Health Science Campus

FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences

Identification and Functional Characterization of Adipogenesis-related

Submitted by: Yu Wu

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

Examination Committee Signature/Date

Major Advisor: Cynthia M. Smas, D.Sc.

Academic Kam Yeung, Ph.D. Advisory Committee: Ronald L. Mellgren, Ph.D.

William T. Gunning, Ph.D.

Beata Lecka-Czernik, Ph.D.

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

Date of Defense: October 14, 2008

Identification and Characterization of

Adipogenesis Related Genes

Yu Wu University of Toledo Health Science Campus 2008

i DEDICATION

This work is dedicated to my father, Zixing Wu, and my mother, Zhiping Yan,

for their great love, and to my husband Yang Lu, for his continual support, encouragement and love.

ii ACKNOWLEDGEMENTS

I want to express my most deep gratitude to my advisor, Dr. Cynthia M. Smas for Her guidance, understanding, patience, and encouragement in the past four years. Her infectious enthusiasm and unlimited zeal have always been the major driving forces during my graduate studies. She encouraged me to develop independent thinking and research skills, which prepared me for future challenges.

I would like to extend my thanks to the distinguished faculty members who served on my committee: Dr. Kam C. Yeung, Dr. Ronald L. Mellgren, Dr. William

T. Gunning III, Dr. Beata Lecka-Czernik and Dr. Ana Maria Oyarce. I have benefited greatly from their advices.

This dissertation would not be realized without the help of my colleagues and

friends at the University of Toledo. The thanks will go to Dr. Ji Young Kim,

Shengli Zhou, Junho Lee, Kun Liu and Kristin Tillison for their friendship, technical advice and emotional support. They have turned my journey through graduate school into a pleasure. I also want to thank all of my friends at the

University of Toledo for their consistent encouragement, support and trust during my graduate studies.

I also want to give a hearty thank to the personnel who keeps the

Biochemistry and Cancer Biology Department together as a cohesive unit. I wish to thank Dr. William Anthony Maltese for his leadership. And our department

iii secretaries Jenifer Zak, Melody Knotts, Anna Chlebowski and Mary Ann

Schuster, deserve the sincerest thanks for their hardworking to keep the department operating in an efficient manner.

In addition, I would like to thank Dr. Cynthia M. Smas, Dr. Kam C. Yeung and

Dr. Ronald L. Mellgren for assisting in pursuing my post-doctoral position.

Last, but certainly not least, I would like to thank my family for their support and care. They have always been the source of courage for me to face any difficulties.

iv TABLE OF CONTENTS

Dedication …………………………………………………………………………….ii

Acknowledgments …………………………………………………………………...iii

Table of Contents ……………………………………………………………………v

Introduction …………………………………………………………………………..1

Literature ……………………………………………………………………………..7

Manuscript #1 ……………………………………………………………………….53 Wdnm1-like, a New Adipokine with a Role in MMP-2 Activation

Manuscript #2 ……………………………………………………………………….107 Identification and Characterization of TSC-36 as a Preadipocyte

Manuscript #3 ……………………………………………………………………….167 Differential screening identifies transcripts with depot-dependent expression in white adipose tissues

Manuscript #4……………………………………………………………………….. 220 Generation and Characterization of mBAP-9, a New Brown Preadipocyte Cell Line, Reveals Differentiation-Dependent Regulation of Cmbl and Atabh, Two Novel α/β Hydrolases

Manuscript #5 ………………………………………………………………………..280 Expression and regulation of transcript for the novel transmembrane Tmem182 in the adipocyte and muscle lineage

Discussion and Summary…………………………………………………………. .307

Conclusions ………………………………………………………………………….315

Bibliography ………………………………………………………………………… 319

Abstract ……………………………………………………………………………….346

v INTRODUCTION

Obesity is a major and continuing public health problem in the United

States and in many industrialized nations. It is a risk factor for a number of pathological disorders, such as cardiovascular disease, hypertension, non-insulin dependent diabetes, cancer, gallbladder disease and atherosclerosis (Gregoire et al., 1998b; Kahn et al., 2006; Ogden et al., 2006; Van Gaal et al., 2006).

Obesity occurs when energy intake exceeds energy expenditure and in humans obesity is defined by body mass index (BMI). BMI is calculated as weight in kilograms divided by the square of body height in meters. A person with a BMI between 25 and 29.9 is defined as overweight, and a BMI equal to or great than

30 is obese.

White adipose tissue (WAT) is a unique organ designated to store triglyceride and maintain energy homeostasis. Adipose tissue is also recognized as a dynamic endocrine organ, that synthesizes and secretes a number of soluble factors such as leptin, adiponectin, resistin and a variety of cytokines including TNFα (tumor necrosis factor α) (Maeda et al., 2002; Spiegelman and

Flier, 1996; Steppan et al., 2001). Adipocytes are formed from preadipocyte precursors and this process of differentiation is termed adipogenesis.

Adipogenesis is important for the understanding and control of obesity (Ntambi and Young-Cheul, 2000). In addition to preadipocytes and adipocytes, adipose tissue also consists of many other cell types including small blood vessels, nerve tissue and fibroblasts (Geloen et al., 1989). Within adipose tissue, it is difficult to

1 distinguish preadipocyte precursors from fibroblasts due to the lack of molecular

markers for preadipocytes. Additionally, even though the mesodermal pluripotent

fibroblasts have the ability to give rise to cartilage, smooth muscle and

preadipocyte cell types, the full developmental linage of the preadipocyte from

fertilized egg is to date ill-defined. It is therefore difficult to study adipogenesis in vivo and adipogenesis has been studied primarily by using in vitro models. The

3T3-L1 cell line is one of the most well established cell culture models of

adipogenesis. 3T3-L1 preadipocytes resemble fibroblasts morphologically, and

have the ability to differentiate to mature adipocytes upon treatment with an

adipogenic cocktail composed of dexamethasone (Dex) and

methylisobutylxanthine (Mix) (Green and Kehinde, 1974, 1975a; Green and

Meuth, 1974).

The upregulation of gene expression during adipogenesis has been

extensively characterized. The adipogenic transcription factors C/EBPs

(CCAAT/enhancer binding ) and PPARγ (peroxisome proliferator-

activated receptor-gamma) play key roles in the transcriptional cascade during

adipogenesis (Gregoire, 2001b; Gregoire et al., 1998b; Rajala and Scherer,

2003). Upon the treatment with the adipogenic cocktail, C/EBPβ and C/EBPδ are the earliest transcription factors induced by Mix and Dex respectively. Then

C/EBPβ and C/EBPδ act to transactivate two master transcriptional regulators of adipogenesis, PPARγ and C/EBPα. These are central to induce multiple genes that function to facilitate and maintain adipogenic differentiation and phenotype

(Rosen and Spiegelman, 2001; Tontonoz et al., 1994b). In addition, other

2 transcription factors, such as KLFs (krüppel-like zinc finger transcription factors),

ADD1/SREBP (adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein), ERRα (estrogen-related receptor α) and

STAT (signal transducers and activators of transcription), all participate in the regulation of adipogenesis (Kim et al., 1998b).

Here we report a novel adipokine secreted by adipocytes named Wdnm1- like, which is a distant member of the whey acidic protein/four-disulfide core

(WAP/4-DSC) family. We identified Wdnm1-like in an Affymetrix DNA microarray study aimed at discovering new gene expression patterns in adipogenesis.

Wdnm1-like is a novel 6.8 kDa secreted protein. Its transcript is upregulated over

20,000-fold during white preadipocyte and brown preadipocyte cell differentiation.

In 3T3-L1 adipocytes, TNFα treatment leads to increased Wdnm1-like transcription expression. Several members of the WAP/4-DSC family have demonstrated roles as proteinase inhibitors. Given the importance of matrix metalloproteinase (MMP) activity and matrix remodeling in respect to adipogenesis, we investigated Wdnm1-like effects on MMP activity; we demonstrate that Wdnm1-like functions to enhance MMP-2 activity.

In contrast to transcriptional upregulation of genes during adipogenesis, decreases in gene expression that occur during adipocyte differentiation are far less studied. The characterization of such genes is important for elucidating the molecular definition and developmental lineage of preadipocytes. Several molecules with highly differential level expression in preadipocytes vs. mature adipocytes have been identified. These include preadipocyte factor-1 (Pref-1),

3 Wnts, GATA-2 and GATA-3. Pref-1 is a transmembrane molecule with EGF-like

repeats and is markedly downregulated in 3T3-L1 adipogenesis. However, Pref-1

expression is extremely low in adipose tissue (Smas et al., 1997). Wnt10b and

GATA-2, 3 are downregulated during differentiation and act as adipogenesis inhibitors (Ross et al., 2000; Tong et al., 2000).

We identified TSC-36 (TGF-β1 stimulated clone 36) as a novel adipocyte differentiation-dependent downregulated gene via differential screening of DNA filter arrays. TSC-36 encodes a secreted protein and its RNA expression is dramatically downregulated from high to nearly undetectable levels during white and brown adipogenesis. TSC-36 protein levels in cells and culture media also decrease during 3T3-L1 differentiation. TNFα elevates TSC-36 RNA and protein level in 3T3-L1 adipocytes. The decrease of TSC-36 in adipogenesis may be in part due to the suppression of the TSC-36 promoter activity by PPARγ and

KLF15. The function of TSC-36 in adipogenesis warrants additional exploration.

Distinct adipose tissue compartments termed “depots” are differentially associated with disease risks. The intra-abdominal adipose tissue (i.e. visceral fat) is more closely correlated with metabolic complications compared to subcutaneous adipose tissue (SAT). This has been attributed, at least in part to the direct drainage of free fatty acids and adipokines into the portal circulation

(Bjorntorp, 1990). In addition, different gene expression patterns in visceral adipose tissue (VAT) and SAT are presumed to be an important contributing

factor for the higher health risks associated with excess VAT. We employed

suppression subtractive hybridization (SSH) by using subtractive cDNA libraries

4 from murine subcutaneous (SC) or intra-abdominal epididymal (EP) white

adipocytes to identify WAT depot-enriched transcripts. The results revealed that

Boc and other 7 transcripts are enriched in EP by at least 3-fold compared to SC

adipocytes. We also identified a dramatic enrichment in SC white adipocytes for

transcript(s) of the (Mups), small secreted proteins with

pheromone functions that are members of the lipocalin family. A further

assessment of Boc and Mups expression patterns was conducted in murine

tissues, an obese mouse model and in various adipogenesis models.

In addition to WAT, mammals also have brown adipose tissue (BAT). The

developmental origin(s) of brown adipocytes is not clear; BAT serves primarily to

dissipate energy instead of storing it (Lowell and Flier, 1997). Our laboratory has

generated the mBAP-9 cell line, derived from preadipocyte precursors present in

murine BAT, as a new in vitro model for the study of brown adipogenesis.

We have characterized the phenotype and molecular definition of mBAP-9 cells.

Moreover, we have utilized this new cell line to determine the expression of ten

novel adipocyte differentiation-dependent genes, and characterized Cmbl

(carboxymethylenebutenolidase-like) and a new adipose tissue α/ hydrolase

Atabh in additional detail. Our research demonstrates that the mBAP-9 cell line is a useful research tool to further explore the molecular and cellular aspects of

brown adipogenesis.

Tmem182, a novel predicted transmembrane protein, is the gene we

observed to exhibit the highest fold upregulation in mBAP-9 adipogenesis (45-

fold) during our characterization of the mBAP-9 cell line. We further characterized

5 the expression and regulation of Tmem182 transcript in adipocytes and adipose

tissues. Tmem182 transcript is highly expressed in adipose tissue, whereas its

expression is 10-fold to 20-fold enriched in WAT vs. BAT. In BAT of ob/ob mice,

Tmem182 transcript is ~3-fold upregulated compared to wild type. Tmem182

transcript expression is upregulated during differentiation in various in vitro

models of white adipogenesis and is dramatically downregulated by TNFα

treatment of adipocytes. Intriguingly, we found that Tmem182 transcript is also

upregulated ~770-fold during the differentiation of C2C12 myoblasts. These data suggest that Tmem182 may play important role in both adipogenesis and myogenesis.

.

6 LITERATURE

Adipose Tissue is the Major Site for Energy Storage

Fatty Acid Synthesis

Fatty acid synthesis takes place in the cytosol, in humans this occurs

primarily in the liver, as well as in adipose tissue and mammary gland during

lactation. The precursors are malonyl-CoA carboxylated from acetyl-CoA, with

acetyl CoA formed from pyruvate in the mitochondria. The barrier for acetyl CoA

entering the cytosol is bypassed by citrate. Citrate is first synthesized in the

mitochondrial matrix by the condensation of acetyl CoA with oxaloacetate and

transported to the cytosol. Then it is cleaved by ATP-citrate lyase to give rise to acetyl CoA. The formation of malonyl CoA is a committed step in fatty acid

synthesis which is catalyzed by acetyl CoA carboxylase (ACC). This enzyme is

crucial to regulate . The intermediates in fatty acid synthesis

are linked to the sulfhydryl groups of an acyl carrier protein (ACP). The

elongation of saturated long-chain fatty acid is catalyzed by an enzyme system

termed . Fatty acid synthase is a homodimer; each subunit

contains three domains. Domain 1, the substrate entry and condensation unit,

contains acetyl transferase, malonyl transferase, and β-ketoacyl synthase

(condensing enzyme). Domain 2, the reduction unit, contains the acyl carrier

protein, β-ketoacyl reductase, dehydratase, and enoyl reductase. Domain 3, the

palmitate release unit, contains the thioesterase. The different catalytic sites are

linked together to form a multifunctional enzyme complex. Fatty acid synthesis

7 occurs via four recurrent reactions; condensation, reduction of acetoacetyl ACP, dehydration and reduction of crotonyl ACP. The unsaturated fatty acids are formed by a combination of elongation and desaturation reaction accomplished by accessory enzymes (Berg, 2002).

Fatty Acid Degradation Fatty acids are fuel molecules and stored as triacylglycerols. The bile salts, which are synthesized from cholesterol in the liver and secreted from the gall bladder, can facilitate the incorporation of dietary into micelles. Pancreatic lipases then digest the triacylglycerols into free fatty acids and monoacylglycerol.

The digestion products are transported to the intestinal epithelium across the

plasma membrane. In the intestinal mucosa cells, fatty acids and

monoacylglycerol are resynthesized to triacylglycerols and packaged into

lipoprotein transport particles called chylomicrons. The particles are primarily

transported into adipose tissue and muscle via the lymphatic system and blood.

Triacylglycerols are degraded into fatty acids and monoacylglycerol by

membrane-bound lipoprotein lipases prior to crossing the membranes of muscle

and adipose tissues. After being transported into cells, triacylglycerols are

resynthesized and stored. Muscle can oxidize the triacylglycerols as an important

energy source. To utilize fat as energy source, the first step is to hydrolyze

triacylglycerols by hormone sensitive lipase (HSL). This adipose tissue lipase can

be activated by epinephrine, norepinephrine, glucagon, and adrenocorticotropic

hormone. Seven-transmembrane-helix (7TM) type receptors in adipocytes

8 mediate the effects of these hormones to activate adenylate cyclase. The

increased level of cyclic AMP triggers protein kinase A to phosphorylate HSL.

Thus these hormones are prolipolytic. In addition to HSL, ATGL (adipose tissue

triglyceride lipase) has recently recognized as a novel triglyceride lipase and it is highly enriched in adipocyte (Jenkins et al., 2004; Villena et al., 2004;

Zimmermann et al., 2004). ATGL possesses highly specific triacylglycerol lipase activity and also exhibits phospholipase and transacylase activity (Jenkins et al.,

2004; Zimmermann et al., 2004). ATGL transcript can be downregulated by insulin and TNFα in 3T3-L1 adipocytes (Kim et al., 2006b). After hydrolyzation, free fatty acids are transported by serum albumin in a soluble form in blood and utilized by other tissues as energy. Fatty acids are oxidized in mitochondria; because oxidation is on the  carbons, this process is termed -oxidation. β-

oxidation occurs via four recurrent steps; oxidation, hydration, oxidation and

thiolysis. For every cycle, one molecule of FADH2, NADH and acetyl CoA are formed. Acetyl CoA can be oxidized by citric acid cycle (Berg, 2002).

Regulation of Fatty Acid Metabsolism Hormone Sensitive Lipase (HSL) The molecular mechanisms underlying the regulation of lipolysis are not known in detail. The activation of hormone sensitive lipase (HSL) is controlled through phosphorylation. Treatment of adipocytes with catecholamines

stimulates phosphorylated HSL to translocate from cytoplasm to the surface of

droplets (Blaak, 2003). Other studies indicate that HSL binding to the lipid

9 droplet in intact cells relies of function of perilipins. Perilipins are a family of phosphoproteins that coat the surfaces of intracellular lipid storage droplets.

Phosphorylated perilipin may serve as a docking protein for HSL. During adipogenesis, perilipins can be phosphorylated in response to increased intracellular cAMP concentration, allowing the phosphorylated HSL free access to the lipid droplet (Souza et al., 1998).

Acetyl CoA Carboxylase (ACC)

Acetyl CoA carboxylase (ACC) plays an essential role in controlling fatty acid metabolism. It catalyzes the production of malonyl CoA from acetyl CoA.

ACC is switched off by phosphorylation and activated by dephosphorylation.

AMP-dependent protein kinase (AMPK) phosphorylates ACC to convert it to an inactive form. AMPK is activated by AMP and inhibited by ATP. The dephosphorylation of ACC is performed by protein phosphatase 2A. Epinephrine and glucagon can activate protein kinase A, which suppresses phosphatase 2A, thereby indirectly inhibiting ACC activation. On the other hand, insulin promotes

ACC dephosphorylation. Hence, ACC can be inhibited by glucagon and epinephrine and activated by insulin (Berg, 2002).

AMP Kinase AMPK is a heterotrimeric serine/threonine protein kinase complex. AMPK is a crucial sensor for cellular energy charge and responds to AMP and an increased AMP/ATP ratio. In addition, the activation of AMPK is also involved in

10 the induction of fatty acid oxidation by leptin in skeletal muscle (Minokoshi et al.,

2002). Moreover, known pharmacologic AMPK modulators such as metformin or

AICA-riboside (AICAR, an AMP analog) can reduce fatty acid synthesis and

facilitate fatty acid oxidation by activating AMPK in liver or in muscle-derived cells

(Winder and Hardie, 1999; Zhou et al., 2001).

Fatty Acid Synthase (FAS)

FAS catalyzes long-chain fatty acid synthesis. Theoretically, inhibition of

FAS could attenuate fatty acid synthesis and reduce fat mass. However, FAS

inhibition might also result in an accumulation of cellular malonyl CoA. Malonyl

CoA can inhibit the fatty acid oxidation which is facilitated by carnitine palmitoyl

transferase 1 (CPT1). Thereby the inhibition of FAS might enhance fat

accumulation in select tissues (Kusunoki et al., 2006).

Adipose Tissue is also a Secretory Organ

Adipose tissue is not only an energy storage site but is also now

recognized as an active endocrine and paracrine organ. It synthesizes and

secretes a number of soluble factors that are involved in regulation of a variety of metabolic processes including energy homeostasis, fatty acid and glucose metabolism, food intake and blood pressure control.

TNFα

11 The transcript and protein level of TNFα (tumor necrosis factor alpha) is

elevated in adipose tissue of obese rodents as well as in humans (Hotamisligil et al., 1995; Hotamisligil et al., 1993; Kern et al., 1995). Both the adipocyte and stromal-vascular fraction of WAT can secrete TNFα, however the latter is the

predominant source in obese WAT due to the high degree of macrophage

infiltration into the SVF of WAT that are responsible for high TNFα expression in the obese adipose tissue (Fain et al., 2004; Hotamisligil et al., 1993; Weisberg et al., 2003). The mechanism for the recruitment and stimulation of macrophages in adipose tissue may be the induction of chemo-attractant signals that occurs upon increased adipocyte cell death during adipose tissue expansion (Cinti et al.,

2005). Another hypothesis is that dietary n-6 polyunsaturated fatty acids can be pro-inflammatory factors and enhance cytokine expression (Schmitz and Ecker,

2008). TNFα binds to two distinct receptors; TNFR1 and TNFR2. TNFR1 mediates the majority of signals induced by TNFα in adipose tissue (Cawthorn et al., 2007; Sethi et al., 2000; Uysal et al., 1998). After TNFR1 is activated, the

TNFR-associated death domain protein (TRADD) binds to the TNFR1-death domain (TNFR1-DD) and recruits downstream adapter proteins including Fas- associated death domain protein (FADD), receptor-interacting protein 1 (RIP1),

TNFR-associated factor 2 (TRAF2) and MAPK (mitogen-activated protein kinase)-activating death domain protein (MADD). Consequently multiple downstream signaling pathways such as nuclear factor-kappa B (NF-κB) and

MAPK cascades are transactivated by these adapter molecules (Chae and Kwak,

2003; Jain et al., 1999b; Kim et al., 2005; Ryden et al., 2002; Suzawa et al.,

12 2003). TNFα functions to induce insulin resistance in adipocytes by inhibiting

transcript expression of the factors required for insulin-stimulated glucose uptake,

such as insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and glucose

transporter GLUT4 (Ruan et al., 2002a; Ruan et al., 2002b; Stephens et al.,

1997). The original triggers of this inhibition are ascribed to suppression of

PPARγ by activated NF-κB (Ruan et al., 2002a). In addition, MAP4K4 (protein

kinase mitogen activated protein kinase kinase kinase kinase 4) mediated JNK

signaling is also involved in TNFα induced insulin resistance by suppressing

PPARγ and GLUT4 transcript level (Tang et al., 2006). Since adipogenesis is driven by the master transcriptional regulators PPARγ and C/EBPα, another effect of TNFα is to inhibit adipogenic differentiation and promote adipocyte dedifferentiation. Moreover, TNFα regulates the secretion of other adipokines, for instance, it suppresses adiponectin and resistin in 3T3-L1 adipocytes (Fasshauer et al., 2001, 2002) but promotes leptin release (Kirchgessner et al., 1997).

Leptin

Leptin was first identified in 1994 by positional cloning of the gene responsible for the genetic defection in the obese (ob/ob) mouse (Zhang et al.,

1994). The product of the obese (ob) gene, leptin, is a hormone synthesized

most abundantly by adipocytes, but it is also expressed in other tissues, for

instance, skeletal muscle, stomach and placenta (Bado et al., 1998; Masuzaki et

al., 1997; Wang et al., 1998). Leptin binds to its receptors in the hypothalamus to

inhibit appetite and reduce body fat mass (Campfield et al., 1996; Schwartz et al.,

13 1996). There are at least six alternatively spliced forms of the leptin receptor (ob-

r) gene in mice (Lee et al., 1996). In human, four leptin receptor variants are

present with an identical extracellular ligand binding domain but different

cytoplasmic carboxyl domains (Barr et al., 1999). In general, leptin receptor isoforms comprise three categories: long, short and soluble. Upon leptin signal stimulation, the long form, OB-Rb, activates the JAK-STAT (Janus kinase-signal transducers and activators of transcription) signaling pathway to induce specific gene transcription (Ghilardi et al., 1996). The short form OB-Ra has no JAK-

STAT activation function due to the lack of the box 2 motif in the intracellular domain, which is required for the tryosine phosphorylation of JAK. The soluble receptor (sOB-R) circulates in human blood and represents the main leptin binding activity (Lammert et al., 2001). Leptin exerts its central anorexigenic effects through the modulation of neuropeptide Y-containing neurons and pro-

opiomenlanocortin (POMC)-containing neurons in the arcuate nucleus of the

hypothalamus. This centrally mediated weight-reducing effects of leptin prompted

development of leptin as therapeutic drug to treat obesity. However

administration of leptin caused weight loss only in a marginal subset of obese

patients (Heymsfield et al., 1999). Leptin action in peripheral tissues is less

known. In isolated murine and human T lymphocytes, leptin functions to reverse

immunosuppression and enhance cell proliferation (Howard et al., 1999; Lord et

al., 1998; Martin-Romero et al., 2000). In addition, leptin may exert its metabolic

effects by inhibiting insulin secretion from pancreatic islets (Emilsson et al., 1997;

14 Fehmann et al., 1997) and stimulating muscle oxidation of fatty acids (Muoio et

al., 1997).

Resistin

Three independent research groups discovered resistin; Steppan et al.

discovered it as the anti-diabetic drug thiazolidinediones (TZDs) downregulated

protein (Steppan et al., 2001), Kim et al. reported it as a novel secreted protein

specifically expressed in white and brown adipose tissues in murine (Kim et al.,

2001) and Holcomb et al. revealed it as a protein associated with pulmonary

inflammation, also named as FIZZ1 (Holcomb et al., 2000). Resistin is a 12.5-

kDa cysteine-rich peptide that can homodimerize (Raghu et al., 2004), the

resistin receptor has not yet been identified. Serum resistin level is enhanced in diet-induced and genetically obese mice and consequently impairs glucose tolerance via antagonizing insulin action (Steppan et al., 2001). Human resistin

shares 59% homology with that of murine at the amino acid level. In contrast to murine, human resistin is expressed in multiple tissues with the highest level in bone marrow and with low expression in adipose tissue (Patel et al., 2003).

Several genetic analyses of resistin in human demonstrate association of single nucleotide polymorphisms (SNPs) at the resistin with obesity or insulin sensitivity. However, other reports are not supportive of this result (Steppan and

Lazar, 2004). Further studies of large populations will be necessary to elucidate the role of resistin in humans.

15 Adiponectin

Adiponectin was identified independently by four research groups as a specific secreted adipokine from adipocytes (Hu et al., 1996; Maeda et al., 1996;

Nakano et al., 1996; Scherer et al., 1995). Adiponectin consists of an N- collagenous domain and a C-terminal globular domain that mediates its bioactivities (Pajvani et al., 2003). In mice, there are at least two types of adiponectin receptors, AdipoR1, which is predominantly expressed in muscle, and AdipoR2 that is expressed mainly in liver. In humans, however, AdipoR1 and

R2 are ubiquitously expressed. Adiponectin may relieve insulin resistance in rodent muscle via the interaction between AdipoR1 and the insulin receptor, therefore enhancing insulin signal transduction (Yamauchi et al., 2003). In addition, adiponectin also acts to protect against diet-induced atherosclerosis and the development of cardiovascular disease (Berg and Scherer, 2005).

Others

Visfatin was found as an adipokine which is highly expressed in visceral vs. subcutaneous adipose tissue. It mimics insulin effects to lower blood glucose via activation of the insulin receptor. However, it does not function through the insulin-like growth factor-2 receptor and its plasma level remains similar upon fasting or feeding in mice (Fukuhara et al., 2005). Angiotensinogen is the precursor of angiotensin II and may be involved in the angiogenesis and the development of hypertension related to obesity (Ahima and Flier, 2000; Emanueli and Madeddu, 2002). Plasminogen activator inhibitor 1( PAI-1) is enriched in

16 omental fat compared with subcutaneous fat (Alessi et al., 1997). PAI-1 knockout mice gain more weight and adipose tissue cellularity in the high-fat diet induced obesity in mice (Morange et al., 2000).

With the identification of growing number of adipokines secreted by adipose tissue, the modulation effects of adipose tissue contribute to peripheral metabolism will be better understood.

Developmental Lineage of Adipocyte

WAT is believed to originate from mesoderm, which also gives rise to smooth muscle and cartilage. BAT may also have a mesodermal origin. Studies showed that when the mesoderm from a 9-day-old rat embryo was engrafted below the adult rat kidney capsule, a tumor containing only BAT was formed

(Loncar, 1992). Mesenchymal stem cells (MSCs) derived from postnatal bone marrow can be the common precursor of several different lineages including adipocytes, osteoblasts, chondrocytes, and myoblasts. In spite of unknown intermediate stages between MSCs and mature adipocytes, it is believed that adipoblasts have the capacitiy to give rise to both white and brown adipocytes dependent on different stimulatory conditions. In rodents, WAT can develop a considerable number of brown adipocytes when chronically exposed to cold and expression of the BAT marker gene UCP-1 can also be detected from in vitro differentiated primary white adipocytes (Cinti, 2005; Klaus et al., 1995).

On the other hand, preadipocytes and adipocytes may exhibit heterogeneity even within the same depots. Several reports indicated that there

17 are at least two populations of adipocytes in a fat pad. Mice with fat-pad specific

knockout of insulin receptor (FIRKO) or hormone-sensitive lipase (HSL) have two

different populations of adipocytes with distinct cell volumes rather than even

size (Bluher et al., 2002; Fortier et al., 2005). Further characterization of the

different adipocyte populations from FIRKO mice indicated they had differential

gene and protein expression patterns (Bluher et al., 2004). Another study

showed two preadipocyte subtypes arising from a single human preadipocyte

clone, one could differentiate and replicate better and expressed higher level of

adipocyte transcription factors, but responded less well to TNFα-induced

apoptosis in comparison to the second subtype (Tchkonia et al., 2005). BAT is

now also considered to possibly arise from a precursor cell distinct from that for

white adipocytes. By microarray analysis of primary preadipocytes, Timmons et

al. discovered that brown preadipocytes exhibit a myogenic transcriptional

signature including expression of MyoD, myogenin and Myf5; suggesting that

BAT may share a common precursor cell type with that of skeletal muscle

(Timmons et al., 2007)

Adipocyte Differentiation

Increase in WAT mass can occur by adipocyte hypertrophy and

hyperplasia and this expansion process was thought to be continuously occurring throughout the lifespan. However, a recent report revealed that in both lean and obese adults, even after marked weight loss such as following bariatric surgery, the number of fat cells stays constant despite ongoing adipocyte turnover,

18 suggesting that adipocyte number is set during childhood and adolescence

(Spalding et al., 2008).

It is difficult to study preadipocyte differentiation in vivo because adipose tissue consists of various types of cells, approximately one third are adipocytes and the remaining are a mixture of blood vessels, nerve tissue, macrophages, fibroblast-like interstitial cells and preadipocytes (Ntambi and Young-Cheul,

2000). To date there are no precise tools and markers to distinguish preadipocytes from fibroblasts in vivo, therefore studies of preadipocyte differentiation have been carried out primarily using in vitro models.

Preadipocyte cell lines can be categorized into two groups; pluripotent precursors and unipotent preadipocytes. For instance, C3H10T1/2 fibroblasts are derived from C3H mouse embryos and can give rise to adipocyte, muscle and cartilage cell types upon treatment by 5-azacytidine, an agent that is a chemical analogue of cytosine and inhibits DNA methylation (Taylor and Jones, 1979).

Other pluripotent fibroblast cell lines including Balb/c 3T3 (Sparks RL, 1986),

1246 (Jiang et al., 1992), RCJ3.1 (Bellows and Heersche, 2001) and CHEF/18 fibroblasts (Sager and Kovac, 1982) are all capable of differentiating into multiple cell lineages. The advantage of using these cell lines is that they serve as good models to understand the process of cell fate determination. On the other hand, the adipocyte cultures derived from these cell lines may not have a high degree of homogeneity.

In the category of unipotent preadipocyte cell lines, 3T3-L1 and 3T3-

F422A are derived from disaggregated Swiss 3T3-mouse embryos (Green and

19 Kehinde, 1976; Green and Meuth, 1974); TA1 (Chapman et al., 1984) and 30A5

(Konieczny and Emerson, 1984) are sublines from 5-azazytidine treated

C3H10T1/2 cells; Ob1771 was obtained from epididymal fat pad of the leptin

deficient obese mouse model-C57BL/6J ob/ob (Negrel et al., 1978). The 3T3-L1

cell line is one of the most extensively utilized and characterized culture models

to study adipogenesis in vitro. 3T3-L1 preadipocytes are morphologically

indistinguishable from fibroblasts. Post-differentiation mature 3T3-L1 adipocytes represent most of the characteristics of adipocytes in vivo. The advantage to using 3T3-L1 cells is the relatively high level of differentiation homogeneity. On the other hand, since it is derived from embryo, its cellular origin and lineage relationship to preadipocytes in adult WAT in vivo remains undetermined.

The in vitro differentiation process typified by 3T3-L1 can be delineated into four stages: (1) Post-confluent proliferation. (2) Confluence and growth arrest.

Initial growth arrest is achieved through cell-cell interact. However, studies have demonstrated that growth arrest but not cell confluence/cell contact is required for differentiation. When confluent 3T3-F442A cells were switched to growth in methylcellulose-stabilized suspension cultures, adipogenic conversion progressed successfully (Pairault and Green, 1979). Additionally, primary rat preadipocytes still differentiate into mature adipocytes when plated at very low density in serum free conditions (Gregoire et al., 1998b). The master transcriptional regulators C/EBPα and PPARγ are not only essential for the activation of adipocyte-specific genes after hormone stimulation, but are also involved in this indispensable growth arrest process. Activation of C/EBPα in

20 adipoblasts causes a direct cessation of cell growth (Umek et al., 1991) and

ligand-activated PPARγ is sufficient to induce growth arrest in fibroblasts and

adipoblasts (Altiok et al., 1997). (3) Hormonal induction and clonal expansion.

Differentiation of 3T3-L1 is typically achieved by treatment with a combination of

dexamethasone (Dex), a synthetic glucocorticoid agonist and

methylisobutylxanthine (Mix), a cAMP phosphodiesterase inhibitor. Sometimes

insulin is also added as a component of adipogenic cocktail to activate insulin-

like growth factor 1 (IGF-1). During the adipogenic cocktail treatment, cells undergo at least one round of DNA synthesis and clonal amplification. Inhibition of DNA synthesis in 3T3-F442A and Ob1771 cells can abolish differentiation

(Amri et al., 1986; Kuri-Harcuch and Marsch-Moreno, 1983). On the other hand, human primary preadipocytes differentiate without mitotic clonal expansion, suggesting that this post-confluent cell division may not be absolutely required for adipocyte development (Entenmann and Hauner, 1996). (4) Permanent growth arrest and terminal differentiation. Temporal changes in expression of numerous genes are involved in this stage. The crucial gene products are summarized below.

The Adipogenic Transcriptional Cascade

PPARγ

PPARγ was first recognized as a binding protein of the adipose-specific

enhancer of the adipocyte fatty acid-binding protein 2 (aP2) gene (Graves et al.,

1992). It belongs to the nuclear hormone receptor (NR) family and is now

21 recognized as the master regulator of adipogenesis. PPARγ contains an N-

terminal A/B domain which harbors a ligand-independent transcriptional

activation function 1 (AF1); followed by a DNA binding domain (DBD) that

includes two zinc finger motifs which facilitate interaction with PPAR response

element (PPRE) in target gene promoters (Nolte et al., 1998). The PPARγ C-

terminus is the ligand-dependent domain called ligand-binding domain (LBD) that

determines heterodimerization by partnering with the retinoid X receptors (RXRs).

PPREs are direct repeats of an AGGNCA half-site separated by a 1-

spacer (DiRenzo et al., 1997; Juge-Aubry et al., 1997). There are two PPARγ

isoforms, γ1 and γ2, which differ only in their first exon. PPARγ2 contains an

additional 30 amino acids at its N-terminus. PPARγ1 exhibits widespread

expression such as adipose tissue, breast, colon, liver, while PPARγ2 is

expressed almost exclusively in adipose tissue. The anti-diabetic

thiazolidinediones (TZD) drugs have high affinity for PPARγ (Lehmann et al.,

1995). The cyclopentanoic prostaglandin derivative 15-deoxy-∆ 12, 14-PGJ2

(15d-PGJ2) is one potential natural ligand (Kliewer et al., 1995). The role of

PPARγ as the master regulator in adipogenesis was revealed by the observation that forced expression of PPARγ and application of a PPARγ ligand stimulates adipogenesis of NIH-3T3 and other fibroblast cell lines (Hu et al., 1995; Shao and

Lazar, 1997; Tontonoz et al., 1994b). Its crucial role was further confirmed by the observation that C/EBPα cannot induce adipogenesis in the absence of PPARγ

(Rosen et al., 2002). The functional difference between PPARγ1 and 2 in

adipogenesis is still ambiguous. When endogenous PPARγ2 is selectively

22 repressed by engineered zinc finger repressor proteins (ZFPs) which target the

PPARγ2-specific promoter, 3T3-L1 cell differentiation is diminished by

approximately 50%. If both PPARγ1 and 2 expression are blocked, adipogenesis

is completely repressed. Exogenous rescue of PPARγ2 can restore adipogenesis,

whereas reintroduction of PPARγ1 had no rescue effect on differentiation (Ren et

al., 2002). In contrast, study of PPARγ-/- murine embryonic fibroblasts indicated

that both PPARγ1 and 2 stimulate differentiation; PPARγ2 exhibits enhanced

ability to induce differentiation and responds to lower ligand concentration

(Mueller et al., 2002b). PPARγ2 knockout mice apparently develop normally

except for an overall retardation in WAT development and the in vitro

differentiation of MEFs (mouse embryo fibroblasts) from these knockout mice is

markedly reduced (Zhang et al., 2004). However, a second PPARγ2 knockout

mouse model has normal WAT development but is more insulin resistant

compared with wild-type (Medina-Gomez et al., 2005). In addition to its role in

adipose tissue development, PPARγ also plays a pivotal role in mature

adipocytes. Introduction of a dominant negative mutant of PPARγ in mature 3T3-

L1 adipocytes leads to de-differentiation (Tamori et al., 2002). Conditional knockout of PPARγ in adipocytes of adult mice causes adipocyte death followed by replenishment with newly formed PPARγ-positive adipocytes (Imai et al.,

2004).

C/EBP Family

23 The C/EBPs are a family of transcription factors characterized by a highly

conserved basic-leucine zipper (bZIP) domain at the C-terminus. In the very early

stage of adipocyte differentiation of 3T3-L1, C/EBPβ and C/EBPδ expression is

increased in response to Mix and Dex treatment respectively, and lead to

induction of C/EBPα (Cao et al., 1991). C/EBPβ and δ are crucial for adipogenesis. MEFs from CEBPβ-/- mice do not undergo mitotic clonal expansion

and consequently their differentiation is abolished (Tang et al., 2003).

Furthermore, C/EBPβ and δ double knockout mice do not accumulate lipid

droplets in the interscapular region whereas wild-type mice develop BAT by 20 h

after birth. Additionally, adult C/EBPβ and δ deficient mice have reduced

epididymal fat pad weight compared with wild-type mice. C/EBPβ and δ are

reported to promote adipocyte differentiation by transactivating C/EBPα and

PPARγ. In C/EBPβ and δ deficient MEFs, induction of PPARγ and C/EBPα is

impaired (Rosen and MacDougald, 2006). However, in vivo studies showed that

the expression levels of PPARγ and C/EBPα remain normal in the adipose tissue

of C/EBPβ and δ double knockout mice. Co-expression of C/EBPα and PPARγ is

not sufficient to rescue the adipogenesis defect in C/EBPβ and δ deficient mice

(Tanaka et al., 1997). C/EBPα plays important roles in adipose tissue

development but its expression is not restricted to fat; liver and placenta also

exhibit a high level of C/EBPα (Birkenmeier et al., 1989). Block of C/EBPα

expression by introduction of antisense RNA to 3T3-L1 cells inhibits their

differentiation (Tao and Umek, 2000) and ectopically overexpressed C/EBPα is

sufficient to induce adipogenic conversion of 3T3-L1 (Lin and Lane, 1994).

24 These results indicate that C/EBPα is both necessary and sufficient for 3T3-L1 differentiation. C/EBPα knockout mice have severe hypoglycemia and it leads to prenatal lethality. This can be rescued by breeding with a transgenic line wherein

C/EBPα expression is driven in liver using an albumin enhancer/promoter. These

conditional C/EBPα knockout mice express C/EBPα in liver but not in other

tissues. These mice lack subcutaneous, peri-renal, and epididymal white adipose

tissue, but have BAT (Linhart et al., 2001). In C/EBPα null mice in which C/EBPβ

substituted for C/EBPα expression in tissues, the liver functions normally but fat

accumulation in WAT is significantly impaired (Chen et al., 2000).

KLFs

The krüppel-like zinc finger transcription factors (KLFs) are also involved

in adipogenesis (Li et al., 2005; Mori et al., 2005). KLFs are DNA-binding

transcriptional regulators containing Cys2/His2 zinc fingers. KLFs play diverse

roles during differentiation and development (Black et al., 2001; Dang et al.,

2000). 16 members of the KLF family have been identified. They all bind to very

similar “GT-box” or “CACCC element” sites on DNA (Bieker, 2001; Black et al.,

2001). Several KLF family members are involved in adipogenesis. KLF15 is

significantly induced during 3T3-L1 differentiation. Inhibition of KLF15 represses

adipogenesis and PPARγ expression but does not affect the expression of

C/EBPβ (Mori et al., 2005). KLF15 is also highly expressed in myocytes in vivo.

Overexpression of KLF15 in adipocytes or C2C12 myoblasts leads to enhanced

insulin-sensitive GLUT4 expression (Gray et al., 2002). KLF4 expression appears

25 within 30 min after exposure of 3T3-L1 preadipocytes to the adipogenic cocktail and binds directly to C/EBPβ promoter. Knockdown of KLF4 blocks differentiation and represses C/EBPβ expression (Birsoy et al., 2008). KLF5 knockout mice exhibit a markedly decreased differentiation of WAT. Overexpression of KLF5 in

3T3-L1 cells promotes differentiation and inhibition of KLF5 abolishes the adipogenic process. C/EBPβ and δ are able to transactivate KLF5 (Oishi et al.,

2005). KLF6 is known as a repressor of the proto-oncogene Delta-like 1

(Dlk1)/pref-1 which inhibits adipogenesis. Inhibition of KLF6 markedly reduces adipocyte differentiation (Li et al., 2005).

In addition to those KLFs which promote adipogenesis, other members of

KLF family are negative regulators of differentiation. KLF2/lung krüppel-like factor

(LKLF) is highly expressed in preadipocytes. Its constitutive overexpression inhibits PPARγ, C/EBPα, and ADD1/SREBP1c but not C/EBPβ and δ (Banerjee et al., 2003). In a tet-responsive 3T3-L1 model, re-expression of KLF2 at physiological levels inhibits differentiation partially via rescue of Pref-1 levels (Wu et al., 2005). Overexpression of KLF7 in 3T3-L1 preadipocytes leads to marked repression of differentiation (Kanazawa et al., 2005).

Other Transcription Factors Promoting Adipogenesis

In addition to PPARγ, C/EBPs and KLFs, there are reports of more than

100 other transcription factors that are linked to positive regulation of adipogenesis. The zinc finger-containing transcription factor Krox20 is highly enriched in adipose tissue and can be induced very early during 3T3-L1

26 differentiation. Overexpression of Krox20 in 3T3-L1 preadipocytes promotes

differentiation through the transactivation of C/EBPβ (Chen et al., 2005). Three

O/E genes (O/E-1, -2, and -3) are upregulated during 3T3-L1 differentiation. 3T3-

L1 cells with ectopically overexpressed O/E-1 have enhanced differentiation and

inhibition of O/E-1 partially represses adipogenesis (Akerblad et al., 2002).

ADD1/SREBP1c belongs to the basic helix-loop-helix leucine zipper

(bHLH-LZ) family. Inhibition of ADD1/SREBP1c markedly reduces 3T3-L1

differentiation. ADD1/SREBP1c may stimulate preadipocyte differentiation by

producing an endogenous ligand for PPARγ (Kim and Spiegelman, 1996; Kim et

al., 1998b). ADD1/SREBP1c also mediates insulin induction of the fatty acid

synthase (FAS) gene and leptin gene via transactivating their promoters (Kim et

al., 1998a). ADD1/SREBP1c knockout mice exhibit normal WAT development

but reduced fatty acid synthesis in livers (Shimano et al., 1997). However, a

mouse model with adipose tissue specific overexpression of SREBP-1c driven by adipocyte-specific aP2 enhancer/promoter shows retarded WAT differentiation,

fatty liver, insulin resistance and diabetes (Shimomura et al., 1998).

Liver X receptors (LXRs) are nuclear hormone receptors. They regulate

cholesterol, fatty acid and glucose homeostasis in liver and macrophages. LXR

has been shown to enhance lipogenesis. Activated LXRα binds to the promoters of PPARγ and ADD1/SREBP1c to increase gene expression and enhance differentiation (Seo et al., 2004). However in contrast to this finding, another report indicated that LXR agonists do not influence 3T3-F442A or 3T3-L1 preadipocyte differentiation (Hummasti et al., 2004). The reason for this

27 discrepancy is still not clear. Another LXR family member LXRβ is essential for

adipocyte size enlargement. LXRβ deficient mice have reduced lipid

accumulation in adipose tissue and impaired glucose intolerance due to reduced insulin secretion (Gerin et al., 2005).

Fu et al. carried out a qPCR analysis to characterize all 49 members of the mouse nuclear receptor superfamily during differentiation of 3T3-L1 cells. 30 out of the 49 nuclear receptors are expressed during adipogenesis, indicating the complexity of the transcriptional cascade of adipogenic conversion (Fu et al.,

2005).

Transcriptional Cofactors

Nuclear cofactors contribute to transcriptional modulation by directly changing chromatin structure or acting as scaffolds to recruit chromatin modifiers.

CBP (cAMP response element binding protein (CREB) binding protein) is a co- activator for C/EBPs and PPARs. CBP functions to hyperacetylate histones and recruit other co-activators such as SRC-1 (steroid coactivator-1), thereby facilitating interaction between the promoter DNA and transcription factors to stimulate transcription initiation. Cbp+/- mice have sharply decreased WAT weight

with enhanced glucose tolerance and insulin sensitivity (Yamauchi et al., 2002).

The SWI/SNF chromatin remodeling complex is known as a co-activator of

erythroid krüppel-like factor (EKLF) to modulate β-globin transcription. Studies have shown that C/EBPα transactivation element III (TE-III) binds the SWI/SNF complex to collaborate with C/EBPα and TBP/TFIIB interaction motifs during

28 adipogenesis in a SWI/SNF dependent manner. The inhibition of the ATPase

subunits of the SWI/SNF complex blocks adipogenesis (Pedersen et al., 2001b;

Salma et al., 2004). The TRAP220 (thyroid hormone receptor-associated

proteins 220 subunit) and PRIP (PPARγ-interacting protein) are both known as

co-activators of PPARγ2. Neither TRAP220-/- or PRIP-/- fibroblasts are able to

differentiate to adipocytes (Ge et al., 2002). In contrast, the NCoR (nuclear

receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid

hormone receptors) are recruited by PPARγ in the absence of ligand to abolish

adipogenesis. Knockdown of NCoR and SMRT in 3T3-L1 contributes to greater

cell differentiation (Yu et al., 2005).

Hormonal Adipogenesis Regulators

Insulin has a remarkable influence on adipogenesis. Insulin promotes preadipocyte differentiation and enhances lipid accumulation in adipocytes

(Girard et al., 1994). In preadipocytes insulin acts predominantly through the insulin like growth factor-1 (IGF-1) receptor. IGF-1 exists abundantly in fetal

bovine serum, thereby 3T3-L1 preadipocytes are able to proceed to differentiate

in serum free media supplemented with IGF-1 (Schmidt et al., 1990). Mice with

fat-specific disruption of the insulin receptor gene (FIRKO mice) show reduced

fat tissue (Bluher et al., 2002). The downstream components of insulin/IGF-1

signaling also play important roles in adipogenesis. Studies in brown

preadipocyte cell lines indicate that each of the four insulin receptor substrates

(IRS-1, 2, 3 and 4) act differently to mediate signaling by the insulin receptor and

29 the IGF-1 receptor. The expression levels of IRS-1 and IRS-2 are relatively high before and after differentiation, IRS-3 transcript is extremely low in preadipocytes and increased during differentiation, whereas IRS-4 is largely absent in adipocyte differentiation. Brown preadipocyte cell lines derived from each of the four IRS knockout (KO) mice exhibit different differentiation abilities in the order of IRS-4

KO> IRS-2 KO>IRS-3 KO> IRS-1 KO. Double knockout of IRS-1 and IRS-3 completely blocks adipogenesis (Tseng et al., 2004). The downstream serine/threonine kinase Akt/PKB that functions in insulin signaling is essential for cell growth, survival and metabolism. Mice lacking Akt/PKB develop insulin resistance (Garofalo et al., 2003).

Estrogen and its receptors including estrogen receptor (ER) α and β are crucial factors in adipose tissue development and regulation in females as well as males. Females have an overall higher body fat percentage, especially subcutaneous fat, than males. This is due to increased adipocyte number and size. This sexually dimorphic adiposity is determined by hormones including estrogen. Additionally, estrogen level and adipose ER expression varies with different physiological states for each individual (Cooke and Naaz, 2004). Both

ERα and β are expressed in preadipocyte, adipocyte and other cell types in adipose tissue including endothelium and smooth muscle cells, and macrophages (Dieudonne et al., 2004; Gulshan et al., 1990; Orimo et al., 1993;

Pedersen et al., 2001a; Venkov et al., 1996). Furthermore, ER is also widely distributed in hypothalamus and liver, suggesting that estrogen can not only directly regulate adipose tissue, but also has indirect effects through influence on

30 whole body energy balance and metabolism. Ovariectomy of mice or menopause in women leads in increased adipose mass, especially visceral fat (Wade et al.,

1985) and this can be reversed by estrogen replacement (Mohamed and Abdel-

Rahman, 2000). Estrogen downregulates adiposity via suppressing LPL

(lipoprotein lipase), which promotes lipogenesis in adipocytes (Hamosh and

Hamosh, 1975; Homma et al., 2000). On the other hand, estrogen enhances lipolysis by inducing hormone-sensitive lipase (HSL) and epinephrine which both promote lipolytic effects (Ackerman et al., 1981; Palin et al., 2003). In central control aspects, ERα knockout mice have markedly increased fat tissue due to decreased energy expenditure but do not exhibit hyperphagia (Heine et al., 2000).

Estrogen also has effects on other organs such as liver. Aromatase knockout

(ArKO) mice, which are estrogen deficient due to lack of the enzyme for endogenous estrogen synthesis, develop aging-related obesity in both males and females. Only male ArKO mice exhibit increased hepatic triglycerides and hepatic steatosis due to enhanced fatty acid uptake; this can be reversed by administration of estrogen (Hewitt et al., 2004).

Preadipocyte Marker Genes

In contrast to transcriptional upregulation of genes during adipogenesis, decreases in gene expression that occur during adipocyte differentiation are far less studied. Some of these may also be useful as preadipocyte markers to facilitate preadipocyte identification and characterization since it is difficult to distinguish preadipocytes from fibroblast cells in vivo. 31 Preadipocyte factor (Pref-1) is a transmembrane protein with epidermal

growth factor (EGF)-like repeats and belongs to the Notch/Delta/Serrate family.

Pref-1 is cleaved at the extracellular domain to generate a soluble 50 KDa form.

Pref-1 is highly expressed in 3T3-L1 preadipocytes and is almost undetectable in

adipocytes. Downregulation of Pref-1 is required for adipogenesis since

constitutive expression of Pref-1 inhibits adipose conversion (Smas et al., 1997).

In addition, repression of Pref-1 by antisense RNA promotes preadipocyte

differentiation (Smas et al., 1999). MEFs from Pref-1 null mice exhibit enhanced

ability to differentiate to adipocytes (Kim et al., 2007c; Moon et al., 2002).

Transgenic mice with adipose tissue specific overexpression of the 50 KDa

soluble Pref-1 develop hypertriglyceridemia, impaired glucose tolerance and

significantly reduced total fat pad weight (Lee et al., 2003).

Wnt signaling also acts to inhibit adipogenesis. Wnt10b is highly enriched

in preadipocyte cultures. It binds to the frizzled receptor to activate β-catenin

dependent signaling and leads to inhibition of adipogenesis (Ross et al., 2000).

Transgenic mice overexpressing Wnt10b have a decline of total body fat and less lipid accumulation in adipose depots even when fed a high fat diet (Longo et al.,

2004). Wnt1, a Wnt/β-catenin signaling activator, also can inhibit adipogenesis

(Moldes et al., 2003). Wnt6 and Wnt10a have been shown to participate the regulation of brown adipocyte differentiation (Tseng et al., 2005; Tseng et al.,

2004).

GATA-2 and GATA-3 are GATA-binding transcription factors. Their

mRNAs are downregulated during white adipocyte differentiation. Constitutive

32 expression of GATA-2 and GATA-3 traps cells at the preadipocyte stage and

such cultures are refractory to the effects of the adipogenic cocktail (Tong et al.,

2000).

Despite much progress in cell culture models, the process of the

emergence of functionally distinct preadipocytes from mesodermal cells during embryonic development remains obscure. Thus elucidation of the molecular definition and developmental lineage of preadipocytes is essential to understand adipogenesis. We have identified TSC-36 (TGF-β1 stimulated clone 36) as a

novel preadipocyte gene. TSC-36 has been studied sporadically in various other

settings but to date has not been described in adipogenesis. Current knowledge of TSC-36 is summarized blow.

TSC-36

Identification of TSC-36

TSC-36 was first isolated by Shibanuma et al. through differential

screening to identify TGF-β1 (transforming growth factor β1) responsive genes in mouse osteoblastic MC3T3-E1 cells (Shibanuma et al., 1993). TGF-β1 is a member of the TGF-β subfamily, which includes a large number of polypeptide growth factors. Other family members are the activin/inhibin subfamily, the bone morphogenetic protein/decapentaplegic subfamily and the 60A subfamily

(Kingsley, 1994). In the TGF-β subfamily, there are at least three genes that share structural homology and similar biological functions with TGF-β1, these are

TGF-β2, -β3, -β5 (Kingsley, 1994). TGF-β1 has numerous effects in diverse

33 biological settings including inhibition of cell proliferation, regulation of cell

differentiation, upregulation of extracellular matrix proteins and proteinase inhibitors (Laiho et al., 1986; Moses, 1992; Moses and Serra, 1996; Roberts et al., 1986; Shull et al., 1992; Siegel and Massague, 2003; Tucker et al., 1984).

TSC-36 is one of the six clones identified to be induced by TGF-β1 in MC3T3-E1 cells with expression levels increased about eight-fold.

Nucleotide sequence analysis reveals that the coding sequence of the murine TSC-36 transcript consists of 918 base pairs and encodes a secreted protein with 306 amino acids which contains a putative N-terminal signal sequence (amino acids 2-16). In vitro translation shows a molecular weight of

~35 KD, however secreted TSC-36 is ~38 KD. It was predicted that this difference might due to post-translational N-glycosylation as TSC-36 has four potential N-glycosylation sites (Shibanuma et al., 1993). Hambrock et al. carried out structural characterization of TSC-36 (Hambrock et al., 2004). This study revealed that TSC-36 has a like domain, an extracellular domain with two EF hand motif and a von Willebrand factor type C domain. Based on sequence analysis, TSC-36 is a member of the SPARC (secreted protein acidic and rich in cysteine, also referred to as osteonectin and basement membrane-40

(BM40)) family. However, the member of the SPARC protein family, TSC-36

appears to share the least similarity with SPARC. SPARC is a calcium and

collagen-binding extracellular matrix (ECM) glycoprotein (Lane and Sage, 1994).

SPARC is involved in many biology processes. It functions to abrogate focal

adhesion, regulate growth factor activation, inhibit cell-cycle and modulate tissue

34 remodeling (Brekken and Sage, 2000). Intriguingly, SPARC-null mice exhibit

increased subcutaneous and epididymal adipose tissue with elevated levels of

serum leptin than wild-type. However, there is no significantly difference of their

overall body weights in comparison with wild-type (Bradshaw et al., 2003). TSC-

36 also has homology to follistatin, within its cysteine rich region (Shibanuma et

al., 1993). Follistatin functions to inhibit follicle-stimulating hormone (FSH)

secretion via binding to activin, the latter belongs to the TGF-β super family and

positively regulates FSH (Phillips and de Kretser, 1998). Mass spectrometry

analysis revealed that secreted TSC-36 exists in two charge isoforms designated

isoform A with a molecular mass of 42.3 KD and isoform B with a molecular

mass of 41.9 KD. The mass of both isoforms are higher than the calculated

molecular mass of 33 KD, indicating the presence of post-translational

modification. Studies have shown that N-glycosylation of TSC-36 protein occurs

at asparagines in position Asn142, Asn173, Asn178 but not the fourth possible asparagine, Asn221; O-glycosylation may also present. The size variation

between two isoforms is attributed to differing sialic acid content in the N-glycan

(Hambrock et al., 2004).

The rat homolog of TSC-36, named follistatin-related protein (FRP), was

isolated from a chemically induced rat glial brain tumor cell line, C6 glioma cells,

by Zwijsen et al. due to its abundance in condition media (Zwijsen et al., 1994).

In this study the identification of the human TSC-36 homolog was also reported.

Rat and human FRPs encode 306 and 308 amino acid proteins respectively, and share a high degree of amino acid sequence similarity with murine TSC-36: 93%

35 between rat and human, 96% between mouse and rat. The concentrated conditioned media from transfected COS cells which contained rat FRP was tested in a growth-inhibition assay of CCl-64 mink lung epithelial cells, but no evidence was found to support a modulatory role for the FRP protein in TGF-β1 induced growth inhibition.

TSC-36 in Embryonic Development

There are a series of reports that describe involvement of TSC-36/Flik

(follistatin-like) in avian and xenopus dorsalization and neutralization. In embryonic gastrulation, an “organizer” is a region with induction ability that can determine differentiation direction of other regions or tissues. In amphibians,

Spemann and Mangold first demonstrated that upon transplantion to the ventral side of the host, the dorsal blastoporal lip acts as “organizer” to induce a new host originated neural structure from the epiderm region (Spemann and Mangold,

1924). In amniotes, the anterior tip of the primitive streak which is termed

“Hensen’s node” was also demonstrated to develop to notochord and to induce the surrounding host embryonic regions to form new structures via transplantation to an extra-embryonic or ventrolateral host site (Storey et al.,

1992; Storey et al., 1995; Waddington, 1932; Waddington, 1933). This new notochord formation process is also called neural induction or dorsalization.

During chick embryonic gastrulation, TSC-36/Flik is expressed in both mesoderm and ectoderm. After Hensen’s node is grafted into the peripheral position of blastoderm, TSC-36/Flik expression appears in the newly induced neural axis,

36 suggesting that it is involved in the neural induction and dorsalization process

(Patel et al., 1996). In another study of chick embryo somite maturation, TSC-

36/Flik expression is highest in the myotome of the somite and the neural tube is

required for its expression. The authors postulated that TSC-36/Flik may play a

role during myocyte differentiation because of the similar expression pattern of

TSC-36/Flik and MyoD during avian somitogenesis (Amthor et al., 1996). When

TSC-36/Flik is inhibited by antisense oligodeoxynucleotides at the gastrula stage, the embryo exhibits deficient axial patterning and holoprosencephaly due to attenuation of dorsalising and neural-inducing signals and consequent

inadequate sonic hedgehog signaling (Towers et al., 1999).

Xenopus TSC-36 is named follistatin-related protein (xFRP), which

exhibits approximately 70% identity to mouse, human and chicken TSC-36

protein. xFRP is expressed throughout the gastrulation process and is localized

in the Spemann organizer, notochord, neural floor plate, hypochord and somite; it

may also modulate neural induction in Xenopus embryonic development

(Okabayashi et al., 1999).

In mouse embryonic development, TSC-36/FRP is expressed in the

primitive streak of which the anterior part is equivalent to Hensen’s node or

organizer in avian and amphibians. At later stages, TSC-36/FRP is expressed in

the somites (De Groot et al., 2000). During mouse embryo development, TSC-

36/FRP has also been found to be expressed in airway epithelia in lung as well as in collecting ducts and in the nascent nephron epithelia in kidney (Adams et al., 2007).

37 In macaques the TSC-36 gene is also termed occ1. TSC-36/occ1 is expressed in macaque visual neurons. It is highly expressed in the primary visual cortex (V1) of macaque and marmoset but not in mouse, rabbit and ferret.

Intriguingly, its expression is sensory-input dependent in macaque but sensory- input independent in mice (Takahata et al., 2008; Takahata et al., 2006; Tochitani et al., 2001).

TSC-36 in tick Haemaphysalis longicornis has been identified and it has effects on tick oviposition (Zhou et al., 2006)

TSC-36 and Cancer

The research group that first cloned TSC-36 from mouse osteoblastic

MC3T3-E1 cells reported that TSC-36 transcript level is decreased dramatically in several v-Ki-ras transformed MC3T3 cell lines compared to untransformed cells (Shibanuma et al., 1993). Similarly, TSC-36 expression is also downregulated when NIH/3T3 cells are transformed with ras (DT cells), whereas

TSC-36 is upregulated to almost normal levels in a flat revertant form isolated from DT cells (C11 cells). However, if the flat revertant cells are induced from DT cells by transfection of K-rev-1 (WT cells), there is no upregulation of TSC-36, which suggested that TSC-36 may play roles in negative regulation of cell growth

(Shibanuma et al., 1993). TSC-36 transcript level is also markedly downregulated in v-myc-transformed NIH cells but not in v-raf transformed ones. Though raf is a ras inhibitor, it has no effect on TSC-36 expression. Ras may cause the reduction of TSC-36 via raf-independent pathways (Mashimo et al., 1997). These results

38 suggested that the expression of TSC-36 transcript can be suppressed specifically by oncogenes and TSC-36 may have effects on the phenotypic alterations of transformed cells. Further studies regarding the relationship between TSC-36 and tumorigenesis revealed that TSC-36 transcript is undetectable in small cell lung cancer (SCLC) cell lines but is expressed in some non-small cell lung cancer (NSCLC) cell lines. Because of the higher tumor aggressiveness of SCLC than NSCLC, the authors postulated that TSC-36 may have antiproliferation function. To address this, TSC-36 was transfected into

NSCLC PC-14 cells, which express very low level of TSC-36 protein, and a stable transfected cell line with TSC-36 overexpression was established. These cells have longer doubling time, lower colony formation abilities and lower rate of

DNA synthesis than the controls. Moreover, TSC-36 overexpression caused a cell shape alteration from round to spindle-like, indicating increased cell adhesion

(Sumitomo et al., 2000). These data demonstrated that overexpression of TSC-

36 can inhibit the growth of human lung cancer cells. In addition, another study identified TSC-36 as a gene downregulated by activator protein-1 (AP-1), which is a transcription factor required for transformation by many oncogenes. Re- expression of TSC-36 inhibited cell invasion (Johnston et al., 2000). One study of p130Cas (Cas) deficient fibroblasts also supported the idea that TSC-36 might be involved in inhibition of cell migration. Cas is a docking protein for many signaling molecules such as FAK and PI3K. Cas-/- fibroblasts exhibit impaired stress fiber formation and cell migration. TSC-36 is one of the genes found to be upregulated in Cas deficient fibroblasts (Nakamoto et al., 2002). Moreover, a recent study

39 reported downregulation of TSC-36 transcript in human endometrial and ovarian

cancers. Additionally, overexpression TSC-36 in ovarian cancer cell line

Ovca420 and endometrial cancer cell line AN3CA impairs cell proliferation and promotes apoptosis. Cell migration and invasion is also attenuated due to TSC-

36 induced downregulation of MMP-2 (Chan et al., 2008). Several other tumor microarray studies also identified TSC-36 as a candidate tumor-suppressor gene.

For instance, it is downregulated in mouse islet carcinomas (Hodgson et al.,

2001) and metastatic clear-cell renal-cell carcinoma (ccRCC) (Tan et al., 2008).

In a breast tumorigenesis study, TSC-36 was also found to be upregulated by the tumor suppressor BRCA1 (Bae et al., 2004). To date the only contradictory report to the idea of a tumor suppressor function of TSC-36 is a recent study which indicates that TSC-36 is an upregulated gene in a glioblastoma (GBM) microarray study and it was suggested to be a hallmark of poor prognosis of

GBM (Reddy et al., 2008). Overall, most of these reports suggest that TSC-36 may play role in tumorigenesis as tumor-suppressor gene and demonstrate that it is downregulated in various types of tumor cells.

TSC-36 and Rheumatic Diseases

TSC-36/FRP (follistatin-related protein) was cloned as a novel autoantigen in systemic rheumatic diseases. The anti-FRP antibody is detected in the sera and synovial fluid from rheumatoid arthritis (RA) more frequently than in other systemic rheumatic diseases such as osteoarthritis (OA) and systemic lupus erythematosus (SLE). The erythrocyte sedimentation rate (ESR) and serum C-

40 reactive protein level (CRP), the major indexes that reflect inflammation, are

markedly higher in anti-FRP antibody-positive RA patients than in the negative

patients (Tanaka et al., 1998). Introduction of exogenous TSC-36/FRP into the

rheumatoid synovial cell line E11 leads to the inhibition of cell growth (Ehara et

al., 2004). Moreover, in vivo study revealed that intraperitoneal injections of

recombinant TSC-36/FRP had significant ameliorative effects on the arthritis

(Kawabata et al., 2004). Further study revealed the possible mechanism

underlying these effects. TSC-36/FRP was showed to inhibit the protein levels of

MMP-1, MMP-3 and prostaglandin E2 in both human and rabbit synovial cell lines,

and this effect can be abrogated by autoantibodies to TSC-36/FRP. MMP-1 and

MMP-3 play important roles in RA physiology and pathology due to their abilities to degrade interstitial collagens and non-collagenous proteins (Tanaka et al.,

2003). In addition, transcript comparison of TSC-36/FRP-treated and untreated mice by microarray shows that TSC-36/FRP downregulates the expression of c- fos, ets-2, IL-6, MMP-3, and MMP-9, which may involved in synovial inflammation and joint destruction (Kawabata et al., 2004). Intriguingly, another report demonstrated that TSC-36 is a substrate of MMP-2 and can be cleaved to several fragments by MMP-2 (Dean et al., 2007). Therefore, TSC-36 may act as an ameliorative factor for RA by inhibiting specific MMPs and other inflammatory factors in synovial cells. However, a later study by Miyamae et al. reported TSC-

36 as a pro-inflammatory molecule. Overexpression of TSC-36 in macrophages leads to enhanced levels of cytokines such as IL-1β, TNFα, and IL-6. This study also reported that administration of TSC-36 in mouse paws exacerbates

41 collagen-induced arthritis (Miyamae et al., 2006). This result is contradictory to that of the study reported by Kawabata et al., which indicated downregulation of cytokines by TSC-36 (Kawabata et al., 2004). The reason for the discrepancy is not yet clear.

TSC-36 Functions in Muscle and Heart

Several studies have reported that TSC-36 also functions to regulate the physiology of smooth muscle cells and cardiomyocytes. TSC-36 has been shown to inhibit proliferation and migration of vascular smooth muscle cells (VSMCs).

VSMC proliferation is the major characteristic of neo-intimal hyperplasia that causes in-stent restenosis. Treatment with daidzein ameliorates neo-intimal proliferation and also induces TSC-36 expression in vitro and in vivo (Liu et al.,

2006). Another microarray study comparing two different beef cattle breeds also identified TSC-36 as a downregulated gene during fetal bovine longissimus muscle differentiation (Lehnert et al., 2007). In a study of heart-specific calsequestrin (CSQ)-overexpressing transgenic mice, TSC-36 and SPARC transcripts were found to be upregulated in heart. CSQ is a high capacity Ca2+ binding protein in the cardiac sarcoplasmic reticulum (SR). These transgenic mice develop concentric cardiac hypertrophy and subsequently dilated cardiomyopathy (Ihara et al., 2002).

Intriguingly, Rosenberg et al. revealed a novel molecular avenue to investigate the mechanism of TSC-36 regulation in muscle cells by utilizing microRNA analysis. MyoD (myogenic differentiation) is a key transcriptional

42 regulator of muscle differentiation. TSC-36 is a gene that suppressed by MyoD in

C2C12 myocytes, meanwhile a second gene named Utrn is also inhibited by

MyoD. The authors found murine microRNA-206 (Mmu-mir-206) is markedly

induced by MyoD, additionally, both TSC-36 and Utrn are targets of Mmu-mir-

206 (Rosenberg et al., 2006). MicroRNAs (miRNAs) are single-strand RNAs of

19-25 nucleotides that originate from endogenous hairpinshaped transcript precursors. miRNAs target mRNAs by binding to the 3'-untranslated regions (3'-

UTR) and result in mRNAs degradation or repression of translation. miRNAs

have been demonstrated to function in many biological process such as cell

proliferation, differentiation, hematopoiesis and apoptosis (Ambros, 2004; Bartel,

2004). In our study of TSC-36, by searching of Sanger miRBase sequence

database we noted that the 3’-UTR of human TSC-36 contains the precursor

transcript of human microRNA-198 (Hsa-mir-198). Interestingly, human TSC-36

is also one of the targets of Hsa-mir-198. Further research regarding microRNA

function and TSC-36 may facilitate the understanding of TSC-36 regulation.

The latest reports regarding TSC-36 and myocyte illustrated that Akt can

induce TSC-36 expression in myocyte. The serine/threonine protein kinase Akt

mediates PI3K (phosphatidylinositol-3 kinase) signaling and is an important factor to promote cardiac myocyte growth and survival. TSC-36 is a novel upregulated target for Akt in a heart specific inducible Akt1 transgenic mouse model. Ectopic expression of TSC-36 in neonatal rat ventricular myocytes (NRVMs) protects the cells from hypoxia/reoxygenation stress in an Akt dependent manner.

Knockdown of TSC-36 by siRNA in NRVMs exacerbates hypoxia/reoxygenation

43 stress-induced apoptosis. Furthermore, an in vivo study demonstrated that intravenously administration of adenoviral expressing TSC-36 leads to elevated serum TSC-36 levels and subsequently protects myocardium from ischemia and reperfusion induced apoptosis (Oshima et al., 2008).

Very recently, the same research group reported another study of the relationship of TSC-36 and Akt in muscle. Overexpression of Akt in skeletal muscle promotes myocyte hypertrophy and angiogenesis. The authors generated muscle-specific Akt1 transgenic mice and identified TSC-36 as an Akt induced gene in gastrocnemius muscle. This study further revealed that TSC-36 is secreted by C2C12 myoblasts and is upregulated by muscle ischemia.

Intramuscular injection of adenoviral vector expressing TSC-36 promotes flow recovery and capillary density in animal hindlimbs under ischemic condition. This effect is due to TSC-36 stimulated endothelial nitric oxide synthase (eNOS) phosphorylation and consequently enhanced revascularization, treatment with an

Akt or eNOS inhibitor can abolish the effect (Ouchi et al., 2008).

Myoblast cells and preadipocytes are known to have same mesodermal origin (Ntambi and Young-Cheul, 2000), the knowledge of TSC-36 functions in myocyte may contribute to a better understanding of the role of TSC-36 in adipogenesis.

Other TSC-36 Functions

TSC-36 can be upregulated by estrogen and tamoxifen in CDO7F cells, an osteoblastic cell line positive for estrogen receptor (Ohashi et al., 1997). In a

44 study regarding the relationship between fibroblast growth factor 9 (Fgf9) and

short bowel syndrome, TSC-36 together with p-ERK and follistatin appear to be

markers of intestinal mesenchymal stem cells (iMSCs). These p-

ERK/follistatin/TSC-36 expressing iMSCs can suppress differentiation of

intestinal myofibroblasts in co-culture (Geske et al., 2008).

Brown Fat Differentiation

Mammals contain not only WAT which mainly functions to store

triglyceride and secrete adipokines, but also brown adipose tissue (BAT) which

burns fat and generates heat. Brown adipocytes contain more mitochondria and

store less triglyceride, present in multiple small lipid droplets instead the

unilocular lipid droplet of white adipocytes. In rodents, BAT is concentrated in the

interscapular fat pad and is most abundant in the neonatal period, acting to protect the newborn from the cold environment. In the human fetus and newborn,

BAT is concentrated in the thoracic cavity surrounding the great vessels and in

the axillary, cervical, perirenal and periadrenal regions (Cannon and Nedergaard,

2004). During the post-neonatal period, BAT is generally observed to significantly

decrease in mass and to rarely exist in adults except in patients that develop

pheochromocytoma, a catecholamine secreting tumor (Lean et al., 1986).

However, recent studies indicted that small amounts of BAT persists in the regions mentioned above in the adult (Nedergaard et al., 2007). In addition, the expression of UCP-1 (uncoupling protein-1), an exclusively brown adipocyte marker transcript, is found within adult human WAT. The UCP-1 protein is

45 located in the inner mitochondrial membrane and uncouples ATP synthesis to

dissipate the proton gradient and thus generate heat (Cannon and Nedergaard,

2004).

PPARγ and C/EBPs are the common master transcriptional regulators that

control both brown and white adipogenesis. The synthetic PPARγ agonists of the

thiazolidinedione (TZD) class promote brown preadipocyte differentiation robustly

and the administration of TZDs to rats results in marked accumulation of

interscapular BAT (Tai et al., 1996). Furthermore, several studies indicated that

after treatment with potent PPARγ ligands such as rosiglitazone, white

adipocytes in both the in vivo and in vitro setting showed more “browning” characteristics including increased mitochondrial mass and significantly

enhanced lipid oxidation (Wilson-Fritch et al., 2003; Wilson-Fritch et al., 2004).

These PPARγ ligands induce upregulation of mitochondrial genes including

UCP-1, Cox (cytochrome c oxidase), Cox8b (subunit VIIIb) and Cox7a1 (subunit

VIIa1). C/EBPs also play essential roles in brown adipogenesis. C/EBPα

knockout and C/EBPβ, δ double knockout mice both show a phenotype of reduced accumulation of lipid droplets in brown adipocytes and decreased UCP-

1 expression (Tanaka et al., 1997; Wang et al., 1995).

A distinct nuclear factor that is involved in brown adipocyte formation is

PGC-1α (PPARγ coactivator-1α). PGC-1α is highly enriched in BAT in contrast to WAT. PGC-1α can be markedly induced in brown fat and skeletal muscle upon cold stimulation. Ectopic overexpression of PGC-1α in white adipocytes results in

enhanced mitochondrial biogenesis and UCP-1 expression. In addition, PGC-1α

46 levels can be regulated by β-adrenergic agonists and by cAMP, agents that

induce UCP-1 expression and adipocyte differentiation. These observations

indicate that PGC-1α is a critical factor for adaptive thermogenesis in BAT, but is

not essential for directing of adipogenesis toward the brown lineage (Rosen and

Spiegelman, 2000).

Recently PRDM16 was identified via global transcription factor analysis as

a central master regulator which determines brown adipocyte formation.

PRDM16 is highly expressed in brown adipocytes but is not expressed in white

adipocytes. Upon introduction of PRDM16 into white preadipocytes, it robustly

induces the expression of BAT-specific genes including PGC-1α and UCP-1 and

also stimulates mitochondrial biogenesis. In vivo overexpression of PRDM16 in

WAT stimulates a conversion towards a BAT phenotype. Knockdown of PRDM16 in a brown adipocyte cell line leads to a depletion of brown adipocyte

characteristics (Seale et al., 2007). PRDM16 interacts with CtBP-1 (C-terminal-

binding protein-1) to form a complex to repress white adipocyte gene expression.

On the other hand, PRDM16 recruits PGC-1α to strongly activate brown

adipocyte genes (Kajimura et al., 2008). These results demonstrate that

PRDM16 plays a pivotal role in brown adipogenesis fate determination.

Depot Specific Characteristics of Various White Adipose Depots

Human WAT is distributed throughout the body with the subcutaneous

depots located in the abdomen, thighs and buttocks. The visceral depots are

around the omentum, intestine, epididymis and peritoneal region. Among these

47 distinct depots, some are responsive to sex hormones, for instance, the fat pad in

breasts and thighs. The fat on the neck and upper back is more sensitive to

glucocorticoids, as evidenced in Cushing’s disease. Visceral depots are more

sensitive to catecholamines and express higher level of glucocorticoid receptor,

therefore the lypolysis and fatty acid turnover frequency is higher than

subcutaneous depots. Visceral fat also expresses higher interleukin-6 and

plasminogen activator inhibitor-1 than subcutaneous depots, factors that are tightly related to inflammation and cardiovascular risk (Gesta et al., 2007). On the

other hand, visceral fat expresses lower levels of adiponectin, a factor that that

promotes insulin sensitivity (Wajchenberg et al., 2002). Additionally, the

subcutaneous depot is more responsive to insulin induced antilipolytic effects

(Engfeldt and Arner, 1988). These facts may all contribute to the observation that

increased visceral fat is related to higher risk of diabetes and metabolic syndrome compared to subcutaneous depots.

MMPs and Extracellular Matrix Remodeling in Adipogenesis

Extracellular matrix (ECM) remodeling is essential in many biological

processes, for instance, embryogenesis, tissue differentiation, cell migration and

tumor growth. The matrix metalloproteinases (MMPs), which belong to the zinc-

dependent proteinases family, play central roles in ECM remodeling and

degradation. There are 23 MMPs in human and 24 in mice (Page-McCaw et al.,

2007). Generally speaking, MMPs consist of a catalytic domain and a propeptide domain. A conserved Cys residue in the propeptide domain ligates the zinc ion

48 located in active-site to inhibit catalysis. MMPs can be categorized into six subgroups according to their primary substrate specificities: collagenase, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs) and other MMPs (Sternlicht and Werb, 2001). Except for MT-MMPs which have transmembrane domains or a glycosylphosphatidylinositol linkage to anchor them to membrane, most MMPs are soluble (Page-McCaw et al., 2007). The tissue inhibitors of metalloproteinases (TIMPs) regulate MMP activities to maintain homeostasis of normal cellular environments and also exert other biological functions in an MMP-independent manner. There are 4 members of the

TIMP family, TIMP1, 2, 3 and 4. Although most studies have focused on the hormonal and transcriptional regulation of adipogenesis, the MMP system has been demonstrated to be essential in adipocyte differentiation (Lilla et al., 2002).

During adipogenic conversion, fibroblast-like preadipocytes release adhesion to the ECM and key alterations in cell morphology and volume occurs with lipid accumulation. It is generally regarded that there is a trend of upregulation for many MMPs in obese adipose tissue. Assessment of MMP transcript levels in the genetic obese models ob/ob and db/db mice and in a diet-induced obesity model

(AKR mice), the transcript levels of MMP-2, MMP-3, MMP-12, MMP-14, MMP-19 and TIMP1 are significantly enhanced in the obese adipose tissues, compared to wild type counter part (Chavey et al., 2003). In addition, MMP-3, MMP-11, MMP-

12, MMP-13, MMP-14 and TIMP1 transcripts are upregulated in adipose tissue of high-fat diet induced obese mice, in contrast with mice on a standard-fat diet

(Maquoi et al., 2002). Furthermore, mice on a high-fat diet that receive the broad-

49 spectrum MMP inhibitor galardin exhibit a marked reduction of subcutaneous and

gonadal fat pad weights (Lijnen et al., 2002a). MMP-2 and MMP-9 are among the

best-characterized members of the MMP system in regard to adipogenesis.

MMP-2 and MMP-9 are secreted by human adipose tissue and upregulated

during murine preadipocyte differentiation. Treatment with MMP-2 and/or MMP-9

inhibitors such as batimastat, Ilomastat or captopril significantly impaired in vitro

adipogenic conversion (Bouloumie et al., 2001; Croissandeau et al., 2002).

Contradictory to in vitro oberservations, several in vivo studies indicated that when examined at 14-d post-natal, the adipose tissue weight of MMP-2 and

MMP-9 null mice are similar to wild type (Colnot et al., 2003; Itoh et al., 1997;

Kato et al., 2001; Larsen et al., 2003). However, Van Hul and Lijnen recently reported that compared with wild type littermates (MMP-2+/+ mice), MMP-2-/- mice gain less body weight and have less adipose tissue on a high-fat diet (HFD). This difference can attributed to the adipocyte hypotrophy noted in MMP-2-/- mice, which suggested that MMP-2 may contribute to adipose tissue development by enhancing adipocyte volume on a HFD (Van Hul and Lijnen, 2008). MT1-

MMP/MMP-14 plays on important role in MMP-2 activation by cleaving the MMP-

2 prodomain. In vivo study indicates that MT1-MMP is required for WAT development (Chun et al., 2006).

Obesity and Inflammation

There are extensive data which support the concept that metabolism and

immunity function in a highly integrated manner. Starvation and malnutrition can

50 cause immunosuppression and increase susceptibility to infection, whereas

obesity leads to development of immunoactivation-linked diseases, such as

diabetes and atherosclerosis. A key player in the relationship of inflammation and metabolism are macrophages. Adipose tissue consists of adipocytes and many other cell types including macrophages. The adipose depots of obese humans and mice are infiltrated with a high number of macrophages (Cancello et al.,

2005; Curat et al., 2006; Weisberg et al., 2003; Xu et al., 2003). In many ways the biological characteristics and functions of macrophages and adipocytes are highly overlapped. Macrophages can accumulate lipid and convert to foam cells in some atherosclerotic lesions (Auwerx, 1991). In addition, macrophages express “adipocyte marker genes” such as FABP4 and PPARγ (Makowski et al.,

2001; Tontonoz et al., 1998). On the other hand, proliferating 3T3-L1 preadipocytes show phagocytotic ability and are positive for MOMA-2, a monocyte-macrophage lineage marker (Cousin et al., 1999). When primary mouse preadipocytes or 3T3-L1 cells are injected into the peritoneal cavity of nude mice, the cells develop phagocytic activity and express several macrophage-specific antigens (Charriere et al., 2003). Adipocytes also express classic “macrophage genes” including TNFα, IL-6 and also MMPs (Bouloumie et al., 2001; Hotamisligil et al., 1993; Makowski et al., 2001; Tontonoz et al., 1998).

TNFα is a highly studied cytokine and adipokine secreted by adipocytes and which plays a pivotal role in obesity related insulin resistance and diabetes. IL-6 deficient mice develop obesity (Wallenius et al., 2002). A number of other

51 adipokines including leptin, adiponectin and resistin participate in the immune response.

52 MANUSCRIPT # 1

Wdnm1-like, a New Adipokine with a Role in MMP-2 Activation

Yu Wu and Cynthia M. Smas

Am J Physiol Endocrinol Metab. 2008 Jul;295(1):E205-15. Epub 2008 May 20.

Department of Biochemistry and Cancer Biology and Center for Diabetes and Endocrine Research University of Toledo Health Science Campus Toledo, OH 43614, USA

Running Title: Novel Adipokine Wdnm1-like

Please address correspondence to:

Cynthia M. Smas, D.Sc. Department of Biochemistry and Cancer Biology and Center for Diabetes and Endocrine Research University of Toledo Health Science Campus Toledo, OH 43614, USA Phone: 419-383-4131 FAX: 419-383-6228 e-mail: [email protected]

53 ABSTRACT

White adipose tissue (WAT) functions in energy storage and as an endocrine

organ. DNA microarray analysis led us to identify Wdnm1-like, a distant member

of the whey acidic protein/four-disulfide core (WAP/4-DSC) family, as a

differentiation-dependent gene in white and brown adipogenesis. Wdnm1-like is

a novel 6.8 kDa protein and Western blot analysis reveals secretion into culture

media. Wdnm1-like transcript is selectively expressed in adipose tissue and liver

and is enriched ~500-fold in white adipose depots vs. brown. Cellular fractionation of WAT demonstrates Wdnm1-like expression is restricted to the adipocyte population. Studies in 3T3-L1 preadipocytes, an in vitro model of white

adipogenesis, indicate Wdnm1-like transcript increases within 6 h of adipogenic induction and an ~17,000 fold increase by day 7. Dramatic upregulation of

Wdnm1-like also accompanies white adipogenesis of ScAP-23 preadipocytes and primary preadipocytes. TNF treatment of 3T3-L1 adipocytes increased

Wdnm1-like transcript level 2.4-fold and was attenuated by pretreatment with the p38 MAP kinase inhibitor SB203580. A number of WAP/ 4-DSC family proteins function as protease inhibitors. This, taken with the role of extracellular remodeling in adipogenesis, led us to address effects of Wdnm1-like on matrix metalloproteinase (MMP) activity. Gelatin zymography of HT1080 fibrosarcoma cells transfected with a Wdnm1-like expression construct revealed markedly increased levels of active MMP-2. Our findings identify a new member of the adipocyte “secretome” that functions to enhance MMP-2 activity. We postulate that Wdnm1-like may play roles in remodeling of the extracellular milieu in

54 adipogenesis as well as in tumor microenvironments where adipocytes are key stromal components.

55 KEY WORDS: Adipocyte, adipogenesis, four disulfide core, secreted factor, adipose tissue, extracellular matrix.

56 INTRODUCTION

The adipocytes of white adipose tissue (WAT) are the major site for storage of

excess energy and these triglyceride stores are mobilized to meet the energy

needs of the organism. Adipose tissue is now also recognized as an endocrine

organ with synthesis and secretion of a variety of soluble factors, many of which

are adipocyte-derived, including leptin, resistin, adiponectin, retinol binding

protein-4 and TNFα and other cytokines (Ailhaud, 2006; Scherer, 2006; Trujillo

and Scherer, 2006). Adipocyte secreted factors function in a variety of

processes including systemic energy balance, inflammation, and adipogenesis

and/or adipose tissue development. Clearly, a full characterization of the

adipocyte "secretome" (Trayhurn and Wood, 2004) will greatly facilitate our

understanding of communication between the fat cell and the extracellular

environment and may provide inroads for the control of the detrimental effects of

excess adipose mass.

Adipocytes make up from one-third to two-thirds of the cell population found in adipose tissue. The remaining cells, collectively referred to as the

stromal-vascular fraction, includes endothelial cells, nerve cells, macrophages,

fibroblast-like interstitial cells, preadipocytes, and likely other as yet to be identified cell types (Ailhaud et al., 1992). In adipose tissue development mature adipocytes arise from the differentiation of preadipocyte precursors present in adipose tissue, in a process that occurs throughout the lifespan (Ailhaud, 2006;

Gregoire, 2001a; MacDougald and Mandrup, 2002; Rosen and MacDougald,

2006). For the past decades, in vitro preadipocyte cell lines such as 3T3-L1

57 (Green and Kehinde, 1975b) have been used extensively to define genes central

to the adipocyte phenotype (Ross et al., 2002; Soukas et al., 2001b).

Adipogenesis is accompanied by upregulation of genes that encode molecules

central to adipose tissue function including those critical in lipogenesis, lipolysis, lipid transport, and hormone signaling (Gregoire, 2001a; Gregoire et al., 1998b).

A variety of in vitro and in vivo studies have determined that the peroxisome

proliferator-activated receptor γ (PPAR γ), a member of the ligand-activated

steroid hormone receptor family, is a master transcriptional regulator of the

adipogenic program (Lazar, 2005; Mueller et al., 2002a; Rosen, 2005; Rosen and

MacDougald, 2006). Studies have also illustrated the important contribution of

the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators

and other transcriptional signals to adipogenesis (Farmer, 2006; Rosen, 2005;

Rosen and MacDougald, 2006). In addition to nuclear signals, signals from the

extracellular environment, such as those involved in extracellular matrix (ECM)

remodeling, are important in adipocyte differentiation and adipose tissue

development (Gregoire et al., 1998b; Sorisky, 1999).

During an ongoing study aimed at uncovering new gene expression

patterns in brown adipogenesis, we identified a novel and to date

uncharacterized secreted factor, Wdnm1-like, as a differentiation-dependent

adipocyte-enriched transcript. Although initially found based on its upregulation

during in vitro brown adipogenesis, we have discovered that Wdnm1-like is

expressed ~500 fold higher in WAT than in BAT. Given their potential to

adipose tissue function and systemic energy balance, there is keen interest in the

58 characterization of novel adipocyte secreted factors. Such factors may also have

roles in the stromal microenvironment of tumor cells, wherein adipocytes

frequently occur. In this study we have characterized the expression, regulation,

and function of this novel secreted factor and demonstrate a role for Wdnm1-like

in enhancing matrix metalloproteinase-2 (MMP-2) activity.

MATERIALS AND METHODS

Adipocyte Differentiation and Cell Culture Treatments

3T3-L1 cells (American Type Culture Collection, Manassas, VA) were

propagated in DMEM containing 10% calf serum. For adipocyte differentiation,

3T3-L1 cells were treated at two days post-confluence with DMEM containing

10% FBS in the presence of the adipogenic inducers 0.5 mM

methylisobutylxanthine (MIX) and 1 μM dexamethasone (Dex) for 48 h.

Adipogenic agents were then removed, and growth of cultures continued in

DMEM containing 10% FBS. For treatment of 3T3-L1 adipocytes with TNFα and

various pharmacological inhibitors, following serum-starvation for 6 h, 3T3-L1

adipocytes were pretreated with either 20 M SB203580, 100 nM wortmannin, 1

μM rapamycin (Sigma-Aldrich, St. Louis, MO), or DMSO vehicle for 1 h and stimulated with 10 ng/ml of TNFα for 16 h in the presence of the indicated inhibitors, or incubated with inhibitors only for the same time period.

ScAP-23 cells were maintained in DMEM containing 10% calf serum and passaged prior to reaching confluence. For adipocyte differentiation, preconfluent ScAP-23 preadipocytes were cultured in DMEM containing 10%

59 FBS in the presence of the adipogenic inducers 0.5 mM MIX, 1 μM Dex, 17 nM

insulin, and 0.2 mM indomethacin for 70 h. These agents were then removed and cultures were maintained in DMEM containing 10% FBS and 17 nM insulin for an additional 4 days.

For culture and differentiation of primary white adipocytes, WAT was collected from C57BL/6 mice and digested with 1 mg/ml of type I collagenase for

40 min with shaking at 37C. Following digestion, material was filtered through a

300 micron pore size nylon mesh (Sefar America Inc., Depew, NY) and filtrate

centrifuged at 2,000 rpm for 5 min. Floating adipocyte fraction was removed and

the pellet of stromal-vascular cells was resuspended in DMEM containing 10%

FBS and plated. Upon confluence cells were either harvested as preadipocytes

or cultured in differentiation media consisting of DMEM containing 10% FBS, 0.1

M Dex, 0.25 mM MIX, and 17 nM insulin for three days, at which time

differentiation media was removed and cultures were maintained in DMEM

containing 10% FBS and 17 nM insulin for an additional 4 days.

For differentiation of the brown preadipocyte cell line, referred to herein as

WT-BAT and obtained from C.R. Kahn (Joslin Diabetes Foundation, Harvard

Medical School, Boston, MA), the method was as previously described (Kim et al.,

2006a; Klein et al., 2002b). Briefly, cells were cultured to confluence in DMEM

containing 10% FBS, 20 nM insulin and 1 nM triiodotyronine (differentiation

medium per Kahn and colleagues (Klein et al., 2002b)). Confluent cells were

incubated in this medium supplemented with 0.5 mM MIX, 0.5 μM Dex, and

60 0.125 mM indomethacin for 48 h at which point cultures switched to

differentiation medium for an additional 5 days.

RAW264.7 murine macrophages were plated at a density of 4106 cells/

well of a 6-well plate in DMEM with 10% FBS media (growth media) and 24 h

post-plating, media was changed to growth media with or without100 ng/ml LPS.

After a 4 h incubation, cells were harvested for total RNA preparation.

RNA Preparation, Northern Blot Analysis, and Real-Time PCR

RNA was purified using TriZol Reagent (Invitrogen Corp.) according to

manufacturer’s instruction. For studies of Wdnm1-like expression in murine

tissues, 8 wk old C57BL/6 or ob/ob male mice were utilized, with all animal

treatments conducted with approval of the University of Toledo Health Science

Campus Institutional Animal Care and Use Committee. Fractionation of whole adipose tissue into adipocyte and stromal-vascular fraction was via collagenase digestion and differential centrifugation, as previously described (Kim et al.,

2006a). For Northern blot analysis, 5 μg of RNA was fractionated in 1% agarose- formaldehyde gels in MOPS buffer and transferred to Hybond-N membrane (GE

Healthcare, Piscataway, NJ). Blots were hybridized in ExpressHyb solution (BD

Biosciences Clontech, Palo Alto, CA) with the indicated 32P-labeled murine cDNA

probes. After washing, membranes were exposed at -80C to Kodak Biomax film with a Kodak Biomax intensifying screen. All lanes shown in a single image were run on the same blot, however in some instances lanes were rearranged or removed for economy and/or clarity of presentation. 61 For real-time PCR analysis, total RNA was subject to purification with an

RNeasy RNA purification kit with DNase I treatment (Qiagen Corp., Valencia, CA) for elimination of all sources of DNA and 5 g used for first strand cDNA synthesis with SuperScript II RNase H-reverse transcriptase (Invitrogen Corp.) and an oligo(dT)-22 primer. Real-time PCR was conducted with an ABI 7500

Real-time PCR System. Target cDNA levels were analyzed by SYBR green- based real-time PCR in 25 l reactions containing 1X SYBR Green PCR Master

Mix (Applied Biosystems, Foster City, CA), 100 nM each forward and reverse primers, and 10 ng of cDNA. PCR was carried out over 40 cycles of 95°C 15 sec,

60°C for 30 sec, and 72°C for 40 s with initial cycle of 50°C for 2 min and 95°C for 10 min to activate AmpliTaq Gold DNA polymerase. Sequences of the real- time PCR primers used in this study, their annealing positions to the respective

GenBank RefSeq sequences, the PCR product sizes, and intron information are provided in Supplemental Table I. Primers were designed using Accelrys DS

Gene software and whenever possible amplify from two different exons. In the few cases wherein primers could not be designed in two separate exons, we conducted reactions minus reverse transcriptase control reactions. For these, we have never observed any PCR product signal only an extremely low degree of background signal the same as that for water only reactions. After the 40th cycle, a dissociation curve over the range of 60 to 95oC was generated for each

PCR reaction with a sharp melting peak, indicating a single unique species of

PCR product, was observed in every case. Expression of each gene was

normalized against Gapdh transcript level. In all cases the same amount of input 62 RNA/cDNA was used in side-by-side comparisons and in most cases the Gapdh

level for the samples under comparison differed by 1or fewer cycles and in no

case was the difference greater than 2 cycles. The cycle threshold value was

generated using ABI PRISM 7500 SDS software version 1.2 and exported to an

Excel spreadsheet to calculate fold differences. In samples where transcript

expression was not evident after 40 PCR cycles, a value of 40 cycles was

assigned in order to calculate a delta Ct value and estimate fold differences.

Such instances are noted in the respective figure legend. Analyses were performed in triplicate.

Affymetrix DNA Array Studies

Affymetrix 430A Gene Chips were hybridized with material prepared from

5 μg of total RNA. Duplicate RNA samples from brown preadipocytes or brown

adipocytes at 7 days post-induction of differentiation were used for transcriptional

profiling studies. The Keck Microarray Facility at Yale University (New Haven CT)

carried out processing of DNA arrays according to standard protocols. An Excel

file containing the complete set of fold-changes generated using the R program

in the Affymetrix affylmGUI package and employing robust multiarray averaging

is provided as supplemental information.

Preparation of a Murine Wdnm1-like Expression Construct

For preparation of a murine Wdnm1-like expression construct, termed

Wdnm1-like-HA, in which a C-terminal HA epitope tag was fused in-frame to the

63 Wdnm1-like coding sequence, a full-length sequence-verified mouse Wdnm1-like

cDNA I.M.A.G.E clone (American Type Culture Collection) was used as template

for PCR-based cloning. Primers sequences fore PCR cloning were: 5' PCR

primer (5'- GCCGAATTCCTCGAGTCCTTCAGCAGCAGCATGAAG-3') and the

3' PCR primer (5'-

GGCGATATCGTCGACTTAAAGAGCGTAATCTGGAACATCGTATGGGTAGTTT

GTAGTACAGATGTGACT-3'). A 5' EcoRI site and a 3' EcoRV site were

incorporated into respective primers to facilitate directional cloning into pcDNA3.1

vector (Invitrogen Corp.).

Transfections and Western Blot Analysis of Wdnm1-like Protein Expression

For assessment of secretion of Wdnm1-like into culture media, 293T cells were transfected with the Wdnm1-like-HA expression construct or empty vector using Lipofectamine 2000 (Invitrogen Corp.). Culture media and cell lysates were collected for analysis at 48 h post-transfection. Media was centrifuged at

5000 rpm for 3 min and cells were harvested by lysis in TNN (+) buffer (10 mM

Tris pH8.0, 120 mM NaCl, 0.5% NP-40, 1mM EDTA, supplemented with a protease inhibitor cocktail). Lysates were incubated on ice for 30 min with intermittent vortexing, supernatant collected via centrifugation, and protein content determined (Bio-Rad Laboratories). 1/150 of total media (20 μl of a total

3 ml) or 1/150 of total cell lysate (~6 μg of 900 μg total protein yield) were size- fractionated on 15% SDS-PAGE gels for Western analysis and proteins transferred onto P-seq Immobilon membranes (Millipore Corp). For signal

64 detection, membranes were blocked by a 1 h incubation in 5% nonfat milk/0.5%

Tween 20 in PBS and then incubated with a 1:2000 dilution of anti-HA primary

antibody (Covance Research Products, Princeton NJ) for 1 h followed by three

10 min washes. Secondary antibody was goat anti-mouse at a dilution of 1:2000

for 1 h followed by three 10 min washes. All washes were in 0.5% Tween 20 in

PBS. Signal was detected by ECL Plus enhanced chemiluminescence (GE

Healthcare) and exposure to X-ray film.

In vitro Translation

A TNT Quick Coupled Transcription/Translation Kit (Promega Corp.) was

employed for in vitro transcription and translation. Reactions programmed with either the Wdnm1-like HA construct or with empty vector were incubated at 30oC for 90 min and 10 μl or 2 μl or of the respective reaction was analyzed on 10%

SDS-PAGE Tricine gels, followed by overnight transfer to Immobilon P-Seq membrane (Millipore Corp.). Membranes were processed for Western blot analysis with anti-HA antibody as described in the section “Transfections and

Western Blot Analysis of Wdnm1-like Protein Expression”.

Gelatin Zymography Assay

5 HT1080 cells were plated at a density of 6.25 x 10 per 60 mm dish. The

day after plating cells were transfected with the Wdnm1-like-HA expression

construct or empty pCDNA3.1 vector using Lipofectamine and Plus Reagent

(Invitrogen Corp.). At 4 h post-transfection, media was removed and 2.5 ml of 65 serum-free media added. Media was harvested at 48 h after transfection,

centrifuged for 3 min at 5000 rpm to remove cell debris, and used fresh for

zymography. For zymograph analysis (Hawkes et al., 2001), 15 μl media was

mixed with 7.5 μl of 3X sample buffer (125 mm Tris-HCl pH 6.8, 8% SDS, 50%

glycerol, 0.02% Coomassie blue) and gels lanes loaded without prior heat

denaturation of samples. Samples were fractionated in 10% SDS-PAGE

zymograph gels that contained 0.1% (w/v) gelatin. After electrophoresis, gels

were treated for renaturation by incubation in 2.5% Triton X-100 for 30 min at

room temperature and then transferred to development solution (0.05 M Tris-HCl

pH 8.8, 5 mM CaCl2, 0.02% NaN3) and incubated overnight at 37C. Gels were

incubated in staining solution (40% methanol, 10% acetic acid, 0.5% Coomassie blue) for 4 h and destained in fixing/destaining solution (40% methanol, 10%

acetic acid) until bands of gelatinase activity were clearly visible. For each

experiment, transfections and assays were carried out on a minimum of duplicate

independent samples and the overall study from transfection through

zymography was conducted three times. Data was documented with digital

photography and representative data shown.

RESULTS

Wdnm1-like is a Novel Secreted Protein of Adipocytes

We initially identified upregulation of murine Wdnm1-like in adipogenesis

during an unrelated DNA microarray study designed to determine the effects of

retrovirally-driven ectopic expression of a novel gene, SMAF1, on brown

66 adipocyte conversion of WT-BAT preadipocytes. For this we had generated an

empty vector control sample set of preadipocytes that had undergone G418 drug

selection for the PLNCX2 retroviral vector and a sample set following their

differentiation to brown adipocytes. To our knowledge, global gene expression

assessment on the adipogenic conversion of WT-BAT cells has not been

previously determined. Kahn and coworkers, the source of the WT-BAT cell line,

however, have reported on the differentiation-dependent expression of similarly

derived brown preadipocyte cell lines which were null for distinct insulin receptor

substrate (IRS) genes (Tseng et al., 2005). In the course of our analyses we

compared gene expression data for brown preadipocytes and after their

differentiation to adipocytes. We found that 173 genes of the ~22,000 on the

array were upregulated 10-fold or greater during brown adipogenesis in this in vitro model. We chose several well-characterized adipocyte genes and 20 other genes that had not yet been assessed in either brown or white adipogenic conversion and validated their respective fold increases upon adipogenesis using real-time PCR. These data are presented in Table I. We note here that in the time since we initially carried out PCR validation of gene expression changes, the adipocyte-related function of several of these genes have been reported. The endoplasmic reticulum oxidoreductase Ero1-like has been demonstrated to regulate secretion of adiponectin (Qiang et al., 2007). Rarres2/chemerin, with a role in adaptive and innate immunity, is produced by adipocytes to regulate adipogenesis and adipocyte metabolism (Bozaoglu et al., 2007; Goralski et al.,

2007). Thus additional examination of these microarray data, with the complete

67 data set provided as supplementary information, is likely to yield further insights

into important factors in adipogenesis.

Of the novel genes identified in our microarray comparisons, one caught

our particular attention because: 1.) It increased ~300 fold during brown

adipogenesis, thus making it the eleventh-most upregulated gene on the array;

and 2.) Its protein sequence suggested it encoded a novel small secreted factor.

This gene was designated as expressed sequence tag (EST) 1100001G20Rik on the microarray. BLAST searches determined that this EST was the murine homolog of the rat Wdnm1-like gene. The single publication to date on Wdnm1- like describes its identification during a SAGE analysis study of gene expression, with enrichment of Wdnm1-like transcript reported in the stem-cell rich limbal region vs. the central corneal epithelium in rats (Adachi et al., 2006). These authors termed this new gene Wdnm1-like based on what they regarded as similarity in protein sequences to a second largely uncharacterized rat gene,

Wdnm1 (Adachi et al., 2006). Wdnm1, named for West-mead DMBA8 non- metastatic cDNA clone, also termed Expi, was initially identified as a transcript

whose expression was decreased in a non-metastatic clone of the mammary

adenocarcinoma cell line DMBA-8 vs. expression in a metastatic clone (Dear et

al., 1988). As described below, Wdnm1-like and Wdnm1 are members of the

whey acidic protein/4-disulfide core (WAP/4-DSC) protein family.

We obtained and sequenced-verified a cDNA I.M.A.G.E clone for murine

Wdnm1-like and as shown in Figure 1A, the transcript is comprised of 434 bases

and encodes a protein of 63 amino acids with a calculated molecular mass of

68 6,792. Start and stop codons are present at nucleotides 88 and 279, respectively.

As depicted in Figure 1B, a C-terminal region of murine Wdnm1-like has a limited

degree of similarity with the WAP/4-DSC motif (Bouchard et al., 2006), ProDom database designation PD026912 (Servant et al., 2002). The consensus WAP/4-

DSC motif consensus sequence consists of 8 cysteines (numbered 1-8) in the order of C1- (Xn) - C2- (Xn) - C3 (X5) - C4- (X5) - C5,C6 - (X3-5) - C7 - (X3-4) -

C8, where X is any amino acid and n represents any number of residues

(Simpson and Nicholas, 2002). Multiple members of this protein family have been identified in humans. As shown in Figure 1B, Wdnm1-like has conservation of 6 of the 8 cysteines comprising the WAP/4-DSC motif, as well as conservation of other residues found in this motif. The remaining region of the Wdnm1-like protein sequence lacks obvious homologies with other proteins.

The murine Wdnm1-like gene spans 5.7 Kb and consists of three exons

(Figure 1C) with the first intron separating theWAP/4-DSC type motif region of the Wdnm1-like from the unique N-terminal half of the protein. While nearly all genes encoding WAP/4-DSC proteins are clustered on murine 2 corresponding to human 20q12-20q13.1 (Bouchard et al., 2006), Wdnm1-like is present on murine chromosome 11. It is located in a region between a gene cluster for five chemokines including Ccl4 and that for the hypothetical protein

Heatr6. Interestingly, an ~45 Kb region of murine chromosome 11 includes genes for Wdnm1-like, Wdnm1/Expi and for a third novel WAP/4-DSC protein with similarity to Wdnm1, designated NP001075426. This region is depicted in

Figure 1D. Wdnm1 transcript is induced upon mammary gland involution and is 69 occasionally used as a molecular marker in studies of mammary gland biology

(Baik et al., 1998). Although Wdnm1 has been postulated as a tumor suppressor gene (Steeg, 1989), its biology has not been well studied and its function is unknown. No information is available regarding expression of the gene for

NP001075426. The sequence for these three related proteins is shown in Figure

1E. As this alignment illustrates, Wdnm1-like has limited

(~30% identity) with either Wdnm1/Expi or NP001075426, whereas a high degree sequence homology (~70% identity) is evident between Wdnm1/Expi and

NP001075426. As previously stated, Wdnm1-like lacks two of the eight cysteines that comprise a singleWAP/4-DSC motif (shown by upward arrows in

Figure 1E), whereas both Wdnm1/Expi (Dear and Kefford, 1991) and

NP001075426 have all eight (bolded).

Wdnm1-like Encodes a Novel 6.7 kDa Secreted Protein

To begin studies on the Wdnm1-like protein, we assessed the size of the

Wdnm1-like primary translation product with coupled in vitro transcription and translation using the Wdnm1-like-HA construct as template; the HA tag adds an additional ~1 kDa to the protein mass. Western analysis in Figure 2A shows that lysate programmed with this template resulted in a major protein species migrating at ~8 kDa, in good agreement with the mass predicted from the primary amino acid sequence of Wdnm1-like. Kyte-Doolittle hydrophobicity analysis indicated that the N-terminus of Wdnm1-like was enriched in hydrophobic residues, consistent with the presence of a signal sequence (Figure 2B). To

70 determine if Wdnm1-like protein was a secreted factor, the Wdnm1-like-HA

construct or empty vector control was transiently transfected into 293T cells and

media or cell lysates harvested at 24 h post-transfection. The Western blot in

Figure 2C, wherein a similar percentage of the total volume collected of cell

lysate or media was analyzed, indicates that the majority of Wdnm1-like protein

is in the culture media. That the molecular mass found for the Wdnm1-like

expressed in mammalian cells appears the same as that of the Wdnm1-like

primary translation product (first two lanes of Figure 2A) indicates that the protein

does not undergo the types of post-translational modifications, such as

glycosylation, that would be predicted to result in measurable alterations in

protein mass.

Wdnm1-like Transcript Expression is Restricted to Adipose Tissue and

Liver

To garner further information on the in vivo tissue expression pattern of

Wdnm1-like we used real-time PCR analysis of multiple murine tissues. Figure

3A indicates that the Wdnm1-like transcript is highly restricted in expression to

WAT and liver; it is present in these tissues at levels several hundred times higher than in the other tissues examined. We next compared Wdnm1-like transcript level in brown adipose tissue (BAT, B) and three distinct WAT depots, subcutaneous (SC, S), epididymal (EP, E) and retroperitoneal (RP, R) (Figure 3B, leftmost panel), and determined that it is expressed in SC WAT at levels ~500 times higher than that detected in BAT. It is also more than 100-fold higher in EP

71 and RP WAT relative to BAT. In this regard, Wdnm1-like expression is similar to

that for resistin (Retn, middle panel of Figure 3B), in contrast to stearoyl co-A

desaturase 1 (SCD1) and adipocyte fatty acid binding protein (aFABP),wherein

more uniform expression across the adipose depots are noted (Figure 3B,

rightmost panel).

To determine which cellular fraction of adipose tissue expresses Wdnm1-

like transcript, murine WAT was separated into the non-adipocyte stromal

vascular fraction and the adipocyte-containing fraction. Real-time PCR analysis

indicates that Wdnm1-like transcript expression is highly enriched in the

adipocyte fraction (Figure 3C, leftmost panel), similar to the adipocyte marker transcript SCD1 (Figure 3C, middle panel), and in contrast to the SVF enrichment

of collagen 1A1 (Figure 3C, rightmost panel). We also compared the levels of

Wdnm1-like transcript in wild type mice versus the ob/ob genetic model of murine

obesity. Figure 3D shows a 1.5-fold increased transcript expression in the EP

WAT and a 3.3-fold increase in BAT. In liver, ob/ob levels of Wdnm1-like

transcript are decreased to 60% of that found for wild type animals.

Given the close chromosomal location of Wdnm1-like with Wdnm1/Expi

and NP001075426, and their sequence similarities, we also determined the

expression of these two transcripts in the same murine tissue panel used in

Figure 3C. We found that Wdnm1 was highly enriched in salivary gland, present

at levels ~2000-fold higher than in any other of the tissues examined (data not

shown). Wdhm1 is often used as a molecular marker in studies of the mammary

gland; we do not know at this time the relative expression level in mammary vs.

72 salivary gland for Wdnm1 transcript. The level for transcript for NP001075426

was nearly undetectable in the tissues examined, precluding an accurate

assessment of differential expression (data not shown).

Differentiation-Dependent Upregulation of Wdnm1-like in White

Adipogenesis

The above data indicates that Wdnm1-like is highly enriched in adipocytes

of WAT. We next conducted Northern blot analysis to examine the expression of

Wdnm1-like during a daily time course of in vitro adipogenesis of 3T3-L1

preadipocytes to white adipocytes. The 3T3-L1 cell line is a highly characterized

and frequently utilized cell culture model of adipogenic conversion wherein

fibroblastic 3T3-L1 preadipocytes convert to mature lipid-laden adipocytes

following a two day treatment with an adipogenic induction cocktail comprised of

FBS, Dex and MIX. The Northern blot analysis in Figure 4A shows that the

Wdnm1-like transcript is first detected very early in adipogenesis, by1 day post- onset of the differentiation program. Its level increased until day 4 and was sustained through day 6. Upregulation of Wdnm1-like transcript is detected earlier than that for two markers of terminal adipocyte differentiation, SCD1 and aFABP, whose expression is first detected at day 3 and is not apparent prior to the removal adipogenic differentiation agents. We also utilized real-time PCR to quantitate Wdnm1-like transcript expression in preadipocytes vs. adipocytes for

3T3-L1 cells and in a second model of in vitro white adiopgenesis recently

developed in this laboratory, ScAP-23. ScAP-23 is an immortalized preadipocyte

73 cell line derived from preadipocytes present in murine WAT. 3T3-L1 adipocyte conversion is accompanied by an ~17,000 fold increase in Wdnm1-like transcript level and ScAP-23 adipogenesis by an ~6000 fold increase in Wdnm1-like like expression (Figure 4B). Figure 4C indicates that Wdnm1-like is also upregulated during in vitro differentiation of primary murine cultures of the preadipocyte- containing stromal-vascular cell population of adipose tissue (left panel). The right panel of Figure 4C confirms effective adipose conversion of the primary cultures, with increased transcript levels for the adipocyte markers aFABP and

SCD1. Given that the Northern blot analysis in Figure 4A indicated upregulation of Wdnm1-like transcript by day 1 post-induction of differentiation, we conducted a second time course study focusing on the first two days of adipogenesis.

Figure 4D illustrates that increased levels of Wdnm1-like transcript occur within 6 h of treatment with Dex and MIX, and that this reaches 78-fold by 12 h and

~2300-fold by 24 h. This dramatic early upregulation is in contrast to the relatively minor changes noted for FABP and SCD1 transcripts. On the other hand, increased levels of transcript of PPARγ2 are found within 6-h post induction and levels continue to increase through 48 h. That this was not discernable in the Northern blot in Figure 4A may be perhaps due the fact that the Northern analysis detects both the γ1 and γ2 forms of PPAR It remains to be determined whether the coincident increase in PPARγ2 and Wdnm1-like transcript in early adipogenesis indicates possible PPARγ2-mediated transcriptional regulation of Wdnm1-like.

74 Regulation of Adipocyte Wdnm1-like Transcript Expression

To investigate signals that might regulate Wdnm1-like transcript level in adipocytes, we first individually tested the effects of the components of the standard adipogenic cocktail on expression of Wdnm1-like transcript. We find that only Dex and MIX in combination results in increased Wdnm1-like transcript levels (Figure 5A). This is first evident at day 1 and is readily apparent at day 2 and day 5. The finding that neither individual component of the adipogenic differentiation cocktail leads to detectable levels of Wdnm1-like transcript further serves to tie expression of this gene to the early events of adipogenesis.

We next treated 3T3-L1 adipocytes with TNFα, a cytokine that impacts adipocyte transcriptional downregulation of the key adipocyte transcription factors PPARγ (Xing et al., 1997) and C/EBPα(Jain et al., 1999a; Stephens and

Pekala, 1992). We find that TNFα treatment of 3T3-L1 adipocytes increases the level of Wdnm1-like transcript by 2.4-fold (p<0.001) (Figure 5B). While neither the PI 3-kinase inhibitor wortmannin nor the mTOR inhibitor rapamycin had an inhibitory effect, pretreatment with the p38 MAP kinase inhibitor SB203580 blocked TNFα -induced upregulation of the Wdnm1-like transcript (Figure 5B).

This consistent with several recent reports that the effects of TNFα on cell types such as osteoblasts, monocytes, and human umbilical vascular endothelial cells can be mediated via p38 MAP kinase and with the limited reports on the role of p38 MAP kinase in the effects of TNFα on adipocyte gene expression (Pandey et al., 2005; Trujillo et al., 2006). The expression pattern of Wdnm1-like in murine tissues, with restriction to WAT and liver, and its upregulation by TNFα, is

75 somewhat reminiscent of that for several acute phase reactants that are secreted

products of adipocytes such as haptoglobin and serum amyloid A3 (Friedrichs et

al., 1995; Sjoholm et al., 2005; Trayhurn and Wood, 2004). We also addressed

whether LPS, a pro-inflammatory agent which acts in macrophage activation

could regulate levels of Wdnm1-like transcript. Figure 5C shows that a 4 h

treatment of RAW264.7 murine macrophages results in a robust upregulation of

Wdnm1-like transcript, suggestive of a possible role for Wdnm1-like in the

inflammatory response.

Ectopic Expression of Wdnm1-like Leads to Increased Levels of Active

MMP-2

Several proteins of the WAP/4-DSC family, for example elafin and

secretory leukocyte proteinase inhibitor (SLPI) (Devoogdt et al., 2004), have demonstrated roles as proteinase inhibitors (Bouchard et al., 2006) with wide ranging impact on physiology. Given that Wdnm1-like possesses only a single

WAP/4-DSC type motif wherein only 6 of the 8 cysteines are present, it appears

to be only distantly related to other WAP/4-DSC proteins. We nonetheless

hypothesized that perhaps it might have a similar proteinase inhibition function.

An important class of extracellular proteases that function in adipocyte

differention and adipose tissue are the matrix metalloproteinases (MMPs)

(Bouloumie et al., 2001; Bourlier et al., 2005; Chavey et al., 2003; Chun et al.,

2006; Croissandeau et al., 2002; Demeulemeester et al., 2005; Lijnen et al.,

2002a). MMPs play key roles in governing cell-matrix interactions by their ability

76 to degrade ECM components. This in turn has multiple cellular consequences, such as for example, the release of growth factors from the ECM (Green and

Lund, 2005). We therefore hypothesized that Wdnm1-like might affect MMP activity. To test this we utilized HT1080 human fibrosarcoma cells, which are commonly used in MMP functional studies as they produce readily detectable levels of the gelatinases MMP-2 and MMP-9. The Wdnm1-like-HA expression construct or an empty vector control plasmid was transiently transfected into

HT1080 cells and media collected for analysis at 48 h post-transfection. Figure

6A demonstrates effective ectopic expression of Wdnm1-like transcript by real- time PCR (left panel) and by Western blot analysis of culture media for Wdnm1- like protein using anti-HA antibody (right panel). The lower portion of Figure 6B shows the result of gelatin zymography analysis of duplicate cultures of HT1080 cells transfected with either empty vector control or the Wdnm1-like-HA expression construct. These data indicate that expression of Wdnm1-like leads to increased levels of the intermediate and active forms of MMP-2 and a reduction in the level of pro-MMP-2. No alterations in MMP-9 activities were noted (Figure 6B, upper portion). We used real-time PCR to assess transcript level for MMP-2 and MT1-MMP( also known as MMP-14), the latter is a key regulator of MMP-2 activation via its action to remove the MMP-2 pro-peptide.

While a degree of variation is found across the experimental replicates, these data indicate no appreciable alteration in the level of neither MMP-2 nor MT1-

MMP/MMP14 transcript as a result of Wdnm1-like expression. This suggests that Wdnm1-like does not cause elevated MMP-2 activity via enhanced

77 expression of the MMP-2 or MT1-MMP/MMP14 gene, but rather by an as yet

undefined novel mechanism.

Discussion

The matrix metalloproteinase systems are key for matrix remodeling that

accompanies many developmental and physiological processes, including

adipogenesis and obesity (Alvarez-Llamas et al., 2007; Klimcakova et al., 2007;

Trayhurn and Wood, 2004). Adipose tissue growth occurs by both hyperplasia

and hypertrophy and these events involve remodeling of the cellular and

extracellular environment. The robust upregulation of Wdnm1-like in early

adipogenesis is consistent with Wdnm1-like having a regulatory role in the

process. Our functional studies of Wdnm1-like indicates that one way in which

this novel adipokine may exert effects in a paracrine or autocrine manner in

adipose tissue/adipocytes is via an impact on MMP-2 activity. Adipose tissue in

vivo and adipocytes from in vivo and in vitro sources produce a number of MMPs

(Bouloumie et al., 2001; Bourlier et al., 2005; Chavey et al., 2003; Chun et al.,

2006; Croissandeau et al., 2002; Demeulemeester et al., 2005; Lijnen et al.,

2002a). While a comprehensive assessment of expression and the effects of all

members of the MMP family has not been done in adipogenesis/adipose tissue,

the level of either activity, protein, or transcript for a number of MMPs has been demonstrated to be altered during adipogenesis (Bouloumie et al., 2001; Bourlier et al., 2005; Chavey et al., 2003; Chun et al., 2006; Croissandeau et al., 2002;

Demeulemeester et al., 2005; Lijnen et al., 2002a). While it is generally regarded

78 that the obese state is accompanied by an overall shift to increased matrix

degradation (Chavey et al., 2003; Lijnen et al., 2001; Maquoi et al., 2002)

knockout of individual MMPs have also been found to promote a diet-induced obesity phenotype (Lijnen et al., 2002b; Pendas et al., 2004). For example,

MMP-11/stromelysin-3 (Lijnen et al., 2002b) and MMP-19 (Pendas et al., 2004) null mice are prone to increased levels of nutritionally induced obesity, with effects on adipogenesis and fat cell hypertrophy, respectively. Studies in vitro with broad spectrum and specific MMP inhibitors as well as blocking antibodies have demonstrated that blocking MMP activities inhibits adipogenesis

(Bouloumie et al., 2001; Bourlier et al., 2005; Chavey et al., 2003; Christiaens and Lijnen, 2006; Croissandeau et al., 2002; Maquoi et al., 2002). In regard to the gelatinases MMP-2 and MMP-9, their expression has been reported to increase during adipogenesis and they have been shown to be important for adipogenic conversion in vitro (Bouloumie et al., 2001; Bourlier et al., 2005;

Chavey et al., 2003). In vivo studies show that MMP inhibitors diminish adipose tissue accumulation (Demeulemeester et al., 2005; Lijnen et al., 2002a).

However, no obvious alterations in adipocyte cellularity were observed for mice null for MMP-2, MMP-3, or MMP-9, suggesting in vitro observations may not hold in the in vivo setting. However, to our knowledge, the response of such animals to dietary obesity or other manipulation was not examined (Chun et al., 2006). In regard to the function we describe for Wdnm1-like in the activation of MMP-2,

MT1-MMP/MMP14 has a major role in the activation of MMP-2 and has been demonstrated to be key adipose tissue development in vivo. Mice null for this

79 membrane-anchored collagenase were observed to have poorly developed adipose tissue (Chun et al., 2006). Thus Wdnm1-like, with its early upregulation in adipogenesis, may be important for initial ECM remodeling events that would favor adipogenesis; given its sustained expression in adipocytes may also be important in adipocyte-ECM interactions.

The global transcriptional response of 3T3-L1 adipocytes and adipose tissue to TNFα treatment was studied by Lodish and coworkers who reported suppression of many adipocyte genes and the activation of expression of many preadipocyte genes (Ruan et al., 2002a; Ruan et al., 2002b). Given this, one might make the assumption that TNFα would likewise decrease expression of

Wdnm1-like transcript in 3T3-L1 adipocytes; in contrast we observed upregulation. A common theme that emerged from the Lodish report was the central role of TNFα mediated gene regulation in the promotion of insulin resistance via downregulation of adipocyte genes that are key for the storage or uptake of free fatty acids or glucose (Ruan et al., 2002a; Ruan et al., 2002b).

Many of these encoded proteins function in the promotion or attenuation of lipolysis and lipogenesis, respectively (Ruan et al., 2002a; Ruan et al., 2002b).

Thus, one would not necessarily expect a diminution of adipocyte Wdnm1-like transcript expression upon TNFα treatment, as there is no evidence that Wdnm1- like has a role in insulin responsiveness or energy metabolism. Moreover, while a large percentage of adipocyte and preadipocyte genes are coordinately decreased or increased upon TNFα treatment of adipocytes, respectively, this is clearly not a hard and fast rule. Expression of transcript for the preadipocyte

80 marker gene Pref-1 is not upregulated in TNFα treated 3T3-L1 adipocytes (Xing

et al., 1997) and transcript for adipocyte-expressed haptoglobin increases upon

TNFα treatment of 3T3-L1 adipocytes (Ruan et al., 2002a). We have yet to

define the transcriptional underpinnings of the TNFα upregulation of Wdnm1-like.

However in their study, Lodish and coworkers report that NF-κB activation is

obligatory for the suppression of adipocyte-specific and for the activation of

preadipocyte genes by TNFα (Ruan et al., 2002a). It is possible that TNFα

upregulation of Wdnm1-like in adipocytes does not fall under the domain of this transcription factor. The transcriptional decrease of adipocyte genes by TNFα has also been ascribed to its ability to decrease transcript levels for the key adipogenic transcription factor PPARγ (Xing et al., 1997). While our studies have not directly addressed whether Wdnml-like transcription is controlled by PPARγ, we report herein that upregulation of Wdnm1-like transcript is a very early event in adipogenesis. As such it is possible that PPARγ may not be a key player in the transcriptional signals underlying adipocyte-specific expression of Wdnm1- like. Of interest in regard to the function we report herein for Wdnm1-like is the observation by Lodish and coworkers that a subset of the TNFα-regulated genes encodes proteins involved in extracellular matrix and cell adhesion (Ruan et al.,

2002a). We thus postulate that since Wdnm1-like modulates MMP-2 activity, and perhaps possesses other similar functions as well, increased expression of

Wdnm1-like transcript and the encoded protein may be important in mediating cell-matrix interactions that occur both in early adipogenesis and in the

81 morphological alterations that accompany TNFα treatment-induced lipolysis and

dedifferentiation of mature adipocytes.

Adipocytes and their secreted products are components of the stroma

present in many organs and tissues and they are coming to light as key

contributors to tumor microenvironment (Schaffler et al., 2007). Adipocytes are also present in bone marrow (Gimble et al., 1996), a common site of tumor cell metastasis (Caers et al., 2007; Clarke and Brown, 2007). MMPs are also key in

cancer biology wherein they play important roles in tumor cell metastasis and invasion (Noel et al., 2008), here a central player appears to be MMP-2 and its membrane bound activator MT1-MMP/MMP14 (Cao et al., 2005; Cao et al.,

2008). We find that elevation of Wdnm1-like results in increased levels of active

MMP-2, the latter a key regulator of cancer cell metastasis, leading to the hypothesis of a pro-tumorigenic effect for Wdnm1-like. A number of well-studied

WAP/4-DSC family proteins, namely, elafin, SLPI (also known as antileukoproteinase 1), WFDC1/HE4, and WFDC2/p20 have described roles in cancer growth and progression (Bouchard et al., 2006). Whether Wdnm1-like has a role in cancer remains to be assessed. We postulate that Wdnm1-like is a new important extracellular factor secreted by adipocytes. Wdnm1-like may impact adipose tissue development and/or adipogenesis as well as function as an important link between adipocytes and the promotion of a tumor cell microenvironment conducive to invasion and metastasis. Future studies from this laboratory will address the mechanism(s) underlying the function and regulation of adipocyte-secreted Wdnm1-like.

82 Acknowledgments

We thank Dr. Ji Young Kim for generously providing RNA samples. This work was supported by NIDDK/NIH grant 5R21DK064992 to C.M. Smas.

Disclosures

None

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91 Figure 1. Characteristics of Wdnm1-like protein sequence. A. Sequence of the 434 bp Wdnm1-like transcript and the encoded 63 amino acid protein (bolded) are shown. The italicized region of the protein sequence corresponds to the region of WAP/4-DSC homology. The underlined G in the protein sequence indicates the position of the first intron. B. Alignment of the WAP/4-DSCtype region of the Wdnm1-like protein with a consensus WAP/4-DSC sequence

(ProDom PD026912). C. Intron-exon arrangement of the murine Wdnm1-like gene. White boxes represent untranslated region and black boxes the translated region of the transcript. Thin dashed lines indicate introns. D. Location of the murine gene for Wdnm1-like and two additional WAP/4-DSC type genes on murine chromosome 11. E. Alignment of protein sequence for the three related

WAP/4-DSC type proteins present in a cluster on murine chromosome 11. The canonical cysteines of the WAP/4-DSC motif are shown in bold typeface, those not found in Wdnm1-like are indicated with upward arrows.

Figure 2. Wdnm1-like encodes a novel 6.7 kDa secreted protein. A.

Western blot analysis of in vitro translation products of HA-tagged Wdnm1-like expression construct or empty vector (EV). 10 l or 2 l of a 1:10 dilution of in vitro translation products was subjected to Western blot analysis performed using anti-HA antibody. Positive control (+) is media from Wdnm1-like transfected

HT1080 cells. Molecular mass markers in kDa are shown at right. B.

Hydrophobicity analysis of Wdnm1-like protein sequence. Wdnm1-like protein sequence was subjected to Kyte-Doolittle hydrophobicity analysis using DS Gene

93 increase leptin production in human adipose tissue: role for p38 mitogen-

activated protein kinase. J Clin Endocrinol Metab 91: 1484-1490, 2006.

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insulin receptor substrates and necdin. Nat Cell Biol 7: 601-611, 2005.

64. Xing H, Northrop JP, Grove JR, Kilpatrick KE, Su JL, and Ringold GM.

TNF alpha-mediated inhibition and reversal of adipocyte differentiation is

accompanied by suppressed expression of PPARgamma without effects on Pref-

1 expression. Endocrinology 138: 2776-2783, 1997.

92 2.1 software with a window size setting of 10. The hydrophobicity score is shown

on the y-axis and the amino acid number on the x-axis. C. Western blot analysis

of Wdnm1-like protein expression in cells and culture media. 293T cells were transfected with an HA-tagged Wdnm1-like expression construct or empty vector

(EV) and a portion of the cell lysate or conditioned media (as described in

Materials and Methods) was analyzed by Western blot analysis using an anti-HA antibody. Coomassie blue gel staining (for media) or membrane re-probed with anti-cylcophilin A antibody (for cell lysate) are shown as loading controls. Arrows in A and C indicate signal for Wdnm1-like protein. Lanes comprising the single panels shown in A or C were generated from the same Western blot exposure, however some lanes have been removed and/or rearranged for clarity and/or economy of presentation. Minor adjustments to brightness and/or contrast were utilized for better visualization.

Figure 3. Adipose tissue is a primary site of Wdnm1-like expression in vivo.

A. Real-time PCR assessment of Wdnm1-like transcript level in a panel of murine tissues. Transcript level in muscle tissue was set to a value of 1. * indicates p<0.001 for WAT or liver vs. the other tissue samples. WAT, white adipose tissue; and Sal. gland, salivary gland. B. Expression of Wdnm1-like and selected adipocyte marker transcripts in adipose tissue depots. Brown adipose tissue (B), subcutaneous (S), epididymal (E) and retroperitoneal (R) WAT cDNA were used for real-time PCR analysis for transcript level of Wdnm1-like (left pane), for resistin (Retn, middle panel) or stearoyl-CoA desaturase 1 (SCD1) and

94 adipose fatty acid-binding protein (aFABP, right panel). The transcript level in

BAT was set to a value of 1. * indicates p<0.001 for Wdnm1-like transcript level

in S, E, and R compared to the B depot. C. Cellular fractionation of WAT. cDNA derived from the cells of the stromal vascular fraction (SVF) or adipocyte fraction

(AF) of SC WAT was used for real-time PCR analysis of Wdnm1-like (leftmost panel). Transcript levels of SCD1 (middle panel) and collagen 1A1 (Col1A1, right panel) are shown to validate effective fractionation of WAT. The transcript level in SVF was set as a value of 1 for the left and middle panels and for AF for the right panel. * indicates p<0.001 for AF vs. SVF. D. Real-time PCR analysis of

Wdnm1-like transcript in wild type C57BL/6 mice (WT) and ob/ob mice. The value in the respective WT depot was set to 1, and * indicates p<0.002 for ob/ob vs. wild type. S, E, and B are as defined for Figure 3B.

Figure 4. Wdnm1-like transcript increases early and is sustained throughout 3T3-L1 adipocyte differentiation. A. Daily time course of transcript expression. RNA was harvested from post-confluent 3T3-L1 preadipocytes prior to induction of adipogenesis (0) and at indicated daily time points after Dex/MIX induction of adipogenesis. 5 μg of total RNA was analyzed by Northern blot using Wdnm1-like, SCD1, aFABP, and PPARγ radiolabelled cDNA probes. For

Wdnm1-like signal, shorter (S) and longer (L) autoradiographic exposure times are shown. EtBr staining of rRNA is shown as a gel loading control. Minor adjustments to brightness and/or contrast were utilized for better visualization. B.

Real-time PCR analysis of Wdnm1-like transcript levels in 3T3-L1 (left panel) or

95 ScAP-23 (right panel) preadipocytes (P) and adipocytes (A). Value in the respective preadipocyte sample was set to 1 and * indicates p<0.001 for A vs. P.

The signals for the 3T3-L1 and ScAP-23 preadipocyte samples was undetectable after 40 PCR cycles and was set to a value of 40, as described in Materials and

Methods, such that fold changes could be calculated. C. Upregulation of

Wdnm1-like transcript (left panel) in primary murine preadipocytes (P) or following differentiation to adipocytes (A). Right panel shows levels of the adipocyte markers SCD1 and aFABP in either preadipocytes (P) or adipocytes

(A), confirming effective adipogenesis of primary cultures. Transcript levels in preadipocytes was set to value of 1 and * indicates p<0.001 for preadipocytes vs. adipocytes. D. Transcript analysis in early adipogenesis. Real-time PCR for the indicated transcript was conducted on samples harvested at 24 h hours prior to adipogenic induction (-24), at the time of adipogenic induction (0), or post- adipogenic induction (6 h, 12 h, 24 h, 36 h, and 48 h). For each transcript, the level in the respective time 0 sample was set to 1. For those samples whose values are not evident from the graph, the numbers directly above the respective x-axis indicate fold induction.

Figure 5. A. Regulation Wdnm1-like transcript by components of the adipogenic cocktail, TNFα and LPS. A. Regulation of Wdnm1-like transcript expression by components of the adipogenic cocktail. Post-confluent 3T3-L1 cells (0) were treated with 1 μM Dex (D) or 0.5 mM MIX (M) or 1 μM Dex and 0.5 mM MIX (D/M) in combination for the indicated number of days. Northern blot

96 analysis was performed on 5 μg total RNA using a radiolabeled Wdnm1-like probe. For Wdnm1-like signal, shorter (S) and longer (L) autoradiographic exposures are shown. EtBr staining of rRNA is shown as a gel loading control.

Minor adjustments to brightness and/or contrast were utilized for better visualization. B. TNFα upregulates Wdnm1-like transcript in 3T3-L1 adipocytes.

3T3-L1 adipocytes were pretreated with either DMSO vehicle (V), or the indicated pharmacological inhibitors of intracellular signaling pathways and after

1 h TNFα was added to cultures at 10 ng/ml, as described in “Materials and

Methods”. After 16 h RNA was analyzed for Wdnm1-like transcript level by real- time PCR. Value in vehicle-treated adipocytes was set to 1. * indicates p<0.001 for TNFα vs. vehicle (V) and for TNFα vs. SB+TNFα; # indicates p>0.05 for

WM+TNFα vs. TNFα alone and for RAP+TNFα vs. TNFα alone; ** indicates p<0.001 for SB vs. TNFα, and ## indicates p>0.05 for WM and RAP vs. vehicle

(V). SB, SB203580; WM, wortmannin, and Rap, rapamycin. C. LPS upregulates

Wdnm1-like transcript in RAW264.7 murine macrophages. RNA was harvested from control cells (-) or those treated for 4 h with LPS (+), with duplicate samples shown. Transcript level for Wdhm1-like was undetectable in the (-) samples, and as described in Materials and Methods, a value of 40 cycles was assigned to calculate fold differences. * indicates p<0.001 for (+) vs. (-) samples.

Figure 6. Secreted Wdnm1-like increases levels of active MMP-2. A. Left

Panel: Ectopic expression of Wdnm1-like transcript in HT1080 cells by real-time

PCR (left). Right Panel: Ectopic expression of Wdnm1-like protein in HT1080

97 cells by Western blot. Cell lysate (for RNA) or media (for protein) was harvested at 48 h post-transfection of HT1080 cells with either empty vector (EV) control or the Wdnm1-like-HA expression construct and subjected to Western blot analysis using anti-HA antibody. Coomassie Blue gel staining is shown as a loading control. B. Gelatin zymography analysis of media from HT1080 cells transfected with Wdnm1-like-HA expression construct or empty vector (EV) control. Media was collected 48 h post-transfection and analyzed on 0.1% gelatin zymograph gels as described in “Materials in Methods”; data is presented as a reverse image. Representative result of a minimum of three independent experiments is shown. Location of the pro-, intermediate (int) and active MMP-2 are indicated at right. D. Real-time PCR analysis of transcript levels of MMP-2 (left panel) and

MT1-MMP/MMP-14 (right panel) in cells transfected with an empty vector (EV) or the Wdnm1-like-HA expression construct. For A-C, duplicate samples derived from independent transfections are shown. For A and C, the leftmost EV sample was set to 1. Lanes comprising panels shown for either A (right panel) or for B were generated from the same Western blot exposure (for B) or zymograph gel

(for C), however some lanes have been removed and/or rearranged for clarity and/or economy of presentation. Minor adjustments to brightness and/or contrast were utilized for better visualization.

98 Figure 1

A. TTGCTGCCTAGCTCAGTGGGTGAAGAGGTTTAAATTCTGGCAGCTCTTGGCCCTGCTGCG TATTGGCTAACATCCTTCAGCAGCAGCATGAAGCTAGGAGCCTTCCTTCTGTTGGTGTCC -M--K--L--G--A--F--L--L--L--V--S- CTCATCACCCTCAGCCTAGAGGTACAGGAGCTGCAGGCTGCAGTGAGACCTCTGCAGCTT -L--I--T--L--S--L--E--V--Q--E--L--Q--A--A--V--R--P--L--Q--L- TTAGGCACCTGTGCTGAGCTCTGCCGTGGTGACTGGGACTGTGGGCCAGAGGAACAATGT -L--G--T--C--A--E--L--C--R--G--D--W--D--C--G--P--E--E--Q--C- GTCAGTATTGGATGCAGTCACATCTGTACTACAAACTAAAAACAGCTTCTACCTGGAAAA -V--S--I--G--C--S--H--I--C--T--T--N--*-...... AAAAATGTGTCTGTTTGGAGCTCTGTGACCAAGAAAACAGTTGAAAATGGAGGCCATGTA TGGAGATTACAAGCAGCACAGTGGAGTGGGACAAGGAGTTGTTTCTTTTAATAAATCATT AATGTAAAAGTCTC (434)

B. mWdnm1-like: LGTCAELCRGDWDCGPEEQCVSIGCSHI-C C C D DC C S GC C PD026912: MSSCPNQCQTDSDCPGNKKCCSNGCGRKQC C.

5.7 Kb

.1 .2 .3 D. 1 1 1 2 mChr 11 B B B B

Scale: 0 40 80 120 kb

Ccl4 Expi Heatr6

NP001075426 Wdnm1-like E. mWdnm1-like: MKLGAFLLLVSLITLSLEVQELQAAVRPL------Q 30 mWdmn1/Expi: MKTATVFVLVALIFMTMTTAWALSNPKEK--PGACPKPPP 38 NP001075426: MKTATVLFLVALITVGMNTTYVVSCPKEFEKPGACPKPSP 40

mWdnm1-like: LLGTCAELCRGDWDCGPEEQCVSIGCSHICTTN-- 63 mWdnm1/Expi: SFGTCDERCTGDGSCSGNMKCCSNGCGHACKPPVF 73 NP001075426: SVGICVDQCSGDGSCPGNMKCCSNSCGHVCKTPVF 75

99 Figure 2

) 0 ) 1 2 ( ( e e ik ik -l -l 1 1 ) 0 ) m m 1 2 n n ( ( d d V V A. (+) W W E E

15 14.4 10 6.5

B. 3 S 2 C 1 O 0 R E -1 -2 1 20 40 60 Amino Acid Position

C. Media Cells

1 1 1

1

m m

m m

n n

n n

e e

e e

d d

d d

k k

k k

V V V V

i i

i i

l l

l l

E E E E

- -

-

-

W W

W W Ab:HA

Coomassie Ab: Blue Cyclophilin A

100 Figure 3

A. 12 2 10 * 8

6 * 4 Wdnm1-like Wdnm1-like Transcript x 10 x Transcript 2 0 r n e n g y t s e e l d i n r i v c n a e a t AT i le s a r u n s W L p u l B L id e S g He T M l. K a S B. 600 100 Retn 8 SCD1 * aFABP 500 80 7 400 6 60 5 300 4 40 3

200 Transcript Transcript Wdnm1-like Wdnm1-like * 2 100 * 20 1 0 0 0 BS ER B S E R B S E R

10 C. 25 40 * 20 * 8 * 30 15 6 20

10 SCD1 4 Col1A1 Transcript

Wdnm1-like Wdnm1-like 5 2 10 0 0 0 SVF AF SVF AF SVF AF

D. 4 WT * 3 ob/ob 2 * Transcript Wdnm1-like Wdnm1-like 1 * 0 SEBLIVER101 Figure 4

A. 0 1 2 3 4 5 6

Wdnm1-like (S)

Wdnm1-like (L)

SCD1

aFABP

PPAR

rRNA

B. 3 3

25 10 20 * * 15 10 5 Wdnm1-like Wdnm1-like 5 Transcript x 10 Transcript x 10 0 0 P A P A 3T3-L1 ScAP-23 C. aFABP 80 180 * * SCD1 140 60 100 40 60 20 * Transcript Transcript Wdnm1-like 20 0 0 P A P A P A

102 Figure 4

D. 25 3 20 Wdnm1-like 15 X 10 10 5 1.1 1.0 7.6 78 0

15 SCD1 10 FABP 5 0

Level Transcript 160 PPAR 120 80 40 2.0 1.0 0 -24 0 6 12 24 36 48 h

103 Figure 5

A. Day 1 Day 2 Day 5 0 D M DM D M DM D M DM

Wdnm1-like (S)

Wdnm1-like (L)

rRNA

B. 4 # # 3 * 2

Transcript Wdnm1-like 1 ## ## ** 0 V TNF SB WM RAP SB WM RAP

+ TNF

* C. * 2 12 10 8 * 6 4

Wdnm1-like 2

10 X Transcript 0 LPS: (-) (-) (+) (+)

104 Figure 6

A. EV Wdnm1-like

4 20 Ab:HA 16 12 8 4 Media

Wdnm1-like x 10 x Wdnm1-like 0 EV Wdnm1- like

B. EV Wdnm1-like

pro-MMP-9

pro-MMP-2

int-MMP-2

active MMP-2

C.

4

4

1 1 -

- 4 P

1.6 P

t t

t t

M M

p p

p

p

i i

i i

2

2 3

r r

M M r r

-

- 1.2

c c c

c

P P

s s

s s

P/ P/

n n M

M 2 n

n 0.8

M M

a a

a a

M M

r r

r r

M M

T T

- - T

T 0.4 1

1 1 T

T M 0 M 0 EV Wdnm1- EV Wdnm1- like like

105 TABLE I PCR Validation of Select Genes Upregulated during in vitro Brown Adipogenesis

Fold Gene Name Unigene Increase 12 Ero1l ERO1-like Mm.387108 13 Arl4a ADP-ribosylation factor-like 4A Mm.12723 14 Itga6 Integrin alpha 6 Mm.225096 19 Scd1 Stearoyl CoA-desaturase 1 Mm.267377 19 Sh2b2 SH2B adaptor protein 2 Mm.425294 24 Bnip3 BCL2/adenovirus E1B interacting protein 1, NIP3 Mm.378890 24 Selenbp1 Selenium binding protein 1 Mm.196558 28 Pex11a Peroxisomal biogenesis factor 11a Mm.20615 33 Lama4 Laminin, alpha 4 Mm.258065 34 Nudt7 Nudix -type motif 7 Mm.27889 53 Rgs2 Regulator of G-protein signaling 2 Mm.28262 55 Cebpa CCAAT enhancer binding protein alpha Mm.349667 150 Aldh6a1 Aldehyde dehydrogenase family 6, subfamily A1 Mm.247510 520 Lrg1 Leucine-rich alpha-2-glycoprotein 1 Mm.348025 657 Slc2a4 Solute carrier family 2, member 4 Mm.10661 810 Lamb3 Laminin, beta 3 Mm.435441 1,040 Rarres2 Retinoic acid receptor responder 2 Mm.28231 1510 Fabp4 Fatty acid bindng protein, adipocyte Mm.582 2,230 Mb Myoglobin Mm.404074 4,260 Mrap Melanocortin 2 receptor accessory protein Mm.22455 18,700 Pparg PPAR gamma 2 Mm.3020 45,000 Adipoq Adiponectin Mm.3969

106

MANUSCRIPT # 2

Identification and Characterization of TSC-36 as a Preadipocyte Gene Yu Wu, Ji Young Kim and Cynthia M. Smas

Department of Biochemistry and Cancer Biology and Center for Diabetes and Endocrine Research University of Toledo Health Science Campus Toledo, OH 43614, USA

Running Title: Novel Preadipokine TSC-36

Please address correspondence to:

Cynthia M. Smas, D.Sc. Department of Biochemistry and Cancer Biology and Center for Diabetes and Endocrine Research University of Toledo Health Science Campus Toledo, OH 43614, USA Phone: 419-383-4131 FAX: 419-383-6228 e-mail: [email protected]

107

ABSTRACT Obesity is a major public health problem in the United States. The

identification and functional characterization of preadipocyte-expressed genes is

a relatively new research area in regard to the study of adipogenesis. We

hypothesize that downregulation of select preadipocyte genes may be a control point for adipogenesis. Through differential hybridization screening of nylon DNA arrays we identified TSC-36 (TGF-β1-stimulated clone 36) as a gene that is

highly expressed in 3T3-L1 preadipocytes and downregulated to nearly

undetectable levels in 3T3-L1 adipocytes. We determined that TSC-36 is a

preadipokine secreted by preadipocytes. The expression of TSC-36 mRNA is

markedly decreased during the time course of both white and brown

adipogenesis. TSC-36 protein level is also dramatically reduced during adipocyte

differentiation. TSC-36 transcript and protein levels are increased by TNFα

treatment of 3T3-L1 adipocytes. In 3T3-L1 preadipocytes, TSC-36 expression is

dramatically downregulated by the demethylating agent 5-azacytidine. To assess

TSC-36 downregulation at the transcriptional level during differentiation, we

generated luciferase reporter constructs containing various portions of the 5’

flanking region of the TSC-36 gene. Transient transfection studies reveal these

sequences exhibited promoter activity in both 3T3-L1 and brown preadipocytes.

In addition, we determined that both PPARγ and KLF15 (krüppel-like zinc finger

transcription factor 15) downregulate the activities of the TSC-36 luciferase

108 promoter constructs. Our data indicate that TSC-36 expression may be a distinct feature of preadipocytes.

KEY WORDS: Preadipocyte, adipogenesis, differentiation, obesity, TSC-36, secreted factor, adipose tissue.

109 INTRODUCTION

Obesity can lead to multiple of health problems such as non-insulin

dependent diabetes mellitus (NIDDM), hypertension, cardiac infarction and some

type of cancers (Ntambi and Young-Cheul, 2000). White adipose tissue is a

unique organ that regulates the balance between energy storage and release to maintain energy homeostasis according to nutritional status. Additionally, adipose tissue is now recognized as a dynamic “secretory” endocrine organ. A number of soluble factors and cytokines including leptin, adiponectin, resistin and

TNFα (tumor necrosis factor α) can be synthesized and secreted by whole

adipose tissue and /or adipocytes (Maeda et al., 2002; Spiegelman and Flier,

1996; Steppan et al., 2001). In addition to white adipose tissue, mammals also

have brown adipose tissue. Brown adipose tissue serves primarily to dissipate

energy instead of storing it (Lowell and Flier, 1997).

Adipogenesis is the process of the formation of new adipocytes from

preadipocyte precursors, and is thus important for the understanding and control

of obesity. The full developmental program of adipose tissue from fertilized egg

has not been clearly dissected. Studies have shown that preadipocytes,

chondrocytes, or muscle cells share the common mesodermal originated

pluripotent precursors (Taylor and Jones, 1979). However, fat tissue is a

combination of adipocytes, small blood vessels, nerve tissue, fibroblasts,

preadipocytes and other undefined cell types (Geloen et al., 1989).

Preadipocytes and fibroblasts are morphologically indistinguishable. Moreover,

110 the early molecular events that determine the commitment of precursor

mesenchymal cells to the adipocyte lineage remain unknown. All these facts

have made it difficult to study preadipocyte differentiation in vivo. Therefore

adipogenesis has been studied primarily by using in vitro cell culture models. The

3T3-L1 cell line was isolated from disaggregated Swiss mouse embryo by Green and colleagues. 3T3-L1 preadipocytes differentiate to mature adipocytes upon treatment with adipogenic cocktail composed of dexamethasone and methylisobutylxanthine. 3T3-L1 cells are the best established model of in vitro

adipogenesis and have been extensively exploited for studying the molecular

mechanisms of adipogenesis (Green and Kehinde, 1974, 1975a; Green and

Meuth, 1974).

To date, increases in gene expression that accompany adipogenesis have

been well characterized. The adipogenic transcription factors C/EBPs

(CCAAT/enhancer binding proteins) and PPARγ (peroxisome proliferator-

activated receptor-gamma) play key roles in the transcriptional cascade during

adipogenesis (Gregoire, 2001b; Gregoire et al., 1998b; Rajala and Scherer,

2003). C/EBPβ and C/EBPδ are early transcription factors that are induced after

exposure to the adipogenic cocktail, and they mediate the expression of both

PPARγ and C/EBPα (Rosen and Spiegelman, 2001; Tontonoz et al., 1994b).

C/EBPα then cooperates with PPARγ to induce a significant adipogenic response

(Hu et al., 1995). PPARγ was first recognized as a binding protein of the adipose-

specific enhancer element in the aP2 (adipocyte fatty acid-binding protein 2)

gene (Graves et al., 1992). The cyclopentanoic prostaglandin derivative 15d-

111 PGJ2 (15-deoxy-∆12, 14-PGJ2) is one potential natural ligand of PPARγ (Kliewer

et al., 1995). The role of PPARγ as a master regulator in adipogenesis has been

revealed by observation that forced expression of PPARγ and the application of

PPARγ ligand can stimulate adipogenesis in many fibroblast cell lines (Hu et al.,

1995; Shao and Lazar, 1997; Tontonoz et al., 1994b). Its crucial role is further

confirmed by the observation that C/EBPα cannot induce adipogenesis in the

absence of PPARγ (Rosen et al., 2002). In vivo studies indicate that homozygous

PPARγ-deficiency is lethal during mouse embryogenesis (Tontonoz and

Spiegelman, 2008). A tetraploid-rescued PPARγ-null pup lacking both WAT and

BAT exhibited lipodystrophy and multiple hemorrhages (Barak et al., 1999). In addition, ADD1/SREBP (adipocyte determination and differentiation factor

1/sterol regulatory element-binding protein) also has a regulatory role in adipogenesis. It has been suggested that ADD1/SREBP stimulates preadipocyte

differentiation by producing an endogenous PPARγ ligand (Kim et al., 1998b).

The functional significance of other transcription factors in adipogenesis including

STAT (signal transducers and activators of transcription) have also been

demonstrated (Hummasti et al., 2004; Stewart et al., 1999).

In contrast, only a relatively few molecules with high expression levels in

preadipocytes but relatively low levels in mature adipocytes, such as Pref-1

(preadipocyte factor-1) (Smas et al., 1997), Wnts (Ross et al., 2000), GATA-2

and GATA-3 (Tong et al., 2000) have been studied in detail. Pref-1 is highly

expressed in 3T3-L1 preadipocyte but is markedly lower albeit still found in the

preadipocyte population in the in vivo setting. Downregulation of Pref-1 is

112 required for adipogenesis since constitutive expression of Pref-1 inhibits adipose conversion (Smas et al., 1997). Pref-1-null mice exhibit general growth retardation, skeletal malformation and obesity resulted from accelerated weight gain (Moon et al., 2002). MEFs from Pref-1 null mice exhibit enhanced ability to differentiate to adipocytes (Kim et al., 2007c; Moon et al., 2002). Transgenic mice with adipose tissue specific overexpression of the 50 KDa soluble Pref-1 develop hypertriglyceridemia, impaired glucose tolerance and significantly reduced total fat pad weight (Lee et al., 2003). Wnts signaling also acts as an adipogenesis inhibitor. In the canonical Wnt signaling pathway, upon Wnt ligands binding to transmembrane receptors of the Frizzled family, the downstream target such as

β-catenin is stabilized and translocates to the nucleus to regulate the expression of Wnt target genes. Overexpression of Wnt or stabilizing free cytosolic β-catenin both block the adipogenesis (Ross et al., 2000). GATA-2 and GATA-3 are GATA- binding transcription factors and their transcript expression levels are downregulated during white adipocyte differentiation but are relatively low in BAT.

Constitutive expression of GATA-2 and GATA-3 traps cells at the preadipocyte stage and such cultures are refractory to the effects of the adipogenic cocktail

(Tong et al., 2000).

The latest publication regarding the location of white preadipocyte progenitor cells in vivo demonstrates that the progenitors reside in the vasculature of adipose tissue (Tang et al., 2008b). Additionally, Rodeheffer et al. reported that a subpopulation of early adipocyte progenitor cells (Lin(-

):CD29(+):CD34(+):Sca-1(+):CD24(+)) are capable to form a normal WAT depot

113 when injected into the residual fat pads of A-Zip lipodystrophic mice (Rodeheffer et al., 2008). However, the process of emergence of the functionally distinct preadipocytes from mesodermal stem cells remains obscure. Thus, the further elucidation of the molecular definition and developmental lineage relationships of preadipocytes is essential to a full understanding of adipogenesis.

Through differential hybridization screening of nylon DNA arrays we identified several genes that were highly expressed in 3T3-L1 preadipocytes and very strongly downregulated to nearly undetectable levels in 3T3-L1 adipocytes.

One of these genes was TSC-36 (TGF-beta1-stimulated clone 36). TSC-36 was first isolated by Shibanuma et al. in a differential screening for TGF-β1-induced genes in the mouse osteoblastic MC3T3-E1 cell line. TSC-36 is a secreted glycoprotein belonging to the BM-40/SPARC/osteonectin protein family

(Shibanuma et al., 1993). Members of this protein family function as regulators of cell interactions with the extracellular milieu during development and in response to injury (Bradshaw and Sage, 2001). For example, SPARC is secreted by adipocytes and its expression is strongly elevated in adipocytes of obese mice

(Tartare-Deckert et al., 2001). The adiposity of SPARC-null mice is increased but their overall body weight does not change significantly (Bradshaw et al., 2003).

TSC-36 is one of the family members with the least protein sequence similarity to

SPARC.

TSC-36 plays role in embryonic development. The chick TSC-36 orthologue, also named Flik for follistatin-like, is expressed in the dorsomedial

114 compartment of the somite and myotome (Amthor et al., 1996) and is involved in

dorsalization and neural induction (Patel et al., 1996; Towers et al., 1999). TSC-

36 also plays a role in some types of cancer; it can inhibit proliferation of human

lung cancer cell lines (Sumitomo et al., 2000). TSC-36 transcript is upregulated

by BRCA1 (breast cancer susceptibility gene 1), which is a tumor suppressor in

breast (MCF-7) cancer cell line (Bae et al., 2004). Preadipocytes and myocytes

are both thought to arise from mesenchymal stem cell. TSC-36 has ameliorative

effects on joint inflammation in a mouse model of arthritis by downregulating

disease–promoting genes such as c-fos and IL-6 (Kawabata et al., 2004).

Interestingly, TSC-36 also has effects on myocytes. Treatment with recombinant

TSC-36 inhibits vascular smooth muscle cell (VSMC) proliferation and migration

(Liu et al., 2006). MyoD has been shown to suppress TSC-36 expression in

C2C12 myocytes via upregulation of microRNA-206 (mir-206) which targets the

TSC-36 3’-UTR (Rosenberg et al., 2006). In a very recent report, TSC-36 was identified as a novel upregulated target of Akt in an inducible Akt1 transgenic mouse model. In vitro and in vivo ectopically expressed TSC-36 protects the neonatal rat ventricular myocytes (NRVMs) and myocardium from hypoxia/ischemia stress, respectively, in an Akt dependent manner (Oshima et al., 2008). Furthermore, the same research group also reported that TSC-36 stimulated endothelial nitric oxide synthase (eNOS) phosphorylation and consequently enhanced revascularization to protect myocytes during ischemic conditions. Treatment with an Akt or eNOS inhibitor abolished this effect (Ouchi et al., 2008).

115 To date there are no studies that establish the link between TSC-36 and

adipocyte differentiation. Our investigations on the expression and regulation of

TSC-36 in adipogenesis may contribute to a more complete picture for this

molecule as well as illuminate aspects of the preadipocyte phenotype.

MATERIALS AND METHODS

Cell Culture and Adipocyte Differentiation

3T3-L1 cells (American Type Culture Collection, Manassas, VA) were

maintained in DMEM with 10% calf serum (CS). For differentiation, cells were

treated at 2 days post-confluence with DMEM supplemented with 10% fetal

bovine serum (FBS), 0.5 mM 3-isobutyl-1-methylxanthine (Mix), and 1 µM

dexamethasone (Dex) for 48 h. After induction, cells were maintained in 10%

FBS medium. ScAP-23 cells were maintained in DMEM containing 10% CS and

passaged prior to reaching confluence. For adipocyte differentiation,

preconfluent ScAP-23 preadipocytes were cultured in DMEM containing 10%

FBS in the presence of the adipogenic inducers 0.5 mM Mix, 1 µM Dex, 17 nM

insulin, and 0.2 mM indomethacin for 70 h. These agents were then removed

and cultures were maintained in DMEM containing 10% FBS and 17 nM insulin

for an additional 4 days. The AD3.7AC cell line was obtained from Dr. B. Lecka-

Czernik (University of Toledo, Health Science Campus, Toledo, OH). Their

culture and adipogenic induction is the same as for ScAP-23. For culture and

differentiation of primary white adipocytes, white adipose tissue collected from

male C57BL/6 mice or Sprague Dawley rats was digested with 1 mg/ml of type I

116 collagenase for 40 min at 37C with shaking. Following digestion, material was

filtered through a 300 micron pore size nylon mesh (Sefar America Inc., Depew,

NY) and filtrate centrifuged at 2,000 rpm for 5 min. The floating adipocyte

fraction was removed and the pellet of preadipocyte-containing stromal-vascular

cells was resuspended in DMEM containing 10% FBS and plated. Upon

confluence cells were either harvested or subjected to culture in differentiation

media that consisted of DMEM containing 10% FBS, 0.5 M Dex, 0.25 mM Mix,

and 17 nM insulin for three days. After this, media was removed and cell cultures

were maintained in DMEM containing 10% FBS and 17 nM insulin for an

additional 4 days. For differentiation of a brown preadipocyte cell line (termed herein WT-BAT) obtained from C.R. Kahn (Joslin Diabetes Foundation, Harvard

Medical School, Boston, MA), cells were cultured to confluence in DMEM supplemented with 10% FBS, 20 nM insulin and 1 nM triiodothyronine (T3).

Confluent cells were incubated in differentiation medium that contained 0.5 mM

Mix, 0.5 µM Dex and 0.125 mM indomethacin for 2 days. After induction, cells were maintained in medium supplemented with 20 nM insulin and 1 nM T3 for an additional 4 days. The culture and adipogenic induction of mBAP-9 cells is as for

WT-BAT. COS cells and NIH-3T3 cells were cultured in DMEM supplemented with 10% FBS.

For treatment of 3T3-L1 preadipocytes with various agents, confluent 3T3-

L1 preadipocytes were maintained in fresh DMEM with 10% CS for 24 h as

control or with 10 nM retinoic acid; 2 ng/ml basic fibroblast growth factor (b-FGF);

10 ng/ml interleukin 6 (IL-6); 100 ng/ml bone morphogenetic protein 4 (BMP4); 117 200 μM indomethacin; 10 μM forskolin; 0.5 mM Mix; 200 ng/ml insulin; 10 nM

sodium butyrate; 0.36 ng/ml rhEGF (recombinant human epidermal growth

factor); 1 mM 5-azacytidine, 10 nM T3 (triiodothyronine); 100 ng/ml VEGF

(vascular endothelial cell growth factor); 1 µM dexamethasone; 1 nM cAMP

(cyclic adenosine monophosphate); 5 μM tamoxifen; 100 ng/ml LPS

(lipopolysaccharide); 10 ng/ml TNFα; 1 μM TSA (trichostatin A); 5 μg/ml PPARγ ligand 15-deoxy-∆12, 14-prostaglandin J2. For treatment of 3T3-L1 adipocytes with TNFα, cells were incubated with 10 ng/ml TNFα for the indicated times. For assessment of TSC-36 regulation by components of the adipogenic cocktail, post-confluent 3T3-L1 preadipocytes were treated with 1 µM Dex or 0.5 mM Mix or the combination of Mix and Dex for 48 h. For 5-azacytidine time course analysis, 3T3-L1 preadipocytes were treated with 1 mM 5-azacytidine or control for the indicated times.

Transfections and Western Blot Analysis

For transfection, COS cells were seeded at 6x105 cells/100 mm dish. 10

µg of TSC-HA-pcDNA or pcDNA vector was transfected using the DEAE-dextran

method. For Western blot analysis culture media and cell lysates were collected

for analysis at 48 h post-transfection. Media was centrifuged at 5000 rpm for 3

min and cells harvested by lysis in TNN (+) buffer (10 mM Tris pH 8.0, 120 mM

NaCl, 0.5% NP-40, 1 mM EDTA, supplemented with a protease inhibitor cocktail).

Lysates were incubated on ice for 30 min with intermittent vortexing, supernatant

collected via centrifugation, and protein content determined (Bio-Rad

118 Laboratories). To examine TSC-36 protein expression in medium during 3T3-L1

cell differentiation, culture medium was harvested daily from the same well of

cells in a 6-well plate, with medium changed 24 h pre-harvest. For assessment of

TNFα induced TSC-36 in 3T3-L1 adipocytes, cells were incubated with or without

10 ng/ml TNFα for 24 h or 48 h. To determine TSC-36 protein half life, 3T3-L1

preadipocytes were treated with 5 µg/ml of cycloheximide and harvested at

indicated time points after treatment. For TSC-36 protein expression after 5-

azacytidine treatment, 3T3-L1 preadipocytes were treated with or without 1 mM

5-azacytidine for 24 h and culture media harvested. Proteins were separated in a

10% SDS-PAGE gel under reducing conditions and electroblotted onto

polyvinylidene fluoride (PVDF) membrane (Millipore Corp.). For signal detection,

membranes were blocked 1 h in 5% nonfat milk/0.5% Tween 20 in PBS and then

incubated with primary antibody for 1 h followed by three 10 min washes.

Secondary antibody was incubated for 30 min followed by three 10 min washes.

All washes were in 0.5% Tween 20 in PBS. Signal was detected by ECL Plus

enhanced chemiluminescence (GE Healthcare) and exposure to X-ray film. Anti-

mouse TSC-36 antibody was purchased from R&D Systems.

In vitro Translation

A TNT Quick Coupled Transcription/Translation Kit (Promega Corp.) was

employed for in vitro transcription and translation. Reactions programmed with

either the TSC-36 HA construct or with empty vector were incubated at 30oC for

90 min and 25 μl of a 1:100 dilution of the respective reaction was analyzed on

119 10% SDS-PAGE gels, followed by overnight transfer to polyvinylidene fluoride

(PVDF) membrane (Millipore Corp.). Membranes were processed for Western

blot analysis with anti-HA antibody as described in the section “Transfections and

Western Blot Analysis”.

RNA Preparation, Northern Blot Analysis, DNA Array Hybridization and

Real-Time PCR

Total RNA was extracted with TriZol Reagent (Invitrogen Corp) according to manufacturer's instructions. For TSC-36 expression in murine tissues, 8 wk old

C57BL/6 male mice were utilized, with all animal treatments conducted with approval of the University of Toledo Health Science Campus Institutional Animal

Care and Use Committee. Fractionation of whole adipose tissue was performed by digestion with 1 mg/ml of type I collagenase for 40 min with shaking at 37C.

Following digestion, material was filtered through a 300 micron pore size nylon mesh (Sefar America Inc., Depew, NY) and filtrate centrifuged at 2,000 rpm for 5 min. Floating adipocyte fraction was collected as adipocyte fraction (AF) and the pellet was collected as stromal-vascular fraction (SVF).

For array analysis, duplicate mouse ResGen GeneFilter Arrays (Invitrogen

Corp.) containing 5,184 genes, were processed according to manufacturer's instructions. Filters were hybridized with [α-33P] dATP labeled cDNA probes

synthesized from 8 μg of total RNA from 3T3-L1 preadipocytes and adipocytes.

For Northern blot analyses, 5 µg of total RNA was electrophoresed in 1%

agarose formaldehyde gels in MOPS buffer and transferred to Hybond-N nylon

120 membrane (GE Healthcare, Piscataway, NJ). Hybridizations were performed

under high stringency conditions in Express Hyb (BD Biosciences Clontech),

according to the manufacturer's directions. Probes were labeled with [α-32P] dATP using a Megaprime DNA labeling system (Amersham Biosciences). After washing, membranes were exposed at -80C to Kodak Biomax film with a Kodak

Biomax intensifying screen. Murine TSC-36, FABP (fatty acid binding protein),

PPARγ, Pref-1 (preadipocyte factor-1), Retn (resistin), SCD1 (stearoyl-CoA desaturase 1), SMAF1 (small adipocyte factor 1), UCP1 (uncoupling protein 1),

KLF15 (Krüppel-like factor 15) and 36B4 cDNAs were used as DNA probes.

For real-time PCR analysis, total RNA was subject to purification with an

RNeasy RNA purification kit with DNase I treatment (Qiagen Corp., Valencia, CA) and 5 g used for first strand cDNA synthesis with SuperScript II RNase H- reverse transcriptase (Invitrogen Corp.) and an oligo(dT)-22 primer. Real-time

PCR was conducted with an ABI 7500 Real-time PCR System. Target cDNA levels were analyzed by SYBR green-based real-time PCR in 25 l reactions containing 1X SYBR Green PCR Master Mix (Applied Biosystems, Foster City,

CA), 100 nM each forward and reverse primers, and 10 ng of cDNA. Sequences of real-time PCR primers used were: murine Gapdh (5’-GGCAAATTCAACGGCACAG-3’ and 5’-

CGGAGATGATGACCCTTTTG -3’); murine TSC-36 (5’-

ATGTGCCGTCACAGAGAAGG-3’ and 5’-GCAGTGCCCATCATAATCAACC-3’).

Expression of TSC-36 was normalized against Gapdh transcript level. In all cases the same amount of input RNA/cDNA was used in side-by-side 121 comparisons and the Gapdh level for samples differed by 2 or fewer cycles. The

cycle threshold value was generated using ABI PRISM 7500 SDS software

version 1.2 and exported to an Excel spreadsheet to calculate fold differences.

Statistical analyses were conducted using single factor ANOVA.

Construction of Expression Vectors for HA Tagged TSC-36

A TSC-36 CDNA clone was obtained from ATCC (I.M.A.G.E. ID: 2647002).

We used the primer (5' GCC AAG CTT CCG GAC CCG AGC ACG ATG TGG3’)

and (5' GGC GTC GAC TTA AAG AGC GTA ATC TGG AAC ATC GTA TGG

GTA GAT CTC TTT GGT GTT CAC CTT3’) to generate the coding region of

murine TSC-36 with a c-terminus HA tag by PCR. The primers introduced the

restriction sites 5’ Hind III and 3’ Sal I (underlined). The Hind III/Sal I restricted

PCR products were purified and inserted into Hind III and Sal I sites of pcDNA3.1.

Luciferase Assay

To prepare luciferase reporter constructs containing regions of the mouse

TSC-36 promoter, murine liver genomic DNA was subject to PCR with primers designed based on murine TSC-36 genomic flanking region sequence available through the Ensembl database (www.ensembl.org). For the -280/+60 promoter

construct, PCR was conducted with a promoter proximal primer (5’ GGC CTC

GAG TGG CGG CAG CGA GTT AGA GGC) that included an Xho I site

(underlined) spanning through 60-base pair (bp) of the TSC-36 transcript in

combination with a promoter distal primer (5’ GGC ACG CGT TGA TTT CTG

122 TGA TTT CCC CCG C) including an Mlu I site (underlined). For the -350/+60

promoter construct, PCR was conducted with the same promoter proximal primer

as for the -280/+60 promoter construct and a promoter distal primer (5’ GGC

ACG CGT CGA GTT CCTTCT GTT ACC CAC) including an Mlu I site

(underlined). For the -791/+60 promoter construct, PCR was conducted with a

promoter proximal primer (5' GGC AGA TCT TGG CGG CAG CGA GTT AGA

GGC 3') including a Bgl II site (underlined) and a promoter-distal primer (5' GGC

GGT ACC GAA AGA GAT TGG GGA TCC ACAC 3') including a Kpn I site

(underlined). For the -3922/+22 promoter construct, PCR was conducted with a

promoter proximal primer (5’ GGC ACG CGT TTA TCA CCA GGC TCC GAG

GAA G 3’) including an Mlu I site (underlined), and a promoter distal primer (5’

CAG CCG GTA CCA ATG CAC ACG CAT TGC CAC) including Kpn I site

(underlined). The resulting PCR products were ligated into the corresponding restriction sites of the pGL2-Basic vector (Promega Corp, Madison, WI).

Promoter constructs were confirmed by complete sequencing.

To assess transcriptional activation of the murine TSC-36 promoter,

promoter-luciferase constructs were transfected into 3T3-L1 and WT-BAT

preadipocytes using Lipofectamine 2000 (Invitrogen Corp). For assessment of

PPARγ regulation, NIH-3T3 cells were utilized for transfection using

Lipofectamine 2000. As indicated, transfections included murine PPARγ and

murine RXRα expression constructs; these were kindly provided by R. Evans

(The Salk Institute for Biological Studies, La Jolla, CA). A PPARγ responsive

promoter construct, 3XPPRE-Luc, also provided by R. Evans, was utilized as a

123 positive control to determine functionality of PPARγ/RXRα constructs. At 24 h

post-transfection, cells were treated with 10 μM PPARγ ligand 15d-PGJ2

(Caymen Chemical Corp., Ann Arbor, MI) for 24 h. For assessment of regulation

by KLF15, TSC-36 promoter-luciferase constructs were transfected into 3T3-L1 and WT-BAT preadipocytes along with a KLF15 expression construct or with empty pcDNA vector using Lipofectamine 2000 (Invitrogen Corp). Firefly and renilla luciferase activities were determined at 48 h post-transfection using a dual luciferase reporter assay system and a Turner Industries luminometer (Promega

Corp.). Statistical analyses were conducted using single factor ANOVA.

RESULTS

Characterization of TSC-36 as Preadipocyte Marker Gene

To identify and characterize distinct gene expression patterns that define preadipocytes, we undertook DNA array analysis of 5,184 genes utilizing murine gene filter arrays. Differential screening was conducted via hybridization with cDNA probes prepared from 3T3-L1 preadipocyte or 3T3-L1 adipocyte total RNA.

Several genes were found to be preadipocyte-enriched, one of these genes was

TSC-36, which was originally isolated as TGF-β stimulated clone in mouse osteoblastic MC3T3-E1 cells (Shibanuma et al., 1993). The differential signal for

TSC-36 is shown in Figure 1A. To confirm this result, Northern blot analysis was conducted using 3T3-L1 preadipocyte and adipocyte RNA. SCD1 (Stearoyl-CoA desaturase 1) cDNA was shown as adipocyte marker. Consistent with differential

124 screening, TSC-36 transcript was dramatically decreased to nearly undetectable

levels in 3T3-L1 adipocytes (Figure 1B).

To assess the expression pattern of TSC-36 transcript during white

adipogenic conversion, Northern blot analysis was conducted using various

models representing white adipocyte differentiation. First, 3T3-L1 adipogenesis was examined with RNA collected at indicated times during differentiation. TSC-

36 RNA expression was diminished at day 2 and was decreased dramatically by

day 3. TSC-36 was nearly undetectable upon full differentiation to mature

adipocytes at day 7 (Figure 2A). Here, Pref-1 was used as a preadipocyte marker

and aFABP was used as an adipocyte marker to assess adipose conversion. We

extended out study of differentiation-dependent downregulation of TSC-36

transcript to other important models of white adipogenesis, including assessment

of adipogenic conversion of primary cells and different cellular fractions of

adipose tissue. These are particularly important to assess as significant

differences of gene expression has been reported between in vitro 3T3-L1

adipogenesis vs. gene expression in isolated adipocytes compared to the

preadipocyte-containing SVF (Soukas et al., 2001a). The preadipocyte-

containing stromal vascular fraction (SVF) was isolated from rat adipose tissue,

digested with collagenase, and cells plated and at confluence were differentiated

with adipogenic agents Dex and Mix. Dramatic downregulation of TSC-36

transcript occurred during differentiation of rat primary preadipocytes to

adipocytes (Figure 2B). Increased SCD1 and SMAF1 transcript levels indicate

effective differentiation of these primary cultures. Moreover, Figure 2C illustrates

125 that TSC-36 transcript was not detected in the adipocyte fraction (AF) of murine adipose tissue that does not contain preadipocytes, but was highly enriched in the preadipocyte-containing SVF. For this study, SCD1, SMAF1 and resistin

(Retn) were used as markers of the adipocyte cell population.

It is well known that increased visceral adipose tissue (VAT) is linked to a higher risk of diabetes and cardiovascular diseases compared to subcutaneous adipose tissue (SC), suggestive of functional heterogeneity of adipocytes (Gesta et al., 2007). Here we examined TSC-36 transcript expression in intra-abdominal epididymal white adipose tissue (EP WAT) and in subcutaneous white adipose tissue (SC WAT). In SC and EP WAT, TSC-36 transcript is enriched over 50-fold in the SVF vs. AF. The TSC-36 level in EP SVF is approximately 2-fold higher than that in SC SVF, indicating TSC-36 is a depot differentially expressed preadipocyte gene (Figure 2D).

The regulation of TSC-36 in brown adipogenic conversion has not been previously addressed. We therefore determined if in vitro brown adipogenesis is accompanied by a reduction of TSC-36 transcript level. Figure 2E demonstrates that TSC-36 RNA expression was readily detected in brown preadipocytes (WT-

BAT) but abolished by 3 days post-induction of adipocyte differentiation. The expression of the brown adipocyte-specific transcript uncoupling protein 1 (UCP1) at day 8 is a marker of brown adipocytes. 3T3-L1 preadipocyte RNA was used as positive control for TSC-36 signal. Pref-1, which is well known as a 3T3-L1 preadipocyte marker, was undetectable in brown preadipocytes.

126 In addition to the well studied 3T3-L1 cell line, our laboratory has developed a new model of in vitro white adipogenesis, ScAP-23. ScAP-23 is an immortalized preadipocyte cell line derived from preadipocytes of murine SC

WAT (Kim et al., 2007b). We have also obtained a murine bone marrow preadipocyte cell line AD3.7 AC from Dr. B. Lecka-Czernik (University of Toledo,

Health Science Campus, Toledo, OH), whose development and characterization at the molecular level has been previously reported (Lecka-Czernik et al., 1999).

For brown adipogenesis, in addition to the WT-BAT cell line established by Kahn and colleagues, we have established the mBAP-9 cell line derived from preadipocyte precursors present in murine BAT (manuscript in preparation).

Future assessment of these adipogenic models may allow the development of a criteria by which to further refine identification of preadipocyte specific genes.

TSC-36 transcript showed significant downregulation in all of these white and brown adipogenesis models tested (Figure 2F), indicating that TSC-36 is a robust and reliable preadipocyte marker gene.

We have examined TSC-36 transcript level in a wide sample of murine tissues and found a degree of TSC-36 RNA expression in most tissues except liver and seminiferous tubules. Among the other tissues, we detected that lung, heart and skeletal muscle express the highest levels of TSC-36 transcript, which is in good agreement with other studies (Adams et al., 2007; Liu et al., 2006;

Mashimo et al., 1997). Here we show for the first time that SC WAT has highest expression of TSC-36 among the tissues we examined (Figure 3).

127 Expression Pattern of TSC-36 Protein during Adipogenesis

To begin studies on the TSC-36 protein, we assessed the size of the TSC-

36 translation product by in vitro transcription and translation using the TSC-36-

HA construct as template; the HA tag adds an additional ~1 kDa to the protein

mass. Western analysis in Figure 4A shows that the in vitro translation product

encoded by TSC-36-HA construct migrates at ~37 kDa, in good agreement with

the mass predicted from the amino acid sequence of TSC-36.

To characterize a commercially available TSC-36 antibody, TSC-36-HA or

empty vector was transfected into COS cells. Cell lysate and culture media were

collected for Western analysis. Figure 4B shows TSC-36-HA protein was

successfully detected by anti-HA antibody in cell lysates as well as in media,

consistent with its description as a secreted protein. Figure 4C indicates anti- mouse TSC-36 antibody readily detects TSC-36 protein ectopically expressed in both COS cell lysates and culture media. The molecular mass found for secreted

TSC-36 in media appears larger than that found in cell lysates or the primary translation product, indicating of post-translational modifications. This is consistent with a report on the glycosylation of TSC-36 (Hambrock et al., 2004).

To determine the TSC-36 protein half life, 3T3-L1 preadipocytes were treated with cycloheximide and harvested at different time points after treatment.

The Western blot in Figure 4D shows that the TSC-36 protein level began to decrease 2 h after treatment and declined sharply by 4 h, indicative of a half-life between 2 and 4 h.

128 To explore TSC-36 protein expression during adipogenesis, 3T3-L1 cell

lysates and culture media were collected prior to induction of adipogenesis as

confluent preadipocytes (day 0), or at daily times post-induction of differentiation

with the standard adipogenic cocktail. An anti-PPARγ antibody was used to show

efficient differentiation of 3T3-L1 cells. TSC-36 protein level in cell lysates was

downregulated upon treatment with adipogenic agents (Figure 5A). Figure 5B

illustrates that TSC-36 is secreted into culture media as a “preadipokine” by 3T3-

L1 cells. The TSC-36 protein level in media declined significantly by D3,

coincident with withdrawal of the adipogenic cocktail. We note that TSC-36

protein levels in media decrease earlier than those in the cell lysate. This may

due to either reduced secretion from the cell or accelerated degradation of the

TSC-36 protein in media.

Regulation of TSC-36 Expression

To investigate signals that might regulate TSC-36 transcript level in

preadipocytes, we first individually tested the effects of the components of

standard adipogenic cocktail on expression of TSC-36 transcript. We find that

only Dex and Mix in combination results in downregulation of TSC-36 transcript

levels in 3T3-L1 preadipocytes (Figure 6A).

It is well known that TNFα is a contributing cause of the insulin resistance seen in obesity and obesity-linked diabetes. Moreover, TNFα suppresses

adipocyte-specific genes and activates select preadipocyte genes in 3T3-L1 cells

(Ruan et al., 2002a). Figure 6B indicates that TSC-36 transcript level was

129 upregulated by TNFα treatment in 3T3-L1 adipocytes. However, Pref-1 transcript

level remains largely constant after TNFα treatment. Decreased SMAF-1 and

PPARγ transcript expression indicate de-differentiation of the adipocytes, as expected. The Western blot results demonstrate that the protein levels of TSC-36 in cell lysate (Figure 6C) and culture media (Figure 6D) are also enhanced by

TNFα treatment of adipocytes.

To investigate how TSC-36 transcript expression is modulated in preadipocytes, a number of different agents relevant to adipogenesis were tested.

3T3-L1 preadipocytes were treated for 24 h with indicated agents and RNA

samples harvested for Northern blot with a TSC-36 probe (Figure 6E). Among all agents tested, 5-azacytidine was found to dramatically downregulate TSC-36 transcript. This result was repeated by triplicate experiments presented in Figure

6F. The time course experiment of 5-azacytidine treatment showed TSC-36

began to decline at 4 h and was sharply decreased at 12 h post-treatment. The

transcript level of a control, 36B4, remained constant even at 48 h post-treatment

(Figure 6G). Secreted TSC-36 transcript in 3T3-L1 preadipocyte media was also

diminished at 24 h after 5-azacytidine treatment (Figure 6H). 5-azacytidine is a

chemical analogue of the cytosine nucleoside. The incorporation of 5-azacytidine

into DNA leads to the inhibition of methyltransferase activity to result in

demethylation. It has been shown that in cultures of Swiss 3T3 and C3H10T1/2

mouse cells treated with 5-azacytidine, three new mesenchymal phenotypes

characterized as muscle cells, biochemically differentiated adipocytes and

130 chondrocytes emerged (Taylor and Jones, 1979). We hypothesize that TSC-36

may be involved in cell fate determination of the preadipocyte lineage.

Functional Analysis of the TSC-36 Promoter Region

To date, there is no information regarding the transcriptional control of the

TSC-36 promoter. Two regions of the 5' flanking regulatory region of TSC-36,

791 bp and 3922 bp, were used to generate luciferase reporter constructs

(Figure 7A). 3T3-L1 (Figure 7B) and WT-BAT (Figure 7C) preadipocytes were

transfected with these luciferase reporter constructs or empty pGL2-basic vector.

The relative luciferase activities of the two TSC-36 promoter constructs are both

markedly elevated compared with the pGL2-basic negative control.

PPARγ is a master transcription factor that induces expression of a

number of adipocyte genes. To assess if downregulation of preadipocyte genes

can also be mediated by PPARγ, the TSC-36 luciferase reporter constructs were

utilized in cotransfection studies by expression of PPARγ in the presence or

absence of its obligate heterodimerization partner RXRα, in NIH-3T3 cells. 24 h prior to harvest, cells were treated with H2O or 10 μM 15-deoxy-∆12, 14-

prostaglandin J2, a PPARγ ligand. Cell lysates were assayed at 48 h post-

transfection. As shown in Figure 7D, compared to the luciferase activities of the

pcDNA control groups, PPARγ alone or with various combinations of ligand or

RXRα significantly downregulated luciferase reporter activities of the -3922/+22, and -791/+60 TSC-36 promoter constructs. The decreases of luciferase reporter

131 activities of the two promoters are in the order of PPARγ+ RXRα +ligand>

PPARγ+ RXRα > PPARγ+ligand> PPARγ.

In addition to the well characterized C/EBP and PPARγ transcription factors, studies over the last several years indicated that the krüppel-like zinc finger transcription factors (KLFs) are involved in adipogenesis (Li et al., 2005;

Mori et al., 2005). KLFs are DNA-binding transcriptional regulators containing

Cys2/His2 zinc fingers that play diverse roles during differentiation and development (Black et al., 2001; Dang et al., 2000). 16 members of KLF family have been identified and they all bind to very similar “GT-box” or “CACCC element” sites on DNA (Bieker, 2001; Black et al., 2001). KLF15 is reported to induce PPARγ expression at an early stage of adipogenesis and overexpression of KLF15 induces adipocyte maturation and GLUT4 expression (Mori et al.,

2005). We therefore examined if KLF15 had effects on the TSC-36 promoter.

The Northern blot in Figure 7E shows that the KLF15 transcript was upregulated while TSC-36 was decreased during 3T3-L1 differentiation. By promoter analysis using MatInspector software, we identified four potential KLF15 biding sites within 4000 bp of the upstream of the TSC-36 transcription start site. Three of these sites are closely grouped at the -280 to -350 bp region in a tandem manner and a fourth is located between -70 and -86. Therefore we designed two additional luciferase reporter constructs, -350/+60 LUC containing all four sites and -280/+60 LUC only containing the most proximal site, as illustrated in Figure

7F. In both 3T3-L1 (Figure 7G) and WT-BAT (Figure 7H), the relative luciferase

132 activities of all three promoter constructs, -280/+60 LUC, -350/+60 LUC and -

791/+60 LUC, are markedly downregulated by KLF15.

Discussion

Extensive studies have led to the establishment of a transcriptional

cascade model of adipogenesis that includes the sequential activation of C/EBPs

and PPARγ (Graves et al., 1992; Rosen et al., 2002; Tontonoz et al., 1994b).

The expression of these well known transcription factors are increased during adipogenesis. However, decreases in gene expression that occur during adipocyte differentiation are far less studied. The characterization of such genes is important for elucidating the molecular definition and developmental lineage of preadipocytes. These preadipocyte enriched genes are important in adipogenesis in at least in two aspects: (1) they can serve as preadipocyte markers to contribute to a better understanding of preadipocyte lineage formation from mesodermal cells, and (2) they may function as inhibitory factors to reduce adipocyte differentiation.

Through differential hybridization screening of nylon DNA arrays we identified TSC-36 as a gene highly expressed in 3T3-L1 preadipocytes and downregulated to nearly undetectable levels in 3T3-L1 adipocytes. Data from microarray studies indicate that the gene expression pattern in preadipocytes and adipocytes in vitro and in vivo is considerably more complex than previously appreciated; some gene regulators associated with in vivo vs. in vitro adipogenesis are overlapping, but a large number of genes are highly

133 differentially expressed in vitro vs. in vivo (Soukas et al., 2001b). We therefore examined TSC-36 transcript in SVF and AF of adipose tissue as a model closely related to in vivo white adipogenesis, TSC-36 transcript is detected exclusively in the preadipocyte-contained SVF. Primary preadipocyte differentiation is another important model to represent in vivo adipogenesis. As expected, TSC-36 transcript level also decreased during adipogenesis of primary preadipocytes. In addition to white adipose tissue, mammals also have brown adipose tissue.

Brown adipocytes are rich in mitochondria and exist in the interscapular fat pad of rodents. In contrast, humans have a significant amount of brown adipose tissue only in the neonatal period. Some studies have suggested that small numbers of brown adipocytes are present within human white fat depots

(Champigny and Ricquier, 1996; Garruti and Ricquier, 1992). Though it is difficult to accurately assess the contribution of brown fat cells to the overall metabolic rate in adult humans, there is no doubt that these cells exist. They may represent a potentially important target whose study may lead to effective obesity treatments by allowing excessive energy intake to be released as heat rather than stored as triglyceride. TSC-36 transcript is highly expressed in brown preadipocytes but cannot be detected after the 3-day adipogenic induction period.

In addition, TSC-36 transcript level is markedly downregulated in several other in vitro white and brown adipogenesis models.

TNFα has been recognized as a link between obesity and insulin resistance. Treatment of mature 3T3-L1 adipocytes with TNFα results in a degree of morphological, biochemical, and transcriptional dedifferentiation to

134 preadipocytes. A number of adipocyte-expressed genes, for example those that

are critical for insulin responsiveness, are downregulated concomitant with

phenotypic alteration. On the other hand, select preadipocyte genes are

upregulated (Ruan et al., 2002a). TSC-36 evidences a tight link to the preadipocyte phenotype as its expression by closely tracked with the dedifferentiation caused by TNFα. In contrast to TSC-36, Pref-1, which has been studied previously as a 3T3-L1 preadipocyte marker, is neither expressed in brown adipocytes nor regulated during TNFα treatment of 3T3-L1 adipocytes.

Together, these results demonstrate that TSC-36 might be a reliable preadipocyte signature gene to recapitulate common traits of not only in vivo and in vitro white adipogenesis, but also in vitro brown adipogenesis.

At protein level, by Western blot we showed that TSC-36 is a secreted protein. Therefore if TSC-36 has any impact on adipogenic cell differentiation, it might exert such effects through an autocrine or paracrine signaling pathway. On the other hand, members of the SPARC protein family have been demonstrated to contribute to the organization of the extracellular matrix (ECM), for instance via growth factor modulation, cell adhesion inhibition, and proteinase inhibition

(Bradshaw and Sage, 2001). Furthermore, TSC-36 was first identified as a TGF-

β1 stimulated transcript; TGF-β1 has significant effects on ECM-related proteins through the induction of proteinase inhibitors and the suppression of proteinases that degrade matrix proteins (Shibanuma et al., 1993). Matrix remodeling is thought to be essential for adipogenesis. During ECM remodeling adipogenic cells release their cell-ECM adhesion and alter morphology (Lilla et al., 2002).

135 Therefore we postulate that TSC-36 might affect adipogenesis through regulating

ECM remodeling.

5-azacytidine was shown to dramatically downregulate TSC-36 transcript and protein in 3T3-L1 preadipocytes. It is known that precursor cell types (Swiss

3T3 and C3H10T1/2 cells) can undergo differentiate to muscle, cartilage and fat cells after treatment with 5-azacytidine (Taylor and Jones, 1979). In fact, adipose tissue itself can be a source of pluripotential stem cells. The adipose stromal compartment contains putative stem cell populations which can differentiate in vitro towards the osteogenic, adipogenic, myogenic, neuronal and chondrogenic lineages when treated with lineage-specific factors (Zuk et al., 2002). Studies also showed that in the absence of Wnt, which is a preadipocyte-expressed gene, myoblast cells can be differentiated to adipocytes (Ross et al., 2000). Thus we postulate that select preadipocyte genes, including TSC-36, might play essential roles in cell fate decisions.

Our preliminary data indicates that PPARγ downregulates the TSC-36 gene promoter. Inspection of the DNA sequence through -3922 bp of the TSC-36

5' flanking region using MatInspector software indicates that PPARγ/RXRα consensus binding sites are present at -2278 to -2298 , -2923 to -2943, and -

3720 to -3740. However, the responsiveness of the -791/+60 construct suggests the possibility that non-canonical PPARγ binding sites might also exist within this sequence. Given the central role of PPARγ in adipogenesis, further characterization of the molecular details underlying TSC-36 downregulation will clarify the specific mechanisms of how PPARγ governs the expression of this

136 unique preadipocyte gene. Furthermore, with such information, hypothesis might be established for identifying a common mechanism(s) for the differentiation– dependent downregulation of gene expression during adipogenesis.

Additionally, KLF15 also downregulated the TSC-36 promoters. It has been demonstrated that inhibition of KLF15 can reduce PPARγ expression and block conversion of 3T3-L1 preadipocytes to adipocytes. Moreover, PPARγ2 promoter activity can be synergistically elevated by KLF15 and C/EBPα in 3T3-

L1 adipocytes (Mori et al., 2005). Our results indicate that KLF15 can downregulate the TSC-36 promoter, although further studies are needed to determine whether KLF15 exerts these effects directly on the TSC-36 promoter or indirectly through PPARγ. The facts strongly suggest that KLF15 and PPARγ are both responsible for the differentiated-dependent downregulation of TSC-36 gene expression. Therefore, the overall picture of preadipocyte-gene downregulation is likely complex.

A recent publication revealed that TSC-36 is an Akt induced factor in cardiomyocytes (Oshima et al., 2008). Akt is a key regulator for cardiac myocyte development. Akt transgenic mice with cardiac-specific TSC-36 expression showed increased cardiac hypertrophy, coronary angiogenesis and VEGF

(vascular endothelia growth factor) secretion (Shiojima et al., 2005). Oshima et al. identified TSC-36 transcript to be upregulated in the hearts of Akt transgenic mice. This report showed that TSC-36 is secreted by neonatal rat ventricular myocytes (NRVMs) and that overexpression of TSC-36 protects NRVMs from hypoxia/reoxygenation-induced apoptosis. Intriguingly, ectopically expressed

137 TSC-36 elevates the levels of phosphorylated Akt as well as those of the downstream targets of Akt, phosphorylated mTOR and Foxo. The other important myocardiac protector, ERK phosphorylation is also promoted by TSC-36

(Oshima et al., 2008). Following this initial report, the same research group published another second study on TSC-36 and skeletal muscle (Ouchi et al.,

2008). Akt overexpression in skeletal muscle promotes myocyte hypertrophy and angiogenesis. The authors generated muscle-specific Akt1 transgenic mice and identified TSC-36 as an Akt-induced gene in gastrocnemius muscle. TSC-36 is upregulated in conditions of muscle ischemia and subsequently promotes revascularization. In vitro study of human umbilical vein endothelium cells

(HUVECs) indicates that TSC-36 facilitates endothelial network formation, promotes cell migration and suppresses endothelia apoptosis. Overexpression of

TSC-36 in HUVEC leads to enhanced phosphorylation of Akt and its downstream targets eNOS (endothelia nitric oxide synthase) and GSK-3β, but TSC-36 has no effects on ERK in this case.

Given the facts that myocytes and preadipocytes have the same mesodermal origin, these two reports may reveal novel directions for functional characterization of TSC-36 in adipogenesis (Brady et al., 1998; Kim et al., 2007a).

The PI3-kinase-Akt signaling pathway is activated by insulin and other growth factors and plays important roles in adipocyte differentiation. Suppression of PI3- kinase by dominant negative mutants or inhibitors including LY294002 and wortmannin blocks 3T3-L1 adipogenic conversion (Sakaue et al., 1998; Xia and

Serrero, 1999). ERK has been shown to play different roles in adipogenesis in a

138 time-dependent manner. ERK is required at the early stage to promote mitotic clonal expansion and later on it needs to be turned off to maintain activation of

PPARγ (Bost et al., 2005). Foxo, a downstream target of Akt, is a negative factor in differentiation. Overexpression of constitutively active Foxo1 increases expression of the cell cycle inhibitor, p21, and abolishes differentiation (Nakae et al., 2003). In addition, Foxo1 also binds to the promoter of PPARγ as a repressor as well as attenuates PPARγ transcriptional activity (Armoni et al., 2006; Dowell et al., 2003). Glycogen synthase kinase-3 (GSK-3), another downstream factor in

Akt signaling, is involved in Wnt pathways. Inhibition of GSK-3 leads to activation of the Wnt pathway and subsequently suppresses differentiation (Longo et al.,

2004; Ross et al., 2000). Additionally, TSC-36 was shown to stimulate phosphorylation of nitric oxide synthase in vascular endothelium (Ouchi et al.,

2008). Nitric oxide (NO) is a highly reactive free radical and functions to modulate cell migration, differentiation, proliferation and development (Kuzin et al., 1996;

Patel et al., 2000). Adipose tissue has the capacity to produce NO (Ribiere et al.,

1996). The production of NO from preadipocytes is enhanced during the first 2 days of in vitro differentiation (Yan et al., 2002). There are two isoforms of NOS

(nitric oxide synthase) termed inducible NOS (iNOS) and endothelial NOS

(eNOS). eNOS may be the major NOS involved in adipogenesis regulation given the fact that specific iNOS inhibitors have no significant effects on NO production and adipogenic conversion (Yan et al., 2002). The RNA and protein expression levels of eNOS are increased in SC WAT in human obese subjects.

Diphenyliodonium (DPI), a NOS inhibitor, has the ability to inhibit lipolysis in

139 adipocytes (Gaudiot et al., 2000). On the other hand, the NO releasing reagent,

hydroxylamine (HA), and NOS substrate L-arginine (Arg) both promote

differentiation of rat primary preadipocytes (Yan et al., 2002).

Together, the facts indicate that Akt and ERK signaling as well as their downstream targets play complicated roles in adipocyte differentiation. Whether

TSC-36 functions through Akt and ERK signaling in adipogenesis needs to be clarified. By performing gain- or loss-of-function studies of TSC-36, the phosphorylation of Akt, ERK, Foxo and GSK-3, eNOS as well as the NO level in cell culture models or in animal models can be examined. Additionally, the inhibitors or agonists of Akt or ERK signaling can be applied to further investigate the role of TSC-36 in adipogenesis. Our preliminary analysis indicated that the

PI3-kinase inhibitor LY294002 showed no effects on TSC-36 transcript expression in 3T3-L1 preadipocytes (data not shown). The angiogenesis in adipose tissue should also be investigated if the TSC-36 transgenic or knockdown mice are available because of the tight association of angiogenesis and adipogenesis.

In summary, we demonstrate that TSC-36 is a new important preadipokine secreted by preadipocytes. Future studies from this laboratory will address the mechanism(s) underlying the function and regulation of TSC-36 in adipogenesis.

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147 Figure Legends

Figure 1. TSC-36 is a gene highly expressed in preadipocytes, but almost

undetectable in adipocytes. A. Initial differential screening identification of

TSC-36 as a novel gene enriched in 3T3-L1 preadipocytes. Shown is the portion

of the DNA filter array indicating the specific TSC-36 signal (arrow) in 3T3-L1

preadipocytes (Pre) and lack of signal in 3T3-L1 adipocytes (Adi). B. TSC-36

downregulation after 3T3-L1 adipogenic conversion. 3T3-L1 cells were harvested

before induction of adipogenesis (Pre) and 7 d after induction (Adi). Northern blot

analysis was performed with murine TSC-36 and SCD1 cDNA probes. The EtBr

staining of rRNA is shown as a gel loading control.

Figure 2. TSC-36 RNA expression is decreased in white and brown

adipogenesis. A. TSC-36 downregulation during 3T3-L1 differentiation. 3T3-L1

cells were harvested prior to induction of adipogenesis (day 0) and at indicated

intervals post-induction of adipogenic differentiation with the standard adipogenic

cocktail protocol. Northern blot shows transcript level for TSC-36, Pref-1 and

aFABP. B. TSC-36 transcript downregulation during adipogenic conversion of rat

primary preadipocytes. RNA samples were collected at indicated times post-

induction of differentiation and analyzed by Northern blot using murine TSC-36,

SCD1 and SMAF1 cDNA probes. C. TSC-36 transcript in stromal vascular fraction (SVF) and adipocyte fraction (AF) of murine subcutaneous adipose tissue. Northern blot shows transcript level for TSC-36, SMAF1, SCD1 and Retn.

D. Real-time PCR assessment of TSC-36 transcript level in cell fractions of white 148 adipose tissue (WAT). cDNA derived from the cells of the stromal vascular

fraction (SVF) or adipocyte fraction (AF) of subcutaneous (SC) or epididymal (EP)

WAT was used for real-time PCR analysis of TSC-36. The transcript level in SC

AF was set as a value of 1. * indicates p<0.001 for SC SVF vs. SC AF and EP

SVF vs. EP AF. # indicates p<0.001 for EP SVF vs. SC SVF. E. TSC-36 RNA

expression is decreased in brown adipogenesis. WT-BAT preadipocytes were

harvested at confluence (day 0) and adipocytes were harvested at day 3 and day

8. 3T3-L1 preadipocytes at confluence without induction (day0) are positive

control. Northern blot analysis was performed with murine UCP1, TSC-36, Pref-1

and aFABP probes. F. Real-time PCR analysis of TSC-36 transcript levels in preadipocytes (Pre) and adipocytes (Adi) of 3T3-L1, ScAP-23, AD3.7AC, mBAP-

9, WT-BAT cell lines. Value in the respective preadipocyte sample was set to 1

and * indicates p<0.001 for Pre vs. Adi. For panel A, B, C, E, The EtBr staining of

rRNA is shown as a gel loading control.

Figure 3. TSC-36 transcript level in a panel of murine tissues. 5 g of total

RNA from indicated mouse tissues were analyzed by Northern blot using murine

TSC-36 cDNA probe. The EtBr staining of rRNA is shown as a gel loading control.

Figure 4. TSC-36 encodes a secreted protein. A. Western blot analysis of in vitro translation products of HA-tagged TSC-36 expression construct or empty vector. 25 l of a 1:100 dilution of in vitro translation products was subjected to

Western blot analysis using anti-HA antibody. Positive control (+) is media from 149 TSC-36 transfected COS cells. Molecular mass markers in kDa are shown at

right. B, C. Vector only or TSC-36-HA expression construct was transfected into

COS cells. Culture media and cell lysates were analyzed by Western blot using

anti-HA (B) or anti-TSC-36 (C) primary antibody. D. TSC-36 protein half life.

3T3-L1 preadipocytes were treated with 5 µg/ml of cycloheximide and harvested

at different time points after treatment. Western blot analysis was performed

using anti-TSC-36 antibody. The membrane was stripped and re-probed with

anti-β tubulin as a loading control.

Figure 5. Western blot analysis of TSC-36 protein expression during 3T3-L1

cell differentiation. A, B. TSC-36 downregulation in 3T3-L1 cell lysates (A) and

culture media (B) during differentiation. 3T3-L1 cells and culture media were

harvested prior to induction of adipogenesis from confluent preadipocytes (day 0), or daily post-induction of differentiation. Western blot was performed using anti-

TSC-36 primary antibody. For panel A, the membrane was stripped and re- probed with anti-PPAR primary antibody as an adipocyte marker and anti-PPIA

(peptidylprolyl isomerase A) primary antibody as a loading control. For panel B, the gel was stained with Coomassie Blue to assess even sample loading.

Figure 6. Regulation of TSC-36 expression. A. TSC-36 transcript regulation by adipogenic cocktail components. Post-confluent 3T3-L1 preadipocytes were treated with 1 µM Dex or 0.5 mM Mix or both Mix and Dex for 48 h. Northern blot

was performed using murine TSC-36 cDNA probes. Duplicate sets of samples

150 are shown. B. TSC-36 transcript level in 3T3-L1 adipocyte during treatment with

TNFα. Mature 3T3-L1 adipocytes were treated with 10 ng/ml TNFα. RNA

samples were collected at indicated times. Northern blot was performed using

murine TSC-36, SMAF1, PPARγ and Pref-1 cDNA probes. C and D, TSC-36

protein level in cell lysate (C) as well as in media (D) in 3T3-L1 adipocyte after

treatment with TNFα. Mature 3T3-L1 adipocytes were treated with 10 ng/ml

TNFα for 24 h or 48 h. Duplicate sets of samples are presented. For panel C,

samples were run in a parallel gels and blotted with anti-PPIA antibody as a

loading control. For panel D, the gel was stained with Coomassie Blue solution to

assess even sample loading. E. Effects of various agents on TSC-36 transcript expression. Confluent 3T3-L1 cells were treated with the indicated agents for 24 h. RNA samples were collected and analyzed by Northern blot using murine

TSC-36 cDNA probe. The abbreviations used are: b-FGF, basic fibroblast growth

factor; IL-6, interleukin 6; BMP4, bone morphogenetic protein 4; rhEGF,

recombinant human epidermal growth factor; T3, triiodothyronine; VEGF,

vascular endothelial cell growth factor; cAMP, cyclic adenosine monophosphate;

LPS, lipopolysaccharide; TSA, trichostatin A. F. Triplicate experiments of 5-

azacytidine treatment were performed. Northern blot was performed using

murine TSC-36 cDNA probe. G. The regulation of TSC-36 transcript expression

during a time course of 5-azacytidine treatment. 3T3-L1 preadipocytes were

treated with 1 mM 5-azacytidine or control for indicated time. Northern blot was

performed using a murine TSC-36 probe and a 36B4 probe control. H. Secreted

TSC-36 protein expression decreased after 5-azacytidine treatment. 3T3-L1

151 preadipocytes were treated by 1 mM 5-azacytidine or control for 24 h. Culture media was collected for Western blot analysis by using anti-TSC-36 antibody.

The gel was stained with Coomassie Blue solution to show the even loading. For panel A, B, E, F, G, the EtBr staining of rRNA is shown as a gel loading control.

Figure 7. Promoter activity of murine TSC-36 5’ flanking region. A.

Schematic diagram of TSC-36-luciferase constructs. The solid line represents the TSC-36 promoter region and the green-hatched box represents the luciferase reporter gene. The numbers to the left indicate the 5' terminal nucleotide of the

TSC-36 5' flanking region contained in the corresponding reporter constructs.

The numbers above the white box indicate the 3' terminal nucleotide of the reporter constructs. B, C. Promoter activity of TSC-36 5’ flanking region in preadipocytes. 3T3-L1 (B) and WT-BAT (C) preadipocytes were transfected with the luciferase reporter construct containing 791 bp or 3922 bp of the 5’ flanking region of the TSC-36 gene or empty pGL2-Basic vector. *indicates p<0.001 for comparison with empty pGL2-Basic vector. D. NIH-3T3 cells were transfected with indicated promoter constructs in the presence or absence (black bars) of cotransfection with PPAR/RXRα 24 h prior to harvest, cells were treated with 10

μM 15d-PGJ2, a PPAR ligand. Cell lysates were assayed at 48 h post- transfection. **indicates p<0.001 for comparison with empty pGL2-Basic vector.

*indicates p<0.001 for comparison with the absence of PPAR/RXRα (black bar).

# indicates p<0.05 for comparison with the absence of PPAR/RXRα (black bar).

E. KLF15 upregulation during 3T3-L1 differentiation. 3T3-L1 cells were harvested 152 prior to induction of adipogenesis (day 0) and at indicated intervals post-induction of adipogenic differentiation. Northern blot shows transcript level for KLF15 and

TSC-36. The EtBr staining of rRNA is shown as a gel loading control. F.

Schematic diagram of TSC-36-luciferase constructs containing putative KLF15 binding sites. The black squares illustrate putative KLF15 binding sites. The numbers above black triangles indicate the positions of putative KLF15 binding sites. The green-hatched box represents the luciferase reporter gene. The numbers to the left indicate the 5' terminal nucleotide of the TSC-36 5' flanking region contained in the corresponding reporter constructs. The numbers above the white box indicate the 3' terminal nucleotide of the reporter constructs spanning through the TSC-36 transcript. G, H. Promoter activities of TSC-36 5’ flanking region and regulation by KLF15 in preadipocytes. 3T3-L1 (G) and WT-

BAT (H) preadipocytes were transfected with the luciferase reporter construct containing 280 bp, 350 bp or 791 bp of the 5’ flanking region of the TSC-36 gene, with KLF15 or pcDNA as control. * indicates p<0.001 and # indicates p<0.05 for comparison with cotransfection of pcDNA (black bar). Firefly luciferase activities were corrected against the value for cotransfection of pRL-null renilla luciferase control. Data represent the mean  S.D. from a minimum of triplicate transfections. The results were determined statistically significant by single factor

ANOVA.

153 FIGURE 1

A. Pre B. Pre Adi TSC-36 SCD1

TSC-36 TSC-36

rRNA Adi

154 FIGURE 2

B. Primary Rat Preadipocytes A. 3T3-L1 0 2 3 5 7 d 0 2 5 7 d

TSC-36 TSC-36

Pref-1 SCD1

aFABP SMAF1

rRNA rRNA

C. SVF AF D. 160 SMAF1 SC EP # 140 * 120 SCD1 100 80

Retn 60 * 40

Transcript TSC-36 20 TSC-36

0 SVF AF

rRNA

155 FIGURE 2

E. Brown 3T3-L1

0 3 8 0 d

UCP1

TSC-36

Pref-1

aFABP

rRNA

F. Pre Adi

120 * * * * * 100

80

60

40

20

(%) Level Transcript TSC-36

0 3T3-L1 ScAP-23 AD 3.7 AC mBAP-9 WT-BAT 156 FIGURE 3

s le u b e h s u n c e AT y n i u a T l e s t t i n r e . c W n t g r i e s m m T n a e e y m s d s a t o C A i u e r iv pl h t e u n S B K Te L H B L S I T S S M

TSC-36

rRNA

157 FIGURE 4

A

A. A

H H -

-

6 6 3

3

- - C

C

S S

Vector T (+) T

50

37

B. Media Cell C. Media Cell

l

A

A

A

A

o

r

H

H H

H

t

-

-

-

-

6

6

6

6 n

r

r r

r

3

3

3

3

o

o

o

o

o

-

-

-

-

t

t

t

t

c

C

C

C

C

c

c c

c

A

e

e

e

e

S

S

S

S

V

V

T V

T T

H T V

50 50 37 37

Ab: HA TSC-36

D. Cycloheximide 0 0.5 1 2 4h

Ab: TSC-36

Ab: β-tubulin

158 FIGURE 5

0 1 2 3 4 5 6 7 8 d A.A

37 Ab:TSC-36

Ab:PPARγ Ab:PPIA

B 0 1 2 3 4 5 6 7 8 d

Ab:TSC-36 37

Coomassie Blue

159 FIGURE 6

IX IX /M /M x x x x e IX e e IX e A. 0 D M D D M D

TSC-36

rRNA

B. 0 1 6 12 24 36 h

SMAF1

PPAR

TSC-36

Pref-1

rRNA

160 FIGURE 6

C. 02448h

Ab:TSC-36

Ab:PPIA

D. 24 h 48 h

TNFα --++ --++

Ab:TSC-36

Coomassie Blue

E.

Ligand

ic acid ic methacin F α

inoino 6 Azacytidine et et - γ PPAR TNF VEG Control T3 cAMP Tamoxifen LPS TSA LPS Dex Insulin Sodium butyrate Sodium 5 Sodium butyrate Sodium Indo Mix rhEGF b-FGF BMP4 Forskolin Control R R IL- Control

161 FIGURE 6

F. Control 5-Azacytidine

G. 5-Azacytidine Control 0 1 2 4 6 12 24 48 0 1 2 4 6 12 24 48 h

TSC-36

36B4

rRNA

162 FIGURE 7

A.

pGL2-Basic vector LUC

+60 -791/+60 LUC -791 LUC

+22 -3922 -3922/+22 LUC LUC

B. C. 1.4 * 5 1.2 4.5 * 4 1 3.5 0.8 3 * 2.5 * 0.6 2 0.4 1.5 1 0.2 0.5

Relative Luciferase Activity 0 Relative Luciferase Activity 0 pGL2-Basic -791/+60 LUC -3922/+22 LUC pGL2-Basic -791/+60 LUC -3922/+22 LUC

163 FIGURE 7

D. 1.4 **

1.2 ** pcDNA 1 PPARγ # PPARγ+ligand PPARγ+RXRα 0.8 PPARγ+RXRα+ligand

0.6 * 0.4 * * * Relative LuciferaseRelative Activity 0.2 * * * 0 pGL2 -791/+60 LUC -3922/+22 LUC

3T3-L1 E.

0 1 2 3 5 7 d

KLF15

TSC-36

rRNA

164 FIGURE 7

F. -311 ~ -327 -287 ~ -303 -70 ~ -86 -70 ~

+60 -791/+60 LUC LUC -309-324 ~ +60 -350/+60 LUC LUC

+60 -280/+60 LUC LUC

165 FIGURE 7

G. 0.45 w/pcDNA 0.40 w/KLF15 0.35

0.30

0.25

0.20

0.15 * Luciferase Activity 0.10 * * 0.05

0.00 Relative pGL2 -280/+60 LUC -350/+60 LUC -791/+60 LUC

H. 0.9 w/pcDNA 0.8 w/KLF15 0.7

0.6

0.5 0.4 * * 0.3 #

0.2

0.1

0

Relative Luciferase Activity pGL2-280/+60 LUC -350/+60 LUC -791/+60 LUC

166 MANUSCRIPT # 3

Differential screening identifies transcripts with depot-dependent expression in white adipose tissues

Yu Wu#, Ji Young Kim#, Shengli Zhou and Cynthia M. Smas*

# Equal first co-authorship contribution

BMC Genomics. 2008 Aug 22;9:397

Department of Biochemistry and Cancer Biology University of Toledo Health Science Campus Toledo, OH 43614 USA

Please address correspondence to:

Cynthia M. Smas D.Sc. (*Corresponding author) Department of Biochemistry and Cancer Biology University of Toledo Health Science Campus Toledo, OH 43614 USA Phone: 419-383-4131 FAX: 419-383-6228

YW: [email protected] JYK: [email protected] SZ: [email protected] CMS: [email protected]

167 Abstract Background - The co-morbidities of obesity are tied to location of excess fat in the intra-abdominal as compared to subcutaneous white adipose tissue

(WAT) depot. Genes distinctly expressed in WAT depots may impart depot- dependent physiological functions. To identify such genes, we prepared subtractive cDNA libraries from murine subcutaneous (SC) or intra-abdominal epididymal (EP) white adipocytes.

Results - Differential screening and qPCR validation identified 7 transcripts with 2.5-fold or greater enrichment in EP vs. SC adipocytes. Boc, a component of the hedgehog signaling pathway demonstrated highest enrichment

(~12-fold) in EP adipocytes. We also identified a dramatic enrichment in SC adipocytes vs. EP adipocytes and in SC WAT vs. EP WAT for transcript(s) for the major urinary proteins (Mups), small secreted proteins with pheromone functions that are members of the lipocalin family. Expression of Boc and Mup transcript was further assessed in murine tissues, adipogenesis models, and obesity. qPCR analysis reveals that EP WAT is a major site of expression of Boc transcript. Furthermore, Boc transcript expression decreased in obese EP WAT with a concomitant upregulation of Boc transcript in the obese SC WAT depot.

Assessment of the Boc binding partner Cdon in adipose tissue and cell fractions thereof, revealed transcript expression similar to Boc; suggestive of a role for the

Boc-Cdon axis in WAT depot function. Mup transcripts were predominantly expressed in liver and in the SC and RP WAT depots and increased several thousand-fold during differentiation of primary murine preadipocytes to

168 adipocytes. Mup transcripts were also markedly reduced in SC WAT and liver of

ob/ob genetically obese mice compared to wild type.

Conclusions - Further assessment of WAT depot-enriched transcripts may

uncover distinctions in WAT depot gene expression that illuminate the

physiological impact of regional adiposity.

Background In addition to its role in energy storage and mobilization, white adipose

tissue (WAT) is an important endocrine organ that synthesizes and secretes

various hormones and adipokines, a number of which impact systemic energy

balance [1-4]. Various studies in humans and rodents have illuminated

distinctions in the physiology, lipolytic response, gene expression and other

aspects of adipocytes present in different WAT depots [5-20]. These

observations have led to the suggestion that individual WAT adipose depots are

best regarded as separate "miniorgans" [10]. These distinctions, and their

molecular underpinnings, are gaining in importance with the realization that it is

the anatomical location of excess adipose tissue that appears to underlie the health impact of obesity, and that interventions targeting reduction of intra- abdominal fat mass can effectively combat obesity-related diseases [15, 21-25].

Several recent studies have sought to identify gene expression distinctions among preadipocytes or adipocytes of different WAT adipose depots [19, 20].

However, a complete and defining picture of WAT depot dependent gene expression, as well as the underlying regulatory events governing depot- dependent gene expression, is yet to be identified.

169 In order to identify WAT depot-enriched transcripts, we undertook preparation and screening of murine suppressive subtractive hybridization (SSH) cDNA libraries enriched for genes expressed in either SC or EP murine adipocytes. Our studies reveal that transcripts for Mups, major urinary protein members of the lipocalin superfamily with pheromone function, exhibit a surprisingly distinctive pattern of transcript expression in WAT depots with dramatic upregulation noted for subcutaneous (SC) WAT and retroperitoneal (RP)

WAT vs. the epididymal (EP) intra-abdominal WAT depot. SSH screening also identified 7 transcripts with enriched expression in EP adipocytes vs. SC adipocytes. Of these, Boc, an immunoglobulin superfamily member that functions in the hedgehog signaling network, exhibited the highest degree of differential expression.

Methods

Animal use and cellular fractionation of murine adipose tissues

All animal treatments were conducted with the approval of the University of Toledo Health Science Campus Institutional Animal Care and Use Committee.

Mice were purchased from The Jackson Laboratory. For Northern blot and qPCR analyses of murine tissues, including in distinct adipose depots, 8-wk old

C57Bl/6J male mice were utilized. For studies of gene expression in obese vs. wild type mice, we used 8-wk old male mice that were ob/ob homozygous on a

ob/ob C57Bl/6J background (strain designation, B6.V-Lep ) or wild type C57Bl/6J

170 mice generated from breeding of ob/+ heterozygotes. Fractionation of whole

adipose tissue into adipocyte fraction (AF) and stromal-vascular fraction (SVF)

was via collagenase digestion and differential centrifugation, as previously

described [26-28], starting with pooled tissue of 6 mice. Resultant cell fractions

were either used directly for RNA preparation, or in the case of primary culture

differentiation studies (see below) SVF cells were plated and cultured in DMEM with 10% FCS.

RNA preparation and transcript analysis

For analysis of transcript expression in murine adipose depots, the SC, retroperitoneal (RP), and EP WAT and interscapular brown adipose tissue (BAT)

were dissected from four individual 8-wk old male C57Bl/6J mice. Tissues were

minced, frozen in liquid nitrogen, and homogenized in TriZol reagent using a

polytron. Other murine tissues were similarly processed. Total RNA was purified

using TriZol reagent according to manufacturer’s instruction (Invitrogen Corp.).

For Northern blot analysis, 5 μg of total RNA was fractionated in 1% agarose-

formaldehyde gels in MOPS buffer and transferred to Hybond-N membrane (GE

Healthcare, Piscataway, NJ). Blots were hybridized in ExpressHyb solution (BD

Biosciences) for 1 h at 65C with the indicated randomly primed 32P-dATP-

labeled cDNA insert probes. After washing for 20 min at 65C with 1% SDS in

1X SSC and for 30 min at 65C with 0.1% SDS in 0.1X SSC, membranes were

exposed at -80C to Kodak BioMax film with a Kodak BioMax intensifying screen.

Northern blot analysis was conducted in duplicate and representative data is 171 shown. All lanes shown as a single autoradiographic image were run on the

same blot, however in some instances lanes may have been reordered or

removed for economy and/or clarity of presentation.

For reverse-transcription and quantitative analysis of gene expression by

qPCR, total RNA was subject to purification using an RNeasy kit with DNase I

treatment (Qiagen Corp., Valencia, CA) and cDNA synthesized with SuperScript

II RNase H(-) reverse transcriptase (Invitrogen Corp.) using an oligo (dT)-22-mer

primer. SYBR green-based qPCR was conducted with an ABI 7500 Real-Time

PCR System (Applied Biosystems, Foster City, CA). Reaction conditions were

1X SYBR Green PCR Master Mix (Applied Biosystems), 100 nM each forward

and reverse primers, and 10 ng of cDNA. PCR was carried out over 40 cycles of

95°C for 15 sec, 60°C for 30 sec, and 72°C for 34 sec with an initial cycle of 50°C

for 2 min and 95°C for 10 min. All primers were designed to span intron locations and qPCR assays were conducted in triplicate. Primer sequences used were:

Gapdh, 5'-GGCAAATTCAACGGCACAG-3' and 5'-

CGGAGATGATGACCCTTTTGG-3'; 36B4 (gene name: acidic ribosomal

phosphoprotein P0/Arbp) , 5'-GAGACTGAGTACACCTTCCCAC-3' and 5'-

ATGCAGATGGATCAGCCAGG-3'; Boc, 5'-AAACAGCAGTGAGGCGAAC-3' and

5'-CACTTGGCAGGAGTCAGAAC-3’; Cdon, 5'-

TAACATACTGAGCCCCCCACAG-3' and 5'-CACTACCATCGTCCAGCTTTCG-3';

Mup1, 5'-AAGAACAAGCAAAGGGGCTGGG-3' and 5'-

ACACAGCAGCAGCAGCATCTTC-3'; Mup1/2, 5'-

ACTGACCCTAGTCTGTGTCC-3' and 5'-AGCCTTTTCTGTTTTGTCAGC-3';

172 Tuba1, 5’-GCAGCCGCGAAGCAGCAAC-3’ and 5’-

CCATGTTCCAGGCAGTAGAGCT-3’; Serping1, 5’-

GTCCAAATTCCTGCCCACTTAC-3’ and 5’-TCAGTTCCAGCACTGTCTCG

-3’; Timp4, 5’-TGGAAAAGTCTTCATCCATCTG-3’ and 5’-

GGTACATGGCACTGCATAG-3’; Col4a2, 5’-ACACTGTGGACTTACCAGG-3’ and 5’-CCAGGAAATCCAATGTCACC; H6pd, 5’-

AGAAGAGCAGTGCCATCCTG-3’ and 5’-TCGATGTGGACAAGGACACC-3’;

Fos, 5’-CCCCAAACTTCGACCATGATG-3’ and 5’-

AGTTGGCACTAGAGACGGAC-3’.

Specific transcript expression was normalized against respective Gapdh and

36B4 signals and fold differences calculated. Detection Gapdh or the 36B4 signals between compared sample sets rarely differed by more than one or two cycles. Graphical data is presented for transcript expression levels calculated by correction to either Gapdh or 36B4 internal control transcripts; these values are readily apparent in the respective graph. However, for clarity of presentation of data in the text, fold differences are presented as the average of the Gapdh and the 36B4 corrected values. The p values stated in the text are applicable to data generated with either correction for the Gapdh or 36B4 internal controls and only those data that meet the criteria of statistically significant differential transcript expression upon correction with both Gapdh and 36B4 are discussed in the text.

173 Suppression subtractive hybridization (SSH) and differential screening of

SSH cDNA libraries

We employed the SSH method to generate subtractive cDNA libraries for

transcripts enriched in SC WAT adipocytes or EP WAT adipocytes. SC WAT

and EP WAT were collected from six 8-wk old male C57Bl/6J mice. The SC

WAT and EP WAT from individual animals was pooled and fractionated into

adipocyte and SVF cell fractions via collagenase digestion as previously

described [26-28] and total adipocyte RNA isolated using TriZol. A PCR-Select

cDNA Subtraction Kit (BD Biosciences, Palo Alto, CA) was employed according

to manufacturer instructions, to generate an SC adipocyte and an EP adipocyte

SSH library, starting from 5 μg of total RNA. The resultant pools of PCR products consisting of double stranded cDNAs were subcloned into the pGEM-T vector (Promega) and transformed into DH5α E. coli to create SC SSH and EP

SSH plasmid-based libraries as E. coli stocks.

The SC adipocyte SSH cDNA library and EP adipocyte SSH cDNA library were screened by differential hybridization of high-density nylon cDNA arrays. Arrays were prepared by robotic spotting of PCR-amplified inserts of

SSH library clones via contract arrangement with the German Resource Center for Genome Research (RZPD, http://www.imagenes-bio.de). The SSH library we

prepared was sent to RZPD as glycerol stock; RZPD plated the library and

robotically picked individual colonies and PCR amplified clone inserts using PCR

primers for sequences flanking the pCR2.1-TOPO vector (Invitrogen Corp.) multi-

cloning site. PCR-amplified inserts were spotted in duplicate from the SSH SC

174 adipocyte and the SSH EP adipocyte libraries to generate high density nylon

arrays, which were returned to us for differential screening. For this, membranes

were prehybridized at 65C for 1 h in ExpressHyb solution containing 20X SSC

and 50 μg of salmon sperm DNA and hybridized overnight at 65C using 33P- dATP-labeled reverse-transcribed probes synthesized from 8 μg of EP adipocyte total RNA or SC adipocyte total RNA. Following hybridization, membranes were washed four times in 2X SSC /0.5% SDS at 65C for 20 min each, followed by two 20 min washes in 0.2X SSC /0.5% SDS at 65C, after which they were exposed at -80C to Kodak BioMax film with a Kodak BioMax intensifying screen.

Signals were analyzed visually and candidate differentially expressed cDNAs were sequenced. Both the fractionated material used to generate the SSH library and that used to screen the library was validated for fractionation into adipocyte and stromal fractions based on expression of marker transcripts for these two fractions. The adipocyte fraction was determined to be positive for transcript expression of SCD1 and negative for TSC-36, a marker we have identified for the SVF fraction of adipose tissue [27]; the opposite pattern was observed for the SVF. The adipocyte fraction was also negative for macrophages and endothelial cells based on the lack of signal for emr1/F80 and von Willebrand factor transcripts, respectively.

Adipocyte differentiation

3T3-L1 cells (American Type Culture Collection, Manassas, VA) were propagated in DMEM supplemented with 10% calf serum. For differentiation, 175 3T3-L1 cells were treated at two days post-confluence with DMEM supplemented with 10% FCS in the presence of the adipogenic inducers 0.5 mM methylisobutylxanthine (MIX) and 1 μM dexamethasone for 48 h. Adipogenic agents were then removed, and growth of cultures continued in DMEM containing 10% FCS. At five days post-induction of differentiation, adipocyte conversion had occurred in approximately 90% of the cells, as judged by lipid accumulation and cell morphology.

Murine primary preadipocyte SVF cultures were prepared from SC WAT of

8-wk old C57Bl/6J male mice, as described under Animal Use and Cellular

Fractionation of Murine Adipose Tissues, above. Cells were propagated in

DMEM supplemented with 10% FCS. For differentiation, cultures were treated at two days post-confluence with DMEM supplemented with 10% FCS in the presence of the adipogenic inducers 0.5 mM MIX, 1 μM dexamethasone, 0.2 mM indomethacin, and 170 nM insulin for 72 h. Adipogenic agents were then removed and growth of cultures continued in DMEM containing 10% FCS.

Primary cell differentiation was analyzed on two sets of cultures with essentially the same results.

Results

Differential enrichment of Boc and Cdon transcripts in EP adipocytes and

EP WAT

Given the relationship between regional adiposity and health morbidities we set out to identify transcripts that evidenced enriched expression in specific

176 WAT depots by creating and screening SSH cDNA libraries designed to be

enriched for transcripts present in EP or SC adipocytes. Genes that were

identified in our SSH screening showing differential transcript expression in EP

adipocytes vs. SC adipocytes were validated by qPCR. Of these, Boc

demonstrated the greatest degree of transcript enrichment in EP adipocytes vs.

SC adipocytes. Boc is a binding partner for Cdon, also known as cell adhesion molecule-related/down-regulated by oncogene [29, 30]. Boc and Cdon are both immunoglobulin superfamily members that are components of the hedgehog signaling pathway [29, 30]. qPCR analysis in Figure 1A shows that Boc transcript is enriched an average 12-fold (p<0.001) in EP adipocytes vs. SC adipocyte and an average 32-fold (p<0.001) in the EP SV cell fraction vs. SC SV cell fraction. When intact adipose tissue is assessed, Boc transcript shows an average 27-fold (p<0.001) enrichment in EP WAT vs. SC WAT (Figure 1B). Boc transcript level similar to that for SC is noted in RP WAT and BAT. These data suggest that it is not solely the adipocytes in EP adipose tissue that are enriched for Boc transcript, but that enrichment is also found for cell type(s) in the SV fraction. We next examined whether a similar pattern of expression might be noted for transcript for the Boc binding protein Cdon. We find that Cdon transcript is also enriched in EP WAT vs. SC WAT, although this is only noted for the SV fraction (Figure 1C) or intact WAT (Figure 1D), and not for isolated adipocytes (Figure 1C). As Boc and Cdon transcripts were both detected in adipocytes we also tested whether their transcript expression level was altered in adipogenesis by assessing levels in 3T3-L1 cells, a well characterized model of

177 in vitro adipocyte differentiation, and in the in vitro differentiation of primary preadipocytes to adipocytes. In both cases, the levels of Boc and Cdon transcripts were not appreciably different in preadipocytes vs. in vitro differentiated adipocytes (data not shown).

Since neither the expression of transcripts for Boc nor Cdon had been previously assessed in adipose tissue, but the hedgehog pathway plays a role in fat formation [31-33], we next determined transcript expression in wild type and obese (ob/ob) murine adipose tissues by qPCR (Figure 2). We find that alteration of Boc transcript level occurs in each of the 4 depots examined, with ob/ob mice showing upregulation of Boc transcript in SC, RP and BAT, and downregulation in EP (Figure 2A). Thus we find that for ob/ob, Boc transcript depot-dependence in SC vs. EP WAT is opposite to that observed for wild type

WAT depots, namely SC ob/ob WAT shows the highest degree of expression of

Boc transcript is an average 2.5 times (p<0.001) that found in ob/ob EP WAT.

For Cdon, upregulation of transcript is noted in ob/ob vs. WT for the SC and BAT depots of an average 1.6-fold (p<0.01) and 4-fold (p<0.001), respectively (Figure

2B). To our knowledge, only limited assessment of murine tissue expression patterns have been reported for Boc and Cdon transcript, and studies assessing expression in adipose tissue vs. other tissues have not been carried out. To determine if EP WAT is a dominant site of expression of these genes in vivo, we used qPCR to compare Boc and Cdon transcript levels in kidney, testis, lung, heart, brain, spleen, muscle and liver with that for EP WAT (Figure 3). EP WAT was chosen for this comparison, since it expressed the highest level of Boc

178 transcript of the four adipose depots we had examined (Figure 1). Of the nine

murine tissues examined, EP WAT was the site of highest expression of Boc

transcript (Figure 3A) and it was among the highest site for expression of Cdon

transcript (Figure 3B). Future work from this laboratory will address the

functional role Boc and Cdon may play in distinct WAT depots.

Data for the other 6 genes that we identified as enriched in EP vs. SC

adipocytes are presented in Additional file 1. However, these do not meet a

criteria of statistically significant (p<0.01) of EP vs. SC depot enriched expression

for isolated adipocytes as well as in whole EP vs. SC WAT. For example, Fos is only minimally expressed in whole WAT of either the SC or EP depot, but its level is dramatically elevated in response to the isolation procedure per se, as has

been described for a number of genes [34].

Differential screening reveals highly enriched expression of Mup

transcripts in the SC WAT depot

Our analyses of differential hybridization of SSH SC library clones

revealed that approximately 50 of the cDNA clones with increased expression in

SC adipocytes vs. EP adipocytes contained sequences corresponding to major urinary protein. Major urinary proteins (Mups) are small acidic molecules with molecular mass of ~19 kDa that belong to the lipocalin superfamily [35].

Lipocalins share a novel conserved calyx-shaped β-barrel structure [36-44] and

proteins in this family are proposed to serve dual molecular functions in the

transport of lipophilic molecules and in the regulation of cell homeostasis [45].

179 Mups exist as a complex array of protein isoforms generated from the multigene

Mup gene family present on murine chromosome 4 [46]; they are present in serum and are the major protein constituent of urine in the mouse [47]. The Mup gene family includes functional genes, pseudogenes and silent genes [48-57]; our recent analysis of the Ensembl database (www.ensembl.org) indicated 44 gene sequences in this family. Only a handful of Mup genes and gene products have been characterized in any detail, mainly Mup1 - Mup5 [58]. The most recent studies of Mup transcripts expression were conducted roughly two decades ago [48-57], when the extent of gene sequence similarity and complexity of the Mup gene family was likely not fully appreciated. Mup gene expression has not been reported to any extent in the intervening time period. In retrospect, it is unclear if single specific Mup transcript species, or rather sets of

Mup transcripts, were truly under study in earlier reports.

An extremely high degree of identity is found in the sequences of various

Mup transcripts with difference in sequence among members of the Mup multigene family often occurring as only scattered single or several base variations [59]. As such, the nearly identical nucleotide sequences of a number of different Mup gene products, particularly those with a high degree of identity with Mup1 and Mup2, render a number of Mup transcripts virtually indistinguishable by either Northern blot or PCR-based methods. Nonetheless, in an attempt to more precisely investigate the nature of differential Mup transcript expression, we designed PCR primer pairs that would be predicted to distinguish the gene products of Mup1 and of 4 Mup1-related genes from that for

180 other Mup sequences; Mup1 and a subset of Mup1-related genes contain a unique region of ~40 nucleotides at the 5' end of the respective transcript(s). The qPCR data employing the Mup1 PCR primer set on fractionated WAT tissues is shown in Figure 4A and indicates higher Mup transcript expression in the SC depot compared to the EP depot. The signal detected with the Mup1 primer set is an average 560 times higher (p<0.001 in SC SV fraction than in the EP SV fraction) and an average 100-fold higher (p<0.001) in the SC AD fraction than in the EP AD fraction. To further discern adipose tissue expression of Mup transcripts, we utilized a second primer set, designated Mup1/2. Based on our assessment of the Mup multigene family, the Mup1/2 primer set is predicted to detect the same set of Mup transcripts detected by the Mup1 primer set, and 11 additional Mup transcripts, including Mup2. Compared with the signal from the

Mup1 primer set, wherein transcripts are highly enriched in the SC SV fraction, transcripts detected with the Mup1/2 primer set are greatly enriched in the SC AD fraction. Here, the SC AD fraction signal is an average 70-fold higher (p<0.001) than the SC SV fraction. Enrichment of signal in the SC vs. the EP depot is also evident. The signal in the SC AD fraction is an average 880-fold higher (p<0.001) than in the EP AD fraction and the SC SV fraction signal is an average 1100-fold higher (p<0.001) than the EP SV fraction.

When levels in whole adipose tissue are examined with the Mup1 primer set (Figure 4C), compared to EP WAT, an average 13-fold higher (p<0.001) level of transcript is noted for SC WAT and an average 48-fold higher (p<0.001) for the

RP depot. Interestingly, although RP WAT is also intra-abdominal in location, it

181 nonetheless expresses Mup transcript(s) at an order of magnitude that is similar to that noted for the SC WAT depot. Figure 4D shows that, similar to our findings with the Mup1 primer set, the Mup1/2 primer set detects enrichment of Mup transcripts in SC WAT and RP WAT. However, here we find that compared with the EP WAT, an average 43-fold higher (p<0.001) level of transcript expression is noted for SC WAT and an average 270-fold higher (p<0.001) level of transcript expression for the RP WAT depot (p<0.001). Although the overall pattern of Mup transcript expression noted with the Mup1 and Mup1/2 primer sets is similar, these data also suggest a greater degree of depot-differential Mup transcript expression is found within that population of transcripts detected with the Mup1/2 primer set. We also conducted Northern blot analysis on SC and EP WAT of four individual mice using the Mup1 sequence as probe and included hybridization for the adipocyte marker transcripts aFABP and SCD1 for comparison purposes.

Due to the high degree of sequence similarity and transcript size among various

Mups, this analysis would be predicted to examine a population of various Mup transcripts. The Northern blot in Figure 4E (top panel) indicates clearly higher expression of signals detected by the Mup1 probe in the SC WAT depot, with a dramatically lower signal for EP WAT. The lower panel of Figure 4E reveals that in fractionated SC and EP WAT, it is the SC adipocytes that show the highest expression of signals detected by the Mup1 probe.

Since our data illustrated enrichment of Mup transcript in adipocytes vs.

SV fraction cells, wherein preadipocytes are found, we next examined whether upregulation of Mup transcripts accompanied adipogenic conversion. qPCR

182 analysis with the Mup1 and Mup1/2 primer sets revealed low levels of transcript expression in 3T3-L1 preadipocytes that were not appreciably altered during their conversion to adipocytes (Figure 4F and 4G). Therefore we next tested the differentiation-dependent expression of Mup transcripts using primary preadipocyte cultures prepared directly from murine SC WAT, and which might therefore be more reflective of the in vivo setting. Use of the Mup1 primer set, shown in Figure 4F, indicates an average 8-fold (p<0.001) increase occurs during adipogenesis of primary cultures. Use of the Mup1/2 primer set (Figure 4G), reveals an average 5200-fold (p<0.001) increase in level of transcript(s) detected by this primer set.

Dysregulation of Mup transcript expression in WAT of ob/ob genetically obese mice

To examine whether Mup transcript(s) expression was altered in obesity we utilized the ob/ob genetic model of murine obesity and compared expression with that of wild type mice. qPCR data obtained with the Mup1 and Mup1/2 primer sets are shown in Figures 5A and 5B. For the Mup1 primer set, compared with SC WAT and RP WAT from ob/ob mice, we find an average 10-fold

(p<0.001) higher transcript level in wild type SC WAT and an average 26-fold

(p<0.001) higher level for wild type RP WAT. For the Mup1/2 primer set, compared with SC WAT and RP WAT of ob/ob mice, we note an average 60-fold

(p<0.001) higher transcripts expression for wild type SC WAT and an average

230-fold (p<0.001) higher level of transcripts for wild type RP WAT (p<0.001).

The Northern blot wherein the Mup1 sequence was used as a probe (Figure 5C)

183 reveals a lack of Mup transcript signal in SC, EP, RP and BAT of WAT of ob/ob

mice. Our findings indicate that Mup transcript(s) evidence differential

expression not only across WAT depots, but also in respect to a well-established

genetic model of murine obesity. That we find a differential degree of Mup transcript(s) enrichment in C57Bl/6J wild type and ob/ob mice when we use the

Mup1 primer set vs. the Mup1/2 primer set suggests that within the Mup multigene family there are distinctions regarding the influence of obesity on the degree of differential expression of particular Mup transcript(s).

Mup transcript expression in murine tissues

Previous studies indicated that Mup transcript expression appeared particularly enriched in livers and also in other select tissues with secretory function; adipose tissue is now recognized as a secretory organ [1, 60]. To examine the relative expression of Mup transcript(s) in adipose tissues vs. other

murine tissues, we conducted qPCR and Northern blot analysis. SC WAT was

chosen as a positive control for these comparisons since it expressed readily

detected levels of Mup transcripts both by qPCR and Northern blot (Figures 4

and 5). Figure 6 shows qPCR analysis for the level of transcript(s) expression

detected with the Mup1 (Figures 6A and 6B) or Mup1/2 primer set (Figures 6C

and 6D). The Mup1 primer set (Figure 6A) detects an average 350-fold (p<0.001)

higher signal in liver vs. SC WAT and the Mup1/2 primer set (Figure 6C) an

average 25-fold (p<0.01) enrichment in liver compared to SC WAT. Figures 6B

and 6D assess Mup transcript(s) expression in SC WAT and a panel of other

184 murine tissues. For the Mup1 primer set (Figure 6B), a roughly similar level of

expression is noted for SC WAT and all other tissues with the exception of

spleen, which evidences minimal expression. In contrast, a different pattern of

Mup transcript(s) expression is noted with the Mup1/2 primer set (Figure 6D),

with SC WAT evidencing the highest expression. The differential nature of the

signals obtained with the Mup1 and Mup1/2 primer sets suggests that among the

multiple Mup transcript populations we detect herein, a number of individual Mup

transcripts likely evidence distinctive patterns of tissue restricted expression.

The Northern blot in the upper panel Figure 7 reveals that while liver tissue

exhibits strongest signal detectable upon hybridization with the Mup1 probe, that

of the tissues tested SC WAT is also a predominant site of expression of Mup

transcripts. Since we note that the highest signal detected by the Mup1 probe

are in liver, we also examined whether the level of these transcript was reduced

in the liver of ob/ob, has we had previously found for SC and RP adipose tissue

in such mice. The Northern blot in the lower panel of Figure 7 indicates a

marked reduction of Mup transcripts in the liver of ob/ob vs. wild type mice.

Discussion The link between regional adipocyte burden and health morbidities is becoming increasingly apparent and thus it is key to reach beyond studies of adipogenesis per se, into studies that describe and explain gene expression in either the adipocyte and/or non-adipocyte cellular component of specific WAT depots. Such studies, however, are dependent on the discovery and validation

185 of model genes that show a robust adipocyte depot-dependent phenotype. Our

data herein and the work of others [19, 20, 61] have identified genes with

differential pattern(s) of expression in regard to WAT depot expression. Kahn

and colleagues recently used DNA microarray chips to assess transcript

expression in adipocytes and SVF cells from murine SC WAT and EP WAT

depots [19]. They identified 197 transcripts that met their criteria of differential

expression for both adipocytes and SVF from SC WAT vs. EP WAT; the vast

majority of the reported genes were altered less than 3-fold [19]. Interesting

insights into the nature of WAT depot-dependent gene expression were, however,

revealed in their further analysis of WAT depot-dependent expression of 12

embryonic development and pattern specification genes [19]. In these cases, the depot-dependence appeared cell autonomous in nature and was also observed for human WAT samples. Furthermore, the transcript levels of a subset of the 12 genes correlated with waist-to-hip ratio and/or body mass index, two established indices of human regional adiposity [19]. These workers also found differential

expression of Mup transcripts in the SC WAT depot (74-fold higher in SC vs. EP);

however as Mup transcript(s) were not among the small subset of developmental and/or patterning genes that were the focus of their study, its expression was not

further validated or examined. Additionally, both our study and theirs observed

increased expression of enrichment of tubulin alpha 1 transcript in the EP white

adipose cells and tissue depot compared to that for SC [19]. It should also be

kept in mind that these studies have examined transcript levels and whether the

corresponding protein levels show differential expression remains to be

186 determined. That we did not identify a larger set of differentially-expressed

genes in common with the Kahn study [19], may be due to relative differences in

sensitivity and/or comprehensiveness of the experimental methods employed. It

is unlikely to be due to sample preparation as we have since assessed our RNA

preparations for three depot-enriched transcripts identified by Kahn and

coworkers [19] and found results similar to those they reported. Namely, levels

of transcripts for Tbx15 and Shox2 were markedly higher in EP WAT than SC

WAT and that for Phldb2 was higher in SC WAT than EP WAT (data not shown).

Uncovering gene expression distinctions across WAT depots have the potential to elucidate the underlying mechanism(s) for the development and/or function of specific adipose depots, and cell types therein, and their relation to disease. Comparison of the transcriptomes of visceral vs. subcutaneous adipocytes from WAT might ultimately provide therapeutic interventions that target visceral adipocytes while sparing white adipocytes in other WAT depots.

While these types of gene expression studies are relatively straightforward, they also carry an important caveat. It has been clearly documented that the standard collagenase isolation procedure, which is necessary to separate whole adipose tissue into adipocyte and other cellular fractions, in itself, results in marked alterations in gene expression [34]. This is attributed in part to the impact of released cytokines, such as TNFα, and other factors from adipose tissue cell components on adipocyte and/or SVF cell gene expression [34]. Such concerns apply to our investigation as we produced and screened our subtracted libraries using the isolated adipocyte component of SC and EP WAT depots. However,

187 we utilized both fractionated and whole adipose tissue samples for the detailed

qPCR validation of depot-differential expression, and found that differential

expression of Boc and Mup occurred both when isolated cell fractions and whole

adipose tissue depots were assessed. On the other hand, the expression level

of Fos transcript (see Additional file 1) is 1000 times higher in the fractionated

cell samples vs. intact adipose tissue; Fos is therefore an example of a gene

whose expression is dramatically altered due to the collagenase digestion

protocol. Another concern that arises in regard to qPCR studies is that transcript

expression is calculated relative to an internal control standard [62-64]; which by

definition is expressed at a consistent level regardless of experimental conditions

or cell/tissue types under study. For example, actin is described to decrease

during adipogenesis, and as such would not be an applicable internal control in

such studies [65]. We show herein that, overall, our differential gene expression data for EP vs. SC WAT depot at the cell and tissue level is of a similar

magnitude when either Gapdh or acidic ribosomal phosphoprotein P0 (36B4) is

used as an internal standard. This suggests that our findings are of a robust

nature and not solely reflective of variation in expression of a single given internal

control transcript across the analyzed samples.

While we do not at this time know the regulatory mechanisms behind the

reduction of Mup transcript expression in ob/ob mice, it is of interest to note that

reduced fertility occurs in the ob/ob mouse [66, 67]. Mups are lipocalins that function as pheromones, either alone or when bound to small hydrophobic molecules [68, 69] and are important in reproductive cycle of rodents where

188 urine-derived signals control sexual attraction, mating and puberty onset [70-73].

As pheromones, Mup proteins control mating behavior and puberty onset in mice; reduced Mup transcript levels in ob/ob mice may conceivably be related to their infertility phenotype [66, 67]. In this regard Mup expression, at least in mice, may be a molecular avenue whereby fat mass or fat distribution might impact mating and fertility. While it is unfortunate that the nearly similar sequence of a number of the Mup genes precludes a precise gene-by-gene analysis of each individual

Mup transcript in this complex multigene family [48-57], nonetheless future studies on the nature and adipose depot specificity of the Mup gene(s) promoter regions may allow a more precise mapping and understanding of Mup gene expression and regulation in distinct WAT depots. There are no known close functional or sequence analogs of Mups in humans, with the odorant binding proteins the most closely related human proteins [74, 75]. However, several lipocalin family members play roles in murine and/or human adipose tissues. For example, lipocalin-2, also known as neutrophil gelatinase associated lipocalin

(NGAL) transcript and protein increases during in vitro adipogenesis of 3T3-L1 preadipocytes and is abundantly expressed in adipose tissue [76-78]. Circulating lipocalin-2 concentrations positively correlate with adipocyte mass, hypertriglyceridemia, hyperglycemia and insulin resistance [78-80]. The lipocalin retinol binding protein 4 (RBP4) has recently been reported to be a marker for abdominal fat mass in humans [81] and some studies have suggested a role for

RBP4 in the pathogenesis of type 2 diabetes [82-87].

189 In the case of EP WAT, we find Boc transcript to show differential

enrichment in EP vs. SC WAT when compared in purified adipocytes, stromal

vascular fraction, as well as in intact WAT depots. Boc acts as a receptor for

sonic hedgehog and is important for the guidance of commissural axons [88].

The Cdon and Boc complex also mediates cell-cell interactions between muscle precursors to promote myogenesis [89]. Hedgehog signaling is a very early

event in the onset of adipogenesis [33]. Since myocytes and adipocytes are

believed to share the same mesodermal progenitor cell type [90] and the

hedgehog signaling pathway has been demonstrated to have an important role in

fat formation [31-33], it is possible that Boc is involved in adipogenesis and/or

adipocyte function. To our knowledge our findings on Boc transcript expression

in WAT depots and the upregulation of Boc transcript in ob/ob WAT are the first

to suggest a role for Boc in adipose tissue. At the least, our observations

indicate that the function of Boc, and possibly its binding partner Cdon, should be

considered in models that address the role of the hedgehog pathway in adipose

tissue.

Conclusions It is possible that additional dissection of the mechanisms underlying the

enrichment of Mup transcripts in the SC WAT depot and Boc transcript in the EP

WAT depot may lead to novel insights on the molecular mechanisms governing

gene expression in distinct WAT depots, for which very little knowledge currently

exists. Studies along these lines may ultimately, for example, result in the design

190 of promoter constructs that would allow for transgenesis or knockout studies to be conducted in a WAT depot-dependent manner. Future analyses of the transcriptional control of WAT depot specific gene regulation may also lead to key insights into regional adiposity and pinpoint WAT depot-specific therapeutic intervention targets in the fight against obesity and its complications.

191 Abbreviations

FCS – fetal calf serum; PCR – polymerase chain reaction; qPCR – quantitative polymerase chain reaction; WAT – white adipose tissue; BAT – brown adipose tissue; SC – subcutaneous; EP- epididymal; RP – retroperitoneal; SCD – stearoyl

CoA desaturase; aFABP – adipocyte fatty acid binding protein; MIX – methylisobutylxanthine; SSH – suppressive subtractive hybridization; Mup – major urinary protein; Boc- brother of Cdon.

192 Authors’ contributions

Y.W. conducted all qPCR studies and analysis excepting the tissue expression. J.Y. prepared and screening the SSH library and did initial assessment of Mup transcript level by Northern blots. S.Z. conducted qPCR for tissue specific expression. C.M.S wrote the text, oversaw all experiments, and was responsible for planning and data analysis at all phases of the project. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by an NIDDK NIH grant to C.M.S.

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205 Figure Legends

Figure 1. Expression of transcripts for hedgehog signaling components

Boc and Cdon in adipose depots. A. qPCR assessment of transcript levels in

SVF and AD fractions of SC and EP WAT using the Boc primer set. B. qPCR assessment of Boc transcript in whole SC, EP, RP or BAT adipose tissue. C.

qPCR assessment of transcript levels in SVF and AD of SC and EP WAT using

the Cdon primer set. D. qPCR assessment of Cdon transcript in whole SC, EP,

RP or BAT adipose tissue. For A-D, the left panels show data corrected against

Gapdh and the right panels show data corrected against 36B4 as internal control

for qPCR analysis; values stated in the text are the average of the Gapdh-

corrected and 36B4-corrected data for each comparison. In A and B, the level in

SC AD was set to a value of 1. SVF, stromal vascular fraction; AD, adipocyte fraction. In B and D, the level in SC WAT was set to a value of 1. For A, * indicates p<0.001 for EP SVF vs. SC SVF and for EP AD vs. SC AD and # indicates p<0.001 for EP SVF vs. all others (both panels). For B, * indicates p

<0.001 for EP vs. all others. For C, * indicates p<0.001 for EP SVF vs. all others

(both panels). For D, * indicates p<0.001 for EP vs. all others (left panel) and EP vs. SC and BAT (right panel).

Figure 2. Expression of Boc and Cdon transcript in wild type and ob/ob

tissues. qPCR assessment of transcript level in wild type C57Bl/6J (WT) and

ob/ob SC WAT, EP WAT, RP WAT and BAT depots using the Boc primer set (A)

and the Cdon primer set (B). For A and B, the left panels show data corrected

206 against Gapdh and the right panels show data corrected against 36B4 as internal

control for qPCR analysis; values stated in the text are the average of the

Gapdh-corrected and 36B4-corrected data for each comparison. The level in WT

BAT was set to a value of 1. For A, * indicates p<0.001 for comparisons of WT

vs. ob/ob samples for each of SC, EP, RP and BAT and # indicates p<0.005 for

ob/ob SC vs. all other ob/ob samples, and **, P<0.005 for WT EP vs. all other

samples (both panels). For B, * indicates p<0.001 for WT vs. ob/ob BAT WT

(both panels) and for WT vs. ob/ob for SC and EP depots left panel only.

Figure 3. Tissue distribution of Boc and Cdon transcript expression. qPCR analysis using the Boc (A) or the Cdon (B) primer set. For A and B, data was corrected against Gapdh (black fill) and or 36B4 (white fill) as internal control for qPCR analysis; values stated in the text are the average of the Gapdh-corrected and 36B4-corrected data for each comparison. For A, * indicates p< 0.001 for

EP WAT compared with all tissues except the Gapdh-corrected value for lung.

For B, * indicates p< 0.001 for EP WAT compared with all tissues except the

Gapdh-corrected value for lung and the 36B4-corrected value for brain. For A and B, the level in kidney was set to a value of 1.

Figure 4. Differential enrichment of Mup transcripts in the SC WAT depot.

A. qPCR assessment of transcript levels in SVF and AD fractions of SC and EP

WAT using the Mup1 primer set. SVF, stromal vascular fraction; AD, adipocyte fraction. B. qPCR assessment of transcript levels in SVF and AD of SC and EP

207 WAT using the Mup1/2 primer set. C. qPCR assessment of whole SC, EP, RP or

BAT adipose tissue using the Mup1 primer set. D. qPCR assessment of whole

SC, EP, RP or BAT adipose tissue using the Mup1/2 primer set. For A the EP

SVF signal level was set to a value of 1 and for B the EP AF signal was set to 1.

For A, * indicates p<0.001 for SC SVF vs. EP SVF and for SC AD vs. EP AD,

and # indicates p<0.01 for SC SVF vs. all others. For B, * indicates p<0.001 for

SC SVF vs. EP SVF and for SC AD vs. EP AD, and # indicates p<0.001 for SC

AD vs. all others. For C and D the signal level in BAT was set to 1 and *

indicates p<0.001 for SC or RP compared with EP and with BAT. E. Upper panel

shows Northern blot analysis of depot-dependent gene expression in either SC

WAT or EP WAT depots of four individual male C57Bl/6J mice using 32P dATP- labeled Mup1, aFABP or SCD1 hybridization probes. Lanes 1-4 and lanes 5-8 represent SC and EP WAT tissue from mouse #1, #2, #3 and #4, respectively.

Lower panel shows Northern blot analysis of fractionated SC and EP WAT. SV, stromal vascular fraction; AF, adipocyte fraction. Ethidium bromide staining of rRNA is shown as gel loading control. F. and G. qPCR assessment for 3T3-L1 or primary cultures of preadipocytes (Pre) and adipocytes (Adi) using the Mup1 (F) or the Mup1/2 (G) primer sets. The level of transcript expression in primary preadipocytes was set to a value of 1. For F and G, * indicates p<0.001 for primary adipocytes vs. all others. For A-D, F and G, the left panels show data corrected against Gapdh and the right panels show data corrected against 36B4 as internal control for qPCR analysis; values stated in the text are the average of the Gapdh and 36B4-corrected data for each comparison.

208

Figure 5. Reduced expression of Mup transcript(s) in WAT of ob/ob mice. A. qPCR assessment of transcript level in wild type (WT) and ob/ob (ob) SC WAT and RP WAT depots using the Mup1 primer set. B. qPCR assessment of transcript level in WT and ob SC WAT and RP WAT depots using the Mup1/2 primer set. For each graph, the signal in the respective WT tissue was set to a value of 100. For A and B the left panel shows data corrected against Gapdh and the right panel show data corrected against 36B4 as internal control for qPCR analysis; values stated in the text are the average of the Gapdh-corrected and 36B4-corrected data for each comparison. For A and B, * indicates p<0.001 for WT SC vs. ob SC and for WT RP vs. ob RP. C. Northern blot analysis of 5 μg of total RNA from the indicated WAT depot or BAT from WT or ob/ob mice. Blot was hybridized to Mup1, aFABP or SCD1 32P dATP-labeled probes. Ethidium bromide staining of rRNA is shown as a gel loading control.

Figure 6. qPCR analysis of tissue distribution of Mup transcript(s) expression. qPCR analysis using the Mup1 primer set (A and B) or the Mup 1/2 primer set (C and D). For A and C the SC WAT level was set to a value of 1.

For B and D the transcript level in spleen was set to a value of 1. For A - D, data was corrected against Gapdh (black fill) and or 36B4 (white fill) as internal control for qPCR analysis; values stated in the text are the average of the Gapdh- corrected and 36B4-corrected data for each comparison. For A and C, *

209 indicates p<0.001 for liver vs. SC WAT. For D, * indicates p<0.05 for SC WAT vs.

all others.

Figure 7. Northern blot analysis of Mup transcript expression in murine tissues. Northern blot analysis of a panel of C57Bl/6J murine tissues hybridized

with a 32P dATP-labeled Mup1 probe. Lower panel shows Northern blot analysis

of liver tissue from two wild type (WT) and two ob/ob mice. Ethidium bromide

staining of rRNA is shown as a gel loading control.

Additional files

Additional file 1 Format: tiff Description: Additional transcripts enriched in EP Adipocytes vs. SC Adipocytes

210 Figure 1

211 Figure 2

212 Figure 3

213 Figure 4

214 Figure 4

215 Figure 5

216 Figure 6

217 Figure 7

218 Additional file 1

219

MANUSCRIPT # 4

Generation and Characterization of mBAP-9, a New Brown Preadipocyte Cell Line, Reveals Differentiation-Dependent Regulation of Cmbl and Atabh, Two Novel α/β Hydrolases

Ji Young Kim#, Yu Wu#, Shengli Zhou, and Cynthia M. Smas*

# Equal authorship contribution

Department of Biochemistry and Cancer Biology Medical University of Ohio Toledo, OH 43614 USA

Running Head: Novel Genes in mBAP-9 Brown Adipocyte Differentiation

* To whom correspondence should be addressed:

Cynthia M. Smas, D.Sc. (* Corresponding author) Department of Biochemistry and Cancer Biology Medical University of Ohio Toledo, OH 43614 USA Phone: 419-383-4131 FAX: 419-383-6228 e-mail: [email protected]

220 ABSTRACT Brown adipose tissue (BAT) is responsible for non-shivering

thermogenesis through the uncoupling action of the mitochondrial protein Ucp1.

We have established mBAP-9, a new brown preadipocyte cell line, generated

from interscapular BAT of C57BL/6 mice. mBAP-9 preadipocytes undergo

spontaneous adipocyte differentiation that is manifested by the accumulation of

multiple lipid droplets and which is enhanced by exposure to dexamethasone, 3-

methyl-1-isobutylxanthine, indomethacin, insulin and triiodotyronine. Induction of

Ucp1 and PPAR transcripts during BAP-9 adipogenesis, expression of PGC-1

transcript, and increased mitochondrial DNA content is indicative of a brown

adipocyte lineage. Isoproterenol and forskolin upregulate Ucp1 transcript

expression in mBAP-9 adipocytes, illustrating effective β-adrenergic and cAMP-

mediated signaling. An in silico database screening followed by real-time PCR

transcript analysis of mBAP-9 preadipocytes and adipocytes resulted in the

identification of 10 adipocyte differentiation-dependent genes that were

upregulated 4-fold or greater during mBAP-9 adipose conversion. Two of these

encoding putative hydrolases, carboxymethylenebutenolidase-like (Cmbl), a novel gene with homology to Psuedomonas putida dienelactone hydrolase, a

new adipose tissue α/ hydrolase (Atabh) were further studied. Cmbl and Atabh

are induced 24-fold and 13-fold, respectively, during in vitro adipogenesis of

mBAP-9 preadipocytes and are regulated by the β-adrenergic agonist

isoproterenol. Transcripts for Cmbl and Atabh are expressed in BAT and in white

adipose tissue (WAT) in vivo, and evidence differential expression in obese

221 adipose tissue(s). We conclude that the mBAP-9 preadipocyte cell line we have developed is a novel and useful tool for detailed molecular studies on differentiation in the adipocyte lineage.

222 Keywords

Gene expression, mitochondria, differentiation, Ucp1, 3T3-L1, adipose tissue

223 Introduction

White adipose tissue (WAT) and brown adipose tissue (BAT) play central roles in energy balance (Flier, 2004; Klaus, 2004; Lowell and Spiegelman, 2000;

Spiegelman and Flier, 2001). WAT is the primary site of storage of excess energy intake in the form of triglycerides and it mobilizes this as free fatty acids to meet energy demands. WAT also secretes a number of key hormones and other factors, collectively defined as adipokines, some of which act in systemic energy balance and in the pathophysiology of obesity (Coppack, 2001; Gregoire, 2001a).

BAT is found only in mammals and has the ability to generate heat through adaptive thermogenesis for the maintenance of body temperature (Cannon and

Nedergaard, 2004; Himms-Hagen et al., 2000; Lowell and Spiegelman, 2000;

Sell et al., 2004), notably in hibernating species and human infants. Compared with white adipocytes, brown adipocytes store less triglyceride and in vivo the lipid droplets of brown adipocytes are characterized by a multilocular appearance vs. the central single lipid droplet of in vivo white adipocytes. A key characteristic of brown adipocytes is the high content of large mitochondria packed with cristae that contain Ucp1, a brown adipocyte-specific inner mitochondrial membrane protein that acts as a proton leak that uncouples ATP synthesis and thus generate heat (Cannon and Nedergaard, 2004). As studied in rodents, chronic stimulation of -adrenergic receptors by cold exposure or -agonist treatment leads to the increased expression of Ucp1, the recruitment of brown adipocytes and hyperplasia of BAT, and to the appearance of brown adipocytes within WAT

(Cinti, 2005; Klingenspor, 2003). The therapeutic potential of the brown

224 adipocyte is highlighted by studies in rodents which indicate that an enhancement of brown adipocyte cell number in WAT can lead to reversal of diabetes and of diet-induced and genetic obesity (Almind et al., 2007; Cinti, 2005;

Collins et al., 2004; Collins et al., 1997; Ghorbani and Himms-Hagen, 1997;

Guerra et al., 1998). In contrast to rodents, where BAT occupies a distinct interscapular anatomical region, in adult humans brown adipocytes appear to be diffusely distributed; as such assessing their contribution to human energy balance is challenging (Cannon and Nedergaard, 2004); however it is now appreciated that the brown adipocyte is an important contributor to human energy balance (Cinti, 2006; Lowell and Spiegelman, 2000). The study of mechanisms that contribute to the formation and function of brown adipocytes may lead to therapeutic strategies targeting energy homeostasis, and the deregulation thereof, in obesity (Tiraby and Langin, 2003).

Adipogenesis is a complex and highly integrated process and it is influenced by a large number of cellular and environmental signals. It is accompanied by morphological alterations, lipid accumulation, expression of lipogenic and lipolytic enzymes, and the establishment of sensitivity to insulin

(Gregoire, 2001a; Gregoire et al., 1998a; MacDougald and Mandrup, 2002;

Rosen and Spiegelman, 2000). Two transcription factors that are central to the conversion of preadipoctyes to adipocytes are the peroxisome proliferators- activated receptor γ (PPARγ) and the CCAAT/enhancer binding proteins

(C/EBPs) (Rosen and Spiegelman, 2000). These and other types of signaling events ultimately result in terminally differentiated mature adipocytes from

225 preadipocyte precursors (MacDougald and Mandrup, 2002; Mueller et al., 2002b;

Rosen et al., 2002; Rosen and Spiegelman, 2000). PPARγ2 is selectively

expressed in white and brown adipocytes in vitro and in vivo (Barak et al., 1999;

Rosen et al., 1999) and its transcript is upregulated early during the time course of adipocyte differentiation (Tai et al., 1996; Tontonoz et al., 1994a; Tontonoz et al., 1994b). C/EBPβ and C/EBP are induced very early in adipogenesis and activate PPARγ, with PPARγ and C/EBPα acting in concert to propel the adipogenic differentiation program in both the white and brown adipocyte lineage

(Darlington et al., 1998; Tanaka et al., 1997; Tontonoz et al., 1994b; Wu et al.,

1996; Wu et al., 1995). A key distinction of brown adipocytes is that they express

PGC-1α, an important transcriptional coactivator of the nuclear receptor PPARγ

that is central to the transcriptional and cellular program that defines brown

adipocytes (Puigserver and Spiegelman, 2003; Puigserver et al., 1998;

Spiegelman et al., 2000). PGC-1α is involved in activation of thermogenesis and

oxidative metabolism in brown fat and in muscle and its expression is

dramatically elevated in BAT upon cold exposure (Puigserver, 2005; Puigserver

and Spiegelman, 2003; Puigserver et al., 1998). Adrenergic stimulation leads to

PGC-1α-mediated transactivation of genes for mitochondrial biogenesis,

respiration and thermogenesis (Puigserver, 2005; Puigserver and Spiegelman,

2003; Puigserver et al., 1998). BAT is enriched for expression of not only PPARγ

and PPARδ but also PPARα (Valmaseda et al., 1999). In addition to the cAMP

Response Element Binding (CREB) protein family, both PPARγ and PPARα are involved in the expression and regulation of the brown adipocyte-specific gene

226 Ucp1(Sears et al., 1996). A number of other signals such as the p160 coactivators SRC-1 and p/CIP (Wang et al., 2006) and the retinoblastoma and p107 proteins (Scime et al., 2005), are involved in brown fat development.

Despite the clear distinctions in gene expression for brown vs. white adipocytes,

as well as apparently preadipocytes (Timmons et al., 2007), these two adipocyte

cell types share many gene expression in common. Additionally data also points

to a degree of plasticity between the brown and white adipocyte lineages (Orci et

al., 2004; Shimomura et al., 1998; Tiraby and Langin, 2003; Tiraby et al., 2003).

In the present study we have generated the mBAP-9 cell line, a new in

vitro model system for the study of brown adipogenesis. mBAP-9 cells are

derived from preadipocyte precursors present in murine BAT. We have

determined the culture conditions that effectively support their adipocyte

conversion and have evaluated the time course of phenotypic and molecular

differentiation of mBAP-9 preadipocytes to mature brown adipocytes. Moreover,

we have utilized this new cell line to determine the adipocyte differentiation-

dependent expression of ten novel genes, and characterized two of these in

additional detail. Our studies indicate that mBAP-9 cells will serve as a novel

and useful research tool to further delineate important aspects of brown

adipogenesis and gene expression.

Materials and Methods

Cell Isolation and Immortalization

227 All animal use was with the approval of the University of Toledo Health

Science Campus Institutional Animal Care and Use Committee. For culture of

BAT stromal-vascular cells, interscapular BAT was dissected from 3 wk old male

C57BL/6 mice. Tissue was rinsed in sterile HBSS, minced with scissors, and

digested with 0.1 mg/ml of type II collagenase (Sigma-Aldrich, St. Louis, MO) for

30 min at 37C with constant agitation. Following digestion, material was filtered

through 250-micron pore size nylon mesh (Sefar America, Inc., Depew, NY) and

filtrate was centrifuged at 2,000 rpm for 5 min. The pellet of stromal-vascular

cells containing preadipocytes was resuspended in DMEM with 10% FCS and

plated at low density to propagate individual clonal cell colonies for auto-

immortalization. Medium was changed every 3-4 days and after 4 months the

isolated clones were trypsinized, expanded as distinct cell lines, and tested for

adipocyte differentiation capacity. We chose one clone that demonstrated robust

growth and a high degree of adipocyte conversion for further study. We refer to it

herein as mBAP-9 (mouse Brown Adipose Precursor cells clone 9). At the time

of the studies reported herein, mBAP-9 cells had been grown continuously in

culture for at ~125 doublings.

Adipogenic Differentiation of mBAP-9 and 3T3-L1 Cells

mBAP-9 cells were maintained in DMEM supplemented with 10% FCS,

1% penicillin/streptomycin and 1% glutamine and passaged prior to reaching

confluence. To induce differentiation of mBAP-9 preadipocytes to adipocytes, cells were cultured to near-confluence in DMEM with 10% FCS, 20 nM insulin

228 and 1 nM triiodotyronine (adipocyte medium), followed by culture in adipocyte

media supplemented with 0.5 mM 3-methyl 1-isobutylxanthine (MIX), 0.5 μM dexamethasone (Dex), and 0.125 mM indomethacin (differentiation medium) for

3 days. This was then replaced with adipocyte medium, which was replenished every two days. 3T3-L1 cells were obtained from American Type Culture

Collection, Manassas, VA and propagated in DMEM supplemented with 10% calf serum. For differentiation, 3T3-L1 cells were treated at two days post-confluence with DMEM supplemented with 10% FCS in the presence of the adipogenic inducers 0.5 mM methylisobutylxanthine (MIX) and 1 μM dexamethasone for 48 h. Adipogenic agents were then removed and growth of cultures continued in

DMEM containing 10% FCS. At five days post-induction of differentiation of either mBAP-9 or 3T3-L1 cells, adipocyte conversion had occurred in approximately 90% of the cells, as judged by lipid accumulation and cell morphology. For TNFα studies, day 7 3T3-L1 adipocytes were cultured in the presence or absence of 10 ng/ml TNFα for 24 h.

For Oil Red O staining, cells were rinsed twice with PBS and fixed in 10% formaldehyde solution in PBS for 1 h. An Oil Red O stock solution of 0.5% in isopropanol was diluted 3:2 with water and filtered through Whatman #4 filter paper. Fixed cells were stained for 1 h at room temperature. Cells were rinsed three times with distilled water and images captured using an Olympus IX70 inverted microscope equipped with a digital camera. To determine gene expression in response to exogenous agents, fully differentiated day 7 mBAP-9

229 adipocytes were serum-starved for 16 h and exposed to either isoproterenol (10

μM), forskolin (10 μM), or dibutyryl cAMP (500 μM or 1 mM).

Assessment of Transcript Levels

RNA was isolated using TriZol Reagent (Invitrogen Corp., Carlsbad, CA)

according to manufacturer’s instruction. For studies of transcript expression in

murine tissues, 8 wk old C57BL/6 or ob/ob male mice were utilized. For Northern

blot analysis, total RNA was fractionated in 1% agarose-formaldehyde gels in

MOPS buffer and transferred to Hybond-N membrane (Amersham Biosciences,

Piscataway, NJ). Blots were hybridized in ExpressHyb solution (BD Biosciences

Clontech, Palo Alto, CA) with the indicated 32P-labeled cDNA probes prepared

using a random-primed labeling kit (Promega Corp., Madison, WI). After high stringency washing, membranes were exposed to Kodak Biomax film with a

Kodak Biomax intensifying screen at -80C. For reverse-transcription and PCR analysis, total RNA was subject to purification using an RNeasy kit with DNase I treatment (Qiagen Corp., Valencia, CA) and cDNA synthesized with SuperScript

II RNase H(-) reverse transcriptase (Invitrogen Corp.) using an oligo (dT)-22 primer. For semi-quantitative PCR, products were sampled at the indicated cycle number and analyzed on ethidium bromide-stained agarose gels. Semi- quantitative PCR was performed with the following primer pairs: PPAR1, 5'-

GGACTGTGTGACAGACAAGATTTGA-3' and 5'-

CTGAATATCAGTGGTTCACCGC-3';

230 PPAR2, 5'- GTGAAACTCTGGGAGATTCTCC-3' and 5'-

CTTCAATCGGATGGTTCTTCG-3'; C/EBPα, 5' - ATAAAGCCAAACAACGCAAC

-3' and 5'- AAACCATCCTCTGGGTCTC - 3'; Ucp1, 5'-

TATCATCACCTTCCCGCTG-3' and 5'- TGAGTCGTAGAGGCCAATC-3';

PGC-1α, 5'- TTTGATGCACTGACAGATGG-3' and 5'-

TGCTCTTCGCTTTATTGCTC-3';

PPARα, 5'- AGGCAGATGACCTGGAAAG-3' and 5'-

TCCCCACATATTCGACACTC-3'; Gapdh, 5'-GGCAAATTCAACGGCACAG-3'

and 5'-CGGAGATGATGACCCTTTTGG-3';

Pref-1, 5'-GTGACAAGTGTGTAACTGCC-3' and 5'-

CAAGCCCGAACGTCTATTTC-3'.

For real-time and semiquantitative analysis of Cmbl and Atabh transcript primer

sets used were: Cmbl, 5'-TGTCCCAGACTTCTTTGTGG-3', and 5'-

TCAAGACAGCATCAACCTCC-3'; Atabh, 5'’- GCACCAGAAAATCTCTCTCAGC-

3' and 5'-ACAGAACAGGGTCTCCTCAAAC-3'. Sequence information for real-

time PCR primer sets for all other genes are available upon request. Unless

otherwise indicated, for real time PCR, 10 ng of cDNA was used for cell samples

and 50 ng for tissue samples.SYBR green-based real-time PCR was conducted

with an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA).

Reaction conditions were 1X SYBR Green PCR Master Mix (Applied Biosystems),

100 nM each forward and reverse primers, and cDNA template. PCR was

carried out over 40 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 34

sec with an initial cycle of 50°C for 2 min and 95°C for 10 min. For real-time PCR

231 analysis, specific transcript expression was normalized against respective Gapdh

transcript signal and fold differences calculated. In cases where transcripts were

not detected by 40 cycles, the Ct was set to equal 40 so that the degree of fold

differences could be presented. These cases are noted in the relevant figure legends.

Determination of Mitochondrial DNA Content

Genomic DNA was prepared from triplicate samples of mBAP-9

preadipocytes and adipocytes using the PureLink Genomic DNA preparation kit

(Invitrogen). Equal mass of DNA was analyzed for mitochondrial genomic DNA

content with ND1 (5'-ACCATTTGCAGACGCCATAA-3' and 5'-

AATTGTTTGGGCTACGGCT -3) and Cox2 (5'-TCTCCCCTCTCTACGCATTC-3'

and 5'-ACGGATTGGAAGTTCTATTGGC -3) primers, and for chromosomal DNA

using primers for the18S gene (5'-TCGAGGCCCTGTAATTGGAA-3' and 5'-

CCCTCCAATGGATCCTCGTT -3)'. Real time PCR was conducted using the

conditions stated above, and the ND1:18S ratio and the Cox2:18S ratio

calculated.

Results

Adipose Conversion of mBAP-9 Cells

mBAP-9 cells were established from a single clone of a low-density plating

of the stromal-vascular fraction cell component of BAT without any artificial

immortalization process. We first assessed the ability of mBAP-9 cells to

232 undergo spontaneous adipogenesis, Figure 1A. For this, mBAP-9 cells were

maintained in growth medium only (DMEM containing 10% FCS) and observed

through 7 days post-confluence. Proliferating mBAP-9 cells (D0) demonstrate

fibroblast morphology during routine culture (Fig.1A, upper panel). After 3 days

post-confluence (D3), small refractive lipid droplets are present in some cells. By

day 5 (D5) these are increasingly apparent, depicted by arrows in the respective photomicrographs. At day 7 (D7) post-confluence, ~3%-5% of the cells had converted to an adipocyte morphology with obvious multilocular lipid droplet accumulation and cell rounding. In an attempt to increase the extent of adipose conversion, we tested the effects of the adipogenic inducing agents, MIX, Dex,

and indomethacin on mBAP-9 adipogenesis. For this, mBAP-9 preadipocytes

were first grown to confluence in adipocyte medium (which contains insulin and

T3) and then cultured for 3 days in differentiation medium followed by incubation

in adipocyte medium (as described in Materials and Methods). Typical cell

morphology was documented under phase contrast microscopy from the

preadipocyte stage (D0) through 7 days post-induction of adipogenesis, Figure

1B. Under these conditions, mBAP-9 cells began to accumulate readily

discernable multilocular lipid droplets in the cytosol from day 3 post-induction (D3)

and most cells showed clear evidence of adipocyte conversion by day 5 post-

induction (D5). At day 7 (D7), ~90% of the cells in the cultures evidenced lipid

droplets. Compared to spontaneous adipose conversion, the adipogenic

induction protocol accelerated the time course of adipogenesis and markedly

increased the percentage of cells in the culture that differentiated. We used this

233 as the standard protocol for differentiation of mBAP-9 cells in subsequent studies.

Figure 1C shows proliferating mBAP-9 preadipocytes and a low magnification

(middle) and macroscopic view of culture plate (right) of mBAP-9 cells that were

differentiated as in Figure 1B. The inset in the middle panel shows Oil Red O

staining for intracellular lipid of a single mBAP-9 adipocyte. Overall, three

striking morphological characteristics are evident for mBAP-9 adipocytes both in

the live and stained cultures: 1.) They have a prominently visible centrally located

nucleus; 2.) The nucleus is ringed with multiple layers of numerous small lipid

droplets; and 3.) Although mBAP-9 adipogenesis is accompanied by conversion

of the fibroblastic preadipocyte to a more rounded appearance, mBAP-9

adipocytes evidence somewhat flattened morphology. They have obvious

attachments to the culture surface at their cell periphery; moreover, these

peripheral regions of the cells clearly lack any visible lipid droplets.

Expression of Adipocyte Marker Genes and PPARγ Transcript Isoforms

during mBAP-9 Adipogenesis

For initial assessment of adipose conversion of mBAP-9 cells at the

molecular level, we determined the expression of several standard adipocyte

marker transcripts expressed by both brown and white adipocytes. SCD1,

aFABP, GLUT4 and lipoprotein lipase (LPL) transcript levels were measured at the preadipocyte stage (day 0) and at 3, 5, and 7 days post-adipogenic induction by Northern blot analysis (Figure 2A) and by real-time PCR (see below). These

data indicate that each of these transcripts is dramatically induced during the

234 time course of mBAP-9 adipogenic conversion. SCD1, aFABP, and GLUT4

transcripts are not detected in preadipocytes (day 0). For SCD1 and aFABP, a

maximal level of transcript upregulation is noted by day 3. The insulin-

responsive glucose transporter GLUT4 shows only moderate upregulation at day

3 but marked induction is noted by day 5. The LPL transcript is detectable in

preadipocytes (day 0); this is consistent with LPL being one of the very earliest

molecular indicators of adipose conversion, emerging when preadipoctyes first

attain confluence (Ailhaud et al., 1992; Semenkovich et al., 1989). Our findings at the transcript level are in line with the morphological changes presented in

Figure 1B, in that by day 3 post-induction, cells are both accumulating lipid droplets and expressing adipocyte marker transcripts. We also determined expression of select marker transcripts in mBAP-9 preadipocytes and adipocytes using real time PCR and determined upregulation (p<0.001) for

adipsin/complement factor D (Cfd) ( ~2800-fold), adiponectin (15-fold),

haptoglobin (30-fold), aFABP (55-fold), LPL (8-fold), Scd1 (115-fold), resistin

( ~940-fold), Glut4 (~540-fold), and ATGL (45-fold), data not shown. Figure 2B

indicates that while mBAP-9 adipogenesis is accompanied by markers of lipid

accumulation and other adipocyte marker transcripts, the expression levels of

these genes in mBAP-9 adipocytes is of similar order of magnitude as that

expressed in BAT rather than WAT; this is particularly evident in the case of

adipsin, adiponectin, haptoglobin and resistin.

In addition to transcriptional upregulation, a subset of preadipocyte-

expressed transcripts are decreased in abundance during adipogenic conversion.

235 In some specific instances, downregulation of preadipocyte genes appears

integral to the differentiation program such that when their downregulation is

blocked, adipogenesis is inhibited (Harp, 2004). One of these genes is Pref-1,

which encodes an EGF repeat-containing transmembrane protein that undergoes

processing to create a soluble inhibitor of adipogenesis (Moon et al., 2002; Smas

et al., 1997; Smas and Sul, 1993). The expression of Pref-1 transcript is

abolished upon adipocyte differentiation of 3T3-L1 preadipocytes (Smas and Sul,

1993), a highly characterized in vitro model of white adipocyte differentiation.

Data indicates that Pref-1 transcript level is also decreased during in vitro brown

adipogenesis (Tseng et al., 2005). We also used real-time PCR to assess

transcript expression of Pref-1 and a second preadipocyte marker, Gata-3, in

mBAP-9 preadipocytes vs. adipocytes. Adipocyte differentiation was

accompanied by a decreased (p<0.001) expression of transcripts for pref-1 (5-

fold) and Gata-3 (10-fold). In addition to preadipocytes, which remain ill-defined

in vivo, additional cell types are present in adipose tissue; these include

endothelial cells, neuronal cells, and macrophages. We find by real-time PCR

that mBAP-9 preadipocytes do not express appreciable levels of transcripts for

the endothelial markers vascular endothelial growth factor receptor Flt1and Von

Willebrand Factor (vWF), the neuronal markers neuron specific enolase (Nse)

and tubulin β3 (Tubβ3), nor the macrophage marker cd68 (Figure 2C). This further highlights the preadipocyte/adipocyte lineage of mBAP-9.

We next addressed the expression of PPARγ, the master adipogenic transcriptional regulator, during mBAP-9 adipogenesis. The Northern blot in

236 Figure 3A shows that a minimal level of PPARγ transcript is detected in mBAP-9

preadipocytes (day 0); this is increased by day 3 post-induction, and reaches

maximal level(s) at day 5 and day 7. The low but detectable level of PPARγ

transcript in mBAP-9 preadipocytes (day 0) may underlie the ability of mBAP-9 cells to undergo a degree of spontaneous adipose conversion, as was shown in

Figure 1A. PPARγ transcript has two isoforms arising by alternate start codon

usage, with the PPARγ2 form described as adipocyte-specific (Tontonoz et al.,

1994a; Tontonoz et al., 1994b). We conducted semiquantitative RT-PCR

assessment using primer sets that distinguish PPARγ1 and PPARγ2 transcripts.

As shown in Figure 3B, it is only the level of the adipocyte specific PPARγ2 form

that increases during adipocyte differentiation of mBAP-9 preadipocytes; this is detected from day 3 onward, real time PCR analysis of mBAP-9 preadipocytes and adipocytes indicated a 2-fold (p<0.001) upregulation of PPARγ2. We also determined expression of C/EBPα transcript during mBAP-9 adipogenesis.

Figure 3C reveals that while C/EBPα transcript is detected in preadipocytes, its level is increased in mBAP-9 adipocytes. Overall, our data is wholly consistent with expectations for an adipogenesis in vitro cell culture model in that we show generation of lipid-droplet morphology, the upregulation of adipocyte-enriched transcripts and the downregulation of a preadipocyte-specific transcript.

Brown Adipocyte Marker Gene Expression and Regulation in mBAP-9 cells

While many adipocyte genes are expressed in both white and brown

adipocytes, under normal conditions it is brown adipocytes that are regarded to

237 specifically express the mitochondrial protein Ucp1 (Cannon and Nedergaard,

2004; Unami et al., 2004). The brown adipocyte phenotype is also intimately tied to expression of PPARγ and PGC-1α with the latter important for mitochondrial biogenesis (Puigserver, 2005; Puigserver and Spiegelman, 2003; Puigserver et al., 1998). We examined the expression of these three genes in mBAP-9 preadipocytes and adipocytes, shown in Figure 4. Semiquantitative PCR assessment indicates Ucp1 and PPARα transcript is upregulated during mBAP-9 adipogenesis (Figure 4A and Figure 4B with real time PCR analysis indicating a

17-fold increase (p<0.001) in UCP1 transcript level (data not shown). Figure 4B also shows semiquantitative PCR data for levels of PGC-1α transcript are present in mBAP-9 preadipocytes and adipocytes and real time PCR analysis indicates a 4-fold increase (p<0.001) during mBAP-9 adipogenesis (data not shown). An additional brown adipocyte specific transcript measured by real time

PCR, Cide-a, and was expressed at levels ~280-fold higher in mBAP-9 adipocytes than preadipocytes (p<00.1), data not shown. We also assessed the relative content of the mitochondrial genome, as a marker for mitochondrial content that reflects mitochondrial biogenesis that occurs during brown adipocyte conversion. We find a ~17-fold increase (p<0.001) in mitochondrial DNA content per cell when the ratio of the ND1 mitochondrial gene to the 18S chromosomal gene was determined, and a ~20 fold increase (p<0.001) when the Cox2 gene was used as a marker for mitochondrial DNA content (data not shown). This is indicative of mitochondrial biogenesis, an important aspect of brown adipocyte differentiation.

238 To determine if Ucp1 in mBAP-9 adipocytes is subject to β-adrenergic

regulation cells were treated with 10 μM isoproterenol for 4 h. Isoproterenol, a non-subtype selective β-agonist, increases Ucp1 mRNA expression in primary brown adipocyte cultures (Cao et al., 2004; Rehnmark et al., 1990) and in in vitro

differentiated HIB-1B brown adipocytes (Puigserver et al., 1998). As shown by

the semi-quantitative PCR assessment in Figure 4C, Ucp1 transcript expression

is markedly elevated under these conditions. This indicates that mBAP-9

adipocytes are responsive to β-adrenergic-mediated signals, a pathway

intimately tied to the brown adipocyte phenotype. cAMP-mediated signals have

been well-documented to regulate Ucp1 gene expression in brown adipocytes

(Cao et al., 2004; Rim and Kozak, 2002; Yubero et al., 1998). We examined

whether cAMP-regulated pathways are utilized in the regulation of Ucp1 gene

expression in mBAP-9 adipocytes by employing forskolin or dibutyryl cAMP

treatment and determine that both of these agents markedly elevate Ucp1

transcript levels (Figure 4C). Together these findings indicate that the

characteristics of mBAP-9 cells are consistent with a brown adipocyte lineage

from a phenotypic, molecular and functional standpoint. We note, however, that

the level of each of these three transcripts mBAP-9 brown adipocytes is

considerably lower than that detected in vivo, as shown by comparison with the

BAT sample. This difference observed between levels of brown adipocyte

marker transcript expression in vitro vs. in vivo has also been reported for other

cell culture models of brown adipogenesis (Benito et al., 1993; Rohlfs et al., 1995;

Ross et al., 1992).

239

mBAP-9 Adipogenesis in Accompanied by Downregulation of Transcripts

for Multiple Members of the Matrix Metalloprotease (MMP) Family

In Figure 1 we observed that while mBAP-9 adipocytes are rounded in

shape compared to mBAP-9 preadipocytes, they nonetheless maintain obvious

regions at the cell perimeter wherein they remain attached to the culture surface;

these regions are devoid of lipid droplets. As an initial investigation into the

relationship between adipocyte differentiation and cell shape in mBAP-9 cells, we

assessed the differentiation-depending expression of transcripts for the matrix

metalloproteases (MMPs). Several MMPs have been reported as differentially

regulated during in vitro differentiation of the 3T3-L1 white preadipocyte cell line and also in obesity (Boudreau and Weaver, 2006; Bouloumie et al., 2001;

Chavey et al., 2003; Chun et al., 2006; Demeulemeester et al., 2005). They are

regarded as important for remodeling of the three dimensional extracellular matrix during adipose tissue development and adipocyte hypertrophy. However to our knowledge investigation of MMPs in neither brown adipose tissue nor brown adipogenesis has not been reported. We thus carried out a comprehensive assessment of transcript expression for each of the 24 known murine Mmps in mBAP-9 preadipocytes and adipocytes. Figure 5 shows that while mBAP-9 preadipocytes express a number of MMPs, these are for the most part dramatically decreased in expression in mBAP-9 adipocytes. On the other hand, with the exception of an ~2-fold increase in Mmp28 transcript, none of the

MMPs used in our comprehensive analysis were more highly expressed in

240 mBAP-9 adipocytes than preadipocytes. Transcripts for Mmp1a, Mmp1b, Mmp7,

Mmp9, Mmp10, Mmp12, Mmp13, Mmp16, Mmp17, Mmp18, Mmp20, Mm 21,

Mmp24, Mmp25, and Mmp27 were under the detection limit. Additional analyses

of Mmp function in this cell culture model will be needed in order to discern which

MMP expression patterns may underlie the morphological alterations that

accompany mBAP-9 adipogenesis. Additionally analysis of Mmp expression in

other models of brown adipogenesis and brown adipose tissue would be needed

and to determine which Mmp expression patterns are common to brown

adipogenesis per se vs. those that are unique to the mBAP-9 cell line.

Identification of New Adipocyte Differentiation-Dependent Transcripts

We next wished to utilize the mBAP-9 model system to uncover novel transcripts that may be implicated in adipocyte differentiation and/or metabolism.

For this we turned to in silico analysis of the Novartis GNF SymAtlas Database

(www.symatlas.gnf.org; Mouse Gene Atlas GNF1M, gcRMA), wherein microarray data is available for gene expression studies across a wide panel of murine tissues, including BAT (Su et al., 2002). Assessment of this database was carried out with the expression value for BAT set at > 10 times the median, yielding 229 candidate genes. We then examined the individual in silico tissue

specific gene expression patterns of each of these candidate genes for

indications of BAT enrichment relative to the numerous other murine tissues

analyzed therein. As we were primarily interested in genes for novel metabolic

functions, those that were already characterized to a greater or lesser extent

241 were not considered further. This in silico approach led us to empirically address

10 candidate genes for enriched expression in mBAP-9 adipocytes vs.

preadipocytes using real time PCR. Results for these analyses are presented in

Table I Among the 5 candidate genes with a putative enzymatic function in

metabolism, the two with the highest fold upregulation, Cmbl and a novel α/β

hydrolase herein designated Adipose Tissue α/β Hydrolase (Atabh), were chosen

for further assessment. The semi-quantitative PCR data in Figure 6A reveals

that the Cmbl transcript is markedly increased in expression level at day 3 post-

induction of differentiation of mBAP-9 preadipocytes to adipocytes, with

essentially the same level of Cmbl transcript found at day 5 and day 7 post-

induction of adipocyte differentiation. A very similar pattern differentiation-

dependent upregulation is noted for Atabh transcript.

We next conducted assessment of transcript levels by real-time PCR to

quantitatively compare expression these two transcripts in mBAP-9

preadipocytes, adipocytes, and in BAT. Brown adipogenesis of mBAP-9 cells

leads to a 24-fold increase (p<0.001) in expression level of Cmbl transcript

(Figure 6B, left panel) and a 13-fold increase (p<0.001) for Atabh transcript

(Figure 6B, right panel). For Cmbl and Atabh, mBAP-9 cells express slightly

higher levels than BAT. A 1.3-fold higher level is noted for Cmbl transcript

(Figure 6A) in mBAP-9 adipocytes vs. BAT (p<0.01) and a 1.6-fold higher level is

found for Atabh transcript (Figure 6A) in vitro vs. in vivo (p<0.05). Given that

adipocytes comprise from one-third to two-thirds of the cell numbers in adipose

tissue, we conclude that mBAP-9 adipocytes vs. brown adipocytes in vivo

242 express similar magnitudes of each of these 2 novel transcripts. Figure 6C

reveals that a 4 h treatment of mBAP-9 adipocytes with isoproterenol and

forskolin results a decrease of ~50% for Cmbl under these conditions whereas

Atabh is upregulated 2-fold by isoproterenol. These data suggest that

expression of Cmbl and Atabh may be influenced by signaling pathways key to

brown adipocyte function.

Sequence Properties of the Putative Hydrolases Encoded by Cmbl and

Atabh

Cmbl is identified in the SymAtlas database as 2310016A09Rik, and

corresponds to Unigene cluster Mm.28108, wherein it is defined as

carboxymethylenebutenolidase-like. The sequence of the murine Cmbl protein

(GenBank number NM_181588) with the dienelactone hydrolase homology

region underlined shown in Figure 7A. Cmbl is a previously unstudied gene

whose protein shows homology to the dienelactone hydrolase enzyme family,

InterPro domain ILPR002925 (Mulder et al., 2005), a subfamily of the α/β

hydrolase fold protein superfamily. Use of the NCBI GenBank database reveals that the gene for Cmbl encodes a 245 amino acid protein of a calculated

molecular mass of 27,902. The dienelactone hydrolase family numbers several hundred members across a spectrum of microbial species (Mulder et al., 2005).

It, however, appears that Cmbl is the sole member of IPR002925 among mice, humans, and other higher species.

243 Dienelactone hydrolases are best studied in regard to the aerobic

degradation of a wide range of aromatic compounds encountered as

environmental pollutants by microorganisms (Frantz et al., 1987a). They play a

crucial role in chlorocatechol degradation via the modified ortho-cleavage

pathway wherein the successive degradation/modification of these toxins occurs

which ultimately results in production of one of a few central dihydroxylated

intermediates, such as halocatechols (Frantz and Chakrabarty, 1987). These are

then substrate(s) for cleavage of the aromatic ring by either the ortho-cleavage

pathway or the 3-oxoadipate cleavage pathway, wherein a series of subsequent

reactions leads to the formation of dienelactones (Frantz and Chakrabarty, 1987).

Dienelactones are hydrolyzed to maleylacetate by dienelactone hydrolase which

is ultimately shuttled into various metabolic routes (Frantz and Chakrabarty,

1987). Murine Cmbl shows a 25% amino acid identity and a 41% amino acid

similarity over a 185-residue stretch (underlined region of Figure 7A) to the

Pseudomonas putida dienelactone hydrolase clcD, a protein that has been functionally demonstrated to hydrolyze maleylacetate (Frantz and Chakrabarty,

1987). Figure 7B shows an alignment of the region of amino acid homology between murine Cmbl protein and the P. putida clcD protein. Murine Cmbl possesses a pentapeptide sequence of Q-G-Y-A-A that precedes a cysteine residue and which is thought to be near the active site of clcD (Frantz et al.,

1987b) and related enzymes (Ngai et al., 1987; Yeh and Ornston, 1984). The

nature of the enzymatic activity that is encoded by Cmbl and its role in adipocyte metabolism remains to be identified.

244 The second gene of interest is a previously unstudied novel gene,

1300007F04Rik, which is predicted to be a α/β fold hydrolase, InterPro database

designation IPR012020 (Mulder et al., 2005). It is represented by UniGene

cluster Mm.432526 with GenBank designation NM_026185. This gene is

designated in GenBank as encoding a hypothetical protein LOC67477, which we

name herein Atabh. Atabh encodes a 459 amino acid protein with a calculated

molecular mass of 51,176. The Atabh protein sequence is shown in Figure 7C.

Hydropathy analysis indicates the presence of an N-terminal putative signal

sequence (bolded, Figure 7C). The α/β hydrolase fold homology region of Atabh

encompasses amino acids 88-386 (underlined, Figure 7B). The α/β fold motif is

found in a wide range of enzymes with various catalytic functions. These

enzymes act on different types of substrates, and often lack any obvious

sequence homologies, but share an overall structural similarity of an open

twisted β-sheet surrounded on both sides by α-helices. They also usually possess a catalytic triad consisting of a nucleophile and an acid (Ollis et al.,

1992). The closest homology to the Atabh protein found in GenBank is for a lung α/β hydrolase-1 (Labh1), also known as Abhd1, a transcript expressed in lung whose function is not characterized (Edgar and Polak, 2002). Atabh and

Labh1 exhibit a ~27% amino acid identity over a 318 amino acid region that comprises the α/β hydrolase fold region.

Tissue Distribution of Cmbl and Atabh Transcript

245 We next determined the overall tissue expression profile of Cmbl and

Atabh via real-time PCR analysis on a panel of 10 murine tissues. Figure 8A reveals that all tissues tested express a detectable level of Cmbl transcript.

Kidney, testis, WAT, and liver each show similar levels of Cmbl transcript

(p>0.05); compared to the other six tissues analyzed, these four tissues are sites of enriched Cmbl transcript expression (p<0.05). BAT evidenced an intermediate level of expression with the level of Cmbl transcript in WAT 5.6 times that of BAT

(p<0.001). In the case of Atabh (Figure 8B), the transcript was significantly enriched in WAT, BAT, liver, and lung vs. the other six tissues tested (p<0.05).

Furthermore, WAT and BAT expressed significantly higher Atabh transcript level than any other tissue tested (p<0.05). That WAT and BAT are the two highest sites of Atabh transcript expression argues that Atabh may be particularly important in adipocyte metabolism. These data suggest that although we initially identified Cmbl and Atabh as brown adipocyte differentiation-dependent transcripts, their encoded proteins would be predicted to play a role in WAT as well as in BAT.

To begin to address if expression levels of either Cmbl or Atabh may be tied to adipocyte physiology, we assessed transcript levels in BAT and WAT from wild type C57BL/6 mice and ob/ob mice, the latter a well-studied murine model of genetic obesity due to leptin mutation. The level of Cmbl transcript is not significantly different in wild type vs. ob/ob BAT (Figure 8C). For WAT a 67% reduction (p<0.001) in Cmbl transcript level in the ob/ob epididymal depot and a

41% decrease in the subcutaneous depot for ob/ob mice is noted. In the case of

246 Atabh, a 75% reduction (p<0.005) in ob/ob BAT is found. For WAT, a 72%

reduction (p<0.005) of Atabh transcript occurs in ob/ob epididymal depot and a

54% reduction (p<0.005) in the subcutaneous depot (Figure 8D).

Discussion

In this work we have generated a new cell culture model for the study of

brown adipocyte conversion. mBAP-9 adipogenesis results in cells that manifest key characteristics of brown adipocytes. As might be anticipated, the differentiation of mBAP-9 preadipocytes to adipocytes is accompanied by the upregulation of genes previously described to be expressed in common in both brown and white adipocytes, namely SCD1, aFABP, GLUT4 and LPL and others

(Gregoire et al., 1998a; Rosen and Spiegelman, 2000) and the downregulation of preadipocyte marker genes. mBAP-9 adipogenesis also involves the upregulation of the adipocyte-specific form of the master transcriptional regulator of brown and white adipogenesis, PPARγ2, which in concert with PGC-1α, has

been demonstrated to be key to the brown adipocyte differentiation program

(Puigserver and Spiegelman, 2003; Puigserver et al., 1998; Spiegelman et al.,

2000). The low but detectable levels of PPARγ2 and PGC-1α in mBAP-9 preadipocytes may underlie the degree of spontaneous adipogenesis we observe for these cells that occurs post-confluence. mBAP-9 adipocytes have with multiple small lipid droplets with a somewhat flattened shape with distinct contact regions with the culture surface. As far as we are aware, mature adipocytes of other in vitro cell culture models of both white and brown adipogenesis generally

247 exhibit a rounded appearance. Our observations that mBAP-9 cells do not

effectively convert to a fully rounded phenotype, but rather maintain peripheral

areas of attachment to the culture surface, may indicate that mBAP-9 cells might

be a unique and useful tool in which to address the relationship(s) between lipid

droplet accumulation and cell shape change during adipogenesis. It remains to

be determined whether the fact that a large subset of matrix metalloprotease

transcripts is markedly decreased in mBAP-9 adipocytes vs. preadipoctyes is

causally related to the distinct cell shape of mBAP-9 adipocytes.

Brown adipocyte cell lines have been key to discerning important

molecular pathways that function in the adipogenesis and metabolism of this

unique cell type, as well as to address the inter-relationships and commonalities

of the white vs. brown preadipocyte/adipocyte lineages. Existing models include

HIB-1B cells, generated from a brown fat tumor of transgenic mice wherein

adipocyte-specific expression of SV40 T-antigen was driven by the aP2/aFABP

promoter (Ross et al., 1992); T37i, derived from a hibernoma developed in a

transgenic mouse in which the expression of SV40 T-antigen was placed under

the control of the proximal promoter of the human mineralocorticoid receptor

gene (Zennaro et al., 1998); MB4, generated from SV40 T-antigen

immortalization of rat fetal brown adipocytes (Benito et al., 1993); B7-4 from a

brown fat tumor of a transgenic mouse carrying a SV40 T-antigen gene under

control of a mouse urinary protein promoter (Kozak and Kozak, 1994); HB2 derived from the stromal-vascular fraction of interscapular BAT of mice deficient of a tumor-suppressor gene p53 (Irie et al., 1999); and those developed by Kahn

248 and colleagues by retroviral infection of brown preadipoctyes of newborn mice with SV40 T-antigen (Klein et al., 2002a). In contrast to these cell lines, to our knowledge, mBAP-9 is unique in that they arose via spontaneous immortalization, as opposed to manipulation of growth-regulatory/control gene(s). While neither of these immortalization routes is necessarily preferred to the other, it is clearly advantageous to have multiple models in which to study an in vivo process in the in vitro setting. In the brown adipocyte cell lines where it has been assessed it appears that the level of Ucp1 and/or PPARα transcript expression is relatively low in these in vitro differentiation conditions vs. that found in BAT in vivo (Benito et al., 1993; Rohlfs et al., 1995; Ross et al., 1992; Valmaseda et al., 1999). The lower relative expression of brown adipocyte transcripts in the currently available in vitro differentiated brown fat cell culture models suggest that while in vitro brown adipogenesis can encompass many aspects of brown adipocyte phenotype, they do not yet appear to fully recapitulate the brown adipocyte phenotype to the extent it is expressed by brown fat cells in BAT in vivo. This likely reflects the intimate relationship between brown adipocyte thermogenic response and whole organism physiology and/or energy balance. Our data herein indicate that mBAP-9 cells are no exception to this phenomenon, in that although Ucp1 transcript is clearly induced during the conversion of mBAP-9 preadipocytes to brown adipocytes, it fails to attain the level of magnitude of expression found in BAT in vivo. Importantly, however, Ucp1 transcript in mBAP-

9 adipocytes is positively regulated by two of the central pathways implicated in

249 Ucp1 regulation in vivo, β-adrenergic responsiveness and cAMP-mediated signaling.

The generation and characterization of mBAP-9 as a new cell culture model of brown adipogenesis led us to identify Cmbl and Atabh as a new adipocyte differentiation-dependent genes, which are upregulated in both brown and white adipogenesis. While the liver is the major site of detoxification, there is sporadic data indicating that adipose tissue/adipocytes also express detoxifying enzymes (Yoshinari et al., 2006; Yoshinari et al., 2004).Given its protein homology with the clcD dienelactone hydrolase of P. putida, Cmbl may be a mechanism whereby adipocytes, which are known to be a reservoir for lipid- soluble toxins (Obana et al., 1981; Viravaidya and Shuler, 2004; Wolff et al.,

1979a; Wolff et al., 1979b; Wolff et al., 1982), can detoxify various aromatic compounds, and possibly use the respective end products in energy metabolism.

The role of Atabh in adipocytes also remains to be determined. The α/β hydrolase fold structure of the protein implies it likely possesses enzymatic activity. Our observations that expression of Atabh transcript is particularly enriched in WAT, BAT, and liver suggests Atabh possess an enzymatic function that is important in select aspects of metabolism shared between these tissue types. The functional analysis of these two new differentiation-dependent adipocyte genes will be addressed in ongoing studies from this laboratory. We anticipate that future studies aimed at identifying additional characteristics and novel gene expression patterns that define the brown adipocyte lineage will be

250 significantly facilitated by comparative studies in multiple models of brown adipogenesis, including mBAP-9 cells.

251 Grants

This work was funded through a NIH NIDDK grant to C. M. S.

Disclosures

None

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264 Figure Legends

Figure 1. Adipogenic differentiation of mBAP-9 cells. (A) mBAP-9 cells were maintained in DMEM/10% FCS and observed by phase contrast microscopy for morphological indications of spontaneous differentiation to adipocytes. Arrows in

D3 and D5 images indicate developing lipid droplets in early stage adipocytes.

(B) mBAP-9 cells were grown in DMEM/10% FCS, 20 nM insulin and 1 nM triiodotyronine and treated with the addition of 0.5 mM 3-methyl 1- isobutylxanthine, 0.5 M Dex, and 0.125 mM indomethacin for 3 days. Adipocyte differentiation was observed by phase contrast microscopy before (D0) and after

(D3, D5, D7) induction of differentiation. (C) View of proliferating mBAP-9 cells

(left panel) and Oil Red O stained cells (microscopic view, middle panel and macroscopic view of a 60 mm culture dish, right panel). The inset in the middle panel shows a representative image of a D7 adipocyte with intracellular lipid stained with Oil Red O.

Figure 2. Differentiation-dependent expression of adipocyte and preadipocyte marker transcripts. (A) RNA was harvested from mBAP-9 cells before differentiation (day 0) and at the indicated time points post-induction of differentiation. Northern blot analysis was performed on 5 μg of total RNA with

32P-labeled mouse SCD1, aFABP, GLUT4, and LPL probes. The rRNA of the

EtBr stained gel is shown below the respective autoradiogram as a loading control. (B) RNA was harvested from mBAP-9 adipocytes, BAT or WAT and real

265 time PCR carried out using 10 ng of cDNA and the indicated specific primer set.

(C) RNA was harvested from mBAP-9 preadipocytes (P) and adipocytes (A) and analyzed by real time PCR using 10 ng of cDNA and a specific primer set for

Pref-1 or Gata-3. (D) Expression of endothelial (Flt1 and vWF), neuronal (Nse and tubulin β3) and macrophage (cd68) markers. Real-time PCR was conducted on RNA from mBAP-9 preadipocytes or from a positive control RNA. Positive

RNA controls used were: lung RNA for Flt1 and vWF; brain RNA for Nse and tubulin β3; spleen RNA for cd68. The value in the respective positive control

RNA sample was set to 100%, as shown. The broken Y-axis and positive control bar indicates discontinuity of Y-axis value linearity.

Figure 3. Expression of PPARγ and C/EBP transcript during mBAP-9 adipogenesis. (A) RNA was harvested from mBAP-9 cells before differentiation

(day 0) and indicated time points post-induction of adipocyte differentiation.

Northern blot analysis was performed on 5 μg total RNA using 32P-labeled

PPARγ probe. The rRNA of the EtBr stained gel is shown below the autoradiogram as a loading control. (B) RNA was harvested from mBAP-9 cells before differentiation (day 0) and at the indicated time points post induction of adipocyte differentiation. Semiquantitative PCR was performed with 10 ng of cDNA using specific primer sets for PPARγ1 and PPARγ2. PPARγ1, PPARγ2, and Gapdh amplification reactions were sampled at the indicated cycle number and analyzed in EtBr-agarose gels. (C) RNA was harvested from mBAP-9 preadipocytes (P) and adipocytes (A) and semiquantitative PCR was performed

266 with 10 ng of cDNA using a C/EBP primer set. C/EBP and Gapdh amplification reactions were sampled at the indicated cycle number and analyzed in EtBr-agarose gels.

Figure 4. Expression and regulation of brown adipocyte markers transcripts. RNA was harvested from mBAP-9 preadipocytes (Pre), adipocytes

(Adi), and BAT. (A) Semiquantitative PCR was performed with 20 ng of cDNA using a Ucp1 primer set. (B) Semiquantitative PCR was performed using a specific primer sets for PPARα or PGC-1α. For (A) and (B), Ucp1, PPARα,

PGC-1α, and Gapdh amplification reactions were sampled at the indicated cycle number and analyzed in EtBr-agarose gels. (C) mBAP-9 adipocytes were serum-starved for 16 h and stimulated with isoproterenol (Iso, 10 M, treatment

#1), forskolin (Fsk, 10 M, treatment #2) or dibutyryl cAMP (cAMP 0.5 mM, treatment #4 or 1 mM, treatment #5) for 4 h. Control cultures were incubated in serum-free (SF, treatment #1) media for a total of 20 h or maintained in regular growth media (R, treatment #6). Ucp1 transcript level was analyzed by semiquantitative PCR with Ucp1 and Gapdh reactions sampled at the indicated cycle number, shown below the respective panel.

Figure 5. Downregulation of MMP Transcripts in mBAP-9 Adipogenesis.

Real time PCR analysis was carried out on mBAP-9 preadipocyte (Pre) and adipocyte (Ad) samples and the level in the respective preadipocyte samples set to 100. Analysis was done for transcripts for all 24 murine Mmps. Those that are 267 not shown were undetectable in both preadipocyte and adipocyte samples after

40 cycles. Those with an * were samples wherein the adipocyte level was undetectable at 40 cycles, for purposes of representing fold differences in expression in preadipocyte vs. adipocyte of these samples, the cycle number was set at 40. * indicates preadipocyte and adipocyte values are significantly different (p<0.001). The Mmps not shown in the graph (Mmp1a, Mmp1b, Mmp7,

Mmp9, Mmp10, Mmp12, Mmp13, Mmp16, Mmp17, Mmp18, Mmp20, Mm 21,

Mmp24, Mmp25, and Mmp27) were below detection level in both preadipocytes and adipocytes.

Figure 6. Expression and regulation of Cmbl and Atabh transcript in mBAP-9 cells. (A) Differentiation-dependent expression. RNA was harvested from mBAP-9 cells before differentiation (day 0) and at indicated time points after differentiation. Semiquantitative RT-PCR was performed using specific primer sets for Cmbl and Atabh. Cmbl and Atabh amplification reactions are shown at

32 cycles. Samples of the Gapdh reactions are shown at 25 cycles. Products were analyzed in EtBr-agarose gels. Multiple cycle sampling and analysis (data not shown) indicated the reactions depicted were within the exponential range of

PCR amplification. (B) Quantitative assessment of Cmbl and Atabh transcripts in mBAP-9 brown adipogenesis and in BAT. Relative Cmbl (left panel) and Atabh

(right panel) transcript level in mBAP-9 preadipocytes (P), adipocytes (A), and

BAT (B). Real-time PCR was performed as described in Materials and Methods.

Values were normalized to Gapdh expression level and transcript expression in

268 preadipocytes set at 1. Statistical analysis was carried out with single-factor

ANOVA. For B (left panel) *, p <0.001 for mBAP-9 Pre vs. mBAP-9 Adi and **,

p<0.01 for mBAP-9 Adi vs. BAT. For B (right panel) *, p <0.001 for mBAP-9 Pre

vs. mBAP-9 Adi and #, p<0.05 for mBAP-9 Adi vs. BAT. (C) Regulation by

isoproterenol and forskolin. mBAP-9 adipocytes were serum-starved for 16 h and stimulated with isoproterenol (Iso, 10 M) or forskolin (Fsk, 10 M) or dibutyryl cAMP (cAMP 0.5 mM or 1 mM) for 4 h. Control cultures were incubated in serum-free (SF) media for a total of 20 h. Transcript levels were analyzed by real time PCR for Cmbl and Atabh as described in Materials and Methods.

Values were normalized to Gapdh expression level and transcript expression in

SF set at 1. Statistical analysis was carried out with single-factor ANOVA. For

Cmbl ##, p <0.005 for Iso and Fsk vs. SF; for Atabh *, p <0.001 for Iso vs. SF.

Figure 7. Characteristics of Cmbl and Atabh protein sequences (A) Amino acid sequence of murine Cmbl. The underlined region indicates the region of homology of Cmbl with the dienelactone hydrolase protein family, IPR002925.

(B) Alignment murine Cmbl (MOUSE) and the clcD protein of P. putida (PSEPU) proteins showing amino acid identities and similarities (+). Dashes indicate gaps in the alignment. The bold underlined region of the PSEPU sequence indicates a pentapeptide sequence of the clcD protein implicated in its enzymatic activity. (C)

Amino acid sequence of murine Atabh. The underlined region indicates the α/β hydrolase fold region and bold typeface denotes a predicted N-terminal signal sequence. For (A-C), the numbers at left indicate amino acid positions.

269

Figure 8. Expression of Cmbl and Atabh transcripts in various murine tissues. RNA was harvested from the indicated murine tissues and Cmbl (A) and Atabh (B) transcript level was analyzed by real-time PCR as described in

Materials and Methods. Values were normalized to Gapdh expression level and transcript expression in lung set at 1. Statistical analysis was carried out with single-factor ANOVA followed by Tukey post-hoc test. For (A) ##, p <0.005 for kidney, testis, WAT and liver vs. all other tissues. For (B) #, p <0.05 for WAT,

BAT, liver and lung vs. all other tissues.

Figure 9. Expression of Cmbl and Atabh transcripts in ob/ob adipose tissue. RNA was harvested from BAT, epididymal (EP) WAT and subcutaneous (SC) WAT of wild type (wt) and ob/ob (ob) mice and real-time PCR for Cmbl (A) and Atabh (B) was performed as described in Materials and

Methods. Values were normalized to Gapdh expression level and transcript expression in wt set at 1. Statistical analysis was carried out with single-factor

ANOVA. For (A) *, p<0.001 for wt EP vs. ob EP and ##, p<0.005 for wt SC vs. ob SC. For (B) ##, p<0.005 for wt BAT vs. ob BAT; wt EP vs. ob EP; and for wt

SC vs. ob SC.

270 Figure 1

271 Figure 2

272 Figure 3

273 Figure 4

274 Figure 5

275 Figure 6

276 Figure 7

277 Figure 8

278 Figure 9

279

MANUSCRIPT # 5

Expression and regulation of transcript for the novel transmembrane protein Tmem182 in the adipocyte and muscle lineage

Yu Wu and Cynthia M. Smas

BMC Res Notes. 2008 Sep 19;1(1):85. [Epub ahead of print]

Department of Biochemistry and Cancer Biology and Center for Diabetes and Endocrine Research University of Toledo Health Science Campus Toledo, OH 43614, USA

Please address correspondence to:

Cynthia M. Smas Department of Biochemistry and Cancer Biology and Center for Diabetes and Endocrine Research University of Toledo Health Science Campus Toledo, OH 43614, USA Phone: 419-383-4131 FAX: 419-383-6228 e-mail: [email protected]

280 Abstract

Background: White adipose tissue is not only an energy storage organ; it also

functions as an endocrine organ. The coordination and integration of numerous

gene expression events is required to establish and maintain the adipocyte

phenotype. Findings: We previously observed a 45-fold upregulation for a

transcript encoding a novel predicted transmembrane protein, Tmem182, upon

brown preadipocyte to adipocyte conversion. Here we use real-time PCR

analysis to further characterize Tmem182 transcript expression in the adipocyte

lineage. Analysis across a panel of 10 murine tissues revealed highest

Tmem182 transcript expression in white adipose tissues (WAT), with 10-fold to

20-fold higher levels than in brown adipose tissue (BAT). Tmem182 transcript

expression is ~3-fold upregulated in BAT of genetically obese (ob/ob) mice vs.

wild type C57BL/6. Analysis of three in vitro models of white adipogenesis

indicates markedly enriched expression of Tmem182 transcript in adipocytes vs.

preadipocytes. Compared to 3T3-L1 preadipocytes, a 157-fold higher level of

Tmem182 transcript is detected at 3 day post-induction of adipogenesis and an

~2500-fold higher level in mature 3T3-L1 adipocytes. TNFα treatment of 3T3-L1 adipocytes resulted in a ~90% decrease in Tmem182 transcript level. As skeletal muscle and heart were also found to express Tmem182 transcript, we assessed expression in C2C12 myogenesis and observed a ~770-fold upregulation upon conversion of myoblasts to myocytes. Conclusions: WAT is the most prominent site of Tmem182 transcript expression and levels of transcript for Tmem182 are altered in adipose tissues of ob/ob mice and upon exposure of 3T3-L1

281 adipocytes to the proinflammatory cytokine TNFα. The dramatic upregulation of

Tmem182 transcript during in vitro adipogenesis and myogenesis suggests

Tmem182 may function in intracellular pathways important in these two cell types.

282 Findings

Background - White adipose tissue (WAT) is the major site for storage of excess energy and these triglyceride stores are mobilized to meet the energy needs of the organism. Adipose tissue is now also recognized as an endocrine organ with synthesis and secretion of a variety of soluble factors such as leptin, resistin, adiponectin, retinol binding protein-4 and TNFα [1-4]. Adipocytes make up from one-third to two-thirds of the cell population found in adipose tissue, with endothelial cells, nerve cells, macrophages, fibroblast-like interstitial cells and preadipocytes, and perhaps other cell types, comprising the remaining stromal- vascular component [5]. Mature adipocytes form as the result of the differentiation of preadipocyte precursors present in adipose tissue [6-10].

Established preadipocyte cell lines such as 3T3-L1 [11] have been an extensively

used in vitro model to define genes central to the adipocyte phenotype [12, 13].

Adipogenesis is accompanied by increased transcription of genes that encode

proteins key to adipocyte function, for example lipogenesis, lipolysis, lipid

transport, and hormone responsiveness [7, 14, 15]. In vitro and in vivo studies

have uncovered a pivotal role for peroxisome proliferator-activated receptor γ

(PPARγ), a member of the ligand-activated steroid hormone receptor family, in

the adipogenic program [8, 10, 16-19]. Studies have also illustrated the

important contributions of the CCAAT/enhancer-binding proteins (C/EBPs) and

other transcriptional signals to adipogenesis [8, 10, 20].

Method - Culture of cell lines and adipogenic conversion for 3T3-L1,

ScAP-23 and wt-BAT, for the fractionation of adipose tissues, and for culture and

283 differentiation of murine preadipocytes from subcutaneous (SC) WAT was as

described [21, 22]. C2C12 cells were maintained and passaged as subconfluent

cultures in DMEM with10% FBS. For differentiation, cultures at 70% confluence

were switched to DMEM with 2% horse serum and 10 μg/ml insulin, and were

cultured under these conditions for 7 days. For treatment of 3T3-L1 adipocytes

with TNFα and various pharmacological inhibitors the method was as described

[23-25]. After serum-starvation for 6 h, 3T3-L1 adipocytes were pretreated with either 50 µM LY294002, 50 µM PD98059, 20 µM SB203580, 100 nM wortmannin,

1 µM rapamycin (Sigma-Aldrich, St. Louis, MO), or DMSO vehicle for 1 h and

then cultured in 10 ng/ml of TNFα for 16 h in the presence of inhibitors. RNA

was purified using TriZol Reagent (Invitrogen Corp.) according to manufacturer’s

instruction. For studies of Tmem182 transcript expression in murine tissues, 8

wk old C57BL/6 or ob/ob male mice were utilized, with all animal treatments

conducted with approval of the University of Toledo Health Science Campus

Institutional Animal Care and Use Committee.

Real-time PCR analysis was as previously described [23, 24, 26]. For

this, total RNA was subject to purification with an RNeasy RNA purification kit

with DNase I treatment (Qiagen Corp., Valencia, CA) and 5 g used for first

strand cDNA synthesis with SuperScript II RNase H-reverse transcriptase

(Invitrogen Corp.) and an oligo(dT)-22 primer. Real time PCR was conducted

with an ABI 7500 Real Time PCR System. Target cDNA levels were analyzed by

SYBR green-based real-time PCR in 25 l reactions containing 1X SYBR Green

PCR Master Mix (Applied Biosystems, Foster City, CA), 100 nM each forward 284 and reverse primers, and 10 ng of cDNA. Analyses were performed in triplicate

and expression of each gene was normalized against Gapdh transcript level.

The cycle threshold value was generated using ABI PRISM 7500 SDS software version 1.2 and exported to an Excel spreadsheet to calculate fold differences.

Sequence of PCR primers used were: Tmem182 (5'-

ACACCAATCAGCCACCATCC-3' and 5'-GCCACGGTAAATAATTGCGGAG-3');

Gapdh (5’-GGCAAATTCAACGGCACAG-3’ and 5’-

CGGAGATGATGACCCTTTTG-3’); and Myogenin (5’-

GCCATCCAGTACATTGAGC-3’and 5’-GTAAGGGAGTGCAGATTGTG-3’).

Primers were designed to span introns.

Properties of Tmem182 gene and sequence - During the

characterization and utilization of a new in vitro model of brown adipogenesis, mBAP-9, we previously identified ten new genes with increased expression in mBAP-9 adipocytes vs. preadipocytes (manuscript in preparation). Of the ten genes identified, the gene found to exhibit the highest fold upregulation in mBAP-

9 adipogenesis (45-fold), encoded an uncharacterized novel predicted transmembrane protein termed Tmem182. Its transcript expression and regulation, however, was not further characterized at the time. Tmem182 is represented by UniGene Mm.334678 [GenBank: NM_001081198]. It encodes a wholly novel 229 amino acid protein with a calculated molecular mass of 25,845.

The protein sequence of Tmem182 is shown in Figure 1A. The Kyte-Doolittle analysis of Tmem182 protein sequence identifies four putative membrane- spanning regions (Figure 1B, and underlined and bolded in Figure 1A), indicative

285 of an integral membrane topology. Database analysis (NCBI Homologene) indicates that Tmem182 homologs are found in human (90%), dog (87 %), rat

(87%) chick (71%), with numbers in parentheses indicating percent amino acid identity to the murine protein. Homologs are also present in pig, horse, monkey and zebrafish.

Tissue distribution of Tmem182 transcript expression - We determined transcript expression of Tmem182 in a panel of murine tissues using real-time PCR. As shown in Figure 2A, of the tissues examined, subcutaneous

WAT is the most enriched site of transcript expression (p<0.001), with expression in muscle, heart and lung at levels from ~10% to ~50% of that in WAT. Lower relative levels of Tmem182 transcript are found in kidney, spleen, testis, brain and liver. We also examined Tmem182 transcript level in brown adipose tissue

(BAT) and three distinct WAT depots, subcutaneous (SC), epididymal (EP) and retroperitoneal (RP), Figure 2B. We find that each of the three WAT depots have a similar magnitude of expression of Tmem182 transcript with from ~10- to ~20- fold higher levels (p<0.001) than that found in BAT.

To begin to address the modulation of Tmem182 transcript levels in regard to the pathophysiology of adipocytes, we compared transcript expression in BAT and WAT from wild type C57BL/6 mice and ob/ob mice, the latter a well- studied murine model of genetic obesity. For Tmem182 transcript, we find a slight increase (~1.7-fold, p<0.001) in ob/ob WAT for the SC depot (Figure2C).

Furthermore, compared to wt BAT, we find a 6.3-fold upregulation (p<0.001) of

Tmem182 transcript level in ob/ob vs. wt BAT, suggestive of a role for

286 dysregulation of Tmem182 in the obese state (Figure 2C). It would thus appear that in the ob/ob genetic model, BAT shifts to a level of Tmem182 transcript expression that is more similar to that found in WAT.

Differentiation-dependent expression of Tmem182 transcript in adipogenesis - We originally identified Tmem182 during the characterization of a new brown adipocyte in vitro cell culture model, termed mBAP-9, wherein we found Tmem182 transcript to be upregulated 45-fold during the adipogenic conversion of these cells from preadipocytes to mature adipocytes (manuscript in preparation). Our tissue expression studies, however, revealed that although

BAT is a site of expression of Tmem182 transcript, the most dominant site of expression in vivo is WAT. We therefore determined if Tmem182 transcript evidenced differentiation-dependent expression in various in vitro models of white adipogenesis. The best characterized cell culture model of adipogenesis is

3T3-L1, these cells were developed ~ 30 years ago [11] and undergo differentiation from preadipocytes to mature white adipocytes following treatment with the adipogenic agents Dex and MIX. Real-time PCR analysis indicates that

3T3-L1 adipocytes express ~ 2500-fold higher levels of Tmem182 transcript than

3T3-L1 preadipocytes (Figure 3A). The increase in transcript expression is first noted at day 3, with a 157-fold increase noted. In addition to the 3T3-L1 model, we examined Tmem182 transcript expression in two other models of white adipogenesis. ScAP-23 cells are a cell line established in this laboratory derived from preadipocytes present in murine SC WAT, in contrast to the embryonic derivation of 3T3-L1 cells. As shown in Figure 3B, adipogenesis of ScAP-23

287 cells leads to a 75-fold increase in levels of Tmem182 transcript. Figure 3C

shows that adipocyte conversion of primary cultures of murine stromal vascular

fraction cells from SC WAT, wherein preadipocytes are found, is accompanied by

an ~22-fold increase in Tmem182 transcript. Although we find differing

magnitudes of upregulation of Tmem182 transcript across the adipogenesis

models that we assessed, in all cases a significant increase in Tmem182

transcript level accompanies adipocyte conversion.

Regulation of Tmem182 transcript by TNFα - We determined the

regulation of Tmem182 transcript following incubation of in vitro differentiated

3T3-L1 white adipocytes with TNFα. TNFα is a proinflammatory cytokine central to adipose tissue pathophysiology and acts to suppress expression of many adipocyte genes; elevated TNFα levels are associated with obesity [27-31].

TNFα  treatment of adipocytes promotes lipolysis and dedifferentiation [32-34], with the latter ascribed TNFα-mediated transcriptional downregulation of the key adipocyte transcription factors PPARγ [35] and C/EBPα [36, 37]. Studies of

TNFα effects on Tmem182 transcript expression were carried out using treatment with TNFα alone, or by treating cells with TNFα after adipocytes had been pretreated with pharmacological inhibitors including those for intracellular signaling pathways with a role in adipocyte gene expression and/or function. For example, reports indicate a role for p38 MAP kinase in the effects of TNFα on adipocyte gene expression [38, 39] and the TNFα effects on the transcript expression of the novel adipocyte lipase ATGL are attenuated by pretreatment with PD-98059, LY-294002, or rapamycin, suggesting involvement of the p44/42

288 MAP kinase, PI 3-kinase, and p70 ribosomal protein S6 kinase signals [40]. As shown in Figure 4, TNFα treatment results in an ~90% decrease (p<0.005) in transcript level for Tmem182 transcript. We postulated that identification of the intracellular signaling pathway(s) involved in the TNFα-mediated decrease might be one step towards gaining insights into Tmem182 function. For example,

TNFα might exert its inhibitory effects by the same intracellular signaling mechanism(s) for a particular subset of genes that share similar and/or related functions in adipocytes. However, the data in Figure 4 reveal that none of the tested pharmacological inhibitors blocked the effects of TNFα, suggesting that

TNFα-mediated diminution of Tmem182 transcript expression occurs via a signaling pathway(s) that is not reliant on p38 MAP kinase, p44/42 MAP kinase, p70 S6 or PI 3-kinase.

Differentiation-dependent expression of Tmem182 transcript in myogenesis - The tissue profiling study in Figure 2 indicates that muscle is also a site of enriched expression of Tmem182 transcript. Adipocytes and myocytes are thought to arise in development from a shared mesenchymal stem cell precursor [8, 41]. The annotation for the GenBank entry

[GenBank:NM_001081198] for Tmem182 indicates it was among the transcripts identified by Kuninger and coworkers in a study of novel genes induced during growth factor-mediated muscle cell survival and differentiation [42]. However, neither the regulation of Tmem182 transcript expression during myogenesis, nor any mention of Tmem182, was in their published report [42]. To determine if

Tmem182 was induced during muscle differentiation we utilized the C2C12

289 model of in vitro myogenesis [43]. As is shown in the left panel of Figure 5,

Tmem182 transcript is markedly upregulated during the conversion of C2C12

myoblasts to myotubes. A 10-fold increase (p<0.001) in transcript level is noted

one day post induction of differentiation and cultures at 7 days post myogenic

induction express ~770 times higher (p<0.001) levels of Tmem182 transcript

compared to the level in C2C12 day 0 myoblasts. The right panel of Figure 5

shows the expression of transcript for myogenin, a muscle regulatory gene that

serves as a marker for myogenesis [43, 44].

Summary - The primary amino acid sequence of Tmem182 predicts an

evolutionarily conserved novel transmembrane protein. Tmem182 protein sequence lacks homologies with previously defined protein families and

Tmem182 function is currently unknown. Enrichment of Tmem182 transcript in

WAT, alteration in obesity, differentiation-dependent upregulation in

adipogenesis and regulation by TNFα suggests that expression of Tmem182

may be integral to the adipocyte phenotype. Interestingly, Tmem182 transcript is

also enriched in muscle tissue and it is markedly upregulated during in vitro

myogenesis of C2C12 myoblasts to myocytes. This suggests Tmem182 may

function in cellular pathways shared by adipocytes and myocytes but not by their

respective precursor cell types. Future studies will further examine the in vitro

and in vivo regulation and the function of Tmem182 in adipocytes and muscle

cells.

290 Abbreviations

PCR, Polymerase chain reaction; FBS, Fetal bovine serum; WAT, White

adipose tissue; BAT, Brown adipose tissue; PPAR, Peroxisome

proliferator-activated receptor; C/EBP, CCAAT/Enhancer binding protein;

Dex, Dexamethasone; Mix, Methylisobutylxanthine; PD, PD98059; SB,

SB203580; LY, LY294002; WM, Wortmannin; Rap, Rapamycin

Competing interests

None

Authors’ contributions

Y. W. conducted all real-time PCR experiments. C. M. S. carried out the analysis in Figure 1, wrote the manuscript and conceived of the study design.

Both authors contributed to data analysis and interpretation, and have read and approved of the manuscript.

Acknowledgements

We thank Dr. J.Y. Kim for kindly providing adipocyte RNA samples.

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Figure legends

Figure 1. Sequence analysis predicts a transmembrane localization for

Tmem182 protein. (A) Amino acid sequence of murine Tmem182. The numbers at right indicate amino acid positions. The four putative membrane- spanning regions are underlined. (B) Results of Kyte-Doolittle analysis with a window size of 10, indicating 4 putative transmembrane regions. Hydrophobicity score is on the y-axis and amino acid residue position on the x-axis.

Figure 2. White adipose tissue enrichment of Tmem182 and dysregulation in ob/ob adipose tissues. (A) Tmem182 transcript expression in various murine tissues. RNA was harvested from the indicated murine tissues and

Tmem182 transcript level was analyzed by real-time PCR as described in

Methods. Values were normalized to Gapdh expression level and transcript expression in lung was set to a value of 1. Statistical analysis was carried out with one-way ANOVA followed by Tukey post-hoc test. ** indicates p <0.001 for

SC WAT vs. all other tissues. * indicates p <0.01 for muscle, heart, and lung vs.

298 all other tissues. (B) Tmem182 transcript expression in wild type brown and

white adipose tissues and (C) Expression of Tmem182 transcript in wild type (wt)

vs. ob/ob white and brown adipose tissues. RNA was harvested from

subcutaneous (SC) WAT, epididymal (EP) WAT and brown adipose tissue (BAT)

of wild type (wt) and ob/ob (ob) mice and real-time PCR for Tmem182 transcript

was performed as described in Methods. Values were normalized to Gapdh

transcript expression level. Transcript expression in BAT was set to a value of 1

for (B) and wt was set to a value of 1 for (C). Statistical analysis was carried out

with single-factor ANOVA. For (B), * indicates p<0.001 for SC, EP, and RP vs.

BAT. For (C), * indicates p<0.001 for ob BAT vs. wt BAT and for ob SC vs. wt

SC.

Figure 3. Tmem182 is a differentiation-dependent gene in white adipogenesis. (A) Tmem182 transcript expression during a time course of 3T3-

L1 differentiation. 3T3-L1 RNA was harvested daily at the indicated days preceding (D-1) and during differentiation. Tmem182 transcript level was analyzed by real-time PCR as described in Methods. Values were normalized to

Gapdh transcript expression level. For D-1, D0, D1, and D2, a value of 40 cycles was assigned to calculate a delta Ct value and transcript expression in D-1 sample was set to a value of 1. * indicates p<0.001 for D3, D4, D5, D6 and D7 vs. all other samples. (B) Tmem182 transcript expression in ScAP-23 preadipocytes (Pre) and adipocytes (Adi) and (C) Tmem182 transcript expression in primary murine subcutaneous preadipocytes (Pre) and adipocytes

299 (Adi). RNA was harvested from the indicated cultures before and after

differentiation and Tmem182 transcript level was analyzed by real-time PCR as

described in Methods. Values were normalized to Gapdh transcript expression

level and transcript expression in Pre set to a value of 1. Statistical analysis was

carried out with single-factor ANOVA. For (B) and (C), * indicates p<0.001 for

Pre vs. Adi.

Figure 4. TNF downregulates Tmem182 transcript expression in 3T3-L1

adipocytes. After serum-starvation for 6 h, 3T3-L1 adipocytes were incubated with either DMSO vehicle (V), 50 µM PD98059 (PD), 20 µM SB20358 (SB), 50

µM LY294002 (LY), 100 nM wortmannin (WM), or 1 µM rapamycin (Rap) for 1 h and then either treated or untreated with 10 ng/ml of TNF for 16 h. RNA

samples were harvested and Tmem182 transcript level was analyzed by real-

time PCR as described in Methods. Values were normalized to Gapdh transcript

expression level and transcript expression in vehicle alone was set to a value of

1. Statistical analysis was carried out with single-factor ANOVA. * indicates

p<0.001 for indicated samples vs. V alone (first column). Treatment with

inhibitors alone failed to appreciably alter expression for Tmem182 transcript

(data not shown).

Figure 5. Tmem182 is a differentiation-dependent gene in C2C12

myogenesis. (A) Tmem182 transcript expression during a time course of

C2C12 myogenic differentiation. C2C12 RNA was harvested at the time of 300 induction of differentiation (0) or at indicated time points thereafter; numbers below graphs indicate days post-induction. Tmem182 (left panel) and myogenin

(right panel) transcript levels were analyzed by real-time PCR as described in

Methods. Values were normalized to Gapdh transcript expression level and the value in the day 0 sample set to 1. Statistical analysis was carried out with single-factor ANOVA. * indicates p<0.001 for indicated values compared to day

0.

Additional files

None

301 Figure 1

302 Figure 2

303 Figure 3

304 Figure 4

305 Figure 5

306 DISCUSSION AND SUMMARY

This dissertation is focused on identification and characterization of novel genes in adipogenesis from several angles.

In the first part of the work, we report that Wdnm1-like is a new adipokine secreted by adipocytes. Adipose tissue is not only a site for triglyceride storage but is also an endocrine organ. Adipose tissue derived hormones and cytokines directly contribute to the pathogenesis of obesity-related disorders such as diabetes and cardiovascular diseases. We identified Wdnm1-like as a highly upregulated adipocyte gene in a brown adipogenesis microarray study, however it is also significantly elevated in white adipogenic conversion and is highly enriched in WAT vs. BAT. Wdnm1-like is dysregulated in visceral WAT and brown adipose depots of ob/ob mice in comparison with wild type mice. In ob/ob mice, the absence of leptin has effects on both energy intake and expenditure and leads to morbid obesity. Visceral adipose tissue is much more closely related to obesity-linked diabetes and metabolic syndrome, as compared to subcutaneous. On the other hand, in ob/ob mice, the BAT marker gene UCP-1 expression is downregulated due to reduced thermogenesis (Commins et al.,

1999). Wdnm1-like may be involved in leptin related metabolic inefficiency and insulin resistance.

The upregulation of Wdnm1-like transcript occurs within 6 h of treatment with adipogenic cocktail during 3T3-L1 cell differentiation and only the combination of Dex and Mix has the ability to enhance the expression of Wdnm1- like transcript. This upregulation of Wdnm1-like emergence parallels the 307 upregulation of PPARγ transcript. Further studies can be conducted to determine

if Wdnm1-like is transactivated by PPARγ in early adipogenesis.

In 3T3-L1 adipocytes, TNFα treatment increases Wdnm1-like transcript

level and this is possibly mediated by p38 MAP. TNFα suppresses many adipocyte genes and activates a large number of preadipocyte genes (Ruan et al.,

2002a; Ruan et al., 2002b). However, we observed upregulation of Wdnm1-like by TNFα treatment. There are several possible explanations; (1) TNFα is

reported to downregulate those adipocyte genes that are closely related to free

fatty acid or glucose uptake, thus subsequently promoting insulin resistance.

However we have not found any Wdnm1-like response upon insulin treatment in adipocytes (data not shown). (2) A small subset of preadipocyte and adipocyte genes are reported to not be regulated by TNFα. For example, the expression level of the well-known preadipocyte marker gene Pref-1 remains constant during

TNFα treatment of 3T3-L1 adipocytes (Xing et al., 1997), and the adipocyte

expressed gene haptoglobin increases upon TNFα treatment of 3T3-L1

adipocytes (Ruan et al., 2002a). (3) The suppression of adipocyte-specific and

activation of preadipocyte genes by TNFα is mediated by the activation of NF-κB.

Whether Wdnm1-like is a downsteam target of NF-κB action remains to be

determined. (4) TNFα also downregulates the master transcriptional regulator

PPARγ to decrease expression of PPARγ-target genes in adipocytes as

mentioned above, whether Wdnm1-like is transactivated by PPARγ is currently

unknown.

308 Intriguingly, we found that LPS has the ability to stimulate Wdnm1-like transcript expression in RAW 264.7 macrophages. Metabolism and immunity are integrated systems and adipose tissue consists of adipocytes and many other cell types including macrophages. Macrophage infiltration can regulate adipocyte differentiation via paracrine activities. Adipogenesis of human primary preadipocytes is impaired by treatment with macrophage-conditioned medium

(Lacasa et al., 2007). The fact that Wdnm1-like is expressed in both macrophages and adipocytes reinforces the concept of highly overlapping biological characteristics and functions of macrophages and adipocytes and suggests a possible role for Wdnm1-like in the inflammatory response.

Finally, we demonstrated that Wdnm1-like functions to enhance the levels of active MMP-2, and showed that this is not through regulation of either MMP-2 or MT1-MMP/MMP14 transcript levels. MMPs are key for matrix remodeling in adipogenesis. MMP-2 activity is increased during preadipocyte differentiation and is markedly induced in obese adipose tissues; inhibition of MMP-2 activity significantly reduces differentiation (Bouloumie et al., 2001; Chavey et al., 2003;

Derosa et al., 2008). However, 14-day old MMP-2 knockout mice shows normal adipose tissue development, indicating the various MMP-2 functions in different environment setting (Itoh et al., 1997; Kato et al., 2001). In contrast, MT1-

MMP/MMP-14, an important activator for MMP-2, is required for white adipose tissue development in vivo (Chun et al., 2006). We postulate that Wdnm1-like may affect the active MMP-2 levels by regulating the protein levels of MMP-2,

MT1-MMP/MMP-14 or TIMP2, the latter is a key molecule important in MMP-2

309 activation. Further studies regarding Wdnm1-like function on the role of MMPs in

adipogenesis will clarify these effects.

Adipocytes and their secreted factors are now recognized as key

contributors to tumor microenvironment (Schaffler et al., 2007). Co-injection of

tumor cells and adipocytes increases tumor cell motility, migration, and

angiogenesis (Iyengar et al., 2003). Adipocyte-secreted collagen VI can support early mammary tumor progression in vivo (Iyengar et al., 2005). MMPs are well

known to play central roles in tumor cell metastasis and invasion (Noel et al.,

2008). Therefore Wdnm1-like may function as an important link between

adipocytes and the promotion of tumor cell invasion and metastasis.

In the second part of the work, we focused on identifying novel markers for preadipocytes. This work revealed TSC-36 is a preadipokine secreted by preadipocytes. TSC-36 transcript level is markedly downregulated in all white and brown adipogenesis models tested. TSC-36 protein expression is also decreased during the time course of adipogenic conversion.

TSC-36 promoter activity can be downregulated by the master transcriptional regulator PPARγ and its heterodimerization partner retinoid X receptor α (RXRα). TSC-36 is also named follistatin like-1 (FSTL1). It has very limited homology to follistatin, an inhibitor of follicle-stimulating hormone (FSH). A recent study reported that the transcript level of follistatin is downregulated by

rosiglitazone-activated PPARγ in intestinal epithelial cells in vivo. This

downregulation is mediated by Sp1, which dramatically upregulates the follistatin

promoter activity, but this regulation can repressed by PPARγ. Furthermore,

310 knockdown of Sp1 can rescue the rosiglitazone effects on the follistatin gene.

The follistatin promoter also contains binding sites for AP (activator protein) -1

and 2, however the authors did not find the regulation of the follistatin promoter

by AP-1 and 2 (Necela et al., 2008). TSC-36 has been shown to be

downregulated by AP-1 in FBR-v-fos (FBR) transformed fibroblasts. Re-

expression of TSC-36 inhibits cell invasion (Johnston et al., 2000). The possible

regulatory relationship between PPARγ, AP-1, Sp1 and TSC-36 can be further addressed.

Recent publications from the K. Walsh laboratory revealed that TSC-36 is a novel upregulated target of Akt. Akt is an important factor for promotion of cardiac myocyte growth and survival. Transgenic mice with targeted cardiac expression of Akt develop concentric cardiac hypertrophy and subsequently dilated cardiomyopathy, TSC-36 transcript is upregulated in cardiomyocytes of

Akt transgenic mice. Overexpression of TSC-36 protects cardiomyocytes from

ischemia and hypoxia/reoxygenation induced apoptosis both in vivo and in vitro

(Oshima et al., 2008). Their study also revealed that TSC-36 is secreted by

C2C12 myoblasts and is upregulated under conditions of muscle ischemia.

Intramuscular injection of an adenoviral vector expressing TSC-36 promoted flow

recovery and capillary density in animal hindlimbs under ischemic conditions.

This effect is due to TSC-36 stimulated endothelial nitric oxide synthase (eNOS)

phosphorylation and consequently enhanced revascularization in vascular

endothelium (Ouchi et al., 2008). The latest publication regarding the location of

white preadipocyte progenitor cells in vivo demonstrates that the progenitors

311 reside in the vasculature of adipose tissue (Tang et al., 2008a). TSC-36 expression levels in these preadipocyte progenitor cells and whether TSC-36 plays a role in Akt signaling in these progenitor cells during development in the adipose tissue vasculature environment are the questions that need to be addressed in future studies.

To explore the possible mechanisms underlying the differential functions of visceral vs. subcutaneous adipose depots, we utilized subtractive cDNA libraries from murine subcutaneous (SC) or intra-abdominal epididymal (EP) white adipocytes to identify the genes distinctly expressed in different depots.

Major urinary proteins (Mups) are enriched in SC adipocytes in comparison with

EP. Mups are known to function in scent signaling and to act as carrier proteins involved in binding and releasing pheromones in rodents (Beynon and Hurst,

2004). Mups are also used as chemosignals for individual recognition (Cotton,

2007). There are rare reports regarding Mups and adipogenesis. In a study focused on the effects of short-term dietary restriction in mice with diet-induced obesity, Mup1 was the only gene found to be significantly downregulated in SC

WAT of both lean and obese mice following dietary restriction weight loss (van

Schothorst et al., 2006). On the other hand, dietary restriction ameliorates high plasma free fatty acids and blood glucose levels significantly. Thus Mups can be possibly involved in obesity-related metabolic diseases.

In contrast to Mups, Boc and other 7 transcripts are enriched in EP at least 3-fold greater than SC adipocytes. Cdon, the binding partner of Boc, is also enriched in the EP WAT vs. SC WAT, although this is only noted for the SV

312 fraction or intact WAT. Boc and Cdon bind specifically to sonic hedgehog (Shh) and are candidate receptors of Shh (Okada et al., 2006). Shh signals not only play role in invertebrate and vertebrate development, but also regulate adipose tissue formation. In Drosophila, overexpression of hedgehog specifically in fat- body blocks fat development. In mammalian cell culture models such as 3T3-L1 cells and pluripotent mesenchymal cells, activation of Shh signals also impairs adipogenesis. Shh signals are upstream of PPARγ and result in inducing multipotent mesenchymal precursors to undergo osteogenesis. This effect may be mediated by Hedgehog signal-induced GATA-2 (Suh et al., 2006). Therefore

Boc and Cdon may be involved in adipocyte formation and function. Future research of regional adiposity and specific gene regulation and function thereof may lead to a further understanding of adipose depot specific physiology.

Here we also report on the mBAP-9 cell line as a novel brown preadipocyte cell line. mBAP-9 is unique because it is derived from preadipocyte precursors present in murine BAT via spontaneous immortalization. By utilizing mBAP-9 cells, we identified ten novel adipocyte genes including two putative new hydrolases, Cmbl (carboxymethylenebutenolidase-like) and a new adipose tissue

α/ hydrolase Atabh. In our studies on TSC-36, mBAP-9 together with other several in vitro culture models served as models in which to validate candidate preadipocyte marker genes. Our research demonstrates that the mBAP-9 cell line is a useful research tool to further explore brown adipogenesis as well as to utilize to identify preadipocyte and adipocyte marker genes.

313 Tmem182 is a novel predicted transmembrane protein found to exhibit a

45-fold upregulation in mBAP-9 adipogenesis during our characterization of mBAP-9 adipogenesis. We further characterized the expression and regulation of

Tmem182 transcript. Tmem182 transcript is highly enriched in the adipocyte fraction of adipose tissue and upregulated several hundred fold during white and brown adipogenesis. Intriguingly, Tmem182 transcript is also upregulated ~770- fold during C2C12 myogenesis. Adipocytes and myocytes are believed to have a common mesenchymal stem cell precursor (Rosen and MacDougald, 2006).

Inhibition of myogenesis via expression of dominant negative MKK3 (mitogen- activated protein kinase kinase-3) in C2C12 myoblasts directs the trans- differentiation of the cells into adipocytes (Yeow et al., 2001). In addition, recent research demonstrates that brown preadipocyte precursors express Myf5, a myogenic specific marker. PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16) is a critical determinant in the cell fate of multipotent mesenchymal precursors. PRDM16 is positively correlated with brown adipogenesis. Brown adipose tissue lacking PRDM16 exhibits abnormal morphology and increased expression of muscle-specific genes (Seale et al., 2008). Based on the observation that Tmem182 is markedly upregulated in adipogenesis and myogenesis, Tmem182 may play role in the adipocyte and myocyte differentiation process.

314 CONCLUSION

1. Wdnm1-like is a novel 6.8 kDa secreted protein. Wdnm1-like is a

differentiation-dependent gene in white and brown adipogenesis. Wdnm1-like

transcript is enriched ~500-fold in white adipose depots vs. brown and is

restricted to the adipocyte population. Wdnm1-like transcript is upregulated

within 6 h of adipogenic induction and reaches an ~17,000 fold increase by

day 7 of 3T3-L1 adipogenesis. TNF treatment of 3T3-L1 adipocytes

increases Wdnm1-like transcript level 2.4-fold and this can be attenuated by

pretreatment with the p38 MAP kinase inhibitor SB203580. Ectopic

overexpression of Wdnm1-like in HT1080 fibrosarcoma cells markedly

increases active MMP-2 levels. Our findings identify a new member of the

adipocyte “secretome” that functions to enhance MMP-2 activity. We

postulate that Wdnm1-like may play roles in remodeling of the extracellular

milieu in adipogenesis as well as in tumor microenvironments where

adipocytes are key stromal components.

2. TSC-36 is identified as a preadipocyte gene which is highly expressed in 3T3-

L1 preadipocytes and downregulated to nearly undetectable levels in

adipocytes. We determined that TSC-36 is a preadipokine secreted by

preadipocytes. The expression of TSC-36 mRNA was markedly decreased

during both white and brown adipogenesis. TSC-36 protein level is also

markedly reduced during differentiation. PPARγ and KLF15 downregulate

315 TSC-36 promoter activities. TSC-36 transcript and protein levels are

increased in 3T3-L1 adipocytes after TNFα treatment. In 3T3-L1

preadipocytes, TSC-36 expression is downregulated by 5-azacytidine.

Together, our data indicate that TSC-36 expression might be a distinct feature

of preadipocytes.

3. To identify genes distinctly expressed in WAT depots which may impart

depot-dependent physiological functions, we undertook preparation and

screening of murine suppressive subtractive hybridization (SSH) cDNA

libraries enriched for genes expressed in either SC or EP murine adipocytes.

Boc, a component of the hedgehog signaling pathway demonstrated highest

enrichment (~12-fold) in EP adipocytes. Boc transcript expression decreases

in obese EP WAT with a concomitant upregulation of Boc transcript in obese

SC WAT depot. As a Boc binding partner, the expression of Cdon was also

assessed in adipose tissue and adipose tissue cell fractions. We also

identified a dramatic enrichment in SC adipocytes vs. EP adipocytes and in

SC WAT vs. EP WAT for transcript(s) for the major urinary proteins (Mups),

small secreted proteins with pheromone functions that are members of the

lipocalin family. Mup transcripts were predominantly expressed in liver, SC

WAT and RP WAT. Mups transcript levels increase several thousand-fold

during differentiation of primary murine preadipocytes to adipocytes. Mup

transcripts were also markedly reduced in SC WAT and liver of ob/ob mice

compared to wild type. Further assessment of WAT depot-enriched

316 transcripts may aid in our understanding of the physiological impact of

regional adiposity.

4. mBAP-9, a new brown preadipocyte cell line, was established from

interscapular BAT of C57BL/6 mice. mBAP-9 preadipocytes undergo

spontaneous adipocyte differentiation that is manifested by the accumulation

of multiple lipid droplets and which is enhanced by exposure to

dexamethasone, 3-methyl-1-isobutylxanthine, indomethacin, insulin and

triiodotyronine. The induction of Ucp1, PPAR and PGC-1 transcripts and

increased mitochondrial DNA content during mBAP-9 adipogenesis indicate a

brown adipocyte lineage for mBAP-9. Isoproterenol and forskolin upregulate

Ucp1 transcript expression in mBAP-9 adipocytes, illustrating effective β-

adrenergic and cAMP-mediated signaling. 10 adipocyte differentiation-

dependent genes that were upregulated 4-fold or greater during mBAP-9

adipogenesis were identified. Two of these encoding putative hydrolases,

carboxymethylenebutenolidase-like (Cmbl), a novel gene with homology to

Psuedomonas putida dienelactone hydrolase, a new adipose tissue α/

hydrolase (Atabh) were further studied. We conclude that the mBAP-9

preadipocyte cell line is a novel and useful tool for detailed molecular studies

of brown adipogenesis.

5. Tmem182 is a novel predicted transmembrane protein found to exhibit 45-fold

upregulation in mBAP-9 adipogenesis. Further characterization of Tmem182 317 transcript expression revealed that it is enriched in WAT. Tmem182 transcript expression is ~3-fold upregulated in BAT of ob/ob mice vs. wild type.

Tmem182 transcript is markedly upregulated in various in vitro models of white adipogenesis. TNFα treatment of 3T3-L1 adipocytes results in a ~90% decrease of Tmem182 transcript level. Strikingly, Tmem182 transcript is also upregulated ~770-fold during myogenesis of C2C12 cells. This suggests that

Tmem182 may function in intracellular pathways important in both adipogenesis and myogenesis.

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345 ABSTRACT

This dissertation is focused on identification and characterization of novel

genes in adipogenesis from different angles.

Wdnm1-like was identified as a novel 6.8 kDa adipokine secreted by

adipocytes. Wdnm1-like is a differentiation-dependent gene in white and brown

adipogenesis. TNF treatment of 3T3-L1 adipocytes increases Wdnm1-like

transcript level 2.4-fold and this can be attenuated by pretreatment with the p38

MAP kinase inhibitor SB203580. Ectopic overexpression of Wdnm1-like in

HT1080 fibrosarcoma cells markedly increases active MMP-2 level.

TSC-36 was identified as a preadipocyte gene that is highly expressed in

3T3-L1 preadipocytes and downregulated to nearly undetectable levels in 3T3-L1 adipocytes. TSC-36 is therefore a preadipokine secreted by preadipocytes.

PPARγ and KLF15 downregulate TSC-36 promoter activities. TSC-36 transcript and protein levels are increased in 3T3-L1 adipocytes after TNFα treatment. In

3T3-L1 preadipocytes, TSC-36 expression is downregulated by 5-azacytidine.

In a study to identify genes distinctly expressed in specific WAT depots and which may impart depot-dependent physiological functions, Boc transcript demonstrated a 12-fold enrichment in EP adipocytes. Expression of transcript for the Boc binding partner, Cdon was also assessed in adipose tissue and cell fractions thereof. We also identified a dramatic enrichment in SC adipocytes vs.

EP adipocytes and in SC WAT vs. EP WAT for transcript(s) for the major urinary proteins (Mups).

346 We established and characterized mBAP-9 which is derived from interscapular BAT of C57BL/6 mice as a new brown preadipocyte cell line. 10 adipocyte differentiation-dependent genes that are upregulated 4-fold or greater during mBAP-9 adipogenesis were identified. Two of these encoding putative hydrolases, Cmbl and Atabh were further studied.

Tmem182 is a novel predicted transmembrane protein found to exhibit 45- fold upregulation in mBAP-9 adipogenesis. Further characterization of Tmem182 transcript expression revealed that Tmem182 transcript is markedly upregulated in various in vitro models of white adipogenesis. Moreover, Tmem182 transcript is also upregulated ~770-fold during the C2C12 cells myogenesis, which suggests Tmem182 may function in intracellular pathways important in both adipogenesis and myogenesis.

In summary, this dissertation reveals novel genes distinctly expressed in adipocyte, preadipocyte or in specific WAT depots and also demonstrates mBAP-9 as a new brown preadipocyte cell line. This work will contribute to define a more complete picture of adipogenesis.

347