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Endocrinology Vol. 142, No. 2 551-556
Copyright © 2001 by The Endocrine Society

ARTICLES

Albumin Regulates Induction of Peroxisome Proliferator-Activated Receptor-{gamma} (PPAR{gamma}) by 15-Deoxy-{Delta}12–14-Prostaglandin J2 in Vitro and May Be an Important Regulator of PPAR{gamma} Function in Vivo1

Eric C. Person, Leslie L. Waite, Robert N. Taylor and Thomas S. Scanlan2

Graduate Group in Biophysics (E.C.P.), Departments of Pharmaceutical Chemistry (T.S.S.), Cellular and Molecular Pharmacology (T.S.S.), and Obstetrics, Gynecology, and Reproductive Sciences (L.L.W., R.N.T.), University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Thomas S. Scanlan, Department of Pharmaceutical Chemistry, University of California, Box 0446, San Francisco, California 94143-0446. E-mail: scanlan{at}cgl.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We observed that serum contains a factor(s) that inhibits the induction of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) by 15-deoxy-{Delta}12,14-PGJ2 (15dJ2). Ten percent FBS reduces 15dJ2 induction of PPAR{gamma} from over 150-fold to less than 15-fold in EP-JEG cells, a stably transfected choriocarcinoma cell line that expresses endogenous PPAR{gamma}. By contrast, rosiglitazone, an unrelated pharmacological agonist of PPAR{gamma}, is not inhibited by serum in this cell line. We have identified the inhibitory principal in serum as albumin. Serum albumin binds 15dJ2 with a dissociation constant of 870 ± 70 nM, effectively reducing the concentration of 15dJ2 available to PPAR{gamma}. Heat treatment of serum abolishes the inhibition, providing a way to test eicosanoid compounds independently of albumin’s inhibitory effect. It is reasonable to assume that 15dJ2 or structurally similar compounds or metabolites are the endogenous activators of PPAR{gamma}. Therefore, albumin may be an important regulator of PPAR{gamma} function in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME proliferator-activated receptors (PPARs) are ligand-inducible transcription factors that belong to the nuclear receptor superfamily (for reviews, see Refs. 1 and 2). There are three different subtypes ({alpha}, ß/{delta}/NUC1, and {gamma}), each with distinct ligand specificities and expression patterns. In humans, PPAR{gamma} is expressed mainly in brown and white adipose tissue, but is also present in the intestine, skeletal muscle, liver, heart, bone marrow stromal cells, placenta, and spleen (1, 3). PPAR{gamma} is the major regulator of adipocyte differentiation and is important in glucose and lipid metabolism.

The antidiabetic drugs known as thiazolidinediones are some of the highest affinity synthetic activators of PPAR{gamma}. Rosiglitazone (BRL 49653, Avandia) was the first identified high affinity PPAR{gamma} ligand and is still one of the most potent ligands, with a Kd of 43 nM and an EC50 of 80 nM for PPAR{gamma}-mediated activation of reporter genes controlled by a PPAR{gamma} response element (4).

Several natural compounds, including metabolites found in oxidized low density lipoproteins, long chain fatty acids, and 15-deoxy-{Delta}12,14-PGJ2 (15dJ2), have been proposed as endogenous activators of PPAR{gamma} (5, 6, 7, 8, 9, 10). Of these, 15dJ2 is the most potent natural activator, with a EC50 of 2 µM for PPAR{gamma}-mediated activation of reporter genes controlled by a PPAR{gamma} response element and a Ki for competitive binding with rosiglitazone of 2.5 µM (6). Although none of these natural ligands has conclusively been shown to be an activator of PPAR{gamma} in vivo, it is reasonable to assume that the high affinity natural activator would be structurally similar to these reported fatty acids.

Albumin is the major protein component of human blood, present at about 40 mg/ml (for reviews, see Refs. 11, 12, 13). Although often regarded as a generic stabilizing protein, it has been shown to play many diverse roles. It is a major contributor to the osmotic blood pressure and is involved in the maintenance of blood pH. It binds many fatty acids and small molecules with Kd values of 100 nM to 100 µM, plays a major role in the transport of fatty acids that would otherwise be insoluble, and protects cells from polyunsaturated fatty acid-induced injury (14, 15). It is involved in the degradation of some PGs and the stabilization of others (16, 17, 18, 19, 20, 21). In fact, 15dJ2 was first reported as an in vitro degradation product of PGD2 incubated with plasma or serum albumin (16, 18).

In this study we report that serum albumin inhibits 15dJ2 induction of PPAR{gamma} by sequestering 15dJ2 in the medium, making it unavailable for binding to and activation of PPAR{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unless otherwise specified, all chemicals were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich Corp. (St. Louis, MO). 15dJ2 was obtained from Cayman Chemical Co. (Ann Arbor, MI). Rosiglitazone (BRL 49653) was synthesized following published procedures (22, 23, 24, 25). All forms of BSA and human serum albumin (HSA) were purchased as powders, dissolved in calcium- and magnesium-free PBS, and then sterile-filtered to give a 500-µM stock solution. FBS was obtained from HyClone Laboratories, Inc. (Logan, UT) or Omega Scientific (Tarzana, CA), and human male AB serum was obtained from Omega Scientific.

Cell culture
EP-JEG cells (3) were cultured at 37 C with 5% CO2 in Eagle’s MEM with Earle’s salts containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were passaged no more than 10 times after being thawed from a common stock.

Treatment of cells with serum and albumin preparations
EP-JEG cells were washed with PBS, incubated with a minimal volume of trypsin for 4 min at room temperature, then washed off the plates using medium. After counting cells using a hemocytometer, cells were pelleted at 1000 x g for 3 min. Cells were then resuspended in fresh medium, and plated onto 12-well plates at a density of 8 x 104 cells/well. After overnight incubation, cells were washed with PBS and then treated in triplicate for 24 h with medium containing 1 µM 15dJ2, 1 µM rosiglitazone, or 0.1% vehicle, and serum or albumin preparations as specified for each experiment. Rosiglitazone was delivered in DMSO. 15dJ2 was delivered in either ethanol or DMSO. We observed no difference in activity among ethanol, DMSO, or medium alone (data not shown).

Luciferase activity assay
Cleared cell lysates were analyzed using a luciferase reporter gene assay (1 814 036, Roche, Indianapolis, IN). Briefly, cells were washed with 1 ml PBS and then lysed with 100 µl of the supplied lysis buffer at room temperature for 15 min. Lysate was used to wash wells and was then transferred to microcentrifuge tubes and cleared for 6 min at maximum speed. Fifty microliters of the cleared lysate were analyzed for luciferase activity. An aliquot of the cleared lysate was used to determine protein concentration. This was performed using the bicinchoninic acid protein assay (23225, Pierce Chemical Co., Rockford, IL). After subtraction of the luciferase activity observed in lysis buffer alone, the luciferase activity was normalized to the protein concentration before averaging and calculation of relative activation as described in the text.

Serum heat treatment
Serum was heat treated immediately before use by placing serum in a sterile Dounce tissue grinder in an 80 C water bath for 20 min. Bovine serum remained liquid during this heat treatment, whereas human serum solidified, resembling a hard-boiled egg. Two or more volumes of medium were added to assist in homogenizing and suspending the solids, which were then diluted to a final concentration of 10% in medium.

HPLC
Reverse phase HPLC was performed using Rainin (Emeryville, CA) HPXL Pumps, an Altech (Deerfield, IL) Nucleosil 100 (C18) 10u 4.6 x 250-mm column with a 7.5-mm guard column, and a Varian (Walnut Creek, CA) ProStar 330 Photo Diode Array Detector controlled by a Varian Star Chromatography Workstation. The mobile phase was composed of Fisher HPLC grade water and acetonitrile with 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The column was equilibrated in water for at least 4 min before samples were loaded. Samples, dissolved in PBS, were injected using a 500-µl sample loop and run in a mobile phase of water for 5 min, ramped linearly over a period of 30 min to 100% acetonitrile, followed by 100% acetonitrile for 10 min. Under these conditions 15dJ2 elutes at approximately 33.2 min with a peak width of 10 sec.

Binding assays
A fixed concentration of 2 µM 15dJ2 was incubated overnight at 4 C with a range of concentrations of either BSA fraction V or HSA fraction V (1 nM to 100 µM) in calcium- and magnesium-free PBS with a total volume of 400 µl. The incubations were transferred to Microcon 30K cut-off Centrifugal Filter Devices and spun in an Eppendorf (Hamburg, Germany) 5417C microcentrifuge for 12 min at full speed to separate 15dJ2 free in solution from the albumin-bound fraction. The flow-through was stored at -80 C until 10 min before injecting 250 µl into the HPLC. Observed peak areas were converted to concentrations using a calibration curve generated by passing 15dJ2, at concentrations ranging from 50 nM to 2 µM in calcium- and magnesium-free PBS in the absence of albumin through the same protocol.

To accurately fit the experimental data we derived an equation for a single binding site allowing for conditions where the total ligand (LT) rather than the total protein (PT) is held constant, while allowing for depletion of both the free protein (Pf) and free ligand (Lf).

(I)


(II)


(III)
Starting with the equation for dissociation (Eq IGo), we substituted for free protein using the expression PT = Pf + PL, where PL is the concentration of the protein-ligand complex. We then substituted for bound complex using the expression LT = Lf + PL. After rearrangement, this gives a quadratic equation (Eq II) that can be solved for free ligand (Eq III) and then fit to the experimental data using the curve-fitting algorithm Kaleidagraph version 3.08d.

Cell growth and toxicity assay
EP-JEG cells were plated into 12-well plates as described above. After allowing cells to attach for 2 h, the medium was removed, and cells were washed with PBS before treatment in triplicate with medium containing 10% FBS, 10% heat-treated FBS, or no serum. The same medium formulations were also tested with 1 µM 15dJ2 to check for relative ligand toxicity. After 2 h, 1 day, or 2 days, cells were washed twice with PBS to remove loose cells and incubated with 200 µl trypsin at room temperature for 3 min. Cells were washed off plates with 800 µl medium containing FBS, pelleted in a microcentrifuge for 3 min, resuspended in 100 µl medium, and then counted in a hemocytometer.

Statistics
Data were derived from multiple independent experiments and are presented as the mean ± SD of triplicate points. Comparisons among different treatments were analyzed using repeated measures ANOVA with Fisher post-hoc tests and statistically significant differences were accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We recently reported that treatment with serum from pregnant women leads to increased PPAR{gamma} expression and activation in both transiently transfected JEG-3 cells and EP-JEG cells (3). While further characterizing the activity of pregnancy serum, we found that human male and nonpregnant female sera as well as FBS contain a factor(s) that reduces the level of PPAR{gamma} activation by 15dJ2. To characterize this inhibitory response we have used EP-JEG cells (3), a JEG-3-derived stable cell line containing a synthetic PPAR response element-driven luciferase reporter. Both JEG-3 and EP-JEG cells express high levels of endogenous PPAR{gamma}, making these cell lines useful for studying native regulation of PPAR{gamma}.

We then looked at varying serum levels to determine their effects on the luciferase reporter. EP-JEG cells were treated with 1 µM 15dJ2 and FBS levels ranging from 0–10%. At low serum concentrations (<1%), we observed from 150- to 700-fold induction by 15dJ2, a substantial increase over the 2- to 15-fold induction seen with higher concentrations of FBS (Fig. 1Go). In contrast, the induction by rosiglitazone was not affected by the serum concentration. Sera from men and nonpregnant women also inhibited induction of PPAR{gamma} by 15dJ2, at similar concentrations (data not shown).



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  		Figure 1. Serum titration. EP-JEG cells were incubated with medium containing 1 µM 15dJ2 ({blacksquare}) or 1 µM rosiglitazone ({triangleup}) and the indicated amount of FBS. The fold induction was calculated relative to 10% FBS with 0.1% vehicle.

 
To investigate the properties of the factor(s) in serum responsible for inhibiting 15dJ2 induction, we investigated the effects of two heat treatments on the activity of serum. Heat treatment of 56 C for 30 min is commonly used to inactivate complement and prepare serum for use in cell culture. A more rigorous heat treatment of 80 C for 20 min is suggested for heat inactivation of some heat-resistant restriction endonucleases. We incubated EP-JEG cells with 1 µM 15dJ2, 1 µM rosiglitazone, or 0.1% vehicle and FBS; FBS treated at 56 C for 30 min; or FBS treated at 80 C for 20 min. Heat treatment at 56 C for 30 min showed a similar level of induction as that of untreated serum, whereas the inhibition of 15dJ2 induction was absent when serum is treated at 80 C for 20 min (Fig. 2Go). Rosiglitazone activity was unaffected by heat treatment of the serum. Heat-treated serum allowed induction at similar levels to the induction by 15dJ2 in serum-free medium. Similar results were observed in transient transfection reporter gene assays using HeLa cells with a cytomegalovirus-driven hPPAR{gamma} expression plasmid and the tk-PPRE3-LUC reporter, although these assays showed lower levels of both induction and inhibition (data not shown). From this point we use the term heat-treated FBS (HTFBS) to refer to serum heated at 80 C for 20 min.



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  		Figure 2. Serum heat treatment. EP-JEG cells were incubated with medium containing 0.1% (vol/vol) vehicle alone ({square}), 1 µM 15dJ2 ({blacksquare}), or 1 µM rosiglitazone () and the indicated preparation of FBS. Fold induction was calculated relative to 10% FBS with 0.1% vehicle.

 
The heat treatment experiment suggested that a serum protein(s) with thermal lability between 56–80 C was responsible for the inhibition of 15dJ2 induction. Albumin, the most plentiful serum protein, has a reported thermal stability of 60-65 C (26), consistent with this observation, and is reported to catalyze the formation of 15dJ2 from PGD2 in vitro (16, 18). To determine whether serum albumin had a role in the inhibition of 15dJ2 induction, we treated EP-JEG cells with 15dJ2 and one of four commercial preparations of BSA at 30 µM, a concentration comparable to the albumin content of 10% FBS, as reported in the manufacturer’s analysis. Both serum-free medium and medium with 10% FBS were included as controls. Each BSA preparation reduced the 15dJ2 induction to a level comparable to that of 10% serum (Fig. 3Go). HSA (30 µM) gave the same results as BSA in these experiments (data not shown).



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  		Figure 3. Screening of albumin preparations. EP-JEG cells were incubated with medium containing 1 µM 15dJ2 and no serum ({square}), 10% FBS ({blacksquare}), or 30 µM of the indicated preparation of BSA (). Fold induction was calculated relative to 10% FBS with 0.1% vehicle.

 
To determine whether albumin inhibits 15dJ2 induction in a dose-dependent manner similar to serum, we treated EP-JEG cells with 1 µM 15dJ2, 10% HTFBS, and BSA fraction V levels ranging from 0–38 µM. BSA was able to inhibit 15dJ2 induction in a dose-dependant manner with an IC50 of approximately 2 µM (Fig. 4Go).



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  		Figure 4. Albumin titration. EP-JEG cells were incubated with medium containing 1 µM 15dJ2, 10% heat-treated FBS, and the indicated concentrations of BSA fraction V. Fold induction was calculated relative to 10% HTFBS, 30 µM BSA fraction V, and 0.1% vehicle.

 
These results suggest that albumin is responsible for the inhibition of 15dJ2 induction of PPAR{gamma} by serum. To determine whether albumin interacted with 15dJ2 directly, we incubated 2 µM 15dJ2 with either BSA fraction V or HSA fraction V (concentrations ranging from 1 nM to 100 µM) and isolated free ligand as described in Materials and Methods. HPLC analysis showed an albumin concentration-dependent reduction in the free 15dJ2 concentration (Fig. 5Go). This result suggests that albumin interacts directly with 15dJ2.



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  		Figure 5. Albumin binding. The concentration of free 15dJ2 was measured after incubation of 2 µM 15dJ2 with the indicated range of BSA fraction V ({diamond}) or HSA fraction V (•) as described in Materials and Methods. The curve fits for a single binding site give Kd values of 870 ± 70 nM (BSA) and 1.2 ± 0.2 µM (HSA).

 
We next asked whether albumin was simply binding 15dJ2 and sequestering it from medium or whether it was chemically transforming 15dJ2 into an inactive metabolite. To distinguish between catalytic and binding activities, we tested the reversibility of albumin’s effect on 15dJ2 activity. Heat treatment at 80 C for 20 min after overnight incubation of 15dJ2 in FBS at 37 C gave similar levels of induction as 15dJ2 in serum-free medium or medium with heat-treated FBS (data not shown), suggesting that albumin’s effect is reversible. To determine whether albumin had catalytic activity we used a published serum extraction protocol (18) followed by HPLC analysis. We observed no new products after incubation of 15dJ2 with FBS relative to 15dJ2 with the PBS control (data not shown). These experiments suggest that albumin inhibits 15dJ2 induction of PPAR{gamma} by reversibly binding 15dJ2, effectively reducing the free 15dJ2 concentration. Fitting the experimental data to the equation for a single binding site derived in Materials and Methods gives equilibrium dissociation constants (Kd) of 870 ± 70 nM (BSA) and 1.2 ± 0.2 µM (HSA) for albumin binding of 15dJ2 (Fig. 5Go). Figure 6Go is a synopsis of Figs. 1Go, 4Go, and 5Go that shows the correlation of the albumin levels required to reduce the free 15dJ2 concentration in the binding experiments with the levels of serum or albumin required to inhibit 15dJ2 induction of PPAR{gamma} in the EP-JEG cell assay experiments.



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  		Figure 6. Overlay plot. The data from Figs. 1Go, 4Go, and 5Go are plotted together. The serum titration from Fig. 1Go ({blacksquare}) and the albumin titration from Fig. 4Go (•) on the left axis. The binding curve from Fig. 5Go is shown ({diamond}) on the right axis. The leftmost data points on the serum and albumin titration curves are broken off to show that they contain no serum or albumin, respectively. The serum levels from Fig. 1Go, shown on the top axis, were converted to micromolar concentrations of BSA using the BSA levels reported in the manufacturer’s analysis.

 
As albumin is known to protect cells from polyunsaturated fatty acid-induced injury (14, 15), we evaluated cell growth and ligand toxicity in EP-JEG cells. Media with or without 1 µM 15dJ2 were incubated with FBS, HTFBS, or no serum, and the number of attached cells was quantified as a function of time. After 2 days, no significant differences were observed in the growth rate of cells treated with FBS or HTFBS, whereas cells treated with serum-free medium showed no significant growth. After 2 days of treatment with 1 µM 15dJ2, cells treated with FBS showed comparable growth as cells treated with FBS and no ligand. After 2 days treatment with 1 µM 15dJ2 in HTFBS we observed no increase in the number of attached cells and a substantial number of detached dead cells, whereas treatment with 1 µM 15dJ2 and serum-free medium resulted in almost no surviving cells after 2 days (Fig. 7Go).



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  		Figure 7. EP-JEG cell growth. EP-JEG cells were treated with medium containing untreated FBS, HTFBS, or no serum. Attached cells, in ten thousands, were counted after 2 h ({square}), 1 day ({blacksquare}), or 2 days ().

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work we have shown that albumin interacts directly with 15dJ2, a PPAR{gamma} agonist, effectively inhibiting induction of PPAR{gamma} by 15dJ2. We see induction by 15dJ2 of 150- to 700-fold at serum levels below 1%, whereas at higher serum concentrations we found only 5- to 20-fold induction by 15dJ2. Heat treatment of serum at 80 C for 20 min produced induction levels comparable to those seen with no serum, indicating that a heat-labile serum factor(s) inhibits induction of PPAR{gamma}. It should be noted that the observed induction levels in serum-free medium are extremely high, and we are not aware of any PPAR{gamma} reporter systems that use full-length PPAR{gamma} that provide such high levels of ligand induction. Moreover, our system used endogenous PPAR{gamma} and a physiologically relevant response element. Bishop-Bailey et al. reported serum-dependent changes in PPAR{gamma} activity in transiently transfected endothelial cells (ECV-304) (27), but found only 10-fold activation in the absence of serum.

Differences in cell growth in different serum preparations cannot account for the observed differences in 15dJ2 induction. The cell growth experiment shows a significant decrease in cell growth when cells are treated with HTFBS and 1 µM 15dJ2 relative to treatment with FBS with 1 µM 15dJ2. The decrease in cell number would lead to a lower observed luciferase activity, so decreased cell growth could not be responsible for the observed increased 15dJ2 induction. It has been reported that 15dJ2 and PPAR{gamma} activation can induce many cell types to differentiate or withdraw from the mitotic cycle and undergo apoptosis (27, 28, 29, 30, 31, 32, 33, 34, 35, 36). Induction of these pathways was observed in 10% FBS at 15dJ2 concentrations higher than the 1 µM that we used in our experiments. Our findings suggest that a lower total concentration of 15dJ2 may activate the apoptosis and cell cycle withdrawal pathways when albumin is unable to reduce the free 15dJ2 concentration. The activation of one or both of these pathways is probably responsible for the observed changes in cell growth.

Albumin is the serum factor responsible for inactivation of 15dJ2 induction of PPAR{gamma}. Albumin preparations reproduced the ability of serum to inhibit 15dJ2 induction, regardless of the method of purification, at concentrations comparable to those in serum that inhibited 15dJ2 induction of PPAR{gamma}. Along with the observation that thermally denatured albumin was unable to inhibit induction, these data suggest that an active conformation of albumin is required to inhibit 15dJ2 induction of PPAR{gamma}.

Albumin plays an important role in regulating the PPAR{gamma} response in these cells by inhibiting the extreme levels of induction observed in its absence. Based on albumin’s role in the transport and metabolism of fatty acids, the two most likely mechanisms of inhibition are by albumin either sequestering 15dJ2 or converting 15dJ2 to a less active form. In our assays, albumin’s interaction with 15dJ2 in vitro was reversible, and analysis by HPLC showed no new products formed after incubation of 15dJ2 with serum. This leaves binding as the most likely mechanism for the in vitro inhibition of 15dJ2 induction by albumin.

Albumin inhibits 15dJ2 induction of PPAR{gamma} by reversibly binding 15dJ2 and reducing the free concentration available to activate PPAR{gamma}. By measuring the free ligand concentration as a function of the albumin concentration we were able to measure an equilibrium dissociation constant for albumin and 15dJ2 of 870 ± 70 nM, assuming a single binding site. This measured value is comparable to the reported Ki of 2.5 µM for 15dJ2 displacement of rosiglitazone binding to PPAR{gamma} (6). With similar binding constants, the much larger pool of albumin would leave little or no 15dJ2 bound to PPAR{gamma} and significantly reduce the observed induction. The free ligand concentrations observed in the binding assay correlate well with the reporter gene activity observed at the corresponding albumin concentrations in the serum and albumin titration experiments. The levels of protein that would be required to inhibit activation through a binding mechanism along with the observation that albumin purified by several methods shows the same activity suggest that albumin, rather than a trace impurity, is responsible for the activity.

Albumin may also affect the observed activities of other PPAR{gamma} ligands. Albumin has been reported to bind a wide range of fatty acids, including several known PPAR{gamma} activators (11), making it likely that albumin will bind many of the other fatty acids and eicosanoids that activate the receptor, including the putative endogenous activator(s). Albumin binding of these fatty acids has the potential to produce effects on the activity of the ligands similar to those observed with 15dJ2. As the in vivo circulating albumin levels are approximately 100-fold higher than the albumin level required to inhibit 15dJ2 induction of PPAR{gamma} in vitro, it is likely that the albumin level in vivo is sufficient to affect induction by endogenous PPAR{gamma} activators. Regardless of whether the endogenous ligand for PPAR{gamma} is 15dJ2 or a similar fatty acid, it is likely that albumin will bind it, and that through this binding interaction, albumin will play an important role in the in vivo regulation of its transport, metabolism, and availability to bind PPAR{gamma} and activate transcription.

By binding potential activators and altering their observed activity, albumin might have an effect on the selection of ligands from screening experiments for PPAR{gamma}. This effect could also have implications for the testing and screening of ligands for other nuclear receptors, as a wide range of endogenous, xenobiotic, and pharmaceutical compounds are known to bind to albumin (11). We have recently observed that albumin inhibits the induction of estrogen and thyroid hormone receptors by some synthetic ligands, where heat treatment of serum shows release of this inhibition without affecting the growth rate of the cells used in the assays (unpublished data).

Heat treatment of serum removes albumin’s inhibition of ligand induction without compromising cell growth or exacerbating ligand toxicity. Although albumin’s reduction of activity probably parallels the induction we would expect to see with the high albumin levels in vivo, the ability to separate receptor activation and serum protein binding in these assays could prove useful. It would give important insights into the potential pharmacology of ligands for nuclear receptor targets as well as allowing the design of drugs that are not prone to these affects.

Several diseases, including cirrhosis of the liver, nephrotic syndrome, protein-losing enteropathy, malnutrition, and acquired immunodeficiency syndrome (37, 38), are associated with quantitatively reduced levels of serum albumin. Other disorders, such as preeclampsia (14, 39), coronary heart disease (40, 41), and diabetes (42), have been shown to manifest qualitative changes in isoforms of serum albumin that can alter lipid and lipoprotein transport. Among these, the most extensively studied is the pregnancy syndrome preeclampsia. In this condition, elevated circulating concentrations of free polyunsaturated fatty acids bind to and shift the pI of plasma albumin from 5.6 to 4.8 (15). Given our observations that albumin reduces the activity of 15dJ2 and potentially decreases the activity of endogenous PPAR{gamma} ligands, quantitative or qualitative changes in circulating albumin could affect the regulation of PPAR{gamma}-activated genes in these diseases.


   Footnotes
 
1 This work was supported by grants from the NSF (MCB-9513300; to E.C.P. and T.S.S.), the NIH [HD-08567 (to L.L.W.) and HD-30367 (to R.N.T.)], and the Sandler Foundation. Back

2 Alfred P. Sloan Research Fellow. Back

Received August 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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