Interferon-gamma -induced Regulation of Peroxisome Proliferator-activated Receptor gamma  and STATs in Adipocytes*

Kyle J. Waite, Z. Elizabeth Floyd, Patricia Arbour-Reily, and Jacqueline M. StephensDagger

From the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

Received for publication, August 29, 2000, and in revised form, October 24, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interferon-gamma (IFN-gamma ) is known primarily for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. To further investigate the effects of IFN-gamma on fat cells, we examined the effects of this cytokine on the expression of adipocyte transcription factors in 3T3-L1 adipocytes. Although IFN-gamma regulated the expression of several adipocyte transcription factors, IFN-gamma treatment resulted in a rapid reduction of both peroxisome proliferator-activated receptor (PPAR) protein and mRNA. A 48-h exposure to IFN-gamma also resulted in a decrease of both CCAAT/enhancer-binding alpha  and sterol regulatory element binding protein (SREBP-1) expression. The short half-life of both the PPARgamma mRNA and protein likely contributed to the rapid decline of both cytosolic and nuclear PPARgamma in the presence of IFN-gamma . Our studies clearly demonstrated that the IFN-gamma -induced loss of PPARgamma protein is partially inhibited in the presence of two distinct proteasome inhibitors. Moreover, IFN-gamma also inhibited the transcription of PPARgamma , which was accompanied by a decrease in PPARgamma mRNA accumulation. In addition, exposure to IFN-gamma resulted in a substantial increase in STAT 1 expression and a small increase in STAT 3 expression. IFN-gamma treatment of 3T3-L1 adipocytes (48-96 h) resulted in a substantial inhibition of insulin-sensitive glucose uptake. These data clearly demonstrate that IFN-gamma treatment results in the development of insulin resistance, which is accompanied by the regulation of various adipocyte transcription factors, in particular the synthesis and degradation of PPARgamma .



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The adipocyte plays an active role in a variety of physiological and pathological processes regulating energy metabolism. The recent consideration of adipose tissue as an endocrine organ that secretes a variety of unrelated bioactive molecules has broadened our understanding of adipocyte function to exceed its previously considered passive role in lipid metabolism. A number of cell lines are available for studying adipocytes. The 3T3-L1 cell line differentiates under the controlled conditions of cell culture from fibroblasts, or preadipocytes, to cells with the morphological and biochemical properties of adipocytes (1, 2). The 3T3-L1 adipocytes are comparable with native adipocytes as they have the ability to accumulate lipid, respond to insulin, and secrete leptin. The major transcription factors involved in adipocyte gene regulation include peroxisome proliferator-activated receptor gamma , proteins belonging to the CCAAT/enhancer-binding protein family, and adipocyte determination and differentiation-dependent factor 1, also known as sterol regulatory element-binding protein (reviewed in Refs. 3 and 4).

Recent studies have also suggested that the signal transducer and activator of transcription (STAT)1 family of transcription factors may also be important in fat cells. The STAT family of transcription factors is comprised of seven family members (STATs 1, 2, 3, 4, 5A, 5B, and 6) that, in response to the stimulation of various receptors, mainly those for cytokines, are phosphorylated on tyrosine residues, which causes their translocation to the nucleus. Each STAT family member shows a distinct pattern of activation by cytokines and upon nuclear translocation can regulate the transcription of particular genes in a cell- or tissue-specific manner (5). In fat cells, the expression of STATs 1, 5A, and 5B is highly induced during differentiation and correlates with lipid accumulation (6, 7). The regulation of STAT expression has also been investigated in NIH 3T3 cells ectopically overexpressing C/EBPs beta  and delta , a condition that results in adipogenesis (8). In these studies, the expression of STATs 1, 5A, and 5B was induced in a PPARgamma ligand-dependent fashion during adipogenesis (9). STATs 3 and 6 are also expressed in adipocytes, but the expression of these proteins does not change during differentiation. However, the tyrosine phosphorylation of STAT 3 occurs following the induction of differentiation, and studies with antisense STAT 3 suggest that this protein may be important in adipogenesis (10). Although the functions of STATs in fat cells have not been identified, numerous studies suggest that these transcription factors may be important regulators of adipocyte gene expression.

Interferon-gamma (IFN-gamma ) is primarily known for its roles in immunological responses but also has been shown to affect fat metabolism and adipocyte gene expression. In adipocytes, IFN-gamma treatment results in a decrease of lipoprotein lipase activity and increased lipolysis (11). In 3T3-F442 adipocytes, exposure to IFN-gamma results in a decreased expression of lipoprotein lipase and fatty acid synthase. Also in various rodent preadipocyte cell lines, IFN-gamma inhibits the differentiation of preadipocytes (12-14). We have recently shown that acute IFN-gamma treatment of cultured and native adipocytes results in a dose- and time-dependent activation of STATs 1 and 3 (15). Exposure of adipocytes to IFN-gamma results in the tyrosine phosphorylation and nuclear translocation of STATs 1 and 3 in fat cells. Because IFN-gamma has effects on adipocyte gene expression, we examined the effects of this cytokine on the expression of a variety of adipocyte transcription factors.

Although we observed that IFN-gamma affected the expression of a number of adipocyte transcription factors, the most profound effect of IFN-gamma was on the expression of PPARgamma . PPARgamma is a member of the nuclear hormone superfamily and exists as two isoforms, PPARgamma 1 and PPARgamma 2, which are transcribed from the same gene by the use of alternative promoters (16). PPARgamma 2 is 30 amino acids longer than PPARgamma 1 and is largely adipocyte-specific. Although expressed in a variety of other tissues, PPARgamma 1 is also predominately expressed in fat (17). Thiazolidinediones (TZDs) are high affinity synthetic ligands of PPARgamma and have recently been shown to affect the degradation of this transcription factor (18). Our studies with IFN-gamma also indicate that PPARgamma is targeted to the proteasome for degradation, but this is not the only mechanism for the substantial effect that IFN-gamma has on PPARgamma expression. Our findings indicate that a newly identified inhibitor of PPARgamma expression, IFN-gamma , results in a substantial loss of PPARgamma expression by regulating two cellular events as follows: 1) targeting PPARgamma to the proteasome for degradation and 2) inhibiting the synthesis of PPARgamma . Prolonged IFN-gamma treatment of 3T3-L1 adipocytes also results in the development of insulin resistance and regulation of other adipocyte transcription factors and supports the hypothesis that PPARgamma is involved in conferring insulin sensitivity.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Dulbecco's modified Eagle's medium (DMEM) was purchased from Life Technologies, Inc. Bovine and fetal bovine serum was obtained from Sigma and Life Technologies, Inc., respectively. Murine interferon-gamma (IFN-gamma ) was purchased from Roche Molecular Biochemicals. Actinomycin D was purchased from Calbiochem. Cycloheximide was purchased from Sigma. The nonphospho STAT antibodies were either monoclonal IgGs purchased from Transduction Laboratories or polyclonal IgGs from Santa Cruz Biotechnology Inc. A highly phospho-specific polyclonal antibody for STAT 1 (Y701) was provided by Quality Controlled Biochemicals. PPARgamma was a mouse monoclonal antibody from Santa Cruz Biotechnology Inc. SREBP-1, C/EBPalpha , and ERK1/ERK2 were rabbit polyclonal antibodies from Santa Cruz Biotechnology Inc.

Cell Culture-- Murine 3T3-L1 preadipocytes were plated and grown to 2 days postconfluence in DMEM with 10% bovine serum. Medium was changed every 48 h. Cells were induced to differentiate by changing the medium to DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin. After 48 h this medium was replaced with DMEM supplemented with 10% fetal bovine serum, and cells were maintained in this medium until utilized for experimentation.

Preparation of Whole Cell Extracts-- Monolayers of 3T3-L1 adipocytes were rinsed with phosphate-buffered saline and then harvested in a nondenaturing buffer containing 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 µM leupeptin, and 2 mM sodium vanadate. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4 °C for 15 min. Supernatants containing whole cell extracts were analyzed for protein content using a BCA kit (Pierce) according to the manufacturer's instructions.

Preparation of Nuclear/Cytosolic Extracts-- Cell monolayers were rinsed with phosphate-buffered saline and then harvested in a nuclear homogenization buffer (NHB) containing 20 mM Tris (pH 7.4), 10 mM NaCl, and 3 mM MgCl2. Nonidet P-40 was added to a final concentration of 0.15%, and cells were homogenized with 16 strokes in a Dounce homogenizer. The homogenates were centrifuged at 1500 rpm for 5 min. Supernatants were saved as cytosolic extract, and the nuclear pellets were resuspended in 0.5 volume of NHB and centrifuged as before. The pellet of intact nuclei was resuspended again in 0.5 of the original volume of NHB and centrifuged again. A small portion of the nuclei was used for trypan blue staining to examine the integrity of the nuclei. The majority of the pellet (intact nuclei) was resuspended in an extraction buffer containing 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol. Nuclei were extracted for 30 min on ice and then placed at room temperature for 10 min. Two hundred units of DNase I were added to each sample, and tubes were inverted and incubated an additional 10 min at room temperature. Finally, the sample was subjected to centrifugation at 15,000 rpm at 4 °C for 30 min. Supernatants containing nuclear extracts were analyzed for protein content.

Gel Electrophoresis and Immunoblotting-- Proteins were separated in 5, 7.5, or 12% polyacrylamide (acrylamide from National Diagnostics) gels containing sodium dodecyl sulfate (SDS) according to Laemmli (19) and transferred to nitrocellulose (Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol. Following the transfer, the membrane was blocked in 4% milk for 1 h at room temperature. Results were visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Pierce).

RNA Analysis-- Total RNA was isolated from cell monolayers with TriZOL (Life Technologies, Inc.) according to the manufacturer's instruction with minor modifications. For Northern blot analysis, 20 µg of total RNA was denatured in formamide and electrophoresed through a formaldehyde-agarose gel. The RNA was transferred to Zeta Probe-GT (Bio-Rad), cross-linked, hybridized, and washed as previously described (20). Probes were labeled by random priming using the Klenow fragment (Promega) and [alpha -32P]dATP (PerkinElmer Life Sciences).

Determination of 2-Deoxyglucose-- The assay of 2-[3H]deoxyglucose was performed as previously described (21). Prior to the assay, fully differentiated 3T3-L1 adipocytes were serum-deprived for 2-4 h. Uptake measurements were performed in triplicate under conditions where hexose uptake was linear, and the results were corrected for nonspecific uptake and absorption determined by 2-[3H]deoxyglucose uptake in the presence of 5 µM cytochalasin B (Sigma). Nonspecific uptake and absorption were always less than 10% of the total uptake.

Nuclei Isolation and Run-on Transcription Assays-- Following exposure of fully differentiated adipocytes to IFN-gamma for 1 h, the cell monolayers (six 10-cm plates per time point) were washed once with ice-cold phosphate-buffered saline and nuclei were isolated, and run-on transcription assays were performed as we have previously described (20).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The expression of adipocyte transcription factors was examined following a time course of IFN-gamma treatment on fully differentiated 3T3-L1 adipocytes. As shown in Fig. 1, immunoblotting of whole cell extracts demonstrated that IFN-gamma treatment resulted in a significant decrease in PPARgamma 2 (upper band) and -gamma 1(lower band) within 24 h and resulted in a notable decline in C/EBPalpha . The expression of STATs 1 and 3 increased following a 24-h IFN-gamma treatment. The expression of STATs 5A, 5B, and 6 was not regulated by exposure to IFN-gamma treatment. Also, the expression of SREBP-1 decreased after a 48-h treatment. The spliced 67-kDa form of SREBP-1 was similarly decreased with IFN-gamma treatment (data not shown).



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Fig. 1.   The effects of IFN-gamma on the expression of adipocyte transcription factors. Whole cell extracts were prepared from fully differentiated 3T3-L1 adipocytes following a treatment with 100 units/ml IFN-gamma for 0, 24, 48, 72, or 96 h. Cells were treated every 24 h with a fresh bolus of IFN-gamma . Extracts were prepared as described under "Experimental Procedures." One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. The molecular mass of each protein is indicated to the left of the blot in kilodaltons. The detection system was horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Pierce). This is a representative experiment independently performed three times.

As shown in Fig. 1, a 24-h treatment of IFN-gamma resulted in a substantial loss of PPARgamma 2 and -gamma 1 protein expression. Therefore, we examined the effects of IFN-gamma over a 24-h time course. Whole cell extracts were isolated from fully differentiated 3T3-L1 adipocytes that were treated with IFN-gamma for the various times indicated in Fig. 2. Interestingly, IFN-gamma resulted in a substantial loss of PPARgamma 2 and -gamma 1 expression within 6 h. In addition, we observed a striking increase in STAT 1 expression between 8 and 12 h and a small increase in STAT 3 during this time period. There was no change in STAT 5A during this time course. JAK 1, the kinase that activates STAT 1 in adipocytes, increases slightly with IFN-gamma treatment. In addition, fatty acid synthase expression decreased with IFN-gamma treatment.



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Fig. 2.   IFN-gamma treatment results in a rapid loss of PPARgamma expression in adipocytes. Whole cell extracts were prepared from fully differentiated 3T3-L1 adipocytes following a treatment with 100 units/ml IFN-gamma as indicated at the top of the figure. One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. The molecular mass of each protein is indicated to the left of the blot in kilodaltons. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed three times. (JAK1, the kinase that activates STAT1 in adipocytes.

Clearly, an analysis of whole cell extracts reveals a substantial loss of PPARgamma 2 and -gamma 1 expression in adipocytes following IFN-gamma treatment. However, it was unclear whether IFN-gamma had any effect on the amount of PPARgamma proteins present in the adipocyte nucleus. To further examine the inhibition of PPARgamma by IFN-gamma , we performed another time course in which adipocytes were fractionated to isolate cytosolic and nuclear extracts. As shown in the top panel of Fig. 3, the majority of PPARgamma 2 and -gamma 1 protein was present in the nucleus, and the amount of nuclear PPARgamma protein was substantially reduced after 6 h. A darker exposure of this blot indicates the presence of PPARgamma proteins in the cytosol in untreated adipocytes and cells that were exposed to IFN-gamma for 30 min. However, following a 6-h or greater IFN-gamma treatment, there was no detectable PPARgamma 2 or -gamma 1 in the cytosol and a significant loss of both PPARgamma isoforms in the nucleus. We also observed an increase in STAT 1 in the cytosol between 6 and 12 h and the presence of activated STAT 1 in the nucleus following a 30-min treatment with IFN-gamma . Detection of the phosphorylated form of STAT 1 was performed with an antibody specific for phosphorylation on tyrosine 701 (STAT 1 Y701). Analysis with either one of these STAT 1 antibodies demonstrates the presence of STAT 1 in the nucleus following a 30-min IFN-gamma stimulation. However, the STAT 1 Y701 antibody is more sensitive, and we observed this protein in the nucleus even after a 12-h IFN-gamma treatment. We have previously reported that STAT 5A is present in the nucleus of adipocytes under basal conditions (15), and IFN-gamma treatment does not cause a redistribution of this protein. Therefore, STAT 5A (Fig. 3, bottom panel) is shown to indicate the even loading of both cytosolic and nuclear samples.



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Fig. 3.   IFN-gamma treatment results in a decrease of nuclear PPARgamma and an increase in cytosolic STAT 1 in adipocytes. Cytosolic and nuclear extracts were isolated from fully differentiated 3T3-L1 adipocytes following treatment with IFN-gamma as indicated at the top of the figure. One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed two times.

The rapid loss of PPARgamma 1 and -gamma 2 proteins in the presence of IFN-gamma suggested that the PPARgamma proteins may be labile. Therefore, we examined the decay of PPARgamma and STATs in 3T3-L1 adipocytes. Whole cell extracts were isolated from 3T3-L1 adipocytes at various times following the addition of 5 µM cycloheximide (+CH) or ethanol (-CH), a vehicle control. As shown in Fig. 4, the inhibition of protein synthesis resulted in the loss of PPARgamma by 12 h with over half of the protein decayed by 6 h. A log plot of the remaining protein versus time was used to estimate the half-life of PPARgamma and of adipocyte-expressed STAT proteins. The estimated half-life of these proteins is indicated in Fig. 4 and is an average calculation of three independent experiments. PPARgamma 1 and -gamma 2 are labile compared with the STAT proteins, which have half-lives at least twice as long as the PPARgamma proteins. We also investigated the effect of IFN-gamma on PPARgamma in the presence of cycloheximide. Because of experimental variability, it was difficult to quantitate the decrease in the half-life of the PPARgamma proteins in the presence of IFN-gamma . However, in each experiment, the decay of the PPARgamma was quicker in the presence of IFN-gamma , as shown in Fig. 5. Adipocytes were treated with 5 µM cycloheximide in the presence or absence of IFN-gamma , and whole cell extracts were isolated at 0, 1, and 4 h. As shown in Fig. 5, the decay of both PPARgamma 2 and -gamma 1 is increased in the presence of IFN-gamma with a complete loss of PPARgamma 1 at 4 h.



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Fig. 4.   The turnover of PPARgamma and adipocyte-expressed STAT proteins. Whole cell extracts were prepared from 3T3-L1 adipocytes following various periods of treatment with 5 µM cycloheximide (+CH) or ethanol (-CH), a vehicle control. One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed three times.



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Fig. 5.   PPARgamma turnover is increased in the presence of IFN-gamma . Whole cell extracts were prepared from 3T3-L1 adipocytes following various periods of treatment with 5 µM cycloheximide or ethanol in the presence or absence of IFN-gamma . One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed three times.

TZD treatment has also been shown to decrease PPARgamma expression. Therefore, we compared the effects of IFN-gamma and englitazone (ENG), a TZD, on the expression of PPARgamma in adipocytes. As shown in Fig. 6, fully differentiated adipocytes were exposed to IFN-gamma or ENG alone or in combination. In the first combination, adipocytes were treated with IFN-gamma 1 h prior to the addition of englitazone. In the second combination, adipocytes were treated with englitazone 1 h prior to the addition of IFN-gamma . For each combination, whole cell extracts were isolated 5 h after initiation of the experiment. These results demonstrate that the combination of both inhibitors of PPARgamma expression resulted in an even greater decrease in PPARgamma expression than one agonist alone.



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Fig. 6.   The IFN-gamma -induced increase of PPARgamma expression is even greater in the presence of englitazone. Whole cell extracts were prepared from fully differentiated 3T3-L1 adipocytes following a 5-h treatment of IFN-gamma , ENG, IFN-gamma  + ENG (added 1 h after the addition of IFN-gamma ), and ENG + IFN-gamma (added 1 h after the addition of ENG). One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. CTL, control.

The results of the cycloheximide experiments in Fig. 5 suggest that the decay of PPARgamma 2 and -gamma 1 is increased in the presence of IFN-gamma . Therefore, we examined PPARgamma expression in the presence of proteasome inhibitors. As shown in Fig. 7, a 6-h treatment of either epoxomicin or lactacystin had little effect on the levels of PPARgamma 2 or -gamma 1 protein. A 6-h IFN-gamma treatment resulted in a substantial loss of PPARgamma protein, but the IFN-gamma -induced loss of PPARgamma 2 and -gamma 1 was inhibited in the presence of either epoxomicin or lactacystin. Notably, the presence of these two different proteasome inhibitors did not restore PPARgamma 2 and -gamma 1 to the levels found in untreated adipocytes, suggesting that protein degradation is only one manner in which IFN-gamma regulates PPARgamma expression. In the presence of the two proteasome inhibitors, there were no differences in the levels of any STATs or ERK1/ERK2. Interestingly, the IFN-gamma -induced increase in STAT 1 was blunted in the presence of epoxomicin or lactacystin, suggesting that the IFN-gamma -induced increase in STAT 1 may be dependent on the degradation of some protein(s).



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Fig. 7.   The IFN-gamma -induced decrease of PPARgamma is partially inhibited in the presence of proteasome inhibitors. Whole cell extracts were prepared from fully differentiated 3T3-L1 adipocytes following a 6-h treatment of either 100 nM epoxomicin or 5 µM lactacystin in the presence or absence of IFN-gamma (100 units/ml). One hundred µg of each extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. Samples were processed and results were visualized as described in the legend to Fig. 1. This is a representative experiment independently performed three times. CTL, control.

These experiments indicate that, in addition to having an effect on the turnover of the PPARgamma proteins, there is presumably another means by which IFN-gamma causes a decrease in PPARgamma expression. Therefore, we examined the effect of IFN-gamma on PPARgamma mRNA accumulation. As shown in Fig. 8, a 2-h IFN-gamma treatment resulted in a substantial loss of PPARgamma mRNA. Northern blot analysis cannot distinguish between the two forms of PPARgamma . A decrease in C/EBPalpha and GLUT4 was also observed following a 20-h IFN-gamma treatment. In addition, we observed an increase in the levels of both C/EBPbeta and C/EBPdelta following an IFN-gamma treatment. A notable decrease in aP2/422 was observed after a 12-h treatment with IFN-gamma . The expression of glycerol phosphate dehydrogenase (GPD), a gene whose expression is elevated in adipocytes, was substantially decreased following a 20-h treatment with IFN-gamma . Following a 24-h IFN-gamma treatment, there was also a slight decline in adipsin mRNA. The hybridization of beta -actin is shown to represent the even loading of the samples.



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Fig. 8.   IFN-gamma treatment results in a rapid loss of PPARgamma mRNA and a decrease in expression of other adipocyte markers. Total RNA was isolated from fully differentiated 3T3-L1 adipocytes following treatment with IFN-gamma as indicated at the top of the figure. Twenty µg of total RNA was electrophoresed, transferred to nylon, and subjected to Northern blot analysis. This is a representative experiment independently performed two times. GPD, glycerol phosphate dehydrogenase.

Because the IFN-gamma -induced loss of PPARgamma mRNA was relatively rapid, we predicted that the decay of the PPARgamma mRNA would be brief compared with C/EBPalpha . Therefore, we investigated the turnover of these two transcription factor mRNAs. Total RNA was isolated from cells at various times after treatment with actinomycin D. As shown in Fig. 9A, the PPARgamma mRNA decayed rapidly compared with the C/EBPalpha mRNA. We estimated the half-life of the PPARgamma mRNA to be less than 3 h. We also examined the decay of PPARgamma in the presence of IFN-gamma to determine whether this growth factor had any effect on the stability of the PPARgamma mRNA. We found that the decay of PPARgamma mRNA was not altered in the presence of IFN-gamma as indicated in Fig. 9B. These results strongly suggested that IFN-gamma would have an effect on the transcription of PPARgamma .



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Fig. 9.   PPARgamma mRNA is more labile than C/EBPalpha mRNA in adipocytes, and the decay of these mRNAs is not affected by IFN-gamma . A, total RNA was isolated from fully differentiated 3T3-L1 adipocytes following treatment with 5 µg/ml actinomycin D for the various periods of time indicated at the top of the figure. Control samples (-actinomycin D) were isolated at the start and finish of the experiment. B, total RNA was isolated from fully differentiated 3T3-L1 adipocytes following treatment with 5 µg/ml actinomycin D for the various periods of time indicated at the top of the figure in the absence or presence of IFN-gamma . In each experiment, 20 µg of total RNA was electrophoresed, transferred to nylon, and subjected to Northern blot analysis for C/EBPalpha and PPARgamma . This is a representative experiment independently performed two times.

To determine whether the IFN-gamma -induced changes in PPARgamma and C/EBPalpha mRNA accumulation shown in Fig. 8 were attributable to the effects on synthesis, we measured the transcription rates of these genes in nuclei isolated from control and IFN-gamma -treated adipocytes. Fully differentiated 3T3-L1 adipocytes were exposed to cycloheximide (±IFN-gamma ) for 1 h. As shown in Fig. 10, a substantial suppression of both PPARgamma and C/EBPalpha was observed following IFN-gamma treatment, indicating that the effect of IFN-gamma on the transcription of these genes was independent of new protein synthesis. IFN-gamma had no effect on beta -actin transcription (data not shown).



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Fig. 10.   IFN-gamma treatment results in a suppression of PPARgamma and C/EBPalpha transcription in adipocytes in a manner that is independent of new protein synthesis. Nuclei were isolated from fully differentiated adipocytes that were exposed to 5 mM cycloheximide in the presence or absence of IFN-gamma for 1 h. Nuclei were subjected to run-on analysis, and the autoradiogram displayed isa representative of an experiment performed twice with independent preparations of nuclei. CTL, control.

IFN-gamma is known to have effects on both lipolysis and lipogenesis, so we investigated the effect of this growth factor on basal and insulin-sensitive glucose uptake. As shown in Table I, serum-deprived 3T3-L1 adipocytes had a 6.7-fold increase in glucose uptake following a 10-min treatment of 100 nM insulin. After a 24-h treatment of IFN-gamma , cultured adipocytes were still responsive to insulin (6.13-fold increase). However, following a 48-h treatment of IFN-gamma , when both PPARgamma and C/EBPalpha were substantially decreased (Fig. 1), there was a discernible decrease in insulin-stimulated glucose uptake (4.42-fold increase). Exposure to IFN-gamma for 72 and 96 h had no effect on basal glucose uptake but resulted in a substantial decrease in insulin-sensitive glucose uptake. Following a 96-h IFN-gamma exposure, there was only a 2.2-fold increase following insulin treatment. IFN-gamma treatment for more than 96 h did not result in a further decline of insulin-sensitive glucose uptake (data not shown). The IFN-gamma -induced effects on insulin sensitivity do not appear to be a result of any significant lipid loss as there were no distinguishable differences in Oil Red O staining from control and IFN-gamma -treated (96 h) adipocytes (data not shown).


                              
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Table I
The effect of IFN-gamma on insulin-sensitive glucose transport



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IFN-gamma affected the expression of many adipocyte transcription factors, including PPARgamma 2 and -gamma 1, C/EBPalpha , C/EBPbeta , C/EBPdelta , SREBP-1, STAT 1, and STAT 3. However, the most profound effect of IFN-gamma was on PPARgamma expression. These studies have also revealed that both the PPARgamma mRNA and protein are labile compared with other adipocyte transcription factors. IFN-gamma treatment of adipocytes leads to a decrease in PPARgamma that is the result of the inhibition of transcription coupled with an increase in the degradation of PPARgamma 2 and -gamma 1. Interestingly, recent studies have revealed that thiazolidinedione treatment of the 3T3-F442A adipocytes results in a reduction of PPARgamma protein that is distinct from mRNA regulation (18). In that study, the data indicated that the TZD treatment of adipocytes resulted in the ubiquitination of PPARgamma and subsequent degradation that was dependent on the proteasome complex (18). These results are comparable with the effects we observed with IFN-gamma . In our studies, two distinct proteasome inhibitors affected PPARgamma 2 and -gamma 1 protein levels in the presence of IFN-gamma but had little effect in adipocytes lacking cytokine stimulation. In summary, both TZD and IFN-gamma treatments of adipocytes appear to target PPARgamma to the proteasome for degradation. Moreover, our observations on the lability of PPARgamma proteins suggest that PPARgamma turnover is an important event.

The inhibition of proteasome activity in the presence of IFN-gamma did not restore the PPARgamma proteins to normal cellular levels, and we observed a potent effect of IFN-gamma on PPARgamma transcription and mRNA accumulation. These results have led us to hypothesize that IFN-gamma -induced STAT 1 activation in fat cells may be responsible for the transcriptional suppression of PPARgamma . Cross-talk between STATs and PPARs has been demonstrated in liver cells (22). We are currently initiating studies to identify the IFN-gamma -sensitive element in the PPARgamma promoters and determine whether STAT 1 is directly involved in the transcriptional suppression of PPARgamma . Although STATs are generally thought to be transcriptional activators, there is evidence that this family of transcription factors can also act as repressors of transcription (23). These studies have led us to hypothesize that IFN-gamma -induced STAT 1 dimers directly bind to the PPARgamma promoters and result in an inhibition of transcription.

The effects of IFN-gamma on PPARgamma degradation are less expected. Numerous studies have shown that serine phosphorylation of PPARgamma on Ser112 by mitogen-activated protein kinases (ERK1/ERK2 and stress-activated protein kinase/c-Jun NH2-terminal kinase) results in a strong suppression of PPARgamma activity (24-27), which in part appears to involve ligand binding (28). Our previous studies in the 3T3-L1 adipocytes have demonstrated that IFN-gamma resulted in both STAT 1 and STAT 3 tyrosine phosphorylation and nuclear translocation (15). However, unlike other cytokines, IFN-gamma did not result in the activation of ERK1/ERK2 in adipocytes. Therefore, it does not appear that ERK1/ERK2-induced serine phosphorylation of PPARgamma could be involved in the effects of IFN-gamma that we observed on PPARgamma degradation. Our results are supported by the findings of Spiegelman and co-workers (18), which indicate that the phosphorylation of PPARgamma on Ser112 is not required for its down-regulation. However, we have not examined the role of serine phosphorylation in the IFN-gamma -induced PPARgamma degradation or the effect of IFN-gamma on the activation of c-Jun NH2-terminal kinase in adipocytes.

Although the mechanism by which IFN-gamma directs PPARgamma to the proteasome for degradation is not known, it is clear that the turnover of PPARgamma is further increased when both IFN-gamma and a PPARgamma ligand are present. Perhaps IFN-gamma could either modulate the phosphorylation state of PPARgamma or have an effect on the synthesis of an endogenous PPARgamma ligand. Alternatively, IFN-gamma -induced PPARgamma degradation could occur via a pathway that is independent of ligand-induced degradation. It is interesting to note that the analysis of PPARgamma mutants by the Spiegelman laboratory demonstrated that the TZD-induced PPARgamma decay was not strictly dependent on its transcriptional activity but was dependent upon the ligand-gated activation function (AF-2) domain. In these studies, ligand binding and the activation of the AF-2 domain not only increased the transcriptional function of PPARgamma but also induced ubiquitination and subsequent proteasomal degradation.

Unlike TZDs, which are insulin sensitizers, IFN-gamma treatment of adipocytes resulted in a condition of insulin resistance, as measured by insulin-sensitive glucose uptake and a decrease in the expression of adipocyte genes, such as GLUT4, aP2/422, GPD, and adipsin. PPARgamma has been implicated in the regulation of systemic insulin sensitivity, and some PPARgamma mutations are associated with severe insulin resistance and diabetes mellitus (29). In our studies, the most profound effect of IFN-gamma was on PPARgamma expression, which was significantly decreased after only 6 h. Interestingly, we did not observe any substantial effects on insulin-sensitive glucose uptake even after a 24-h treatment of IFN-gamma despite the dramatic loss of PPARgamma expression. Following a 48-h treatment, we did observe a substantial inhibition of insulin-sensitive glucose uptake. At this time, there was also a marked effect on C/EBPalpha expression. These studies suggest that the loss of PPARgamma may be insufficient to confer insulin resistance in 3T3-L1 adipocytes. However, the low levels of PPARgamma observed after a 24- and 48-h IFN-gamma treatment may be sufficient levels of PPARgamma expression to account for the insulin responsiveness of these cells. Alternatively, the primary role of PPARgamma may be to regulate the expression of other transcription factors, such as C/EBPalpha . Nonetheless, the increase in PPARgamma turnover and the inhibition of PPARgamma synthesis induced by IFN-gamma are prominent because of the relatively rapid decay of both the PPARgamma mRNA and the protein. Because the regulation of PPARgamma is the first observed effect of IFN-gamma on adipocyte transcription factor expression, this event is likely very important in the development of IFN-gamma -induced insulin resistance. IFN-gamma treatment also results in a decrease of GLUT4, aP2, GPD, and adipsin expression in adipocytes. However, there is no notable difference in the morphology of the cells, and there is no observable difference in Oil Red O staining from untreated 3T3-L1 fully differentiated adipocytes and those that have been treated for 96 h with IFN-gamma (data not shown). In conclusion, the tightly controlled regulation of PPARgamma synthesis and degradation that we observed in the presence of IFN-gamma suggests that the cellular levels of PPARgamma are a meaningful effector of gene expression.


    FOOTNOTES

* This work was supported by Grant R01DK52968-02 from the National Institutes of Health and by a Career Development Award from the American Diabetes Association (to J. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biological Sciences, Louisiana State University, 508 Life Sciences Bldg., Baton Rouge, LA 70803; Tel.: 225-388-1749; Fax: 225-388-2597; E-mail: jsteph1@unix1.sncc.lsu.edu.

Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M007894200


    ABBREVIATIONS

The abbreviations used are: STAT, signal transducer and activator of transcription; C/EBP, CCAAT/enhancer-binding protein; ERK, extracellular signal-regulated kinase; TZD, thiazolidinedione; DMEM, Dulbecco's modified Eagle's medium; ENG, englitazone; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PPAR, peroxisome proliferator-activated receptor; SREBP-1, sterol regulatory element-binding protein.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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