THE EFFECTS OF LEPTIN, GIP AND CLENBUTEROL ON BONE MARROW GENE

EXPRESSION

by

SARAH N. LACKAY

(Under the Direction of Clifton A. Baile)

ABSTRACT

Administration of Leptin, GIP, and Clenbuterol results in increased adipogenesis and

decreased osteoblastogenesis in mice and rats. Microfluidic technology was used to analyze

changes in expression of 23 genes chosen for their association with adipocytes, osteoblasts, and

varying growth factors. Comparison of injection location showed that there was no significant

difference between LV, ARC and VMH injection of Leptin on gene expression in rats.

Treatment with GIP decreased overall gene expression according to Tukey’s test (p<0.0001).

However, varying doses of GIP elicited no different effect on gene expression, implying a

threshold level of GIP that must be met to induce a decrease in gene expression. Leptin also

decreased overall gene expression relative to control (p<0.0001). Gene expression in rats treated

with Leptin was significantly lower than gene expression in rats treated with GIP 1.0 µg

(p=0.0059) and GIP 10 µg (p=0.0131). This suggests that Leptin has a more potent effect on

bone marrow than does GIP. Treatment with Clenbuterol also elicited a decreased expression of

bone marrow genes (p<0.0001).

INDEX WORDS: Adipogenesis, Osteogenesis, Bone Marrow, Leptin, GIP, Clenbuterol

THE EFFECTS OF LEPTIN, GIP AND CLENBUTEROL ON BONE MARROW GENE

EXPRESSION

by

SARAH N. LACKAY

B.A., Vassar College, 2004

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2006

© 2006

Sarah N. Lackay

All Rights Reserved

THE EFFECTS OF LEPTIN, GIP AND CLENBUTEROL ON BONE MARROW GENE

EXPRESSION

by

SARAH N. LACKAY

Major Professor: Clifton A. Baile

Committee: Steven L. Stice

Roger Dean

Electronic Version Approved:

Maureen Grasso

Dean of the Graduate School

The University of Georgia

May 2006

iv

DEDICATION

I dedicate this thesis to my parents, Thomas and Carole Lackay, whose love and support

have made all my accomplishments possible.

v

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Clifton A. Baile, who has provided me with everything I

needed to finish my graduate work here at the University of Georgia. Thanks to Steven Stice and

Roger Dean for their valuable input. I would also like to thank Diane Hartzell, for organizing

our experiments and making all protocols available to me. Special thanks to Ji Lin, without his

help I would still be trying to isolate RNA. Also thanks to Jiuhua Duan, Qiang Li, Wei Zhang,

Hye- Kyeong Kim, and Yang-Ho Choi for assisting with all of the experiments and data

collection. Kara Huff and Roger Nielsen, you have both been so important to me and all of my

work, thank you for answering all of my many questions and so selflessly taking the time to help

me learn so many techniques. Thank you Dorothy Hausman for all the skills you have taught me

over the past year. Thank you Mark Hamrick for your valuable input. Thank you also to

Stephanie Chirello for doing everything necessary to get my reagents to me in time, and for

putting up with me always being late on time sheets. Very special thanks to Mark Froetschel for

helping me to get to UGA in the first place, and to Robin Harvey for helping me to always get

my assistantship money.

Thanks to all of my friends, especially Frank West, Matt Bechard, Brenda Darby, and

Brent Jackson. Without all of our study sessions, parties, trips to the airport and lunches I

wouldn’t have enjoyed graduate school at all. Remember, the difference between a good friend

and an extraordinary friend is just that little extra!

Thanks to Kelly Robbins, without whom I would have always been hungry, poor, and

lonely. The love and support you have given me was crucial to my success. Thank you for

always saying the right thing and keeping me positive and on-track.

vi

Thanks to Ripley who always cuddled me and cheered me up when I was stressed, and to

Hanna, who always kept me moving forward (by running behind me and biting my ankles).

Thanks to my grandfather for his love and support over the years.

Thank you to my Aunt Anne-Marie and my Uncle Bill Simonitis, and for all of their fun

and zany good times over the years. You are ingrained in my sense of family, and I know that I

am a better person because of the influence you have both had on my life. Uncle Bill- you

always said, “Don’t work hard, work smart.” I’ll at least try the second part!

Thank you to my grandmother, Mary Simonitis. Without our daily soup and sandwich

growing up, I would have never been inquisitive enough to pursue a research degree. She

remains the smartest woman I know, and can still answer any vocabulary question I ask.

Thank you to my sister. You have been my constant role model. My successes in life are

in no small part due to the fact that I try to be like you. You are and will always be my best

friend, and the person whose advice I most respect.

And most importantly, thank you to my parents. Dad, your sacrifices throughout the

years have always inspired me to push myself further and do that little extra to achieve success.

I can always overcome my fears and do more than I thought possible by following your example.

Mom, your constant support and ability to make every day special, and your unending

selflessness made me who I am today. Just like you.

vii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS…..………………………………..………………………………….v

LIST OF TABLES………………..………………………..…………………………………......ix

LIST OF FIGURES……….…………..………………..…………………………….…………...x

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW……...……………………………....1

2 ADIPOCYTE DIFFERENTIATION…...………………………………………………....3

3 BONE FORMATION and LEPTIN………..…………..………………………………....7

Bone Formation………………………..……………………………………….....7

Leptin……………………………………..………………………………..……...9

Leptin and Bone……………………………..…………………………..……….11

4 DISEASES ASSOCIATED WITH BONE MARROW…..……………………..………14

Diabetes ………………………………………………..………………..……….14

Osteoporosis………………………………………………..…………….....……17

Obesity…………………………………………………………..……….....……22

5 MICROFLUIDIC TECHNOLOGY and GENE OVERVIEW……………..………..…..25

viii

6 EFFECT OF INJECTION OF LEPTIN AND GLUCOSE DEPENDANT

INSULINOTROPIC POLYPEPTIDE ON BONE MARROW ADIPOCYTE

APOPTOSIS AND OSTEOGENESIS IN RATS………………………..………..……..36

Abstract…..………………………………………………………………..……..37

Introduction…..…………………………………………………………..………38

Materials and Methods…...………………………………………………..……..38

Results………………………..…………………………………………..………42

Discussion………………………..…………………………………………..…..49

Conclusion…………………………..…………………………………………...52

7 CLENBUTEROL, A BETA-2 ADRENERGIC AGONIST, INCREASES MURINE

BONE MARROW POTENTIAL FOR ADIPOGENESIS ………………………….53

Abstract………………………………………………………………………54

Introduction………………………………………………………………..…55

Materials and Methods………………………………………………….........55

Results………………………………………………………………………..57

Discussion……………………………………………………………………58

Conclusion…………………………………………………………………...65

8 SUMMARY………………………………………………………………………….66

REFERENCES…………………………………………………………………………………..68

APPENDICES………...…………………………………………………………………………77

A Rat Gene Names and Alternative Symbols………………………………………78

B Glossary……………………………………………………….…………………79

ix

LIST OF TABLES

Page

Table 6.1: The LS Mean RQ values were significantly lower for both 10.0 µg Leptin and

GIP doses of 0.1 µg, 1.0 µg, and 10.0 µg, as compared to aCSF treatment……...........43

Table 6.2: p values for linear contrasts between treatment groups, numbered as follows:

1) aCSF 2) GIP 0.1 µg 3) GIP 1.0 µg 4) GIP 10.0 µg and 5)Leptin 10 µg.………...45

Table 6.3: LS Mean RQ Values for LV and VMH treatments of Leptin…………..…….……...46

Table 6.4: p-values for the linear contrast of VMH and LV treatments of Leptin. 1) LV

0.0 µg 2) LV 0.05 µg 3) LV 1.25 µg 4) VMH 0.0 µg 5) VMH 0.05 µg...….………...47

Table 6.5: LS Mean RQ Values for VMH and ARC injections of Leptin……………..………...47

Table 6.6: p-values for linear contrasts of ARC and VMH Leptin treatments…………..............48

Table 7.1: LS Mean RQ values for varying doses of Clenbuterol fed to ICR mice over a

21 day treatment period…………… ………………………………….…………..58

Table 7.2: Genes tested for based on association with adipocytes, osteoblast, and growth

factors…………………………………………………………………………….....……......59

x

LIST OF FIGURES

Page

Figure 6.1: LS Mean RQ Values for aCSF, 0.1 µg GIP, 1.0 µg GIP, 10 µg GIP and 10 µg

Leptin…………………………………………………………………...…………......44

Figure 6.2: LS mean RQ values for LV and VMH treatments of Leptin………………..……...46

Figure 6.3: LS mean RQ values for ARC and VMH injections of Leptin………………….........48

Figure 7.1: LS Mean RQ values for varying doses of Clenbuterol fed to ICR mice over a

21 day treatment period ……………………………………………………..………..57

1

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

There are several diseases influenced by the delicate balance between adipogenesis and

osteoblastogenesis in bone marrow. At least 10.3 million Americans are diagnosed with diabetes

mellitus, and an additional 5.4 million are estimated to have undiagnosed diabetes (Grundy et al.,

1999). Current estimates indicate that fifty percent of women over age 45, and ninety percent of

women over age 75 are affected by osteoporosis, and osteoporosis affects more than two million

American men. One in five men over the age of 65 will fracture their hip, spine, wrist, or ribs

due to osteoporosis (Sedlak et al., 2000). One third of the American population is now

considered obese (Spiegelman and Flier, 2001). It is estimated that 300,000 people die annually

in the Unites States as a result of obesity. Most of these deaths are due to obesity’s contribution

to the development of diabetes, hypertension, cardiovascular disease, and cancer. It is therefore

imperative to not only identify which genes are affected by these conditions, but also to learn

more about possible treatments useful for restoring the bone resorption – bone formation

balance.

Leptin is a 16 kD protein, and is the product of the ob gene. A mutation of this gene

results in obesity in the ob/ob mouse. Prouteau et al. (2006) found that changes in leptin

expression were significantly correlated with changes in bone resorption marker in response to

weight loss and regain. Glucose dependant insulinotropic polypeptide (GIP) is an insulinotropic

agent with a stimulatory effect on insulin release and synthesis in the pancreas. Recently, GIP

receptors and GIP mRNA have been found in bone, and administration of GIP to ovariectomized

2

mice prevented bone loss. Clenbuterol is a β2-adrenergic receptor (β2-AR) agonist that has been

shown to decrease body fat and increase muscle mass with oral administration in rodents.

Studying the effects these compounds have on bone marrow gene expression may shed some

light on the complex pathways connecting adipogenesis to obesity, osteoporosis, and diabetes.

3

CHAPTER 2

ADIPOCYTE DIFFERENTIATION

An adipocyte is defined as the animal connective tissue cell specialized for the synthesis

and storage of fat. Adipocytes are bloated with goblets of triglycerides, and their nucleus is

displaced to one side of the cell. In mammals there are two distinct types of adipose tissue, white

adipose tissue (WAT) and brown adipose tissue (BAT). These two tissues have opposite

functions. BAT utilizes lipids to generate heat (thermogenesis), while WAT stores excess

energy as triglyceride in lipid droplets. During periods of food intake, energy is stored in WAT,

to be mobilized during periods of food shortage. However, as WAT increases, BAT decreases

and no BAT has been identified in human adults (Tong and Hotamisligil, 2001).

BAT contains a high number of mitochondria as well as an uncoupling protein that leaks

the proton gradient across the mitochondrial membrane to generate a futile cycle to produce heat

(Tong and Hotamisligil, 2001). Spiegelman calls the mitochondria a cellular furnace where fuels

derived from fatty acids and glucose are oxidized and energy is either stored in ATP or released

as heat. As electrons are passed down the energy gradient of the electron transport chain,

protons are pumped out of the inner matrix of the mitochondria, generating an electrochemical

gradient across the inner mitochondrial membrane. These protons then do one of two things.

First, they might reenter the mitochondrial matrix through ATP synthase, driving the synthesis of

ATP (coupled respiration). Or, they might “leak” back across the inner mitochondrial membrane

(uncoupled respiration). In uncoupled respiration, accelerated via uncoupling proteins (UCPs),

energy is released as heat because leaks disrupt the cycle and result in fuel oxidation in the

absence of work (Spiegelman et al., 2001). Mice genetically reduced in BAT are prone to

4

obesity. This is not true for mice deficient in UCP-1. Mice lacking UCP-1 do have a heightened

sensitivity to cold, however (Spiegelman et al., 2001).

In recent years, however, the adipocyte is gaining recognition as a secretory organ, and

not just an energy reserve. This is primarily due to the discovery of Leptin, the obese gene

product that is secreted by mature adipocytes as a gauge of lipid stores. It has since been found

that several proteins are produced by adipocytes, such as adipsin, acylation stimulation protein

(ASP), adipocyte complement-related protein (Acrp30/AdipoQ), tumor necrosis factor-α

(TNFα), and migration inhibitory factor (MIF). In addition to these immune system related

proteins, vascular function-related proteins such as angiotensinogen and plasminogen activator

inhibitor type 1 (PAI-1) are also secreted from adipocytes. Therefore, the adipocyte can be seen

as an endocrine cell in addition to its existing role as a paracrine/autocrine cell (Gregoire, et al.,

1998).

In preadipose cells, growth arrest is necessary for adipocyte differentiation.

CCAAT/enhancer binding protein α (C/EBP-α) and PPAR-γ are involved in this growth arrest

and have been shown to transactivate adipocyte specific genes. After this growth arrest,

preadipocytes must receive the appropriate signals to differentiate into adipocytes. Preadipose

cell lines undergo at least one round of DNA replication and cell doubling. Retinoblastoma

proteins pRB, p107, and p130 bind to E2F/DP to inactivate growth-promoting transcriptional

activities. A family of growth-arrest specific (Gas) proteins Gas6, Gas1, and Gas3 are also

expressed in preadipocytes (Gregoire et al., 1998).

Early signs of differentiation include: expression of lipoprotein lipase mRNA, induction

of C/EBP-α and PPAR-γ, conversion from fibroblastic to spherical shape, decrease in actin and

tubulin expression, increase in secretion of type IV collagen, decrease in amount of pericellular

5

Fibronectin, and a dramatic decrease in Pref-1. Pref-1 is abundant in preadipocytes and is

though to maintain the preadipose phenotype (Gregoire et al., 1998).

ADD1/ SREBP1 can accelerate adipogenesis when coexpressed in fibroblasts expressing

PPAR-γ. Interestingly ADD1/SREBP1 can also activate a broad program of genes involved in

fatty acid and triglyceride metabolism in both fat and liver. ADD1/SREBP1 controls seven

enzymes of fatty acid metabolism. Expression of ADD1/SREBP1 is regulated by feeding,

fasting, and insulin (Spiegelman et al., 2001).

The GATA transcription factors have been found to be critical in the early stages of

adipogenesis. Members of the GATA family share a highly conserved zinc finger DNA binding

domain and bind specifically to a consensus DNA sequence . GATA-2 and GATA-3 are the

dominant members of this family in WAT. Expression of both of these factors is down regulated

at the onset of adipocyte differentiation. Constitutive expression of these factors inhibits

adipogenesis, via the inhibition of PPARγ2 and protein-protein interaction with the C/EBP

family (Tong and Hotamisligil, 2001).

Id proteins are nuclear helix-loop-helix proteins without a basic domain. Hence they

cannot bind DNA. Instead they form heterodimers with other bHLH proteins to regulate

differentiation and development. Id2 and Id3 are expressed in preadipocytes but suppressed in

differentiated form. CHOP-10, a member of the C/EBP family, is expressed in preadipocytes

and downregulated after hormonal induction. CHOP-10 forms a heterodimer to prevent C/EBPβ

from binding the C/EBPα gene promoter. AEBP-1, a transcription factor with carboxypeptidase

activity, and AP-2α, a transcriptional repressor of the C/EBPα promoter, are both expressed in

preadipocytes and downregulated in mature adipocytes. There are also several soluble factors

affecting differentiation. Wnt proteins, highly conserved secreted signaling molecules that

6

regulate cell–cell interactions during embryogenesis, inhibit adipocyte differentiation. Pref-1 is

expressed in preadipocytes, but constitutive expression prevents adipocyte conversion (Tong and

Hotamisligil, 2001).

In cultured preadipocyte cells, insulin, IGF-1, glucocorticoids, and cAMP-generating

agents have all been shown to promote terminal differentiation. Upon stimulation with these

agents, cells undergo several rounds of proliferation and then a growth arrest. During this

period, C/EBP-β and C/EBP-δ have a temporal rise in expression. This is followed by an

increase of expression in PPAR-γ and C/EBP-α. Both of these factors are part of a positive

feedback loop, inducing each others expression. The cooperative action of PPAR-γ and C/EBP-

α drives the expression of several genes necessary for generation and maintenance of adipocytes,

including aP2, PEPCK, glycerophosphate dehydrogenase, fatty acid synthase, acyl CoA

carboxylase, Glut4, and insulin receptor. Interestingly, coexpression of PPAR-γ and C/EBP-α

can even transdifferentiate myoblasts into adipocytes. Embryonic stem cells deficient in PPAR-γ

can not be differentiated into adipocytes and do not perform adipose tissue formation in chimeric

mice (Tong and Hotamisligil, 2001).

Other factors and events included in terminal differentiation include sensitivity to insulin,

increase in lipogenesis, increase in the activity, protein and mRNA levels for enzymes involved

in triacylglycerol metabolism, increases in glucose transporters and insulin receptor number, loss

of β1-adrenergic receptors and an increase in β2- and β3- subtypes. Terminal differentiation also

requires the synthesis of aP2, FAT/CD36, perilipin, monobutyrin, adipsin, Acrp30/AdipoQ, PAI-

1, angiotensinogen II, and Leptin (Gregoire et al., 1998). ADSF/Resistin, on the other hand, is a

product of adipocytes, but not preadipocytes (Tong and Hotamisligil, 2001).

7

CHAPTER 3

BONE FORMATION AND LEPTIN

Bone Formation

There are several different types of bone growth. Growth in bone length (endochondral

ossification) involves two steps, the addition of cartilage tissue to the growth plates at the

proximal and distal ends of long bones, and the transformation of the cartilaginous scaffold into

bone tissue in the adjacent metaphyses. Growth in bone width (modeling) involves osteoblasts

on the periosteal surface of a bone cortex and osteoclasts on the endocortical surface of the

cortex. Osteoblasts increase the outer circumference of bone by depositing bone matrix and later

mineralizing it (Schoenau et al., 2004). Osteoclasts resorb bone, increasing the size of the

marrow cavity. During modeling, osteoclasts usually remove less bone tissue than is deposited

by osteoblasts. Bone maintenance (remodeling) is the continuous turnover of existing bone,

consisting of successive cycles of bone resorption and formation on the same bone surface.

Osteoclasts remove a small quantity of bone tissue which is later replaced by osteoblasts. This

remodeling process renews the bone tissue and helps to prevent tissue damage from

accumulating. Without bone remodeling, maintenance of blood calcium levels, mechanical

support to soft tissues, and protection for the brain and spinal cord would not be possible (Harada

and Rodan, 2003).

Bone formation begins with chemotaxis of osteoblast precursors to sites of resorption

defects. A proliferation of these precursors then matures into osteoblasts. When mature, these

osteoblasts are capable of laying down mineralized bone matrix, expressing the structural

proteins of the bone matrix, and then mineralizing the bone matrix. Many growth regulatory

factors are involved in this prolonged highly coordinated process. These factors are incorporated

8

into the bone matrix, released in active form when the bone is resorbed, and are available locally

to control events in bone formation. It is theorized that these factors are released as a cascade to

control osteoblast precursor proliferation and chemotaxis, osteoclast apoptosis, and osteoblast

differentiation (Mundy 2002). However, bone formation is much slower than bone resorption.

Bone resorbed in 2-3 weeks will take at least three months to rebuild (Harada and Rodan, 2003).

Bone formation is thought to be regulated by the CNS and the hormone Leptin (Johnson

and Tabin, 1997). There are several factors that regulate bone resorption and formation.

Calcium mobilization requires bone destruction, and therefore invariably causes bone resorption.

Athletic activity and weight bearing exercise increases bone mass. Estrogen also preserves bone,

possibly as a result of an evolutionary need of calcium stores for embryonic skeletal

development (Harada and Rodan, 2003).

It is important to remember that bone density has a different definition than the physical

density defined by Archimedes (mass of a body divided by its volume). Bone density is defined

as the degree to which a radiation beam is attenuated by a bone, as judged from a two-

dimensional projection image (areal bone density) (Schoenau et al., 2004).

Bone mineralization is the incorporation of minerals such as calcium and phosphorous

into organic bone matrix after it has been synthesized and deposited by osteoblasts. Bone

mineral content is the mass of mineral per unit bone length. Mineralization can only occur

where bone matrix has previously been deposited. A decrease in bone mineralization can occur

by two mechanisms. Either not enough organic matrix has been deposited, or not enough

mineral has been incorporated into the matrix (osteomalacia and osteopenia, respectively)

(Schoenau et al., 2004).

9

Leptin

Leptin is a 16 kD protein, and is the product of the ob gene. A mutation of this gene

results in obesity in the ob/ob mouse. Leptin is secreted mainly by white adipose tissue, and its

regulation on food intake and body weight occurs via negative feedback to the hypothalamic

nuclei. It has been found to reduce food intake and increase energy expenditure, among other

things. Most importantly for the following experiments though, is Leptin’s effect on human

marrow stromal cells. It has been found that Leptin enhances stem cell differentiation to

osteoblasts and inhibits differentiation to adipocytes. Thomas et al (1999) found that Leptin did

not affect hMS2-12 cell proliferation but did increase mRNA protein levels of alkaline

phosphatase (AP), type 1 collagen, osteocalcin, and lipoprotein lipase. Leptin decreased mRNA

levels of adipsin and Leptin. While mineralized bone matrix increased by approximately 59%

during Leptin treatment, lipid droplet formation decreased by about 50 %. Interestingly, they

found that Leptin did not affect PPARγ2 or Cbfa1 expression. PPAR γ2 is a transcription factor

involved in the adipocyte pathway and Cbfa1 is a factor in the osteoblast pathway. From these

results it was concluded that Leptin acts on human marrow stromal cells to enhance

differentiation into osteoblasts, and inhibit differentiation into adipocytes.

Removal of the Leptin signal caused by food restriction exceeds the rate at which food

stores are reduced. This reduction in Leptin is responsible for a complex neural response similar

to that seen during starvation. Absence of the Leptin signal promotes obesity by creating an

internal impression of starvation. Reintroduction of energy supplies, however, reverses this

starvation process and raises Leptin levels. Leptin is limited in its abilities as an anti-obesity

hormone, however, as large increases in fat mass limit the ability of Leptin to suppress food

10

intake. Leptin also affects peripheral tissues such as muscle and liver, suppressing the

accumulation of triglyceride and contributing to insulin resistance.

The most clinically relevant circuit in the brain for expressing Leptin receptors and

responding to Leptin with neuropeptides and changes in energy expenditure is the Leptin-

regulated central melanocortin pathway. In this pathway Leptin acts through ObRb receptors in

two distinct populations of neurons in the arcuate nucleus. The first population expresses the

feeding–inducing neurons NPY and AgRP. The second population expresses mRNAs encoding

anorexigenic peptides, cocaine and amphetamine related transcript (CART) and α-MSH. Leptin

reduces the expression of the first population and induces the expression of the second. AgRP

and α-MSH are antagonistic ligands for a common receptor, the melanocortin 4 receptor

(MCR4). Activation by MSH reduces food intake, while suppression of MCR4 by AgRP

increases feed intake and decreases hypophagic response to Leptin. Gene deletion of MCR4

causes obesity, as does heterzygosity for the knockout allele.

In a study on a family (HD) with a strong prevalence of early onset morbid obesity,

Clèment et al. (1998) found that subjects with a homozygous mutation in the Leptin receptor

gene were morbidly obese, had no pubertal development, and had a reduced secretion of growth

hormone and thyrotropin. These results suggest that Leptin is necessary not only for regulation

of body weight, but also for sexual maturation and secretion of growth and thyrotropic-

hormones, implicating it as a critical link between energy stores and hypothalamic pituitary

function.

11

Leptin and Bone

Leptin expression has been shown to be related to many diseases involving bone

resorption and formation. Leptin levels have been found to be decreased in several diseases,

including leukemia and lymphoma (Nilgun, et al., 2005). Agras et al. (2005) found that elevated

Leptin levels are associated with increased bone mass at the lumbar sites in renal transplant

recipients, indicating a bone-sparing effect in renal patients. An inverse relationship between

Leptin and bone mineral density was also found in chronic liver patients (Ormarsdottir, et al.,

2001).

Prouteau et al. (2006) found that changes in Leptin expression were significantly

correlated with changes in bone resorption marker in response to weight loss and regain.

Osteocalcin and total plasma protein remained unaffected by weight loss. However, a 4±0.5%

body weight regain induced a 276% increase in Leptin levels and an 18% increase in insulin.

Jansson (2006) has shown that leukemia inhibitory factor (LIF) treatment of

ovariectomized (OVX) mice caused a decrease in weight of white fat depots, brown fat mass,

and serum Leptin levels. Treatment with LIF did not, however, affect trabecular bone mineral

density or femur length.

Stavropoulou et al. (2005) examined the role of Leptin in osteoporosis by correlating

Leptin levels with N-telopeptide of collagen type 1 (NTx) and osteocalcin levels before

ovariectomy at 20,40 , and 60 days after operation. By day 20 post-operation, levels of NTx,

osteocalcin, and Leptin were significantly increased. Bone markers and Leptin levels remained

constant until day 40, and decreased (insignificantly) on day 60. Leptin was significantly

correlated with bone markers after ovariectomy, indicating that alterations in Leptin levels

during the progression of osteoporosis follow changes in bone markers.

12

Kishida et al. (2005) studied the effect of Leptin on bone development, specifically

endochondral ossification at the growth plate. They found that Leptin was localized in

prehypertrophic chondrocytes in normal mice, and that Ob-Rb was localized in hypertrophic

chondrocytes in both normal and ob/ob mice. Growth plates of the ob/ob mice were more fragile

than normal mice, with disturbed columnar structure, decreased type X collagen expression, less

organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin

administration in ob/ob mice led to increase in femoral and humeral lengths and decrease in the

proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. Leptin

also eliminated the matrix mineralization of chondrocytes in both ob/ob and wild type mice.

Leptin suppressed apoptosis, cell growth and matrix calcification. Their results show that Leptin

is associated with several events in the terminal differentiation of chondrocytes.

Chan et al. (2005) found a potential therapeutic use for Leptin in patients with

hypothalamic amenorrhea. Hypothalamic amenorrhea and anorexia nervosa are associated with

low Leptin concentrations. Chan et al. found that Leptin can restore ovulatory menstrual cycles,

and improve reproductive, thyroid, and IGF hormones and bone markers in hypothalamic

amenorrhea.

Javaid et al. (2005) found a strong positive correlation between umbilical venous Leptin

concentration and each of whole body bone mineral contents and estimated volumetric bone

density, whole body lean mass, and whole body fat mass. Indicating not only an predicative

value for Leptin in size of neonatal skeleton and volumetric mineral density, but also maternal fat

stores as a determinant on fetal bone accrual via variation in fetal Leptin concentrations.

Rigaux et al. (2005) studied a group of 31 men with severe brain injuries, and compared

serum factors influenced by bone metabolism between patients with and without heterotrophic

13

bone formation. Groups with heterotrophic bone formation had significantly higher serum

alkaline phosphates levels and significantly lower serum Leptin levels. These results indicate

that the antiosteogenic effect of Leptin mediated by hypothalamic neurons may be impaired by

hypothalamic damage related to severe brain injury.

Hjeltness et al. (2005) studied the variations in Leptin levels of tetraplegic patients and

found that fasting tetraplegic subjects had Leptin levels four times those of controls. In

tetraplegia plasma Leptin levels were negatively correlated with total lean mass but correlated

positively with total fat mass. A circadian variation in plasma Leptin concentrations was more

evident in tetraplegia than in controls. If Leptin metabolism is impaired among these patients, a

distortion of thermogenesis and energy expenditure would occur, explaining the risk of

metabolic syndrome and osteoporosis among tetraplegics.

Satoh et al. (2004) found that while plasma Leptin levels are increased in obese type 2

diabetes patients, adiponectin levels are decreased, suggesting a role for Leptin to adiponectin

ratio as a potential atherogenic index in these patients.

There are several medical conditions linked to abnormal Leptin levels. A microfluidic

card quantifying expression of genes involved in Leptin physiology could serve as a possible

diagnostic tool for several diseases, including but not limited to diabetes, osteoporosis and

cancers such as leukemia.

14

CHAPTER 4

DISEASES ASSOCIATED WITH BONE MARROW

Diabetes

Diabetes mellitus is defined as a relative or absolute lack of insulin leading to

uncontrolled carbohydrate metabolism. At least 10.3 million Americans are diagnosed with

diabetes mellitus, and an additional 5.4 million are estimated to have undiagnosed diabetes

(Grundy et al., 1999). There are several types of diabetes, but the three most common are type 1,

type 2 and gestational diabetes. Patients with type 1 diabetes, also known as insulin-dependant

diabetes mellitus (IDDM), do not produce insulin. This case is most common in younger

patients. The exact cause of this type of diabetes is unknown, but genetic factors seem to play a

major role in its occurrence. It is thought that juvenile onset diabetes may be an autoimmune

response to pancreatic β-cells. Type 2 diabetes patients (non-insulin-dependant diabetes

mellitus, NIDDM), are resistant to the insulin they produce, leading to impaired insulin

utilization. In this form of diabetes, hyperinsulinemia cannot account for the body’s resistance.

In adult onset diabetes there seems to be no immunological component, as there is in juvenile

onset cases, but there is an association with obesity. Gestational diabetes develops only in

pregnant women with no previous history of diabetes. It is defined as glucose intolerance,

usually noticed between the 24th

and the 28th

weeks of pregnancy. Usually, the blood glucose

level returns to normal after delivery. This form of diabetes is usually not a serious condition for

the mothers’ health, but can cause fetal and newborn death.

The American Heart Association recognizes diabetes as an independent risk factor for

cardiovascular disease with complications such as coronary heart disease, stroke, peripheral

arterial disease, retinopathy, and possible neuropathy and cardiomyopathy. Insulin resistance is

15

common in patients with type 2 diabetes, and these patients are likely to have several metabolic

risk factors including atherogenic dyslipidemia, hypertension, elevated plasma glucose, and a

prothrombotic state. Also common in patients with diabetes is diabetic nephropathy. This

serious and progressive disease indicates damage to the kidney, followed by a decline in the

glomular filtration rate. In patients with progressive renal insufficiency, as is commonly the case

in individuals with type 2 diabetes, end-stage renal disease ensues and dialysis or transplantation

become necessary to sustain life. Mortality rates for patients with diabetes who are on renal

dialysis exceed 20% per year (Grundy et al., 1999).

Recently a connection has been found between plasma Leptin levels and diabetes.

Specifically, plasma Leptin levels have a strong association with cardiovascular risk factors such

as insulin resistance, metabolic syndrome, and inflammatory markers (Reilly et al., 2004). The

risk of atherosclerotic cardiovascular disease increases 2-4 fold in type 2 diabetes mellitus.

Because diabetes is associated with increased adiposity, there are usually elevated levels of

plasma Leptin, secreted by an excess of adiposity. It is believed that peripheral actions of Leptin

such as endothelial activation and migration, smooth muscle cell proliferation and calcification,

and the activation of monocytes and adaptive immune responses may promote atherosclerosis in

obese patients with type 2 diabetes mellitus. Leptin receptors have been found on atherosclerotic

lesions. It has been found that Leptin is a predictor of cardio vascular disease events (Wallace et

al., 2001). Reilly et al. found that, in addition to elevated plasma Leptin levels, patients with

type 2 diabetes also had increased coronary artery calcification, and that this calcification

increased across Leptin quartiles. Female gender, waist size, body mass index, systolic blood

pressure, and plasma levels of insulin and C-reactive protein were all positively associated with

Leptin. Increased plasma Leptin levels have also been found in women with gestational

16

diabetes, indicating an association of Leptin with parameters of glucose metabolism (Kautzky-

Willer et al., 2001). Interestingly, Muzzin et al. (1996) found that treatment of the ob/ob mouse

with Leptin resulted in the normalization of serum insulin levels, glucose tolerance, and dramatic

reductions in food intake and body weight, implicating Leptin as a possible treatment for both

obesity and diabetes.

Diabetes has also been linked to problems occurring in the bone marrow. Diabetic bone

marrow mononuclear cells have an impaired capacity to differentiate into endothelial progenitor

cells in vitro and to participate in vascular like structure formation (Tamarat, et al., 2004). These

deficient bone marrow cells are involved in a condition known as bone marrow mononuclear cell

dysfunction, which may contribute to the abrogated post-ischemic revascularization reaction

associated with diabetes. In patients with diabetes collateralization is insufficient to overcome

the loss of blood flow through occluded arteries, leading to ischemia.

Diabetes has been found to be related to osteoporosis, as type 1 diabetes is associated

with decreased bone mass, increased fracture risk, and delayed healing. It has also been

suggested that the biomechanical integrity of diabetic bone may be impaired. A decreased bone

turn over, due to impaired osteoblastic maturation and function is thought to play a part in the

osteopenia associated with diabetes. Apoptosis of osteoblastic cells has been found to be

enhanced in diabetic bone. This decrease in bone turn over is due to decreased bone formation,

not resorption. IGF-1, osteocalcin, and bone specific alkaline phosphatase are decreased in

adolescence with type 1 diabetes. Patients with type 2 diabetes do not have low bone mass,

thought to be an effect of the additional weight these patients accumulate. Also, insulin has been

shown to increase proliferation and function of osteoblasts, and an insulin deficiency suppresses

osteoblastic activity. Therefore, increased levels of insulin found in insulin-resistant diabetics

17

may actually improve osteoblastic function. Also, elevated levels of androgens in obese women

may contribute to bone maintenance. However, despite a higher bone mineral density, there may

be an increased fracture risk associated with type 2 diabetes (Schneider and Shapses, 2003). An

accumulation of bone without the proper turnover via participation of osteoclasts may be

responsible for this.

Diabetes has also been linked to several types of cancer, including digestive-tract tumors.

In a study on over one million Koreans, Jee et al. (2005) found that a higher fasting serum

glucose level is associated with higher death rates from all cancers combined. For men, higher

fasting glucose level was specifically associated with cancers of the esophagus, liver, pancreas,

colon/rectum, stomach, and the bladder, as well as with leukemia. Fasting serum glucose level

was inversely associated, however, with prostate cancer. For women, a higher fasting glucose

level was associated with cancers of the pancreas, liver, lung breast, and cervix.

Osteoporosis

Osteoporosis is defined as a reduction in bone mass, leading to fractures after minimal

trauma. It is characterized by a decrease in bone density and an enlargement of bone spaces

producing porosity and brittleness. Unlike the cause of bone fractures in diabetes, many forms

of osteoporosis are caused by reduced activity of osteoclasts, resulting in defective remodeling of

bone and increased bone density. A common cause of this reduced osteoclast activity is a proton

pump or chloride channel defect. Both of these pumps are responsible for acidifying the bone

surface beneath osteoclasts (Steward, 2003). Again, contrary to the negative health risks

associated with obesity in a diabetic patient, obesity serves a protective purpose and is a negative

risk factor for osteoporosis. In support of this, trabecular bone and adipose tissue content in the

bone marrow are inversely related in human disuse osteoporosis (Thomas et al., 1999).

18

However, little is known about the pathophysiology of osteoporosis, and cases are

classified as either “primary” or “secondary” on the basis of loosely defined clinical associations.

In primary osteoporosis no other associated disease state is present. This is the most common

form of the disease and patients include post-menopausal women. Primary osteoporosis is also

common in thin Caucasian women. Primary osteoporosis occurs frequently in patients with

chronic diseases accompanied by low body weight, such as anorexia nervosa and chronic

inflammatory bowel disease. In secondary osteoporosis an associated disease state is recognized,

although the exact connection of the two diseases is poorly understood. This secondary form of

osteoporosis is frequently linked to corticosteroid therapy. Osteoporosis is also sometimes

classified as either high-turnover or low-turnover. Low-turnover osteoporosis is conducive for

using specific treatments to selectively inhibit bone resorption or stimulate bone formation

(Mundy, 2000).

Osteoporosis is especially prevalent in post-menopausal women, and current estimates

indicate that fifty percent of women over age 45, and ninety percent of women over age 75, are

affected by this disease. Yamauchi et al. (2001) found that plasma Leptin levels were associated

with bone mineral density and the presence of vertebral fractures in post-menopausal women.

Osteoporosis does not only affect elderly women, however. Leptin has been found to be

a regulator of bone mineral density in men, as well (Sato et al., 2001). Osteoporosis affects more

than two million American men. One in five men over the age of 65 will fracture their hip,

spine, wrist, or ribs due to osteoporosis (Sedlak et al., 2000).

Also, there are rare cases of osteoporosis presenting itself in children. Idiopathic juvenile

osteoporosis is present in children and adolescents, and resolves after the onset of puberty

19

(Kauffman et al., 2001). However, these cases are usually attributed to a vitamin D deficiency or

calcium malabsorption leading to hyperparathyroidism.

Important to the physiology of osteoporosis is the fact that osteoclast functional activity,

and not osteoclast formation, is increased in primary involutional osteoporosis (Jevon et al.,

2003). Therefore the best treatment would be one that either inhibits this increase in activity,

stimulates osteoblast activity to catch up to the increase in osteoclast activity, or does both.

Among the markers involved in osteoporosis, MCSF is required for osteoclast differentiation.

MCSF production by osteoblasts inhibits osteoclast apoptosis. Therefore, MCSF may increase

bone resorption by elongating the lifespan of osteoclasts. RANK is expressed by osteoclast

precursors, while RANKL is expressed by differentiated osteoblasts (Jevon et al., 2003).

Op/op mice have a defect in the gene encoding MCSF (CSF1) but treatment of human

patients with MCSF is of no benefit. The TCIRG1 gene is mutated in 50% of human cases with

MIOP. The oc/oc mouse bears a 1.6 kb deletion in this gene. TCIRG1 gene encodes the

osteoblast-specific subunit of the vacuolar H(+)-ATPase (Steward, 2003). It has also been found

that a polymorphism at the Sp1 site of the Col1A1 locus can act as a marker for osteoporotic

fracture (McGuigan 2000).

Several drugs currently exist for the treatment of osteoporosis, divided into three classes:

bone resorption inhibitors, bone formation stimulators, and those that accomplish both. Bone

resorption inhibitors include estrogen, calcitonin, and bisphosphates. Flavanoids have received

recent attention for their ability to act as inhibitors of bone resorption. Stimulators of bone

formation include anabolic agents such as fluoride, low dose parathyroid hormone, strontium

ranelate, statins and peptide growth factors. Additionally several molecular targets have been

identified in the osteoclast resorption pathway, such as CBFA1, a critical transcription factor

20

expressed uniquely by osteoblasts and centrally involved in osteoblast differentiation. If CBFA1

is not expressed, a complete lack of bone formation will result. Bone morphogenic proteins are

among the growth factors identified as having a role in bone formation. The BMPs enhance

osteoblast differentiation. BMP2 acts as an autocrine factor in osteoblast proliferation, and is

expressed by differentiating normal osteoblasts. BMP2 enhances the expression of the structural

proteins in the bone matrix, such as type-1 collagen, osteopontin, osteocalcin, and bone

sialoprotein. It also enhances CBFA1. The colony-stimulating factor-1 receptor and RANK

ligand have both been found to be involved in osteoclastic bone resorption (Mundy 2002).

Steward (2003) notes that the most serious consequences of osteoporosis occur in the

nervous system, where cranial nerves, blood vessels and the spinal cord are compressed by either

gradual occlusion or lack of growth of skull foramina. As a result, patients can develop several

complications, including optic, facial and trigeminal neuropathies, strabismus, hearing loss,

dysarthria, hydrocephalus, cerebral atrophy and developmental delay. Infantile osteoporosis is

associated with a high mortality rate, as a result of bone marrow encroachment. However, 70%

of infants receiving bone marrow transplants from matched sibling donors attain long-term

disease free survival. Hence, diagnosis is the largest obstacle to overcome in these patients, and

a way to monitor progress of treatment would be a valuable asset. There are six variants of

autosomal recessive OP and two major variants of autosomal dominant OP (ADO).

Vacuolar proton pump deficiency causes visual failure, hypocalcaemia, fractures, mild to

severe hematological impairment, hepatosplenomegaly, failure to thrive, poor growth and

reduced life expectation. It is associated with an increased bone density, modeling deformities,

pseudorickets, and plentiful osteoclasts.

21

ClC-7 chloride pump deficiency causes severe osteoporosis and retinal degeneration, and

is associated with a normal numbers of osteoclasts.

Carbonic anhydrase isoenzyme type 2 deficiency causes short stature, psychomotor

retardation, renal tubular acidosis, optic atrophy and visual loss, and minor hematological

impairment and is associated with intracranial calcification.

Neuronopathic OP, also known as OP and infantile neuroaxonal dystrophy is

accompanied by gross hepatosplenomegaly, severe anemia, thrombocytopenia, high arched

palate, gum hypertrophy, hyperreflexia, clonus, opisthotonos, fisting, poor head control,

irritability, and early death. This form of the disease is associated with mild to severe cerebral

atrophy.

OP with agenesis of corpus callosum suffers the same conditions and is a possible variant

of neuronopathic OP, and is differentiated by the absence of the corpus callosum.

Dysosteosclerosis is associated with fractures, developmental delay, visual and hearing

loss, osteomyelitis of the mandible, macular atrophy of the skin, and flattened fingernails.

Radiological and pathological features include platyspondyly, sclerotic long bones, retarded

white matter myelination, and intracerebral calcification.

The two types of dominant OP are autosomal dominant OP types I and II. Type I is

associated with pain, hearing loss, and osteosclerosis in the cranial vault, but not an increased

fracture rate. Type 2 is associated with fractures, scoliosis, hearing loss, optic atrophy, facial

palsy, thickening of the vertebral end plates and skull base, and bone-within-bone/endbone

structures.

Because osteoporosis affects such a wide variety of people, a possible treatment for this

disease would be a lucrative enterprise. Although several studies have reported Leptin having no

22

effect on bone physiology, there are an equal or perhaps greater number of papers reporting a

Leptin effect on bone mineral density. A microfluidic card could help to determine if Leptin is

involved in osteoblastogenesis.

Obesity

Obesity is defined as an increase in body weight beyond the limitation of skeletal and

physical requirements, as the result of an excessive accumulation of fat in the body. One third of

the American population is now considered obese (Spiegelman and Flier, 2001). It is estimated

that 300,000 people die annually in the Unites States as a result of obesity. Most of these deaths

are due to obesity promotion of diabetes, hypertension, cardiovascular disease, and cancer.

Obese patients are almost all hyperleptinemic, but because the hypothalamus is unable to

recognize the Leptin signal body weight is not reduced normally (Leptin resistance) (Lustig et

al., 2004). It is known that both bone mass and serum Leptin levels are increased in obesity.

Obese patients are also almost uniformly hyperinsulinemic. Because insulin stimulates Leptin

production from adipocytes, weight loss regimens that cause lipolysis lead to Leptin suppression.

A reduction in insulinemia would therefore improve Leptin sensitivity and promote weight loss

(Lustig et al., 2004).

The central nervous system (CNS) regulates energy balance and body weight via several

different mechanisms. First, it influences behavior, such as intake and activity. Second it

influences autonomic nervous system activity, impacting energy expenditure and metabolism.

Lastly it affects the neuroendocrine system, influencing the secretion of hormones such as

growth hormone, thyroid, cortisol, insulin, and sex steroids. Leptin is involved in all of these

pathways (Spiegelman and Flier, 2001).

23

Behavior, including physical activity and feed intake, is affected by the central nervous

system. Both short and long term control systems exist in the regulation of feeding behavior.

Short term control involves neural and endocrine factors such as cholecystokinin (CCK) that are

generated during ingestion. Long term signals report the status of energy stores. Leptin is one of

these signals, reporting on the current amount of adipose tissue (Spiegelman and Flier, 2001).

Several studies have been done on mice to investigate the relationship between Leptin

and obesity. In ob/ob mutant mice, the truncated and biologically inactive Leptin protein results

in obesity. This obesity syndrome is corrected, however, by administration of Leptin. In db/db

mice the signaling form of the Leptin receptor, ObRb, is deleted, resulting in resistance to

endogenous and exogenous Leptin (Spiegelman and Flier, 2001).

Lustig et al. (2004) showed that the ratio of resting energy expenditure (REE) to Leptin

concentration correlates negatively with changes in insulin area under the curve (IUAC) to oral

glucose tolerance testing. The authors used pharmacological agents to suppress insulin secretion.

These results support the hypothesis that hyperinsulinemia may be a cause of Leptin resistance.

Because several obese subjects lost weight after insulin impairment, reduction of insulinemia

may be a method to promote weight loss and increase insulin sensitivity. The REE: Leptin ratio

may also be a useful clinical indicator of Leptin sensitivity.

Clèment et al. (1998) studied a family with a strong prevalence of morbid obesity and

found that a mutation in the human Leptin receptor gene causes both obesity and pituitary

dysfunction. Their results imply that Leptin is required for body weight regulation, sexual

maturation, and for the secretion of growth and thyrotropic-hormones. This demonstrates

Leptin’s role as a critical link between energy stores and hypothalamic pituitary function in

humans.

24

Hotamisligil et al. (1996) found that another gene, aP2, was central to the pathway

linking obesity to insulin resistance. aP2 encodes the adipocyte fatty acid binding protein, and

mice with a null mutation in aP2 failed to develop insulin resistance or diabetes, and they also

failed to express TNF-α, a molecule that may link obesity to insulin resistance.

25

CHAPTER 5

MICROFLUDIC TECHNOLOGY AND GENE OVERVIEW

Each of these disease states has associated with it changes in the expression of genes

related to adipocytes, osteoblasts, and several other types of cells. In order to detect these

changes 23 genes were selected for a card applicable to mice, and an equivalent 23 genes were

selected for a card applicable to rats. Genes were chosen based on their ability to be detected in

a mixed cell environment, and to express genes specific to one type of cell, i.e. osteoblasts,

adipocytes, etc.

Gamma-carboxyglutamic acid protein (BGLAP, also known as Bone Gamma-

Carboxyglutamic Acid Protein; Bone Gla Protein, BGP; Osteocalcin) is a small highly conserved

molecule associated with the mineralized matrix of the bone. Osteocalcin is the only gene that is

expressed in osteoblasts, but not in other extracellular matrix (ECM) –producing cell types. The

murine osteocalcin gene contains a cis-acting element (OSE2) in its promoter that binds only to

osteoblast nuclear protein Osf2 and confers osteoblast-specific activity on a heterologous partner.

It can therefore be inferred that an increase in BGLAP expression would signal induction of

osteoblastogenesis.

Osteoclasts, the multinucleated cells involved in bone resorption, degrade inorganic and

organic components of bone in local areas subjacent to the matrix attachment site. In mice,

inactivation of the Tcirg1 (T Cell Immune Regulator- 1) gene causes osteoclast-rich

osteoporosis. Osteoporosis is the result of a decreased level of osteoclast remodeling, without

which osteoblasts build up forming a bone that is denser, yet more likely to fracture. Hence a

decreased level of Tcirg1 is indicative of a buildup of osteoblasts, and the onset of osteoporosis.

26

CCL27 (Chemokine, CC motif, Ligand 27) is a chemokine involved in T-cell mediated

skin inflammation. Mice injected with human CCL27 intradermally had a dose-dependant

expression of IL2, CCR10 and LFA1A. Treatment with glucocorticosteroid or anti-Ccl27

reduced skin thickness and leukocyte recruitment in contact hypersensitivity and atopic

dermatitis mouse models. It can therefore be inferred that a decreased level of expression of

Ccl27 would indicate reduced skin thickness and immune response, whereas an increased

expression would signal increased immune response.

Colagen1A1 (Collagen of skin tendon and bone, Alpha chain 1) has been linked to both

osteoporosis and osteogenesis imperfecta. A gene polymorphism in the Sp1 binding site is

associated with osteoporotic fracture.

Wang et al. (1990) showed that when BMP2A produced by recombinant DNA techniques

was implanted into rats, bone formation occurred by day 14. BMP2 protein induced medulla-

blastoma cell apoptosis, whereas BMP2 antagonist blocked both retinoid and BMP2-induced

apoptosis. Cheng et al. (2003) measured the ability of 14 human BMPs to induce osteogenic

transformation in a mouse pluripotential stem cell line, a mouse mesenchymal stem cell line, and

a human osteoblastic cell line. BMP was able to stimulate alkaline phosphatase activity in

mature osteoblasts, and BMP2 was among the few BMPs able to induce all markers of osteoblast

differentiation in pluripotential and mesenchymal stem cells.

Runt related transcription factor 2 (RUNX2, also known as CBFA1) encodes a protein

that binds to an osteoblast-specific cis-acting element (OSE2) in the promoter of osteoclacin.

Ducy et al. (1997) showed that Cbfa1 is an osteoblast specific transcription factor and a regulator

of osteoblast differentiation. Komori et al. (1997) generated mice with a mutated Cbfa1 locus

and found that mice homozygous for the mutation died immediately after birth, with complete

27

lack of ossification of the skeleton. Immature osteoblasts and osteoclasts appeared at the

perichondrial regions, but neither vascular nor mesenchymal cell invasion was observed in

cartilage, suggesting intramembranous and endochondral ossification were completely blocked,

and that Cbfa1 has an essential role in osteogenesis.

Transcription factor Sp7 (Specific Protein Sp7, Osterix) is a zinc finger transcription

factor of the SP gene family and a putative master regulator of bone cell differentiation.

Nakashima et al. (2002) identified from mouse progenitor cells the transcription factor osterix

(Osx), which is specifically expressed in all developing bones. In the same study by Nakashima,

Osx-null mice were found to have no bone formation, despite expression of Runx2.

PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) perhaps deserves some

additional review, due to the depth of its association with diabetes, obesity, adipocyte

differentiation and cancer. The PPARs are members of the nuclear receptor subfamily of

transcription factors. Elbrecht et al. (1996) found that PPARγ is expressed at high levels in

adipocytes and at much lower levels in bone marrow, spleen, testis, brain, skeletal muscle and

liver. The highest levels of PPARγ are found in adipose tissue and spleen, with much lower

levels in the kidney, liver, small intestine, and muscle (Fajas, et al., 1997) There are in fact three

isoforms of PPARγ: 1, 2, and 3; however, PPARγ 1 and PPARγ 3 give rise to the same protein.

Insulin stimulates the ligand-independent activation of PPARγ1 and PPARγ2. Obesity and

nutritional factors, on the other hand, influence only the expression of PPARγ2 in human

adipocytes (Deeb et al., 1998).

Biochemical studies have shown that FMOC-L-leucine (F-L-leu), a PPARγ ligand that

binds to the ligand binding domain of a PPARγ molecule, induces a particular allosteric

configuration of PPARγ leading to a modified pattern of target intervention which improves

28

insulin sensitivity in normal, diet-induced glucose-intolerant, and diabetic db/db mice. However,

adipogenic activity is decreased with F-L-leu binding. It has therefore been inferred that F-L-leu

activates some (insulin sensitization) but not all (adipogenesis) PPARγ -signaling pathways

(Rochhi et al., 2001) Nakamichi et al. (2003) found that overexpression of PPARγ in a mouse

insulinoma cell line inhibits glucose-stimulated proinsulin biosynthesis and insulin release.

Elbrecht et al. (1996) while studying the molecular cloning and expression of cDNAs encoding

human PPARγ1 and PPARγ2, found that the antidiabetic activity of thiazolidinediones (TZDs) is

mediated through the activation of PPARγ 1 and PPARγ 2.

Tontonoz et al. (1995) found that PPARγ 2 regulates adipocyte expression of the

phosphoenolpyruvate carboxykinase gene. Further demonstrating the effect of PPARγ on

adipose tissue, Tong et al. (2000) suppressed PPARγ causing constitutive GATA2 and GATA3

expression, trapping cells at the preadipocyte stage. Lapsys et al. (2000) found that lipoprotein

lipase (LPL), muscle carnitine palmitoyltransferase-1, and fatty acid binding protein (FABP)

correlated significantly with PPARγ expression. These three genes are highly involved in lipid

metabolism, and hence these results suggest PPARγ activators may regulate fatty acid

metabolism in both skeletal muscle and adipose tissue. In examining the relationship between

body mass index and PPARγ isoform expression in freshly isolated human adipocytes, Sewter et

al. (2002) found a strong inverse relationship between the two, indicative of an autoregulatory

mechanism holding the expansion of individual adipocytes in states of positive energy balance.

Rosen et al. (2002), in order to examine the relationship of PPARγ and C/EBPα as transcription

factors in adipogenesis, created an immortalized cell line of fibroblasts lacking PPARγ. They

found that C/EBPα has no ability to promote adipogenesis without PPARγ, indicating a single

pathway of fat cell development exists in which PPARγ and C/EBPα participate. Furthermore

29

these results imply that PPARγ is the proximal effector of adipogenesis. Lastly, Patsouris et al.

(2004), in a study of hepatic glycerol metabolism, showed that PPARγ directly regulates glycerol

metabolism in adipose tissue.

PPARγ has also been shown to have roles in various cancers and immunological

pathways. Mueller et al. (1998, 2000) has shown the role of PPARγ in both metastatic breast

adenocarcinomas–where PPARγ induces terminal differentiation of malignant breast epithelial

cells, as well as in prostate carcinomas-where PPARγ exerts an inhibitory effect on the growth of

cancer. Welch et al. (2003) in a study on the regulation of lipopolysaccharide and IFN-γ target

genes in macrophages, came to the conclusion that PPARγ has a physiological role in regulating

both native and acquired immune responses. Ameshima et al. (2003) found that loss of PPARγ

expression characterizes an abnormal, proliferating, apoptosis-resistant endothelial cell

phenotype. In support of this conclusion, Bruemmer et al. (2003) found that activation of

PPARγ leads to apoptosis and growth arrest in vascular smooth muscle cells.

PPARγ has also been shown to have an effect on bone metabolism in vivo. Akune et al.

(2004) studied the effect of PPARγ insufficiency on osteogenesis. PPARγ -/- embryonic stem

cells failed to differentiate in to adipocytes, but did spontaneously differentiate in to osteoblasts.

However, reintroduction of PPARγ restored adipogenesis to wild type levels. PPARγ +/- mice

displayed high bone mass due to increased osteoblastogenesis but normal osteoblast and

osteoclast functioning. This effect was not mediated by insulin or Leptin.

Fatty acid binding protein, which expression was found to correlate significantly with

PPARγ (Lapsys et al., 2000), is part of a family of highly homologous cytosolic proteins. Fatty

acid binding proteins are small cytoplasmic proteins that are highly tissue specific (Hotamisligil

et al., 1996). Hotamisligi’s experiments with aP2 deficient mice on high-fat, high-caloric diets

30

were key to concluding that aP2 is central to the pathway linking obesity to insulin resistance.

aP2 deficient mice in this study gained more weight than control mice while developing obesity,

but did not develop insulin resistance or diabetes, nor did they express TNFα in adipose tissue.

Resistin (Retn) is a protein found in the white, but not brown adipose tissue of mice.

Resistin, like PPARγ, is also related to diabetes. Type 2 diabetes, characterized by target-tissue

resistance to insulin- is strongly associated with obesity. Steppan et al. (2001) found that resistin

is actually a signaling molecule secreted from adipocytes, and that it may be the hormone that

links obesity to diabetes. Resistin is expressed during adipocyte differentiation but is

downregulated in mature adipocytes exposed to TZDs. Resistin levels in mouse serum are

increased dramatically in both genetic and diet-induced obesity, and administration of resistin

impairs glucose tolerance and insulin action in normal mice. McTernan et al. (2002) found that

abdominal fat depots showed a 418% increase in resistin mRNA expression compared to the

thigh, and that this expression of abdominal fat may indicate the increased risk of type 2 diabetes

associated with central obesity. Verma et al. (2003) found a possible link between resistin

expression and cardiovascular disease in the metabolic syndrome during their studies on human

endothelial cells.

Factor D, also known as adipsin, is a serine protease secreted by adipocytes into the

bloodstream, and is deficient in several obesity models. White et al. (1992), in their study of

human adipsin expression, found that adipose tissue is the major site of synthesis of human

adipsin mRNA. Xu et al. (2001) as well as White found that excretion of adipsin from human

macrophages may indicate a role in immune system biology.

Delta-drosophila like factor 1, also known as preadipocyte factor 1 (PREF1), is a

regulator of adipocyte differentiation, and is a member of the epidermal growth factor like family

31

of proteins. Smas and Sul (1993) found that PREF1 mRNA was expressed highly in

preadipocytes, but was not expressed during differentiation of cultured preadipocytes to

adipocytes. Adipsin has also been found by Laborda et al. (1993) to possibly be involved in

neuroendocrine differentiation. Jensen et al. (1994) found that adipsin, which they termed fetal

antigen 1, colocalizes with insulin to the insulin secretory granules of the beta cells within the

islets of Langerhans. Lee et al. (2003) generated transgenic mice which expressed PREF1 in

adipose tissue only. They found that there was a substantial decrease in fat pad weight, and that

the adipose tissue had reduced expression of adipocyte markers and adipocyte-secreted factors.

PREF1 however, as a preadipocyte factor, was increased in these transgenic mice, suffering from

hypertriglyceridemia, decreased glucose tolerance and insulin sensitivity. Transgenic mice

created to express PREF1 in the liver only also had a decrease in adipose mass and marker

expression, indicating an endocrine (hormonal) pathway of PREF1 function.

AE Binding Protein 1 (AEBP1; Aortic Carboxypeptidase-Like Protein, ACLP) is a

secreted protein that associates with the extra-cellular matrix (ECM). It is a gene whose cDNAs

are found exclusively in osteoblast and adipose tissue libraries (Ohno et al., 1996). Zhang et al.

(2005) found that AEBP1 expression is terminated in terminally differentiated, non-proliferative

adipocytes. They found that overexpression of AEBP1 during a high-fat diet regime induced

massive obesity in female transgenic mice, suggesting a sex-specific susceptibility to obesity via

an estrogen signaling pathway.

Gata3 is an enhancer-binding protein containing a zinc finger domain. Tong et al. (2000)

showed that murine Gata3, like Gata 2, is specifically expressed in white adipocyte precursors

and, consequently, that their down-regulation indicated terminal differentiation. As mentioned

earlier, PPARγ mediates suppression of differentiation during constitutive expression of Gata2

32

and Gata3. Furthermore, Gata3 deficient embryonic stem cells have an enhanced capacity to

differentiate into adipocytes, while defective Gata3 expression is associated with obesity. Gata3

has also been found to be involved in Th2 cytokine gene expression (Zheng and Flavell,1997),

and it has been suggested that inhibition of Gata3 is a possible treatment for asthma and other

hypereosinophilic diseases ( Zhang et al., 1997).

Colony Stimulating Factor 1 (CSF1) is a protein involved in osteoclastogenesis. Dobbins

et al. (2002) found that a mutation is CSF1 causes osteopetrosis in the toothless (tl) rat,

suggesting that CSF1 is a growth factor required for osteoclast differentiation and activation.

Van Wesenbeeck et al. (2002) confirmed that the tl rat is CSF1-null.

Tumor Necrosis Factor Receptor Superfamily, Member 11A (TNFRSF11A, also known

as Receptor Activator of NF-Kappa-B,RANK; Osteoclast Differentiation Factor Receptor,

ODFR; PDB2 Gene) is a type 1 trans-membrane protein with 4 extracellular cystiene-rich

pseudorepeats (Anderson et al., 1997). This osteoclast differentiation factor mediates an

essential signal for osteoclastogenesis. Furthermore, TNFRSF11A is a ligand for

osteoprotegerin (OPG), a secreted protein that inhibits osteoclastogenesis (Nakagawa et al.,

1998). Li et al. (2000) found that in addition to mediating OPG ligand effects on bone resorption

and remodeling, TNFRSF11A has a role in the physiological and pathological effects of

calciotropic hormones and proresorptive cytokines.

MADS Box Transcription Enhancer Factor 2, Polypeptide C (MEF2C) is a regulatory

protein involved in myogenesis. Transcripts of MEF2C have been found in skeletal muscle and

brain. Unlike MEF2A, which is involved in induction of muscle differentiation, Breitbart et al.

(1993) found that MEF2C is likely involved with maintenance of the differentiated state. Chen

et al. (2000) supported this hypothesis, proposing a model involving the interaction of NCOA2,

33

myogenin, and MEF2C in the regulation of muscle specific gene expression. Interestingly, Lin

et al. (1997), in an experiment involving a known mutation of MEF2C, found that MEF2C is an

essential regulator of cardiac morphogensis and right ventricle development.

Thy-1 T-Cell Antigen (THY1, also known as Theta Anitgen and CD90 Anitgen,CD90) is

a major cell surface glycoprotein characteristic to T-Cells, and involved in cell-cell interactions

(Raff, 1971; Letarte-Muirhead et al., 1975). It is expressed on fibroblasts, brain cells, and some

T Cells. Greenspan and O’Brien (1989) found that murine Thy1 stimulates neurite outgrowth in

neonatal sympathetic ganglion neurons. Abeysinghe et al. (2003) found that Thy1 expression,

like THBS1, SPARC, and fibronectin, was upregulated in nontumorigenic clones.

Thrombospondin 1 (THBS1, also known as TSP1) is a homotrimeric secreted

glycoprotein that associates with the extracellular matrix and has potent angiogenic activity. It is

associated with the platelet membrane and has a role in platelet aggregation. However, THBS1

is not limited to platelets. It is synthesized and secreted by a variety of cells for incorporation

into the ECM. THBS also binds heparin, sulfatides, fibrinogen, fibronectin, plasminogen, and

type V collagen. Volpert et al. (2002) showed that THBS1 was a natural inhibitor of

angiogenesis which derived its specificity for remodeling vessels from its dependence on Fas/Fas

ligand mediated apoptosis to block angiogenesis. Bocci et al. (2003) supported this finding with

their own results, indicating that TSP1 is a secondary mediator of the antiangiogenic effects of at

least some low-dose metronomic chemotherapy regimens. Thakar et al. (2005) found that TSP1

is a regulator of ischemic damage in the kidney, playing a role in the pathophysiology of

ischemic renal failure. Another role of THBS1 is its involvement in normal lung homeostasis

(Lawler et al. 1998).

34

Thrombomodulin (THBD, also known as THRM, Thrombophilia Due to

Thrombomodulin Defect, Included) is an endothelial cell surface glycoprotein that is bound by

thrombin in a 1:1 complex, altering its specificity towards several substrates and allowing it to

become a physiologic anticoagulant. Isermann et al. (2003) found that disruption of the mouse

thrombomodulin gene leads to embryonic lethality caused by a defect in the placenta. Activated

coagulation factors induced cell death and growth inhibition of placental trophoblast cells by 2

distinct mechanisms; conversion of fibrinogen to fibrin, and engagement of protease-activated

receptors.

Myogenic Differentiation Antigen 1 (MyoD1 also known as MYOD; Myogenic Factor 3,

MYF3) is a helix-loop-helix protein expressed only in skeletal muscle and its precursors, which

stabilizes its commitment to myogenesis by activating its own transcription. Mice with a null

mutation in MyoD or Myf5 have normal skeletal muscle, but a mutation in MyoD combined with

mutant Myf5 causes a complete lack of skeletal muscle, leading to death (Rudnicki et al., 1993).

These results indicate that either of these genes is required for the determination of skeletal

myoblasts, their propagation, or both during embryonic development.

B-Cell CLL/Lymphoma 2 (BCL2 also known as Oncogene b-cell leukemia 2, Leukemia,

Chronic Lymphatic , Type 2 ,Included; Follicular Lymphoma, Included) is a integral inner

mitochondrial membrane protein involved in apoptosis of B lymphocytes. BCl2 deficiency

causes selective apoptosis of melanocyte stem cells, but not differentiated melanocytes

(Nishimura et al., 2005). Overexpression of BCL2 blocks the apoptotic death of a pro-B-

lymphocyte cell line (Hockenberry et al., 1990). Farlie et al. (1995), in an experiment with

transgenic mice expressing BCL2 under the control of the neuron-specific enolase promoter,

35

found thatBCL2 expression can protect neurons from cell death during development. Therefore,

BCL2 may have a role in survival of neurons as well as apoptosis.

Caspase 3, Apoptosis Related Cysteine Protease (CASP3, also known as PARP Cleavage

Protease, Apopain, CPP32) is an enzyme responsible for poly (ADP-ribose) polymerase (PARP)

inactivation in mammalian cells during apoptosis (Nicholson et al.,1995). Casp3 is also the main

caspase responsible for amyloid-beta 4A precursor protein (APP) cleavage during apoptosis.

This is consistent with Casp3 elevation in the failing neurons of Alzheimer disease brains. This

indicated a dual role of Casp3 in proteolytic processing of APP. Casp3 has also been found to be

associated with HD-associated cell death (Li et al., 2000) as well as the promotion of myogenesis

(Fernando et al., 2002).

36

CHAPTER 6

EFFECT OF INJECTION OF LEPTIN AND GLUCOSE DEPENDANT INSULINOTROPIC

POLYPEPTIDE ON BONE MARROW ADIPOGENESIS AND OSTEOGENESIS IN RATS1

___________________________

Lackay, S.N., M. A. Della-Fera, D. L. Hartzell, Y.-H. Choi, C. A. Baile, Q. Li, M. Hamrick, C. Isales,

M. J. Kuhar. To be submitted to Bone

37

ABSTRACT

Glucose dependant insulinotropic polypeptide (GIP) is an incretin hormone with a

stimulatory effect on insulin release and synthesis in the pancreas. Recently, GIP receptors and

GIP mRNA have been found in bone, and administration of GIP to ovariectomized mice

prevented bone loss. GIP receptor knock out mice had reduced osteogenesis and increased bone

marrow fat, despite decreased body fat content. These finding suggest that GIP has an anabolic

effect on bone, as well as on body fat content. Leptin is a cytokine secreted primarily by

adipocytes, which impacts bone formation. Leptin deficient mice had increased bone mineral

density in the spine and treatment with Leptin in these mice reduced bone mass and density. It

has been shown in previous studies that Leptin treatment in Leptin deficient mice resulted in a

dramatic decrease in bone marrow adipocytes. To compare the effects of 4 day

intracerebroventricular (ICV) injections of GIP and Leptin, as well as the difference between

ICV, arcuate nuclei (ARC) and ventromedial hypothalamic nucleus (VMH) injection of Leptin,

on adipocyte apoptosis, real time Taqman RT-PCR (ABI microfluidic cards) was used to

quantitatively compare mRNA levels of selected adipocyte and osteoblast genes in bone marrow

samples from rats. Both ICV GIP (p<0.0001) and ICV Leptin (p<0.0001) significantly

decreased gene expression relative to control. Various doses of GIP were not significantly

different from one another. Location of injection, VMH vs. ICV vs. ARC, also had no

significant effect on gene expression. Due to the 23 genes tested, these data suggest that

treatment with Leptin and varying doses of GIP increase adiposity, and possibly osteolysis in

bone marrow.

Key Words: GIP, Leptin, Bone Marrow, Adipogenesis, Osteogenesis, Microfluidics

38

INTRODUCTION

The bone marrow is a dynamic environment, and the major players in this environment

are adipocytes and osteoblasts. These two groups reside on a molecular seesaw; the proliferation

of adipocytes excludes the differentiation of osteoblasts, and vice versa. Because so many

diseases are influenced by the balance between these two cell types, it is important to find

compounds which can upregulate the differentiation of one of these two groups, thereby

suppressing the differentiation of the other. GIP and Leptin have both been found to decrease

adiposity and to possibly have an anabolic effect on bone. Microfluidic technology was used in

this study to test the expression of 23 genes involved in adipogenesis and osteoblastogenesis, as

well as the expression of several growth factors.

MATERIALS & METHODS

Animals: Male Sprague-Dawley rats (250 – 274 g) were purchased from Harlan, Inc.

(Indianapolis, IN) (2 groups of 22 rats, purchased one week apart). Rats were housed in

individual cages and had access to pelleted standard lab chow and water ad libitum throughout

the study.

Materials. Artificial cerebrospinal fluid (aCSF) was used as the vehicle and control

article and consisted of (g/L) NaCl, 8.66; KCl, 0.224; CaCl2·2H2O, 0.206; MgCl2·6H2O, 0.163;

Na2HPO4·7H2O, 0.214; NaH2PO4·H2O, 0.027. All peptides will be solubilized based on their net

protein content. Rat leptin was purchased from R&D Systems (Minneapolis, MN). Vehicle was

used to dilute the leptin and achieve a concentration of 2.0 mg/mL. Human GIP (H-5645.1000)

was purchased from Bachem (San Carlos, CA).

39

Surgical Procedures. Rats were anesthetized with a 3:2:1 (v/v/v) mixture (1 ml/kg ip) of

ketamine HCl (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA; 100 mg/ml), acepromazine

maleate (PromAce, Fort Dodge; 10 mg/ml), and xylazine (Rompun, Miles, Schawnee Mission,

KS; 20 mg/ml), and the hair in the dorsum of the head as well as the area behind scapular bones

was removed. Each rat was then placed in the stereotaxic instrument (Stoelting, Wood Dale, IL)

and the skin disinfected with betadine solution.

To determine the effect of GIP and leptin when administered to the ICV, VMH, and ARC

rats were sedated and aseptically implanted intracranially with either a 26-guage external guide

cannula (for ARC and VMH, depth of 1.0mm dorsal to injection depth) or 22-guage guide

cannula (for LV, 0.8 mm posterior to the bregma, 1.5 mm lateral to the midline and 3.2 mm

ventral to the surface if the skull, total length 12.80 mm long) based on the atlas of the rat brain.

The cannulas were held in place with four stainless steel machine screws (Plastics One, Roanoke,

VA) and cranioplastic cement (Plastics One, Roanoke, VA) attached to the skull. A 31 gauge

stylet (ARC and VMH, C315DC, Plastics One) or a 28 gauge stylet (LV, C313DC, Plastics One)

was installed in the guide cannula while the rat was not receiving injections. After cannulation a

programmable transponder (IPTT-300TM

, BioMedic Data Systems, Inc., Seaford, DE) for

telemetry was implanted under the skin using a needle-syringed typed injector. To control post-

surgical pain rats were given 1.1 mg/kg banamine at 12 hour intervals. Rats were allowed to

recover for one week, in order for body weights to return to pre-surgical levels.

In order to determine the effect of VMH and ARC locations of injection, injections were

made in conscious unrestrained rats using 31 gauge injector cannulas by sterile PE tubing to a

Hamilton syringe (80500, 705N 50 µl syringe) mounted on a microprocessor controlled syringe

40

pump (KD Scientific Inc., Holliston, MA). Injections of 0.5 µl were administered over a 2.5

minute period.

To determine the effect of ICV location of injection, injections were carried out using an

injector cannula (C3131, Plastics One) that protrudes 1.1 mm below the tip of the guide cannula

and was connected to a Gilmont microsyringe via polyethylene tubing (PE20 Intramedic,

Cat#427406, BD, Sparks, MD). Each injection was administered in the LV over 30 seconds.

Proper cannula placement was verified in two ways. First, during implantation surgery,

backflow of CSF from the tip of the guide indicated correct placement in the ventricle. Second,

an ANG II drinking test was performed at the end of the recovery period, whereby a rat had to

consume at least 5 ml of water in 30 min following an ICV injection of 100 ng ANG II (100

ng/10 µl) solubilized in sterile aCSF; (Sigma, St. Louis, MO) for the cannula to be considered

correctly placed. In the morning of the ANG II test, food was removed. Rats were injected with

ANG II, the time recorded, in the same manner described above and the weight of each water

bottle was measured and recorded. Injection time was recorded as well. After ANG II injection,

rats were returned and water bottles were provided for 30 min, followed by measurement of the

bottles.

Treatments: For the GIP experiment, treatments of 0 (aCSF), 0.1 µg GIP, 1.0 µg GIP, 10

µg GIP and 10 µg rLeptin were administered at 24-hour intervals for 4 days as 10 µl injections.

For the ICV vs. VMH location of injection study, treatments were LV 0, LV 0.05, LV

1.25, VMH 0 and VMH 0.05 µg Leptin administered at 24 hour intervals as 0.5 µl injections for

4 days.

41

For the VMH vs ARC location of injection experiment, treatments were ARC 0.0, ARC

0.05, ARC 0.25, VMH0.0, and VMH 0.25 µg Leptin administered at 12 hour intervals as 0.5 µl

injections for 4 days.

Data Collection: Food intake, feeding behavior and spontaneous activity were measured

automatically. Body weight was measured once daily just prior to injections. Body temperatures

were measured twice daily (prior to ICV injection and approximately 4 h post-injection). Rats

were deeply anesthetized with CO2 before decapitation on day 5 to remove the brain, blood,

gastrocnemius muscle, intrascapular brown fat, retroperitoneal, epididymal, and inguinal white

fat pads and left femora and tibias. After mice were euthanized via CO2 asphyxia and

decapitation, trunk blood was collected and the leg was cut off from the body. The ends of the

tibia and femur were cut off with a diamond saw, and the bone was placed in a microcentrifuge

tube punctured on the bottom by a sterile 18 gauge needle. This tube was then placed in a larger

microcentrifuge tube and then centrifuged for 2 minutes in a 4 degree centrifuge. Afterwards,

the bone was discarded and the marrow, which was transferred to the bottom tube, was put in

liquid nitrogen, until it could be later stored in a -80 degree freezer.

Tissue and Serum Assays. Blood was collected and saved for measurement of leptin,

GIP and insulin concentrations by RIA.

RNA from bone marrow was isolated using an RNeasy Lipid Tissue Kit, and an optional

step of DNase digestion was added to reduce DNase contamination. Samples were then run on a

bioanalyzer to determine RNA quality, quantity and concentration. Using the concentrations

from the bioanalyzer samples were prepared for PCR and cloned to DNA using ABI CDNA

protocol on a thermocycler.

42

Samples in their cDNA form were then prepared for microfluidic analysis, making a final

sample of 4 microliters brought up to volume with 96 microliters water and 100 microliters

master mix. Samples were then loaded into the card. The card was spun two times for one

minute at 1200 RPM, and then loaded into the Applied Biosystem 7900 for analysis.

Statistics. ANOVA and LSD or Tukey’s test was used to determine significance of

differences among treatments. For the unnested analysis the model used was:

yijkl = trti + genej + cardk + trt*geneij + eijkl

where yijkl is the log of the relative quantification of gene j of mouse l receiving treatment i run

on card k. For the nested analysis the model used was:

yijkl = trti + genej + cardk + eijkl

where yijkl is the log of the relative quantification of gene j of mouse l receiving treatment i run

on card k.

Animal Disposition. At the conclusion of the experiment all rats were sedated with CO2

and sacrificed by guillotine for the collection of blood and tissue, and the carcasses incinerated.

Procedures Compliance. All surgical and experimental procedures proposed in this

study were conducted in accordance with the NIH Guidelines and were approved by the Animal

Care and Use Committee for The University of Georgia prior to initiating the studies

RESULTS

General linear model: overall ICV GIP and Leptin treatment effect. To analyze the data, a

general linear model (Tukey’s Test) was used, with treatment, gene, card, and the treatment by

card interaction used as class variables. The dependant variable was a measure of relative

quantification (RQ) of fluorescence detected by the ABI Prism 7900. The RQ value is derived

from the ∆∆Ct value. The ∆∆Ct value is derived from two differences in levels of fluorescence.

43

The first is the difference between level of gene expression and endogenous control (18 s RNA).

The second is the difference in the above between a given sample and the sample used to

normalize the data.

RQ= ∆∆Ct -2

Treatment with GIP and Leptin (ICV) had a significant impact on overall gene expression

(p<0.0001). Because treatment by gene interaction was highly insignificant (p=0.9998),

treatment seems to similarly decrease RQ value on all responsive genes. Treatment with GIP

tended to decrease the least squared mean of the log of RQ, as did treatment with Leptin.

Least Squared Mean RQ Values for GIP and Leptin Treatment

Treatment LS Means

aCSF 3.27829301

G0.1 1.35429362

G1 2.25197459

G10 2.17984856

L10 0.58191797

Table 6.1: The LS Mean RQ values were significantly lower for both 10.0 µg Leptin and GIP

doses of 0.1 µg, 1.0 µg, and 10.0 µg, as compared to aCSF treatment.

44

Mean RQ Value by Treatment

0

0.5

1

1.5

2

2.5

3

3.5

aCSF G0.1 G1 G10 L10

Treatment/Dose

RQ

Valu

e

Figure 6.1: LS Mean RQ Values for aCSF, 0.1 µg GIP, 1.0 µg GIP, 10 µg GIP and 10 µg

Leptin.

Linear contrasts show that control (aCSF) was significantly different from GIP dose of

0.1 µg (p<0.0001) and Leptin 10 µg (p<0.0001). Treatment with Leptin was also significantly

different from treatment with GIP 1 µg (p=0.0059) and GIP 10 µg (p=0.0131). Control was not

significantly different from GIP 1 µg, but approached significance (p=0.0837).

45

P-values for Linear Contrasts between GIP and Leptin Treatment Groups

1 2 3 4 5

1 <.0001 0.0837 0.1042 <.0001

2 0.2575 0.3563 0.4594

3 0.9999 0.0059

4 0.0131

5

Table 6.2: p values for linear contrasts between treatment groups, numbered as follows: 1)

aCSF 2) GIP 0.1 µg 3) GIP 1.0 µg 4) GIP 10.0 µg and 5)Leptin 10 µg

GIP doses of 0.1 µg,1.0 µg, and 10 µg were not significantly different from one another,

indicating that a threshold level must be passed in order to derive changes in gene expression.

However, as GIP 0.1 µg elicited a significant drop in gene expression from control, only small

doses are required to reach this threshold.

Leptin was significantly different from both control and doses of GIP 1.0 µg and 10 µg.

Leptin also evoked the largest decrease in RQ value of all the treatments.

General linear model: by gene analysis. The general linear model run by gene did not

have enough power to distinguish significant changes in gene expression.

Effect of location on gene expression. A general linear model found that there was no

significant difference of location of injection of bone marrow gene expression (p=0.1804) There

was also no significant difference between the different doses of a given treatment, indicating

that a threshold value needed to elicit a change in bone marrow gene expression was not met. It

46

cannot be determined from this study if differing doses above that threshold level would cause

greater changes in gene expression.

Effect of Location on Marrow Gene Expression

0

0.2

0.4

0.6

0.8

1

1.2

LV 0 µg LV 0.05 µg LV 1.25 µg VMH 0 µg VMH 0.05 µg

Treatment

LS

Mean

RQ

Valu

e

Figure 6.2: LS mean RQ values for LV and VMH Treatments of Leptin

LS Mean RQ Value for LV and VMH Injection of Leptin

Treatment LS Mean RQ Value

LV 0 0.914375

LV 0.05 1.014758

LV 1.25 0.951324

VMH 0 0.603189

VMH 0.05 1.027067

Table 6.3: LS Mean RQ Values for LV and VMH treatments of Leptin

47

Linear Contrast p-values for ICV and VMH Injections of Leptin

1 2 3 4 5

1 0.9737 0.9993 0.2770 0.9664

2 0.9737 0.9938 0.1886 1.0000

3 0.9993 0.9938 0.2882 0.9905

4 0.2770 0.1886 0.2882 0.1894

5 0.9664 1.0000 0.9905 0.1894

Table 6.4: p-values for the linear contrast of VMH and LV treatments of Leptin. 1) LV 0.0 µg

2) LV 0.05 µg 3) LV 1.25 µg 4) VMH 0.0 µg 5) VMH 0.05 µg

A study comparing the effect of VMH and ARC location was also performed. This study

found that treatment with Leptin was significant (p=0.0015), but location was not. While

injection with Leptin did lower overall expression as compared to both VMH and ARC controls,

the VMH and ARC least squared RQ means were not significantly different from one another.

Comparison of Least Squared RQ Value for VMH and ARC Injections of Leptin

Leptin Dosage (µg/day) and Location LS Mean RQ Value

ARC 0.0 3.18720709

ARC 0.05 1.11555325

ARC 0.25 1.12107061

VMH 0.0 2.69120325

VMH 0.25 0.85329449

Table 6.5: LS Mean RQ Values for VMH and ARC injections of Leptin

48

p-values for ARC and VMH treatments of Leptin

ARC 0.0 ARC 0.05 ARC 0.25 VMH 0.0 VMH 0.25

ARC 0.0 0.0166 0.0248 0.8521 0.0074

ARC 0.05 0.0166 1.0000 0.0039 0.9677

ARC 0.25 0.0248 1.0000 0.0111 0.9621

VMH 0.0 0.8521 0.0039 0.0111 0.0017

VMH 0.25 0.0074 0.9677 0.9621 0.0017

Table 6.6: p-values for linear contrasts of ARC and VMH Leptin treatments

LS Mean RQ Values for VMH and ARC

Injections of Leptin

0

0.51

1.5

2

2.53

3.5

ARC 0.0 ARC 0.05 ARC 0.25 VMH 0.0 VMH

0.25

Location and Dose (in micrograms/day)

LS

Mean

RQ

Valu

e

Figure 6.3: LS mean RQ values for ARC and VMH injections of Leptin

49

DISCUSSION

The general linear model implied a decrease in the expression of all genes affected by

treatment with GIP and Leptin. Increasing doses of GIP elicited no larger decrease in response.

However, treatment with Leptin caused a decrease from the least squared means of both control

(aCSF) and GIP treatments. This data shows that while genes respond similarly to GIP and

Leptin, Leptin has a more potent effect on bone. This decrease in genes most likely corresponds

to increased adipogenesis, as explained below, agreeing with the results of Li et al. (2005) who

found that treatment with GIP increased marrow adipocyte density, while osteoclast density was

unaffected by either GIP or Leptin. In Li’s study, ICV Leptin reduced body weight and fat mass,

while ICV GIP only decreased weight gain but did not alter fat mass. Also, while ICV Leptin

decreased food intake and water intake in rats, ICV GIP did not. This could explain Leptin’s

more potent effect on bone marrow, as compared to GIP. In Li et al.’s study, serum Leptin was

decreased by both Leptin and GIP, while insulin and glucagon were unaffected. Machinal-

Quelin et al. (2001) found that Leptin increased proliferation and differentiation of primary

cultured preadipocytes from the subcutaneous area, in a mechanism involving MAPK, AP1 and

STAT3. It is possible that the decreased fat pads cause a decrease in serum Leptin, and the body

compensates for this lack in fat stores by increasing adipogenesis in the bone marrow.

Gata3 is specifically expressed in white adipocyte precursors, and down regulation of this

gene sets the stage for terminal differentiation. This effect is regulated through the suppression

of PPARΓ. DLK1 is also a regulator of adipocyte differentiation, and similarly, its expression is

abolished in mature adipocytes. FABP4 is involved in lipid transport and storage. Because of

the overall decrease in gene expression, these data suggest that there is increased adipocyte

differentiation after treatment with GIP and Leptin. However, at the time the RNA samples were

50

taken, adipocytes had not yet matured to a stage capable of secreting factors and assisting in lipid

production and transport.

Both BBC3 and Casp2 were down-regulated, indicating a decreased apoptotic activity

among cells in bone marrow.

Osteoblastic marker BGLAP is expressed in the mineralized matrix of bone cells, and its

down-regulation would indicate a decreased differentiation from pre-osteoblast to osteoblast.

Bradshaw et al. (2003) showed that SPARC-null mice have greater deposits of

subcutaneous fat and larger epididymal fat pads in comparison with wildtype mice. Therefore a

downregulation of SPARC implies an opportunity for increased adipogenesis. Bradshaw et al.

(2003) also proposed that SPARC limits the accumulation of adipose tissue in mice in part

through its demonstrated effects on the regulation of cell shape and production of the

extracellular matrix. Therefore a downregulation of SPARC would suggest increased adipocyte

size and/or number.

High expression of CTSK denotes bone resorption. Inaoka et al. (1995) cloned a human

cDNA for CTSK using a probe for the previously isolated rabbit sequence. Highest expression

was noted in osteoarthritic hip bones and especially in an osteoclastoma. The authors proposed

that CTSK may be an important component of human osteoclastic bone resorption whose

pathologies include osteoporosis and osteoarthritis. Because CTSK is down-regulated, it is

possible that instead of bone resorption occurring, along with adipogenesis, the fat cells are

merely increasing in size but not number.

Annexin is involved in bone mineral metabolism. Suarez et al (1993) found that rat

osteoblasts express annexin and that these proteins play an important role in bone formation by

virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with

51

cytoskeletal elements, and/or regulate phospholipase A2 activity. Annexin’s down regulation

also implies that there is decreased osteoblast activity, perhaps due to increased adipogenesis.

Bi et al. (2005) showed that the extracellular matrix protein, biglycan (Bgn), plays an

important role in the differentiation of osteoblast precursors. Bgn is involved in regulating

osteoclast differentiation through its effect on osteoblasts and their precursors. Bi et al. showed

that osteolysis occurred more rapidly and extensively in Bgn deficient mice compared to wild

type (WT) mice. Therefore a decrease in expression of this gene would imply increased

osteolysis, and therefore the opportunity for further adipogenesis.

CD36 is expressed by osteoblastic cells, and Carron et al, 2000 found that CD36 may be

a receptor responsible for the promotion of bone resorption by TSP-1. A decrease in this genes’

expression, therefore, could indicate either a decreased osteoblastogenesis or decreased

resorption due to osteoclasts.

All other genes tested were growth factors, EGF, FGF14, MADH1, SRA1, TNF, and

VEGF. EGF is an epidermal growth factor whose overexpression leads to decreased

chondrocytic activity and a build up of osteoblasts. It is known that FGF14 is a fibroblast growth

factor expressed in the central nervous system. MADH1, also known as TGFB1, has a wide

range of biological effects, including cell growth, apoptosis, matrix production and

differentiation. SRA1 is transcriptional co-activator of steroid nuclear receptors. TNF, tumor

necrosis factor, has effects on lipid metabolism, coagulation, insulin resistance, and endothelial

function. VEGF, vascular endothelial growth factor, promotes the proliferation of capillary

endothelial cells. While several genes relating to the functions stated above would have to be

tested, it is possible that the effects of GIP and Leptin reach far beyond the osteoblast-adipocyte

seesaw.

52

CONCLUSION

Our data suggest that treatment with GIP and Leptin causes increased adiposity in the

bone marrow, either by increased proliferation or enlargement of individual adipocytes. The

genes tested that are normally expressed in bone marrow may have decreased expression due to

decreased osteogenesis, or even osteolysis. The data also show that location of Leptin injection

(ICV, ARC or VMH) has no effect on gene expression.

These data are contradictory to previous findings that treatment with Leptin and GIP

decrease adiposity and have an anabolic effect on bone marrow. However, as a gene by gene

analysis could not be done, it is possible that the overall decreasing effect found in this study is

due to an overwhelming response from one subset of genes, and hardly any response from

another.

If this is the case, then it is possible that there is no response from adipocyte genes, but a

decrease response from osteoblast genes, or vice versa. Alternatively, a downregulation in

growth factor gene expression and no change in adipocyte and/or osteoblast genes could also

explain the overall decrease in gene expression. Further studies will have to be done to

determine the effect of these treatments on specific genes in the bone marrow.

53

CHAPTER 7

CLENBUTEROL, A BETA-2 ADRENERGIC AGONIST, INCREASES MURINE BONE

MARROW POTENTIAL FOR ADIPOGENESIS

____________________________

Lackay, S.N., M. J. Azain, D. L. Hartzell, C. A. Baile, M. A. DellaFera, M. Hamrick, T. D. Pringle. To be

submitted to Bone

54

ABSTRACT

Clenbuterol is a β2-adrenergic receptor (β2-AR) agonist that has been shown to decrease

body fat and increase muscle mass with oral administration in rodents. While Clenbuterol has

been shown to increase adipose tissue apoptosis, few studies have been done on the effect of β2-

AR agonists on bone marrow. In order to further examine bone marrow response to β2-AR

agonists, mice were fed diets containing Clenbuterol in doses of 2, 20, and 200 ppm. Real time

Taqman RT-PCR (ABI microfluidic cards) was used to quantitatively compare mRNA levels of

selected adipocyte and osteoblast genes in bone marrow samples from mice. Clenbuterol

treatment increased the body weight gain by 51.69% (2 ppm), 69.73% (20 ppm), and 68.18%

(200 ppm). Clenbuterol had no effect on bone mineral density, bone mineral content, and area of

total bone, or on lean tissue, fat tissue, and percent fat. A Tukeys test on dose effect over all

genes showed that treatment with Clenbuterol resulted in a significant decrease in gene

expression (p<0.0001). However, the individual doses were not significantly different from one

another, implying a threshold level of Clenbuterol is responsible for the changes in gene

expression measured. Due to the decrease in the adipogenesis markers chosen, GATA2, DLK1,

LEP, RTN, FABP4, and PPARγ, it is likely that Clenbuterol’s effect is increased adipogenesis.

The overall decrease in the osteoblastic markers chosen, ANXA5, BGLAP, BGN, CG36,

SPARC, MMP11, CTSK, FN1 and SERPINH1 agrees with increased adipogenesis. We

conclude that, despite the absence of definitive phenotypic differences during the study period,

mRNA changes occurring in the bone marrow are indicative of an increased adipogenesis.

Key Words: Clenbuterol, Microfluidics, Adipogenesis, β2-Adrenergic Receptor Agonist

55

INTRODUCTION

Osteoporosis is a disease affecting many people in today’s society. The dynamic

environment of bone marrow is effected by many compounds, and the balance between bone

formation by osteoblasts and bone resorption caused by adipocytes and osteoclasts can be easily

overturned, especially in the elderly. Several studies have shown that β2-adrenergic receptor

agonists inhibit bone growth and stimulate osteoclastogenesis. Despite this, no studies have

investigated the effect of these β2-AR agonists on bone marrow adipocyte content, which is

inversely related to bone loss in several models of osteoporosis. Clenbuterol is a β2-adrenergic

receptor (β2-AR) agonist that has been shown to decrease body fat and increase muscle mass.

This study will test the effect of several doses of Clenbuterol, 0 ppm, 2ppm, 20 ppm, and 200

ppm, on gene expression of adipocytes and osteoblasts in bone marrow.

MATERIALS AND METHODS

Animals: Five week old male ICR mice (Harlan Research Laboratories, Indianapolis,

IN) were used in this study. Mice were housed separately, provided with ground rodent chow

(ProLab® RMH 2500; Purina Mills, St. Louis, MO) and allowed to acclimate for one week prior

to start of study.

Materials: Control mice were fed a basal diet, ad libitum, of RMH 2500 from Purina

Mills for the duration of the 21 day treatment period. Clenbuterol (Sigma Chemical Company,

St. Louis, MO, item # C5423) was prepared in the basal diet at 2, 20, and 200 ppm for test mice.

Test mice were also fed ad libitum. All mice had access to water ad libitum.

Tissue collection: Mice were injected with calcein (7 mg/kg; Sigma), for the fluorometric

determination of calcium, 10 days and 24 hours before they were euthanized via CO2

56

asphyxiation followed by decapitation. Both femora were dissected free and the proximal and

distal ends removed with a diamond wire saw. The femur was then placed in a pre-pierced (via

18G needle) 0.5 ml microcentrifuge tube within a 1.5 ml microcentrifuge tube. Marrow plugs

were obtained by centrifugation at 12,000 rpm for 2 minutes at 4ºC. The cell pellet was then

transferred to liquid nitrogen and later stored at -80 ºC until used for quantitative RT-PCR with a

microfluidic card. RNA was isolated from the bone marrow sample using the RNeasy Lipid

Tissue Mini Kit (Qiagen.) Samples were then cloned to cDNA and prepared for use in a

microfluidic card. The card was run in the ABI Prism 7900HT Sequence Detection System.

Statistics. ANOVA and LSD or Tukey’s test was used to determine significance of

differences among treatments. For the unnested analysis the model used was:

yijkl = trti + genej + cardk + trt*geneij + eijkl

where yijkl is the log of the relative quantification of gene j of mouse l receiving treatment i run

on card k. For the nested analysis the model used was:

yijkl = trti + genej + cardk + eijkl

where yijkl is the log of the relative quantification of gene j of mouse l receiving treatment i run

on card k.

Animal Disposition. At the conclusion of the experiment all rats were sedated with CO2

and sacrificed by guillotine for the collection of blood and tissue, and the carcasses incinerated.

Procedures Compliance. All surgical and experimental procedures proposed in this

study were conducted in accordance with the NIH Guidelines and were approved by the Animal

Care and Use Committee for The University of Georgia prior to initiating the studies

57

RESULTS

Overall treatment was highly significant (p<0.0001). Control had significantly higher

expression than Clenbuterol doses of 2 ppm (p<0.0001), 20 ppm (p<0.0001) and 200 ppm

(p<0.0001). However, varying doses of Clenbuterol had no significant changes in expression

level. This suggests that Clenbuterol’s effect on bone marrow works via a threshold mechanism.

Once the threshold level of dosage is met, no further treatment with Clenbuterol has a greater

effect on bone marrow cells. Because the treatment by gene interaction was highly insignificant

(p=0.9685), it seems that treatment with Clenbuterol similarly decreases all responsive genes.

LS Mean RQ Values for Varying Doses of Clenbuterol

0

0.5

1

1.5

2

2.5

3

Basal Diet Clenbuterol 2 ppm Clenbuterol 20 ppm Clenbuterol 200 ppm

Clenbuterol Dose

LS

Mean

RQ

Valu

e

Figure 7.1: LS Mean RQ values for varying doses of Clenbuterol fed to ICR mice over a 21 day

treatment period

58

LS Mean RQ Values for Varying Doses of Clenbuterol

Table 7.1: LS Mean RQ values for varying doses of Clenbuterol fed to ICR mice over a 21 day

treatment period

Gene by gene analysis was performed via Tukey’s test, with adjustment for multiple

comparisons via Bonferroni’s test. Using Bonferroni’s test with a variable of 23, p-values

needed to be equal to or less than 0.002 to be significant. After this adjustment, there were no

significant treatment effects in the gene by gene analysis.

DISCUSSION

Overall, Clenbuterol decreases gene expression as compared to the mean. Because of the

highly insignificant gene by treatment interaction, it can be assumed that treatment with

Clenbuterol similarly affect all responsive genes. There were 23 genes tested for changes in

expression, chosen for their association with adipocyte and osteoblast cell types, along with

some growth factor genes.

Clenbuterol Dose

(in ppm)

LS Mean

RQ Value

0 2.75836668

2 1.27183326

20 1.09273148

200 1.08436452

59

Murine Gene Names and Alternative Symbols

Gene Alternative Titles

Caspase 3, Apoptosis Related Cysteine

Protease (CASP3)

PARP Cleavage Protease; Apopain; CPP32

T-Cell Immune Regulator 1 (TCIRG1) ATPase, H+ Transporting , Lysosomal V0

Subunit A Isoform 3; Vacuolar Proton Pump,

Alpha Subunit 3; TIRC7, included; OC116,

included

Chemokine, CC Motif, Ligand 27 (CCL27) Small Inducible Cytokine, Subfamily A,

Member 27 (SCYA27); Il11RA Locus

Chemokine (ILC); Cutaneous T Cell-Attracting

Chemokine (CTACK); Eskine

Collagen, Type 1, Alpha 1 (Col1A1) Collagen of skin, tendon and bone, alpha 1

chain; Col1A1/ PGDFB Fusion Gene, included

Bone Morphogenic Protein 2 (BMP2) Bone Morphogenic Protein 2A (BMP2A)

Runt-Related Transcription Factor 2 (RUNX2) Core-Binding Factor Runt Domain, Alpha

Subunit 1 (CBFA1); AML3 Gene (AML3);

PEBP2 Alpha A; OSF2

Transcription Factor Sp7 (SP7) Specificity Protein 7; Osterix (OSX)

Peroxisome Proliferator- Activated Receptor

Gamma (PPARγ)

PPARγ1, included; PPARγ2, included;

PPARγ3, included; PAX8/PPARγ Fusion

Gene, included

Fatty Acid Binding Protein 4 (FABP4) Fatty Acid Binding Protein, Adipocyte

Resistin (RETN) RSTN; Found in Inflammatory Zone 3 (FIZZ3)

Factor D Complement Factor D (DF); Adipsin (ADN)

Delta, Drosophila, Homolog-Like 1 (DLK1) Preadipocyte Factor 1 (PREF1); Fetal Antigen

1 (FA1); pG2

AE Binding Protein 1 (AEBP1) Aortic Carboxypeptidase Like Protein (ACLP)

GATA Binding Protein 3 (GATA3) Enhancer Binding Protein GATA3

Colony Stimulating Factor 1 (CSF1) Colony Stimulating Factor, Macrophage

Specific (MCSF)

Tumor Necrosis Factor Receptor Superfamily,

Member 11A (TNFRS11A)

Receptor Activator of NF-Kappa B (RANK);

Osteoclast Differentiation Factor Receptor

(ODFR);PDB2 Gene; Trancer

MADS Box Transcription Enhancer Factor 2,

Polypeptide C (MEF2C)

Thy-1 T-Cell Antigen (Thy1) Theta Antigen; CD90 Antigen (CD90)

Thrombospondin 1 (THBS1) TSP1

Thrombomodulin (THBD) THRM; Thrombophilia due to

Thrombomodulin Defect, included

Myogenic Differentiation Antigen (MYOD1) MYOD; Myogenic Factor 3 (MYF3)

B-Cell CLL/Lymphoma 2 (BCL2) Oncogene B-Cell Leukemia 2; Follicular

Lymphoma, included

Leptin (LEP) Obese, Mouse, Homolog of (OB)

Control Gene 18s

Table 7.2: Genes tested for based on association with adipocytes, osteoblast, and growth factors

60

Caspase 3 is the enzyme responsible for the proteolytic breakdown of poly (ADP-ribose)

polymerase (PARP) at the onset of apoptosis (Nicholson et al.,1995). Caspase 3 has also been

found to be involved in osteoblastic differentiation. Caspase 3 inhibitor causes accelerated bone

loss in ovariectomized mice (Miura et al, 2004). Therefore a decrease in caspase 3 expression

could imply not only a decrease in apoptotoic activity, but also a decrease in osteoblastic

differentiation.

T-Cell Immune Regulator 1 (TCIRG1) is a vacuolar protein pump polypeptide necessary

for the acidification of subosteoclastic resorption lacuna. This acidification allows osteoclasts to

degrade inorganic and organic components of bone subjacent to the attachment site. Li et al

(1999) found that inactivation of the Tcirg1 gene caused osteoclast-rich osteopetrosis. A

decrease in TCIRG1 would indicate an increased bone density, not due to increased osteoblast

proliferation, but rather to a decreased capability of osteoclasts to resorb bone. This type of

increased bone density is unhealthy, and when it occurs in infantile malignant autosomal

recessive osteopetrosis, can be fatal.

However, CSF1is a protein involved in osteoclastogenesis as well. Dobbins et al. (2002)

found that a mutation is CSF1 causes osteopetrosis in the toothless (tl) rat, suggesting that CSF1

is a growth factor required for osteoclast differentiation and activation. Therefore, a decrease in

CSF1 expression would indicate a decrease in osteoclast activity. Also, TNFRSF11A is an

osteoclast differentiation factor that mediates an essential signal for osteoclastogenesis.

Furthermore, TNFRSF11A is a ligand for osteoprotegerin (OPG), a secreted protein that inhibits

osteoclastogenesis (Nakagawa et al., 1998).

The skin-associated chemokine CCL27 has a role in T cell-mediated skin inflammation

(Homey et al, 2002). Homey et al. injected mice with human CCL27 intradermally. They found

61

a dose-dependent expression of IL2, CCR10, and LFA1A and that treatment with

glucocorticosteroid or anti-Ccl27 markedly reduced skin thickness and leukocyte recruitment.

Therefore a decrease in CCL27 expression could indicate that treatment with Clenbuterol has a

negative effect on the immune response, decreasing leukocyte recruitment to injured areas, and

impairing the inflammatory response.

Cheng et al (2003) found that BMP2 is one of the few bone morphogenic proteins able to

induce all markers of osteoblast differentiation in pluripotential and mesenchymal stem cells.

Supporting the role of BMP2 in bone formation, Wang et al. (1990) showed that when BMP2A

produced by recombinant DNA techniques was implanted into rats, bone formation occurred by

day 14. Therefore, a decrease in BMP2 would indicate a decrease in bone formation, caused by

decrease osteoblastic differentiation.

Runt Related Transcription Factor 2 (RUNX2) has been described as the master switch in

osteogenesis (Stein et al., 2004). Stein et al. found that RUNX2 controls the integration,

organization, and assembly of nucleic acids and regulatory factors for skeletal gene expression.

Komori et al. (1997) also showed that Runx2 plays a critical role in osteogenesis. Strangely, in a

study on bone marrow stromal cells and primary osteoblast cultures, Geoffrey et al. (2002) found

that overexpression of RUNX2 enhances osteoclast differentiation in vitro and bone resorption in

vivo. Therefore a decrease in this marker would imply decreased osteoblast proliferation and

differentiation, but a large increase leads to osteoclast rich bone resorption.

Gao et al. (2004) found that SP7 is a putative master regulator of bone cell

differentiation. Nakashima et al (2002) found that SP7 null mice had no bone formation.

Nakashima et al found that although mesenchymal cells could invade the mineralized cartilage

matrix, they could not deposit bone matrix. They also found that osteoclasts could also enter the

62

cartilage matrix. These results suggest that a downregulation in the Sp7 gene would indicate a

decreased osteoblasts proliferation and function, but no decrease in osteoclast activity, making

increased bone resorption a possible outcome.

Peroxisome Proliferator-Activated Receptor-Gamma (PPARγ) is involved in adipocyte

differentiation. Tong et al. (2000) showed that murine Gata3 is specifically expressed in white

adipocyte precursors and that its downregulation sets the stage for terminal differentiation.

Steppan et al. (2001) found that resistin is actually a signaling molecule secreted from

adipocytes, and that it may be the hormone that links obesity to diabetes. Resistin is expressed

during adipocyte differentiation but is downregulated in mature adipocytes exposed to TZDs.

Therefore, a decrease in the expression of any or all of the above genes would imply increased

adipocyte differentiation. This increase in adipocyte differentiation supports the previous

findings of possible decrease in osteoblast proliferation and differentiation.

Fatty Acid Binding Protein 4 (FABP4) is a cytosolic protein expressed in a tissue specific

fashion (Hotamisligil et al., 1996). Hotamisligil created a null mutation murine FABP4 gene and

placed these FABP4 deficient mice on a high fat, high caloric diet. While both control mice and

FABP4 deficient mice gained weight while on this diet, FABP4 -/- mice gained more weight.

Therefore, a decrease in FABP4 gene expression may indicate increased adipogenesis.

Adipsin is a serine protease that is secreted by adipocytes into the blood stream, and is

deficient in several animal models of obesity. White et al. (1992) demonstrated its high level of

expression in fat. A decrease in this gene could mean increased adipogenesis.

DLK1 was cloned by Smas and Sul (1993). It is a regulator of adipocyte differentiation

and is expressed highly in preadipocytes. However, upon differentiation, DLK1 expression is

63

completely terminated. A decrease in this genes expression, therefore, indicates increased

adipocyte differentiation.

AEBP1 is a secreted protein that associates with the extra-cellular matrix. It is a gene

whose cDNAs are found exclusively in osteoblast and adipose tissue libraries (Ohno et al.,

1996). Zhang et al (2005) found that AEBP1 expression is terminated in terminally

differentiated, non-proliferative adipocytes. They found that overexpression of AEBP1 during a

high-fat diet regime induced massive obesity in female transgenic mice, suggesting a sex-

specific susceptibility to obesity via an estrogen signaling pathway. Downregulation of this

gene’s expression suggests an increase in terminal differentiation of preadipocytes to adipocytes.

Thy 1 is a major cell surface glycoprotein characteristic to T-Cells, and involved in cell-

cell interactions. THBS1 is a secreted glycoprotein that associates with the extracellular matrix

and has potent angiogenic activity. It is associated with the platelet membrane and has a role in

platelet aggregation. THBD is an endothelial cell surface glycoprotein that is bound by thrombin

in a 1:1 complex, altering its specificity towards several substrates and allowing it to become a

physiologic anticoagulant.

MyoD1 is a helix-loop-helix protein expressed only in skeletal muscle and its precursors,

which stabilizes its commitment to myogenesis by activating its own transcription. MEF2C is a

regulatory protein involved in myogenesis. Transcripts of MEF2C have been found in skeletal

muscle and brain. Breitbart et al (1993) found that MEF2C is likely involved with maintenance

of the differentiated state.

Colagen1A1 has been linked to both osteoporosis and osteogenesis imperfecta. A gene

polymorphism in the Sp1 binding site is associated with osteoporotic fracture. Therefore,

64

decreased expression in this gene may indicate an imbalance between osteoclast resorption and

osteoblast differentiation.

BCL2 is an integral inner mitochondrial membrane protein involved in apoptosis of B

lymphocytes. BCl2 deficiency causes selective apoptosis of melanocyte stem cells, but not

differentiated melanocytes (Nishimura et al.,2005). Overexpression of BCL2 blocks the

apoptotic death of a pro-B-lymphocyte cell line (Hockenberry et al., 1990). Farlie et al. (1995),

in an experiment with transgenic mice expressing BCL2 under the control of the neuron-specific

enolase promoter, found thatBCL2 expression can protect neurons from cell death during

development. Therefore, BCL2 may have a role in survival of neurons as well as apoptosis. A

decrease in BCL2 may not only imply a decrease in apoptosis, but also a decrease in neuronal

survival.

Leptin is a protein that is involved in maintenance of body weight and energy

expenditure. Leptin deficiencies are associated with overweight and obesity. Leptin is secreted

by adipocytes and is used by the body as a means of assessing fat stores. A decrease in Leptin

expression would indicate a decrease in mature adipocyte, or a decrease in adipogenesis.

Analysis of body weight, bone length, bone mineral density, bone mineral content, and

area found that there was no significant difference between treatment groups. However, lean

muscle mass was significantly higher in Clenbuterol treated mice, and intrascapular brown

adipose tissue as well as epididymal, inguinal, and retroperitoneal white adipose tissue were

significantly lower. Percent fat was also significantly lower in Clenbuterol treated mice. This

suggests compensation in the bone marrow for the apparent decrease in fat in other areas of the

body. This agrees with Bonnet et al.’s (2005) results, on bone alterations under β2-agonist

treatments. Bonnet et al found that clenbuterol treatment reduced BMC, femoral length, cortical

65

width, and bone mineral density. Clenbuterol treatment also elicited a lower trabecular number,

connectivity and trabecular bone volume. However, in the same study Bonnet et al found that

Clenbuterol increased muscle mass and reduced fat mass as compared to controls. Clenbuterol

also increased bone resorption marker without any effect on bone formation marker. In a

separate study, Bonnet et al. (2005) found similar results and noted that bone loss contrasts with

the anabolic effect on muscle mass. Armstead et al. (2005) found that clenbuterol significantly

increased body weight and lean mass, and significantly decreased percent body fat, in a dose-

dependent manner. Bone mineral content, bone mineral density, trabecular bone volume

fraction, osteoclast number, mineral apposition rate or mineralizing endocortical and periosteal

surface did not differ among groups in Armstead’s study, suggesting that clenbuterol may

attenuate the response of bone to changes in body weight and muscle mass. Therefore,

Clenbuterol may play a part in allowing the increased adipogensis in bone marrow in respose to

decreased serum Leptin levels.

These results support the present data, indicating an increase in adipocyte differentiation

and a decreased differentiation of osteoblasts in the bone marrow, increasing the risk of fracture

in animals treated with Clenbuterol.

CONCLUSION

The results suggest that treatment with Clenbuterol leads to increased adipogenesis in the

bone marrow. Further studies will have to be performed in order to detect specifically which

genes are affected by Clenbuterol, and the physiological process by which decreased expression

in these genes leads to increased adipogenesis.

66

CHAPTER 8

SUMMARY

Chapters 1-5 provide a review of scientific literature relating to the main thesis topic, the

balance of adipocytes and osteoblasts in bone marrow, and how different treatments affect genes

relating to these two cell types. Various studies have related the 23 selected genes with diseases

caused, at least in part, by the imbalance of bone resorption and bone formation, such as obesity,

osteoporosis, and diabetes.

Chapter 6 is a study on the effects of both GIP and Leptin on gene expression in bone

marrow. Interestingly, GIP and Leptin are both shown to decrease overall gene expression

(P<0.001), implying an increase in adipocyte differentiation. In this study it was also found that

Leptin has a more potent effect on bone than does GIP. It was also shown that a threshold effect

was in place, and that increasing the amount of GIP injected had no further impact on gene

expression. This Chapter also explored the possibility that location of injection may affect the

extent of treatment impact on gene expression. However, results showed that there was no

significant difference in gene expression between ICV and VMH injections of Leptin.

Chapter 7 is a study on the effect of Clenbuterol on bone marrow gene expression. This

study also shows that Clenbuterol treatment decreased overall gene expression, again leading to

increased adipogenesis. This study also showed that there was no difference in gene expression

with increasing dosage of Clenbuterol, implying a threshold effect similar to that seen in the GIP

study.

The implication of this research can be used to further examine gene expression in bone

marrow. Further studies on expression of specific genes would be useful to determine the

pathways by which increased adipogenesis occurs in response to the given treatments. Exploring

67

these pathways would provide useful information and help in the constant search for treatments

for obesity, osteoporosis, and diabetes, as well as several other diseases affected by the

adipocyte-osteoblast balance of bone marrow.

68

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APPENDICES

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APPENDIX A

Rat Gene Names and Alternative Symbols

Gene Alternative Titles

Annexin A5 (ANAXA5) Annexin V (ANX 5), Endonexin II (ENX2),

Placental Anticoagulant Protein I, Vascular

Anticoagulant-Alpha, Lipocortin V, Placental

Prtoein 4, PP4, Anchorin CII

BCL2 Binding Component 3 (BBC3) p-53-Upregulated Modulator of Apoptosis (PUMA)

Gamma-Carboxyglutamic Acid Protein, Bone

(BGLAP)

Bone Gamma-Carboxyglutamic Acid Protein, Bone

Gla Protein (BGP), Osteocalcin

Biglycan (BGN) Proteoglycan-I (PG-1)

Caspase 2, Apoptosis-Related Cysteine Protease

(CASP2)

Neural Precursor Cell Expressed, Developmentally

Downregulated 2 (NEDD2), ICH1

CD36 Antigen (CD36) Leukocyte Differentiation Antigen CD36, Platelet

Glycoprotein IV (GP4), Glycoprotein IIIb (GP3B),

GP IIIb, Thrombospondin Receptor, Collagen

Receptor, Platelet, Fatty Acid Translocase (FAT)

Cathepsin K (CTSK) No alternative titles/symbols

Delta, Drosophila, Homolog-Like 1 (DLK1) Preadipocyte Factor 1 (PREF1), Fetal Antigen 1

(FA1), pG2

Epidermal Growth Factor (EGF) Urogastrone (URG)

Fatty Acid Binding Protein 4 (FABP4) Fatty Acid-Binding Protein, Adipocyte

Fibroblast Growth Factor 14 (FGF14) Fibroblast Growth Factor Homologous Factor 4

(FHF4)

Fibronectin 1 (FN1) FN, Large, External, Transformation-Sensitive

Protein (LETS), FNZ, Included

GATA-Binding Protein 3 (GATA3) Enhancer Binding Protein GATA3

Leptin (LEP) Obese, Mouse, Homolog of (OB)

Mothers Against Decapentaplegic, Drosophila,

homolog of, 1 (SMAD1)

MAD, Drosophila, Homolog of, MADH1, SMA-

and MAD-Related Protein 1 (MADR1), TGF-Beta

Signaling Protein 1 (BSP1)

Matrix Metalloproteinase 11 (MMP11) Stromelysin III (STMY3)

Peroxisome Proliferator-Activated Receptor-

Gamma (PPARG)

PPARG1, Included; PPARG2, Included, PPARG3,

Included; PAX8/PPARG Fusion Gene, Included

Resistin (RETN) RSTN, Found in Inflammatory Zone 3 (FIZZ3)

Serpin Peptidase Inhibitor, Clade H (Heat Shock

Protein 47), Member 1, (Collagen Binding Protein

1) (SerpinH1)

Arsenic-Transactivated Protein 3 (AsTP3), Collagen

Binding Protein 1 (CBP1), Collagen Binding

Protein 2 (CBP2), Heat Shock Protein 47 (HSP47),

Proliferation Inducing Gene (PIG 14), Rheumatoid

Arthritis Antigen A-47 (RA-A47), SERPINH2,

gp46

Secreted Protein, Acidic, Cysteine-Rich (SPARC) Osteonectin (ON)

Steroid Receptor RNA Activator 1 (Sra1) SRA

Tumor Necrosis Factor (TNF) Tumor Necrosis Factor, Alpha (TNFA); Cachectin;

TNF,Monocyte-Derived;TNF, Macrophage-Derived

Vascular Endothelial Growth Factor (VegF) VEGFA, Atherosclerosis, Susceptibility To,

Included

Control Gene 18s

Table 7.2: Genes tested for based on association with adipocytes, osteoblast, and growth factors

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APPENDIX B

GLOSSARY

ACRP30 adipocyte complement-related protein hormone, secreted by adipocytes, regulates

energy homeostasis and glucose and lipid metabolism

Adipocyte An animal connective tissue cell specialized for the synthesis and storage of fat.

Such cells are bloated with globules of triglycerides, the nucleus being displaced

to one side and the cytoplasm seen as a thin line around the fat droplet.

Adipogenesis The production of fat, either fatty degeneration or fatty infiltration; also applied to

the normal deposition of fat or to the conversion of carbohydrate or protein to fat

ADD1 Adducin1, cell-membrane skeletal protein that was first purified from human

erythrocytes and subsequently isolated from bovine brain membranes. Isoforms

of this protein have been detected in lung, kidney, testes, and liver.

ADN Adipsin, a serine protease that is secreted by adipocytes into the bloodstream. It

is deficient in several animal models of obesity, see page 32.

Adrenergic receptor Reactive components of effector tissues, most of which are

innervated by adrenergic postganglionic fibers of the sympathetic nervous system.

Such receptor's can be activated by norepinephrine and/or epinephrine and by

various adrenergic drugs; receptor activation results in a change in effector tissue

function, such as contraction of arteriolar muscles or relaxation of bronchial

muscles; adrenergic receptor's are divided into alpha-receptor's and beta-

receptor's, on the basis of their response to various adrenergic activating and

blocking agents. There are several subtypes of adrenergic receptors, including α1,

α2, β1, β2, and β3. Alpha α receptors are found in the blood vessels and pre- and

post-synaptic nerve terminals. All β receptors activate adenylate cyclase, raising

the intracellular cAMP concentration. The β1 subtype is mainly found in the heart

and in the cerebral cortex, while the β2subtype predominates in the lung and

cerebellum.

AEBP1 AE Binding Protein 1, see page 33.

Agonist A drug that has affinity for and stimulates physiologic activity at cell receptors

normally stimulated by naturally occurring substances, thus triggering a

biochemical response

AGRP Agouti Related Protein, regulates body weight via central melanocortin receptors

Angiotensinogen ANG is formed from a precursor, angiotensinogen, which is produced by

the liver and found in the alpha-globulin fraction of plasma. The lowering of

blood pressure is a stimulus to secretion of renin by the kidney into the blood.

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Renin cleaves from angiotensinogen a terminal decapeptide, angiotensin I. This

is further altered by the enzymatic removal of a dipeptide to form angiotensin II.

Ap2 Adaptor Related Protein Complex 2, the alpha subunit is part of the so-called AP2

coat assembly protein complex which links clathrin to receptors in the coated

vesicles. The alpha adaptins are exclusively found in the endocytic coated

vesicles.

ARC Arcuate nucleus, a nucleus located in the middle hypothalamus in the most ventral

part of the third ventricle near the entrance of the infundibular recess. Its small

cells are in close contact with the ependyma.

ASP Acylation Stimulation Protein, an acute phase reactant; increased synthesis is

induced during acute inflammation. The liver is the main site of synthesis,

although small amounts are also produced by activated monocytes and

macrophages.

BAT Brown Adipose Tissue, a type of adipose tissue present in many newborn or

hibernating mammals. In contrast to white adipocytes (fat cells), which contain a

single, large fat vacuole, brown adipocytes contain several smaller vacuoles and a

much higher number of mitochondria. Brown fat also contains more capillaries

since it has a greater need for oxygen than most tissues.

BCL2 B-Cell CLL/Lymphoma 2, see page 36.

BGLAP Bone Gamma Bone Gamma-Carboxyglutamic Acid Protein, see page 26.

BMP2A Bone Morphogenic Protein 2A, see page 27.

C Reactive Protein This blood test is used as an indicator of acute inflammation. C-reactive

protein is a protein of the pentraxin family, produced by the liver during periods

of inflammation and detectable in serum in various disease conditions particularly

during the acute phase of immune response. Normally C-reactive protein should

be negative in the bloodstream. C-reactive protein is synthesised by hepatocytes

and its production may be triggered by prostaglandin E1 or parogen. It consists of

five polypeptide sub units forming a molecule of total molecular weight 105 kD.

It binds to polysaccharides present in a wide range of bacterial, fungal and other

cell walls or cell surfaces and to lecithin and to phosphoryl or choline containing

molecules. It is related in structure to Serum Amyloid. and C polysaccharide.

Conditions which can cause a positive C-reactive protein include: rheumatoid

arthritis, lupus, pneumococcal pneumonia, rheumatic fever, cancer, tuberculosis

and myocardial infarction. A positive C-reactive protein may also be seen in the

later half of pregnancy and in some who are taking birth control pills.

Calcitonin A 32 amino acid polypeptide hormone that is produced in humans primarily by

the C cells of the thyroid, and in many other animals in the ultimobranchial body

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CASP3 Caspase 3, Apoptosis Related Cysteine Protease, see page 36.

CCK Cholecystokinin, a peptide hormone of the gastrointestinal system responsible for

stimulating the digestion of fat and protein. Cholecystokinin, previously called

pancreozymin, is secreted by the duodenum, the first segment of the small

intestine, and causes the release of digestive enzymes and bile from the pancreas

and gallbladder, respectively. It also acts as a hunger suppressant. Recent

evidence has suggested that it also plays a major role in inducing drug tolerance to

opioids like morphine and heroin, and is partly implicated in experiences of pain

hypersensitivity during opioid withdrawal.

CCL27 Chemokine, CC motif, Ligand 27, see page 27.

CD36 CD 36 Antigen, encodes a deduced 472-amino acid protein, and is the primary

receptor for adhesion of platelets to collagen.

CEBP/α CCAAT/Enhancer Binding Protein α, directly interacts with CDK2 and CDK4

and arrests cell proliferation by inhibiting these kinases. A region between amino

acids 175 and 187 of CEBPA was determined to be responsible for direct

inhibition of cyclin-dependent kinases and caused growth arrest in cultured cells.

CEBPA inhibited CDK2 activity by blocking the association of CDK2 with

cyclins. The activities of Cdk4 and Cdk2 were increased in mouse CEBPα

knockout livers, leading to increased proliferation.

Chemotaxis A response of motile cells or organisms in which the direction of movement is

affected by the gradient of a diffusible substance. Differs from chemokinesis in

that the gradient alters probability of motion in one direction only, rather than rate

or frequency of random motion.

Clenbuterol A substituted phenylaminoethanol that has β2 adrenomimetic properties at very

low doses. It is used as a bronchodilator in asthma.

COL1A1 Collagen of skin tendon and bone, Alpha chain 1, see page 27.

CSF1 Colony Stimulating Factor 1, see page 33.

Db/db Diabetic mice, lacking the leptin receptor

Diabetes A general term referring to disorders characterized by excessive urine excretion

(polyuria), as in diabetes mellitus and diabetes insipidus. When used alone, the

term refers to diabetes mellitus.

Diabetes insipidus Rare form of diabetes in which the kidney tubules do not reabsorb

sufficient water. This can be because (a) either the renal tubules have defective

receptors for antidiuretic hormone (ADH, vasopressin) or (b) a class of aquaporin

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water channel in the collecting duct is defective or (c) there is inadequate ADH

production by the pituitary, leading to the excessive production of dilute urine.

Diabetes mellitus Relative or absolute lack of insulin leading to uncontrolled carbohydrate

metabolism. In juvenile onset diabetes (that may be an autoimmune response to

pancreatic cells) the insulin deficiency tends to be almost total, whereas in adult

onset diabetes there seems to be no immunological component but an association

with obesity.

DP1 E2F Dimerizaition Partner 1, regulates the expression of various cellular

promoters, particularly those involved in the cell cycle. E2F factors bind to DNA

as homodimers or heterodimers in association with dimerization partner DP1.

E2F The transcription factor E2F1 was identified as a DNA binding protein present in

many growth-responsive and growth-promoting genes. E2F family members

regulate these genes, in part, through their interaction with other cellular proteins.

Included among these so-called nuclear pocket proteins are RB1, p107, and p130.

E2F1, E2F2, and E2F3 are known targets of RB1, which inhibits transcription of

target genes. Another member of the E2F family, also called DP-1, forms

heterodimers with E2F1 which stimulate the DNA-binding and RB1-binding

action of E2F1.

Endochondral Growing or developing within cartilage; applied especially to developing

bone.

Estrogen Group of steroid compounds, named for their importance in the oestrus cycle,

functioning as the primary female sex hormone. While estrogens are present in

both men and women, they are usually present at significantly higher levels in

women of reproductive age. They promote the development of female secondary

sex characteristics and are also involved in the thickening of the endometrium and

other aspects of regulating the menstrual cycle. Follicle stimulating hormone

(FSH) and luteinizing hormone (LH) regulate the production of estrogen in

ovulating women. Since estrogen circulating in the blood can feedback to reduce

circulating levels of FSH and LH, some oral contraceptives contain estrogens.

FAT Fat Tumor Suppressor, encodes a transmembrane protein containing 34 cadherin

repeats in association with a number of other motifs. The Drosophila 'fat' locus

encodes a tumor suppressor gene, and recessive (loss-of-function) mutations lead

to hyperplastic overgrowth of the imaginal discs, indicating that contact-

dependent cell interactions may play an important role in regulating growth.

Flavanoids Flavanoids are strong antioxidants which enhance and stimulate enzymes,

preventing the growth and development of cancer cells.

Flourides Organic and inorganic compounds containing the element fluorine. As a halogen,

fluorine forms a monovalent ion (−1 charge). Fluoride forms a binary compound

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with another element or radical. Examples of common fluoride compounds

include hydrofluoric acid (HF), and sodium fluoride (NaF).

FN1 Fibronectin, a glycoprotein of high molecular weight, known as fibronectin or

LETS, was identified on the surface of fibroblasts by labeling with radioactive

compounds or specific antibodies. The protein is absent or greatly reduced in

many transformed cells. LETS is thought to have a role in cell adhesion,

morphology, and surface architecture. Its absence is thought to have a causal role

in the loss of contact inhibition of movement in transformed cells.

GATA Members of this family of DNA-binding proteins recognize a consensus sequence

known as the 'GATA' motif, which is an important cis-element in the promoters

of many genes GATA1 is essential for normal primitive and definitive

erythropoiesis and is expressed at high levels in erythroid cells, mast cells, and

megakaryocytes. GATA2 is expressed in hematopoietic progenitors, including

early erythroid cells, mast cells, and megakaryocytes, and also in

nonhematopoietic embryonic stem cells, see page 33.

GIP Glucose Dependant Insulinotropic Polypeptide, also known as Gastric Inhibitory

Polypeptide, a 42 amino acid hormone that stimulates insulin production and

secretion from the pancreas.

IGF1 Insulin Like Growth Factor 1, part of a family of peptides that play important

roles in mammalian growth and development. IGF1 mediates many of the

growth-promoting effects of growth hormone

ICV Intracerebroventricular Cortex, the locus of administration of drugs or chemicals

into the ventricular system of the brain. Often used in animal studies and

occasionally for the introduction of anti-infectives that do not penetrate the blood-

brain barrier into the brain in humans.

ID2 Id proteins inhibit the functions of basic helix-loop-helix (HLH) transcription

factors in a dominant-negative manner by suppressing their heterodimerization

partners through the HLH domains. Members of the ID family also promote cell

proliferation, implying a role in the control of cell differentiation. Inhibitor of

DNA Binding 2 able to disrupt the antiproliferative effects of tumor suppressor

proteins of the RB family, thus allowing cell cycle progression ID3 is an inhibitor

of E proteins, such as E2A.

LIF Leukemia Inhibitory Factor, candidate regulator of mesenchymal-to-epithelial

conversion during kidney development

Leptin A 16 kD protein that plays a critical role in the regulation of body weight and

energy expenditure

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LPL Lipoprotein Lipase, encodes a protein of 475 amino acids that becomes a mature

protein of 448 residues after cleavage of a signal peptide. Analysis of the

sequence indicated that human lipoprotein lipase, hepatic lipase, and pancreatic

lipase are members of a gene family.

LV Lateral ventricle, a cavity shaped somewhat like a horseshoe in conformity with

the general shape of the hemisphere; each lateral ventricle communicates with the

third ventricle through the interventricular foramen of Monro, and expands from

there forward into the frontal lobe as the anterior horn as well as caudally over the

thalamus as the central part or cella media which, behind the thalamus, curves

ventrally and laterally, then forward into the temporal lobe as the inferior horn;

from the apex of the curve a variably sized posterior horn extends back into the

white matter of the occipital lobe. The large choroid plexus of the lateral

ventricle invades the cella media and the inferior horn (but not the anterior and

posterior horn) from the medial side.

MCR4 Malanocortin Receptor 4, a 333-amino acid protein encoded by a single exon

found to be expressed primarily in the brain; its expression was notably absent in

the adrenal cortex, melanocytes, and placenta.

MEF2C MADS Box Transcription Enhancer Factor 2, Polypeptide C, see page 34.

MIF Migration Inhibitory Factor, the first lymphokine to be Expression of MIF activity

was found to correlate well with delayed hypersensitivity and cellular immunity

in humans. MIF activity could be detected in the synovia of patients with

rheumatoid arthritis. The expression of MIF at sites of inflammation suggested a

role for the mediator in regulating the function of macrophages in host defense.

MSH Muscle Segment Homobox, expressed in the heart valves, mandibular and hyeloid

arches, and limb buds during normal murine development

MyoD1 Myogenic Differentiation Antigen 1, see page 36.

NPY Neuropeptide Y, abundant and widespread peptide in the mammalian nervous

system. It shows sequence homology to peptide YY and over 50% homology in

amino acid and nucleotide sequence to pancreatic polypeptide.

Ob/Ob Obese mice, lacking the leptin gene

Obesity An increase in body weight beyond the limitation of skeletal and physical

requirement, as the result of an excessive accumulation of fat in the body

OB-Rb Leptin receptor, the full-length isoform expressed in abundance in hypothalamus

but also present in islets. It is the only isoform for which biologic activity has

been established.

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Op/op Osteoporotic mice

Ossification The formation of bone or of a bony substance, the conversion of fibrous tissue or

of cartilage into bone or a bony substance

Osteoblast Mesodermal cell that gives rise to bone

Osteoclast Large multinucleate cell formed from differentiated macrophage, responsible for

breakdown of bone

Osteogenesis Production of bone

Osteoporosis (OP) A reduction in the amount of bone mass, leading to fractures after

minimal trauma

OVX Ovariectomized

P107 Encodes a 936-residue protein, comparison with RB1 showed a major

region of homology extending over 564 residues. This region in RB1 is essential

to its growth-controlling function.

P130 Tumor suppressor gene, has a molecular mass of about 120 kD. Essential for

telomere length control in human fibroblasts, with loss of either protein leading to

longer telomeres. RBL2 forms a complex with RAD50 through RINT1 to block

telomerase-independent telomere lengthening.

PAI-1 Plasminogen Activator Inhibitor Type 1, a protein containing 402 amino acids

with a predicted nonglycosylated molecular mass of 45 kD. Cultured human

umbilical vein endothelial cells contain 2 PAI1 mRNA species, both encoded by a

single gene, differing by 1 kb in the 3-prime untranslated region. Plasminogen

activator inhibitor shows structural similarities to angiotensinogen, alpha-1-

antitrypsin, and antithrombin III. Plasminogen activator inhibitor-2 is less similar

to PAI1 than it is to the other proteins of this group. There are at least 3

immunologically distinct plasminogen activator inhibitors (PAIs): placental PAI,

protease nexin, and endothelial-cell-derived PAI. The last is also distinctive for

its beta-mobility in agarose zone electrophoresis and its inhibition of both tissue-

type PA and urokinase-type PA.

Parathyroidhormone Parathyroid hormone (PTH) is secreted by the parathyroid glands

as a polypeptide containing 84 amino acids. It acts to increase the concentration

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of calcium in the blood, whereas calcitonin (a hormone produced by the thyroid

gland) acts to decrease calcium concentration.

Perilipin A hormonally-regulated phosphoprotein that encircles the lipid storage droplet in

adipocytes, the major cellular A-kinase substrate in adipocytes

PPARγ Peroxisome Proliferator-Activated Receptor-Gamma, the peroxisome proliferator-

activated receptors (PPARs) are members of the nuclear hormone receptor

subfamily of transcription factors. PPARs form heterodimers with retinoid X

receptors (RXRs) and these heterodimers regulate transcription of various genes.

There are 3 known subtypes of PPARs, PPAR-alpha, PPAR-delta, and PPAR-

gamma. PPAR-gamma is believed to be involved in adipocyte differentiation, see

page 28.

Pref1 Preadipocyte Factor 1, regulator of adipocyte differentiation, a novel member of

the epidermal growth factor (EGF)-like family of proteins. It was synthesized as

a transmembrane protein with 6 tandem EGF-like repeats. In preadipocytes,

multiple discrete forms of the protein product of 45 to 60 kd were present, owing

in part to N-linked glycosylation. While PREF1 mRNA was abundant in

preadipocytes, its expression was completely abolished during differentiation of

cultured preadipocytes to adipocytes, see page 32.

pRB Retinoblastoma (RB) is an embryonic malignant neoplasm of retinal origin. It

almost always presents in early childhood and is often bilateral. Spontaneous

regression ('cure') occurs in some cases.

Resorption The loss of substance through physiologic or pathologic means, such as loss of

dentin and cementum of a tooth or of the alveolar process of the mandible or

maxilla

RETN Resistin, 108-amino acid FIZZ3 protein, shares an N-terminal signal peptide and a

C-terminal stretch of 10 cysteine residues with identical spacing with the other

FIZZ family members. In situ hybridization analysis detected diffuse expression

of mouse Fizz3 in white but not brown adipose tissue in a variety of organs. See

page 31

RUNX2 Runt Related Transcription Factor 2, also known as CBFA1, see page 27.

Sp7 Transcription Factor Sp7, see page 28.

SREBP1 Sterol Regulatory Element-Binding Transcription Factor 1, protein that controls

cholesterol homeostasis by stimulating transcription of sterol-regulated genes.

Statins Member of a class of hypolipidemic agents, used as pharmaceuticals to lower

cholesterol levels in people at risk for cardiovascular disease because of

hypercholesterolemia.

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Strontium Ranelate An orally active drug that dissociates bone remodeling by increasing bone

formation and decreasing bone resorption

TCIRG1 T Cell Immune Regulator- 1, see page 26.

THBD Thrombomodulin, see page 35.

THBS1 Thrombospondin 1, see page 35.

THY1 Thy-1 T-Cell Antigen, see page 34.

TNFα Tumor Necrosis Factor Alpha, a multifunctional proinflammatory cytokine, with

effects on lipid metabolism, coagulation, insulin resistance, and endothelial

function

TNFRSF11A Tumor Necrosis Factor Receptor Superfamily, Member 11A, see page 34.

VMH Ventromedial Hypothalamus, a nucleus of the middle hypothalamus, the largest

cell group of the tuberal region with small-to-medium size cells

WAT White Adipose Tissue, adipose cells with a scant ring of cytoplasm surrounding a

single large lipid droplet. Their nuclei are flattened and eccentric within the cell.

White adipose tissue serves three functions: heat insulation, mechanical cushion, a

source of energy.