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Journal of Lipid Research, Vol. 47, 450-460, February 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
Methods |
* Division of Endocrinology, Metabolism, and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110
Published, JLR Papers in Press, November 30, 2005
1 To whom correspondence should be addressed. e-mail: pbickel{at}im.wustl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PPAR). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins 
and ß, PPAR, sterol-regulatory element binding protein-1, S3-12, and perilipin. OP9 differentiation is not diminished by maintenance in culture at high cell density or by long periods in continuous culture, thereby facilitating the generation of stable cell lines that retain adipogenic potential. Thus, the unique features of OP9 cells will expedite the study of adipocyte biology.
Abbreviations: ADC, adipogenic cocktail; BMP-4, bone morphogenic protein 4; C/EBP, CCAAT/enhancer binding protein; DN, dominant negative; eGFP-perilipin, perilipin tagged at its N terminus with enhanced green fluorescent protein; GLUT4, glucose transporter 4; IO, insulin oleate; M-CSF, macrophage colony-stimulating factor; PPAR, peroxisome proliferator-activated receptor ; SR, serum replacement; SREBP, sterol-regulatory element binding protein; SSC, side scatter; TAG, triacylglycerol
Supplementary key words bone marrow • flow cytometry • transfection • triacylglycerol • glucose transporter 4 • glucose uptake • peroxisome proliferator-activated receptor • S3-12 • perilipin • 3T3-L1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Three decades ago, Green and colleagues (3–5) reported that a clonal subline of mouse 3T3 cells had a propensity to differentiate into adipocytes when in a "resting state." This 3T3-L1 cell culture model of adipogenesis has been exploited extensively to investigate the mechanisms of adipocyte differentiation, lipid metabolism, insulin signaling, and glucose transport as well as to identify physiologically important adipocyte-secreted proteins, such as adiponectin (6) and resistin (7). Nevertheless, the 3T3-L1 model has significant limitations. First, from the time of initial plating, the generation of 3T3-L1 adipocytes from preadipocytes requires at least 2 weeks (8). Second, if 3T3-L1 cells become confluent and are further propagated or if they are passaged extensively, they no longer differentiate robustly into adipocytes. These issues make culturing of 3T3-L1 cells demanding and limit their utility in the generation of stable cell lines. Third, to our knowledge, it has not been possible to efficiently detect RNAs and proteins from transiently transfected DNA in 3T3-L1 adipocytes. This limitation derives from the facts that most plasmid transfection protocols require subconfluent cells and that levels of RNAs and proteins expressed from such transfections wane before 3T3-L1 cells become adipocytes. Finally, because the 3T3-L1 cell line originated from a single clone and thus has clone-specific traits, it fails to recapitulate the primary cells it models. Thus, an alternative, tractable adipocyte model system is required.
We now report our initial characterization of a new adipocyte cell culture model, OP9 mouse stromal cells, that provides a tractable alternative system for studies of adipocyte biology. The OP9 cell line was established from the calvaria of newborn mice genetically deficient in functional macrophage colony-stimulating factor (M-CSF) (9). OP9 cells are used in coculture to support hematopoietic cell differentiation from embryonic stem cells (9). Specifically, we describe conditions that cause OP9 cells to store TAG, to take up glucose in response to insulin, and to express adipocyte proteins. Unlike 3T3-L1 cells, OP9 cells will robustly differentiate into adipocytes after being confluent and after many passages and long periods in culture. Furthermore, OP9 cells can be differentiated rapidly enough to detect protein expressed from transiently transfected DNA in fully differentiated adipocytes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(catalog no. sc-61); C/EBP ß (catalog no. sc-7962); peroxisome proliferator-activated receptor (PPAR) (catalog no. sc-9000); and sterol-regulatory element binding protein-1 (SREBP-1) (catalog no. sc-367).
Propagation of cells
OP9 cells were generously provided by Dr. Toru Nakano (9). OP9 cells are available from the American Type Culture Collection (Manassas, VA; catalog no. CRL-2749); however, we have not had consistent results differentiating the American Type Culture Collection OP9 cells into adipocytes. OP9 cells were grown in OP9 propagation medium: MEM- with 20% FBS (Premium Fetal Bovine Serum; BioWhittaker, Walkersville, MD; catalog no. 14-051Q), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. In our hands, the optimal differentiation potential of OP9 cells was achieved when cells were replated every 3 days at a density of at least 7,000 cells/cm2. OP9 cells grown in this manner are flat with broad cell processes. OP9 cells maintained at low cell density adopt a spindly morphology and differentiate into adipocytes poorly. 3T3-L1 cells were grown in 3T3-L1 propagation medium: DMEM with 10% bovine serum (Invitrogen; catalog no. 16170-078), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Differentiation of adipocytes
Three methods were used to differentiate preadipocytes into adipocytes as described below.
Serum replacement method
OP9 or 3T3-L1 cells were grown to confluence and then cultured for 2 additional days in either OP9 or 3T3-L1 propagation medium as described above. The cells were then cultured up to 4 more days in serum replacement (SR) medium: MEM- with 15% KnockOutTM SR (Invitrogen; catalog no. 10828-028), 100 U/ml penicillin, and 100 µg/ml streptomycin. For the purpose of studying insulin effects, it is important to change the SR medium to OP9 propagation medium after day 2 of differentiation, because KnockOutTM SR contains very high concentrations of insulin (final concentration of 1.7 µM). In our initial experiments, the SR medium contained 5 ng/ml bone morphogenic protein 4 (BMP-4) (R&D Systems, Inc., Minneapolis, MN). We subsequently found there to be no significant difference in adipocyte differentiation without BMP-4, so all experiments were performed without BMP-4, except for the assays for insulin-stimulated glucose uptake.
Insulin oleate method
OP9 cells were plated at 5,000 cells/cm2. When cells adhered to the plate, the OP9 propagation medium was replaced with insulin oleate (IO) medium: MEM- with 0.2% FBS, 175 nM insulin, 900 µM oleate bound to albumin (5.5:1 molar ratio, prepared as described previously) (14), 100 U/ml penicillin, and 100 µg/ml streptomycin.
Adipogenic cocktail method This method was performed as described previously for 3T3-L1 cells (8). In brief, cells were grown to confluence and then cultured for 2 additional days in 3T3-L1 adipocyte medium: DMEM with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were then cultured for 2 days in DM1: DMEM with 10% FBS, 175 nM insulin, 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were cultured for an additional 2 days in DM2: DMEM with 10% FBS, 175 nM insulin, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. At that point, 3T3-L1 cells were maintained in 3T3-L1 adipocyte medium, and OP9 cells were maintained in OP9 propagation medium.
Labeling lipid droplets with red fatty acid
Adipocytes were incubated for 2 h in IO medium containing 20 µg/ml BODIPY® 12 carbon red fatty acid (Molecular Probes, Invitrogen Detection Technologies; catalog no. D3822, BODIPY®FL C12) delivered from a 20 mg/ml stock dissolved in dimethyl sulfoxide.
Immunofluorescence microscopy
Adipocytes were fixed, stained, and imaged as described previously for 3T3-L1 cells (14).
Live cell imaging
Imaging live OP9 cells on 22 mm coverslips
Thirty-six hours after adding IO medium, the coverslip was washed with PBS, pH 7.4, and the side without cells was dried. A chamber was created by placing two coverslips 18 mm apart and anchoring them to the slide with silicone vacuum grease. Then the coverslip was placed cell-side down spanning the gap between the anchored coverslips. The chamber between the slide and the cell-bearing coverslip was then filled with PBS, and adipocytes were imaged as described previously (14).
Imaging live OP9 cells in culture dishes Cells were imaged in medium with an Eclipse TS1500 inverted microscope (Nikon, Inc.) using the 20x objective. Images were captured with a Coolpix 5000 digital camera (Nikon, Inc.).
Protein assay
Protein was assayed by the bicinchoninic acid method in a microtiter format using BSA as the standard (Pierce Biotechnology, Inc., Rockford, IL).
TAG measurements
Cells were dispersed in PBS with 10 mM EDTA. Cells or increasing amounts of corn oil dissolved in ethanol (up to 40 µg of corn oil) were extracted from the aqueous phase (16). The organic phases were dried and the colorimetric TAG detection reagent (Thermo Electron Corp., Melbourne, Australia; catalog no. 2780-400H) was added to the lipid residue. Samples and the corn oil standards were transferred to a microtiter plate, and absorbance at 540 nm was measured. The TAG mass extracted from the cells was estimated by interpolation on the line that best fit the corn oil standards.
Immunoblotting
Immunoblotting was done as described previously (14). Cells were lysed in HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.5% cholate with Complete Protease Inhibitor Cocktail (Roche Applied Science; catalog no. 1 836 153), and the supernatants (21,000 g for 10 min) were resolved by PAGE and blotted.
Extraction of nuclear proteins
Nuclear protein-enriched supernatants were generated using a commercial kit according to the manufacturer's protocol (Pierce Biotechnology; catalog no. 78833).
Generation of OP9 cell lines stably expressing NLS-LacZ and a PPAR dominant negative mutant
cDNAs encoding ß-galactosidase with a nuclear localization signal (NLS-LacZ) or a FLAG-tagged PPAR dominant negative (DN) mutant (17) were cloned into the 
U3 vector, and pseudotyped retroviral particles were produced and used to transduce subconfluent cells, as described (18). To minimize differences in the cell lines not attributable to the virally expressed proteins, we generated each cell line in parallel.
Glucose uptake assays
Specific uptake of 2-deoxy-D-[3H]glucose into adipocytes in response to insulin was measured essentially as described (19). The fold increase in 2-deoxyglucose uptake in response to insulin was calculated as the 2-deoxyglucose uptake of the insulin-treated adipocytes divided by the 2-deoxyglucose uptake of the untreated adipocytes. Nonspecific uptake in the presence of 10 µM cytochalasin B was subtracted from all values. The SR medium used to initiate OP9 cell differentiation in these studies contained 5 ng/ml BMP-4.
Transfection of OP9 cells
A total of 50,000 OP9 cells were plated in a 35 mm dish. After 18 h, cells were transfected with 4 µg of DNA and 12 µl of Optifect reagent, according to the manufacturer's protocol (Invitrogen; catalog no. 12579-017). After 8 h, transfection medium was replaced with either propagation medium or IO medium as indicated.
Flow cytometry
Cells were transfected as described above and treated as indicated in the legend to Fig. 1. Immediately before scanning, cells were trypsinized and resuspended in Hanks' balanced salt solution with 0.2% bovine serum albumin. A total of 10,000 events from this cell suspension were analyzed with a Calibur cytometer (Beckman-Coulter, Inc.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Adipogenic stimuli cause OP9 cells to acquire adipocyte morphology, accumulate TAG, and express adipocyte marker proteins
We found three treatments that caused OP9 cells to assume the morphology of adipocytes (contain abundant intracellular lipid droplets) and accumulate TAG. The first treatment is the adipogenic cocktail (ADC) method traditionally used to differentiate 3T3-L1 cells. ADC treatment causes OP9 cells to accumulate similar amounts of TAG as 3T3-L1 cells (Fig. 2A). Like 3T3-L1 cells, OP9 cells must be confluent for ADC culturing to cause robust differentiation; preconfluent ADC-treated OP9 cells fail to accumulate TAG and do not assume the appearance of adipocytes (data not shown). In addition, like 3T3-L1 cells, ADC-differentiated OP9 cells form a thick mat of adipocytes that is not conducive to microscopy. Unlike 3T3-L1 adipocytes, which can be dispersed by trypsinization, replated, and effectively imaged by microscopy (13, 14), we were unable to disperse the mat of OP9 adipocytes by trypsinization. Thus, we were unable to take informative images of OP9 adipocytes that had been differentiated by the ADC method.
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supplemented with 900 µM oleate and 175 nM insulin. In contrast to ADC differentiation, IO culturing efficiently differentiates preconfluent OP9 cells (Fig. 1B, panel i). A 3 day incubation in IO medium causes OP9 cells to accumulate at least as much TAG as 3T3-L1 cells at 8 d of ADC culturing (Fig. 2A). The IO method also induces adipocyte morphology, including round cell shape and nuclei displaced to the cell periphery by large perilipin-coated lipid droplets (Fig. 3A, D). For comparison, we treated preconfluent 3T3-L1 cells with IO medium. More than 90% of 3T3-L1 cells die when cultured in IO medium for 48 h, but OP9 cell death in IO medium is much less. However, the few surviving 3T3-L1 cells often display morphology similar to that of primary adipocytes, a large unilocular lipid droplet with a peripherally displaced nucleus. Up to 50% of IO-cultured OP9 cells have unilocular droplets, but the most common morphology is one of multiple droplets of >5–10 µm diameter with the nucleus displaced to the cell periphery. Notably, most IO-cultured OP9 cells contain numerous lipid droplets within 48 h of initiating differentiation, and many cells develop lipid droplets within 18 h (data not shown). Both the IO medium and the SR medium have added insulin and NEFA but little (IO) or no (SR) serum. However, these two methods are qualitatively different. IO medium effectively differentiates subconfluent OP9 cells (Fig. 1B, panel i), whereas SR medium effectively differentiates confluent OP9 cells (Figs. 4A, 5B). SR culturing is more effective when cell density is high, whereas IO culturing is less effective as cell density increases (data not shown).
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OP9 cells have adipogenic transcription factors without exogenous adipogenic stimuli
We investigated by immunoblotting whether the accelerated expression of adipocyte marker proteins in OP9 cells is driven by the early expression of transcription factors known to promote adipogenesis (Fig. 6). OP9 cells that have been confluent for 48 h have high levels of PPAR1, C/EBP-p42, C/EBPß-LAP, and SREBP-1, and the expression of PPAR2 and C/EBP-p30 increases rapidly during differentiation induced by SR culturing. Moreover, preconfluent OP9 cells express easily detectable levels of PPAR1 and C/EBP proteins, as assessed by immunoblotting of nuclear extracts (data not shown). In contrast, PPAR RNA and C/EBP protein isoforms are not detectable in confluent but undifferentiated 3T3-L1 cells (26, 27). These data suggest that OP9 cells are at a later stage in adipogenesis than 3T3-L1 cells.
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is necessary for OP9 differentiation into adipocytes in OP9 cells suggests that the adipocyte differentiation program followed by these cells shares common mechanisms with the well-characterized 3T3-L1 model. To test this hypothesis, we generated OP9 cells that stably expressed either ß-galactosidase or a DN PPAR mutant (17). Retrovirus-mediated stable expression of ß-galactosidase in OP9 cells has no significant effect on TAG accumulation, adipocyte morphology, or adipocyte marker protein expression (Fig. 5). In contrast, stable expression of the DN PPAR in SR-treated OP9 cells inhibits the accumulation of TAG (Fig. 5A), stops all but rare cells from acquiring adipocyte morphology (Fig. 5B), and prevents the induction of adipocyte marker proteins (Fig. 5C). These observations suggest that OP9 adipocyte differentiation is through a PPAR-dependent pathway. Note that there is one well-differentiated adipocyte in the DN PPAR field (Fig. 5B). Such rare cells likely do not express the DN PPAR mutant. We were unable to determine the extent of the viral infection of DN PPAR by microscopy. However, >99% of OP9 cells infected in parallel with retrovirus particles that encoded ß-galactosidase showed ß-galactosidase activity. That OP9 cells infected with the control virus differentiated normally suggests that the failure of the DN PPAR-expressing cells to differentiate is attributable to the expression of the DN PPAR mutant and is not a general effect of viral infection.
Insulin causes OP9 cells to increase glucose uptake
Adipocytes, like skeletal muscle cells, express the insulin-responsive GLUT4. In mice, adipocyte-specific ablation of GLUT4 leads to insulin resistance in both liver and muscle (28), which demonstrates the importance of insulin-stimulated glucose transport in adipocytes for whole body fuel metabolism. Consistent with the precocious expression of adipocyte transcription factors and marker proteins and the robust accumulation of TAG, day 5 OP9 adipocytes are at least as insulin-responsive as day 8 3T3-L1 adipocytes (Fig. 7).
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3T3-L1 preadipocytes are difficult to transfect with naked DNA (31). Investigators have resorted to more cumbersome and cell-toxic methods, such as electroporation or adenoviral transduction (32). Using flow cytometry, we investigated the efficiency of transfection of OP9 cells with an expression plasmid encoding perilipin tagged at its N terminus with enhanced green fluorescent protein (eGFP-perilipin). Preconfluent OP9 adipocytes were transiently transfected with eGFP-perilipin and then cultured in IO medium for 36 h. Flow cytometry indicated that 19 ± 4% (n = 11) of the transfected OP9 cells had increased fluorescence over untreated cells. Furthermore, in the cell preparation that was both transfected and IO-cultured, 
13% of the total events both were in the adipocyte gate and expressed eGFP-perilipin (Fig. 1A, panel iv). These events represented OP9 adipocytes that expressed the transiently transfected protein. Fluorescence microscopy showed that many IO-cultured OP9 cells that expressed eGFP-perilipin contained numerous lipid droplets that were of uniform size and were surrounded by green fluorescence. A representative cell is shown in Fig. 1B, panel iii.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, C/EBP and C/EBPß, and SREBP-1). OP9 cells recapitulate common mechanisms of adipocyte differentiation, as suggested by the failure of OP9 cells to differentiate into adipocytes in the context of stable expression of the DN PPAR mutant.
Lane and Tang (33) have proposed a model of adipogenesis in which multipotent stem cells first commit to the adipocyte lineage to become preadipocytes and then differentiate into "late-stage" preadipocytes before terminal differentiation. In this schema, preconfluent OP9 cells likely represent late-stage preadipocytes, because they already express PPAR1 and C/EBP proteins and because they can differentiate into adipocytes within 48 h.
Several practical features of OP9 cells make this cell line a particularly convenient model for adipocyte studies. First, OP9 adipocytes can be rapidly produced from either preconfluent cells (IO method) or confluent cells (SR and ADC methods). Second, in contrast to 3T3-L1 cells, OP9 cells can be maintained at high density without loss of potential to differentiate into adipocytes. Third, OP9 cells continue to differentiate well even at high passage number. These features permit large numbers of OP9 cells to be conveniently maintained in culture and OP9 adipocytes to be produced for experiments within a few days.
3T3-L1 cells are of limited utility for the generation of cell lines for the stable expression of proteins or RNAs. 3T3-L1 cells must be maintained at low density. Therefore, it is cumbersome to subject large numbers of 3T3-L1 cells to the DNA transduction procedure; this limits 3T3-L1 DNA transduction procedures to only the most efficient methods. Because 3T3-L1 cells only differentiate well for a limited number of passages, stable 3T3-L1 cell lines may not maintain the capacity for robust adipocyte differentiation. OP9 cells can be kept in culture at high density and continue to differentiate well at high passage numbers and after months in continuous culture; thus, OP9 cells do not have these limitations. As an illustration, OP9 cells that stably express a scrambled small interfering RNA have been generated, and these cells maintain the ability to differentiate into adipocytes, as shown by TAG storage and gene expression (S. E. Gale, A. Frolov, X. Han, P. Bickel, L. Cao, A. Bowcock, J. E. Schaffer, and D. S. Ory, unpublished data).
Transient transfection is a powerful tool for the initial exploration of the function and intracellular location of proteins (34, 35). This technique is particularly powerful if the effect can be seen morphologically, because the untransfected cells in the same field serve as a negative control. This concept is demonstrated in Fig. 1B, panels ii and iii, in which OP9 adipocytes that express eGFP-perilipin form smaller and more uniform lipid droplets than cells that do not express eGFP-perilipin. Furthermore, transient transfection also can be used to confirm the biochemical effects of a stably expressed protein. This may be important even when dealing with pools of cells that stably express a protein, because the number of copies and the position of integration of the genes vary between the control and experimental pools. These differences are potentially confounding. Finally, overexpression of some proteins interferes with cellular function to such an extent that it is difficult to generate stable cell lines. For these reasons, the ability to express readily detectable levels of exogenous proteins by transient transfection in OP9 adipocytes is potentially valuable.
OP9 cells were derived from the calvaria of a mouse that lacked functional M-CSF (9). Both the site of extraction and the genetics of the mouse of origin have implications for the adipogenic phenotype of these cells. The site of extraction suggests that OP9 cells were destined to be bone marrow adipocytes. M-CSF is a cytokine that promotes the clonal growth and differentiation of cells of the monocyte-macrophage lineage, and M-CSF deficiency leads to bone overgrowth as a result of the failure of osteoclast maturation (36, 37). M-CSF has also been reported to promote adipocyte hyperplasia when locally overexpressed in rabbit subcutaneous adipose tissue (38). Both M-CSF and its receptor are expressed in human subcutaneous adipocytes (38). Obesity is associated with the infiltration of adipose tissue by macrophages, and these infiltrating macrophages release factors that affect adipocytes and adipogenesis (39). It remains to be determined whether the lack of M-CSF affects OP9 adipocyte differentiation positively, negatively, or not at all. There are at least three independent reports of marrow-derived cells isolated from wild-type mice that differentiate into adipocytes upon hormone treatment (40–42).
The standard model of adipocyte differentiation is that preadipocytes must undergo growth arrest followed by mitotic clonal expansion (43), but the necessity of clonal expansion has been questioned (44–46). Mitotic clonal expansion does not appear to occur during IO culture of OP9 or 3T3-L1 cells. Large perilipin-coated lipid droplets that are characteristic of mature adipocytes begin to appear after 18 h of IO culture, and cell number decreases during IO culture. These observations are consistent with mitotic clonal expansion being dispensable for adipocyte differentiation, but definitive resolution of this important question requires further study.
The rapid and profound effects of IO medium on both OP9 and 3T3-L1 cell morphology suggest an important role for long-chain fatty acids in the promotion of adipocyte differentiation, as has been suggested recently for chicken adipocytes (47). Whether the oleate in the IO medium leads to the generation of ligands for adipocyte transcription factors such as PPARs or acts via other mechanisms remains to be investigated. Given that adipocytes are the primary storage site for long-chain fatty acids and that the storage capacity of each adipocyte is finite, long-chain fatty acids may promote the differentiation of preadipocytes as an adaptive response to increase the overall size of the depot.
The OP9 adipocyte model has great potential to accelerate critically important areas of metabolism research. The robust insulin-stimulated glucose transport displayed by OP9 adipocytes makes this model particularly promising for studies of GLUT4 trafficking and its regulation. We may not have optimized every protocol for OP9 adipocyte differentiation and manipulation. However, the use of OP9 cells by the adipocyte research community should lead to improved methods as each laboratory modifies the procedures we have reported.
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ACKNOWLEDGMENTS |
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retroviruses used in this study, Martha Wolins and Pamela Skinner for help editing the manuscript, Marissa Schoenfish for assistance with immunofluorescence microscopy, Dr. Daniel Ory and Dr. Paul Hruz for critical review of the manuscript, and Amy Boyet of the Washington University School of Medicine, Department of Pathology and Immunology Flow Cytometry Facility, for technical assistance. This work was supported by National Institutes of Health Grants RO1 DK-51836 (P.E.B.) and T32 DK-07296 (N.E.W.). Manuscript received October 20, 2005 and in revised form November 29, 2005.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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