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Adipogenesis: Usefulness of in vitro and in vivo experimental models^1,2

Department of Animal Sciences, University of Illinois, Urbana 61801

	Abstract

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The use of experimental models is the foundation of experimental biology, so it is important to know how much the models can tell us about actual animals. Inconsistent or contradictory results from in vitro models are often associated with the perception that a particular model or results are somehow wrong and therefore cannot tell us anything important about how an animal works. In fact, in vitro conditions do not create new biology. Differences between in vitro and in vivo behavior can only result from the actual cellular repertoire, which provides a powerful tool to uncover new information. Adipose tissue research provides a useful context for examining this issue because the regulation of adipose growth and metabolism has important economic implications for livestock production. Examples are discussed in which either excess skepticism or narrow interpretation of results slowed progress toward our current understanding of adipose biology. Similarly, contemporary examples using genomics are used to suggest that large inconsistencies are still apparent with in vitro methods. Careful consideration of these inconsistencies may provide new insights.

Key Words: Gene Expression • Disheveled • Growth Hormone • Preadipocyte • Sprouty

	Introduction

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As animal scientists we are interested in adipose tissue for two reasons. We want to raise animals with the lowest amount of adipose tissue necessary because the additional feed required for lipid synthesis compared with muscle increases production costs and because fat deters health-conscious consumers. Conversely, we want high amounts of intramuscular adipose tissue because it is associated with meat quality and thus makes animals more valuable. Therefore, understanding adipogenesis and lipid metabolism in order to improve animal production has long been a major goal of animal scientists. In vitro models form part of the foundation of experimental biology and are widely used to study adipogenesis and adipose tissue. When using in vitro models or methods, it is reasonable and critical to ask how much the model can tell about an actual animal. Unfortunately, a comparison of in vitro and in vivo effects is often associated with the perception that a particular model or results are somehow wrong and therefore cannot tell us anything important about how an animal works. In fact, no in vitro condition or any mutation necessary to create an immortalized cell line creates new biology or new pathways. Any change is part of the actual cellular repertoire and divergence between in vitro and in vivo behavior provide powerful ways of uncovering new information. Careful consideration of inconsistencies is likely to provide new insights, whereas an unconsidered disregard of unusual model behavior is likely to represent a missed opportunity. Adipose research provides a useful context for examining issues surrounding this problem because of the important implications that adipose growth and lipid metabolism have for both livestock and humans.

	In Vitro and In Vivo Adipogenesis

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Adipose Tissue

The formation of adipose cells from mesenchymal tissue begins in mid to late gestation as indicated by the morphology of human, cattle, mouse, pig, and rat embryos. The timing of adipose development depends on the species as well as on the adipose depot (Mersmann et al., 1975; Hausman et al., 1980; Hood, 1982; Martin et al., 1998). White adipose tissue expansion takes place rapidly after birth as a result of increased size of existing fat cells and proliferation of preadipocyte (precursor) cells. New fat cells continue to be generated throughout life (Anderson and Kauffman, 1973) particularly after feeding high-carbohydrate or high-fat diets (Faust et al., 1978, 1984; Miller et al., 1984), so understanding the regulation of preadipocyte proliferation is important. In vitro experiments correspond well with in vivo observations on adipose expansion because precursors that differentiate into mature adipocytes can be isolated from various adipose depots of older individuals from several species (Bjorntorp et al., 1982; Kirkland et al., 1990; Hausman et al., 1992). Newly differentiated fat cells are predisposed to fill with lipid and differences between newly differentiated and preceding cells, as indicated by variation in adipose cell size, appear to dissipate relatively quickly (Etherton and Allen, 1980; Whitehurst et al., 1981). In postnatal animals, adipose tissue is not only an important tissue of energy homeostatsis but also an endocrine organ of diverse function (see articles by Hausman and Richardson, 2004; Miner, 2004).

In Vitro Models of Adipogenesis

Traditional in vitro methods include incubations of explant pieces, slices or cells, tissue culture of explants or slices, primary cell culture, and established cell line culture. The common denominator distinguishing these methods from an analytical technique applied to ex vivo samples is that ongoing biological processes are being examined or followed. From this perspective, many genomic technologies are in vitro methods. Transcription in a bullet tube or in a cell culture clearly shares similar degrees of separation from an intact animal.

Explants and slices are straightforward techniques. A slice or small piece of tissue is placed in nutrient media or salt solution, and measurements made after some period of time. Long-term adipose explant cultures have been used as an in vitro model for nearly 100 yr (Foot, 1912), with success characterized by an increase in cell size or outgrowth of proliferating cells. More recently, short-term explants have been used extensively to characterize adipose metabolic regulation, and a considerable body of literature has developed about the technology for slice and isolated cell preparations (Etherton and Chung, 1981; Rule et al., 1988; Mills, 1999). Explants are generally thought to be quite representative of in vivo adipose function because the cells and the connective tissue are intact. However, to obtain consistent results, explant slices must be washed and preincubated with reasonable care. The length of time over which an explant can be incubated before one must be concerned about changes in function is a frequent question. With modern knowledge of how hypoxia, nutrient deprivation, and growth factor deprivation cause apoptosis (Riva et al., 1998; Moley and Mueckler, 2000), it is recognized that preparations incubated longer than a few hours will start to experience irreversible damage unless the tissue is maintained in enriched media. However, this does not mean that explants are stable for long periods even under culture conditions.

An obvious experiment to shed light on the differences between adipose function in vivo and in vitro is simply a direct comparison. However, comparing the differences is no small task because adipose tissue expresses more than 8,000 genes, including more than 120 receptors and 80 secreted proteins and hormones (Yang et al., 2003). It would be surprising if the disruption or tissue trauma associated with in vitro preparation did not result in numerous changes. Adipocytes from newly cultured explants of human subcutaneous adipose tissue rapidly express tumor necrosis factor- (TNF-) and hypoxia-induced factor-1 and downregulate adipocyte-specific proteins, such as hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferator-activated receptor-2 (PPAR), indicating a catabolic response even after the relatively simple and gentle preparation used for explant preparation (Gesta et al., 2003). Differentiation of preadipocyte cell lines in culture alone is associated with large changes in the expression of many hundreds of genes (Guo and Liao, 2000; Gerhold et al., 2002; Jessen and Stevens, 2002). Given the importance and complexity of adipose function as well as the known differences between species, it should not be surprising when variations between adipose function in vivo and in vitro are observed.

Primary Cell Cultures and Cell Lines

Much of the progress in understanding adipocyte biology has depended on experiments using immortal cell lines. The most frequently used preadipocyte cell lines are 3T3-L1 and 3T3-F442A cells. These lines were cloned from heterogeneous Swiss 3T3 cells that had been derived from dissociated near-term mouse embryos (Green and Meuth 1974; Green and Kehinde, 1975, 1976). Less commonly used, the Ob17 cell line was derived from epididymal adipose tissue of adult ob/ob obese mice (Negrel et al., 1978). Once differentiated in vitro, these cell lines have many characteristics of adipose cells in vivo.

At the most fundamental level, differentiation of preadipocytes into adipose cells is defined by the acquisition of a lipid-filled morphology and appropriate hormone responsiveness, signaling pathways and metabolism. Cultured preadipocytes begin to differentiate shortly after the cells become confluent and proliferation rate declines. Key regulatory events of differentiation include the induction of CCAAT/enhancer binding protein-ß (C/EBPß) and C/EBP followed by induction of PPAR and C/EBP, which upregulate adipose functional genes (for reviews, see Hausman et al., 2001; MacDougald and Mandrup, 2002). Differentiation of primary cultures and cell lines is commonly enhanced by treating postconfluent cells with a differentiation cocktail, a combination of insulin, isobutylmethylxanthine (IBMX), and dexamethasone (Rubin et al., 1978), a regime which impacts three separate signal transduction pathways. Insulin, acting through the IGF-I receptor, tends to act differently in primary cultures and cell lines. Insulin enhances the rate of lipid filling in cell lines but typically enhances the fraction of cells that differentiate in primary preadipocytes. Primary preadipocytes may also differ from cell lines in regard to the effects of IBMX and steroids on differentiation (Gregoire et al., 1998). Treatment of 3T3-L1 cells with IBMX or forskolin, which increases cAMP levels and expression of C/EBP, an early gene in adipogenic specification, results in marked increases in lipogenic enzymes and adipose-specific proteins. However, neither forskolin nor IBMX increases differentiation (or proliferation) of primary porcine preadipocytes (Boone et al., 1999) as assessed by lipoprotein lipase (LPL) or glycerol phosphate dehydrogenase. Similarly, the synthetic glucocorticoid dexamethasone is a potent enhancer of 3T3-L1 preadipocyte differentiation, increasing the expression of C/EBP and PPAR (Wu et al., 1996). However, glucocorticoids are clearly conditionally inducers. A glucocorticoid-induced leucine-zipper protein (GILZ) antagonizes adipocyte differentiation; GILZ binds to a C/EBP binding site inhibiting transcription of PPAR2 and LPL (Shi et al., 2003). Moreover, Cushing’s syndrome patients with elevated glucocorticoids usually have visceral obesity but wasting of subcutaneous fat (McPhee et al., 1997). This in vivo consequence may be the result of elevated TGF-ß, which has been shown to suppress the effects of dexamethasone on primary rat preadipocyte differentiation but not on 3T3-L1 preadipocytes (Shin et al., 2003).

Despite these differences, and others, between primary cultures and cell lines, in vivo replacement experiments clearly demonstrate that the essential adipogenic process in cell lines is not abnormal or pathologic. When either 3T3-L1 or Ob17 preadipocyte cell lines are injected into nude mice, the cells develop into adipose tissue that is indistinguishable from normal adipose tissue (Green and Kehinde, 1979, Vannier et al., 1985). Clearly, whatever altered properties these cell lines have does not keep them from apparently normal function, reiterating the observation that all in vitro biology is part of the full cellular repertoire. Differences between primary and cell line cultures may simply reflect that adipose tissue has multiple mechanisms that have a similar end point.

Cross Talk in Signaling Pathways

Adipose tissue response to GH illustrates a classic problem of in vivo and in vitro interpretation. Large numbers of in vivo experiments and current commercial use demonstrate that GH has a potent lipolytic effect in vivo (Dunshea, 1993; Etherton, 2000) and, with chronic, high exposure, GH induces insulin resistance (Holt et al., 2003). However, early in vitro work showed that GH initially had an insulin-like activity and then a weak lipolytic effect only after a lag of some hours (Birnbaum and Goodman, 1979; Goodman, 1981). The immediate effects of GH on naive adipose tissue in culture include increased lipid synthesis, increased glycogen synthesis, and inhibition of catecholamine-stimulated lipolysis. After exposure of adipose tissue to GH for 4 to 6 h, the effects of GH seem to be reversed, with anti-insulin-like effects being observed. Growth hormone also has in vitro effects on preadipocyte proliferation and differentiation that appear inconsistent with in vivo effects. Fetal hypophysectomy of fetal pigs results in decreased fat cell number with an increase in adipocyte size and corresponding lipogenic enzyme activity (Hausman and Hausman, 1993), indicating that GH inhibits preadipocyte proliferation and has weak effects on lipid metabolism during early development. Effects on 3T3 and OB17 cells lines are consistent with in vivo observations as GH decreases proliferation and increases differentiation (Morikawa et al., 1982; Tang et al., 1995; Vierck et al., 1996). In contrast, GH increases the proliferation of rat and human primary preadipocytes (Vassaux et al., 1994; Wabitsch et al., 1996) and inhibits differentiation (Hansen et al., 1998)

Although not obvious early on, the message from this conflicting data was that something unexpected and interesting was happening and it was not an artifact (Nam and Lobie, 2000). In subsequent years, it has become clear that this discrepancy was a result of cross talk in the signaling pathways (Figure 1) shared by insulin and GH as well as other hormones (Dominici and Turyn, 2002). Insulin binding to the insulin receptor activates the receptor tyrosine kinase, which phosphorylates insulin receptor substrate-1 (IRS-1; Saltiel and Kahn, 2001). Phosphorylated IRS-1 initiates several signaling cascades, including the phosphoinositide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) family proteins. A number of proteins are phosphorylated and activated by PI3K, including mammalian target of rapamycin (mTOR), an important nutrient-regulated intracellular kinase (Rohde et al., 2001) that enhances preadipocyte cell clustering and differentiation (Fox et al., 1998; Bell et al., 2000). Mitogen-activated protein kianse can directly phosphorylate PPAR, inhibiting adipogenic activity (Hu et al., 1996). Ligand binding to the GH receptor initiates receptor dimerization and recruitment of janus kinase (JAK) to the receptor. Active JAK can phosphorylate IRS-1 and MAPK, activating similar pathways to insulin. Both receptors also activate additional independent signaling cascades, accounting for differences in hormone action as well as similarities. As intracellular signaling has become an increasing focus of research, it has become clear that many physiological process share common regulatory signal pathways with specificity depending on the specific context of intracellular proteins being expressed (Dumont et al., 2002; Pires-daSilva and Sommer, 2003).

View larger version (45K): [in this window] [in a new window]   Figure 1. In vitro treatment with GH may cause insulin-like effects that are not apparent in vivo. This occurs because of cross talk between the signal transduction pathways for insulin and GH. The receptors for insulin and GH are quite different, the insulin receptor (IR) signaling through an intrinsic tyrosine kinase and the GH receptor (GHR) signaling through a separate janus kinase (JAK2). However, because of structural similarities in phosphorylated (P) tyrosine and serine on downstream components, both insulin and GH can activate the phosphoinositide-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways. Insulin receptor substrate-1 (IRS-1), Ras GTPases (RAS). Modified from Nam and Lobie (2000) and Dominici and Turyn (2002).

In vivo, circulating IL-6 levels are increased by catecholamines (Mohamed-Ali et al., 2001; Vicennati et al., 2002), whereas isolated human adipocytes and differentiated 3T3 adipocytes secret IL-6 in response to micromolar adrenergic agonists, such as isoproterenol and clenbuterol. In vitro, TNF- and GH both increase IL-6 expression in differentiated 3T3 adipocytes (Fasshauer et al., 2003). Stimulation by GH occurs over a time frame of 1 h, relatively shorter than a number of other GH effects. Conversely, overexpression of IL-6 suppresses growth partly through a reduction or GH receptor mRNA (Lieskovska et al., 2002) although this effect has not been demonstrated in adipose tissue.

Adipose Contains Multipotent Stem Cells

An important question for animal scientists is the nature of adipose precursor cells because this information may be useful to restrict proliferation where fat is undesirable and cause proliferation where it is desirable. This is another area in which the interpretation of in vitro experiments may have been misleading. Preadipose cell lines differentiate very uniformly following growth arrest at confluence. Altering differentiation conditions or media may alter the percentage of differentiated cells but has relatively small effects on the properties of an individual cell, such as size and enzyme activity. Primary cells behave similarly except that growth arrest at confluence is not as distinct and primary cells will grow into multiple layers with greatly decreased growth rate following confluence rather than simply becoming arrested. In the past several decades, these observations certainly contributed to a perception that preadipocytes in vivo were a relatively homogeneous population of determined but not committed precursors. However, in vivo, cells are in a three-dimensional matrix and usually have some cell-to-cell contact with a variety of cell types. Regulation associated with confluence, although not artifactual, is more reflective of metastasis than normal growth.

It has become apparent in recent years that both adipose tissue and muscle contain not only lineage-restricted precursors but also a reserve pool of multipotent mesenchymal precursors that can be directed into several lineages. Adipose tissue contains determined preadipocytes that undergo limited proliferation before; differentiation, but preadipocytes may also derive from undetermined precursors (Kras et al., 1999), in fact, mesenchymal stem cells from adipose tissue may be induced to differentiate into other cell types, including cartilage, bone, and muscle (Zuk et al., 2001, 2002). Conversely, mesenchymal precursors in muscle, including the so-called side population, can differentiate into adipocytes (Asakura et al., 2001). Particularly interesting is evidence that muscle satellite cells may largely be undetermined and as capable of forming intramuscular fat as of increasing muscle mass. Satellite cells appear to be much less determined than myoblasts and have increased adipogenic potential with age (Taylor-Jones et al., 2002), consistent with the concept that marbling develops from previously uncommited mesenchymal cells.

The significance of variably committed precursor or stem cells in adipose tissue and muscle is enormous: it opens the door for a wide range of applications, from reconstructing tissues to controlling timing and location of marbling deposition. The information needed to capitalize on this new knowledge begins with further understanding undifferentiated mesenchymal cells, adipocytes, and muscle cells, as explored in the next section.

	Adipose Genomics

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Differential Gene Expression

Genomics are techniques that rely heavily on in vitro biological processes regardless of sample origin. Although powerful and useful, genomics experiments are subject to the same inconsistencies as other in vitro techniques (Alabaster et al., 2002; Livesey, 2002). It is clear that genomics experiments may yield results that are difficult to interpret. However, just as the contradictions of early in vitro experiments with GH, described above, resulted from an unexpected complexity of signaling pathways, careful examination of genomics experiments may yield new information. The following section is an attempt to illustrate some of the future opportunities in this area, which may reflect those of past in vitro research.

The following example utilizes a publicly available tool, Unigene, from the National Center for Biotechnology Information, U.S. National Library of Medicine. Unigene, which is accessible from the same Web site as PubMed, is a system for partitioning GenBank sequences into nonredundant clusters. Digital Differential Display (DDD) is a Unigene component for comparing expression levels in tissue or cells using database information (Wheeler et al., 2003). Sequencing of mRNA provides a gene expression fingerprint for different cell types. Unigene DDD provides a statistical test of which genes have different expression levels between tissues and are therefore likely to contribute to the unique characteristics of each tissue. To further examine differences between mesenchymal cells, DDD was used to compare gene expression between adipose tissue of 4-d-old mice, muscle tissue, and pools of undifferentiated mesenchymal tissue. Mouse information was used in the example simply because of the much larger number of reported genes than that for livestock species.

In this comparison, the most differentially expressed gene was tropomyosin in skeletal muscle, a result that is not particularly surprising. What about comparable expression of classic adipose-specific genes or genes responsible for adipose differentiation? Generally, these were found down the list of differential expression.

Surprisingly, the three most highly differentially expressed genes in adipose (Figure 2) have not been previously associated with adipose tissue development. An unknown cDNA was the most highly differentially expressed adipose gene. The second most highly differentially expressed adipose tissue gene, and seventh overall most differentially expressed, in this example was disheveled 1 (Dvl1, or Dsh), a homolog of the drosophila disheveled gene involved in cell fate determination. The third most differentially expressed adipose tissue gene in adipose was sprouty (Spry) a homolog of a drosophila gene involved in several morphogenic processes. The differential expression of these genes would clearly suggest that Dvl and/or Spry play a role in adipogenesis. However, at the time of this writing, there is only a single publication containing a reference to either protein as well as to adipose tissue. A paper looking at polymorphisms reported that, in the mouse, Dvl maps to chromosome 4 along with Adfp, which encodes an adipocyte differentiation-related protein (Beier et al., 1992). There are no reports of a role for Spry in adipose tissue.

View larger version (26K): [in this window] [in a new window]   Figure 2. An example of how genomics may indicate future research opportunities. This figure is a computer screen from Unigene Digital Differential Display for mouse sequences in GenBank. The screen shows gene expression differences between adipose tissue, muscle tissue and, undifferentiated mesenchyme. The genes with the greatest differential expression in adipose were an unknown cDNA, and homologs for the Drosophila genes, disheveled 1 (Dvl1) and sprouty 1 (Spry1). The decimal numbers and shaded circles in the left-hand columns indicate expression and the A < B expression beneath indicates statistical differences. Large differences in expression level may indicate genes important for development or function of a specific tissue. This display suggests Dvl1 or Spry1 may have an important role in adipogenesis even though very few publications report investigations of these proteins in adipose tissue.

Dishevelled is a component of Wnt signaling pathways (Figure 3). The Wnt family of proteins are paracrine growth factors, highly expressed in preadipocytes, and are potent inhibitors of adipogenesis (Ross et al., 2000). Depending on the context, Wnt signals may cause cell proliferation, apoptosis, cell fate determination, differentiation, or precursor cell maintenance. The Wnt signaling through the frizzled receptors inhibits adipogenesis by blocking the induction of C/EBP and PPAR (Bennett et al., 2002). Disheveled is an upstream component of the intracellular signaling cascade from frizzled, and the outcome of Wnt signaling depends on the path of Dvl signal transmission (Novaka and Dedharb, 1999; Wharton, 2003). Disheveled inactivates glycogen synthase kinase 3 (GSK3), preventing it from phosphorylating ß-catenin with subsequently increased degradation. Therefore, Dvl acts to prolong the activity of ß-catenin, which presumably has a role in suppressing adipogenesis. This is particularly interesting because stabilization of ß-catenin is associated with inhibition of adipogenesis in myoblasts and the age-related increase in adipogenic potential of muscle satellite cells (Taylor-Jones et al., 2002). Wnt signaling through the ß-catenin pathway is also associated with activation of Pax7-mediated myogenic specification of precursor cells following muscle injury (Polesskaya et al., 2003).

View larger version (47K): [in this window] [in a new window]   Figure 3. Intracellular signaling pathway involving disheveled (Dvl), which is differentially expressed in developing adipose tissue. Disheveled is a signaling component for the Wnt family of growth factors. Wnt growth factors are expressed by preadipocytes and are paracrine inhibitors of differentiation. Disheveled is a signaling component from the Wnt receptor, fizzled, that inhibits glycogen synthase kinase 3 (GSK3), thereby protecting ß-catenin against phosphorylation and subsequent ubiquitin (Ub) targeting for proteolytic degradation. Thus stabilized, ß-catenin has greater effects on transcription than it would without Dvl. Modified from Wharton (2003).

Sprouty and Fibroblast Growth Factor

Sprouty proteins (Spry) are both negative and positive modulators of receptor tyrosine kinase (RTK) signaling in several developmentally important RTK-induced signaling pathways (Christofori 2003). Sprouty can act a suppressor of MAPK signaling from epidermal growth factor, fibroblast growth factor (FGF), vascular endothelial growth factor, or platelet-derived growth factor. Binding of ligands to RTK results in tyrosine autophosphorylation of the receptors and signal transduction through subsequent binding of adaptor molecules, such as Grb2, to the phosphorylated tyrosine. Phosphorylated Spry can act as a binding decoy, preventing downstream activation of MAPK. Alternatively (Figure 4), phosphorylated Spry can bind the E3 ubiquitin ligase, Cbl with relatively high specificity. Activated RTK are targeted for degradation by Cbl-mediated ubiquitination (Dikic and Giordano, 2003; Peschard and Park, 2003), so decoy binding of Cbl by Spry prevents receptor degradation, resulting in prolonged RTK signaling.

View larger version (48K): [in this window] [in a new window]   Figure 4. Sprouty (Spry) is differentially expressed in adipose tissue and may enhance receptor tyrosine kinase (RTK) signaling by protecting the receptor from proteolysis. The Cbl family of proteins are E3 ubiquitin ligases, which function as negative regulators of RTK signaling. The Cbl ligases add ubiquitin (Ub) to phosphorylated (-P) receptors targeting them for proteolytic degradation by proteosome. This mechanism degrades ligand-activated, autophosphorylated receptors limiting the duration of growth factor signaling. Sprouty prevents this by acting as a decoy for Cbl ligase reducing receptor ubiquitination. In the presence of Spry, RTK activation by epidermal growth factor (EGF) or fibroblast growth factor (FGF) results in longer signaling through the Raf kinase and mitogen-activated protein kinase (MAPK) signaling cascade, mediated either by phospholipase C (PLC) or Grb2 mediated activation of Ras GTPase (Ras). Modified from Christofori (2003).

Treating 3T3-L1 preadipocytes with FGF induces activation of MAPK signaling, which lasts for at least 12 h (Prusty et al., 2002), much longer than typical for most RTK and therefore consistent with a predicted function of Spry. This enhanced FGF signaling would correspond to Spry acting as a decoy for Cbl. This idea is interesting in light of a report that Cbl helps mediate effects of rosiglitazone (Standaert et al., 2002), a PPAR agonist that increases insulin sensitivity but also enhances adipocyte differentiation (Larsen et al., 2003). It also appears that Cbl is involved in the insulin-mediated regulation of glucose transport (Salteil and Kahn, 2001).

Expression Related to Adipocyte Differentiation

Can examining changes in gene expression during differentiation-cultured cells provide any additional information about the significance of these pathways implicated in adipogenesis by genomics but which lack published information? During 3T3-L1 differentiation, ß-catenin expression goes up (Guo and Liao, 2000), which could be reflective of altered Dvl activity. Unfortunately, however, ß-catenin expression also goes down (Gerhold et al., 2002) in similar circumstances. Because cell lines were at least at some point in the past, a consistent clone, one would not expect inconsistent results in experiments that certainly appear similar from the described culture methods. On the contrary, inconsistency may be more of a rule than exception with these gene expression experiments. Out of three similar reports examining differentiation-related changes in gene expression of 3T3 cells, as many as 100 genes exhibit large changes in expression level in some experiments but not others (Guo and Liao, 2000; Gerhold et al., 2002; Jessen and Stevens, 2002).

This large variation in gene expression between different experiments may be an important message. Although the number of experimental units in each case is effectively one making for questionable statistics, common sense suggests that it is unlikely this level of variation within a clonal cell line can be completely ascribed to experimental errors occurring across three different laboratories. The biology of adipocyte differentiation may be sufficiently complex that there are few completely unique regulatory steps. From a different perspective, this may simply mean adipogenesis is outcome driven, rather than sequence driven. For example, embryonic development proceeds by following a set of process instructions rather than a blueprint, clearly demonstrated by the phylotypic stage of development common to a wide range of species (Gerhart and Kirschner, 1997). It seems reasonable that a process as important as adipogenesis for energy storage would follow a similar flexible strategy.

	Implications

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Our scientific training strongly emphasizes testing whether hypotheses are true or false. However, it is important to remember that inconsistent results obtained among various experiments do not constitute a hypothesis test. Contradictory results may not make sense or be easily explained. It may not be apparent how a cell line functions like a cow, nor may it be clear why different labs get different results. However, this does not necessarily mean the information is wrong or not useful. In fact, it may mean something very interesting is happening and may be an important key to our next level of understanding.

	Footnotes

 
¹ Publication of this symposium paper is supported by USDA/ARS.

² Sincere apologies to colleagues whose relevant work was not included because of space limitations for this manuscript.

³ Correspondence: 1503 S. Maryland Dr. (phone: 217-333-6181; e-mail: Jnova{at}uiuc.edu).

Received for publication July 9, 2003. Accepted for publication September 24, 2003.

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