THE CONTROL OF APOLIPOPROTEIN C-I GENE EXPRESSION DURING
ADIPOCYTE DIFFERENTIATION
by
John M. David
A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences
Spring 2006
Copyright 2006 John M. David All Rights Reserved UMI Number: 1435920
UMI Microform 1435920 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 THE CONTROL OF APOLIPOPROTEIN C-I GENE EXPRESSION DURING
ADIPOCYTE DIFFERENTIATION
by
John M. David
Approved: David C. Usher, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee
Approved: Daniel D. Carson, Ph.D. Chair of the Department of Biological Sciences
Approved: Thomas M. Apple, Ph.D. Dean of the College of Arts and Sciences
Approved: Conrado M. Gempesaw II, Ph.D. Vice Provost for Academic and International Programs ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. David Usher, for providing me with the opportunity to begin my graduate career, his guidance and support of this research project, and the invaluable experience that I gained while working in his laboratory.
I would like to thank my committee members, Dr. Roger Wagner and Dr. William Cain, for their criticism and support.
I would like to thank the past and present members of the Usher laboratory group, including Dr. Donna Maslak, Dr. Robin Davis, and Amanda Peters, for their assistance in the laboratory.
Finally, I would like to thank the other students in the Biological Sciences department that I have interacted with during my academic career, most notably Brady Redmond,
Dianna Willis, and Brian Danysh, for their help and advice in both my research and coursework.
This manuscript is dedicated to my parents, John and Marylou David, for their unconditional love and support.
iii TABLE OF CONTENTS
LIST OF TABLES...... vii
LIST OF FIGURES ...... viii
ABSTRACT...... xi
Chapter
1 INTRODUCTION ...... 1
1.1 Obesity and Disease ...... 1
1.2 The 3T3-L1 Cell Line: An In Vitro Model of Adipogenesis ...... 2
1.3 Regulation of Energy Balance...... 5
1.3.1 Adipokines ...... 6
1.3.2 Cholesterol Balance ...... 9
1.3.3 Inflammation...... 11
1.4 Hypothesis and Experimental Approach...... 13
2 MATERIALS AND METHODS...... 15
2.1 HDL Preparation ...... 15
2.2 Cell Culture ...... 15
2.2.1 3T3-L1 Culture Conditions...... 15
2.2.2 β-cyclodextrin/HDL Treatment ...... 17
2.2.3 Stimulation of PPARγ Activity with Pioglitazone...... 17
iv 2.2.4 Stimulation of PPARγ Activity with Rosiglitazone...... 17
2.2.5 Stimulation LXR Activity with T0901317 ...... 18
2.2.6 Trypan Blue Staining ...... 18
2.2.7 Oil Red-O Staining ...... 18
2.3 Total RNA Isolation and Treatment...... 19
2.4 cDNA Synthesis ...... 20
2.5 Quantitative PCR...... 21
2.5.1 Data Analysis...... 21
2.6 Apolipoprotein C-I ELISA...... 22
3 RESULTS ...... 24
3.1 SAGE Analysis of 3T3-L1 Preadipocytes and Adipocytes ...... 24
3.1.1 The Expression of Genes Important for Lipid Transport...... 26
3.2 The Expression of Apolipoprotein C-I mRNA in 3T3-L1 Adipocytes...... 27
3.3 Apolipoprotein C-I mRNA Expression in Mouse Tissues and Human
Adipocytes...... 29
3.4 The Expression of Apolipoprotein C-I Protein in Human Adipocytes ...... 31
3.5 The Effect of Cholesterol Depletion on 3T3-L1 Adipocyte
Differentiation ...... 31
3.5.1 The Histological Effect of β-cyclodextrin/HDL Treatment on
3T3-L1 Differentiation...... 34
3.5.2 The Effect of β-cyclodextrin/HDL Treatment on Apolipoprotein
C-I mRNA Expression During Adipocyte Differentiation ...... 35
v 3.6 The Role of Transcription Factors in the Regulation of the Apoc1 Gene .....35
3.6.1 The Effect of β-cyclodextrin/HDL Treatment on Pparg and its
Target Genes ...... 36
3.6.2 The Effect of PPARγ Agonists on 3T3-L1 Adipocyte
Differentiation...... 37
3.6.3 The Effect of β-cyclodextrin/HDL Treatment on Srebp1-c,
Srebp2, and their Target Genes...... 39
3.6.4 The Effect of β-cyclodextrin/HDL Treatment on Lxr and its
Target Genes ...... 40
3.6.5 The Effect of the Liver X Receptor Ligand T0901317 on 3T3-L1
Adipocyte Differentiation ...... 41
4 DISCUSSION ...... 43
REFERENCES ...... 87
vi LIST OF TABLES
1 Primer sequences for genes studied in quantitative PCR experiments ...... 55
2 Relative quantitation of gene expression ...... 56
3 SAGE analysis of 3T3-L1 preadipocytes and adipocytes...... 57
4 Cluster analysis of 3T3-L1 preadipocyte and adipocyte SAGE libraries ...... 58
5 SAGE identification of genes that encode for secreted proteins ...... 59
6 SAGE identification of secreted proteins that function in lipid metabolism ...... 60
7 Production of apolipoprotein C-I by cultured human adipocytes ...... 61
8 Viability test results for 3T3-L1 differentiation in the presence of β-
cyclodextrin and HDL ...... 62
9 Viability test results for 3T3-L1 differentiation in the presence of PPARγ and
LXR agonists ...... 63
vii LIST OF FIGURES
1 Phenotypic and genotypic events during 3T3-L1 differentiation ...... 64
2 Light microscope images of 3T3-L1 preadipocytes and differentiated
adipocytes ...... 65
3 mRNA expression of the reference genes Tbp and Hprt1 during 3T3-L1
differentiation ...... 66
4 Apolipoprotein E/C1/C4/C2 linkage group expression in 3T3-L1 preadipocytes
and adipocytes ...... 67
5 mRNA expression time course of adipocyte marker genes during 3T3-L1
differentiation ...... 68
6 mRNA expression time course of apolipoprotein genes during 3T3-L1
differentiation ...... 69
7 Apolipoprotein and adipocyte marker gene expression in murine liver and
adipose tissue...... 70
8 Light microscope images of human preadipocytes and differentiated adipocytes
in culture ...... 71
9 mRNA expression profiles for ADN, APOE, and APOC1 in cultured human
adipocytes ...... 72
10 Apolipoprotein C-I ELISA standard and calibrator curves ...... 73
viii 11 Oil red-O staining of 3T3-L1 preadipocytes and adipocytes...... 74
12 Apolipoprotein C-I mRNA expression during 3T3-L1 differentiation in the
presence of β-cyclodextrin and HDL ...... 75
13 mRNA expression of PParg, Adn, and Cebpa during 3T3-L1 differentiation in
the presence of β-cyclodextrin and HDL...... 76
14 mRNA expression of Pparg, Adn, and Cebpa during 3T3-L1 differentiation in
the presence of pioglitazone ...... 77
15 Apolipoprotein C-I mRNA expression during 3T3-L1 differentiation in the
presence of pioglitazone ...... 78
16 Apolipoprotein E mRNA expression during 3T3-L1 differentiation in the
presence of pioglitazone ...... 79
17 Apolipoprotein C-I and apolipoprotein E mRNA expression during 3T3-L1
differentiation in the presence of rosiglitazone ...... 80
18 mRNA expression of Srebp1-c, Fas, and Scd1 during 3T3-L1 differentiation in
the presence of β-cyclodextrin and HDL...... 81
19 mRNA expression of Srebp2, Ldl-r, and Hmgcr during 3T3-L1 differentiation
in the presence of β-cyclodextrin and HDL ...... 82
20 mRNA expression of Lxr, Abca1, and Apoe during 3T3-L1 differentiation in
the presence of β-cyclodextrin and HDL...... 83
21 mRNA expression of Lxr, Abca1, and Apoe during 3T3-L1 differentiation in
the presence of T0901317...... 84
ix 22 Apolipoprotein C-I mRNA expression during 3T3-L1 differentiation in the
presence of T0901317...... 85
23 Apolipoprotein C-I mRNA expression during 3T3-L1 cholesterol depletion in
the presence of T0901317...... 86
x ABSTRACT
Serial analysis of gene expression (SAGE) was used to generate global gene expression profiles of 3T3-L1 preadipocytes and adipocytes. Our primary interest was in late gene expression, specifically genes that encode secreted proteins, so that we may detect novel adipocyte genes that are related to energy balance and obesity. SAGE detected the expression of apolipoprotein C-I (apoC-I) mRNA in 3T3-L1 adipocytes, but not preadipocytes. ApoC-I, the product of the Apoc1 gene, is a 6.6 kDa protein which is found in plasma associated with chylomicrons, very low-density lipoproteins (VLDLs), and high-density lipoproteins (HDLs). The function of apoC-I and the regulation of its production is not well understood, though studies have suggested that it may interfere with triglyceride-rich lipoprotein clearance, free fatty acid transport into peripheral tissues, and cholesterol ester transfer protein (CETP) activity (Jong, et al., 1999). To elucidate the SAGE findings, the expression of apoC-I was determined by quantitative
PCR and ELISA. Apoc1 mRNA expression was shown to increase during late-phase adipocyte differentiation and positively correlate with lipid accumulation, suggesting that it may be regulated by cellular triglyceride or cholesterol content. Quantitative PCR was used to monitor the expression of Apoc1 mRNA during 3T3-L1 differentiation under cholesterol-depleted conditions, and Apoc1 expression was suppressed by the cholesterol depletion. Lastly, to investigate the role of specific transcription factors in the regulation
xi of the Apoc1 gene, 3T3-L1 cells were treated with synthetic agonists of the peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) during differentiation. Apoc1 mRNA expression was suppressed by the PPARγ agonists pioglitazone and rosiglitazone; however, Apoc1 expression was increased by the LXR agonist T0901317. These results suggest that Apoc1 expression in adipocytes is involved with cholesterol efflux and may be controlled by LXR.
xii Chapter 1
INTRODUCTION
1.1 Obesity and Disease
Obesity is epidemic, affecting 30 percent of US adults, and is linked to increased morbidity and mortality. Obesity predisposes an individual to a number of cardiovascular risk factors, including hypertension and dyslipidemia, and also may lead to coronary heart disease, metabolic syndrome, type II diabetes, sleep apnea, and even some forms of cancer (Orzano and Scott, 2004; Haslam and James, 2005). Central to obesity is adiposity, an increase in adipose tissue mass resulting from an imbalance between caloric intake and expenditure. Increased adipose mass involves both an increase in adipocyte cell size and increase in cell number. Besides adipocytes, adipose tissue contains supporting tissue, the stromal-vascular tissue, composed of preadipocytes, fibroblasts, macrophages, and endothelial cells (Berg, et al., 2005). When adipocytes increase in cell size and number in response to external simuli, they secrete a number of different autocrine, paracrine, and endocrine factors that affect other tissues (Coppack,
2005).
1 1.2 The 3T3-L1 Cell Line: An In Vitro Model of Adipogenesis
The increase in adipocyte cell number seen in obesity is due to the differentiation of preadipocytes in the adipose tissue. In vivo study of adipocyte differentiation is difficult because adipose tissue contains multiple cell types that cannot be easily distinguished. Preadipocytes, which are only a small percentage of cells within adipose tissue, are difficult to isolate because they cannot be distinguished from fibroblasts, making primary cultures unreliable (Ntambi and Kim, 2000).
Most of the research on adipocyte differentiation has been conducted in vitro using established cell lines, such as the 3T3-L1 cell line, that store fatty acids as triglycerides upon stimulation. These cell lines are homogenous populations and can be passaged indefinitely. The 3T3-L1 preadipocyte cell line was derived from Swiss 3T3 mouse embryo fibroblasts. 3T3 cells are pluripotent stem cells of mesodermal origin, and they can be differentiated into committed preadipocytes, cartilage, bone, or smooth muscle by treatment with 5-azacytidine, an inhibitor of DNA methylation (Taylor and
Jones, 1979; Ailhuad, et al., 1992). 3T3-L1 cells, a subclone of the 3T3 cells, are adipocyte precursor cells that have undergone terminal determination to a progenitor cell and have the potential to be converted into adipocytes by using a defined hormone treatment (Ntambi and Kim, 2000). Mature 3T3-L1 adipocytes posses many of the phenotypic characteristics of true adipocytes in tissue, including large lipid droplets.
Studies have shown that when injected into mice, 3T3-L1 preadipocytes differentiate and form fat pads that are indistinguishable from normal adipose tissue (Neels, et al., 2004).
2 3T3-L1 cell differentiation corresponds to a sequence of phenotypic changes that begin with the adipoblast and lead sequentially to the formation of the preadipocyte, the immature adipocyte, and finally the mature adipocyte. Adipoblasts are fibroblast-like, unipotent adipocyte precursor cells that are formed from the determination of the multipotent mesenchymal stem cell. Adipoblasts give rise to preadipocytes in culture upon growth-arrest. Preadipocytes are characterized by an absence of lipid droplets and the expression of early markers of differentiation, such as lipoprotein lipase (LPL) and type VI collagen (Ailhaud, et al., 1992; Ntambi and Kim, 2000).
Differentiation of confluent, growth-arrested 3T3-L1 preadipocytes is induced by treatment with an adipogenic cocktail which contains insulin, dexamethasone, and 3- isobutyl-1-methylxanthine. The events that occur during differentiation are described in
Figure 1. Insulin activates the insulin-like growth factor 1 receptor, dexamethasone stimulates the glucocorticoid receptor pathway, and 3-isobutyl-1-methylxanthine increases the cellular cAMP level, stimulating the cAMP-dependent protein kinase pathway. Approximately 24 hours after induction, the cells reenter the cell cycle and undergo post-confluent mitosis, or clonal expansion. This is initiated by the c-fos, c-myc, and c-jun genes, which are only expressed for six hours after treatment. This mitosis is believed to unwind the DNA and provide the transcriptional machinery with access to genes that are necessary for terminal differentiation and the mature adipocyte phenotype
(Ntambi and Kim, 2000; Lane and Tang, 2005).
The CCAAT/enhancer binding proteins C/EBPβ and C/EBPδ are the first transcription factors to be activated after the induction of differentiation, and are involved
3 in directing the differentiation program. These genes are only active during the first two days of differentiation, beginning within one hour after the addition of the induction cocktail to the cell medium and reaching maximum expression after 24 hours. C/EBPβ expression is primarily stimulated by dexamethasone and C/EBPδ expression is stimulated by 3-isobutyl-1-methylxanthine. C/EBPβ appears to play two roles in adipocyte differentiation – to induce the clonal expansion previously mentioned, and to activate the expression of the key adipogenic transcription factors C/EBPα and PPARγ
(Ntambi and Kim, 2000).
The up-regulation of the sterol regulatory element binding protein 1c (SREBP-1c) gene during early differentiation is also believed to affect PPARγ expression. Insulin heightens the expression of SREBP-1c, which activates genes involved in glucose metabolism and the synthesis of fatty acids (Ntambi and Kim, 2000; Weber, et al., 2004;
Lane and Tang, 2005).
Approximately 48 hours after the induction of differentiation, the cells become growth-arrested and the expression of the “intermediate genes” C/EBPα and PPARγ is activated. PPARγ reaches maximal expression by Day 4 of differentiation, and C/EBPα peaks by Day 5. These two genes act in a synergistic manner and are critical for the progression of the late phases of adipocyte differentiation; they activate specific “late genes” that are responsible for maintaining the adipocyte phenotype, including the glucose transport GLUT-4, the fatty acid binding protein aP2 (a.k.a. FABP4), and the lipid droplet protein perilipin (Ailhaud, et al., 1992; Ntambi and Kim, 2000).
4 The differentiating cells begin to express late markers of differentiation by Day 3.
These markers include lipogenic and lipolytic enzymes and other proteins that function to develop the mature adipocyte phenotype. As motile fibroblasts are converted into sessile adipocytes, a number of cytoskeleton and extra cellular matrix-related genes are down- regulated and the production of actin, tubulin, fibronectin, and proteoglycans is halted.
The cells are considered immature adipocytes at this time; they begin to round up and take on a cuboidal shape, accumulate cytoplasmic lipid droplets, and express both early and late markers of differentiation. By Day 6 of differentiation, the 3T3-L1 cells express the late markers of a mature adipocyte, such as adiponectin, leptin, and resistin, and exhibit further accumulation of lipid (Ailhaud, et al., 1992; Ntambi and Kim, 2000).
1.3 Regulation of Energy Balance
Adipocytes play a key role in energy balance - to store energy in the form of triglycerides (TGs) in periods of energy excess, and release this energy in periods of energy deprivation. The storage and release of triglycerides and fatty acids (FAs) are well-regulated so that energy in the form of TGs is stored immediately after feeding, and released during periods of fasting. Upon eating a meal, TGs are digested to FAs, and these FAs are adsorbed into the intestine. There they are resynthesized as TGs and assembled into chylomicrons, which are released into the lymph and then enter the circulation. Chylomicrons are hydrolyzed by lipoprotein lipase (LPL), located on the capillary endothelial surface, and the resulting FAs are taken up by adipocytes to be stored in TG droplets. During a time of fasting, adipocytes undergo lipolysis and release
5 their TG stores as FAs into the circulation, which increases plasma FA levels and muscle
FA uptake. The movement of FAs to the liver also increases during fasting, fueling hepatic gluconeogenesis and ketogenesis. In extended periods of fasting, glucose is reserved for utilization by the central nervous system, and FAs fuel organs such as the heart, skeletal muscle, and renal cortex. If this regulation of energy storage and release becomes impaired, non-adipose tissues may accumulate FAs or TGs, which can cause toxicity and dysfunction (Yu and Ginsberg, 2005).
The classic view of passive adipocyte function has dramatically changed over the last ten years. In 1994, the discovery of leptin, an adipocyte-derived hormone that acts on the hypothalamus to suppress appetite and other organs to affect energy balance, was a major development in adipocyte research and established adipose tissue as a multifunctional, endocrine organ (Fruhbeck, et al., 2001). Studies have shown that adipocytes are responsive to metabolic signals and are capable of secreting a number of proteins and other factors, collectively termed adipokines, which have important roles in energy balance, metabolism, immunity, and cardiovascular function. There is growing evidence indicating that this secretory function of adipocytes is impaired in obesity, which can lead to obesity-related diseases such as type II diabetes and metabolic syndrome (Hauner, 2004).
1.3.1 Adipokines
Adipocytes control energy balance though the secretion of hormones and other signaling molecules. Leptin, the product of the ob gene, is predominantly produced by
6 adipocytes, although low levels of expression have been detected in other tissues,
including the intestine and brain. Leptin regulates appetite and weight by acting as a
satiety signal; signaling through the hypothalamus, leptin suppresses food intake and
stimulates energy expenditure (Koerner et al., 2005). In normal humans and rodents, the
decrease in plasma leptin is an important signal for the switch between fed and fasted
states. Low leptin stimulates a robust response which includes the stimulation of
appetite, suppression of thyroid, growth, and reproductive hormones, and the inhibition
of thermogenesis and immunity (Ahima, 2005).
Leptin expression is regulated by hormonal and nutritional status. Concentrations
of plasma leptin and leptin mRNA correspond with estimates of obesity. Obesity is
associated with increased production of leptin and high amounts of circulating leptin, but
a lack of appetite suppression. This indicates leptin resistance, and is supported by the
decreased ability of leptin to reduce feeding and weight in obese animals (Fruhbeck et al.,
2001; Ahima, 2005). Adipocyte size is associated with leptin synthesis; larger adipocytes
produce more leptin than smaller adipocytes from the same individual. Leptin production
is also influenced by immune activation, shown by a positive correlation of TNF-α and plasma leptin concentrations (Fruhbeck et al., 2001).
Leptin has diverse functions in addition to appetite and weight control, and has been shown to be involved in reproduction, hematopoiesis, angiogenesis, the immune response, and bone formation. In humans, leptin does not cause pronounced weight- reduction, and mutations for leptin are rarely found in obese people (Koerner, et al.,
2005; Rajala and Scherer, 2005).
7 Adiponectin, a serum protein that is secreted only by adipocytes, was identified
around the same time as leptin, but its significance was not realized until years later.
Adiponectin expression increases during adipocyte differentiation, and its expression in
subcutaneous fat is greater than in omental fat. Circulating adiponectin levels are
decreased in obese individuals, and the reduction of adiponectin has been shown to
precede the onset of cardiovascular disease and type II diabetes. Conversely, weight loss
stimulates adiponectin production, which is associated with increased insulin sensitivity
(Koerner, et al., 2005).
Adiponectin has also been associated with adipogenesis. Overexpression of
adiponectin in 3T3-L1 cells resulted in a more robust and accelerated differentiation of
preadipocytes to adipocytes, along with increased lipid accumulation and insulin
sensitivity. Overall, adiponectin seems to play a protective role in type II diabetes and
cardiovascular disease, and further study of its mechanisms of action and regulation may
lead to the development of therapeutic strategies for obesity-related disorders (Koerner, et
al., 2005).
Resistin is a recently-identified cytokine that is up-regulated during feeding and
adipogenesis in mice, and is down-regulated during fasting and by agonists of PPARγ.
The expression of resistin in mice is restricted to adipose tissue and is positively- correlated with adipose tissue mass. In vivo studies have shown that resistin causes glucose intolerance and hyperinsulinaemia (Aubry, et al., 2005).
In humans, resistin is expressed in a number of cell-types and tissues, including preadipocytes, macrophages, bone marrow, and lung. Resistin expression can be
8 induced by cytokines and endotoxin, and is associated with inflammatory markers that are considered risk factors for cardiovascular disease (Koerner, et al., 2005; Rajala and
Scherer, 2005). Studies have shown a negative correlation between plasma total cholesterol and resistin mRNA levels in human white adipose tissue (Jove, et al., 2003).
The expression and action of resistin differs in mice and humans, and further research is needed to define its role in insulin resistance and/or inflammation.
1.3.2 Cholesterol Balance
Adipocytes secrete important regulators of reverse cholesterol transport such as
LPL, apolipoprotein E (apoE), and cholesterol ester transfer protein (CETP). Reverse cholesterol transport (RCT) is the process by which excess cellular cholesterol is removed from cells and transferred to lipoprotein particles that are transported to the liver, where the cholesterol can be catabolized. The first step in RCT involves the release of free cholesterol (FC) from the plasma membrane of cells to extracellular acceptor particles. There are several methods by which this can occur, including aqueous diffusion of FC to a phospholipid-containing acceptor, efflux to small HDL particles by receptors such as scavenger receptor class B type 1 (SR-B1), and also the release of FC to apolipoproteins. Small HDLs and apolipoproteins may serve as cholesterol shuttles, which transport the FC from the cell membrane to lipoproteins that act as cholesterol sinks, such as HDL. HDL particles increase in size when surface cholesterol is converted to cholesterol esters (CEs) by lecithin-cholesterol acyltransferase (LCAT), allowing more
9 cholesterol to be transferred to the HDL. Large HDL particles are then targeted to the liver (Christian, et al., 1999).
ApoE has been shown to function in RCT as a cholesterol shuttle. ApoE, a structural and functional component of plasma lipoproteins, is associated with all lipoprotein classes except LDL, although most is associated with HDL. The majority of plasma apoE is derived from the liver; however adipocytes and macrophages also synthesize significant levels of apoE (Mak, et al., 2002).
Besides accelerating cholesterol efflux from cells to HDL, apoE is a ligand for the low-density lipoprotein receptor (LDL-R), and mediates the clearance of plasma lipoproteins from the circulation by interacting with this receptor in the liver (Zechner et al., 1991).
CETP, a plasma glycoprotein that is primarily secreted by adipocytes and hepatocytes, mediates the interchange of cholesterol esters and triglycerides between lipoproteins. CETP is expressed in humans, rabbits, and several rodent species, but not mice (Bruce et al., 1998). Specifically, CETP transfers CEs from HDL to VLDL, with the reciprocal movement of TGs. Cholesterol from peripheral tissues is esterified in HDL and subsequently transferred to apolipoprotein B-containing lipoproteins by CETP, which are then removed and the cholesterol catabolized by the liver. Overexpression of CETP reduces plasma HDL (Vassiliou and McPherson, 2004).
LPL is synthesized and secreted by adipocytes and muscle cells. LPL is transported to the capillary endothelial surface, where it hydrolyzes triglyceride-rich lipoproteins (TGRLs), chylomicrons and very low-density lipoprotein (VLDL) (Jin et al.,
10 2002). LPL is activated by apolipoprotein C-II (apoC-II), which resides on the surface of lipoprotein particles. Another apolipoprotein, apoC-III, inhibits apoC-II’s activation of
LPL (Jong, et al., 1999). LPL plays an important role in energy balance; the lipolytic action of LPL produces free fatty acids, which transverse the endothelial cells before being taken up by adipocytes, where they are stored as triglycerides, or by muscle cells where they are used for ATP production. Lipolysis of TGRLs also results in the recycling of surface phospholipids and apolipoproteins from chylomicrons and VLDL to
HDL (Jin et al., 2002).
LPL expression is related to atherosclerosis; LPL deficiency in mice and humans is associated with hypertriglyceridemia and decreased HDL levels. Overexpression of
LPL in adipose tissue has been shown to raise HDL levels and reduce atherosclerosis in mice, rabbits, and rats (Jin et al., 2002).
1.3.3 Inflammation
Obesity is recognized as a state of low-grade inflammation; plasma levels of several cytokines and acute phase proteins associated with inflammation are increased in obese individuals. Similarly adipocytes have been shown to produce a number of inflammation-related proteins, including cytokines, chemokines, and acute phase proteins. The elevated production of inflammation-related proteins is believed to play a role in the development of metabolic syndrome and type II diabetes. Proinflammatory cytokines, such as TNF-α and IL-6, also have important effects on lipid and glucose metabolism (Trayhurn, 2005).
11 Tumor necrosis factor-alpha (TNF-α), first identified in macrophages, is a
cytokine that induces insulin resistance, anorexia, and weight loss. Adipocytes express
the TNF-α cytokine and both receptor subtypes, and there is evidence that the TNF-α system is up-regulated in obese individuals (Hauner, 2004). TNF-α exhibits autocrine and paracrine actions in adipose tissue, including lipolysis, apoptosis, and the regulation of adipokine expression (Trayhurn, 2005). TNF-α inhibits C/EBPα and PPARγ, key transcription factors involved in adipocyte differentiation, resulting in the suppression of adipocyte-specific proteins such as LPL, the fatty acid-binding protein aP2, fatty acid synthetase (FAS), and the glucose transporter GLUT-4 (Fruhbeck, et al., 2001).
Adiponectin may influence the production of inflammatory proteins. Studies have shown an association between adiponectin levels and serum markers of inflammation
(Berg, et al., 2005). Adiponectin decreases the production of tumor necrosis factor-alpha and inhibits the activation of immune cells, thereby attenuating the inflammatory response (Koerner, et al., 2005).
Interleukin 6 (IL-6) is a multi-functional cytokine produced by many cell types, including adipocytes. As much as one-third of total circulating IL-6 is derived from adipose tissue in a normal individual, and omental adipose tissue produces three-fold more IL-6 than subcutaneous adipose tissue (Zhao, et al., 2005). There is a positive correlation between body mass index and IL-6 production. Increased IL-6 production is associated with decreased insulin sensitivity and increased risk of cardiovascular disease.
IL-6 has also been shown to decrease LPL activity, which results in increased
12 macrophage uptake of lipids and leads to the formation of foam cells (Fernandez-Real
and Ricart, 2003; Berg et al., 2005).
Cytokines like TNF-α and IL-6 promote the expression of acute phase proteins by adipocytes. The acute phase response in the body’s initial reaction to injury, infection, or inflammation, and is accompanied by fever, secretion of glucocorticoids, activation of complement, and a drastic change in the level of acute phase proteins (Kurash, et al.,
2004). Acute phase proteins are primarily synthesized in the liver, although the adipocyte has been determined to be a significant source of production. Acute phase proteins secreted by adipocytes include C-reactive protein (CRP), haptoglobin, orosomucoid, and the complement-related proteins complement factor D (adipsin), B, and C3. CRP expression by the liver and adipocytes in response to IL-6 has pro- atherogenic activities on vascular endothelium and smooth muscle (Lin, et al., 2001).
Many of the transcription factors that stimulate the acute phase response in the liver are also expressed abundantly in the adipocyte, such as C/EBPα and C/EBPβ.
While it is difficult to determine the relative contribution of adipocytes to plasma levels of the acute phase proteins, adipocytes have been shown to possess a proinflammatory potential equal or greater than macrophages with respect to a subset of inflammatory markers (Rajala and Scherer, 2005).
1.4 Hypothesis and Experimental Approach
Adipose tissue, along with the liver, muscles, and brain, form an axis for regulating energy balance. The regulation involves adipocytes in two functional states –
13 storing triglycerides during periods of feeding, and releasing fatty acids to the blood during periods of fasting. This process depends on adipocytes receiving proper signals from other tissues and the release of signals by adipocytes which regulate other tissues.
Most gene expression profiling studies have focused attention on the early events that occur during adipocyte differentiation. To identify molecules important for regulating energy balance, we analyzed events occurring late during differentiation by using serial analysis of gene expression (SAGE). SAGE libraries of 3T3-L1 preadipocytes and mature adipocytes (harvested six days after the induction of differentiation) provided a profile of global gene expression for these cells. Our primary goal was to identify secreted proteins that were important in regulating energy balance and thus give insights into obesity, type II diabetes, and atherosclerosis.
Several novel secreted-protein genes were detected by SAGE. To confirm their importance, quantitative polymerase chain reaction (PCR) was used to determine their expression during 3T3-L1 differentiation, in adult mouse adipose tissue, and during the differentiation of a human preadipocyte cell line. Finally, to determine how these candidate genes were regulated, 3T3-L1 cells were differentiated in the presence of antagonists or synthetic transcription factor agonists and gene expression patterns were determined by quantitative PCR.
14 Chapter 2
MATERIALS AND METHODS
2.1 HDL Preparation
HDL was prepared by centrifugation from human plasma. The plasma density was adjusted to 1.21 mg/mL with sodium bromide (NaBr) (Fisher Scientific), and then centrifuged for 43 hours at 50,000 RPM and 15oC. After centrifugation, the lipoprotein
layer was brought to a density of 1.35 mg/mL with NaBr in a final volume of 5 mL. A
0% - 25% NaBr gradient was layered on top of the samples, and then centrifuged at
50,000 RPM and 15oC for 90 minutes. The HDL layer was removed from the centrifuge
tubes and dialyzed against 1 L of 1X PBS (10 mM PBS, pH 7.4) at 4oC overnight, then
replaced with fresh PBS and dialyzed again at 4oC overnight. The concentration of HDL
was determined using absorbance at 280 nm and using 2 mg/mL BSA (Pierce Chemicals)
as a standard.
2.2 Cell Culture
2.2.1 3T3-L1 Culture Conditions
3T3-L1 cells were thawed into Growth Medium 1 (GM1), containing DMEM
(Invitrogen) with 10 % calf serum (Invitrogen), 1% L-glutamine (Mediatech Cellgro),
and 1% penicillin/streptomycin (Mediatech Cellgro), and seeded directly into T75 flasks
15 at a cell count of approximately 3.75 x 104 per flask. Flasks were maintained in 20 mL of
o GM1 at 37 C and 5% CO2, and the medium was changed every 48-72 hours. The cells were passaged upon reaching 60% confluency. Cells were treated with 1.5 mL of 0.25% trypsin (Mediatech Cellgro) and then diluted into 100 mL of GM1, after which 20 mL of the diluted cells were seeded into new T75 flasks. Cells were not passaged more than two times to ensure proper differentiation. After the final trypsin treatment, cells were distributed evenly into 6-well plates at a cell count of approximately 2 x 106 cells per
o well. 6-well plates were maintained in 2 mL of GM1 at 37 C and 5% CO2, with a medium change every 48-72 hours, until confluency was reached.
3T3-L1 differentiation was conducted using a standard 3T3-L1 differentiation protocol, previously described by Yue, et al. (2004) with minor modifications. To assure contact inhibition and the conversion of adipoblasts to preadipocytes, 3T3-L1 cells were induced to differentiate to adipocytes 72 hours post-confluency (Day 0). The cells were washed twice with 1 mL of 1X PBS per well, and 2 mL of inducing medium was added to each well. The inducing medium contained Growth Medium 2 (GM2; DMEM, 10% fetal bovine serum (Invitrogen), 1% L-glutamine, and 1% penicillin/streptomycin) supplemented with the inducing agents (0.5 mM 3-isobutyl 1-methylxanthine, 1 µM dexamethasone, and 10 µg/mL insulin).
On Day 2, 48 hours after the beginning of differentiation, the inducing medium was removed, the cells were washed twice with 1 mL of 1X PBS, and fresh GM2 was added to each well.
16 2.2.2 βββ-Cyclodextrin/HDL Treatment
β-cyclodextrin (Sigma-Aldrich) was prepared at 7.5 mM in deionized water, and added to cell culture medium at a final concentration of 0.75 mM. HDL was added to the same medium at a final concentration of 1 mg/mL protein. This cyclodextrin/HDL- enriched medium (CHEM) was prepared fresh, added to the 3T3-L1 cells on Day 3 of differentiation, and changed daily for 5 days. Treated 3T3-L1 cells were monitored for lipid accumulation and viability.
2.2.3 Stimulation of PPARγγγ activity with Pioglitazone
Pioglitazone (Cayman Chemical) was prepared in a solution of 10 mM pioglitazone in DMSO, and added to cell culture medium at a final concentration 10 µM.
The pioglitazone-enriched medium was prepared fresh, added to 3T3-L1 cells on Day 3 of differentiation, and changed daily for 5 days. Treated 3T3-L1 cells were monitored for lipid accumulation and viability.
2.2.4 Stimulation of PPARγγγ activity with Rosiglitazone
Rosiglitazone (Cayman Chemical) was prepared in 1 mM solution in DMSO, and added to the cell culture medium at a final concentration of 1 µM. The rosiglitazone- enriched medium was prepared fresh, added to the 3T3-L1 cells on Day 3 of differentiation, and changed daily for 5 days. Treated 3T3-L1 cells were monitored for lipid accumulation and viability.
17 2.2.5 Stimulation of LXR activity with T0901317
T0901317 (Cayman Chemical) was prepared as a 3 mM solution in DMSO, and
added to the cell culture medium at a final concentration of 3 µM. The T0901317-
enriched medium was prepared fresh, added to the 3T3-L1 cells on Day 3 of
differentiation, and changed daily for 5 days. Treated 3T3-L1 cells were monitored for
lipid accumulation and viability.
2.2.6 Trypan Blue Staining
Trypan Blue staining was performed to determine cell viability. Cells were
detached from wells of 6-well plates by adding 500 µL of 0.25% trypsin (Mediatech
Cellgro) to each well and rocking the plates for approximately 10 minutes at room temperature. 1.0 mL of GM2 was then added to each well and the cell suspensions were transferred to 1.5 mL Eppendorf tubes. 10 µL of each mixture of 15 µL of cell suspension and 15 µL of Trypan Blue working solution (Invitrogen) was loaded into a hemocytometer (Cascade Biologics) and both viable (non-dyed) and non-viable (blue- dyed) cells were counted using phase-contrast microscopy.
2.2.7 Oil Red-O Staining
Oil red-O lipid staining was performed using the Adipogenesis Assay Kit
(Chemicon International) and following the manufacturer’s protocol. On the last day of
3T3-L1 differentiation, medium was removed from cultured cells, and cells were washed twice with 2 mL of PBS per well of 6-well plates. 1 mL of oil red-O working solution
18 (0.36% oil red-O in 60% isopropanol) was added to each well and the plates were incubated at room temperature for 15 minutes. The staining solution was then removed, and wells were washed with 1 mL of Chemicon Wash Buffer. The cells were examined by phase-contrast microscopy at 200X magnification.
2.3 Total RNA Isolation and Treatment
Total RNA was isolated from 3T3-L1 cells by using TRIzol Reagent (Invitrogen) and following the manufacturer’s protocol. 1 mL of TRIzol Reagent was added to each well of 6-well plates. The plates were incubated at room temperature for 15 minutes while shaking, and the contents of each well were transferred to 1.5 mL Eppendorf tubes.
Phase separation was performed by adding 0.2 mL of chloroform per 1mL of TRIzol reagent in the initital homogenate. Samples were hand-shaken vigorously for 15 seconds and allowed to incubate at room temperature for 3 minutes. Samples were then centrifuged at 12,000 x g at 4oC for 15 minutes, and the resulting colorless aqueous phase, containing RNA, was transferred to a fresh tube. The RNA was then precipitated from the aqueous phase by adding 0.5 mL of isopropanol per 1 mL of TRIzol reagent in the initial homogenate. The samples were incubated at room temperature for 10 minutes and then centrifuged at 12,000 x g at 4oC for 10 minutes to pellet the RNA. After removing the supernatant, the precipitated RNA was washed with 1 mL of 75% ethanol per 1 mL of TRIzol in the initial homogenate, and the pellets were air-dried at room temperature for 5 minutes. The RNA was re-suspended in 20 µL of THE RNA storage buffer (Ambion) and the concentration was determined using absorbance at 260 nm with
19 a BioPhotometer (Beckman Industries).
To remove contaminating DNA from the RNA samples, DNase treatment was
performed by using a DNA-free kit (Ambion) and following the manufacturer’s protocol.
20 µL reaction mixtures typically contained 10 µg of RNA, 2 µL of 10x DNase I buffer,
1 µL of DNase I, and RNase-free water. Samples were then gently mixed and incubated at 37oC for 20-30 minutes. DNase I was inactivated by adding 5 µL of DNase I
Inactivating Reagent to the sample, followed by mixing and incubating the sample for 2 minutes at room temperature. The sample tube was then centrifuged at 10,000 x g for 1 minute to pellet the inactivation reagent. The treated RNA samples were then transferred to new tubes, quantified using absorbance at 260 nm, and then stored at -20oC until
further use.
2.4 cDNA Synthesis
Total RNA isolated from the 3T3-L1 cells was reverse transcribed to cDNA by
using the Omniscript Reverse Transcriptase Kit (Qiagen) and following the
manufacturer’s protocol. 20 µl reaction mixtures typically contained 2 µg of RNA, 2 µl
of 10X Buffer RT, 2 µL of 5 mM dNTP mix, 2 µL of 0.5 µg/µL random hexamer primers
(Promega), and RNase-free water. Reverse transcription was performed at 37oC for 60
minutes and reactions were stored at -20oC until further use.
20 2.5 Quantitative PCR
PCR primers (Table 1) were designed using Primer Express software version 2.0
(Applied Biosystems). The length of the primers was from 17 to 23 nucleotides, the
melting temperature was from 59°C to 61°C, the GC content was from 40% to 60%, and
the expected PCR products ranged from 50 to 150 base pairs.
Quantitative PCR was performed by using an ABI SYBR Green PCR Kit
(Applied Biosystems) and following the manufacturer’s protocol. Reaction mixtures
contained 20 ng of cDNA, 1 µL each of 10 µM forward and reverse primers, 12.5 µL of
2X SYBR Green PCR Master Mix (Applied Biosystems), and 5.5 µL of deionized water.
Reaction mixes were prepared in bulk and component volumes were scaled-up as needed.
25 µl of the mixture was transferred to each well of a 96-well reaction plate (Applied
Biosystems). The reactions were run in an ABI Prism 7000 Sequence Detection System
(Applied Biosystems). The PCR program was initiated at 95°C activate the Taq DNA
polymerase, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C.
Dissociation analysis of the PCR products was performed immediately following the last
cycle of the PCR program, using a temperature range of 60°C to 95°C.
2.5.1 Data Analysis
Quantitative PCR data was analyzed by using ABI Prism SDS software, version
1.0. Samples were obtained from triplicate cultures, and each PCR reactions for each
sample were performed in triplicate wells. The average CT value from triplicate PCR
reactions was calculated and relative quantitation was used to determine relative mRNA
21 levels (Table 2). mRNA levels were calculated relative to the reference genes TATA-box
binding protein (TBP) or hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1).
Data are represented as the means of the triplicate experiments +/- standard deviation
(SD).
2.6 Apolipoprotein C-I ELISA Assay
96-well plates were coated with goat anti-human apolipoprotein C-I polyclonal
antibody (BioDesign). The antibody was diluted to 3 µg/mL in coating buffer (0.2 M
PIPES, pH 6.5), 100 µL of the diluted antibody was added to each well, and the plates
were incubated at 4oC overnight with shaking. Plates were then washed with 1X PBS-
Tween (10 mM PBS, 0.5% Tween 20, pH 7.4) and blocked with 200 µL of ELISA
Diluent (10 mM PBS, 0.05% Tween 20, 1mM Na2EDTA, 5 g/L casein, pH 7.4) per well
at room temperature for 1 hour. After the blocking, plates were washed with 1X PBS-
Tween and the antigen (human plasma (gift from Dr. Daniel Rader, University of
Pennsylvania) or purified apoC-I (BioDesign)) was added to the wells at the desired
concentration. Dilutions of antigen were prepared in ELISA Diluent, 100 µL was added
to each well, and the plates were incubated at 4oC overnight. The next day, after washing
with 1X PBS-Tween, HRP-conjugated goat anti-human apoC-I polyclonal antibody
(BioDesign) was prepared in ELISA Diluent at a 1:2,500 dilution, 100 µL was added to each well, and the plates were incubated at room temperature for 2 hours with shaking.
The plate was then developed using an o-phenylenediamine dihydrochloride (OPD) substrate. One SIGMAFAST OPD tablet (Sigma-Aldrich) was dissolved in 20 mL of
22 deionized water, per plate. The plates were washed with 1X PBS-Tween, 200 µL of substrate was added to each well, and plates were allowed to develop at room temperature for 30 minutes while protected from light. Plates were then analyzed in a plate reader (Dynex Technologies) at 450 nm.
23 Chapter 3
RESULTS
3.1 SAGE Analysis of 3T3-L1 Preadipocytes and Adipocytes
Serial analysis of gene expression (SAGE) libraries were generated for 3T3-L1 preadipocytes and mature adipocytes to develop a “transcriptome,” a quantitative catalog of overall gene expression. SAGE is an unbiased and comprehensive transcript profiling strategy. A SAGE profile is not limited to a pre-determined set of genes, as is microarray technology, and allows for the detection of novel genes. The specific interest of this study was to detect novel adipocyte-expressed genes that are involved in regulating adipocyte energy balance.
3T3-L1 preadipocyte cells (Figure 2A) were differentiated into adipocytes (Figure
2B) using a standard differentiation cocktail which contained insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. SAGE libraries were created using RNA that was isolated from non-differentiated preadipocytes and mature adipocytes six days after the induction of differentiation.
The SAGE libraries detected a number of genes whose expressions were both up- regulated and down-regulated during adipocyte differentiation. A total of 885 genes were up-regulated and 937 genes were down-regulated (Table 3). Approximately 50% of the
24 up-gregulated and down-regulated genes had characterized UniGene annotations, and the rest were either novel expressed sequence tags (ESTs) or unknown.
Cluster analysis was used to organize the SAGE expression data into groups based on gene function. As 3T3-L1 preadipocytes differentiate into adipocytes, their morphology changes from elongated, fibroblast-like cells to rounded, cuboidal cells.
These morphological changes are mirrored in the SAGE cluster analysis of structural changes; a number of genes involved in the cytoskeleton, cell mobility, and extracellular matrix are down-regulated, indicating that the cells became sessile after differentiation
(Table 4A).
There are also a number of metabolic changes that occur during 3T3-L1 differentiation. Genes involved in both fatty acid synthesis and cellular respiration were up-regulated by three- to twelve-fold (Table 4B). Most of the changes were associated with mitochondrial genes – genes involved in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. This corresponds to an overall increase in the number of mitochondria in adipocytes; previous studies have determined that adipocytes contain 19 times more mitochondria than preadipocytes (Kim et al, 2004).
Adipocytes are not only sources for triglyceride storage, but also secrete factors that function in energy balance and inflammation (Ailhaud, 1992). SAGE analysis detected 15 up-regulated and 15 down-regulated genes that code for secreted proteins
(Table 5). Of the previously known genes, the expression of the gene for the cytokine tumor necrosis factor (TNF) was down-regulated in the adipocytes, but other inflammation-related genes were upregulated, including macrophage migration inhibitory
25 factor (MIF). MIF plays a role in the regulation of macrophage function and is an important initiator of other pro-inflammatory cytokines (Shimizu, 2004). Of the new genes, Apoc1, Orm1, and Angptl4 are candidate genes for regulating fatty acid influx to the adipocyte.
3.1.1 The Expression of Genes Important for Lipid Transport
SAGE analysis of 3T3-L1 preadipocytes and adipocytes detected the increased expression of five genes involved in lipid transport – Apoe, Apod, Apoc1, Cd36, and Srb1
(Table 6). Four of the genes, Apoe, Apod, Cd36, and Srb1 have previously been identified; however, Apoc1 gene expression in adipocytes has not. Apolipoprotein C-I
(apoC-I), a 6.6 kDa protein found in plasma, is a constituent of chylomicrons, very-low- density lipoproteins (VLDLs), and high-density lipoproteins (HDLs). ApoC-I is the product of the 4.7-kb Apoc1 gene and is primarily expressed in the liver, though moderate expression occurs in the skin, spleen, lung, and testes (Hoffer, et al., 1993; Jong, et al.,
1999). The Apoe and Apoc1 genes are linked in a 30-kb cluster on murine chromosome 7 and in a 48-kb cluster on human chromosome 19, along with Apoc2 and Apoc4. These four secreted proteins are involved in lipoprotein and lipid homeostasis. The function and regulation of apoC-I is not well understood, though mouse models have suggested that it may interfere with TG-rich lipoprotein clearance, free fatty acid transport into peripheral tissues, and cholesterol ester transfer protein (CETP) activity (Jong, et al.,
1999; Mak, et al., 2002).
26 3.2 The Expression of Apolipoprotein C-I mRNA in 3T3-L1 Adipocytes
We employed a quantitative real-time PCR system to monitor the expression of the Apoe/Apoc1/Apoc4/Apoc2 gene cluster during an eight-day differentiation time course. 3T3-L1 preadipocytes were differentiated into adipocytes, and total RNA was isolated on days 0, 3, 4, 5, 6, and 8 of differentiation. The RNA was DNase-treated and reverse-transcribed into cDNA, and real-time PCR was performed using a SYBR Green-I dye chemistry provided by Applied Biosystems, Inc. Messenger RNA expression levels were determined by relative quantitation using the ∆∆CT method as described in the
Applied Biosystems User Bulletin #2 (1997).
The housekeeping genes Tbp (TATA-binding protein) and Hprt1 (hypoxanthine phosphoribosyl transferase 1) were used as internal references to normalize the expression data. The TBP protein is a component of the transcription factor TFIID, which is involved in the initiation of transcription by RNA polymerase II, and HPRT1 is involved in the purine salvage pathway. Ideally, a reference gene should exhibit constant expression levels among different tissues in an organism, during all stages of development, and should not be affected by experimental treatments. The expression of these genes has been shown to remain constant in studies using neutrophils (Zhang, et al.,
2005). Expression of these genes also remained constant during 3T3-L1 differentiation
(Figure 3). Tbp and Hprt1 expression varied less than two-fold throughout the differentiation time course.
Quantitative PCR data supported the SAGE findings and previous reports that
Apoe expression in Day 6 3T3-L1 adipocytes was significantly increased (Figure 4).
27 Apoc1 expression was also significantly increased in the adipocytes, confirming our discovery that Apoc1 mRNA is expressed in 3T3-L1 adipocytes. The Apoc2 and Apoc4 genes were not expressed in the adipocytes or preadipocytes. This suggests that Apoe and Apoc1 may be coordinately regulated in the adipocyte.
Relative to Tbp expression, Apoe expression is 15-fold higher and Apoc1 50-fold higher. Relative to Day 0 preadipocyte expression, Apoe increased 8.6-fold and Apoc1 increased 300-fold. This difference in Apoc1 relative expression levels when normalized to Tbp or Day 0 preadipocytes is due to differences in sensitivity for the detection of expression of mRNA in preadipocytes. Whereas Apoe appears to have some expression in preadipocytes, Apoc1 does not or it is present at very low levels. Therefore normalizing expression to preadipocyte mRNA levels is essentially normalizing Apoc1 to a background reading, which is the equivalent of dividing by zero. Normalizing expression to Tbp allows for more accurate comparisons of expression between different genes.
The expression of Apoe and Apoc1 mRNA was monitored during an eight-day
3T3-L1 differentiation time course. To verify that the differentiation progressed normally, the expression profiles of the marker genes peroxisome proliferative activated receptor gamma (Pparg) and adipsin (Adn) were also determined. Pparg is a marker of intermediate-phase adipocyte gene expression, and its expression was initially detected on Day 1 and reached a maximum on Day 6 (Figure 5). Adn, a late-phase gene expression marker, showed an increase of expression on Day 3 and continued to increase throughout the rest of the time course (Figure 5). Apoe and Apoc1 both exhibited late-
28 phase expression profiles (Figure 6). Low levels of Apoc1 expression were detected on
Day 4, one day later than Adn, and Apoc1 expression increased between days 3 and 8 by approximately 40-fold.
3.3 Apolipoprotein C-I mRNA Expression in Mouse Tissues and Human
Adipocytes
The 3T3-L1 cell line is derived from mouse embryonic stem cells and has been adapted to an in vitro tissue culture environment. The gene expression profiles of an established cell line may differ from the tissue of origin; the comparison of cell line and tissue gene expression is necessary to determine the significance of the previous findings.
The major source of plasma apolipoprotein E is the liver, but it is also secreted from adipocytes and macrophages. Apoc1 and Apoc2 mRNA has been shown to be expressed in the murine liver and macrophages. Apoc4 mRNA has been tentatively identified in the liver, but the levels of mRNA are extremely low (Mak et al., 2002).
To determine if the genes from the Apoe/Apoc2 gene linkage cluster are expressed in mouse adipose tissue, quantitative PCR experiments were performed. RNA was isolated from the livers and epididymal fat pads of male BALB/c mice. The expression of Apoe, Apoc1, Apoc2, and the adipocyte marker genes Pparg and Adn were compared for the tissues (Figure 7). As expected, expression of Pparg and Adn was only detected in the adipose tissue and Apoc2 expression was detected only in the liver. Apoe and
Apoc1 were expressed in the liver and adipocytes and Apoc4 was not detectable in either tissue. Apoe and Apoc1 mRNA expression was much lower in adipocytes than in the
29 liver. Also, while Apoc1 levels were comparable to levels found in 3T3 L1 cells, Apoe
expression was much higher in adipose tissue than in 3T3 L1 cells. This may in part be
explained by the nature of adipose tissue. Adipose tissue is composed adipocytes and a
stromal vascular network, consisting of preadipocytes, macrophages, and endothelial
cells, which account for about 50% of the tissue mass. In addition to adipocytes,
macrophages are known to produce significant amounts of apoE but very little apoC-I
(Laffite, et al., 2001).
To determine whether APOC1 mRNA expression is synthesized by human adipocytes, pure human adipocyte cultures were used. Human primary preadipocyte and adipocyte cultures were obtained from Zen-Bio, Inc. Preadipocytes were isolated by centrifugation of collagenase-treated subcutaneous adipose tissue from a healthy, non- diabetic female donor with a body mass index (BMI) of 27.6. The preadipocytes were cultured and differentiated into adipocytes using a differentiation medium which contained insulin, dexamethasone, isobutylmethylxanthine, and a proprietary PPARγ agonist. Similar to 3T3-L1 cells, human preadipocytes are elongated and fibroblast-like, and adipocytes are cuboidal and contain lipid droplets (Figure 8).
The expression of ADN, APOE, and APOC1 mRNA in human preadipocytes and
adipocytes was compared using quantitative PCR (Figure 9). RNA samples isolated from
days 0, 3, 6, 9, and 12 of a differentiation time course. The mRNA expression of all three
genes increased during human adipocyte differentiation and their timing, relative to each
other, was similar to what we observed for their orthologs in the 3T3-L1 time course. An
increase of ADN mRNA was detected on Day 3 and reached a maximum of 80-fold on
30 Day 6. APOE mRNA increased significantly on Day 6 and peaked at 30-fold expression on Day 9. APOC1 expression increased to approximately 3-fold on Day 9 and was the same on Day 12. APOC1 mRNA is expressed very late during human adipocyte differentiation, and this observation agrees with the 3T3-L1 expression data; however, the expression levels in human adipocytes were much lower than in the 3T3-L1 cells or mouse adipose tissue.
3.4 Expression of Apolipoprotein C-I Protein in Human Adipocytes
Cell supernatants were harvested from human preadipocytes and mature adipocytes in DMEM medium with and without 10% fetal bovine serum after 48 hours of incubation at 37°C. ELISA assays were performed using a polyclonal goat anti-human apoC-I antibody, and curves were constructed using purified apoC-I protein (calibrator) and human plasma (standard). Calibrator and standard curves had similar slopes (0.872 and 0.859, respectively), indicating that the antibody interacts with purified and lipoprotein-bound apoC-I equivalently (Figure 10). ELISA indicated that apoC-I was present in the supernatant of cultured adipocytes but not of preadipocytes (Table 7). The adipocyte cell supernatant with serum had approximately 10 ng/mL of apoC-I and the adipocyte supernatant without serum had approximately 4.5 ng/mL of apoC-I.
3.5 The Effect of Cholesterol Depletion on 3T3-L1 Adipocyte Differentiation
Apolipoprotein C-I has been shown to affect the activity of CETP and LCAT
(Jong, et al., 1999). As previously mentioned, CETP mediates the interchange of
31 cholesterol esters and triglycerides between lipoproteins and LCAT converts free
cholesterol to cholesterol-ester on HDL particles. Both of these proteins are involved in
reverse cholesterol transport, and their interaction with apoC-I suggests that apoC-I may
also play a role in RCT.
A “shuttle-sink” model for RCT has been proposed that involves small particles as being the initial acceptors of the FC, which would transport the FC from the plasma membrane to a cholesterol sink, such as HDL. The movement of FC between cells and lipoproteins is typically a bi-directional process, and the direction of net movement is determined by properties of the acceptor (Rothblat, et al. 1999).
The process by which cholesterol is removed from cell membranes has been studied using artificial cholesterol acceptors, such as cyclodextrins (Christian, et al.,
1999). Cyclodextrins are cyclic oligosaccharides composed of 6, 7, or 8 glucopyranose units, referred to as α, β, and γ, respectively. These molecules are water-soluble and
contain a hydrophobic cavity that is capable of dissolving non-polar compounds, thus
enhancing their solubility in aqueous solutions (Rothblat, et al., 1999; Kilsdonk, et al.,
1995). β-cyclodextrin has been shown to be very efficient in solubilizing cholesterol, and
its high affinity for cholesterol has made the compound a valuable tool in the study of
cholesterol flux. At high concentrations (5 – 100 mM), β-cyclodextrin can remove cholesterol from the cell at very high rates. When present in low concentrations (≤ 1
mM), β-cyclodextrin can act as a cholesterol shuttle, transporting FC from the plasma
membrane to a lipoprotein-based cholesterol sink (Rothblat, et al., 1999).
32 Various HDL subclasses have also been implicated in cholesterol efflux. In
addition to apoE, small, phospholipid-rich particles have been shown to act as initial
cholesterol acceptors and large HDL particles serve as cholesterol reservoirs, where
cholesterol is converted to cholesterol ester through the action of LCAT. The addition of
both cyclodextrin and large HDL particles to cells in culture shifts the equilibrium to
cholesterol efflux, causing rapid and extensive depletion of cholesterol from cells (Atger,
et al. 1997).
Studies with 3T3-L1 cells have shown that treatment with β-cyclodextrin increased cholesterol efflux five-fold when both β-cyclodextrin and HDL were present in the culture medium (Atger, et al. 1997). Also, the expression of a number of genes during 3T3-L1 differentiation has been shown to be affected by cholesterol depletion.
The genes Srebp2, Hmgcr, and Ldl-r, which are involved in cholesterol biosynthesis and transport, were up-regulated by cholesterol depletion, while the cholesterol transporter
Abca1 was down-regulated. Pparg and Cebpα, which code for key transcription factors
in adipogenesis, were not affected by cholesterol depletion of the 3T3-L1 cells (Le Lay et
al., 2001).
To determine if apoC-I acts similarly to apoE and is involved in cholesterol
efflux, 3T3-L1 cells were treated with β-cyclodextrin and HDL during differentiation.
Relative mRNA expression of Apoc1 was measured and compared to the expression of
Apoe and other genes involved in cholesterol homeostasis.
33 3.5.1 Histological Effect of βββ-cyclodextrin/HDL Treatment on 3T3-L1 Adipocyte
Differentiation
3T3-L1 cells were induced to differentiate into adipocytes, and β- cyclodextrin/HDL-enriched medium, containing 0.75 mM β-cyclodextrin and 1.0 mg/mL
HDL, was added to the cells on Day 3 of differentiation. This treatment was started on
Day 3 so that adipocyte differentiation would progress normally and not be inhibited by the cholesterol depletion. Early and intermediate transcription factors are expressed prior to Day 3, which assures that late genes are capable of being expressed. The treatment continued for 5 days (120 hours) until Day 8 of differentiation, when the adipocytes are considered to be mature.
Lipid accumulation was monitored during the treatment by staining the cells with oil-red O, a lipid-soluble dye. Non-differentiated 3T3-L1 preadipocyte cells remained fibroblast-like, exhibited an elongated shape, and did not accumulate triglycerides (Figure
11A). Untreated mature 3T3-L1 adipocytes had a cuboidal shape and accumulated triglycerides in lipid droplets (Figure 11B). 3T3-L1 cells that were induced to differentiate and also treated with cholesterol depletion medium containing β- cyclodextrin did display a cuboidal phenotype. However, lipid staining revealed that triglycerides did not accumulate within the cells (Figure 11C). The cholesterol depletion treatment inhibited lipid droplet formation in differentiating 3T3-L1 adipocytes.
There was no significant change in viability for 3T3-L1 cells under the various treatments containing both β-cyclodextrin and HDL (Table 8); however, treatments containing only β-cyclodextrin at 0.75 and 1.0 mM concentrations caused complete cell
34 death within 24 hours. HDL functions as a cholesterol sink, and its presence in the culture medium along with β-cyclodextrin allows for a net efflux of cholesterol from the cells without causing injury. High levels of cell viability are maintained through the exchange of cholesterol between HDL and the adipocyte plasma membrane.
3.5.2 The Effect of βββ-cyclodextrin/HDL Treatment on Apolipoprotein C-I mRNA
Expression During 3T3-L1 Differentiation
Apoc1 mRNA expression was detected during 3T3-L1 differentiation by quantitative PCR. Under normal differentiation conditions, a significant increase in
Apoc1 expression is detected on Day 4 and mRNA levels continue to increase during the rest of the time course. During conditions of cholesterol depletion, Apoc1 expression is completely suppressed (Figure 12).
3.6 The Role of Transcription Factors in the Regulation of Apolipoprotein C-I
Cholesterol depletion of 3T3-L1 adipocytes during differentiation suppressed the expression of Apoc1, suggesting that Apoc1 expression may be controlled by cellular cholesterol or triglyceride content. To identify the specific transcription factors that are involved in the regulation of Apoc1 gene expression, the mRNA levels transcription factors that are involved in triglyceride and cholesterol homeostasis, including Pparg and
Lxr, were monitored by quantitative PCR during cholesterol depletion of 3T3-L1 adipocytes. Synthetic agonists of PPARγ and LXR were used to determine if the activation of these transcription factors had a direct effect on Apoc1 expression.
35 3.6.1 The Effect of βββ-cyclodextrin/HDL Treatment on Pparg and its Target Genes
PPARγ is a key adipogenic transcription factor that regulates TG accumulation in
the adipocyte. PPARγ is a member of the nuclear hormone receptor superfamily, and its transcriptional activity requires dimerization with the retinoid X receptor (RXR) and binding to a ligand. Endogenous ligands of PPARγ are poorly characterized, but fatty
acids, eicosanoids, and several synthetic ligands are known to stimulate PPARγ activity.
Activation of PPARγ increases insulin sensitivity by up-regulating the expression of the
glucose transporter GLUT-4 and induces the expression of genes involved in intracellular
fatty acid synthesis and transport. PPARγ has also been shown to increase the expression
of adipsin and affect the expression of inflammatory cytokines such as TNF-α and IL-6
(Li and Glass, 2004; Lazar, 2005).
Quantitative PCR experiments comparing the mRNA expression of Pparγ and its target genes Adn and Cebpa during adipocyte differentiation in the presence of β- cyclodextrin and HDL revealed no significant difference for Pparg compared to the control differentiation 24 hours (Day 4) after cholesterol depletion (Figure 13). However, on Day 8 (120 hours) there was a significant 2-fold decrease in Pparg expression. For the Pparg target gene Cebpa, expression did not differ from the control at either 24 hours
(Day 4) or 120 hours (Day 8). Adn expression increased four-fold after 24 hours of cholesterol depletion (Day 4), but was similar to the control after 120 hours (Day 8).
36 3.6.2 The Effect of the PPARγγγ Agonists on 3T3-L1 Adipocyte Differentiation
Pioglitazone is a member of the thiazolidinethione (TZD) family, insulin
sensitizing agents used to treat type II diabetes. These antidiabetic drugs are ligands for
PPARγ; TDZs aid in the dimerization and activation of PPARγ/RXR heterodimers, which
can translocate to the nucleus and stimulate the expression of various target genes. TZDs
have been shown to improve glucose homeostasis by reducing hepatic glucose output and
increasing peripheral glucose disposal (Bogacka et al., 2004; de Souza, et al., 2001).
The exact mechanism by which TZDs cause insulin sensitivity is not completely
understood, though modulation of adipose tissue energy balance by PPARγ stimulation is
believed to play an important role. Studies suggest that TZDs improve adipose
physiology by enhancing adipocyte differentiation and gene expression, which
coordinately affects lipid metabolism (de Souza, et al., 2001). TZDs may cause adipose
tissue remodeling by inducing the differentiation of preadipocyte precursors in adipose
tissue, thus creating new and more insulin sensitive adipocytes. This remodeling might
also reduce the production of TNF-α and resistin, adipokines that are correlated with
insulin resistance (Bogacka et al., 2004).
To determine if PPARγ affected Apoc1 expression, 3T3-L1 cells were treated with 10 µM pioglitazone on Day 3 of differentiation, and this treatment continued for five days, until Day 8. The pioglitazone-treated cells differentiated normally, as they were morphologically identical to the control cells. Pioglitazone treatment did not cause cytotoxicity. The viability of the pioglitazone-treated cells was determined to be approximately 90% by Trypan blue staining (Table 9).
37 As expected, Pparg did not increase after 24 hours of treatment (Day 4) with pioglitazone. However, expression of the Pparg target gene Adn increased by approximately four-fold in response to the agonist. After five days of pioglitazone treatment, the expression of Pparg and Adn decreased less than 2-fold compared to the control, while Cebpa expression remained unchanged (Figure 14).
There was a significant increase, approximately three-fold, in Apoc1 mRNA levels after 24 hours of pioglitazone treatment. However, Apoc1 mRNA levels in the pioglitazone-treated cells reach a maximum on Day 5 at approximately 13-fold, while
Apoc1 mRNA levels in the control cells continued to increase during the time course and reached 46-fold expression on Day 8 (Figure 15). Though pioglitazone increased Apoc1 expression on Day 4, after 24 hours of treatment, overall Apoc1 mRNA levels during differentiation were suppressed. Apoe expression was unaffected by pioglitazone (Figure
16), although expression decreased but not significantly at Day 8. This suggests that the activation of PPARγ may be suppressing Apoc1 expression.
Rosiglitazone, a different TZD PPARγ agonist, was also used to determine the effect of PPARγ activation on Apoc1 and Apoe expression. 3T3-L1 cells were treated with 1 µM rosiglitazone in the same manner as the pioglitazone treatment; the treatment started on Day 3 of differentiation and continued for five days, and did not damage the cells (Table 9). Similar to the effect of pioglitazone, Apoc1 mRNA levels were suppressed by approximately 4.5-fold on Day 8, while Apoe mRNA levels were not affected (Figure 17).
38 3.6.3 The Effect of βββ-cyclodextrin/HDL Treatment on Srebp-1c, Srebp2, and their
Target Genes
Cholesterol regulates the activity of certain proteins and the expression of genes in adipocytes. The sterol regulatory element binding proteins (SREBPs) are transcription factors that target genes involved in cholesterol and fatty acid synthesis and uptake. Low levels of intracellular cholesterol induce the transport of SREBPs from the endoplasmic recticulum to the golgi apparatus, where they are cleaved and released back into the cytoplasm. Cleaved SREBPs can then migrate to the nucleus and activate their target genes (Brown and Goldstein, 1997).
SREBP-1c is involved in the regulation of adipogenesis and insulin-responsive genes which control lipogenesis and glucose metabolism. The targets of SREBP1-c include FAS and stearoyl-CoA desaturase 1 (SCD1), which are involved in the synthesis of fatty acids and triglycerides, respectively. SREBP2 regulates genes that are involved in cholesterol biosynthesis and transport, such as HMGCoA reductase (Hmgcr) and the low-density lipoprotein receptor (Ldl-r), respectively (Weber, et al., 2004).
The expression of Srebp-1c, Srebp2, and their target genes was examined during
3T3-L1 differentiation in the presence and absence of β-cyclodextrin. The expression of
Srebp-1c and its targets Fas and Scd1 all increased during normal differentiation.
Treatment with β-cyclodextrin/HDL medium had no effect on the expression of Srebp1- c, Fas or Scd1 (Figure 18).
The expression of Srebp2 and its target Hmgcr did not change during 3T3-L1 differentiation, while the expression of another target of Srebp2, Ldl-r, increased.
39 However, unlike Srebp-1c and its target genes, the expression of Srebp2, Ldl-r, and
Hmgcr was significantly increased after 24 hours of treatment with β-cyclodextrin and
HDL (Figure 19). These increased expression levels, compared to the non-treated control, continued throughout the rest of the time course. SREBP2 is a transcription factor that controls cholesterol homeostasis, and it is expected that a state of cholesterol depletion would up-regulate the expression of Srebp2 to maintain a balance in cholesterol synthesis and transport.
The Srebp1-c and Pparg expression results suggest that Apoc1 gene expression is
not regulated by transcription factors that control triglyceride accumulation. To
determine whether transcription factors that control cholesterol efflux affect ApoC1, the
expression of Lxr and its target genes Abca1 and Apoe was examined.
3.6.4 The Effect of βββ-cyclodextrin/HDL Treatment on Lxr and its Target Genes
The liver X receptor α (LXR) is controlled by cellular cholesterol content.
Similar to PPARs, LXRs are members of the family of nuclear hormone receptors and
form heterodimers with RXR. When activated by an oxysterol ligand, this transcription
factor complex can translocate to the nucleus and activate specific genes. Targets of
LXR include the genes for ATP-binding cassette protein A1 (ABCA1), apoE, and other
genes involved in cholesterol catabolism, absorption, and transport (Zhang and
Maseldorf, 2002).
Quantitative PCR revealed that the expression of Lxr, Abca1, and Apoe increased
during normal 3T3-L1 differentiation, but was suppressed during treatment with β-
40 cyclodextrin and HDL (Figure 20). This suppression began on Day 4 (24 hours of treatment) and continued over the rest of the time course. As previously mentioned, the activity of LXR is dependant on an oxysterol ligand, and the cellular concentration of this ligand is decreased during cholesterol depletion (Zhang and Maseldorf, 2002). The decrease in target gene expression may have been due to either the decrease in Lxr expression or its natural agonist, which has been shown to decrease during cholesterol depletion.
3.6.5 The Effect of the Liver X Receptor Ligand T0901317 on 3T3-L1 Adipocyte
Differentiation
T0901317 is a high-affinity agonist of the transcription factor LXR. This synthetic, non-steroidal compound has been shown to act with the LXR/RXR heterodimer and induce the expression of the ABCA1 cholesterol transporter in mice
(Repa, et al., 2000). The activation of LXR in macrophages increased cholesterol efflux by ABCA1 and apoE, and studies have shown T0901317 to be an activator of Lxr and
Apoe in 3T3-L1 adipocytes and mouse adipose tissue, respectively (Laffitte, et al., 2001;
Seo, et al., 2004).
3T3-L1 cells were treated with 3 µΜ T0901317 during differentiation. Like the previous experiments, this treatment began on Day 3 of differentiation and continued for five days, until Day 8 of differentiation, with no significant affect on cell viability (Table
9). The mRNA levels of Lxr, Abca1, and Apoe significantly increased after treatment
41 with T0901317. Lxr and Abca1 expression increased after 24 hours of treatment, while
Apoe expression increased only after 120 hours (Day 8) of treatment (Figure 21).
Apoc1 mRNA expression increased in response to T0901317. 24-hour treatment with T0901317 had no affect on Apoc1 mRNA expression, but there was a substantial increase in expression after five days of treatment similar to what was observed for Apoe
(Figure 22). The relative level of Apoc1 mRNA in T0901317-treated 3T3-L1 cells was approximately four-times greater than that in the control cells. Cholesterol depletion of
3T3-L1 cells with β-cyclodextrin and HDL completely suppressed the expression of
Apoc1; however, T0901317 treatment during the cholesterol depletion restored Apoc1 mRNA expression to the control level on Day 4 and approximately 55% of the control level on Day 8 (Figure 23). Combined with the findings that both Apoc1 and the LXR target genes were suppressed during cholesterol depletion, this result suggests that the expression of Apoc1, like Apoe, is a controlled by the transcription factor LXR.
42 Chapter 4
DISCUSSION
The classical view of the adipose tissue as a passive site for triglyceride storage has changed since the discovery of leptin, an adipocyte-secreted protein that controls appetite by targeting the hypothalamus. The adipocyte is now recognized as an endocrine cell which secretes a number of proteins and signaling factors that act on other tissues.
These secreted factors, termed adipokines, can affect multiple system-wide processes, such as energy balance, metabolism, and immunity, both positively and negatively. The regulation of many adipokines is linked to the size and state of the adipocyte, and an imbalance in adipokine expression can lead to obesity and several other disorders, including type II diabetes, cardiovascular disease, and metabolic syndrome (Fruhbeck, et al., 2001; Hauner, 2004).
SAGE analysis of 3T3-L1 preadipocytes and adipocytes identified a number of genes that were up-regulated in the adipocyte. Approximately 500 up-regulated genes were annotated in the mouse UniGene database, and cluster analysis revealed that these genes were related to cellular metabolism, the cytoskeleton, extracellular matrix, and secretory products. The principal interest of this study was to identify novel candidate
43 genes for obesity, specifically genes that encode secreted proteins that may affect adipocyte lipid accumulation and cell size.
SAGE detected the expression of 15 secreted-protein genes in 3T3-L1 adipocytes, several of which have not been well characterized for adipocytes, including angiopoietin- like 4, orosomucoid 1, and apolipoprotein C-I.
Angiopoietin-like 4 (ANGPTL4) is a known target of PPARγ and is up-regulated in white adipose tissue and the liver during fasting, indicating that it may play a role in lipid metabolism. Intravenous administration of ANGPTL4 into mice increased plasma triglyceride, specifically the fraction of VLDL and chylomicrons, and non-esterified fatty acids up to five-fold. This ANGPTL4-induced hyperlipidemia was shown to result from the ability of ANGPTL4 to significantly inhibit the activity of LPL, thereby decreasing plasma VLDL and chylomicron hydrolysis (Yoshida, et al., 2002). These findings suggest that ANGPTL4 may act to regulate adipocyte uptake of FA by inhibiting LPL.
Orosomucoid (ORM1), also known as α-1 acid glycoprotein (AGP), is a major serum glycoprotein, though its function is not clear. ORM1 is primarily synthesized by the liver, but extra-hepatic synthesis has been reported in leukocytes, human breast epithelium, and microvascular endothelial cells. The expression of Orm1 in adipocytes had not been previously reported. The serum concentration of ORM1 increased up to five-fold during an acute-phase response, and its glycosylation pattern can also change depending on the type of inflammation (Hochepied, et al., 2003). ORM1 plays an important role in maintaining capillary barrier for macromolecules. Studies suggest that
ORM1 decreases capillary permeability by interacting with the endothelial cells on
44 capillary walls and serum proteins. ORM1 has been shown to affect the three- dimensional arrangement of collagen in vitro, indicating that it may affect the organization of molecules on the endothelial surface. Also, ORM1 has a low isoelectric point, and it has been suggested that the binding of ORM1 to the glycocalyx increases the negative charge of the luminal surface of the vessel wall (Sorensson, et al., 1999). The effects that ORM1 has on capillary permeability suggest from our study that it may be capable of inhibiting FA uptake by adipocytes by preventing transcapillary passage of
FA.
SAGE analysis also indicated that the apolipoprotein C-I (apoC-I) gene, Apoc1, was expressed in the 3T3-L1 adipocytes. Previous studies had shown that the liver was the primary site of Apoc1 expression, but that moderate Apoc1 mRNA levels could also be found in the skin, spleen, lung, and testes. However, the expression of Apoc1 in adipocytes had not been previously reported.
ApoC-I is found in plasma, associated with chylomicrons, VLDL, and HDL.
Human APOC1-transgenic mice overexpressing apoC-I were hyperlipidemic; the mice exhibited elevated levels of TGs and cholesterol due to an accumulation of VLDL-size particles in the plasma. To investigate this defect in lipoprotein metabolism, VLDL turnover studies were performed, and the hyperlipidemia in the APOC1-transgenic mice was shown to be due to impaired hepatic uptake of VLDL (Jong et al, 1998).
Overexpression of human APOC1 in mice has also been reported to cause elevated levels of free fatty acids (FFAs) in the plasma, severe hepatic insulin resistance, various skin abnormalities, reduced abdominal adipose tissue mass, and a lack of
45 subcutaneous adipose tissue (Jong, et al, 1998; Jong, et al., 1999). These observations suggest that apoC-I may play a role in epidermal lipid synthesis and/or transport and adipose tissue formation.
Apoc1 knock-out mice exhibited normal serum lipid levels on a chow diet; however, these mice developed hyperlipidemia on a high-fat and high-cholesterol diet.
This result was unexpected, since APOC1-transgenic mice were also hyperlipidemic. In vitro binding experiments indicate that apoC1-deficient VLDL is a poor competitor for
LDL binding to the LDL receptor (LDLR), which mediates clearance of circulating lipoproteins by the liver (Jong, et al., 1997; Jong, et al., 1999). This suggests that apoC-I deficiency may impair receptor-mediated hepatic uptake of VLDL.
VLDL turnover studies were performed to measure the effect that apoC-I deficiency has on VLDL-triglyceride (TG) metabolism. VLDL turnover studies indicated that clearance of labeled VLDL-TG was shown to be reduced in the apoC-I- deficient mice. This clearance was attributed to impaired hepatic uptake, since both
VLDL-TG production and LPL-mediated lipolysis were not shown to be significantly affected by a complete lack of apoC-I (Jong, et al., 1997).
A study in baboons with high plasma HDL levels indicated that the transfer of cholesterol esters from HDLs to LDLs and VLDLs was inhibited by a 4-kDa protein that is very similar to the amino-terminal domain of apoC-I. This suggests that apoC-I may interact with CETP, which transfers cholesterol esters between lipoproteins and may play a role in the onset of cardiovascular disease. Additionally, a synthetic peptide comprising
46 the apoC-I amino-terminal domain was shown to inhibit the activity of CETP in vitro
(Jong, et al., 1999).
To investigate the affect that apoC-I may have on CETP in vivo, transgenic mice expressing human CETP were crossed with apoC-I knock-out mice. The activity of
CETP and the depletion of cholesterol ester from HDL were increased approximately 2- fold in the human CETP/apoC-I knock-out mice. The addition of purified apoC-I to plasma from these mice was able to decrease the rate of cholesterol ester transfer in vitro
(Gautier, et al., 2002).
ApoC-I has also been shown to activate the enzyme lecithin-cholesterol acyl transferase (LCAT), which is involved in converting free cholesterol to cholesterol-ester on HDL particles. Apolipoprotein A-I (ApoA-I) is regarded as the most powerful LCAT activator, and apoC-I was able to activate LCAT to approximately 78% of that of apoA-I
(Jong, et al., 1999; Khovidhunkit, et al., 2001).
Very little is known about the regulation of ApoC-I expression. Apoc1 is found on human chromosome 19 and mouse chromosome 7, clustered with Apoe, Apoc2 and
Apoc4. The expression of this gene cluster in the liver is regulated by two homologous hepatic control regions, termed HCR-1 and HCR-2, located approximately 6- and 18-kb downstream of Apoc1, respectively. Either element can control the expression of all four genes in the cluster; however, HCR-1 seems to primarily direct Apoe and Apoc1 expression, while HCR-2 has a major affect on Apoc4 and Apoc2 expression (Zannis et al., 2001).
47 Apoe is known to be expressed in adipocytes, and this was confirmed by our
SAGE findings. Real-time PCR detected the expression of both Apoc1 and Apoe in mature 3T3-L1 cultured adipocytes, mouse epididymal adipose tissue, and human primary adipocytes supporting the SAGE data. Apoc2 and Apoc4 were not expressed in
3T3-L1 cells or adipose tissue.
The expression of Apoc1 and Apoe was monitored during an eight-day 3T3-L1 differentiation time course, and they were both expressed late during the differentiation.
The expression profiles of Apoc1 and Apoe were compared to Pparg and Adn, markers of intermediate and late gene adipocyte gene expression, respectively, and an increase in
Apoc1 mRNA was detected 24 hours later than Adn mRNA, making Apoc1 one of the latest genes expressed during differentiation. Its expression does not increase until after lipid droplets are observed in the cells.
As mentioned earlier, APOC1 and APOE were also expressed in differentiated human adipocytes. Quantitative PCR detected an increase in both APOC1 and APOE mRNA levels late during a 12-day differentiation time course. APOC1 mRNA up- regulation was detected on Day 9, six days after an increase in ADN mRNA. To determine if the observed increased expression of APOC1 led to an increase in apoC-I synthesis, cell supernatants from human preadipocytes and adipocytes on Day 9 of differentiation were screened for apoC-I protein by ELISA. ApoC-I was found in the supernatants for adipocytes; it was not present in the supernatants from preadipocyte cultures.
48 ApoC-I produced by adipocytes may play a role in either the accumulation of
fatty acids or cholesterol by the adipocyte. ApoC-I over-expression may interfere with
LPL catabolism of VLDL, thus preventing fatty acids from being taken up by adipocyte.
This would agree with previous findings that apoC-I affects VLDL clearance and
increases blood FAs.
If apoC-I controls TG accumulation, then transcription factors that control the
synthesis of TG might also control Apoc1 expression. PPARγ is a member of the
hormone nuclear receptor family and is an important adipogenic transcription factor.
PPARγ specifically targets genes involved in adipocyte differentiaion and TG
accumulation, including Adn, Cebpa, and Srb1. PPARγ is required in adipogenesis, and
ectopic expression of PPARγ in fibroblasts, preadipocytes, and myoblasts results in adipogenesis (Lazar, 2005). The activation of PPARγ requires a ligand; ligands of
PPARγ include fatty acids, eicosanoids, and the synthetic, high-affinity thiazolidinethiones (TZDs), such as pioglitazone and rosiglitazone (Li and Glass, 2004).
The expression of the adipogenic transcription factor PPARγ and its target genes
Adn and Cepba were monitored during the cholesterol depletion time course. As expected
Adn increased 4-fold after 24 hours, while neither Pparg nor Cepba expression was
affected. The PPARγ agonists pioglitazone, on the other hand, led to suppression rather
than stimulation of Apoc1 expression; Apoe expression was unaffected by the treatment.
To confirm this observation, a different PPARγ agonist, rosiglitazone was tested with
identical results. This suggests that activation of PPARγ either directly or indirectly
inhibits apoC-I synthesis. It also indicates that Apoc1 expression is controlled
49 independently of Apoe expression. Since PPARγ reaches maximum expression before
Apoc1 is expressed, this may explain in part why Apoc1 is expressed very late during adipocyte differentiation
Cholesterol is an essential membrane component, found in the lipid bilayer of the vertebrate plasma membrane, which is involved in maintaining membrane fluidity, structural integrity of signal transduction complexes, and vesicular trafficking. In adipocytes, free cholesterol is also associated with a phospholipids-protein monolayer that comprises the lipid droplet boundary. This protective boundary surrounding the triacylglycerol-cholesterol ester core, shields the adipocyte from the potentially toxic lipid (Le Lay, et al., 2003).
As cholesterol is an important constituent of the adipocyte lipid droplet, apoC-I may affect cholesterol balance and thus ultimately limit the size of individual adipocytes.
As mentioned earlier, apoC-I affects the activities of LCAT and CETP, proteins that are involved in RCT. Similarly apoE, which is synthesized in adipocytes, functions in cholesterol efflux by mediating the transfer of excess cholesterol from adipocytes to small HDL particles (Mahley and Rall, 2000).
Studies using 3T3-L1 adipocytes have shown that Apoe mRNA levels increase during adipocyte differentiation and that this expression is regulated by cellular cholesterol content. Apoe expression was shown to correlate with the development of lipid droplets within the cells, and when the cells were cultured in the presence of biotin, which inhibits cholesterol and triglyceride accumulation but not differentiation, Apoe mRNA levels were suppressed. Cholesterol-loading of mature 3T3-L1 adipocytes
50 increased both intracellular free cholesterol content and Apoe mRNA expression, but did
not affect triglyceride concentrations (Zechner, et al., 2001). Cellular free cholesterol
content also regulates apoE synthesis in macrophages. Cholesterol loading increased the
production of apoE, while cholesterol depletion using HDL decreased the production of
apoE (Mazzone, et al., 1987).
To determine the effect of cholesterol depletion on Apoc1 expression, 3T3-L1
cells were treated with β-cyclodextrin and HDL during differentiation. Apoc1 mRNA
levels were completely suppressed during cholesterol depletion, suggesting that cellular
cholesterol levels have an affect on Apoc1 gene expression.
The expression of the transcription factors SREBP1-c and SREBP2, which are
regulated by cholesterol, and their target genes, were also monitored during cholesterol
depletion. Cholesterol depletion had no effect on Srebp1-c or its target genes Fas and
Scd1. These results, in addition to the pioglitazone study, suggest that gene expression
associated with TG accumulation is not affected by genes that control cholesterol
accumulation in the adipocyte once late gene expression has been initiated. On the other
hand, the expression of Srebp-2, which is not induced during the differentiation of 3T3-
L1 preadipocytes, increases significantly upon cholesterol depletion. Srebp-2 target genes, Ldl-r and Hmgcr, do as well. This indicates that cellular levels of cholesterol and oxysterols are being reduced during beta-cyclodextrin cholesterol depletion.
Cellular cholesterol content controls the activity of the transcription factor LXR.
LXR forms a heterodimer with RXR, and the transcriptional activity of this complex requires an oxysterol ligand (Zhang and Maseldorf, 2002). During 3T3-L1
51 differentiation with cholesterol depletion, the levels of Lxr mRNA were unaffected; however, mRNA levels of its targets Abca1 and Apoe were suppressed. The cholesterol depletion reduces the availability of oxysterols for LXR, thereby reducing its ability to activate its target genes.
To determine if LXR controls Apoc1 expression during 3T3-L1 differentiation,
3T3 L1 cells were stimulated with LXR agonist T0901317. Messenger RNA levels of Lxr and its target genes Abca1 and Apoe expression levels were all increased, as well as
Apoc1. Apoc1 expression in T0901317-treated cells after five days of treatment was approximately four times greater than in the control cells. Apoc1 expression in cholesterol-depleted 3T3-L1 cells treated with T0901317 was restored to approximately
55% of the control level.
LXR has been shown to regulate the expression of Apoe in both human and mouse macrophages and adipocytes. This is substantiated by the identification of two enhancer regions, termed multi-enhancer 1 (ME.1) and multi-enhancer 2 (ME.2), which are located 3’ of the Apoe and the Apoc1 gene, respectively. ME.1 and ME.2 each contain a single LXR response element (LXRE), which is a specific DNA sequence that is recognized by the LXR/RXR heterodimer. ME.1 and ME.2 are specific for macrophage and adipocyte control of Apoe, and are distinct from the hepatic controlling elements in the Apoe/c2 gene cluster. T0901317 was able to induce Apoe expression in mouse macrophages and adipocytes, but not in monocytes or the liver (Laffitte et al.,
2001). This indicates that LXR controls tissue-specific expression of Apoe through ME.1 and ME.2, and these enhancer regions may also control the expression of Apoc1.
52 On the basis of these findings, we propose a model for the control of apoC-I expression in adipocytes, and how it may regulate adipocyte cholesterol content and size.
Apoc1 expression is initially inhibited by PPARγ but increases late during adipocyte differentiation as the cholesterol and TG content of the adipocyte increases. During cholesterol depletion, genes controlled by LXR are inhibited. This includes genes associated with cholesterol efflux such as Apoe. As TG accumulation of 3T3-L1 adipocytes also seemed to be impaired, this observation suggests that LXR either directly or indirectly regulates genes associated with fatty acid metabolism and/or transport. The
LXR agonist T0901317 was able to enhance the expression of Apoc1 in a manner similar to Apoe. This result, along with previous studies in macrophages, indicates that Apoc1 expression in adipocytes is involved with cholesterol efflux and may be controlled by
LXR.
ApoE is known to play a role in cholesterol efflux from cells, and recent studies suggest that it interacts with the cholesterol transporter ABCA1 in macrophages. The
ATP-binding cassette transporter (ABC) proteins are known for controlling cholesterol efflux in a number of cell types. An ABCA1-ApoA-I pathway for cholesterol efflux is described for the hepatocytes and macrophages, though ApoA-I is not synthesized by adipocytes. ApoE can independently mediate cholesterol efflux from cells, but cholesterol efflux was shown to be higher in macrophages when both ABCA1 and apoE were present, and ABCA1 expression was required for apoE-mediated efflux when endogenous apoE accumulated extracellularly (Huang et al., 2006).
53 ABCG1, another ABC cholesterol transporter, is most effective in promoting cholesterol transfer to large HDL particles, unlike ABCA1 which prefers lipid-poor HDL
(Huang et al, 2006). ABCG1 is known to control lipid homeostasis in hepatocytes and macrophages. We recently discovered that ABCG1 is expressed in 3T3-L1 adipocytes.
We propose that apoC-I may mediate cholesterol efflux and cell size in adipocytes through an interaction with ABCG1. ApoC-I may associate with HDL particles via
ABCG1, and this apoC1-containing HDL is transported across the endothelial layer into the capillary. These HDL particles may also contain apoC-III, and the combination of both apoC-I and apoC-III may result in apoC-III being transferred to an apoC-II- containing TG-rich lipoprotein. ApoC-III is known to inhibit LPL activation by apoC-II, and this interaction would inhibit the lipolysis of TG-rich lipoproteins, resulting in a local inhibition of FA uptake by adipocytes. This proposed function for apoC-I is consistent with previous studies that show that increased plasma FFA levels and lack of adipose tissue are observed in APOC1-transgenic mice. As mentioned above, ANGPTL4 and
ORM1 are also expressed in adipocytes, and these proteins may act in concert with apoC-
I to reduce FA flux to adipocytes by inhibiting LPL function, impairing FA transport across the endothelial layer, and removing LPL from the luminal side of endothelial cells.
Further studies must be conducted to establish an apoC-I-ABCG1 interaction and also the ability of apoC-III to transfer from an apoC-I-containing HDL to a TG-rich lipoprotein.
54 Table 1. Primer sequences for genes studied in quantitative PCR experiments. Primer sets were designed using Primer Express software version 2.0 (Applied Biosystems). The length of the primers was from 17 to 23 nucleotides, the melting temperature was from 59°C to 61°C, the GC content was from 40% to 60%, and the expected PCR products ranged from 50 to 150 base pairs.
55 Table 2. Relative quantitation of gene expression. Quantitative PCR data was analyzed by relative quantitation. Normalized data is expressed as a fold change between a target gene and a reference gene. “X” is the day of differentiation, “Target Gene” is the gene of interest, and “Reference Gene” is an endogenous control gene used to normalize the results, such as TBP.
Calculated Value Formula
∆CT Avg. CT Value for Day (X), Target Gene - Avg. CT Value for Day (X), Reference Gene -∆C Fold Change 2 T
56 Table 3. SAGE analysis of 3T3-L1 preadipocytes and adipocytes. Expressed genes that were detected in SAGE 3T3-L1 preadipocyte and adipocyte libraries were compiled into a database. “UniGene” refers to the number of genes that had mouse UniGene annotations, and “EST” indicates expressed sequence tags with no specific gene annotation.
Number Number Annotation Upregulated Downregulated UniGene 503 536 EST 355 398 None 27 53 Total 885 987
57 Table 4. Cluster analysis of 3T3-L1 preadipocyte and adipocyte SAGE libraries. SAGE gene expression data was analyzed using a cluster analysis technique. Ontology terms were used to classify genes based on cellular function. (A) Displays genes associated with cell structure and the number up-regulated and down-regulated during 3T3-L1 differentiation. (B) Displays metabolic changes that occurred during differentiation and the number of genes up-regulated and the fold change.
A Number Number Gene Function Upregulated Downregulated Cytoskeleton 10 66 Mobility 2 41 ECM 12 39
B Number Gene Function Upregulated Fold Change Glycolysis 6 3-5 TCA 8 8-12 Oxidative Phosphorylation 54 3-6 Fatty Acid Synthesis 11 3-6 Amino Acid Catabolism 7 4-8 Glutathione Metabolism 4 10-14 Transport Class I 14 3-5 Class II 2 10, 14 Stress Response Anti-oxidants 2 8, 12 Apoptosis 5 3-5
58 Table 5. SAGE identification of genes that encode for secreted proteins. SAGE analysis of 3T3-L1 preadipocytes and adipocyte detected the up-regulation and down- regulation of genes that encode for secreted proteins. “Known” indicates genes that have been previously identified, and “New” indicates genes that were first identified in these libraries, including Apoc1.
Number Number Classification Upregulated Downregulated Secreted 15 15 Known 10 4 New 5 11
59 Table 6. SAGE identification of secreted proteins that function in lipid metabolism. SAGE analysis of 3T3-L1 preadipocytes and adipocytes detected five genes that encode for secreted proteins which function in lipid metabolism. “Tags” indicates the number of times the particular mRNA was detected in our database.
Adipocyte Preadipocyte Gene Tags Tags Apoc1 8 1 Apoe 3 0 Apod 3 0 Cd36 5 0 Srb1 19 0
60 Table 7. Production of apolipoprotein C-I by cultured human adipocytes. Cell supernatants were harvested from human preadipocytes and adipocytes after 48 hours of incubation at 37°C in DMEM with and without 10% fetal bovine serum. ELISA assays were performed using a polyclonal goat anti-human apoC-I antibody. Data are represented as mean +/- SD. “ND” indicates non-detectable levels of protein.
ApoC-I Cell Amount of CV Concentration Supernatant OD Value ApoC-I (ng) (%) (ng/mL) Adipocyte with serum 1.066 +/- 0.012 0.510 +/- 0.012 2.43 10.196 serum-free 0.662 +/- 0.042 0.226 +/- 0.019 8.56 4.516 Preadipocyte with serum 0.152 +/- 0.026 ND N/A ND serum-free 0.124 +/- 0.007 ND N/A ND
61 Table 8. Viability test results for 3T3-L1 differentiation in the presence of βββ- cyclodextrin and HDL. 3T3-L1 cells were cultured with varying concentrations of β- cyclodextrin and HDL. Calculated concentrations and percentages of viable 3T3-L1 cells were determined by hemocytometer counts of trypan blue stained cells on Day 8 of 3T3- L1 differentiation.
Cells Recovered Viable Cells Culture Condition (Cells/mL) (Cells/mL) % Viable Non-differentiated 3.77E+05 3.74E+05 99.27 Differentiation Control 2.50E+05 2.48E+05 98.90 0.75 mM β-CD 0 0 0 1.0 mM β-CD 0 0 0 1 mg/mL HDL Only 2.50E+05 2.48E+05 98.90 0.65 mg/mL HDL Only 7.37E+05 7.04E+05 95.52 1.0 mM β−CD + 1 mg/mL HDL 8.33E+05 8.20E+05 98.35 1.0 mM β−CD + 0.65 mg/mL HDL 3.66E+05 3.41E+05 93.23 0.75 mM β−CD + 1 mg/mL HDL 1.14E+05 1.13E+05 99.27 0.75 mM β−CD + 0.65 mg/mL HDL 3.19E+05 3.11E+05 97.41
62 Table 9. Viability test results for 3T3-L1 differentiation in the presence of PPARγγγ and LXR agonists. 3T3-L1 cells were cultured with the PPARγ agonists pioglitazone and rosiglitazone and the LXR agonist T0901317. Calculated concentrations and percentages of viable 3T3-L1 cells were determined by hemocytometer counts of trypan blue stained cells on Day 8 of 3T3-L1 differentiation.
Cells Recovered Viable Cells Recovered Culture Condition (Cells/mL) (Cells/mL) % Viable Non-differentiated 3.77E+05 3.74E+05 99.27 Differentiation Control 2.50E+05 2.48E+05 98.90 10 µM Pioglitazone 8.13E+05 7.25E+05 89.17 1 µM Rosiglitazone 1.60E+05 1.44E+05 90.00 3 µM T0901317 1.12E+05 1.04E+05 92.86
63 Figure 1. Phenotypic and genotypic events during 3T3-L1 differentiation. (A) Time line of physiological and transcriptional events during 3T3-L1 differentiation. (B) Roles of key early (italicized) and intermediate (bold) transcription factors in the 3T3-L1 differentiation program.
64 A
B
Figure 2. Light microscope images of 3T3-L1 preadipocytes and differentiated adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes using a hormone cocktail containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. (A) Preadipocytes are fibroblast-like and (B) adipocytes are cuboidal in shape and contain lipid droplets. Images were captured using phase-contrast microscopy at 200X magnification.
65 30 Tbp 29 Hprt1
28
27
26 e u l
a 25 V t C 24
23
22
21
20 012345678 Day
Figure 3. mRNA expression of the reference genes Tbp and Hprt1 during 3T3-L1 differentiation. Quantitative PCR was performed using RNA isolated during a 3T3-L1 differentiation time course on days 0, 1, 2, 3, 4, 5, 6, and 8. The CT value indicates the cycle number at which the fluorescence generated in the reactions crosses a threshold line. Data are represented as mean +/- SD.
66 60 Preadipocyte Adipocyte
50 l e
v 40 e L A N
R 30 m e v i t a l
e 20 R
10
* 0 Apoe Apoc1 Gene
Figure 4. Apolipoprotein E/C1/C4/C2 linkage group expression in 3T3-L1 preadipocytes and adipocytes. Quantitative PCR was performed using RNA isolated from 3T3-L1 preadipocytes and differentiated adipocytes. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD. An (*) indicates non-detectable levels of mRNA.
67 18 Pparg
16
14 l e
v 12 e L
A 10 N R m
e 8 v i t a l
e 6 R
4
2
0 0 12345678 Day
160 Adn
140
120 l e v e 100 L A N
R 80 m e v i t
a 60 l e R 40
20
0 012345678 Day
Figure 5. mRNA expression time course of adipocyte marker genes during 3T3-L1 differentiation. mRNA expression levels of Pparg (top) and Adn (bottom) during 3T3- L1 differentiation. Quantitative PCR was performed using RNA isolated during a 3T3- L1 differentiation time course on days 0, 1, 2, 3, 4, 5, 6, and 8. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
68 20 Apoe 18
16
l 14 e v e
L 12 A N
R 10 m e v i 8 t a l e
R 6
4
2
0 012345678 Day
60 Apoc1
50 l e
v 40 e L A N
R 30 m e v i t a l
e 20 R
10
0 012345678 Day
Figure 6. mRNA expression time course of apolipoprotein genes during 3T3-L1 differentiation. mRNA expression levels of Apoe and Apoc1 during 3T3-L1 differentiation. Quantitative PCR was performed using RNA isolated during a 3T3-L1 differentiation time course on days 0, 3, 4, 5, and 8. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
69 10000 Adipose Liver
1000 l e v e L A N
R 100 m e v i t a l e R 10
* * * 1 Adn Pparg Apoe Apoc1 Apoc2 Gene
Figure 7. Apolipoprotein and adipocyte marker gene expression in murine liver and adipose tissue. Quantitative PCR was performed using RNA isolated from BALB/c livers and epididymal fat pads. mRNA levels were normalized relative to the reference gene Tbp. An (*) indicates non-detectable levels of mRNA. Data are represented as mean +/- SD.
70 A
B
Figure 8. Light microscope images of human preadipocytes and differentiated adipocytes in culture. Human (A) preadipocytes and (B) adipocytes were obtained from Zen-Bio, Inc. Preadipocytes were isolated from subcutaneous adipose tissue and differentiated into adipocytes. Images were captured using phase-contrast microscopy at 200X magnification.
71 90 ADN 80
l 70 e v
e 60 L A
N 50 R
m 40 e v i t
a 30 l e
R 20
10
0 024681012 Day
35 APOE 30 l e
v 25 e L
A 20 N R m
e 15 v i t a l
e 10 R
5
0 024681012 Day
4 APOC1 l
e 3 v e L A N
R 2 m e v i t a l e
R 1
0 024681012 Day
Figure 9. mRNA expression profiles for ADN, APOE, and APOC1 in cultured human adipocytes. Quantitative PCR was performed on RNA isolated from human adipocytes on days 0, 3, 6, 9, and 12 of a differentiation time course. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
72 ApoC-I apoC-1 1.4
1.2 ) m
n 1.0 y=0.872x+0.866
0 r2=0.983 5 4
( 0.8 e c
n 0.6 a b r o
s 0.4 b A 0.2
0.0 0.01 0.1 1 10 ng
Plasma 1.6
) 1.4 m n 1.2 0 5 4
( 1.0 e c
n 0.8 a b
r y=0.859x+4.54
o 0.6
s r2 = 0.99 b
A 0.4
0.2 1e-5 1e-4 1e-3 Dilution
Figure 10. Apolipoprotein C-I ELISA standard and calibrator curves. Dilutions of purified apolipoprotein C-I and human plasma were prepared in ELISA Diluent. ApoC-I levels were measured by using a polyclonal goat anti-human apoC-I antibody and absorbance readings were taken at 450 nm. Data are represented as mean +/- SD.
73 A
B
C
Figure 11. Oil red-O staining of 3T3-L1 preadipocytes and adipocytes. Triglyceride accumulation during 3T3-L1 differentiation was monitored by staining with the lipid- soluble dye oil red-O. Images were captured using phase-contrast microscopy at 200X magnification. (A) Non-differentiated 3T3-L1 preadipocyte cells. (B) Mature 3T3-L1 adipocytes on day 8 of differentiation. (C) Cyclodextrin-treated 3T3-L1 adipocytes on day 8 of differentiation.
74 60 Control CD/HDL 50 l e
v 40 e L A N
R 30 m e v i t a l
e 20 R
10
0 012345678 Day
Figure 12. Apolipoprotein C1 mRNA expression during 3T3-L1 differentiation in the presence of βββ-cyclodextrin and HDL. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 3, 4, 5, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of β-cyclodextrin and HDL (CD/HDL). mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
75 Pparg 35 Control CD/HDL 30 l e
v 25 e L
A 20 N R m
e 15 v i t a l
e 10 R
5
0 048 Day
Adn 400 Control 350 CD/HDL l
e 300 v e L 250 A N
R 200 m e v
i 150 t a l e
R 100
50 * 0 048 Day
Cebpa 18 Control 16 CD/HDL
l 14 e v e 12 L A
N 10 R
m 8 e v i t
a 6 l e
R 4
2
0 048 Day
Figure 13. mRNA expression of PParg, Adn, and Cebpa during 3T3-L1 differentiation in the presence of βββ-cyclodextrin and HDL. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of β-cyclodextrin and HDL (CD/HDL). mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
76 Pparg 35 Control Pioglitazone 30 l e
v 25 e L
A 20 N R m
e 15 v i t a l
e 10 R
5
0 048 Day
Adn 400 Control 350 Pioglitazone
l 300 e v e
L 250 A N
R 200 m e v i
t 150 a l e
R 100
50 * 0 048 Day
Cebpa 25 Control Pioglitazone 20 l e v e L
A 15 N R m e
v 10 i t a l e R 5
0 048 Day
Figure 14. mRNA expression of Pparg, Adn, and Cebpa during 3T3-L1 differentiation in the presence of pioglitazone. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of pioglitazone. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD. An (*) indicates non-detectable levels of mRNA.
77 60 Control Pioglitazone 50 l e
v 40 e L A N
R 30 m e v i t a l 20 e R
10
0 012345678 Day
Figure 15. Apolipoprotein C1 mRNA expression during 3T3-L1 differentiation in the presence of pioglitazone. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 3, 4, 5, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of pioglitazone. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
78 2.5 Control Pioglitazone
2.0 l e v e L 1.5 A N R m e
v 1.0 i t a l e R 0.5
0.0 012345678 Day
Figure 16. Apolipoprotein E mRNA expression during 3T3-L1 differentiation in the presence of pioglitazone. Quantitative PCR was performed on RNA isolated from 3T3- L1 adipocytes on days 0, 3, 4, 5, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of pioglitazone. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
79 Apoc1 80 Control 70 Rosiglitazone l
e 60 v e L 50 A N
R 40 m e v
i 30 t a l e 20 R
10
0 048 Day
Apoe 16 Control 14 Rosiglitazone l
e 12 v e L 10 A N
R 8 m e v
i 6 t a l e 4 R
2
0 048 Day
Figure 17. Apolipoprotein C1 and apolipoprotein E mRNA expression during 3T3- L1 differentiation in the presence of rosiglitazone. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of rosiglitazone. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
80 Srebp1-c 8 Control 7 CD/HDL l
e 6 v e L 5 A N
R 4 m e v
i 3 t a l e
R 2
1
0 048 Day
Fas 5 Control CD/HDL 4 l e v e L
A 3 N R m e
v 2 i t a l e R 1
0 048 Day
Scd1 350 Control CD/HDL 300 l e
v 250 e L
A 200 N R m
e 150 v i t a l
e 100 R
50
0 048 Day
Figure 18. mRNA expression of Srebp1-c, Fas, and Scd1 during 3T3-L1 differentiation in the presence of βββ-cyclodextrin and HDL. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of β-cyclodextrin and HDL (CD/HDL). mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
81 Srebp2 16 Control 14 CD/HDL l
e 12 v e L 10 A N
R 8 m e v i 6 t a l e
R 4
2
0 048 Day
Ldl-r 4.5 Control 4.0 CD/HDL
l 3.5 e v
e 3.0 L A
N 2.5 R
m 2.0 e v i t
a 1.5 l e
R 1.0
0.5
0.0 048 Day
Hmgcr 25 Control CD/HDL 20 l e v e L
A 15 N R m e
v 10 i t a l e R 5
0 048 Day
Figure 19. mRNA expression of Srebp2, Ldl-r, and Hmgcr during 3T3-L1 differentiation in the presence of βββ-cyclodextrin and HDL. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of β-cyclodextrin and HDL (CD/HDL). mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
82 Lxr 5 Control CD/HDL 4 l e v e L 3 A N R m
e 2 v i t a l e
R 1
0 048 Day
Abca1 14 Control 12 CD/HDL l e
v 10 e L
A 8 N R m
e 6 v i t a l
e 4 R
2
0 048 Day
Apoe 18 Control 16 CD/HDL
l 14 e v e 12 L A
N 10 R
m 8 e v i t 6 a l e
R 4
2
0 048 Day
Figure 20. mRNA expression of Lxr, Abca1, and Apoe during 3T3-L1 differentiation in the presence of βββ-cyclodextrin and HDL. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of β-cyclodextrin and HDL (CD/HDL). mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
83 Lxr 25 Control T0901317 20 l e v e L
A 15 N R m e
v 10 i t a l e R 5
0 048 Day
Abca1 90 Control 80 T0901317
l 70 e v
e 60 L A
N 50 R
m 40 e v i t
a 30 l e
R 20
10
0 048 Day
Apoe 90 Control 80 T0901317
l 70 e v e 60 L A
N 50 R
m 40 e v i t
a 30 l e
R 20
10
0 048 Day
Figure 21. mRNA expression of Lxr, Abca1, and Apoe during 3T3-L1 differentiation in the presence of T0901317. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of T0901317. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
84 300 Control T0901317 250 l e v
e 200 L A N
R 150 m e v i t a l 100 e R
50
0 048 Day
Figure 22. Apolipoprotein C1 mRNA expression during 3T3-L1 differentiation in the presence of T0901317. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control) and in the presence of T0901317. mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
85 80 Control CD/HDL 70 CD/HDL + T0901317
60 l e v e
L 50 A N
R 40 m e v i
t 30 a l e R 20
10
0 048 Day
Figure 23. Apolipoprotein C1 mRNA expression during 3T3-L1 cholesterol depletion in the presence of T0901317. Quantitative PCR was performed on RNA isolated from 3T3-L1 adipocytes on days 0, 4, and 8 of a differentiation time course, under standard differentiation conditions (Control), cholesterol depletion (CD/HDL), and cholesterol depletion in the presence of T0901317 (CD/HDL + T0901317). mRNA levels were normalized relative to the reference gene Tbp. Data are represented as mean +/- SD.
86 REFERENCES
Ahima, R. S. 2005. The central actions of adipose tissue. Trends in Endocrinology and
Metabolism 16:307-313.
Ailhuad, G., P. Grimaldi, and R. Negrel. 1992. Cellular and molecular aspects of
adipose tissue development. Annual Review of Nutrition 12:207-233.
Atger, V. M., M. de la Llera Moya, G. W. Stoudt, W. V. Rodrigueza, M. C. Phillips,
and G. H. Rothblat. 1997. Cyclodextrins as catalysts for the removal of cholesterol from
macrophage foam cells. Journal of Clinical Investigation 99:773-780.
Berg, S. H. and P. E. Scherer. 2005. Adipose tissue, inflammation, and cardiovascular
disease. Circulation Research 96:939-949.
Bogacka, I., H. Xie, G. A. Bray, and S. R. Smith. 2004. The effect of pioglitazone on peroxisome proliferators-activated receptor-g target genes related to lipid storage in vivo.
Diabetes Care 27: 1660-1667.
Brown, M. S. and J. L. Goldstein. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331-340.
Bruce, C., R A. Chouinard, Jr., and A. R. Tall. 1998. Plasma lipid transfer proteins, high-density lipoproteins, and reverse cholesterol transport. Annual Reviews in Nutrition
18:297-330.
87 Christian, A. E., H. S. Byun, N. Zhong, M. Wanunu, T. Marti, A. Furer, F.
Diederich, R. Bittman, G. H. Rothblat. 1999. Comparison of the capacity of beta-
cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum
lipoproteins. Journal of Lipid Research 40:1475-1482.
Coppak, S. W. 2005. Adipose tissue changes in obesity. Biochemical Society
Transactions 33:1049-1052. de Souza, C. J., M. Eckhardt, K. Gagen, M. Dong, W. Chen, D. Laurent, and B. F.
Burkey. 2001. Effects of pioglitazone on adipose tissue remodeling within the setting of
obesity and insulin resistance. Diabetes 50:1863-1871.
Fernandez-Real, J. M., and W. Ricart. 2003. Insulin resistance and chronic
cardiovascular inflammatory syndrome. Endocrine Reviews 24:278-301.
Fruhbeck, G., J. Gomez-Ambrosi, F. J. Murazabal, and M. A. Burrel. 2001. The
adipocyte: a model for integration of endocrine and metabolic signaling in energy
metabolism regulation. The American Journal of Physiology – Endocrinology and
Metabolism 280:E827-E847.
Gautier, T., D. Masson, M. C. Jong., L. Duverneuil, N. Le Guern, V. Deckert, J. P.
Pais de Barros, L. Dumont, A. Bataille, Z. Zak, X. C. Jiang, A. R. Tall, L. M.
Havekes, and L. Lagrost. 2002. Apolipoprotein CI deficiency markedly augments
plasma lipoprotein changes mediated by human cholesteryl ester transfer protein (CETP)
in CETP transgenic/ApoCI-knocked out mice. The Journal of Biological Chemistry
277:31354-31363.
Haslam, D. W. and W. P. James. 2005. Obesity. Lancet 366: 1197-1209.
88 Hauner, H. 2004. The new concept of adipose tissue function. Physiology and Behavior
83:653-658.
Hochepied, T., F. G. Berger, H. Baumann, and C. Libert. 2003. a1-acid glycoprotein:
an acute phase protein with inflammatory and immunomodulating properties. Cytokine
and Growth Factor Reviews 14:25-34.
Hoffer, M. J., M. M. van Eck, L. M. Havekes, M. H. Hofker, and R. R. Frants. 1993.
The mouse apolipoprotein C-I gene: structure and expression. Genomics 18:37-42.
Huang, Z. H., M. L. Fitzgerald, and T. Mazzone. 2006. Distinct cellular loci for the
ABCA1-dependent and ABCA-1 independent lipid efflux mediated by endogenous
apolipoprotein E expression. Arteriosclerosis, Thrombosis, and Vascular Biology 26:157-
162.
Jin, W., D. Marchadier, and D. J. Rader. 2002. Lipases and HDL metabolism. Trends
in Endocrinology and Metabolism 13:174-178.
Jong, M. C., J. H. van Ree, V. E. H. Dahlmans, R. R. Frants, M. H. Hofker, and L.
M. Havekes. 1997. Reduced very-low-density lipoprotein fractional catabolic rate in apolipoprotein C1-deficient mice. Biochemical Journal 321:445-450.
Jong, M. C., M. H. Hofker, and L. M. Havekes. 1999. Role of apoCs in lipoprotein metabolism. Arteriosclerosis, Thrombosis and Vascular Biology 19:472-484.
Jong, M. C., M. J. J. Gijbels, V. E. H. Dahlams, P. J. J. van Gorp, S. J. Koopman,
M. Ponec, M. H. Hofker, and L. M. Havekes. 1998. Hyperlipidemia and cutaneous abnormalities in transgenic mice overexpressing human apolipoprotein C1. Journal of
Clinical Investigation 101:145-152.
89 Jove, M., A. Planavila, A. Cabrero, F. Novell, E. Ros, D. Zambon, J. C. Laguna, and
M. V. Carrera. 2003. Reductions in plasma cholesterol levels after fenofibrate treatment
are negatively correlated with resistin expression in human adipose tissue. Metabolism
52:351-355.
Juge-Aubry, C. E., E. Henrichot, and C. A. Meier. 2005. Adipose tissue: a regulator of
inflammation. Best Practice and Research – Clinical Endocrinology and Metabolism
19:547-566.
Khovidhunkit, W., J. K. Shigenaga, A. H. Moser, K. R. Feingold, and C. Grunfeld.
2001. Cholesterol efflux by acute-phase high density lipoprotein: role of lecithin:cholesterol acyltransferase. Journal of Lipid Research 42: 967-975.
Kilsdonk, E. P., P. G. Yancey, G. W. Stoudt, F. W. Bangerter, W. J. Johnson, M. C.
Phillips, and G. H. Rothblat. 1995. Cholesterol efflux mediated by cyclodextrins.
Journal of Biological Chemistry 29:17250-17260.
Kim, B., H. Choo, J. Lee, J. Kim, and Y. Ko. 2004. Extracellular ATP is generated by
ATP synthase complex in adipocyte lipid rafts. Experimental and Molecular Medicine
36:476-485.
Koerner, A., J. Kratzsh, and W. Kiess. 2005. Adipocytokines: leptin – the classical,
resistin – the controversial, adiponectin – the promising, and more to come. Best Practice
and Research – Clinical Endocrinology and Metabolism 19:525-546.
90 Kratchmarova, I., D. E. Kalume, B. Blagoev, P. E. Scherer, A. V. Podtelejnikov, H.
Molina, P. E. Bickel, J. S. Andersen, M. M. Fernandez, J. Bunkenborg, P.
Roepstorff, K. Kristiansen, H. F. Lodish, M. Mann, and A. Pandey. 2002. A proteomic approach for identification of secreted proteins during the differentiation of
3T3-L1 preadipocytes to adipocytes. Molecular Cell Proteomics 1:213-222.
Kurash, J. K., C. Shen, and D. Tosh. 2004. Induction and regulation of acute-phase proteins in transdifferentiated hepatocytes. Experimental Cell Research 292:342-358.
Lafitte, B. A., J. J. Repa, S. B. Joseph, D. C. Wilpitz, H. R. Kast, D. J. Mangelsdorf, and P. Tontonoz. 2001. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proceedings of the National Academy of Sciences
98:507-512.
Lane, M. D. and Q. Tang. 2005. From multipotent stem cell to adipocyte. Birth Defects
Research 73:476-477.
Lazar, M. A. 2005. PPARg, 10 years later. Biochimie 87:9-13.
Le Lay, S., C. Robichon, X. Le Liepvre, G. Dagher, P. Ferre, and I. Dugail. 2003.
Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of
3T3-L1 cells. Journal of Lipid Research 44:1499-1507.
Le Lay, S., S. Krief, C. Farnier, I. Lefrere, X. Le Liepvre, R. Bazin, P. Ferre, and I.
Dugail. 2001. Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes. The Journal of Biological Chemistry 276:16904-
16910.
91 Li, A. C., and C. K. Glass. 2004. PPAR- and LXR-dependent pathways controlling lipid metabolism and the development of atherosclerosis. Journal of Lipid Research 45:2161-
2173.
Lin, Y., M. W. Rajala, J. P. Berger, D. E. Moller, N. Barzilai, and P. E. Scherer.
2001. Hyperglycemia-induced production of acute phase reactants in adipose tissue. The
Journal of Biological Chemistry 276:42077-42083.
Mahley, R. W. and S. C. Rall, Jr. 2000. Apolipoprotein E: far more than a lipid transport protein. Annual Reviews in Genomics and Human Genetics 1:507-537.
Mak, P. A., B. A. Lafitte, C. Desrumaux, S. B. Joseph, L. K. Curtiss, D. J.
Mangelsdorf, P. Totonoz, and P. A. Edwards. 2002. Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. The
Journal of Biological Chemistry 277:31900-31908.
Mazzone, T., H. Gump, P. Diller, and G. S. Getz. 1987. Macrophage free cholesterol content regulates apolipoprotein E synthesis. The Journal of Biological Chemistry
262:11657-11662.
Musil, K. J., A. Malmstrom, and J. Donner. 1991. Alteration of proteoglycan metabolism during the differentiation of 3T3-L1 fibroblasts into adipocytes. Journal of
Cell Biology 114:821-826.
Neels, J. G., T. Thinnes, and D. J. Loskutoff. 2004. Angiogenesis in an in vivo model of adipose tissue development. FASEB Journal 18:983-985.
Ntambi, J. M. and Y. Kim. 2000. Adipocyte differentiation and gene expression.
Journal of Nutrition 130:3122S-3126S.
92 Orzano, J. A. and J. G. Scott. 2005. Diagnosis and treatment of obesity in adults: an applied evidence-based review. The Journal of the American Board of Family Practice
17:359-369.
Rajala, M. W. and P. E. Scherer. 2003. Minireview: The adipocyte – at the crossroads of energy homeostasis, inflammation, and atherosclerosis. Endocrinology 144:3765-
3773.
Repa, J. J., S. D. Turley, J. A. Lobaccaro, J. Medina, L. Li, K. Lustig, B. Shan, R. A.
Heyman, J. M. Dietschy, and D. J. Mangelsdorf. 2000. Regulation of absorption and
ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 289:1524-1529.
Rothblat, G. H., M. de la Llera-Moya, V. Atger, G. Kellner-Weibel, D. L. Williams, and M. C. Phillips. 1999. Cell cholesterol efflux: integration of old and new observations provides new insights. Journal of Lipid Research 40:781-796.
Seo, J. B., H. M. Moon, W. S. Kim, Y. S. Lee, H. W. Jeong, E. J. Yoo, J. Ham, H.
Kang, M. G. Park, K. R. Steffensen, T. M. Stulnig, J. A. Gustafsson, S. D. Park, and
J. B. Kim. 2004. Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression. Molecular
Cell Biology 24:3430-3444.
Shimizu, T. 2005. Role of macrophage inhibitory factor (MIF) in the skin. Journal of
Dermatological Science 37:65-73.
Sorensson, J., J. L. Matejka, M. Ohlson, and B. Haraldsson. 1999. Human endothelial cells produce orosomucoid, an important component of the capillary barrier. American
Journal of Physiology 276:H530-H534.
93 Taylor, S. M. and P. A. Jones. 1979. Multiple new phenotypes induced in 10T1/2 and
3T3 cells treated with 5-azacytidine. Cell 4:771-779.
Trayhurn, P. 2005. Endocrine and signaling role of adipose tissue: newe perspectives on
fat. Acta Physiologica Scandinavica 184:285-293.
Vassiliou, G., and R. McPherson. 2004. Role of cholesterol ester transfer protein in
selective uptake of high density lipoprotein cholesterol esters by adipocytes. Journal of
Lipid Research 45:1683-1693.
Weber, L. W., M. Boll, and A. Stampfl. 2004. Maintaining cholesterol homeostasis: sterol regulatory element-binding proteins. World Journal of Gastroenterology 10:3081-
3087.
Yoshida, K., T. Shimizugawa, M. Ono, and H. Furukawa. 2002. Angiopoietin-like 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase.
Journal of Lipid Research 43:1770-1772.
Yu, Y., and H. N. Ginsberg. 2005. Adipocyte signaling and lipid homeostasis: sequelae of insulin-resistant adipose tissue. Circulation Research 96:1042-1052.
Zannis, V. I., H. Y. Kan, A. Kritis, E. Zanni, and D. Kardassis. 2001. Transcriptional regulation of the human apolipoprotein genes. Frontiers in Bioscience 6:D456-D504.
Zechner, R., R. Moser, T. C. Newman, S. K. Fried, and J. L. Breslow. 1991.
Apolipoprotein E gene expression in mouse 3T3-L1 adipocytes and human adipose tissue and its regulation by differentiation and lipid content. The Journal of Biological
Chemistry 266:10583-10588.
94 Zhang, X., L. Ding, and A. J. Sandford. 2005. Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Molecular Biology 6:4.
Zhang, Y. and D. J. Mangelsdorf. 2002. LuXuRies of lipid homeostasis: the unity of nuclear hormone receptors, transcription regulation, and cholesterol sensing. Molecular
Interventions 2:78-87.
Zhao, Y., D. D. Feng, and C. Chen. 2005. Contribution of adipocyte-derived factors to beta-cell dysfunction in diabetes. The International Journal of Biochemistry and Cell
Biology (In Press).
95