiology, several transgenic and

knockout models have been developed. Animals with ubiquitous inactivation of GR die

shortly after death due to atelectasis of lungs. Impaired gluconeogenesis in the liver and

alterations in the HPA activity were also observed in these animals (Cole et al., 1995a).

Since GR can influence transcription by direct and indirect DNA-binding, in order to

further analyze the consequence of these two functions, Reichardt et al (1998) developed

a dimerization-deficient (GRdim) mice. Using the Cre/loxP system, they introduced a

point mutation (A458T) that disrupts the homodimerization, leaving indirect DNA-

binding ability of the receptor intact. Surprisingly, these animals were viable and did not

develop lung atelectasis. Thus, it was concluded that the DNA-binding of GR is not

14 essential for the survival of an organism. Since glucocorticoids are renowned for

regulation of glucose metabolism, mice with liver-specific inactivation of GR were made

to specifically study the involvement of the receptor on liver carbohydrate metabolism

(Opherk et al., 2004). Half of these mutant mice die within two days after birth due to

low blood sugar levels. After 48 hours, there was no mortality noted and animals reached

adulthood without further complications. Their basal glucose levels were normal due to

compensation from counter-regulatory hormones, such as insulin and glucagon.

However, they exhibit hypoglycemia and reduced expression of gluconeogenic enzymes

when subjected to extreme starvation. Hepatic inactivation of GR also reduced

hyperglycemia in streptozotocin-induced diabetes in these animals. This animal model

reveals an important contribution of GR signaling in gluconeogenesis and in the

development of diabetic hyperglycemia.

Tetratricopeptide Repeat Proteins in Glucocorticoid Receptor Complex:

The HSP90-binding co-chaperones frequently found in GR receptor complex include the FK506 binding proteins, FKBP51 and FKBP52, Cyp40 and PP5. Three of these proteins are also categorized as immunophilins (FKBP51, FKBP52, and Cyp40) because of their capacity to bind immunosuppressive drugs, such as FK506 and cyclophilin. Protein phosphatase 5 also binds to FK506 with low affinity but is not considered as an immunophilin. All four proteins possess a common TPR motif which is the hallmark of this group of proteins and they are therefore called TPR proteins (Fig. 4).

They compete for the single binding site generated by two HSP90 molecules (Owens-

Grillo et al., 1995). This allows only one TPR protein to be part of the receptor complex

15 at any given time. The TPR motif is composed of highly degenerate 34 amino acid sequences that fold into a pair of -helical subdomains. The key function of this motif is to provide protein-protein interactions by generating an amphipathic groove allowing other proteins to bind (Hirano et al., 1990; Sikorski et al., 1990). The other known features of TPR proteins include a PPIase domain to which immunosuppressive drugs bind, a PPIase-like region of unknown function, the phosphatase domain of PP5, a calmodulin - binding site on FKBP52. Selective preference of TPR proteins for an individual receptor may depend on many factors, such as their protein structure, cellular availability and affinity towards HSP90 and the receptor itself (Ratajczak et al., 2003).

The following sections briefly describe all four major TPRs that can enter the SR complex and provide evidence to support their role as modulators in receptor functions.

FIGURE 4. FUNCTIONAL DOMAINS OF TPR PROTEINS

16 FKBP51:

FKBP51, a glucocorticoid-induced gene product, is abundantly expressed in numerous tissues (refer to thesis Fig. 7; (Baughman et al., 1997; Yeh et al., 1995). It is also termed p24, FKBP54 or FKBP5 and was first identified as a part of the purified progesterone receptor complex by immunoaffinity chromatography (Smith et al., 1990).

Subsequent studies led to the cloning and characterization of FKBP51 gene (fkbp5). Like other members of the FKBP family, it has a C-terminal TPR domain that binds to the

MEEVD sequence in the C-terminal of HSP90 and an N-terminal PPIase region. It shares

70% sequence similarity and 60% identity with FKBP52, a closely related member of the group. Recent findings from our laboratory show that cellular localization of this protein greatly varies across different cell types. In L929 and WCL2 cells, it is uniformly distributed among cytoplasmic and nuclear compartments, whereas in COS-1 cells it is primarily nuclear (unpublished observations).

In spite of sequence and domain similarities, both FKBP51 and FKBP52 show distinctive properties. Subtle differences in residues outside the common domains give each TPR protein a unique function. For example, despite similar TPR domains, each protein shows different affinities for HSP90. Using an assortment of truncation mutants of FKBP51 and FKBP52 and co-immunoprecipitation studies, it was found that C- terminal regions outside the core TPR domain of each protein significantly influence

HSP90 binding. For FKBP51, unique sequences within the final 30 amino acids were identified that boost its HSP90 binding affinity compared to FKBP52 (Cheung-Flynn et al., 2003).

17 The expression of FKBP51 is regulated by several steroid hormones. Apart from

glucocorticoids (Baughman et al., 1995; Baughman et al., 1997) both androgens (Amler

et al., 2000; Febbo et al., 2005) and progestins (Hubler and Scammell, 2004; Kester et al.,

1997) strongly enhance its expression. FKBP51 gene (FKBP5) has an intron (intron E), located 75kb from the transcriptional initiation site in the 5’noncoding sequence that is responsive to glucocorticoids and progestins (Hubler and Scammell, 2004). Steroidal regulation of TPR proteins and its association with SR may have important physiological and biological implications.

Since the discovery of TPR proteins, many laboratories have made intensive efforts to provide insights into the role these proteins play in steroid receptor physiology.

Development of glucocorticoid resistance in squirrel monkeys has been linked to elevated

FKBP51 expression over FKBP52 in SML (squirrel monkey lymphocytes) and its increased incorporation into GR complexes. Further, this has been shown to reduce the receptor’s binding affinity for hormone (Denny et al., 2000). Although, human FKBP51 shares over all 94% amino acid homology with squirrel monkey FKBP51 and lowers the hormone-binding affinity of the receptor, the inhibitory effect is much more pronounced in the latter. This difference is attributed to differences in the N and C-terminal regions of this protein (Scammell, 2000). Treatment of SML with FK506 or rapamycin resulted in a dramatic induction of GR hormone-binding by causing dissociation of FKBP51 from receptor complexes and preventing its reassembly with the receptor (Denny et al., 2000).

More recently, using a yeast model system, a role of FKBP51 in the transcriptional regulation of GR was determined. Over-expression of both FKBP52 and

FKBP51 showed that FKBP51 did not inhibit GR reporter activity in yeast that lacks

18 FKBP52 but efficiently blocked the potentiation of receptor by FKBP52 (Riggs et al.,

2003). Although molecular mechanisms of the inhibitory effect of FKBP51 are still

unresolved, David Smith and his colleagues (Riggs et al., 2007) were able to identify a

single amino acid residue (leucine 119) within the surface loop of the PPIase region of

FKBP51 that largely contributes to the inhibitory effect. Changing this residue to proline

as seen in FKBP52 confers potentiation activity to this protein. Both PPIase domain and

HSP90- binding ability is required for the inhibitory effect of FKBP51, however, PPIase activity itself is not required.

To better understand the biological relevance of the TPR-SR interactions, mice

deficient in FKBP51 have been created (Yong et al., 2007b) . These animals are viable

and surprisingly show no obvious steroid receptor abnormalities. In fact, the only phenotype observed so far was their inability to gain weight and visceral adiposity when

challenged with a high fat diet (refer to thesis Fig. 8) This is also consistent with the

report of increased expression of FKBP51 during adipocyte differentiation (Baughman et

al., 1997; Yeh et al., 1995). The functional consequence of the expression of this protein

in adipocytes is currently not known. Since GR also stimulates adipogenesis, it is

plausible that elevated FKBP51 expression results from increased GR activity.

FKBP52:

FKBP52 is a high molecular weight immunophilin first identified while creating

an antibody to the EC-1 epitope of PR complex from rabbit uterus (Tai et al., 1986). This

was later found out to be a 59kDa protein and termed as p59. In 1990, using EC1

antibody, Sanchez identified a heat - and chemical stress - inducible (Sanchez, 1990),

19 homologous 56kDa GR-interacting protein in IM-9 cells and relabeled it as HSP56. Two laboratories later discovered this protein to be FK506 binding immunophilin and named as FKBP52 (Davies and Sanchez, 2005). Crystallographic analysis and sequence studies have revealed four major domains for FKBP52. The N-terminal domain has PPIase activity. Immunosuppressive drugs, such as FK506 and rapamycin, bind to this region and inhibit its activity. However unlike FKBP12, FKBP52 does not inhibit calcinerurin when bound to immunosuppressive drugs (Davies et al., 2005; Lebeau et al., 1994). The second domain is PPIase-like region of unknown function. A consensus ATP/GTP binding sequence is located between amino acids 199 and 222 of this region (Callebaut et al., 1992). Phosphorylation of Thr-143 in the hinge region prevents FKBP52 from binding to HSP90 (Miyata et al., 1997). This region also possesses an eight amino acid sequence that is electrostatically complementary to the NLS on the receptor. Antibodies to this region decrease the rate of translocation of hormone-bound GR to nucleus (Czar et al., 1995). The three TPR domains follow the PPIase-like region and this region is responsible for binding to HSP90. Two putative calmodulin binding sites (CaM) containing PEST motifs reside in the C-terminal end of FKBP52 and CyP40 (Massol et al., 1992). FKBP4, the gene that encodes FKBP52, has been mapped to the short arm of chromosome 12. It contains 10 exons and nine introns and span approximately 9kb of genomic DNA. The organization of FKBP4 is quite similar to that of FKBP5 except that the introns are significantly shorter than those of FKBP5 (Scammell et al., 2003).

The role of FKBP52 in nuclear translocation of GR has been well established.

Although exact mechanisms of nuclear transport have not been clearly delineated, increasing evidence supports the role of HSP90-based chaperone machinery in nuclear-

20 cytoplasmic shuttling of steroid receptors (DeFranco, 1999; Liu and DeFranco, 1999).

Association of FKBP52 with the motor protein ,dyenin, and its co-localization along the microtubules support a role in receptor trafficking to the nucleus (Czar et al., 1994).

Interaction of FKBP52 with dyenin occurs via the PPIase domain (Silverstein et al.,

1999). Recently, our laboratory has provided new evidence for a hormone-induced

functional exchange of FKBP51 to FKBP52 within the GR complex along with the

recruitment of dyenin and translocation of the heteromeric complex to the nucleus

(Davies et al., 2002). Association of GR with different FKBPs appears to influence

receptor localization such that FKBP51 favors cytoplasmic retention and FKBP52,

targets the receptor to the nucleus.

Besides transporting receptors to the nucleus, FKBP52 is also important for

modulating certain SR transactivity. This has been demonstrated both in vitro by cell-

based assays and in vivo using FKBP52 knockout animals. Using an in vitro yeast model

system, Riggs et al (2003) showed that FKBP52 potentiated hormone-dependent GR

activity at physiologically relevant concentrations of hormone. This was due to an

increase in receptor hormone-binding affinity and required the PPIase domain and

interaction with HSP90. Based on these findings, they speculated that functional interaction of FKBP52 with HSP90, positions the PPIase domain close to proline residues

in the LBD and trans-cis isomerization of these residues alters the receptor’s hormone-

binding affinity. However, a follow up study to understand the ability of PPIase domain

to change the receptor’s hormone-binding capacity revealed that PPIase activity is not

required for potentiation. In this study, a proline residue found in 119th position, which is

located in a hairpin loop overhanging the catalytic pocket, was identified to confer

21 potentiation activity to the receptor (Riggs et al., 2007). Using MEF cells derived from

FKBP52 knockout animals, we were able to further confirm that this protein is indeed a positive modulator of GR transcriptional activity (refer to thesis Fig. 6).

Physiological functions of FKBP52 were perhaps best studied using knockout mice model. This animal model not only confirmed its role in SR transcriptional activity but also revealed a novel mechanism for tissue-selective SR functions. FKBP52 males showed penile hypospadias and prostate dysgenesis due to compromised AR activity in penis and prostate. Even though FKBP52 is present in primary sex organs, such as testis, these organs were not affected by the loss of this protein. Molecular studies using MEFs derived from knockout embryos revealed that AR hormone-binding and nuclear translocation were not affected (Cheung-Flynn et al., 2005; Yong et al., 2007b). FKBP52 females were sterile due to implantation failure. Analysis of uterine lysates from these animals showed a reduction in PR transcriptional activity but normal ER activity. As in the case of AR, loss of FKBP52 only affected PR activity in certain tissues. Again, loss of this protein did not alter PR hormone-binding affinity (Tranguch et al., 2005; Yang et al., 2006) . Thus in vivo studies, unlike cell-based findings, exposed a unique role of

FKBP52 in SR transcriptional activation, downstream of hormone-binding and nuclear translocation.

Though the majority of reports have been on its association with SR, FKBP52 has several other biological functions independent of SR as well. It has been shown to be involved in Ca2+ influx, copper efflux, cardiac hypertrophy, p53 translocation and IL-2

production (Galigniana et al., 2004a; Gkika et al., 2006; Jamshidi et al., 2004; Krummrei

et al., 2003; Sanokawa-Akakura et al., 2004; Sinkins et al., 2004). In C1866 immune

22 cells, FKBP52 represses the transactivation of interferon regulatory factor-4 by a

mechanism dependent on PPIase activity (Mamane et al., 2000). It also binds single

strand DNA sequences in adeno-associated viral genome to prevent second strand

synthesis (Qing et al., 2001). A role for this protein in the regulation of cholesterol

trafficking was also reported. Cytosolic caveolin, a cholesterol transporter, has been

found to associate with HSP-immunophilin complex to transport newly synthesized

cholesterol to caveolae present in the membranes (Uittenbogaard et al., 1998). Our results

expose a new role of FKBP52 in liver metabolism. Although an independent role of this

protein in metabolism or via other nuclear receptors is not excluded, based on the current

literature, we strongly suspect that these effects are mediated through GR.

Cyp40 and PP5:

Cyp40 was first identified as a part of ER heterocomplexes (Ratajczak et al.,

1993) and was purified using a cyclosporine A affinity column along with Cyp18 from the soluble fraction of calf brain (Kieffer et al., 1992). Its structure is very similar to that

FKBP52 except that it does not possess a PPIase-like region. Like FKBP52, it is heat-

inducible and therefore considered a heat shock protein (Mark et al., 2001). In MCF-7 breast cancer cell lines, Cyp40 is up-regulated in response to estrogen and is thought to play an important role in breast cancer (Kumar et al., 2001). Although primarily seen in association with ER, it has also been reported in small amounts in GR and PR heterocomplexes (Owens-Grillo et al., 1995). Along with FKBP52 and cytosolic caveolin, it forms a chaperone complex that transports newly synthesized cholesterol to caveolae, present in the membrane (Uittenbogaard et al., 1998).

23 Protein phosphatase 5 is ubiquitously expressed in eukaryotic cells and is

localized in both cytoplasmic and nuclear compartments (Chinkers, 2001). It belongs to the class of serine/threonine phosphatases as it contains a serine/threonine phosphatase

domain that has intrinsic phosphatase activity (Cohen, 1997). Reversible phosphorylation

of serine/threonine residues is considered a key regulatory mechanism that controls several cellular pathways and is mediated by the actions of kinases and phosphatases

(Kurosawa, 1994). The phosphatase region of PP5 shares 42-43% similarity with other

serine/threonine phosphatases such as PP1, PP2A and PP2B (Barton et al., 1994). This

protein was first identified based on its TPR interaction with atrial natriuretic peptide

(ANP) in a yeast two-hybrid screen (Chinkers, 1994). Unlike other TPRs, the TPR

domains of PP5 reside in the N-terminal region and may possibly be responsible for its

distinct biological and biochemical functions (Becker et al., 1994; Blatch and Lassle,

1999). PP5 has very low phosphatase activity due to auto-inhibition by its TPR domain

and removal of this domain increases its enzyme activity by 50-fold. Polyunsaturated

fatty acids (PUFA) such as arachidonic acid have been shown to activate PP5 in vitro, although physiological activators have not yet identified for this enzyme (Chen and

Cohen, 1997). However, future studies involving a more comprehensive screening with several other PUFAs may identify a novel link between fatty acid metabolism and PP5.

Interaction of PP5 and GR occurs via the TPR domain of this protein (Silverstein et al., 1997). Over-expression of the TPR domain of a dominant negative PP5 in CV-1 cells inhibits GR induced gene activity (Chen et al., 1996). Nuclear-cytoplasmic shuttling of GR is also thought to be mediated by PP5 due to its interaction with motor protein, dynein (Galigniana et al., 2002). Hormone-binding capacity of GR is shown to be

24 positively affected by the presence of PP5 (Davies et al., 2005). Several lines of evidence suggest its involvement in cell cycle regulation. Using MEF cells derived from PP5

knockout animals, Yong et al (2007a) demonstrated the impact of loss of PP5 as cell

cycle regulation. When exposed to ionizing radiation, PP5-deficient cells display DNA

damage due to improper functioning of ATR - a kinase involved in DNA repair

mechanisms.

Role of Glucocorticoids in Metabolism: An Overview

Glucocorticoids (GCs) and their intracellular receptor (GR) are integral

components of a tightly regulated hormonal system that maintains energy homeostasis in

mammals. Along with other hormones, such as glucagon and insulin, they help to sustain

blood glucose within narrow limits by regulating several crucial intermediary metabolic

pathways. Their insufficiency (Addison’s disease) or excess (Cushing’s syndrome) can

therefore cause serious disorders.

Glucocorticoid hormones, named after their role in glucose homeostasis, are

secreted from the adrenal cortex, under the control of a neuroendocrine feed back system-

the hypothalamic-pituitary-adrenal (HPA) axis. Secretion of GCs is a ‘classic endocrine

response’ to stress (Sapolsky et al., 2000). Excitation of the HPA axis by a stressor

activates the production of hypothalamic corticotrophin-releasing hormone (CRH). CRH

acts on the anterior pituitary to initiate pro-opiomelanocortin (POMC) gene transcription.

Secretion of POMC - encoded adrenocorticotropic hormone (ACTH) precedes its

stimulation of adrenal GC synthesis from adrenal cortex. Glucocorticoids can, in turn,

interfere with CRH and POMC gene transcription at the hypothalamic level and inhibit

25 ACTH secretion from the anterior pituitary, thereby completing the auto-regulatory feedback loop (Malkoski and Dorin, 1999).

Homeostatic control of blood glucose levels during periods of fasting and feeding is accomplished through the counter-regulatory actions of catabolic and anabolic hormones, such as glucagon, glucocorticoids and insulin. This tight control is the net effect of two simultaneously ongoing pathways - glucose production (gluconeogenesis) in the liver and uptake and metabolism by peripheral tissues, such as muscle and adipose. In response to feeding, i.e. when glucose is abundant, pancreatic -cells secrete insulin which stimulates glucose uptake in muscle and adipose. It suppresses gluconeogenesis in the liver and promotes glycolysis (breakdown of glucose) to release energy in the form of

ATP. Insulin also increases the storage of substrates in fat, liver and muscle by stimulating lipogenesis (lipid synthesis) and glycogen synthesis and inhibiting lipolysis and glycogenolysis (break down of fat and glycogen respectively) (Saltiel and Kahn,

2001).

Under fasting conditions when blood glucose levels falls, insulin secretion is repressed and production of another set of hormones, such as glucagon and glucocorticoid is triggered to maintain glucose levels within the limits. During the first

24 h of fasting, blood glucose is mainly maintained by the breakdown of glycogen in the liver, a process mostly regulated by the hormone glucagon. Glucagon is a peptide hormone produced by the -cells of pancreas. After long periods of food deprivation, liver gluconeogenesis becomes the major source of blood glucose (Opherk et al., 2004).

Both glucagon and glucocorticoids initiate hepatic gluconeogenesis from non- carbohydrate sources. These hormones also mobilize free fatty acids from peripheral

26 adipose and amino acids from muscle. Free fatty acids are taken up by the liver for further oxidation (-oxidation) to produce acetyl-coenzyme A (acetyl-CoA). Acetyl-CoA has two fates. It either enters tricarboxylic acid cycle (TCA) and releases energy to support the gluconeogenesis or further condenses to form ketone bodies, an important fuel for the brain. Amino acids are further broken down to lactate or pyruvate to form substrates for gluconeogenesis. A fasting liver is thus gluconeogenic whereas liver of a fed animal is lipogenic (Saltiel and Kahn, 2001). Several dozens of enzymes participate in this complex array of metabolic responses many of which are transcriptionally regulated by glucocorticoids. Glucocorticoid actions on three important metabolically active organs are described in the following sections (Fig. 5).

FIGURE 5. METABOLIC PATHWAYS REUGLATED BY GLUCOCORTICOIDS

27 Liver:

The liver plays a unique role in controlling both carbohydrate and lipid metabolism. The significance of GR in hepatic metabolism was demonstrated by a genome wide analysis of murine livers treated with a glucocorticoid agonist, Dex. This exhaustive study showed several direct and indirect target genes controlled by GR, many of which are important for energy metabolism (Phuc Le et al., 2005). In the liver, GCs control two key branches of metabolism namely gluconeogenesis and lipogenesis.

Glucocorticoids and Gluconeogenesis:

Gluconeogenesis during fasting provides glucose for extrahepatic tissues such as brain and erythrocytes and is considered as the salient feature of the liver. This process can be defined as the synthesis of glucose from non-carbohydrate sources such as pyruvate and glycerol. Several key enzymes convert pyruvate to glucose, through a series of biochemical reactions (Vegiopoulos and Herzig, 2007). The rate of gluconeogenesis is controlled by the activities of unidirectional enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). While PEPCK catalyses the conversion of oxaloacetate to phosphoenolpyruvate, the first rate-limiting step in the gluconeogenesis, G6Pase catalyses the final step, production of glucose from glucose 6- phosphate (G6P) (van Schaftingen and Gerin, 2002). Pyruvate dehydrogenase kinase 4

(PDK4) is another enzyme required to preserve pyruvate for gluconeogenesis. In the fed state pyruvate, the end product of glycolysis, is converted to acetyl-CoA and is either utilized for de novo lipid synthesis or enters the TCA cycle to produce energy. This conversion is carried out with the help of the pyruvate dehydrogenase complex (PDC).

28 During fasting, PDK4 phosphorylates and inactivates PDC thereby channeling pyruvate

towards glucose synthesis (Sugden and Holness, 2002). These three enzymes are

essential for gluconeogenesis and are transcriptionally controlled by GCs. In many

metabolic abnormalities such as obesity and diabetes, their uncontrolled expression by

GCs causes fasting and random hyperglycemia (Walker, 2006).

The primary metabolic target of glucocorticoids during fasting is the liver. During

the first few hours of fasting, blood glucose levels are maintained by the breakdown of

glycogen. However as fasting progresses, glycogen stores are depleted and

gluconeogenesis becomes the main source of blood glucose. Transcriptional enhancement

of gluconeogenic genes by glucocorticoids becomes important at this period to maintain normal glycemia. Consistent with this, mice with liver-specific inactivation of GR display fasting hypoglycemia after 48 h of fasting due to downregulation of

gluconeogenic enzymes (Opherk et al., 2004).

PEPCK:

Expression of PEPCK is positively regulated by glucagon and glucocorticoids and

negatively regulated by insulin. Fasting increases and a carbohydrate-rich meal decreases

its expression levels (Hanson and Reshef, 1997). The PEPCK promoter has been

extensively studied. Its expression is altered by components of diet, such as lipids and

carbohydrates (Chen, 2007) and various hormones (Hanson and Reshef, 1997). It has a

glucocorticoid response unit (GRU) and several distal and proximal accessory units and

all are required for a full glucocorticoid response (Cassuto et al., 2005). The GRU

extends from -455 to -321 and contains three accessory domains known as AF1- AF3

29 (Imai et al., 1993). The AF1 and AF3 sites are occupied by transcription factors, such as

hepatocyte nuclear factor 4 (HNF-4), chicken ovalbumin upstream promoter transcription

factor (COUP-TF), peroxisome proliferator-activated receptor 2 (PPAR2), retinoic acid

receptor and retinoid X receptor. Fork head family of transcription factors (FOXO) and

HNF-3 bind to AF-2 domain (Hanson and Reshef, 1997). Two distal accessory factors, to

which factors like HNF-4 and peroxisome proliferator-activated receptor  (PPAR)

bind, are also a part of the GRU unit (Cassuto et al., 2005). Several coactivators, such as

SRC-1, p300/CBP and p/CIP and PGC-1 also play an important role in the activation of this gene (Kucera et al., 2002; Wang et al., 2004a). Dysregulation of PEPCK due to over- expression of PPAR  coactivator 1 (PGC-1) has been found in type II diabetes and obese patients (Puigserver et al., 1998). During fasting, the expression of PGC-1 is activated by glucocorticoids, which in turn bind to GR through its conserved LXLL domain and increases its activity (Yoon et al., 2001).

G6Pase:

The glucocorticoid receptor controls the expression of G6Pase by binding to the three GREs present in the first 200bp of the promoter along with other transcription factors, such as HNF-1, HNF-4 and FOXO1 that synergistically activate the gene (Lin et al., 1998; Nakae et al., 2001; Vander Kooi et al., 2005). This enzyme is found mainly in liver and kidneys where gluconeogenesis occurs and is also expressed in the -cells of

pancreas (van Schaftingen and Gerin, 2002). The hypothesis that the altered expression of

G6Pase can lead to insulin resistance first emerged from a study in which the catalytic

subunit of the G6Pase was over-expressed in rat hepatocytes using recombinant

30 adenovirus (Seoane et al., 1997). Like other gluconeogenic enzymes, insulin suppresses

its activity (Trinh et al., 1997). In vivo, injection of glucocorticoids only slightly increases its expression in the liver, but in hepatoma cells, GC treatment causes up to 10-fold expression (Nordlie et al., 1965; Schmoll et al., 1996).

PDK4:

Pyruvate dehydrogenase kinase 4 is another enzyme transcriptionally regulated by glucocorticoids. This enzyme phosphorylates PDC and negatively regulates its activity.

PDC catalyzes the oxidation of pyruvate to acetyl-CoA thereby linking glucose

metabolism to fatty acid synthesis and the TCA cycle. In a well fed state, PDC is active

and converts pyruvate (the end product of glycolysis) to acetyl-CoA for fatty acid

synthesis. During starvation and fasting, PDK4 phosphorylates and inactivates PDC to

conserve three carbon compounds (lactate, pyruvate and glycerol) to provide substrates

for gluconeogenesis. Pyruvate dehydrogenase complex is therefore tightly regulated by

its phosphorylation and dephosphorylation status that are controlled by PDKs and

phosphatases, respectively. Phosphorylation on its serine residues inactivate, PDC and

dephosphorylation by phosphatases activates the complex. To date, there are four PDKs

known to exist. All four isoforms are differentially expressed in tissues. PDK4 is highly expressed in heart, liver and kidney and is regulated by both short and long-term

mechanisms. Short-term regulation includes inhibition by pyruvate and NAD+ and

positive stimulation by acetyl-CoA and NADH. The long-term regulation includes

transcriptional control by hormones and other factors (Sugden and Holness, 2006).

The glucocorticoid receptor, when activated, bind to GREs found in the promoter

31 region of PDK4 to transcriptionally activate its expression (Kwon and Harris, 2004).

Mutation of GREs completely abolishes GR-induced PDK4 expression. It has also been reported that FOXO proteins, that bind to the insulin response elements in the PDK4 promoter are required for glucocorticoid response. Insulin, by phosphorylating FOXO proteins suppresses GR’s ability to induce PDK4 expression. Other than glucocorticoids, retinoic acid, fatty acids, PPAR alpha ligands and PGC1- are all known to activate its gene expression (Ma et al., 2005). In diabetes mellitus, induction of PDK4 by glucocorticoids is increased leading to hyperglycemia (Kwon and Harris, 2004).

Role of Glucocorticoids in Metabolic Syndrome:

An increasing body of evidence suggests a role of glucocorticoids in the pathogenesis of type II diabetes. In fact, the strong resemblance between Cushing’s syndrome of endogenous and exogenous glucocorticoid excess and metabolic syndrome made endocrinologists speculate that glucocorticoid levels may be elevated in metabolic syndrome and obesity. Consistent with this, patients with insulin resistance and diabetes show slightly elevated serum levels of glucocorticoids (Walker, 2006). Hyperglycemia is a major hallmark of metabolic syndrome. Several studies reveal a direct role of glucocorticoids in inducing hyperglycemia in metabolic syndrome. Mice with liver- specific inactivation of GR were protected against streptozotocin-induced hyperglycemia and show reduced expression of gluconeogenic enzymes, validating the role of GR in promoting hyperglycemia in diabetes (Opherk et al., 2004). Liver-specific GR antagonism by antisense oligonucleotides and synthetic compounds decreases fasting blood glucose levels, improves glucose tolerance and increases insulin sensitivity in

32 diabetic mice models (Jacobson et al., 2005; Liang et al., 2005; von Geldern et al., 2004).

Short-term treatment of obese db/db mice with RU-486 (a GR antagonist) normalizes postprandial glucose values (Gettys et al., 1997).

Glucocorticoids and Hepatic Lipid Metabolism:

Molecular mechanisms leading to hepatic lipid accumulation in response to

glucocorticoids has been studied in less detail compared to its role in glucose homeostasis

and thus they have remained largely elusive. Abnormal hepatic fat accumulation, also

known as hepatic steatosis or fatty liver has been linked to insulin resistance and

metabolic syndrome. Hepatic steatosis directly impairs intracellular insulin signaling by

activating protein kinase C (PKC) and jun N-terminal kinase 1 (JNK1) pathways (Samuel

et al., 2004). Activation of these enzymes interferes with tyrosine phosphorylation of

insulin receptor substrates (IRS1 and IRS2) and impairs downstream signaling events,

such as insulin-stimulated protein kinase B (Akt) activation and glycogen synthesis.

Using in vivo magnetic resonance imaging and spectroscopy studies, Hwang et al (2007)

further demonstrated that elevated levels of intrahepatic triglyceride (TG) are also

associated with hepatic dysfunction and peripheral insulin resistance. Studies conducted

in bovine hepatocytes and humans also revealed an inverse correlation between increased

liver TG and liver insulin clearance (Kotronen et al., 2007; Strang et al., 1998).

Chronically elevated glucocorticoid levels also lead to aberrant accumulation of

lipid in the liver and may be an important risk factor in the development of fatty liver in

patients with metabolic syndrome. This is further supported by the fact that hepatic

steatosis is one of the pathophysiological feature of Cushing syndrome (Lonardo et al.,

33 2006; Taskinen et al., 1983) . Several in vivo and in vitro results suggest that GCs

increase lipid synthesis in the liver. Glucocorticoid treatment of rats increased hepatic TG

synthesis and lipid accumulation (Cole et al., 1982). This study reported complete

oxidation of fats but repressed ketogenesis in response to glucocorticoid treatment. The

effect of glucocorticoids on lipid synthesis could be partially explained by its ability to

stimulate the production of acetyl-CoA carboxylase, an enzyme that synthesize malonyl-

CoA for de novo lipogenesis. Additionally, GCs act to increase the expression of fatty

acid synthase (FAS), another key enzyme that utilizes malonyl-CoA for lipid synthesis

(Soncini et al., 1995). In several cell-based assays, synthesis of TG was increased in

isolated hepatocytes exposed to Dex (Giudetti and Gnoni, 1998; Mangiapane and

Brindley, 1986). A role of GCs on hepatic lipid accumulation was further corroborated by studies conducted in adrenalectomized rats. These animals when fed high fat diet, showed no hepatic TG accumulation, and this condition was reversed by exogenous GC treatment (Mantha et al., 1999). Mice bearing liver-specific disruption of GR also show reduced serum TG levels (Opherk et al., 2004).

Hepatic TG synthesis from fatty acids and its secretion as Very Low-Density

Lipoprotein (VLDL) particles is a complex process that includes several enzymes and final incorporation to ApoB proteins with the help of microsomal transport proteins

(MTP) (Hussain et al., 2003). Livers of mice treated with Dex increased expression of enzymes that participate in TG synthesis, such as Diacylglycerol O Acyl Transferase 1 and 2 (DGAT1 and DGAT2), but decreased TG hydrolase (TGH) mRNA stability - an enzyme responsible for TG lipolysis. Thus, combination of reduced lipolysis and increased lipid synthesis over-powered the secretion of TG and led to massive

34 accumulation in the liver. According to this study, Dex had no effect on the secretion of

TG by liver (Dolinsky et al., 2004).

In contrast to their opposing effects on most metabolic functions, glucocorticoids synergistically act with insulin to up-regulate lipogenesis. In vitro, glucocorticoids are necessary to potentiate the action of insulin on a number of lipogenic enzymes in rat hepatocytes (Hillgartner et al., 1995). Studies on adrenalectomized rats suggest that glucocorticoids are necessary for the lipogenic response to re-feeding after starvation

(Williams and Berdanier, 1982).

Modulation of local GC levels and actions by 11--hydroxysteroid dehydrogenase

(HSD) enzymes is an active area in current glucocorticoid research. There are two isoenzymes of HSD: 11HSD1 and 11HSD2. Type one converts inactive cortisone to active cortisol and type two does the reverse. Livers of db/db mice show increased expression of 11HSD1 and PEPCK proteins suggesting increased GC activity in murine diabetic models. They also had high serum corticosterone, insulin and blood glucose levels. Treatment of these mice with the glucocorticoid antagonist, RU-486, reduced the phenotype of type II diabetes (Liu et al., 2005). In mice, over-expression of hepatic

11HSD1 resulted in mild insulin resistance, fatty liver, dyslipidemia and increased hepatic lipid synthesis (Paterson et al., 2004).

Studies that show glucocorticoid effects on -oxidation have been largely inconclusive. A study by Agius et al (1986) reported no direct effect of glucocorticoids on carnitine palmitoyl transferase 1 (CPT-1), the essential rate limiting enzyme required for mitochondrial -oxidation. However, PPAR, a transcription factor that regulates the expression of many enzymes involved in -oxidation, itself is transcriptionally activated

35 by GR (Lemberger et al., 1994). Therefore, increased GCs may indirectly increase lipid oxidation by increasing the levels of PPAR. Clearly more studies are needed to demonstrate a definite role of GCs in regulating fatty acid oxidation.

Skeletal Muscle:

Insulin resistance in skeletal muscle is considered to be a key factor that contributes to the pathogenesis of type II diabetes. A strong relationship between intramyocellular TG content and metabolic syndrome have been demonstrated by recent studies in animals and humans. However, the mechanisms by which excess TG in muscle initiate insulin resistance remains controversial (Dresner et al., 1999). Based on studies using isolated perfused rat heart and diaphragm, Randle et al. (1963) first postulated that excess FFA impair glucose metabolism by substrate competition. According to the classical Randle hypothesis, increased FFA oxidation reduces glucose oxidation via allosteric effects of the products of FA oxidation. This includes inhibition of several enzymes, such as phosphofructokinase-1 and also the PDC. However, only few in vitro and in vivo experiments conducted in skeletal muscle were able to actually confirm this hypothesis (Dresner et al., 1999; Roden et al., 1996). Together, these studies suggest that in muscle, FFA have effects on glucose metabolism other than or beyond those postulated by the Randle cycle. Recent studies by Shulman and colleagues have demonstrated a direct link between FFA and insulin signaling. This happens when increased FFA concentration causes a concomitant increase in intracellular levels of diacylglycerol

(DAG) or long chain fatty acyl-CoA which directly stimulates PKC activity. PKC is a serine threonine kinase and phosphorylate serine residues of IRS-1. This reduces tyrosine

36 phosphorylation and impairs downstream receptor signaling (Yu et al., 2002).

Additionally, transgenic mice with inactivation of PKC are protected against lipid - induced defects in insulin action in skeletal muscle (Kim et al., 2004).

In skeletal muscle, glucocorticoids promote protein break down to produce substrates available for gluconeogenesis. Therefore, they represent catabolic hormones and counteract the anabolic effects of insulin signaling in muscle. Consistent with this, muscle atrophy (muscle wasting) is a common symptom in Cushing’s syndrome. A broad range of evidence supports the role of GCs in muscle catabolism and insulin resistance. Several target genes that play an important role in facilitating insulin resistance have been identified by a gene expression profile conducted in skeletal muscle of rats treated with Dex (Almon et al., 2005). In muscle, GCs inhibit glucose uptake, glycogen and protein synthesis. They do so by interfering with several metabolic pathways controlled by insulin. Treatment of rats with Dex resulted in reduced insulin receptor phosphorylation and PI3 kinase activity (Giorgino et al., 1993; Saad et al.,

1993). Both in vitro (C2C12 myotubes) and in vivo (muscles from rat) experiments show

GR’s ability to inhibit Akt phosphorylation (Long et al., 2003; Sandri et al., 2004). When rats were treated with Dex for 5 days, they showed reduced sensitivity to glucose uptake and glucose oxidation (Dimitriadis et al., 1997). Reduced numbers of GLUT4 transporters in response to insulin were also noted in the skeletal muscle of the same animals. Chronic GC treatment has been shown to suppress insulin-stimulated glycogen synthesis by impairing glycogen synthase kinase 3 (GSK-3) phosphorylation (Ruzzin et al., 2005).

37 Glucocorticoids in muscle, while promoting protein break down and amino acid

transport, inhibit protein synthesis. The latter is caused by its ability to interfere with Akt signaling pathway (Vegiopoulos and Herzig, 2007). However, GCs inhibit signaling events downstream of Akt. This was demonstrated in muscle of rats treated with Dex where they showed reduced phosphorylation of components involved in protein synthesis, such as p70S6-kinase and eukaryotic Initiation Factor 4E(eIF4E)-binding protein 1 that resulted in low rate of transcription-initiation and translation (Shah et al.,

2000). In muscle, protein degradation in response to GCs happens via the E3 ubiquitin proteasome degradation pathway. Several proteins that promote ubiquitination such as atrogin-1 and MuRF-1 are increased in response to GCs (Bodine et al., 2001). They also regulate mRNA levels of the subunits of 26S proteasome in fast twitch skeletal muscle

(Combaret et al., 2004). Glutamine synthase, an enzyme that participates in amino acid transport, is an example of a classical GC-inducible gene (Wang and Watford, 2007).

Recently, Myostatin, a protein that inhibits muscle growth was found to be up-regulated by Dex in rat muscle and C2C12 myotubes (Artaza et al., 2002; Ma et al., 2003). This protein inhibits protein synthesis and increases the expression of atrogin and MuRF-1 in skeletal muscle in vivo (McFarlane et al., 2006; Welle et al., 2006). Taken together, these results suggest that GCs promote insulin resistance by counteracting insulin actions at several check points and by promoting protein degradation and amino acid transport. The latter provides abundant substrates for gluconeogenesis and increases blood glucose levels.

Many enzymes that take part in lipid metabolism such as PPAR, PGC-1 and

PDK4 are also directly regulated by GCs in muscle (Smith and Muscat, 2005). Recently,

38 a gene that is responsible for increased energy expenditure, uncoupling protein-3 (UCP-

3), has also been found to be directly activated by GR (Amat et al., 2007). Uncoupling

protein-3 is a member of the mitochondrial membrane transporter, preferentially

expressed in skeletal muscle and favors fatty acid oxidation (MacLellan et al., 2005). It is

also thought to protect muscle against the production of excess reactive oxygen species

(ROS) and to dissipate energy as heat.

Adipose Tissue:

Increased glucocorticoid secretion is associated with central abdominal obesity which is closely linked to metabolic syndrome. In adipose tissue, glucocorticoids differentially affect peripheral and central adipose depots. In the peripheral adipose tissue, they are mostly lipolytic. This is supported by the fact that GCs increase hormone-sensitive lipase

(HSL) mRNA levels in rat adipocytes (Slavin et al., 1994). Hormone-sensitive lipase is an enzyme responsible for the hydrolysis of TG into glycerol and non-esterified FFAs

(NEFA). These fatty acids are released into the blood stream and are taken up by the muscle to produce energy or by liver to undergo oxidation and/or esterification. Increased

GC secretion therefore increases NEFA pools and increased flux of NEFA is positively correlated with metabolic syndrome and insulin resistance (Macfarlane et al., 2008). In rat adipocytes, Dex directly impairs insulin signaling by down-regulating IRS-1, PI3 kinase and PKB and causing a parallel reduction in glucose transport (Buren et al., 2002).

Phosphoenolpyruvate carboxykinase is an enzyme that not only plays an important role in

hepatic gluconeogenesis but also regulates glyceroneogenesis in adipose tissue.

Glyceroneogenesis is the de novo synthesis of glycerol 3-phosphate from glucose that

39 forms the back bone of TG molecules (Reshef et al., 2003). In contrast to its positive

regulation in the liver, GCs negatively regulate PEPCK expression in adipose tissue by

inhibiting CCAT/enhancer binding protein (C/EBP), a transcription factor important for glyceroneogenesis (Olswang et al., 2002). Thus, GC actions in the peripheral adipose

tissue prevent fat storage and stimulate lipolysis.

In the central fat depots, GCs promote adipogenesis and cellular hypertrophy

(Gaillard et al., 1991). A role of GCs in the transcriptional cascade leading to

preadipocyte differentiation is also supported by in vitro studies using 3T3L1 murine

cells and human preadipoctyes. In both cases, treatment of cells with a cocktail

containing Dex and insulin caused differentiation of adipocytes - a process also mediated

by C/EBP transcription factors (Wu et al., 1996). In simple obesity without metabolic

syndrome, plasma cortisol is not elevated. However, the systemic GC actions are

amplified due to tissue-specific expression of 11HSD1. Its activity is increased in

adipose tissue of obese humans and rodents suggesting adipose tissue glucocorticoid

excess (Seckl et al., 2004; Tomlinson and Stewart, 2007). Transgenic mice over-

expressing this protein in adipose tissue exhibit symptoms of metabolic syndrome

(Masuzaki et al., 2001). Similarly, adipose-specific inactivation of 11HSD1 protects

animals from diet-induced obesity, further supporting the role of this protein in

promoting obesity (Kershaw et al., 2005).

Other Steroid Receptors in Metabolism:

In mammals, steroid receptors AR and ER are mostly associated with reproductive

functions. However, an increasing body of evidence now indicates their role in regulating

40 energy metabolism as well. Low levels of androgen and estrogen have been implicated in

insulin resistance and atherosclerosis. A low serum concentration of endogenous androgen is linked with abdominal fat deposition. An age-dependent decrease in serum testosterone and gradual increase in body fat mass is also usually seen in men. Androgen deprivation by castration to treat prostate cancer promotes insulin resistance while testosterone administration in type II diabetic men improves glucose tolerance (Fukui et al., 2007). Understanding the molecular mechanism by which androgen improves insulin sensitivity and glucose levels is an ongoing effort in many laboratories. Transgenic and knockout AR animal models provide great tools to study the metabolic defects mediated

by this receptor. Aging AR - null male (ARKO) mice showed reduced insulin sensitivity and glucose intolerance (Lin et al., 2005). These animals displayed weight gain, hyperinsulinemia, hyperglycemia, hyperleptinemia, hypertriglyceridemia and ectopic TG deposition in liver and muscle. Histological analysis of adipocytes derived from ARKO animals showed them to be significantly larger compared to wild-type counterparts.

Glucose tolerance (GTT) and insulin tolerance test (ITT) revealed insulin resistance in

ARKO mice. This was also correlated with reduced PI3 kinase activity in insulin target

organs such as skeletal muscle and liver. Even though serum leptin levels were higher in

ARKO mice, these animals were leptin-resistant as exogenous leptin treatment failed to reduce their body weight. Consistent with increased lipid accumulation in liver, muscle and white adipose tissue (WAT), mRNAs of several enzymes that participate in lipid synthesis were elevated. Dihydrotestosterone (DHT) replacement therapy failed to reverse the phenotype seen in ARKO mice confirming a direct role of AR in mediating glucose and lipid homeostasis. To better understand the significance of AR in hepatic

41 metabolism, a liver-specific AR knockout mouse was generated (Lin et al., 2008). These

animals, when fed high fat diet, developed steatosis and insulin resistance. Excess liver lipid accumulation was due to reduced fatty acid oxidation and increased lipid synthesis.

Expression of PPAR mRNA levels was down-regulated and sterol regulatory binding protein-1C (SREBP1C), a protein that plays an important role in lipogenesis, was up- regulated. Reduced insulin sensitivity was associated with increased expression of gluconeogenic enzymes and reduced PI3 kinase activity.

With the help of estrogen receptor  (ERKO) knockout and aromatase-deficient knockout (ArKO) mice considerable progress has been made in our understanding of how

ER affects glucose and lipid metabolism. Both male and female ERKO animals were

hyperinsulinemic and hyperglycemic (Bryzgalova et al., 2006). Whole body insulin

sensitivity and glucose uptake by peripheral organs were slightly reduced in these

animals. Insulin resistance was mainly due to defective suppression of glucose production

in the liver. This is in agreement with the finding that estrogens improve insulin

sensitivity by suppressing gluconeogenic enzymes (Ahmed-Sorour and Bailey, 1981).

Microarray analysis of livers of ERKO animals revealed increased expression of

lipogenic enzymes and decreased expression of genes involved in lipid transport. The

aromatase knockout mouse is another important model to understand ER actions in

maintaining energy homeostasis (Jones et al., 2000). These animals lack Cyp19 gene that encodes aromatase. Aromatase catalyzes the final step in the production of endogenous estrogen and ArKO mice therefore lack endogenous estrogen. Both male and female

ArKO mice accumulated intra-abdominal adipose tissue. This was associated with increased adipocyte volume, hyperleptinemia, hyperinsulinemia, hypercholesterolenemia,

42 fatty liver, reduced lean mass and reduced spontaneous physical activity. Although further investigations are needed to unravel the exact molecular and cellular mechanisms, the above mentioned studies clearly demonstrate that androgens and estrogens acting via

AR and ER can significantly influence glucose and lipid metabolism.

43 MATERIALS AND METHODS

Animal Maintenance and Husbandry:

The generation of FKBP51 and FKBP52 mutant mice was reported earlier (Yang

et al., 2006; Yong et al., 2007b). These animals were maintained on a mixed

C57BL/6J:129SvEv background. Since both strains were created independently by two different targeting strategies, separate WT littermates (for each strain) were used as controls. FKBP52 male and female knockouts are infertile and cannot be used for breeding purposes. To generate homozygous animals, a breeding scheme was carried out with heterozygous animals. The yield of FKBP52-/- pups from such breeding was far below the expected Mendellian ratio. In order to generate sufficient age - and sex - matched animals to obtain statistical differences, FKBP52+/- animals were used for experiments. FKBP51-/- animals were fertile and gave birth to homozygous pups.

Animals were housed in a temperature-controlled environment with 12 h dark-light cycle.

All procedures were approved by the Institutional Animal Care and Utilization

Committee of The University of Toledo. Experiments were performed on male mice of two months of age.

Diet:

Under normal conditions mice were fed a standard chow containing 12 kcal% fat

ad-libitum. Two month-old WT and mutant mice were grouped into three separate

groups. One group fed standard chow is used as a control, another for fasting and the

third group was fed a high fat diet (ad-libitum) containing 45 kcal% fat (Research Diets).

During HF diet treatment, body weight of each individual animal was recorded weekly.

44 After four weeks, an over-night fast (16-18 h; food removed at 5:00PM on the day prior

to experiments) was performed on the second set of animals. On the following day, all

groups of mice were anesthetized using sodium pentobarbital (55 mg/kg body weight) at

11:00 AM. After blood collection, animals were euthanized and various organs were

weighed and collected for further analyses.

Metabolic Analyses:

Whole venous blood was drawn from the retro-orbital sinuses to measure various

metabolic analyses. Blood was collected in heparin-coated tubes, kept on ice and spun for

15 minutes to collect plasma. Blood glucose levels were measured using a glucometer

(Accu-check Aviva – Roche Diagnostics). Corticosterone (MP Biomedicals), plasma insulin and C-peptide levels (Linco Research) were measured by radioimmunoassays.

Plasma FFAs (Wako) and TG (Pointe Scientific) were measured by colorimetric assays.

Gel Electrophoresis and Western Blotting:

Extracts were prepared by homogenizing the tissue samples using lysis buffer

[150 mM NaCl, 50 mM Hepes, pH 7.6, 5% sodium azide, Triton X-100 and freshly

added protease inhibitors (Sigma St.Louis, MO)]. Homogenized samples were rotated in

the cold room (40C) for 90 min followed by centrifugation at 20,000 X g for 30 min.

Supernatant was allocated based on equal protein content and resolved on 10%

polyacrylamide SDS gels as described by Laemmli (Laemmli, 1970). Proteins were then

transferred to Immobilon polyvinylidene difluoride (PVDF) membrane. Specific primary

antibodies were used to probe for each protein tested [GR (FiGR monoclonal antibody was a gift from Jack Bodwell, Dartmouth Medical School, Hanover, NH), FKBP51,

45 FKBP52, GAPDH (Santa Cruz), CyP40 (Affinity Bioreagents, PP5 (gift from Michael

Chinkers, University of South Alabama, College of Medicine, Mobile, AL), ApoB48/100

(Chemicon International), FAS (Najjar et al., 2005), MTP (BD Biosciences)]. The blots

were incubated with appropriate secondary antibodies, developed by enhanced chemiluminescence (ECL) and quantified by densitometry.

Northern Blot:

Total liver mRNA was purified using Trizol (Invitrogen Corporation, Carlsbad, CA),

separated by electrophoresis on a formaldehyde gel, and analyzed by Northern blotting.

Using random primed DNA labeling kit, cDNAs for PEPCK, PDK4 and G6Pase were

used to probe Nytran membranes. GAPDH cDNA was also used to normalize for the

amount of mRNA applied. Hybridization was detected on HyBlot autoradiography film

(Denville Scientific Inc.) and quantified by densitometry.

Oil Red O Staining of Liver Samples:

Frozen liver samples were sliced (thickness: 10 microns) using a cryostat. The

sections were stained with Oil Red O (Sigma) and counterstained with hematoxylin

(Fisher Scientific).

Tissue Triglyceride Content:

Hepatic and muscle triglyceride content was measured by extracting lipid from

homogenized samples with CHCl3:CH3OH (2:1, v:v). The samples were dried under N2

and resuspended using ethanol. Tissue Triglyceride levels were measured from the samples by a colorimetric assay (Pointe Scientific).

46 Transient Transfection and Luciferase Reporter Assays:

Mouse Embryonic Fibroblasts (a gift from Dr. Weinian Shou, Indiana University,

School of Medicine, Indianapolis, IN) were cultured in DMEM containing 10% charcoal

stripped calf serum to 90-95% confluency in 6-well plates. They were transiently

transfected with reporter constructs for pGRE2EIB-Luc or PEPCK-luc (gift from Dr.

Darryl Granner, Vanderbilt Medical Center, Nashville, TN) using Lipofectamine 2000.

To normalize transfection efficiency, cytomegalovirus-driven galactosidase reporter was

also co-transfected with reporter constructs. Twenty-four hours post-transfection, cells were treated with 1µM Dex (Sigma Chemical Co, St.Louis, MO) and were harvested

after an additional 24 h. Luciferase assays were performed using a commercially

available kit from Promega (Madison, WI).

Reverse Transcriptase PCR (RT-PCR):

Total RNA was extracted from tissues (soleus muscle) using Trizol (Invitrogen

Corporation) reagent and was treated with 1U of RQ1 RNase-free DNase (Promega)/µg

of RNA. After spectrophotometric quantification, reverse transcription was carried out

with a 1st Strand cDNA Synthesis Kit for RT-PCR (Roche) using oligo (dT) primer as per

manufacturer’s instructions. First strand cDNA was amplified using PCR master mix

(Promega) using specific primers against muscle CPT1 and PDK4. Ribosomal RNA (18s)

was also amplified as an internal control to ensure equal amount of template

concentration between samples. Amplified PCR products were electrophoresised on 1%

agarose gels, visualized using ethidium bromide staining and quantified by densitometry.

47 Statistical analysis:

Data were analyzed with GraphPad Prism using unpaired t-test. P values less than 0.05 were considered to be statistically significant.

48 RESULTS

FKBP51 and FKBP52 animals were fed a diet of 45 kcal% fat that is generally

used to induce obesity in murine models. Since each mutant strain was created independently by a separate targeting strategy, different WT controls generated from their own littermates were used for each mutant. Also as reported previously (Tranguch et al.,

2005; Yang et al., 2006), FKBP52 male and female knockout animals are infertile and

cannot be used for breeding purposes. Hence, any breeding to generate homozygous

animals has to be done by mating heterozygotes. The yield of FKBP52 (-/-) from

breeding +/- animals was far below the expected Mendelian ratio (about 40%). In order to

generate a large pool of sex - and age - matched animals to obtain statistical difference,

experiments were performed on FKBP52+/- animals. When appropriate, MEFs derived

from FKBP52 -/- animals were also used, to further substantiate our conclusions.

FKBP51-null animals show no reproductive abnormalities (Yong et al., 2007) and

knockout pups are viable at birth and follow expected the Mendelian ratio. Unless otherwise stated, male mice of two months age of both strains were used for the study.

As mentioned before, two month-old WT52, FKBP52+/-, WT51 and FKBP51-/- animals were fed a high fat diet for four weeks. During this period, each animal was monitored for weight gain. At the end of the treatment, they were sacrificed for blood and organ collection. In addition to the HF diet, as a different mode of metabolic stress, over- night fasting (16-18 h) was also performed on mutant animals. Lack of a phenotype

(except for the low TG and PDK4 levels) under this condition was somewhat surprising, but could be due to the short duration of fast. During this period (early hours of fasting) euglycemia is mostly maintained by glycogenolysis regulated by glucagon. It is only

49 during prolonged fasting (starvation) that actions of glucocorticoids become necessary to

maintain homeostasis. Therefore, a prolonged fasting of 36-48 h may show a more

dramatic phenotype in these animals. For the most part, similar metabolic in vivo studies

employ a short fast after HF diet treatment to obtain steady state measurements of the

metabolic parameters. However, both fasting and HF diet by themselves regulate the

HPA axis and in turn, glucocorticoid levels. In order to keep these variables separate and

to avoid any overlapping effects, fasting was not performed at the end of the HF diet. The

following sections describe the phenotype of both mutant mice on HF diet and provide

data for molecular mechanisms that support our hypothesis.

FKBP51 and FKBP52 differentially modulate GR transcriptional activity

Although the oppositional effects of FKBP51 and FKBP52 on receptor functions

have been previously reported, most of the studies were done by over-expressing either

TPR protein in cells. For example, over-expression of FKBP51 significantly reduces

GR’s ability to bind hormone, leading to glucocorticoid resistance in New World

primates (Scammell et al., 2001). Riggs et al (2003) confirmed this inhibitory effect of

FKBP51 and further demonstrated a positive regulation by FKBP52, specific for

glucocorticoid receptor ectopically-expressed in yeast. Here, we tested this reciprocal

regulation by a direct approach using MEFs derived from WT and TPR knockout embryos. The MEF cells express GR but lack AR, PR or ER. The effects of FKBP51 and

FKBP52 on GR were measured by transfecting the null TPR MEF cell lines with a GR - responsive pGRE2EIB-Luc minimal reporter. This minimal promoter is composed of two synthetic glucocorticoid response elements driving the expression of a luciferase reporter

50 gene, allowing us to eliminate any possibilities of interaction of other transcription

factors contributing to reporter activity. The cells were treated with Dex, a potent

glucocorticoid agonist (Fig. 6).

FIGURE 6. FKBP52 IS A POSITIVE AND FKBP51 IS A NEGATIVE MODULATOR OF GR TRANSCRIPTIONAL ACTIVITY

GRE-Luc GRE-Luc + Dex 140000    90000

40000 -gal activity   20000 15000 10000  5000  Luciferase/ 0 WT 52 -/- 51 -/-

WT, KO52 and KO51 MEFs were transiently transfected with pGRE2EIB-Luc and -GAL expression vector followed by treatment with 1µM Dex for 24 h. Values represent the means ± S.E.M. of three independent experiments.

In WT cells, as expected, a hormone-induced increase in reporter activity was observed.

In FKBP52 -/- cells, this induction was dramatically reduced, but not completely lost

(Two-fold increase compared to 20-fold increase in WT). In cells lacking FKBP51, the response to Dex was greater than that observed in WT cells. These results confirm that

FKBP51 and FKBP52 exert reciprocal effects on GR transcriptional activity and suggest a modulatory rather than essential role for TPRs in regulating GR function.

51 Expression profile of TPR proteins in metabolic organs

As tissue distribution of proteins greatly influences in vivo events, we examined the expression profile of GR and TPR proteins in various organs that are metabolic targets of GR action. Fig. 7A shows immunoblot analysis of proteins present in the whole tissue extracts of WT male mice. When probed for GR, several bands of approximately

97kDa were found. These are likely multiple GR isoforms. Several isoforms of GR due to alternative transcriptional splicing and translation initiation sites have been reported (Lu and Cidlowski, 2004). Most studies, however, concentrate on one isoform of GR: GR.

Therefore, it would be interesting to see if different isoforms or modifications exist within a particular tissue and if these differences, in turn, translate to different functions.

We found ubiquitous expression of FKBP51 and PP5 in all tissues tested with differences in their levels in each organ. Interestingly, expression of FKBP52 and Cyp40 showed a tissue specific pattern with FKBP52 being expressed only in liver, pancreas and adrenal and Cyp40 in all tissues except pancreas. This tissue - specific expression of TPR proteins perhaps forms an additional basis for receptor selectivity.

Since liver is an important organ for GR control of metabolism, levels of GR and

TPR proteins in WT and FKBP mutant livers were measured by immunoblotting (Fig.

7B). As expected, expression of FKBP51 was absent in the livers of FKBP51-/- and levels of FKBP52 is reduced to half of WT in FKBP52+/- animals. Expression levels of all other TPR proteins were comparable between WT and mutant samples.

52 FIGURE 7. TISSUE DISTRIBUTION OF TPR PROTEINS

Expression levels of GR and TPR proteins were measured in lysates from liver (L), muscle (M), pancreas (P), adipose (Adi) and adrenals (Adr) of wild-type mice (A) as well as from liver lysates of WT, FKBP52+/- and FKBP51-/- animals (B). Analysis was performed by Western-blotting using lysates of equal protein content.

FKBP51 and FKBP52 mutant mice show diverse phenotypes on high fat diet

As stated earlier, we set out to study the role of TPR proteins in GR-controlled metabolism in an environment that typically leads to metabolic derangements in humans.

Consumption of a Western diet rich in fat is widely known to cause metabolic abnormalities (Chew et al., 2006). Therefore, we exposed our mutant FKBP animals to a high fat diet regimen known to produce abnormalities characteristic of metabolic

53 syndrome, such as visceral adiposity, weight gain, and increased glucose and insulin levels.

FIGURE 8. FKBP52 AND FKBP51 ANIMALS SHOW DIFFERENT PHENOTYPES ON HIGH FAT DIET

A B C

D E F

Two month-old male mice were either fed regular diet (RD) or high fat diet (HF) for one month. After the treatment animals were euthanized and organs were collected. A/D, Whole WT52 and FKBP52+/- or whole WT51 and FKBP51-/- animals fed HF diet for one month. B/E, Percent weight gain in WT and 52+/- or WT and FKBP51-/- animals fed RD or HF during one month period. C/F, After one month of RD or HF feeding intra- abdominal (visceral) adipose tissue were weighed and visceral adiposity was expressed as percent of total body weight. Values expressed as mean ± S.E.M. (n = 5-11; ≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups)

Based on our molecular studies in MEF cells, we predicted that FKBP52 mutant mice will have less GR activity and therefore would be protected from the adverse effects of high fat feeding. Conversely, ablation of FKBP51 should create a hyper-active GR,

54 generating mutants that are more susceptible to the diet-induced abnormalities.

Interestingly, the pheonotypes we obtained after a month of chronic high fat treatment were far from what was expected. FKBP52 mutant mice fed HF gained similar weight

and showed the same amount of visceral adiposity compared to WT littermates (Figs. 8A,

8B and 8C). In contrast, FKBP51 -/- animals were protected against diet-induced obesity

and visceral adiposity (Figs. 8D, 8E and 8F).

High fat diet induces hyperglycemia, hyperinsulinemia and decreased insulin clearance in FKBP52, but not FKBP51, mutant mice

As hyperglycemia and hyperinsulinemia are the hallmarks of MS (Chew et al.,

2006), we next examined random glucose, and insulin levels in FKBP mutant animals fed

HF. WT52 animals showed a trend towards increased blood glucose levels in response to

HF, but the difference did not reach statistical significance compared to WT52 animals on a regular diet (Fig. 9A). Although mice fed a high fat diet can develop insulin

resistance or diabetes (Buettner et al., 2007), the occurrence and extent of this response is dependent on many variables, such as duration of diet, age of animals and genetic background. Thus, lack of a strong hyperglycemic response in our WT animals could be due to any one of these factors. In contrast to WT52, FKBP52 mutants fed HF showed a significant elevation of plasma glucose levels, suggesting that FKBP52 loss makes the animals more sensitive to diet-induced hyperglycemia. Under regular diet conditions, no difference in glucose levels between WT52 and FKBP52 mutant animals was noted.

Since hyperglycemia is usually a consequence of hyperinsulinemia (Kahn, 1994), we measured random serum insulin levels (Fig. 9B). WT52 animals on high fat diet

55 showed a moderate rise in insulin levels. Since these animals showed almost normal

glycemia, the rise in insulin levels most likely indicates that our 4-week, high fat diet

regimen is producing the early stages of insulin resistance. Interestingly, FKBP52

mutants on high fat diet showed a greater increase in insulin levels, suggesting a

protective effect of FKBP52 against diet-induced hyperinsulinemia.

FIGURE 9. FKBP52 ANIMALS ON HF DIET SHOW MILD HYPERGLYCEMIA, HYPERINSULINEMIA AND REDUCED INSULIN CLEARANCE

A B C

D E

Both RD and HF fed animals were anesthetized and whole venous blood was drawn from retro-orbital sinuses to measure glucose levels (A), plasma insulin (B), corticosterone (C) and C-peptide (D). Insulin clearance was measured by calculating the C-peptide/insulin ratio (E). Values expressed as mean ± S.E.M. (n = 5-23; ≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups)

In some studies of diet-induced insulin resistance, elevated GC levels are also observed (Walker, 2006). Measurement of plasma corticosterone levels showed no

56 increase for this hormone in WT52 animals subjected to HF, consistent with the lack of hyperglycemia under these conditions (Fig. 9C). However, a significant elevation of corticosterone was seen in FKBP52 mutants fed HF, indicating that the protective effect

FKBP52 extends to this key metabolic factor. Although this result also suggests that the underlying cause of hyperglycemia may be an elevated GC response in mutant mice, further tests of GR hepatic activity in FKBP52 mutant mice do not support this mechanism (see below).

Hyperinsulinemia can result from increased insulin secretion at pancreatic -cells or from decreased insulin clearance at the liver. To better understand the cause of elevated insulin in FKBP52 mutant mice, we tested C-peptide levels as an indicator of insulin secretion. C-peptide is a cleavage product of proinsulin that is secreted in a 1:1 molar ratio with insulin (Michael et al., 2000). In response to HF, a significant increase in

C-peptide secretion was seen in WT52 animals and a trend towards increased secretion in

FKBP52 mutants (Fig. 9D). Importantly, no statistical difference was noted in C-peptide levels between WT52 and FKBP52 mutants fed high fat. These results indicate that insulin secretion is intact and comparable in these animals. To estimate insulin clearance,

C-peptide/insulin molar ratios were calculated (Fig. 9E). In WT52 animals, no difference was noted in insulin clearance in response to high fat. In contrast, insulin clearance was greatly reduced in FKBP52 mutants on high fat. Therefore, we conclude that hyperinsulinemia in FKBP52 mutants is due to impaired clearance.

57 FIGURE 10. LOSS OF FKBP51 DOES NOT AFFECT GLUCOSE OR INSULIN LEVELS

A B C

D E

Both RD and HF fed mice were anesthetized and whole venous blood was drawn from retro-orbital sinuses to measure glucose levels (A), plasma insulin (B), corticosterone (C) and C-peptide (D). Insulin clearance was measured by calculating the C-peptide/insulin ratio (E). Values expressed as mean ± S.E.M. (n = 5-11; ≠ p < 0.05; ≠ ≠ p < 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups)

FKBP51 mutant mice and corresponding WT controls were subjected to the same analysis. In contrast to FKBP52 mutants, FKBP51 (–/–) mice did not develop hyperglycemia in response to HF, and the rise of serum insulin in response to HF was comparable to that seen in WT51 controls (Figs. 10A and 10B). C-peptide secretion was normal and a trend to reduced insulin clearance was noted in both WT51 and FKBP51 mutants fed HF (Figs. 10D and 10E). However, these trends did not reach statistical significance. We therefore conclude that loss of FKBP51, unlike FKBP52, does not make mice more susceptible to diet-induced insulin resistance. In keeping with this

58 conclusion was the lack of hypercorticosteronemia in FKBP51 mutants fed HF (Fig.

10C). Indeed, these animals had a significant reduction of serum corticosterone, which likely results from the protective effects of FKBP51 loss against diet-induced metabolic stress (see Discussion).

Increased hepatic lipid accumulation and altered lipid metabolism in FKBP52 mutant mice

Since high fat diet is known to cause lipid accumulation in the liver (fatty liver or steatosis), leading to impairment of insulin signaling and, ultimately, insulin resistance, we next examined whether deficiency of either TPR protein affects liver lipid accumulation. Fig. 11A shows Oil Red O staining of livers from WT52 and FKBP52 mutants to detect accumulation of neutral lipids. As expected, WT52 animals on HF diet showed an increase in lipid accumulation compared to animals on RD. FKBP52 mutants on HF diet showed a larger number of macro-vesicular lipid vacuoles, as well as vacant, non-stained areas, which could be interstitial depots of fat washed away before staining.

These results were also quantified by measuring hepatic TG content (Fig. 11B).

Consistent with Oil Red O staining, FKBP52 mutants on HF diet showed a significant

increase in their hepatic TG content compared to WT52. Under regular diet conditions,

FKBP52 mutant mice had a greater number of small lipid droplets, but this difference did

not reach significance when TG content was measured. In contrast to FKBP52 mutants,

FKBP51-deficient animals fed HF showed levels of hepatic lipid comparable to those

seen in WT51 controls as measured by both Oil Red O and TG assay (Figs 11C and

11D). As a whole, these results suggest that FKBP52 loss makes mice more susceptible

59 to diet-induced steatosis, while FKBP51 loss does not.

FIGURE 11. INCREASED HEPATIC LIPID ACCUMULATION IN FKBP52 MUTANTS ON HF DIET

A B

C D

Oil Red O staining of liver sections taken from WT and FKBP52+/- (A) or WT and FKBP51-/- (C). B/D, Hepatic TG content was measured from homogenized liver tissues by extracting lipid with CHCl3: CH3OH (2:1, v:v). Values expressed as mean ± S.E.M. (n = 3-5; ≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups)

60 FIGURE 12. EXPRESSION OF PROTEINS IMPORTANT FOR LIVER LIPID METABOLISM

A

B C D

A, Western analysis of proteins important for lipid homeostasis in liver. B, C, D, Quantitation of results in Panel A was achieved by densitometric scanning of films. Values for each protein is expressed as percentage of the maximum value obtained after scanning and represent means ± S.E.M. of four independent experiments. (≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups)

Because lipid accumulation in FKBP52 mutants fed a HF diet was elevated, we wanted to know if this was due to altered expression of proteins that play a role in lipid synthesis or re-esterification, such as MTP, ApoB100 and FAS. The first two are important for the re-esterification process of lipids and the latter contributes to de novo synthesis. There were no differences between WT52 and FKBP52 mutants with respect to MTP or ApoB100 expression under RD and HF diet conditions (Figs. 12A, 12B and

12C). It is currently not known whether MTP or ApoB proteins are direct transcriptional

61 target genes of GR. Fatty acid synthase is regulated by GCs in lung and human adipose tissue (Lu et al., 2001; Rufo et al., 1999; Wang et al., 2004b), but nothing much is known about its regulation by GCs in liver. Hepatic FAS regulation by insulin, however, is well documented (Shimomura et al., 2000). There was a significant induction of FAS in

FKBP52 mutants fed HF diet (Figs. 12A and 12D). Even under RD conditions, FAS expression levels showed a tendency to increase, although it did not reach statistical significance. This result is consistent with our hepatic lipid data (Figs. 11A and 11B) and may be at least one of the contributing factors to elevated steatosis in FKBP52 mutants.

Normal serum triglycerides and free fatty acids but elevated muscle triglyceride content in FKBP52 mutants fed high fat

High serum TG and free fatty acids (FFA) are closely associated with metabolic disorders. Therefore, we measured plasma TG and FFA in both FKBP52 and FKBP51 mutants. In WT animals exposed to HF, TG levels showed either no increase or a non- statistical increase (Fig. 13A). This is further indication that our four-week HF diet regimen is a moderate effector of metabolic syndrome. In FKBP52 mutants fed HF, TG and FFA levels (Fig. 13B) did not change compared to WT. The normal TG level was unexpected, considering the increased lipid accumulation in the livers of FKBP52 mutant animals. Because ApoB100 and MTP levels were normal in FKBP52 mutants fed HF

(Fig. 7A), it is likely that reduced hepatic export of triglycerides cannot account for the lower than expected serum levels of these metabolites. Instead, we tested the hypothesis that FKBP52 mutants undergo redistribution of TGs to peripheral organs, such as muscle or adipose tissue. Fig. 13C shows direct measurement of TG levels in soleus muscle. As

62 expected, WT52 animals on HF diet show a trend towards increased muscle TG

accumulation. But this difference was even more pronounced in FKBP52 mutants.

Therefore, increased uptake of TG from plasma by muscle may, at least in part,

contribute to the normal serum TG levels seen in FKBP52 mutants on a HF diet.

FIGURE 13. FKBP52+/- AMIMALS ON HIGH FAT DIET SHOW NORMAL SERUM TG AND FFA LEVELS BUT HAVE INCREASED MUSCLE TG

A B

C

After one month of HF diet treatment, both RD and HF fed animals were anesthetized and whole venous blood was drawn from retro-orbital sinuses to measure TG (A) and FFA (B) levels. C, Muscle TG content was measured from homogenized muscle tissues by extracting lipid with CHCl3:CH3OH. Values expressed as mean ± S.E.M. (n = 5-23; ≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups

63 FKBP51- deficient mice show reduced serum triglycerides and free fatty acids, as well

as elevated muscle fatty acid metabolism, on both regular and high fat diets

With respect to serum TG and FFA levels, the most striking differences were

noted in FKBP51 mutant mice, which had reduced TG and FFA levels under both regular

diet and HF diet conditions (Fig. 14).

FIGURE 14. FKBP51-/- AMIMALS SHOW REDUCED SERUM TG AND FFA LEVELS BOTH ON RD AND HF DIETs

A B

Two month-old male mice (WT and FKBP52+/-) were divided in to two groups. One group was fed RD and the other a HF diet. After one month, animals were anesthetized and whole venous blood was drawn from retro-orbital sinuses to measure TG (A) and FFA (B) levels. Values expressed as mean ± S.E.M. (n = 5-11; ≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups

These results are in good agreement with the ability of FKBP51 mutants to resist other adverse effects of high fat feeding, such as obesity and insulin resistance (Figs. 8 and 10). Taken as a whole, our results suggest that FKBP51 mutants have a high-energy expenditure phenotype, perhaps due to elevated GR activity. Glucocorticoids are known to increase the expression of proteins important for lipid clearance, such as PPAR and

64 PDK4 (Vegiopoulos and Herzig, 2007), elevation of which is likely to increase

mitochondrial -oxidation. Because hepatic lipid accumulation was comparable to WT animals, we reasoned that liver must not be the site of elevated lipid metabolism in

FKBP51 mutants. Instead, we tested soleus muscle, an important organ regulating energy expenditure (Wende et al., 2007), for the expression of genes important for lipid clearance.

Carnitine palmitoyl transferase-1 (CPT1) is a PPAR-regulated gene required for

-oxidation (Kersten et al., 1999), while PDK4 is a key GR-regulated gene that shunts pyruvate away from lipogenesis and towards gluconeogenesis by inhibiting the mitochondrial PDC (Sugden and Holness, 2006). Fig. 15 shows the RNA expression profile of CPT1 and PDK4 by RT-PCR in soleus muscle.

FIGURE 15. FKBP51-/- AMIMALS SHOW INCREASED CPT1 AND PDK4 LEVELS

A B

RT-PCR analysis of genes important for lipid catabolism. Values represent means ± S.E.M. of three independent experiments. (≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group); *p<0.05; **p<0.01 (Vs WT, between groups)

65 Under high fat diet conditions, PDK4 is significantly elevated in FKBP51

mutants. Even under regular diet PDK4 levels showed a tendency to increase. Expression of CPT-1 was elevated under both conditions in mutant mice. Taken as a whole, the

FKBP51 data are consistent with other animal models showing resistance to diet-induced obesity due to increased energy expenditure. One such model includes ectopic expression of PGC-1 in a variety of tissues that shows increased oxidative capacity in muscle and increased energy expenditure and resistance to obesity (Kamei et al., 2003).

Reduced expression of hepatic gluconeogenic enzymes in FKBP52 mutant mice fed high fat

Since FKBP52 mutants showed increased serum glucose levels under high fat diet conditions, we tested for altered expression of hepatic gluconeogenic enzymes. Fig. 16 shows Northern blot analysis of PEPCK, PDK4 and G6Pase in WT and FKBP52 mutants. In response to over-night fasting, both WT and FKBP52 mutant animals showed about the same level of gluconeogenic enzyme induction, with the exception of PDK4, which was significantly reduced. A more dramatic reduction of all three enzymes was not expected, even in the face of impaired GR activity due to FKBP52 loss, because the major regulator of these enzymes during short fasting intervals is glucagon, rather than

GCs (Opherk et al., 2004). More interesting were the effects on high fat diet, where all three gluconeogenic enzymes showed a trend to up-regulation in WT animals. This was somewhat surprising because there was no corresponding increase in blood glucose levels under these conditions (Fig. 9A). However, it is likely that euglycemia is maintained by a corresponding increase of insulin secretion (Fig. 9B). These results also provide further evidence that high fat diets, most likely acting through fatty acid activated PPAR, have

66 gluconeogenic-promoting effects in the liver (Cassuto et al., 2005; Chen, 2007; Huang et al., 2002; Xu et al., 2007). With this in mind, it was interesting to see reduced expression of the gluconeogenic enzymes in FKBP52 mutants subjected to high fat.

FIGURE 16. REDUCED EXPRESSION OF GR REGULATED GLUCONEOGENIC GENES IN FKBP52+/- ANIMALS FED HF DIET

A

B C D

A, Northern analysis of genes important for glucose homeostasis in liver. B, C, D, Quantitation of results in Panel A was achieved by densitometric scanning of films. Values for each protein is expressed as percentage of maximum value obtained after scanning and represent means ± S.E.M. of four independent experiments. (≠ p< 0.05; ≠ ≠ p< 0.01 (Vs RD, within group) ; *p<0.05; **p<0.01 (Vs WT, between groups)

As a further test of the liver results, GR activity at the PEPCK promoter was tested in MEF cells (Fig. 17). The results show reduced GR activity in FKBP52 KO MEF cells compared to WT. As a whole, these results suggest that loss of FKBP52 leads to

67 reduced GR hepatic activity at gluconeogenic genes. As elaborated in Discussion, we propose that this observation may represent the fundamental defect responsible for lipid accumulation and eventual insulin resistance in the FKBP52 mutants – namely, reduced synergy between PPAR and GR signaling at the gluconeogenic genes, leading to shunting of carbon to lipid rather than glucose anabolism.

FIGURE 17. REDUCED PEPCK REPORTER ACTIVITY IN FKBP52+/- MEF CELLS

WT and KO52 MEFs were transiently transfected with PEPCK-Luc and -GAL expression vector followed by treatment with 1µM Dex for 24 h. Values represent the means ± S.E.M. of three independent experiments.

68 DISCUSSION

In this manuscript, we have analyzed the roles of FKBP51 and FKBP52 in lipid and carbohydrate metabolism under high fat diet conditions. Our results suggest that these steroid receptor co-chaperones differentially control two key branches of metabolism. Our conclusion was based on the following results. FKBP52 mutants on a high fat diet show reduced expression of hepatic gluconeogenic enzymes and increased hepatic lipid accumulation. There was a moderate increase in glucose and insulin levels, probably due to altered liver metabolism. The metabolic organ mainly affected by the loss of FKBP52 appears to be liver. As this TPR is not expressed in muscle and adipose, it is not expected to have a primary defect in these organs. However, we do not exclude the possibility of a secondary effect due to substrate shuffling, such as TG from liver to peripheral organs. In contrast, ablation of FKBP51 had no effect on hepatic glucose or lipid metabolism in mice. The glucose and insulin levels of FKBP51-/- animals were not statistically different from their wild- type counterparts and had similar degrees of hepatic lipid accumulation and expression of gluconeogenic enzymes (data not shown). Instead, we discovered a difference in the expression of genes important for lipid oxidation in their muscle. This was consistent with their inability to gain weight when they consumed high fat diet and low serum TG and FFA levels. Taken together, these results suggest that ablation of FKBP52 primarily affects glucose/lipid metabolism in liver and loss of

FKBP51 alters lipid metabolism in muscle. Thus, TPR proteins appear to preferentially influence select metabolic cascades in specific tissues.

69 Is GR the in vivo target of FKBP activity?

How do TPR proteins control metabolism? Many reports show their association

with steroid receptors and their ability to modulate receptor functions. The roles of

steroid receptors in metabolism are well documented. So it’s reasonable to assume that

TPR proteins control metabolic pathways by regulating SR activities. Of all the steroid

receptors, GR is widely known for its metabolic actions. And as shown by our TPR-

deficient MEF studies, loss of FKBP51 and FKBP52 greatly impact GR transcriptional

activity. Hence, the metabolic phenotypes of TPR mutant mice are most likely due to loss

or gain of GR activity on its target genes. To a lesser extent, other nuclear receptors, in

particular AR and ER, have been shown to control metabolic pathways. In vivo studies

using FKBP52 knockout animals (male and female) show reproductive defects due to

compromised AR and PR activities (Tranguch et al., 2005; Yang et al., 2006). In contrast, no such reproductive defects were noted due to the loss of FKBP51 (Yong et al., 2007b).

Reduced AR and ER activities have been linked to diabetes and insulin resistance (Fukui et al., 2007). Both in vivo and in vitro studies suggest that FKBP52 is not an important player in the ER signaling pathway (Riggs et al., 2003; Yang et al., 2006) and ER is known to down-regulate the expression of gluconeogenic enzymes (Bryzgalova et al.,

2006). Therefore, if there was a defect in ER activity due to loss of FKBP52, we would expect to see an increase in glucose metabolism in the liver. FKBP52+/- animals show reduced expression of gluconeogenic enzymes. On the other hand, high fat fed, liver - specific AR knockout mice showed similar hepatic lipid accumulation as FKBP52+/- mice. Again in contrast to FKBP52, they had increased expression of hepatic gluconeogenic enzymes (Lin et al., 2008). Because GR is known to up-regulate many

70 gluconeogenic enzymes (Vegiopoulos and Herzig, 2007), reduced GR activity can lead to low expression of these genes, as seen in FKBP52 mutants. Hence, we speculate that the reduced expression of gluconeogenic enzymes is due to compromised GR activity.

FKBP51-/- mice appear to have increased fatty acid oxidation in muscle and are

therefore resistant to diet-induced obesity. All three receptors, AR, ER and GR either

directly or indirectly control fatty acid oxidation (Campbell et al., 2003; Lin et al., 2008;

Vegiopoulos and Herzig, 2007). Contrary to FKBP52, in prostate cancer cells, high levels of FKBP51 increase AR activity (Febbo et al., 2005). Hence, loss of FKBP51 should negatively affect AR activity. But in vivo, FKBP51 deficiency had no effect on AR, at least in the organs analyzed (Yong et al., 2007b). Association of FKBP51 with ER has not been shown so far. Hence, loss of this TPR is not likely to affect ER activity. On the other hand, in MEF cells, FKBP51 deficiency increases GR activity. A direct transcriptional effect of GR on many lipid - clearing genes including PDK4 and PPAR

is very well known. Therefore, we hypothesize that increased GR activity due to loss of

FKBP51 may up-regulate expression of these enzymes which, in turn, increases fatty acid

oxidation. Additional studies are clearly needed to dissect how loss of each TPR affects

individual receptor activity in metabolic pathways. But from what is known of steroid

receptor functions in metabolism, we postulate that the metabolic phenotypes of TPR

mutants are most likely due to defects in GR activity.

Relevance to FKBPs to metabolic syndrome

Both FKBP51 and FKBP52 are frequently found in GR complexes and modulate receptor

activities. Therefore, lack of an obvious GR defect in TPR mutants was rather surprising.

71 Global GR-/- mice die shortly after birth due to lung atelectasis, demonstrating GR

functions are essential for the survival of an organism (Cole et al., 1995b). GR can

influence transcription through DNA-binding - dependent and - independent mechanisms.

To understand how these individual mechanisms contribute to overall GR physiology,

dimerization-deficient mice (GRdim) were created (Reichardt et al., 1998). These animals

have a mutation in the GR gene that impairs its ability to dimerize and bind to DNA.

Therefore, GR signaling that requires DNA-binding is completely lost. Even though these

animals show certain abnormalities, they are viable suggesting that DNA-binding of GR

is not essential for survival. This led us to hypothesize that TPR proteins may not be

affecting GR functions globally and perhaps, like other SRs, only in select organ/tissue.

We assumed that since these animals are viable, as in GRdim mice, only transactive

properties of the receptors are affected. Many transactive functions of GR, such as its

ability to regulate many metabolic genes, require DNA-binding. Most of the

transrepressive functions (e.g. anti-inflammatory properties) are carried out by DNA-

binding-independent mechanisms, such as tethering. As glucocorticoids are stress

hormones, we also reasoned that an initial stress event may be necessary to uncover any

defects in GR signaling. This happens to be true for other nuclear receptors as well.

Deletion of genes encoding nuclear receptors does not always produce an apparent

phenotype. For example, disruption of LXR mutant mice shows a phenotype only when

animals were challenged with a high cholesterol diet (Peet et al., 1998). Another example

is the PXR/SXR null mouse, which exhibits a dramatic phenotype only when treated with xenobiotics (Staudinger et al., 2001; Xie et al., 2000; Xie et al., 2001).

72 Since our objective was to learn how TPR proteins mediate GR - controlled

metabolism, we decided that any conventional stress method (such as restraint stress) to

study GR actions would not be appropriate in this scenario. Glucocorticoid actions have

been intrinsically tied to metabolic syndrome and obesity. Indeed, it has been proposed that patients with obesity and metabolic syndrome have increased glucocorticoids or glucocorticoid receptor activation in peripheral organs (Wake and Walker, 2004; Walker,

2006). Treatment of obese, diabetic ob/ob mice for 21 days with glucocorticoid antagonist RU-486 reduces postprandial glucose values and hyperglycemia (Gettys et al.,

1997). A direct involvement of GR in diabetic hyperglycemia was seen in animals with inactive hepatic GR. Blood glucose and gluconeogenic enzymes were markedly reduced in these animals compared to controls when they were made diabetic by streptozotocin injection (Opherk et al., 2004). Metabolic disorders are largely caused by sedentary life and consumption of food rich in saturated fat. Challenging animals with a high fat diet and inducing dietary obesity can therefore be considered as a similar environmental setting. Any outcome from such a study will be meaningful and will help us better understand how TPR proteins regulate glucocorticoid signaling in metabolic disorders.

Thus, the results obtained will further allow us to design better drug targets to treat metabolic syndrome. High fat diet is also considered as a chronic metabolic stimulus. It is known to activate the HPA axis and alters both basal and stress - induced glucocorticoid activity (Brindley et al., 1981; Tannenbaum et al., 1997). Hence, challenging the animals with four-weeks of high fat diet was ideal for our studies. By doing so, we were not only providing a natural environment that causes metabolic syndrome and obesity but also

73 creating a stress event to activate GR responses. Thus, this regimen makes an excellent

background to study the role TPR proteins play in GR metabolic signaling.

Model of diet-induced insulin resistance in FKBP52 mutant animals

As shown in Fig. 18, glucocorticoids control three important aspects of liver metabolism.

They are: gluconeogenesis, lipogenesis and lipid oxidation (-oxidation). Based on the results obtained so far, we hypothesize that the fundamental defect causing insulin resistance in high fat fed FKBP52+/- animals is reduced gluconeogenesis and increased lipid accumulation in the liver (Fig.18).

FIGURE 18. WORKING MODEL FOR DIET INDUCED INSULIN RESISTANCE IN FKBP MUTANT ANIMALS

74 In gluconeogenesis, several key enzymes are transcriptionally controlled by GR.

Phosphoenolpyruvate carboxykinase and G6Pase are the two rate - limiting enzymes in

gluconeogenesis (Vegiopoulos and Herzig, 2007). Pyruvate dehydrogenase kinase 4 is

another important enzyme and is a serine/threonine kinase that phosphorylates PDC.

Pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA for lipid synthesis when pyruvate is abundant in the cells. Pyruvate dehydrogenase kinase 4 phosphorylates and inactivates PDC to conserve three carbon compounds (pyruvate, lactate and glycerol) for gluconeogenesis. For this reason, it is considered as the

‘metabolic switch’ of fuel selection (Sugden and Holness, 2006). Pyruvate is a substrate for both gluconeogenesis and lipid synthesis. We propose that in high fat fed FKBP52 mutants, reduced expression of gluconeogenic enzymes favor conversion of pyruvate to acetyl-CoA and de novo lipogenesis. Low expression of PDK4 further contributes to this

effect. The source of pyruvate can either be glycolysis and/or glycerol. Glycerol that is

abundant in high fat diet can be taken up by the liver to convert to glyceraldehyde 3-

phosphate: an intermediate product in glucose synthesis/oxidation. It can also be utilized

for the reesterification of FFA. Consistent with increased lipid accumulation, an increase

in FAS, the main regulator of fatty acid synthesis was also noted in these animals. Lipid

accumulation could also be due to reduced fatty acid oxidation as GCs control the

expression of PPAR , an important transcription factor in lipid oxidation. Due to altered

lipid metabolism in the liver, primary hepatic resistance develops leading to hyperinsulinemia. Increased hepatic TG accumulation is considered to interfere with several components of insulin signaling (Samuel et al., 2004) causing hepatic insulin resistance. Since gluconeogenic enzymes are reduced in the livers of FKBP52 mutants,

75 hyperglycemia is most likely due to secondary insulin resistance in peripheral organs.

Desensitization of insulin receptors and impairment of downstream signaling due to hyperinsulinemia and/or increased partitioning of TG from liver to peripheral organ could account for this secondary resistance. Our Muscle TG results are in agreement with the latter mechanism. Even though WT animals fed high fat diet also showed moderate increase in insulin levels, lipid accumulation and expression of gluconeogenic enzymes, surprisingly hyperglycemia was not observed in these animals. This indicates that our

four-week high fat diet treatment was a moderate effecter of insulin resistance in these

animals, and that loss of FKBP52 accelerates this response.

Gluconeogenesis, synthesis of glucose from non-carbohydrate sources, is tightly

regulated by insulin and its counter-regulatory hormones glucocorticoids and glucagon.

The underlying cause of hyperglycemia in metabolic syndrome is due to insulin’s

inability to suppress the expression of gluconeogenic genes and a chronic dominance of counter-regulatory hormones (Vegiopoulos and Herzig, 2007). High fat diet and lipid molecules can also elevate gluconeogenic enzymes by activating PPAR. This transcription factor by itself or along with GR increase the expression of genes important for glucose synthesis (Cassuto et al., 2005; Chen, 2007; Huang et al., 2002; Vegiopoulos and Herzig, 2007). This is a possible mechanism for the increased expression of gluconeogenic enzymes observed in WT52 animals on HF. Since PPAR itself is

regulated by GR, loss of GR activity in FKBP52 mutants should also abolish this effect.

Unlike its well-established role in controlling gluconeogenesis, molecular

mechanisms of glucocorticoid dependent lipid synthesis are not very well defined.

However, patients with Cushing’s syndrome or patients receiving long-term GC

76 treatment show dyslipidemia and fatty liver, and GC treatment of isolated hepatocytes increase lipogenesis and VLDL production signifying that GCs favors lipogenesis. This is most likely due to glucocorticoids ability to transcriptionally activate lipogenic enzymes like FAS or acetyl-CoA caroboxylase (Mangiapane and Brindley, 1986). However, to date only few such GC controlled target genes in lipid metabolism have been identified.

Aberrant intrahepatic triglyceride accumulation is also intrinsically tied to metabolic syndrome. Short-term high fat feeding to rats resulted in fatty liver and lipid accumulation. It also directly impaired insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation by activating protein kinase C (PKC) and C-jun N-terminal kinase 1

(JNK1). These serine threonine kinases phosphorylate serine residues that led to decreased tyrosine phosphorylation of insulin receptor substrates and attenuated insulin signaling (Samuel et al., 2004). An inverse relationship between liver TG accumulation and insulin clearance has also been demonstrated in humans and bovine hepatocytes although exact mechanisms are still not understood (Kotronen et al., 2007; Strang et al.,

1998).

While the basis of low gluconeogenesis in FKBP52 mutant animals was easy to decipher, the reasons for increased lipid synthesis seem more complex. We propose three not mutually exclusive mechanisms. First, in liver loss of FKBP52 only affected GR’s ability to control gluconeogenesis leaving lipid metabolism intact. Second, besides GR,

AR has also shown to regulate lipid metabolism. While GR favors lipogenesis, AR reduces it (Lin et al., 2008). So defect in AR activity may increase lipogenesis. Currently it is not known if FKBP52 mutant animals have impaired AR activity in the liver but they do have defects in reproductive organs (Yong et al., 2007b). Hence altered lipid

77 metabolism can also stem from compromised AR activity. Third, increased insulin levels stimulate the production of sterol regulatory element binding protein-1c (SREBP-1c) that activates many proteins important for fatty acid synthesis, such as FAS. Treatment of hepatocytes extracted from WT and mutant animals with GCs, androgens and insulin and a more extensive analysis of genes induced by each hormone in lipid metabolism may be necessary to understand the exact mechanism(s) controlling increased lipogenesis in the

FKBP52 mutants.

Increased lipid accumulation in FKBP52 mutants can also results from reduced fatty acid oxidation as GCs thought to indirectly control -oxidation by regulating the expression of PPAR (Lemberger et al., 1996; Lemberger et al., 1994). Peroxisome proliferator-activated receptor  (PPAR) is a nuclear receptor that is important for the transcription of many enzymes involved in -oxidation, such as CPT1 and acyl-CoA oxidase. Lipolysis of dietary fats or fasting, release a large pool of FFA into the portal circulation. Once inside the liver, FFAs either enter mitochondria for subsequent oxidation to produce acetyl-CoA or re-esterified to TG and secreted as VLDL. Acetyl-

CoA generated by fatty acid oxidation is used as a precursor for fatty acid synthesis or enters TCA cycle to produce reducing equivalents for ATP synthesis (Burgess et al.,

2006). In whole animals, PPAR mRNA is induced in response to physical and metabolic stress and follows a diurnal rhythm which parallels circulating corticosterone

(Lemberger et al., 1996). Consistent with its role in lipid oxidation, PPAR null mice fed high fat diet or fasted for 24 h show massive lipid accumulation in their livers

(Kersten et al., 1999). All the three metabolic pathways i.e. gluconeogenesis, fatty acid oxidation and lipogenesis are interlinked and tightly regulated at multiple levels by

78 substrate availability, transcriptional and translational modifications of enzymes.

Therefore any alteration in flux through one pathway has inevitable impact on others

(Burgess et al., 2006). This fact is clearly demonstrated in livers of FKBP52 mutants on

HF diet.

FKBP52+/- animals show increased serum insulin levels on high fat diet.

Hyperinsulinemia develops when pancreatic -cells produce more insulin to compensate for the resistance or due to reduced clearance or both. In FKBP52+/- animals insulin secretion as measured by C- peptide levels, was similar to WT animals. Insulin clearance was determined by C-peptide and insulin ratio. FKBP52+/- animals on high fat diet showed reduced insulin clearance. Hence we concluded that the hyperinsulinemia was due to a defect in insulin clearance. Large amount of liver fat content has been shown to significantly decrease insulin clearance. The precise biochemical mechanisms whereby fatty acids and cytosolic TGs causes impaired insulin clearance remain poorly understood. Insulin clearance can also happen if there is low expression of proteins that participate in this mechanism. One such candidate protein is carcinoembryonic antigen- like cell adhesion molecule (CEACAM1). It is a transmembrane protein and is up- regulated by dexamethasone, a synthetic glucocorticoid (Najjar et al., 1996). Upon phosphorylation by insulin receptor, it binds indirectly to the receptor and becomes a part of insulin endocytosis complex. The principal mechanism of insulin clearance is receptor mediated endocytosis and degradation and occurs mostly in liver. Liver-specific inactivation of this protein leads to hyperinsulinemia and insulin resistance in mice (Poy et al., 2002). Since its expression may be regulated by GR (Najjar et al., 1996), reduced

79 receptor activity in FKBP52 +/- may cause low expression of this protein leading to impaired insulin clearance. Experiments are currently underway to test this concept.

How does hyperglycemia develop in FKBP52 mutants irrespective of reduced

gluconeogenic enzymes? We postulate that increased lipid synthesis might have caused

secondary peripheral resistance perhaps in the muscle or adipose. We do not suspect

defects in adipose metabolism as its mass did not alter between WT and FKBP52+/-

animals. Increased visceral adipose mass indicates a disordered adipose metabolism

(Lewis et al., 2002). This suggests that skeletal muscle could be the likely target.

Moreover muscle is also the major site of whole body glucose utilization (Wende et al.,

2007). Hence it’s plausible that due to increased lipid synthesis, large amounts of TG

were exported to muscle interfering with glucose uptake and oxidation leading to

elevated blood glucose levels. Increased FFA and TG can impair glucose metabolism in

muscle. Recent studies in humans and animals have also demonstrated a strong

relationship with increased intramuscular TG content and insulin resistance (Petersen and

Shulman, 2002). Consistent with this, we also see a trend towards increase in muscle TG

levels of FKBP52+/- animals on high fat diet. Why we do not see increased plasma TG

is somewhat surprising and may be attributed to increased redistribution of TG from liver

to skeletal muscle. Alternatively, increased insulin levels can desensitize insulin receptors

in the peripheral organs and impair downstream signaling events.

Reasons for resistance to diet-induced obesity in FKBP51-/- mice

We believe that the deficiency of FKBP51 removes the inhibitory effect it has on

the receptor, creating a hyper-active GR, leading to increased expression of its target

80 genes. However, this does not happen to be a whole-body effect since over-activation of

GR has devastating consequences and may cause metabolic syndrome (Vegiopoulos and

Herzig, 2007). FKBP51 knockout animals are otherwise normal except for their inability

to gain weight and visceral adiposity on HF diet, and low TG and FFA levels even under

regular diet conditions compared to their WT counterparts. This implies that GR activity

is affected only in select organs or pathways. Low serum TG and FFA are indicative of increased fat metabolism. Thus increased GR activity due to loss of FKBP51 may up- regulate several GC-induced genes that are important for fatty acid catabolism and energy expenditure leading to the observed phenotype. This mouse model is consistent with other published animal models which exhibit reduced fat storage due to increased fatty acid oxidation and energy expenditure (Bansode et al., 2008; Kalaany et al., 2005). The three major organs that are important for lipid handling are liver, muscle and adipose.

Liver is the major hub of lipid synthesis and TG secretion. Free fatty acids entering the liver undergo complete oxidation to provide energy for glucose and lipid synthesis.

Muscle burns fat for energy production. Adipose tissues are of two types – white and

brown. The major function of white adipose tissue is to store fat and brown adipose tissue

has components that participate in adaptive thermogenesis and energy expenditure (Cinti,

2005).

Oil red o staining on the livers of FKBP51-/- animals on RD and HF were not

different from their WT controls. This suggests that liver lipid metabolism was probably

not altered in mutant mice. Consistent with their normal glucose levels, expression of

hepatic gluconeogenic enzymes were also not changed in FKBP51 knockouts. Taken

together, these results indicate that ablation of FKBP51 unlike FKBP52 had no effect on

81 hepatic GR activity. This tissue specificity was not surprising because similar

observations have also been made with respect to AR and PR activity in reproductive

organs of FKBP52 knock out mice.

To test the hypothesis that loss of FKBP51 increases GR-induced genes in fatty acid catabolism, we analyzed several receptor targeted genes in muscle. Interestingly, two genes - CPT1 and PDK4 that take part in lipid catabolism were increased in the soleus muscle of FKBP51 mutants. Carnitine palmitoyl transferase 1 is a protein that is involved in -oxidation by transporting fatty acyl-CoA across mitochondria. Its expression is regulated by PPAR, which is a known GR target gene. Pyruvate dehydrogenase kinase

4 is controlled by GR and has a key position in skeletal muscle metabolism. It assists in lipid oxidation by inactivating PDC, thereby preventing pyruvate (end product of glycolysis) to enter TCA cycle for complete oxidation (Pilegaard and Neufer, 2004).

Thus, it appears that in FKBP51 mutants due to increased expression of lipid clearing proteins, excess fat consumed is getting oxidized.

The pioneering work by Randle et al (1963) demonstrated that glucose oxidation is decreased in response to increased fatty acid oxidation. This happens due to allosteric

modification of components of glycolytic pathway by the products of fatty acid

oxidation. Since most of the ingested glucose is taken up by the muscle for oxidation or

glycogen storage, any defect in muscle glucose metabolism can severely affect whole-

body glucose homeostasis. If glucose oxidation is impaired in muscle, pyruvate could be

released to the blood and can be utilized for glucose synthesis in the liver. But glucose

oxidation does not appear to be affected in FKBP51-/- animals as blood glucose levels

82 were not altered in both under RD or HF diet conditions. There is also no change in the

expression of hepatic gluconeogenic enzymes.

If glucose oxidation is indeed inhibited by increased PDK4 expression glucose

taken up by the muscle can also be stored as glycogen, leaving blood glucose levels

unaltered. Studies performed using a conditional muscle specific PGC-1 gain of

function mice show a phenotype of increased muscle glycogen content due to increased

fatty acid oxidation and PDK4 expression (Wende et al., 2007). PGC-1 coactivates

several transcription factors that are involved in fatty acid synthesis and mitochondrial

biogenesis. It augments the expression of GLUT4 and PDK4 and therefore increases

glucose uptake and inhibit glycolysis. This directs accumulated G6P towards glycogen

synthesis. Although, it is not completely understood whether GR has any direct effect on glycogen synthesis, analysis of glycogen content or glycogen synthesizing enzymes in

FKBP51 mutants may provide interesting results. Currently we are in the process of conducting a more exhaustive gene expression analysis in muscle and adipose. This also includes expression of proteins important for energy expenditure, such as UCP3, and several proteins that are important for adipose differentiation. As FKBP51 was selectively accumulated during adipose differentiation (Yeh et al., 1995) it would be interesting to see how loss of FKBP51 affects this process in whole animals and test if this is partly responsible for the phenotype.

In summary, our present study demonstrates that FKBP51 and FKBP52 selectively regulate glucocorticoid mediated metabolic pathways. Although glucocorticoids have extensive physiologic importance, its responses are not always uniform and greatly vary even within a single cell. Molecular mechanism underlying

83 tissue-specific regulation of GR is rather complex and not yet completely understood.

Therefore designing drugs that selectively control specific metabolic pathways regulated by GR has provided a challenge for researchers. Few tissue-selective ligands called

Selective Glucocorticoid Receptor Modulators (SGRM.) have been identified, however these drugs mainly target inflammatory pathways than metabolic responses (Rosen and

Miner, 2005). While glucocorticoids have anti-inflammatory properties, they have wide- spread metabolic actions as well. For this reason, their usage as anti-inflammatory drugs often produces side effects and cause metabolic syndrome. Moreover in diabetes and

insulin resistance, over-activation of metabolic pathways by counter-regulatory

hormones, such as glucocorticoids, often produces hyperglycemia and dyslipidemia.

Therefore tissue specific glucocorticoid receptor antagonism has been validated as a

strategy for regulating metabolism in such conditions. Recently, small molecules that

inhibit 11HSD1 – an enzyme that tissue specifically regenerate active cortisol have

emerged as a therapeutic agent. These drugs have been shown to prevent glucocorticoid

regeneration in select metabolic tissues and block glucocorticoid induced hepatic

steatosis and insulin resistance (Hale and Wang, 2008; Hughes et al., 2008; Matfin, 2008;

Tomlinson and Stewart, 2007). Here we show that TPR proteins are part of the complex system that provides tissue specificity to GR in metabolically important organs. Hence these proteins provide excellent drug targets to achieve tissue specific modulation of GR to treat some or all of the features of metabolic syndrome.

84 CONCLUSIONS

1. In the present study, using MEFs derived from WT and TPR (FKBP51 and FKBP52) knockout animals we reconfirm previous in vitro findings that FKBP51 is a negative and

FKBP52 is a positive modulator of GR transcriptional activity.

The main goal of this study was to understand how these proteins affect in vivo GR signaling. For this purpose we used WT and TPR knockout animal models. Lack of overt

GR abnormalities in mutant animals were surprising , but we reasoned that as glucocorticoids are stress hormones, an initial stress event may be necessary to uncover any defects in GR signaling. Therefore we decided to challenge all animals with high fat diet, a well known metabolic stress for a period of four weeks.

2. FKBP52 mutant mice on high fat diet showed similar weight gain and visceral adiposity compared to their WT counter parts but had slightly higher glucose and insulin levels. Further we discovered that the reason for hyperinsulinemia was reduced insulin clearance resulting from excess hepatic lipid accumulation. The underlying cause for this elevated lipid accumulation was substrate redistribution of pyruvate towards acetyl- CoA and fatty acid synthesis. Pyruvate is a common substrate for gluconeogenesis and fat synthesis. Many enzymes important for gluconeogenesis are transcriptionally regulated by glucocorticoids. Loss of FKBP52 greatly impairs GR transcriptional activity and as a result expression levels of all the gluconeogenic enzymes tested were down-regulated in mutant mice. Thus, pyruvate formed from glucose (glycolysis) or excess glycerol (from high fat diet) is now redistributed to de novo lipid synthesis rather than gluconeogenesis.

85 Consistent with this we also see significantly elevated FAS protein levels. Increased TG uptake or down-regulation of insulin receptor by elevated insulin levels in peripheral organs are plausible mechanisms for the development of hyperglycemia in mutant animals.

3. FKBP51 knockout animals developed a different phenotype on high fat diet. These animals were resistant to diet induced obesity and had low serum TG and FFA levels.

This led us to hypothesize that loss of FKBP51 creates a hyper-active GR and increases the expression of genes important for fatty acid oxidation and/ or energy expenditure. Oil red O staining and northern blot analysis revealed that liver lipid and glucose metabolism is not severely affected in FKBP51 mutants. By analyzing a battery of genes important for lipid catabolism, we identified two genes (CPT1 and PDK4) that were up-regulated in mutant animals on high fat diet. This at least in part explains why the mutant animals

have low TG and are resistant to diet induced obesity.

In summary, our results show that glucocorticoid receptor co-chaperone proteins

FKBP51 and FKBP52 differentially regulate important metabolic pathways. To our

knowledge, this is the first in vivo demonstration that FKBP proteins can regulate

metabolic processes. As these proteins are modulators of GR functions, our data has a

great impact on drug development against metabolic abnormalities caused by over-

stimulation of GR.

86 SUMMARY

Steroid receptor complexes consist of a molecule of receptor linked to an HSP90 dimer and one of the four TPR proteins. The four TPRs known to associate with the receptor are FKBP51, FKBP52, Cyp40 and PP5. Since the HSP90 dimer generates only one TPR binding site, four mature complexes can be formed based on the TPR content.

This heterogeneity of the SR complexes resulting from the incorporation of one of the four TPR proteins poses a fundamental question: Does each TPR protein differentially regulate receptor function and in turn SR physiology?

To delineate the role of both FKBP51 and FKBP52 in steroid physiology, mice deficient in each protein were created. Both male and female FKBP52 mice showed defects in reproductive functions due to compromised AR and PR transcriptional activity in distinct organs, whereas no reproductive abnormalities were noted in FKBP51 animals.

Female mice deficient in FKBP52 showed implantation failure due to reduced PR activity in the uterus. Analysis of FKBP52-/- males revealed that these animals are infertile due to hypospadias and dysgenic prostate. Further molecular analysis discovered compromised

AR activity in corresponding organs. Taken together, these studies suggest that TPR proteins control SR actions in a tissue - selective manner.

Both FKBP51 and FKBP52 are frequently seen complexed with GR and in vitro

studies over-expressing either TPR or using MEFs derived from TPR knockout animals

suggest that FKBP51 is a negative and FKBP52 is a positive modulator of GR functions.

However, no overt GR abnormalities were seen in mutant animals. Whole animal GR

knockout, on the other hand is peri-natal lethal. They die shortly after birth due to lung

atelectasis. We reasoned that if FKBP52 or FKBP51 had a global effect on GR activity

87 these mutant animals should also be peri-natal lethal. The fact that these animals are

viable suggests that perhaps like other SRs, TPRs only contribute to GR functions in

certain organs or under certain physiological conditions. In order to uncover defects in

GR signaling, we concluded that an initial stress event may be necessary, as

glucocorticoids are stress hormones whose functions become crucial during stress events

to protect organisms. Therefore, in efforts to understand the role of FKBP51 and FKBP52 in GR physiology, we decided to challenge the mutant animals with well known metabolic stressors: high fat diet and fasting. Surprisingly, fasting of animals for 16-18 h did not show any major metabolic abnormalities in these animals. This could be because only after prolonged food deprivation (starvation) glucocorticoids actions become critical and, in the initial stages of fasting, whole body homeostasis is mostly maintained by hormones like glucagon. Hence, the short duration of fasting we did on our animals may not be sufficient to induce great differences in GR signaling events.

High fat diet is a chronic stimulus that activates HPA axis. In order to examine how high fat feeding affects GR signaling in TPR mutants, both WT and mutant animals were fed a diet that is rich in saturated fats for four weeks. Interestingly, FKBP52 and

FKBP51 mutants displayed entirely different phenotypes under the same diet treatment.

FKBP52 mutants showed similar weight gain and visceral adiposity compared to WT

counterparts, but had slightly higher glucose and insulin levels. In order to determine the

basis of hyperinsulinemia, we measured insulin secretion and insulin clearance. Insulin

secretion as measured by C-peptide levels was found normal between WT and FKBP52

mutants. However, FKBP52 mutant mice on high fat diet had decreased insulin clearance

and, therefore, had higher plasma insulin levels. To understand the cause of reduced

88 insulin clearance, we determined the levels of hepatic lipid accumulation. Increased

hepatic lipid buildup is widely known to cause impaired insulin clearance and insulin

signaling. Oil Red O staining on liver sections and tissue TG levels revealed that

FKBP52+/- on high fat diet had greater lipid accumulation than the WT animals. Further

analysis of proteins important for lipid synthesis showed a significant increase in the

expression of FAS, suggesting elevated lipid synthesis. We speculate that reduced insulin

clearance in FKBP52 mutant mice results from increased liver fat accumulation. The underlying cause for this greater lipid storage was found to be reduced gluconeogenesis.

Glucocorticoids regulate several important enzymes that participate in gluconeogenesis.

Using MEFs cells, we show that loss of FKBP52 greatly impairs GR activity. Consistent with this, several gluconeogenic genes regulated by GR were found to be down-regulated in FKBP52 mutant animals on high fat diet. We hypothesize that reduced gluconeogenic enzymes in FKBP52 mutants redirect pyruvate towards lipid synthesis. Pyruvate is a common substrate for gluconeogenesis and fatty acid synthesis. Under fed conditions

PDC, converts pyruvate to acetyl-CoA that is further used for de novo lipogenesis. PDC activity is negatively regulated by PDK4. We propose two not mutually exclusive explanations for the development of hyperglycemia. First, increased lipid synthesis greatly increases the output of TGs to plasma. As a result, peripheral organs, such as muscle, take up large quantities of TG which greatly impairs insulin signaling and causes hyperglycemia. Second, higher serum insulin levels sensitize the insulin receptor leading to down-regulation of insulin signaling pathways in peripheral organs. Based on our results, we conclude that, under normal conditions, FKBP52 has a protective role and its loss make animals more susceptible to diet- induced insulin resistance.

89 FKBP51 animals on high fat diet were resistant to diet-induced obesity. They had low serum TG and FFA. Plasma glucose and insulin levels were not affected in these animals. They also had similar hepatic lipid accumulation compared to WT animals when fed HF diet. As FKBP51 is a negative modulator of GR functions, we hypothesized that loss of this protein creates a hyper-active GR leading to over-stimulation of several genes important for lipid catabolism and perhaps energy expenditure. Thus, we began analyzing genes important for fatty acid oxidation in the soleus muscle. After initial screening we found that two genes (CPT1 and PDK4) were elevated, corroborating our hypothesis that increased expression of lipid - clearing genes regulated by GR gives FKBP51mutants a greater ability to utilize fat.

Taken as a whole, these results suggest that FKBP proteins play a key role in regulating different branches of metabolism by providing selectivity to receptor functions. Therefore, designing drugs that target TPR proteins rather than the receptor itself might provide an opportunity to control many features of metabolic syndrome with little or no side effects.

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123 ABSTRACT

Glucocorticoids (GCs) are important regulators of carbohydrate and lipid

metabolism, serving to antagonize the actions of insulin. As such, GC antagonists have

held promise in the treatment of diabetes and metabolic syndrome. Yet, widespread and

potent side effects have precluded such usage. To identify new and selective targets of

GC action, we tested the roles of FKBP51 and FKBP52 in glucocorticoid receptor (GR)- controlled metabolism using FKBP - ablated mice. FKBP51 and FKBP52 serve as GR

co-chaperones and we show that loss of FKBP51 increases GR transcriptional activity,

while FKBP52 loss decreases it. Using high fat diet (HF) as a metabolic stress, we have

observed the following. In response to HF, no differences between WT and

FKBP52 (+/–) animals occurred in body weight and visceral adiposity. However, high fat

fed FKBP52 (+/–) mice became hyperglycemic, hyperinsulinemic and showed increased

lipid accumulation in the liver. They also had reduced expression of GR - regulated

gluconeogenic enzymes. We hypothesize that impairment of GR liver activity due to loss

of FKBP52 decreases the expression of gluconeogenic enzymes, leading to substrate

redistribution of pyruvate to de novo lipid synthesis and lipid accumulation. Increased

lipid accumulation in the liver causes reduced insulin clearance and, ultimately, insulin

resistance. In contrast, FKBP51 (–/–) animals fed HF did not become hyperglycemic.

Instead, they were resistant to diet-induced obesity, with greatly reduced visceral

adiposity under HF diet conditions. No difference was noted in the hepatic lipid

accumulation of FKBP51 (–/–) mice on high fat diet. They had normal blood glucose and

insulin levels with all diets, but did show low plasma triglyceride and free fatty acid

levels under these conditions. Consistent with the phenotype, analysis of genes involved

124 in the muscle lipid metabolism revealed elevated expression of CPT-1 and PDK4. Based on the results obtained so far, we are currently investigating the hypothesis that ablation of FKBP51 leads to over-activation of GR in the muscle, thereby increasing the expression of genes regulating lipid oxidation and/or energy expenditure. Our results are the first demonstration that FKBP proteins selectively control metabolic processes.

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