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The Short- and Long-Term Effects of Tumor Necrosis Factor- and BRL 49653 on Peroxisome Proliferator-Activated Receptor (PPAR)2 Gene Expression and Other Adipocyte Genes

The USDA Human Nutrition Research Center on Aging at Tufts University and Division of Endocrinology Tupper Medical Research Institute New England Medical Center Boston, Massachusetts 02111

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Expression of tumor necrosis factor- (TNF) in adipocytes has been reported to correlate with insulin resistance associated with obesity. The thiazolidinediones such as BRL 49653 have been reported to improve insulin sensitivity in obese animals and humans. Although its exact mechanism of action is not known, BRL 49653 has been shown to antagonize some of the inhibitory actions of TNF. BRL 49653 binds and activates the peroxisome proliferator-activated receptor (PPAR2), an important nuclear transcription factor in adipocyte differentiation; however, its regulation of PPAR2 in differentiated adipocytes is unknown. In this paper, we find that BRL 49653 blocked the ability of TNF to down-regulate the expression and transcription of several adipocyte genes, but BRL 49653 did not prevent TNF from down-regulating PPAR2. Moreover, BRL 49653 alone initially decreased the expression of PPAR2 mRNA and protein greatly. After 24 h of treatment in 3T3-L1 adipocytes, BRL 49653 down-regulated PPAR2 by greater than 90% and potentiated the decrease of PPAR2 mRNA by TNF at this time. These unexpected results prompted us to repeat the experiments for a longer time to determine whether BRL 49653 would continue to down-regulate PPAR2. With prolonged BRL 49653 treatment, PPAR2 mRNA expression was not decreased as greatly, and the protein levels were decreased 20–30% below control at 72 h compared to 90% at 24 h. Although BRL 49653 continued to prevent the inhibitory effects of TNF on perilipin and aP2 mRNA, by 72 h, BRL 49653 was not as potent an inhibitor of TNF’s down-regulation of perilipin protein. Since PPAR2 protein was more abundant at this time, these results suggest that the level of PPAR2 protein is not the sole factor that regulates the transcriptional control by BRL 49653.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tumor necrosis factor- (TNF) is a cytokine synthesized and secreted by diverse cells, including macrophages and adipocytes, with pleiotropic effects on many different cells (1). Initially, TNF was proposed to play a central role in the syndrome of cachexia, which is characterized by a loss of both fat and muscle mass, and is associated with cancer, heart failure, and infectious diseases (1, 2). Recently, adipocyte expression of TNF mRNA was found to correlate with insulin resistance in obese mice and people (3, 4, 5). Furthermore, human adipocytes secrete TNF, suggesting that the actions of TNF are mediated through both autocrine and paracrine pathways (6, 7).

TNF has been shown to alter adipose tissue metabolism and decrease the expression of several adipocyte gene products such as aP2 (the fattyacid-binding protein), GLUT 4 (the insulin-responsive glucose transport protein), C/EBP (the CAAT enhancer-binding protein), and PPAR2 (peroxisome proliferator-activated receptor, a nuclear transcription factor) (PPAR2) (8, 9). PPAR2 is critical for the terminal differentiation of precursor cells into adipocytes. Although PPAR2 is expressed in many different cells at low levels, its predominant expression is in adipocytes, where it is expressed early in the differentiation pathway (10). The expression of PPAR2 increases during the differentiation of preadipocytes to adipocytes (10, 11). This transcription factor is activated by fatty acids, peroxisome proliferators, and disparate lipids such as clofibric acid, linoleic acid, and 5,8,11-eicosatetraynoic acid (the synthetic analog of arachidonic acid) as well as the arachidonic acid metabolite, 15 deoxy-^{12, 14}-prostaglandin J₂ (15d-PGJ₂) (12, 13, 14).

The thiazolidinediones, a family of antidiabetic compounds that includes BRL 49653, have been shown to bind strongly to and activate PPAR2 (14). The ability of these compounds to improve insulin sensitivity in obese animals was directly related to their binding affinity for PPAR2 (14). When a PPAR2 ligand is incubated with preadipocytes or fibroblasts that have been transfected with PPAR2, PPAR2 expression increases, and an increase in adipocyte differentiation is seen (14).

The precise mechanism by which PPAR2 regulates adipocyte differentiation has not yet been defined. However, the evidence strongly suggests a role for PPAR2 in the regulation of many adipocyte genes (15).

Previously, TNF was shown to reduce the expression of PPAR2 (9). Zhang et al. (9) suggested that down-regulation of PPAR2 by TNF was an important part of the mechanism by which TNF reduces expression of several other adipocyte genes. With low level constitutive expression of PPAR2, Zhang and co-workers were able to partially prevent TNF from decreasing the expression of aP2 and adipsin in adipocytes, suggesting that the level of PPAR2 expression is important for TNF to exert its effects on adipocyte gene expression.

In this paper, we use the 3T3-L1 adipoblast cell line to examine the mechanisms by which TNF and BRL 49653 interact to regulate adipocyte gene expression in differentiated adipocytes (16). Most other studies have focused on gene expression during differentiation of preadipocytes to adipocytes (17, 18). For the most part, our results concur with previous findings, but some of our results were unexpected and quite surprising. Although BRL 49653 very effectively opposed TNF’s down-regulation of several adipocyte genes such as perilipin (the lipid droplet-associated protein), aP2, and hormone-sensitive lipase (HSL) (the rate-limiting enzyme in lipolysis) during incubation periods of up to 24 h, it did not prevent the down-regulation of PPAR2 at this time. In fact, BRL 49653 alone from 6–24 h decreased PPAR2 mRNA and protein expression in differentiated cells, and, in combination with TNF, decreased PPAR2 expression further. Longer incubation times with BRL 49653 resulted in a smaller down-regulation of the PPAR2 protein but also less inhibition of the effects of TNF on perilipin protein levels. Therefore, the contrasting effects of BRL 49653 and TNF on gene expression in differentiated adipocytes are not simply the result of alterations in the level of PPAR2 expression. Understanding the mechanism by which the thiazolidinediones antagonize TNF’s actions may increase understanding of how the thiazolidinediones such as BRL49653 ameliorate insulin resistance.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Adipocyte Gene Expression Is Modulated Differentially by TNF and BRL 49653
To investigate the effects of TNF and BRL 49653 on adipocyte gene expression, adipocytes were treated with TNF(250 pM) and BRL 49653(10 µM), separately or combined, and total RNA was isolated at different time points. Our initial experiments were carried out for a maximum of 24 h. As shown in Fig. 1, Northern blot analysis was performed using cDNA probes for PPAR2, perilipin, and aP2. Perilipin and aP2 are expressed in adipocytes but not in preadipocytes and were previously reported to be down-regulated by TNF (9, 19). The effects of BRL 49653 on these genes in differentiated adipocytes has not been reported. Our results show that BRL 49653 affects the expression of PPAR 2 differently than these other adipocyte genes, decreasing rather than increasing its expression.

View larger version (57K): [in this window] [in a new window]   Figure 1. Northern Blot Analyses of Mouse PPAR2, Perilipin, and aP2 after Treatment of 3T3-L1 Adipocytes with or without TNF and/or BRL 49653 Total RNA was isolated from 3T3-L1 adipocytes treated with TNF (250 pM), BRL 49653 (10 µM), both, or neither (control) for 1, 6, and 24 h. Twenty micrograms of total RNA from each condition were analyzed by Northern blot analysis. The following cDNA probes were hybridized consecutively to the same blot: A, PPAR2, perilipin A, and aP2. These autoradiograms are representative of three to five experiments. b, Equal loading was ensured by ethidium bromide staining of the rRNA subunits. c, The autoradiograms were quantified by laser densitometry (Molecular Dynamics Personal Densitometer) using Imagequant software (Molecular Dynamics). Data are presented as the mean ± SEM expressed as a percentage of control. All control values were constant throughout the time course.

Incubation with TNF alone resulted in a significant decrease in steady state mRNA of perilipin, PPAR2, and aP2 after 6 h of treatment; the decrease at 1 h was minimal (Fig. 1). Both PPAR2 and perilipin decreased more than 70% by 6 h, whereas the decrease in aP2 mRNA was slightly delayed, but by 24 h it was also decreased by 70%. Thus, TNF greatly reduced the expression of the mRNAs for several adipocyte- specific genes throughout the 24-h time course.

When the effects of BRL 49653 on mRNA levels were examined, the results were unexpected. The effect of BRL 49653 on PPAR2 differed from its effects on perilipin and aP2 mRNA expression in 3T3-L1 adipocytes, especially during the first 24 h of treatment. BRL 49653 induced both perilipin and aP2 mRNA levels about 200% at 24 h with little effect at 1 or 6 h of treatment compared with untreated controls (Fig. 1). Surprisingly, the only message examined that was not induced by BRL 49653 was PPAR2. In fact, the effect of BRL 49653 on PPAR2 mRNA was similar to the effect of TNF; the thiazolidinedione decreased the mRNA of its own receptor; by 6 h BRL 49653 reduced the PPAR2 mRNA by 50%, and this decrease was seen through 24 h.

When TNF and BRL 49653 were incubated together for 6 h, the thiazolidinedione greatly inhibited the ability of TNF to down-regulate perilipin and, to a greater extent, aP2 mRNA, as determined by densitometry (Fig. 1c). After 24 h of coincubation, BRL 49653 completely prevented the ability of TNF ability to decrease aP2, and it prevented the ability of TNF to decrease perilipin mRNA by 50%, as compared with TNF alone. By contrast, BRL 49653 did not prevent TNF from decreasing the level of PPAR2 mRNA expression; in fact, the PPAR2 message declined further in the presence of both agents than with either one alone (Fig. 1). Whereas TNF decreased PPAR2 by 65% at 24 h and BRL 49653 decreased PPAR2 by 50%, together they decreased PPAR2 mRNA by 90% at 24 h. In conclusion, both TNF and BRL 49653 decreased the PPAR2 mRNA throughout the time course, with the greatest effect occurring at 24 h.

TNF Decreases Adipocyte Gene Transcription
The decrease in steady state message of several adipocyte genes by TNF (including the nuclear transcription factor PPAR2) suggested that the effects of TNF were mediated in part at the level of transcription. The effects of TNF on adipocyte gene expression at the level of transcription were examined (Fig. 2). First, 3T3-L1 adipocytes were treated with TNF for 1, 3, or 6 h and nuclei were isolated. The nuclei were subsequently used in a nuclear run-on assay (Fig. 2). TNF dramatically decreased the transcription of PPAR2, perilipin, and HSL by 1 h. Bluescript plasmid without insert was undetected and served as a negative control. Actin transcription decreased slightly at 1 h but increased slightly at 3 and 6 h (2-fold over control levels) consistent with what others have seen (8). We also saw a similar increase in steady-state levels of actin mRNA in the presence of TNF (data not shown). Therefore, the effects of TNF on transcription were specific, affecting all of the adipocyte-specific genes examined.

View larger version (60K): [in this window] [in a new window]   Figure 2. Nuclear Run-on Analysis of 3T3-L1 Adipocytes Treated with or without TNF Nuclei were isolated from 3T3-L1 adipocytes that had been treated for 0, 1, 3, or 6 h with TNF (250 pM). New RNA transcripts were synthesized in the presence of [32P]UTP, the radiolabeled RNA was isolated, and 2 x 106 cpm from each condition were hybridized to cDNA probes (15 µg/slot) previously slot blotted onto a positively charged nylon membrane. The blot was washed, and the signal was detected using a PhosphorImager. Densitometry was performed using ImageQuant from Molecular Dynamics. This blot is representative of three experiments with the same results.

BRL 49653 Inhibits TNF from Decreasing Transcription of Adipocyte Genes Other Than PPAR2

View larger version (76K): [in this window] [in a new window]   Figure 3. Nuclear Run-on Analysis of 3T3-L1 Adipocytes Treated with or without TNF ± BRL 49653 Nuclei were isolated from 3T3-L1 adipocytes treated with or without TNF (250 pM), BRL 49653 (10 µM), or both for 1 h, and the nuclear run-on assay was performed as in Fig. 2, except the radioactive signal was detected on Kodak XAR film instead of the PhosphorImager. Densitometry was performed as previously described, and the results are reported in Table 1. This experiment was performed four times with similar results.

Ectopic expression of the transcription factor C/EBPß, alone or in combination with C/EBP in fibroblasts, followed by treatment with steroids, has been reported to stimulate expression of PPAR2 and adipocyte differentiaton (20). We therefore investigated whether down-regulation of PPAR2 by TNF and BRL 49653 reflected a decrease in the transcription of C/EBPß and C/EBP. The results indicate that the 85–90% reduction in PPAR2 transcription in the presence of TNF and BRL 49653 is not accompanied by significant reductions in C/EBPß or . In fact, C/EBPß and C/EBP transcription levels are actually increased by 38% and 59%, respectively, in the presence of BRL (Fig. 3 and Table 1).

View this table: [in this window] [in a new window]   Table 1. The Effect of BRL and TNF on Adipocyte Gene Transcription

Both TNF and BRL 49653 Reduce PPAR2 Protein Levels
Since both TNF and BRL 49653 separately and together reduce PPAR2 mRNA levels, their effects on protein expression were examined. Consistent with the data from both the Northern and nuclear run-on studies, both TNF and BRL 49653 decreased PPAR2 protein levels (Fig. 4a). With TNF treatment, PPAR2 protein decreased by 45% at 1 h, 70% at 4 h, and greater than 90% at 24 h. BRL 49653 alone reduced PPAR2 protein level by 45% at 1 h and 4 h and by greater than 50% at 24 h. When BRL 49653 was coincubated with TNF, the effects were similar to BRL 49653 alone; by 24 h PPAR2 protein was reduced by greater than 80%. The combination of the two agents did not reduce the expression of PPAR2 protein to a greater extent than either one alone.

View larger version (26K): [in this window] [in a new window]   Figure 4. Western Blot Analyses of PPAR2 and Perilipin Proteins during a 24-h Time Course. 3T3-L1 Adipocytes Were Treated with or without TNF and/or BRL 49653 Protein extracts were isolated from 3T3-L1 cells treated with TNF (250 pM), BRL 49653(10 µM), both, or neither from 1–24 h and analyzed by Western blot on a 10% SDS-polyacrylamide gel. The antibodies were detected using enhanced chemiluminescence and Kodak XAR film. A, The mPPAR2 time course. B, The perilipin time course. C, These blots are representative of three experiments with the same results; the means are represented in the graphs below.

The Arachidonic Acid Metabolite 15d-PGJ₂ Has a Similar Mechanistic Effect to the Synthetic Agent BRL 49653
The arachidonic acid metabolite 15d-PGJ₂ is a naturally occurring ligand for PPAR2. The effects of 15d-PGJ₂ on PPAR2 protein expression were similar to the efects of both TNF and BRL49653. The 15d-PGJ₂ compound caused PPAR2 to decline significantly (Fig. 5). Also, 15d-PGJ₂ or BRL 49653, in combination with TNF, caused a further decline in PPAR2 protein expression compared with any of the three agents alone. PPAR1, which is also recognized by the PPAR antibody, was similarly decreased by 15d-PGJ₂, BRL 49653, and TNF (Fig. 5, lower band). In summary, both 15d-PGJ₂ and BRL 49653 decreased PPAR2 protein expression at 24 h in differentiated adipocytes, which is contrary to their previously reported actions in preadipocytes (21).

View larger version (25K): [in this window] [in a new window]   Figure 5. Western Blot Analysis of PPAR2 in 3T3-L1 Adipocytes Treated with or without 15d-PGJ2 or BRL 49653 in the Presence or Absence of TNF 3T3-L1 adipocytes were treated with TNF (250 pM), BRL 49653(10 µM), 15 d-PGJ2(15 µM), or a combination of TNF with BRL 49653 or 15d-PGJ2 for 24 h, and proteins were extracted as previously described. The proteins were separated in a 10% SDS-polyacrylamide gel, and chemiluminescence was used to detect PPAR2 protein. This antibody also recognizes PPAR1, which is depicted as the lower band in this figure.

The Effects of TNF and BRL 49653 on PPAR2, Perilipin, and aP2 mRNA Expression at 48 and 72 h

View larger version (25K): [in this window] [in a new window]   Figure 6. Northern Blot Analyses of Mouse PPAR2, Perilipin, and aP2 during Prolonged Treatment of 3T3-L1 Adipocytes with or without TNF and/or BRL 49653 Total RNA was isolated from 3T3-L1 adipocytes treated with TNF (250 pM), BRL 49653 (10 µM), both, or neither (control) for 48 or 72 h. The cells were refed each day with serum-depleted media containing 2% BSA containing the appropriate treatment. Twenty micrograms of total RNA from each condition were analyzed by Northern blot analysis. The following cDNA probes were hybridized consecutively to the same blot: A, PPAR2, perilipin A, and aP2. These autoradiograms are representative of three to four experiments. B, Equal loading was ensured by ethidium bromide staining of the rRNA subunits. C, The autoradiograms were quantified by laser densitometry (Molecular Dynamics Personal Densitometer) using Imagequant software (Molecular Dynamics). Data are presented as the mean ± SEM expressed as a percentage of control.

When BRL 49653 Is Added to 3T3-L1 Cells Pretreated for 24 h with TNF, It Prevents Decreases in Perilipin and aP2 mRNA Expression but Not in PPAR2 Expression

View larger version (18K): [in this window] [in a new window]   Figure 7. Northern Blot Analyses of 3T3-L1 Cells Treated with BRL 49653 and/or TNF after a 24-h Pretreatment with TNF The 3T3-L1 cells were preincubated with TNF (250 pM) for 24 h and subsequently refed with either TNF, TNF +BRL, or BRL alone, in serum- depleted medium containing 2% BSA for an additional 48 h. A, Northern blot showing that BRL, either alone or in the presence of TNF, is able to reverse the inhibitory effects of TNF by increasing perilipin and aP2 expression but does not reverse TNF’s inhibition of PPAR2 mRNA levels. B, Blots are representative of three experiments with the same results; the means are represented in the graphs below.

The Effect of TNF and BRL 49653 on PPAR2 and Perilipin Protein Expression at 48 and 72 h

View larger version (24K): [in this window] [in a new window]   Figure 8. Western Blot Analyses of PPAR2 and Perilipin Proteins Extracted from 3T3-L1 Adipocytes Treated with or without TNF ± BRL 49653 for 48 or 72 h Protein extracts were isolated from 3T3-L1 cells treated with TNF (250 pM), BRL 49653(10 µM), both, or neither for 48 or 72 h and analyzed by Western blot on a 10% SDS-polyacrylamide gel. The antibodies were detected using ECL and Kodak XAR film. A, The mPPAR2 time course. B, The perilipin A timecourse. C, Blots representative of three experiments with the same results; the means are represented in the graphs below.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

A direct relationship between high levels of adipocyte secretion of TNF and the insulin resistance of obesity has been strongly suggested (3, 22). BRL 49653 has been effective in ameliorating insulin resistance in obese individuals. The exact mechanisms used by BRL 49653 are unknown, but it appears to have an effect on adipocyte metabolism, and its antidiabetic actions appear to be mediated through PPAR2 (23). The evidence connecting PPAR2 activity to the antidiabetic activities of the thiazolidinediones (TZDs) includes the data showing that the TZDs with the greatest antidiabetic activity also possessed the strongest binding affinity for PPAR2. In addition, non-TZD ligands with strong affinitity to PPAR2 have also been shown to possess antidiabetic activity (23). We therefore examined the effects of TNF and BRL 49653 on the expression of PPAR2 and other adipocyte genes.

In our run-on experiments, we also examined whether TNF or BRL 49653 alters the transcription of the transcription factors, C/EBP_ß and C/EBP. Recent experiments by Wu et. al. (20) have shown that conditional expression of C/EBP_ß and C/EBP in the presence of steroid directly stimulates the appearance of PPAR2, and that if the stimulus for C/EBPß and C/EBP expression is removed, the levels of expression of PPAR2 and aP2 mRNA subsequently fall. The run-on experiments indicated that neither TNF nor BRL 49653 decreased C/EBP/ transcription levels significantly. Thus, our data suggest that the actions of TNF and BRL on PPAR2 expression are probably not mediated through C/EBPß/ in differentiated adipocytes.

In our experiments with 3T3-L1 adipocytes, BRL 49653 prevented the TNF- associated decrease in perilipin A mRNA and protein expression. Interestingly, BRL 49653 was more effective at counteracting the effects of TNF on perilipin protein expression at 24 h of treatment than it was at 6 h, suggesting that BRL 49653 may need time to overcome the actions of TNF. BRL 49653 did not, however, prevent the TNF-associated decrease in PPAR2 expression. In fact, TNF and BRL 49653 together decreased PPAR2 mRNA expression to a greater extent than either agent alone. It is also possible that the PPAR2 mRNA is degraded faster than the PPAR2 protein, which may be more stable during the longer treatments. Recently, Wu et. al. (24) reported that, in adipocyte-like cells, the induction of GLUT 4 mRNA expression (the insulin-dependent glucose transporter) by PPAR2 required 24–48 h of exposure to the TZD, ciglitazone, suggesting that the effects of PPAR2 are not immediate and occur over a period of time.

In addition to altering the transcription of several adipocyte genes, TNF has also been shown to exert posttranscriptional control by increasing mRNA turnover (25, 26). The amount of time it takes BRL 49653 to work maximally may be the result of its actions at may different levels, some transcriptional and some posttranscriptional. Surprisingly, BRL 49653 was less effective in preventing TNF from decreasing perilipin protein (but not mRNA) at 72 h than 24 h, even though PPAR2 protein expression was elevated compared with its 24 h level, suggesting that increased amounts of PPAR2 do not necessarily lead to an increase in BRL 49653 activity.

Since both TNF and BRL 49653 decrease PPAR2 expression, yet have disparate effects on the expression of other adipocyte-specific genes, we speculate that a low level of PPAR2 protein expression may be sufficient for activity. The ability of PPAR2 to exert its actions on gene expression in the presence of one of its ligands, i.e. BRL 49653, may depend on factors other than the amount of PPAR2 present.

Recent studies have also suggested that phosphorylation of PPAR2 by kinases such as mitogen-activated protein kinase reduces its ability to bind to its target DNA binding sequences, and therefore reduces its activity (27). TNF is a weak activator of mitogen-activated protein kinase, and the ability of PPAR2 to bind to its target DNA sequence may be decreased by phosphorylation as a result of the actions of TNF in adipocytes (27). BRL 49653 may then overcome this block. The reduction in mRNA and protein levels of PPAR2 and other adipocyte-specific genes after TNF treatment may be important for the antiadipogenic effects of TNF that could be due to changes in complex interactions secondary to phosphorylation (9).

Recently, a coactivator of PPAR2, called mouse steroid receptor coactivator-1 (mSRC-1), has been isolated. This coactivator binds to PPAR2 in a ligand-independent fashion. When mSRC-1 is coexpressed with PPAR2, and a PPAR2 ligand is present, the transcriptional activity of mPPAR2 increases (28). One may speculate that BRL 49653 is a potent stimulator of this coactivator.

In summary, the work presented in this paper indicates that both TNF and BRL 49653 decrease the transcriptional and translational regulation and expression of PPAR2. However, BRL 49653 antagonizes the actions of TNF on the expression of other adipocyte-specific genes and, alone, induces expression of these genes. Understanding the interactions between TNF and BRL 49653 on adipocyte gene expression may help us determine how the thiazolidinediones enhance insulin sensitivity in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Cell Culture and Differentiation
3T3-L1 fibroblasts (ATTC) were grown to confluence in DMEM containing 10% calf serum. At confluence the cells were treated with the differentiation mix of 10 µM dexamethasone and 5 mM isobutyl-methylxanthine in DMEM containing 10% FBS for 3 days. This was followed by a 2-day treatment of 10 µg/ml insulin in DMEM containing 10% FBS. The cells were then allowed to grow for another 5 days in DMEM/FBS without additional hormones. On day 10 the cells were serum deprived overnight, with DMEM containing 2% BSA or 0.5% calf serum and any treatments commenced the next day. The dose of TNF used was 10 ng/ml, which is equivalent to 250 pM. The dose of BRL 49653 used was 10 µM, previously shown to be the maximum effective dose (13).

Reagents
TNF was purchased from Genzyme Corp. (Cambridge, MA). BRL 49653 was originally purchased from Biomol Corp. (Plymouth Meeting, PA) but recently was generously provided by Hamish Ross from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). 15d-PGJ₂ was purchased from Cayman Chemical (Ann Arbor, MI).

RNA Analysis
RNA was isolated using TRIZOL (GIBCO/BRL Life Technologies, Gaithersburg, MD) following the instructions provided by the manufacturer. Briefly, the 3T3-L1 cells were rinsed twice with PBS and 3 ml of TRIZOL were added to each 100-mm tissue culture plate. The cells were scraped, transferred to a 15-ml tube, and allowed to incubate for 5 min at room temperature. Then, 0.6 ml of chloroform was added, and the tubes were shaken vigorously and centrifuged for 10 min at 4 C. The aqueous phase was removed, and the RNA was precipitated with isopropyl alcohol and centrifuged for 20 min at 4 C. The pellet was washed with 70% ethanol, dried, and dissolved in sterile, distilled water. Northern blot analysis was performed using HYBOND-N nylon transfer membrane (Amersham, Arlington Heights, IL), which was cross-linked, incubated in Express Hyb hybridization solution (CLONTECH, Palo Alto, CA), and hybridized to cDNA probes labeled with [³²P]dCTP using the random primer method (29).

Probes
The cDNA probes used were: murine perilipin A, a generous gift from the laboratories of Drs. Jasmine Gruia-Gray, Alan Kimmel, and C. Londos; murine aP2 was kindly provided by Dr. David Bernlohr; rat HSL was kindly provided by Drs. Cecilia Holm and Michael Schotz; C/EBPß and C/EBP were kindly provided by Dr. Steve Farmer; ß-actin was kindly provided by Dr. Phil Pekala; and murine PPAR was supplied by Drs. Peter Tontonoz and Bruce Spiegelman. The PPAR probe recognizes both PPAR1 and 2; however, the predominant isoform expressed in 3T3-L1 adipocytes is 2.

Two mPPAR2 antibodies were used: one was the generous gift of Dr. Mitchell Lazar and the other was purchased from Affinity Bioreagents (Golden, CO). Both gave similar results.

Western Analysis
For perilipin Western blots, 50 µg of protein from 3T3-L1 adipocytes were separated on a 10% SDS polyacrylamide gel, and transferred to nitrocellulose using a semidry transfer apparatus from Owl Scientific (Woburn, MA). The membrane was blocked overnight in Tris buffer containing 5% BSA, incubated with the same, fresh buffer containing the primary antibody for 1 h, washed, incubated with the horseradish peroxidase-linked secondary antibody, and subjected to enhanced chemiluminescence (ECL) from Amersham. Autoradiography was then performed. With the mPPAR2 antibody, nonfat dried milk replaced the BSA as a blocking agent to avert the formation of background bands resulting from the primary antibody cross-reacting with BSA. Also, 100–150 µg of total protein were loaded in each lane since the PPAR2 protein is much less abundant than perilipin (30).

Perilipin antibodies were generated using a peptide that is identical to amino acids 9–23 of the rat perilipin (31) (CLLDGDLPEQENVLQ) to immunize rabbits (Quality Controlled Biochemicals, Inc., Hopkinton, MA). The serum was subsequently affinity purified with a peptide column and used at a dilution of 1:1500. For HSL, a peptide KDLSFKGNSEPSDSPEMC, based upon the rat (GeneBank) HSL sequence, was used to immunize rabbits (QCB). The antisera were subsequently affinity purified over a peptide column and used for Western blotting at a dilution of 1:1500.

Nuclear Run-On Analysis
This procedure was performed following the protocol of Greenberg and Ziff (32) with minor modifications. Briefly, cells were washed with ice-cold PBS, scraped, and lysed in buffer containing 0.5% NP-40, 10 mM Tris, 10 mM NaCl, 3 mM MgCl₂, 0.001 M dithiothreitol, and 0.25 M sucrose. The nuclei were pelleted, washed once with lysis buffer without NP-40, and quickly frozen in glycerol storage buffer in liquid N₂ until needed. The nuclei were then thawed on ice and incubated with an equal volume of 2x reaction buffer containing cold ribonucleotides and 100 µCi [-³²P]UTP for 30 min at 30 C. The radioactive RNA was isolated through a series of purification steps and then hybridized to cDNA plasmids (10–15 µg/slot) that had been previously slot blotted onto a positively charged nylon membrane. The hybridization was allowed to continue for 2 days, and the membranes were then washed and exposed to x-ray film, or the radioactivity was detected on the PhosphorImager. The film was then scanned with a Personal Densitometer from Molecular Dynamics (Sunnyvale, CA) and was analyzed using Imagequant software provided by the manufacturer.

	ACKNOWLEDGMENTS

 
We would like to thank Drs. Martin Obin, Eric Paulson, and Sandra Souza for their critical reviews of this manuscript. We would also like to thank Dr. Susan Fried for her helpful advice and Mia Yamamoto and Ping Lien for their technical assistance.

	FOOTNOTES

 
Address requests for reprints to: Susan Edelstein Rosenbaum, The USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, and Division of Endocrinology, Tupper Medical Research Institute, New England Medical Center, Boston, Massachusetts 02111.

This work was supported by NIH Grants T32DK-07704 and P30 DK-40561.

{smhd3}Note Added in Proof.

In preliminary studies, S. C. Souza and A. S. Greenberg in our laboratory have found that 5-day treatment of 3T3-L1 cells with TNF plus BRL increases the protein expression of perilipin A approximately 2-fold compared to TNF alone.

Received for publication June 12, 1997. Revision received April 17, 1998. Accepted for publication April 23, 1998.

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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