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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that plays an important role in adipocyte gene expression. Previous studies from our laboratory and others have shown that PPARγ can be ubiquitin modified and targeted to the proteasome for degradation in response to transcriptional activation. In this study, we determined whether other degradation pathways contributed to the tumor necrosis factor α (TNFα)–induced degradation of PPARγ proteins in 3T3-L1 adipocytes.

Methods and Procedures: In these studies, 3T3-L1 cells were studied by performing subcellular fractionations and western blotting after various TNFα treatments. Whole tissue extracts from rat adipose tissue were also used to examine PPARγ degradation.

Results: We observed that TNFα can induce a caspase-mediated degradation of PPARγ proteins in the presence of cycloheximide. The caspase-mediated degradation of both PPARγ1 and PPARγ2 resulted in the generation of a specific 44-kd cleavage product. The specific cleavage product was unaffected by proteasome inhibitors, but was repressed by a general caspase inhibitor. Use of several specific caspase inhibitors revealed that caspase-1 was activated following treatment with TNFα and cycloheximide (CH), and inhibition of caspase-1 blocked the cleavage of PPARγ proteins in cultured adipocytes. In addition, a similar PPARγ degradation product was observed in rodent adipose tissue.

Discussion: In summary, this is the first study to demonstrate that PPARγ levels can be modulated by caspase activity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Peroxisome proliferator-activated receptor γ (PPARγ) plays essential roles in adipocyte differentiation, adipose tissue function, glucose homeostasis, and insulin sensitivity in refs. 1,2. PPARγ belongs to the nuclear receptor superfamily, and it contains two isoforms, PPARγ1 (γ1) and PPARγ2 (γ2) that arise from alternative promoter usage and alternative splicing (3). PPARγ1 lacks the N-terminal 30 amino acids present in γ2. PPARγ1 is expressed at low levels in multiple tissues, whereas γ2 is highly expressed in adipocytes (4). PPARγ knockout mice exhibit placental dysfunction and embryonic lethality (5,6), whereas the adipose tissue specific PPARγ knockout mice exhibit adipose lipodystrophy, elevated lipid deposition in liver, and reduced levels of adipocyte-secreted cytokines (7). Ectopic expression of PPARγ in myoblasts or fibroblasts promotes these nonprecursor cells to differentiate into adipocytes (4,8), further demonstrating that PPARγ is a master regulator of adipocyte differentiation and function.

PPARγ is activated by endogenous arachidonic acid metabolites, such as 15-deoxy-δ 12, 14 prostaglandin J2 (9) and by thiazolidinediones (10). PPARγ binds to DNA as a heterodimer with retinoid X receptor α (11) and the ligand-bound form of PPARγ/retinoid X receptor α can bind to direct repeats of hormone response elements separated by one base, so-called DR-1 sites, such as the aP2 enhancer (4). The negative regulation of PPARγ activity can be achieved by mitogen-activated protein kinases, including extracellular signal-related kinases-1/2 and c-Jun N-terminal kinases mediated phosphorylation at Ser112 (12,13). The covalent modification by small ubiquitin-related modifier protein (SUMOylation) at Lys107 also negatively regulates PPARγ transcriptional activity (14,15,16,17).

The regulation of PPARγ turnover is another important contributor to PPARγ activity. In contrast to other adipogenic transcription factors, such as CCAAT/enhancer binding protein α, and sterol response element binding protein-1, which are stable, PPARγ proteins are very labile in adipocytes (18). Following inhibition of protein synthesis, studies in our lab and others have showed that the ubiquitin-proteasome pathway is important for PPARγ degradation, and the ubiquitin-proteasome mediated decay of PPARγ occurs in the nucleus (19,20).

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine that is well known for its role in insulin resistance in obesity and type 2 diabetes in ref. 21. Chronic TNFα treatment of adipocytes substantially alters gene expression, in part through the nuclear factor-kB pathway, by downregulating the expression of key adipocyte genes (22). Acute treatment of adipocytes with TNFα results in decreased insulin-stimulated insulin receptor substrate-1 tyrosine phosphorylation, PI3 kinase activity, and insulin-sensitive glucose transport (23,24,25). Short- and long-term treatments of TNFα also reduce GLUT4, insulin receptor substrate-1, PPARγ, cyclic-nucleotide phosphodiester (PDE)-3B, and Akt protein levels (26,27,28,29,30). Overall, these findings have shown that TNFα mediates insulin resistance via multiple pathways.

In this study, we examined the role of caspases in the loss of PPARγ proteins induced by TNFα in adipocytes. We observed that TNFα leads to PPARγ degradation, and we found that a specific cleavage product occurs after treatment with various doses of TNFα in the presence of cycloheximide (CH), and the cleavage product appears primarily in the nucleus. Both a general caspase inhibitor and an inhibitor of caspase-1 substantially blocked the specific cleavage of PPARγ. Yet, caspase-3, caspase-8, and caspase-9 inhibitors did not prevent such processing. We also observed that TNFα leads to an increase of active caspase-1 in the nucleus through a decrease of procaspase-1 and an increase of the active caspase-1 P10 subunit. In summary, our findings strongly suggest that in 3T3-L1 adipocytes, PPARγ proteins can be cleaved by caspases under specific conditions, and caspase-1 appears to play a role in this process.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

Materials

Dulbecco's modified Eagle's medium (DMEM) was purchased from Sigma (St. Louis, MO). Bovine and fetal bovine sera were purchased from Sigma and Invitrogen (Carlsbad, CA), respectively. CH was obtained from Sigma. TNFα was purchased from Biosource (Invitrogen, Carlsbad, CA). Lactacystin, Boc-D-FMK (Boc-D), Z-YVAD-FMK (YVAD), Z-DEVD-FMK (DEVD), Z-IETD-FMK (IETD), and Z-LEHD-FMK (LEHD) were purchased from Calbiochem (EMD Biosciences, Gibbstown, NJ). An extracellular signal-related kinase-1 polyclonal antibody, both PPARγ monoclonal and polyclonal antibodies, and a caspase-1 polyclonal antibody were obtained from Santa Cruz (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. Enhanced Chemiluminescence kit was purchased from Pierce (Rockford, IL).

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 sera, 0.5 mmol/l 3-isobutyl-1-methylxanthine, 1 μmol/l dexamethasone, and 1.7 μmol/l insulin. After 48 h, this medium was replaced with DMEM supplemented with 10% fetal bovine sera, and cells were maintained in this medium until used for experimentation. Fully differentiated adipocytes were serum deprived overnight before experimentation in DMEM media containing fatty acid-free 0.3% bovine serum albumin.

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 mmol/l NaCl, 10 mmol/l Tris, pH 7.4, 1 mmol/l (ethylenebis(oxyethylenenitrilo))tetraacetic acid, 1 mmol/l EDTA, 1% Triton-X 100, 0.5% Igepal CA-630 (Nonidet P-40), 1 μmol/l phenylmethylsulphonyl fluoride, 1 μmol/l pepstatin, 50 trypsin inhibitory milliunits of aprotinin, and 10 μmol/l leupeptin, and 2 mmol/l sodium vanadate. Samples were extracted on ice for 30 min and centrifuged at 13,000g at 4 °C for 10 min. Supernatants containing whole cell extracts were analyzed for protein concentration using a bicinchoninic acid kit (Pierce) according to the manufacturer's instructions.

Preparation of cytosolic/nuclear extracts

Monolayers of 3T3-L1 adipocytes were rinsed with phosphate-buffered saline and harvested in a nuclear homogenization buffer containing 20 mmol/l Tris-Cl, pH 7.4, 10 mmol/l NaCl, and 3 mmol/l MgCl2. Nonidet P-40 was added to a final concentration of 0.15%, and the cells were homogenized with 16 strokes in a Dounce homogenizer. The resulting homogenate was centrifuged at 6,000g for 6 min, and the supernatant was saved as cytosolic extract. The nuclear pellet was resuspended in 0.5 volume of nuclear homogenization buffer and centrifuged as before. The nuclear pellet was then resuspended in a nuclear extraction buffer containing 20 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.9, 420 mmol/l NaCl, 0.2 mmol/l EDTA, and 25% glycerol. Nuclei were extracted on ice for 30 min followed by incubation with 200 U of DNase I (Invitrogen) at room temperature for 15 min. Finally, the sample was centrifuged at 13,000g at 4 °C for 10 min. The resulting nuclear extract and the previously obtained cytosolic extract were analyzed for protein concentration using a bicinchoninic acid kit (Pierce) according to the manufacturer's instructions.

Immunoprecipitations of PPARγ from adipocyte extracts

Cells were harvested under nondenaturing conditions and cytosolic and nuclear extracts were isolated as described earlier. After a single freeze-thaw cycle, the protein extracts were preincubated with protein-A agarose and the resulting supernatant was then incubated with 5 μg of the polyclonal anti-PPARγ antibody for 1 h at 4 °C. Protein-A agarose (RepliGen) was added to the mixture and the sample was rotated for an additional hour. Bound PPARγ and any associated proteins were isolated by pelleting this mixture. The pellets were rinsed twice with phosphate-buffered saline and bound proteins were eluted from the agarose by incubating at 100 °C for 10 min after the addition of Laemmli sample buffer. As described below, these samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed using western blotting with a monoclonal PPARγ antibody.

Gel electrophoresis and western blot analysis

Proteins were separated in 5, 7.5, 10, or 12% polyacrylamide (acrylamide from National Diagnostics (Atlanta, GA)) gels containing sodium dodecyl sulfate according to Laemmli (31) and transferred to nitrocellulose membrane in 25 mmol/l Tris, 192 mmol/l glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% fat-free milk at room temperature for 1 h. Results were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence.

Rodent adipose tissue isolation

Animals were euthanized by cervical dislocation, and tissues were immediately removed and frozen in liquid nitrogen. Frozen tissues were homogenized in a buffer containing 150 mmol/l NaCl, 10 mmol/l Tris, pH 7.4, 1 mmol/l (ethylenebis(oxyethylenenitrilo))tetraacetic acid, 1 mmol/l EDTA, 1% Triton-X 100, 0.5% Igepal CA-630, 1μmol/l phenylmethylsulphonyl fluoride, 1 μmol/l pepstatin, 50 trypsin inhibitory milliunits of aprotinin, and 10 μmol/l leupeptin, and 2 mmol/l sodium vanadate. Homogenates were centrifuged for 10 min at 9,000g to remove any debris and insoluble material and then analyzed for protein content. The 6-week-old lean fa/+ were purchased from Harlan (Indianapolis, IN). All animal studies were carried out with protocols which were reviewed and approved by institutional Institutional Animal Care and Use Committees.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

PPARγ protein degradation is induced by TNFα and CH treatment of 3T3-L1 adipocytes

Our previous studies have shown that PPARγ proteins are labile compared to several adipocyte transcription factors (18). In this study, we further explored the mechanism of TNFα-induced decay of PPARγ proteins, as it is known that TNFα treatment of adipocytes causes a reduction in PPARγ levels (28). To examine the degradation of PPARγ proteins, fully differentiated 3T3-L1 adipocytes were treated with CH and the various doses of TNFα indicated in Figure 1. Whole cell extracts were isolated following 2-, 3-, and 5-h treatments. Of the five doses of TNFα studied, we observed a loss of PPARγ1 and γ2 levels following a 5-h treatment with all doses except for the lowest one (0.01 nmol/l). A loss of both PPARγ1 and γ2 levels were observed at 3 h for the two highest doses of TNFα. In addition, all of the TNFα treatments, except for the 0.01 nmol/l dose, resulted in the generation of a band around 44 kd which coincided with the loss of PPARγ proteins. The levels of extracellular signal-related kinase-1 were shown for each analysis to demonstrate even loading of the protein samples in this study.

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Figure 1. Peroxisome proliferator-activated receptor γ (PPARγ) protein degradation is induced by tumor necrosis factor α (TNFα) and cycloheximide (CH) treatment of 3T3-L1 adipocytes. Mature 3T3-L1 adipocytes were treated with 50 μmol/l CH alone (control (CTL)), or in the presence of various doses of TNFα treatment which was added to the cells 15 min after the addition of the CH. Whole cell extracts were harvested at the indicated time points. One hundred microgram of each extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to western blot analysis. The arrows indicate the specific cleavage product of PPARγ. This is a representative experiment independently performed three times. ERK, extracellular signal-related kinase.

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The specificity of the 44-kd band is confirmed by immunoprecipitation analysis

Fully differentiated 3T3-L1 adipocytes were exposed to both TNFα and CH for 4 h before isolation of nuclear and cytosolic extracts. The extracts were used for immunoprecipitation with a polyclonal PPARγ antibody. As shown in Figure 2, a strong signal for PPARγ2 was observed in the nucleus of untreated adipocytes. The band for PPARγ1 was masked by the presence of the antibody heavy chain; however, a 44-kd band was present in the nucleus, and to a lesser extent in the cytosol, of TNFα- and CH-treated adipocytes. We performed the immunoprecipitations with buffer alone (mock) to further indicate the specificity of the band we observed.

image

Figure 2. The specificity of the 44-kd band is confirmed by immunoprecipitation analysis. Mature 3T3-L1 adipocytes were vehicle treated or treated with 50 μmol/l cycloheximide (CH) and 1 nmol/l tumor necrosis factor α (TNFα) for 4 h. Cytosolic and nuclear extract were isolated and 200 μg of each extracts was used for immunoprecipitation (IP) analysis with a polyclonal peroxisome proliferator-activated receptor γ (PPARγ) antibody and western blotting (WB) analysis with a monoclonal PPARγ. One IP contained buffer and all other reagents, except for the extract. This sample is labeled with an M to indicate the mock control. The arrow indicates the specific cleavage product of PPARγ. This is a representative experiment independently performed two times.

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The production of a 44-kd PPARγ degradation product is dependent on caspase activity

Previous studies have shown that PPARγ is targeted to the ubiquitin proteasome under various conditions (18,19,20). So, we examined whether the specific 44-kd degradation product was generated through the ubiquitin proteasome pathway. Fully differentiated 3T3-L1 adipocytes were exposed to TNFα, CH, or both TNFα and CH in the presence of lactacystin, Boc-D, or both of these inhibitors. Whole cell extracts were isolated at two time points after these treatments and the loss of PPARγ proteins was examined. As shown in Figure 3, TNFα or CH alone resulted in a loss of PPARγ1 and a similar decrease in the levels of PPARγ2 at both time points examined. TNFα and CH together resulted in a substantial loss of both PPARγ isoforms that was accompanied by the presence of the 44-kd band; however, the presence of Boc-D, a general caspase inhibitor, attenuated the loss of PPARγ2 and inhibited the generation of 44-kd degradation product. The presence of lactacystin, a specific proteasome inhibitor, attenuated the effect of TNFα and CH on PPARγ2, but did not affect the presence of the 44-kd band. The presence of both the caspase and proteasome inhibitor attenuated the loss of PPARγ2, and the 44-kd degradation product was not present. These results suggest that PPARγ proteins are processed by a caspase(s) when both TNFα and CH were present.

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Figure 3. The production of the 44-kd peroxisome proliferator-activated receptor γ (PPARγ) cleavage product was repressed by caspase inhibition, but not by proteasome inhibition. Mature 3T3-L1 adipocytes were pretreated with 33 μmol/l Boc-D-FMK (Boc-D) or 5 μmol/l lactacystin (Lact) alone, or with both inhibitors for 1 h. Next, the cells were treated with 50 μmol/l cycloheximide (CH) or 1 nmol/l tumor necrosis factor α (TNFα) alone, or with both TNFα and CH. Whole cell extracts were harvested at the indicated time points. One hundred microgram of each extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to western blot analysis. The arrow indicates the specific cleavage product of PPARγ. This is a representative experiment independently performed three times. ERK, extracellular signal-related kinase.

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To determine which caspase(s) are involved in the degradation of PPARγ proteins under these conditions, we used several different caspase inhibitors. Fully differentiated 3T3-L1 adipocytes were exposed to either a general caspase inhibitor, or specific inhibitors for caspase-3, caspase-8, or caspase-9. As shown in Figure 4, CH and TNFα treatment resulted in a loss of both isoforms of PPARγ and the production of a 44-kd band. The generation of this PPARγ degradation product was inhibited by the presence of Boc-D, a general caspase inhibitor, but the inhibitors for caspase-3, caspase-8, or caspase-9 did not block the formation of the 44-kd PPARγ degradation product. A caspase 6 inhibitor, Z-VEID-FMK, was also used and did not block the formation of the PPARγ degradation product in several independent experiments (data not shown).

image

Figure 4. The production of the 44-kd peroxisome proliferator-activated receptor γ (PPARγ) cleavage product was not affected by caspase-3, caspase-8, or caspase-9 inhibitors. Mature 3T3-L1 adipocytes were pretreated with 33 μmol/l Boc-D-FMK (G), or 25 μmol/l Z-DEVD-FM (3), or 25 μmol/l Z-IETD-FMK (8), or 25 μmol/l Z-LEHD-FMK (9) for 1 h. Next, the cells were treated with 50 μmol/l cycloheximide (CH) for 15 min followed by 1 nmol/l tumor necrosis factor α (TNFα) treatment. Whole cell extracts were harvested at the indicated time points. One hundred microgram of each extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to western blot analysis. The arrow indicates the specific cleavage product of PPARγ. This is a representative experiment independently performed three times. ERK, extracellular signal-related kinase.

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Next, we examined the protein sequence of PPARγ to search for caspase catalytic recognition and cleavage sites. We identified a putative tetrapeptide (MVAD) caspase recognition site, close to the N-terminus, at residue D62 in PPARγ1 and residue D92 in PPARγ2 (Table 1). Similar to interleukin-1β, this site contains a hydrophobic amino acid (methionine, M) as the first residue, a valine (V) as the second, and an aspartate (D) as the fourth, which corresponds with caspase-1 specific substrates suggested in previous studies (32). Cleavage at this site in either PPARγ1 or γ2 would result in the production of a 413 amino acid peptide (shown in Figure 5), which would migrate at ∼44 kd. To determine whether caspase-1 affected the cleavage of PPARγ in the presence of TNFα and CH, we treated cells with either a caspase general inhibitor or a specific caspase-1 inhibitor. As shown in Figure 6a, a 5-h treatment with TNFα and CH resulted in the presence of the 44-kd band; however, the presence of this band was inhibited by treatment with a caspase-1 inhibitor (Z-YVAD-FMK) or a general caspase inhibitor (Boc-D). The levels of extracellular signal-related kinase-1 were shown for this experiment to demonstrate even loading of the protein samples. In addition, we examined the subcellular distribution of procaspase-1 and found that this protein was present in both the cytosol and nucleus of 3T3-L1 adipocytes. Moreover, we observed a decrease of procaspase-1 in the nucleus, indicative of its cleavage. The results in Figure 6b also indicate an increase in the P10 subunit of caspase-1 in the nucleus, which is considered an indicator of caspase-1 activation (33). Because PPARγ is primarily located in the nucleus of 3T3-L1 adipocytes (Figure 2 and (19)), we predict that cleavage takes places in the nucleus.

Table 1.  Recognition sites of caspase-1
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Figure 5. The identification of a potential caspase-1 cleavage site in peroxisome proliferator-activated receptor γ1 (PPARγ1) and PPARγ2. A schematic of full-length PPARγ1 and PPARγ2 are shown. The asterisks and the arrow indicate the proposed caspase-1 cleavage sites. The specific tetrapeptide recognition sites at D62 in γ1or D92 for γ2 are in bold characters. The length of the presumed cleavage product is indicated.

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image

Figure 6. Inhibition of caspase-1 blocks the formation of the 44-kd peroxisome proliferator-activated receptor γ (PPARγ) cleavage product and tumor necrosis factor α (TNFα) increases the amount of active caspase-1 in the nucleus. (a) Mature 3T3-L1 adipocytes were pretreated with 33 μmol/l Boc-D-FMK (labeled G for general inhibitor), or 25 μmol/l Z-YVAD-FMK (labeled 1 for caspase-1 inhibitor) for 1 h. Next, the cells were treated with 50 μmol/l cycloheximide (CH) for 15 min followed by 1 nmol/l TNFα treatment. Whole cell extracts were harvested 5 h after TNFα treatment. One hundred microgram of each extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and subjected to western blot analysis. The arrow indicates the specific cleavage product of PPARγ. This is a representative experiment independently performed three times. (b) Mature 3T3-L1 adipocytes were treated with 50 μmol/l CH for 15 min before treatment with 1 nmol/l TNFα. Cytosolic and nuclear extracts were isolated at the indicated time points. One hundred microgram of each extracts was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to western blot analysis. This is a representative experiment independently performed three times. ERK, extracellular signal-related kinase.

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The 44-kd PPARγ degradation product is present in brown and white adipose tissue

To determine whether the degradation band induced by the TNFα and CH treatment of 3T3-L1 adipocytes was of potential physiological relevance, we examined adipose tissue isolated from rodents. As shown in Figure 7, we detected a 44-kd band in brown adipose tissue extracts in two of three tissue samples examined. We also detected the 44-kd band in three of the five epididymal extracts (white adipose tissue) we examined. Each extract was prepared from a different rat.

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Figure 7. A 44-kd peroxisome proliferator-activated receptor γ (PPAR) degradation product is present in rat brown and white adipose tissue. Interscapular brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) was obtained from lean fa/+ (Zucker) rats that were 6 weeks of age. The rats were killed and the fat pads were immediately removed and frozen in liquid nitrogen. Three BAT samples were analyzed and five WAT samples were examined. One hundred and fifty microgram of each tissue extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to western blot analysis. In each blot, the arrow indicates a 44-kd band.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

TNFα has been found to be an important mediator of insulin resistance in obesity and obesity-related type 2 diabetes in refs. 21,34. The actions of TNFα on adipocytes can be mediated by modulating gene expression via nuclear factor-kB (22) and by substantially affecting the expression of several adipocyte proteins (26,27,28,29,30). Our observations that TNFα, in the presence of CH, can directly affect PPARγ protein decay when caspase-1 is active and other finding showing that TNFα can induce Akt protein decay through caspase-6 (30) establishes a new role for TNFα action in the modulation of adipocyte proteins. This concept is supported data showing an improvement of insulin-stimulated glucose uptake following a pretreatment with a general caspase inhibitor in TNFα-treated 3T3-L1 adipocytes (30).

Our data strongly suggest that PPARγ proteins are cleaved by caspases, which adds PPARγ to a growing list of transcription regulators that are cleaved by caspases. These proteins include STAT1 (35), nuclear factor-kB (36), SP-1 (37), GATA-1 (38), and AP-2α (39). Interestingly, all of these transcription regulators have reduced transcriptional regulation activity when they are processed by caspases. Our results indicate that the caspase cleavage site of PPARγ is located near the N-terminus (Figure 5), in which the ligand-independent transactivation domain is located (40). We hypothesize that the cleavage of PPARγ at this site likely affects its transactivating function; however, the low amount of the 44-kd band present in adipose tissue in rats suggests that this form of the protein is highly labile.

The cleavage of PPARγ by caspases is evident following treatment with TNFα and CH, but not with TNFα alone in cultured adipocytes. This observation raises the question of whether this caspase-mediated cleavage contributes to PPARγ turnover under physiological conditions. It has been shown that treatment of cells with both TNFα and CH often leads to cell death through the signaling by TNFα receptors (TNFR) −1 and −2, which leads to the activation of multiple caspases (41,42,43). Although we observed an increase of caspase-1 P10 subunit in the nucleus in response to TNFα treatment (Figure 6b), which indicates the increase of caspase-1 activity, it is noteworthy that a basal level of P10 subunit of caspase-1 without TNFα treatment was present suggesting that activated caspase-1 was present in the nucleus even under basal conditions. The role of caspase-1 in maturation of interleukin-1β and interleukin-1β in monocytes suggests that the principal role of active caspase-1 may not be restricted to apoptosis, but also play a role in nonapoptotic processes in ref. 44. Because TNFα can result in decreased PPARγ expression in the absence of CH, our results suggest that the formation of the 44-kd degradation product is not required for PPARγ degradation. In cultured cells, we have not observed the 44-kd band in the absence of CH. These results might suggest that these experiments are phenomenology, but we have observed the 44-kd band in rat adipose tissue suggesting this cleavage occurs in physiological conditions. In addition, we have performed additional studies which suggest that the 44-kd band is PPARγ by performing immunoprecipitations with specific and nonspecific antibodies. We only observe the 44 kd when using PPARγ, and not PPARα or PPARδ, specific antibodies (data not shown).

In summary, our data strongly suggest that PPARγ proteins can be processed by caspases in TNFα and CH treated 3T3-L1 adipocytes. In this experimental paradigm, the specific cleavage of PPARγ1 and γ2 results in the generation of a 44-kd cleavage product that is partially, if not completely, mediated by the activation of caspase-1. These studies may contribute to a better understanding of the regulation of PPARγ in adipocytes, and possibly facilitate our comprehension of insulin resistance in obesity and diabetes.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES

This work was supported by a Research Award from the American Diabetes Association to J.M.S.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. REFERENCES
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