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Am J Physiol Endocrinol Metab 293: E219-E227, 2007. First published March 27, 2007; doi:10.1152/ajpendo.00695.2006
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Suppression of PPAR-{gamma} attenuates insulin-stimulated glucose uptake by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes

Wei Liao,1 M. T. Audrey Nguyen,1 Takeshi Yoshizaki,1 Svetlana Favelyukis,1 David Patsouris,1 Takeshi Imamura,1 Inder M. Verma,2 and Jerrold M. Olefsky1

1Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California; and 2Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California

Submitted 19 December 2006 ; accepted in final form 22 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) plays a critical role in regulating insulin sensitivity and glucose homeostasis. In this study, we identified highly efficient small interfering RNA (siRNA) sequences and used lentiviral short hairpin RNA and electroporation of siRNAs to deplete PPAR-{gamma} from 3T3-L1 adipocytes to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-{gamma} knockdown prevented adipocyte differentiation but was not required for maintenance of the adipocyte differentiation state after the cells had undergone adipogenesis. We further demonstrate that PPAR-{gamma} suppression reduced insulin-stimulated glucose uptake without affecting the early insulin signaling steps in the adipocytes. Using dual siRNA strategies, we show that this effect of PPAR-{gamma} deletion was mediated by both GLUT4 and GLUT1. Interestingly, PPAR-{gamma}-depleted cells displayed enhanced inflammatory responses to TNF-{alpha} stimulation, consistent with a chronic anti-inflammatory effect of endogenous PPAR-{gamma}. In summary, 1) PPAR-{gamma} is essential for the process of adipocyte differentiation but is less necessary for maintenance of the differentiated state, 2) PPAR-{gamma} supports normal insulin-stimulated glucose transport, and 3) endogenous PPAR-{gamma} may play a role in suppression of the inflammatory pathway in 3T3-L1 cells.

small interfering RNA; lentivirus; peroxisome proliferator activated-receptor-{gamma}

PEROXISOME PROLIFERATOR-ACTIVATED receptor-{gamma} (PPAR-{gamma}), a nuclear receptor, plays a critical role in glucose and lipid metabolism. On ligand binding, PPAR-{gamma} undergoes a conformational change, stabilizing its interaction with Retinoid X Receptor (RXR). The PPAR-{gamma}/RXR heterodimer then recruits a set of coactivators to the promoter regions of target genes, thus stimulating transcription (2, 6). PPAR-{gamma} can also mediate ligand-stimulated transrepression of genes through a peroxisome proliferator response element-independent process in which the PPAR-{gamma} ligand binds to the NCoR/SMRT corepressor complex (32). This interaction prevents the ubiquitination and proteasomal degradation of the corepressor, keeping the gene in a repressed mode.

PPAR-{gamma} also plays an important role in regulating insulin sensitivity and glucose homeostasis (1, 4, 35). Strong evidence for this came from the discovery that insulin-sensitizing drugs, thiazolidinediones (TZDs), are high-affinity ligands for PPAR-{gamma} and exert their effects by modulating gene expression (21). TZDs decrease plasma glucose and improve insulin sensitivity in a wide variety of diabetic and obese rodent models and in humans (20, 24, 25). Recent work on PPAR-{gamma} knockout mice suggests that PPAR-{gamma} may have direct insulin-sensitizing effects in muscle and adipose tissue (8, 9, 30). However, the precise molecular mechanisms are still unclear, although previous reports have shown that activation of PPAR-{gamma} may enhance basal and insulin-induced glucose uptake in 3T3-L1 adipocytes (27, 31).

The importance of PPAR-{gamma} in adipocyte differentiation has been well documented (2, 6). Ectopic expression of PPAR-{gamma} in fibroblasts and muscle precursor cells induces differentiation of these cells into adipocytes, whereas knockout of PPAR-{gamma} in the preadipocyte stage prevents differentiation. Activation of PPAR-{gamma} upregulates genes involved in lipid uptake and storage in adipose tissue (23, 40, 43, 45, 48). However, it is less clear whether PPAR-{gamma} is necessary for maintenance of the adipocyte differentiation state after cells have already undergone adipogenesis.

Insulin resistance is associated with a chronic low-grade inflammatory response characterized by activation of various inflammatory pathway components (10, 37, 41, 50). Evidence for inflammatory responses is quite marked in adipose tissue. Increased adipose tissue macrophage content and proinflammatory gene activation have been well described in obesity and other insulin-resistant states. Presently, it is believed that inflammatory stimuli, such as TNF-{alpha}, activate signaling pathways that cross talk and desensitize insulin signaling, leading to insulin resistance (11–14, 46, 47). TNF-{alpha} is one of several cytokines released by macrophages or adipocytes that can cause insulin resistance. The insulin-sensitizing effects of TZDs may be, in part, explained by their role in repressing inflammation processes. Thus several studies have shown that the PPAR-{gamma} agonists can inhibit macrophage activation and secretion of inflammatory cytokines (19, 36, 49). TZDs also attenuate TNF-{alpha}-induced inhibition of insulin signaling in adipocytes (33, 38).

In this study, we wanted to determine the direct effect of PPAR-{gamma} in adipocytes. After identifying highly efficient small interfering RNA (siRNA) sequences, we used lentiviral short hairpin RNA (shRNA) and electoporation of siRNA to knock down PPAR-{gamma} in adipocytes so as to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-{gamma} knockdown prevents differentiation of 3T3-L1 preadipocytes into adipocytes but is not required for maintenance of the adipocyte differentiation state after the cells have already undergone adipogenesis. We further demonstrate that suppression of PPAR-{gamma} expression reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes. By using dual siRNA strategies, we show that this effect of PPAR-{gamma} deletion is mediated through both GLUT4 and GLUT1. Importantly and interestingly, the early insulin signaling steps were intact in PPAR-{gamma}-deficient adipocytes, whereas these cells displayed enhanced inflammatory response to TNF-{alpha} stimulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Mouse monoclonal antibodies against phosphotyrosine (PY20), PKC-{lambda}, Rab4, red fluorescent protein (RFP), and ERK1/2 monoclonal antibodies were from BD Biosciences (Lexington, KY). Rabbit antibodies against IKK1 [IKK-{alpha}, JNK1/2, phospho-JNK1/2, phospho-Akt (Ser473), phospho-ERK1/2 (Thr202/204), phospho-IKK1/2 (IKK{alpha}/beta, Ser181)] were from Cell Signaling Technology (Beverly, MA). Rabbit antibody against GLUT4 was from Chemicon International (Temecula, CA). Rabbit antibody against GLUT1 was from Abcam (Cambridge, MA). Mouse monoclonal antibodies against green fluorescent protein (GFP), PPAR-{gamma}, and PTEN and rabbit antibodies against insulin receptor-beta (IRbeta), insulin receptor substrate 1 (IRS1), CCAAT/enhancer binding protein (C/EBP)-{alpha}, C/EBPbeta, beta-tubulin, and annexin II were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibody against insulin receptor substrate 2 (IRS2) was from Upstate (Waltham, MA). Goat antibody against aP2 and mouse monoclonal antibody against TNF receptor 1 were from R&D Systems (Minneapolis, MN). Rabbit antibody against actin was from Sigma (St. Louis, MO). DMEM and FBS were from Invitrogen (San Diego, CA). All radioisotopes were from ICN (Costa Mesa, CA). All other reagents were purchased from Sigma.

Design and subcloning of shRNA template into lentiviral vector. A third generation of self-inactivating lentivirus vector containing a cytomegalovirus-driven enhanced GFP reporter and a U6 promoter upstream of cloning restriction sites (HpaI and XhoI) to allow the introduction of oligonucleotides encoding shRNAs (Fig. 1A) was previously described (39). We constructed a control shRNA (siLUC against luciferase) and four shRNA lentiviruses for PPAR-{gamma} (shPPAR-{gamma}1 to shPPAR-{gamma}4) targeting mouse PPAR-{gamma} mRNA (Fig. 1B). Each hairpin consists of a T, a 20- to 21-nt sense sequence, a short spacer (TTCAAGAGA), the antisense sequence, 5 Ts (a stop signal for RNA polymerase III), and an XhoI site (3, 39). The oligonucleotides were annealed and inserted between the HpaI and XhoI sites of the plasmid. Some mutations were introduced in the sense sequence of the hairpin structure to facilitate sequencing and to avoid destruction by bacteria during amplification in bacterial host (26). Correct insertions of shRNA cassettes were confirmed by restriction mapping and direct DNA sequencing.


Figure 1
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  		Fig. 1. Construction of short hairpin peroxisome proliferator-activated receptor-{gamma} (shPPAR-{gamma}) and identification of efficient shPPAR-{gamma} sequences. Schematic representation of short hairpin RNA (shRNA) lentiviral vector (A) and the oligonucleotide sequences for constructing siLUC and shPPAR-{gamma} lentiviruses (B) are shown. 293T cells were cotransfected with shPPAR-{gamma} plasmids together with PPAR-{gamma}-green fluorescent protein (GFP) report plasmid (C) or with PPAR-{gamma}-red fluorescent protein (RFP) report plasmid alone (D). Three days after transfection, cells were lysed for immunoblot analysis of actin, GFP, PPAR-{gamma}-GFP, or PPAR-{gamma}-RFP as indicated. Control 1, reporter plasmid only; control 2, reporter plasmid + shRNA plasmid without hairpin structure; control 3, reporter plasmid + shLUC plasmid (shRNA against luciferase-containing plasmid). SIN-LTR, self-inactivating long terminal repeat; {Psi}, packaging signal; cPPT, central polypurine track; MCS, multiple cloning site; CMV, cytomegalovirus promoter; WRE, woodchuck hepatitis virus response element.

 
Lentivirus production and infection of 3T3-L1 adipocytes. Recombinant lentiviruses were produced by cotransfecting 293T cells with the lentivirus expression plasmid and packaging plasmids using the calcium phosphate method (5, 29, 34). Infectious lentiviruses were harvested at 48 and 72 h posttransfection and filtered through 0.22-µm cellulose acetate filters (28). Recombinant lentiviruses were concentrated by ultracentrifugation (2 h at 50,000 g) and subsequently purified on a 20% sucrose cushion (2 h at 46,000 g). The infectious titer was determined by FACS analysis of GFP positivity in 293T cells. The infection of 3T3-L1 adipocytes (8 days postdifferentiation) with shPPAR-{gamma} lentivirus was carried out by addition of lentivirus into the cell culture with a multiplicity of infection of ~120. The controls were infected with shLUC (shRNA against luciferase-containing plasmid). Four hours after the incubation, the medium was changed.

Construction of GFP and RFP reporter plasmids. For these plasmids, the GFP cDNA in a lentiviral expression plasmid (39) was excised by NheI/EcoRI digestion and replaced with a PCR product in which the GFP or RFP cDNA was flanked by NH2-terminal XbaI/NheI/SmaI sites and a COOH-terminal (His)6 tag and EcoRI/SalI site. To generate the cDNA for PPAR-{gamma}-GFP (or -RFP) fusion protein, the PCR products were restricted with NheI and EcoRI and subcloned into the NheI/EcoRI site of the vector, and a mouse PPAR-{gamma} cDNA preceded by a Kozak consensus sequence flanked with NheI at 5' and SmalI at 3' was inserted. The plasmids were verified by DNA sequencing.

Identification of efficient siRNA sequences. Optimal siRNA targets for PPAR-{gamma} knockdown were identified by cotransfecting 293T cells with PPAR-{gamma} reporter plasmid and shPPAR-{gamma} plasmid by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. Three days posttransfection, reporter gene expression was examined by anti-GFP or anti-RFP immunoblots.

Electroporation of siRNA into 3T3-L1 adipocytes. Five or six days postdifferentiation, 3T3-L1 adipocytes were electroporated with either siGLUT1, siPPAR-{gamma}, or siLUC (HPLC purified from Integrated DNA Technologies; 2.5 nmol per 1 x 107 cells) using the Gene Pulser Xcell (Bio-Rad, Hercules, CA; 250 V and 950 µF), as described previously (22). The siPPAR-{gamma} sequence was identical to the target sequence subcloned into siPPAR-{gamma}3 expression plasmid (Fig. 1). The siGLUT1 target sequence was GGCCGAACCUUCGAUGAGA. Forty-eight hours after electroporation, glucose uptake assays were performed as described (17).

GLUT4 translocation assay. We adapted a modified GLUT4 translocation method by Subtil et al. (42). Briefly, 3T3-L1 adipocytes were electroporated as described above with a plasmid containing dually tagged rat GLUT4 cDNA (HA epitope in the first exofacial loop and GFP at the COOH terminus; a generous gift from Dr. T. E. McGraw), together with the designated siRNAs. The electroporated cells were plated onto coverslip-bottom dishes (MaTek, Ashland, MA). Two days postelectroporation, the adipocytes were treated with the indicated amount of insulin for 20 min and then fixed in 3.7% paraformaldehyde. HA-GLUT4-GFP was stained with mouse monoclonal anti-HA.11 (Covance, Princeton, NJ) followed by Cy3-conjugated secondary antibody (Jackson Immunolabs, West Grove, PA). Fluorescence quantification was performed on a Nikon TE300 inverted microscope using a Nikon x40 numerical aperture 1.3 oil objective, a TILL Photonics II monochromator, and a 12-bit Hamamatsu Orca charge-coupled device camera using Simple PCI software (Hamamatsu, Bridgewater, NJ). At least 20 images were taken for each treatment, in which transfected cells were selected based on GFP expression. The data were summarized from three independent experiments and are shown as means ± SE.

Cell culture. Culture and differentiation of 3T3-L1 cells were described previously (16, 18). In brief, the cells were grown and maintained in high-glucose DMEM containing 10% FBS in a 10% CO2 environment. The cells were allowed to grow for 2 days postconfluency and then differentiated by the addition of IBMX (500 µM), dexamethasone (25 µM), and insulin (4 µg/ml) for 4 days. The medium was changed every 3 days.

Immunoanalyses, 2-[3H]deoxyglucose uptake, and oil red O staining. For immunoblotting, the cells were lysed in a buffer containing 25 mM Tris·HCl (pH 7.4), 0.5 mM EGTA, 25 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 0.2 mM leupeptin, 1 mM benzamidine, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and rocked for 40 min at 4°C. Insoluble material was removed by centrifugation at 12,000 g for 10 min at 4°C. Cell lysates were separated by SDS-PAGE for immunoblot analysis. The proteins were detected by enhanced chemiluminescence with horseradish peroxidase-labeled secondary antibodies.

The procedure for 2-[3H]deoxyglucose (2-DOG) uptake was described previously (17). 3T3-L1 adipocytes were serum starved for 3 h, and the cells were stimulated with insulin for 20 min at 37°C. Glucose uptake was determined after the addition of 2-[3H]DOG (0.1 µCi, final concentration of 0.1 mM) in Krebs-Ringer-phosphate-HEPES buffer (10 mM HEPES, pH 7.4, 131.2 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 2.5 mM NaH2PO4) for 10 min at 37°C.

Fat staining with oil red O was described previously (7). 3T3-L1 cells were fixed with 3.7% formaldehyde for 10 min and then stained with oil red O for 1 h, followed by washing with 70% methanol and water.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of efficient siRNAs targeting mouse PPAR-{gamma}. We constructed PPAR-{gamma}-GFP and PPAR-{gamma}-RFP reporter plasmids by fusing mouse PPAR-{gamma} cDNA with GFP or RFP for identification of efficient siPPAR-{gamma} sequences. Transfection of 293T cells with the reporter plasmids showed high expression of PPAR-{gamma}-GFP and PPAR-{gamma}-RFP, as observed under fluorescent microscope. Immunoblot analysis with anti-GFP, anti-RFP, and anti-PPAR-{gamma} antibodies showed that the corresponding PPAR-{gamma} fusion proteins were correct in size (data not shown). We then selected four siRNA target sequences against the PPAR-{gamma} gene and designed and subcloned shPPAR-{gamma} templates into the expression plasmids; 293T cells were cotransfected with the PPAR-{gamma} reporter plasmid (-GFP or -RFP) together with different shPPAR-{gamma} plasmids (Fig. 1B). Control cells were either transfected with the reporter plasmid, the reporter plasmid plus empty expression plasmid, or the reporter plasmid plus shLUC plasmid. The plasmid itself had no effect on PPAR-{gamma}-GFP expression (Fig. 1C) or on PPAR-{gamma}-RFP expression (Fig. 1D) (compare lane 2 vs. lane 1 within the controls). The shLUC controls also had no effect on PPAR-{gamma}-GFP expression (Fig. 1C) or on PPAR-{gamma}-RFP expression (Fig. 1D) (compare lane 3 vs. lane 1 within the controls). All four shPPAR-{gamma} constructs designed were highly efficient, and reporter proteins were knocked down to undetectable levels by three of them, as identified by immunoblot analyses (Fig. 1, C and D). Actin expression was not altered by any of the shRNA plasmids.

PPAR-{gamma} knockdown blocks differentiation of 3T3-L1 preadipocytes into adipocytes. 3T3-L1 preadipocytes were infected with control (shLUC) or shPPAR-{gamma} lentivirus, followed by initiation of differentiation by adding a standard differentiation mix (IBMX, dexamethasone, and insulin) to the cell cultures. 3T3-L1 preadipocytes infected with the control shLUC lentivirus differentiated normally, whereas the cells infected with any of the shPPAR-{gamma} lentiviruses differentiated very poorly (Fig. 2A), with shPPAR-{gamma} 3 lentivirus being the most inhibitory. Immunoblot analyses of lysates (prepared 4, 8, and 13 days after differentiation) showed that PPAR-{gamma} was markedly reduced in the cells infected with shPPAR-{gamma} lentiviruses (Fig. 2B). The C/EBP{alpha}, C/EBPbeta, IRbeta, and IRS2 proteins were reduced markedly, and aP2 was barely detectable. IRS1 was also moderately reduced. Conversely, expression of other proteins, including PKC-{lambda}, actin, and annexin II, were not altered by shPPAR-{gamma} lentivirus (Fig. 2B).


Figure 2
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  		Fig. 2. shPPAR-{gamma} lentivirus blocks 3T3-L1 differentiation into adipocytes. 3T3-L1 preadipocytes were infected with control (shLUC) or shPPAR-{gamma} lentivirus. Cells were grown 2 days postconfluency and then differentiated with a differentiation cocktail for 4 days. On day 13 postdifferentiation, GFP expression (A, top row) and the phase contrast images of same areas (A, 2nd row) were taken. Lipid droplet staining with oil red O was performed for gross photography (A, 3rd row) followed by nuclei staining with hemotoxylin for microphotography (A, bottom row) under microscope. B: immunoblot analyses were performed on days 4, 8, and 13 postdifferentiation.

 
We further tested the effects of the shPPAR-{gamma} lentivirus in the absence or presence of rosiglitazone, a specific PPAR-{gamma} ligand, which can independently induce adipocyte differentiation. As shown in Fig. 3A, rosiglitazone induced differentiation of 3T3-L1 preadipocytes into adipocytes in the control cells, whereas this effect was completely abolished in the shPPAR-{gamma} lentivirus-infected cells. Immunoblot analyses show that rosiglitazone can induce the expression of PPAR-{gamma}, aP2, C/EBP, IRbeta, and IRS2 (but not IRS1) in the control cells (Fig. 3B, lane 2 vs. lane 1). Rosiglitazone-induced expression of these proteins was completely blocked in shPPAR-{gamma} lentivirus-infected cells (Fig. 3B, lane 4 vs. lanes 1 and 2). IRS1 was also moderately decreased in the shPPAR-{gamma} lentivirus-infected cells, independent of rosiglitazone treatment (compare lane 3 vs. lane 1 and lane 4 vs. lane 2). Other proteins tested, including PKC-{lambda}, ERK, PTEN, and annexin II, were not altered by rosiglitazone treatment or by PPAR-{gamma} knockdown (Fig. 3B). Furthermore, the expressions of aP2 and c/EBP{alpha}, two well-described PPAR-{gamma} direct target genes, were not altered, suggesting that no other PPAR isotype compensated for PPAR-{gamma} deletion.


Figure 3
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  		Fig. 3. shPPAR-{gamma} lentivirus block rosiglitazone (Rosi)-induced adipogenic differentiation. 3T3-L1 preadipocytes were infected with control (shLUC) or shPPAR-{gamma} lentivirus. Cells were grown to 2 days postconfluency and then differentiated by exposure to rosiglitazone (1 µM) for 13 days (medium was changed every 3 days). Thirteen days postdifferentiation, GFP expression (A, top row) and the phase-contrast images of same areas (A, 2nd row) were taken. Lipid droplet staining with oil red O was performed for gross photography (A, third row) followed by nuclei staining with hemotoxylin for microphotography (A, bottom row) under microscope. B: immunoblot analyses were performed on day 13 postdifferentiation.

 
PPAR-{gamma} knockdown does not induce adipocyte dedifferentiation. Although it is known that PPAR-{gamma} is an essential transcription factor for adipogenesis, it is less clear whether PPAR-{gamma} is important in maintaining the adipocyte differentiation state after the cells have already undergone adipogenesis. To address this question, we differentiated 3T3-L1 adipocytes using the standard differentiation cocktail (IBMX, dexamethasone, and insulin). After 3T3-L1 differentiation, cells were infected with shPPAR-{gamma} lentivirus. Six days after shPPAR-{gamma} lentiviral infection in the fully differentiated 3T3-L1 adipocytes, expressions of PPAR-{gamma} and Cbl-associated protein (a PPAR-{gamma} regulated gene) were markedly decreased in shPPAR-{gamma}-infected cells (Fig. 4A). In addition, GLUT4, IRbeta, and IRS2 proteins appeared to be slightly decreased, whereas expressions of other proteins, including those characteristic of differentiated adipocytes (aP2 and C/EBP), Rab4, and annexin II, were not altered by PPAR-{gamma} knockdown. There was also no evidence that the PPAR-{gamma}-deficient adipocytes had undergone dedifferentiation. The microscopic appearance of the shPPAR-{gamma}-infected cells remained the same as that of control, and the lipid content as determined by oil red O staining was comparable between the PPAR-{gamma}-deficient and control adipocytes (Fig. 4A).


Figure 4
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  		Fig. 4. Effect of PPAR-{gamma} knockdown on insulin-stimulated glucose uptake in adipocytes. For A, 3T3-L1 adipocytes (8 days postdifferentiation) were infected with control (shLUC) or shPPAR-{gamma} lentivirus. Six days postinfection, cells were either lysed for immunoblot analyses or images were captured from live cells under the fluorescent microscope (GFP expression, top row; phase contrast of same area, middle row), followed by fixation of cells and staining with oil red O for gross photos (bottom row), or the cells were stimulated with indicated amounts of insulin for 20 min followed by 2-[3H]deoxyglucose (2-DOG) uptake. The data are summarized from 4 independent experiments and presented as means ± SE. For the B–D, 6 days postdifferentiation, the adipocytes were also electroporated with small interfering RNA (siRNA) as indicated (2.5 nmol per 1 x 107 cells) using the Gene Pulser Xcell (250 V and 950 µF). Two days postelectroporation, cells were lysed for immunoblot analysis or stimulated with insulin (100 ng/ml) for 20 min for 2-[3H]DOG uptake. In B, the cells were electroporated with siLUC (control) or siPPAR-{gamma}. The data are summarized from 8 independent experiments (means ± SE; n = 8). {dagger}P < 0.005 compared with control; *P < 0.05 compared with control. In C, the cells were electroporated with either control (siLUC), siPPAR-{gamma}, siGLUT1, or both siGLUT1 and siPPAR-{gamma} (siG1+siP{gamma}) for 2-[3H]DOG uptake (mean ± SE; n = 4). *P < 0.0001 compared with control; {dagger}P < 0.0025 compared with siGLUT1. In D, 3T3-L1 preadipocytes were infected with shLUC or shGLUT4 lentivirus and then differentiated. Six day postdifferentiation, adipocytes were electroporated with siLUC or siPPAR-{gamma} for 2-[3H]DOG uptake (mean ± SE; n = 4). shG4+siP{gamma}, shGLUT4 cells electroporated with siPPAR-{gamma}. *P < 0.05 compared with control; {dagger}P < 0.0001 compared with control; {ddagger}P < 0.005 compared with shGLUT4. In C and D, insulin-stimulated 2-[3H]DOG uptake was calculated from difference between 2-[3H]DOG uptake in the presence of insulin and the basal 2-[3H]DOG uptake.

 
Suppression of PPAR-{gamma} attenuates insulin-stimulated glucose transport by affecting both GLUT1 and GLUT4. To determine whether PPAR-{gamma}-deficient adipocytes show impaired glucose uptake in response to insulin, control and shPPAR-{gamma} lentivirus-infected adipocytes were stimulated with increasing amounts of insulin followed by measurement of 2-DOG uptake. As showed in Fig. 4A, insulin-stimulated 2-DOG uptake was reduced in lentivirus shPPAR-{gamma}-transduced cells compared with control lentivirus-transduced cells. We also knocked down PPAR-{gamma} expression by electroporating 3T3-L1 adipocytes with PPAR-{gamma} siRNA that had the identical target sequence as the lentiviral shPPAR-{gamma}. As shown in Fig. 4B, siPPAR-{gamma} decreased PPAR-{gamma} expression by ~80%, and insulin-stimulated glucose uptake was significantly reduced.

Our group (22) recently demonstrated that both GLUT1 and GLUT4 are important transporters for insulin-stimulated glucose uptake in 3T3-L1 adipocytes and that knockdown of both GLUT1 and GLUT4 completely abolished insulin-stimulated glucose transport. Thus GLUT1, GLUT4, or both must account for the decreased insulin-stimulated glucose uptake in PPAR-{gamma}-deficient cells. To assess this, we performed dual knockdown by silencing both GLUT1 and PPAR-{gamma}. The rationale for this is that, if PPAR-{gamma} deficiency decreases insulin-stimulated glucose uptake via a GLUT1 mechanism, then knockdown of GLUT1 should abolish the inhibitory effect of PPAR-{gamma} on glucose transport.

Adipocytes were electroporated with siLUC, siGLUT1, siPPAR-{gamma}, or siGLUT1 plus siPPAR-{gamma}. Insulin-stimulated glucose uptake was markedly reduced with either siPPAR-{gamma} or siGLUT1 (Fig. 4C), and basal glucose uptake was markedly reduced by siGLUT1 (22) (data not shown). Dual siRNAs (siGLUT1 and siPPAR-{gamma}) further decreased insulin-stimulated glucose uptake. These data indicate that decreased functionality of both GLUT1 and GLUT4 is involved in the impairment of insulin-stimulated glucose uptake induced by PPAR-{gamma} deficiency. This was further supported by experiments with dual knockdown of GLUT4/PPAR-{gamma}. Insulin-stimulated glucose uptake was markedly reduced by either siPPAR-{gamma} or siGLUT4, and the combination of siRNAs (siGLUT4 and siPPAR-{gamma}) further decreased insulin-stimulated glucose uptake (Fig. 4D).

Effect of PPAR-{gamma} knockdown on insulin signal phosphorylation cascades and GLUT4 translocation. Multiple experiments showed that GLUT4 but not GLUT1 levels were reduced in PPAR-{gamma}-deficient cells (Fig. 5A). To further define the effects of PPAR-{gamma} silencing on insulin action in 3T3-L1 adipocytes, we examined GLUT4 translocation. We employed a single cell GLUT4 translocation assay (42) to directly quantify the amount of GLUT4 targeted to the plasma membrane on insulin stimulation. This technique relies on the transient transfection of a GLUT4 construct tagged with an HA epitope in the first extracellular loop and GFP in the intracellular COOH-terminal region (HA-GLUT4-GFP) into cells. For a given cell, the extent of GLUT4 translocation to the plasma membrane can be quantitated by normalizing the immunofluorescent labeling of the extracellular HA epitope against total cellular GFP content. As illustrated in Fig. 5B, basal GLUT4 translocation was comparable between control cells and PPAR-{gamma}-deficient cells. GLUT4 translocation in response to maximal insulin stimulation (100 ng/ml) in PPAR-{gamma}-deficient cells (~13-fold increase) was also similar to that of control cells. However, at submaximal insulin stimulation (3 and 10 ng/ml), GLUT4 translocation was modestly but significantly reduced compared with control.


Figure 5
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  		Fig. 5. Effect of PPAR-{gamma} knockdown on insulin and TNF-{alpha} signaling in adipocytes. A: adipocytes were electroporated with control (siLUC) or siPPAR-{gamma}. Two days postelectroporation, cells were lysed for immunoblot analyses of GLUT1, GLUT4, and annexin II. The immunoblots were scanned and quantitated with NIH Image software. Data were summarized from 4 independent experiments and represented as % of control. Annexin II was not altered by siPPAR-{gamma} (not shown). *P = 0.05 compared with control. B: experiments were performed to measure translocation of exogenous dually tagged HA-GLUT4-GFP in 3T3-L1 adipocytes that were electroporated with either HA-GLUT4-GFP plus siLUC (control) or HA-GLUT4-GFP plus siPPAR-{gamma}. Two days posttransfection, cells were stimulated with 0, 3, 10, or 100 ng/ml insulin for 20 min. GLUT4 translocation kinetics was assayed. Data were summarized from 3 independent experiments and are represented as mean ± SE. *P < 0.05 compared with control; {dagger}P = 0.07 compared with control. C: 3T3-L1 adipocytes were transduced with control (shLUC) or shPPAR-{gamma} lentivirus. Six days postinfection, the cells were stimulated with indicated amounts of insulin for 5 min and then lysed for immunoblot analyses. D: 3T3-L1 adipocytes (day 6 postdifferentiation) were electroporated with control siRNA (siLUC) or siPPAR-{gamma}. Two days posttransfection, the cells were incubated with or without chronic insulin stimulation (100 ng/ml) for 20 h followed by an acute 5 min insulin stimulation as indicated. The cells were then lysed for immunoblot analyses. E: adipocytes were electroporated with control (siLUC) or siPPAR-{gamma}. Two days postelectroporation, cells were incubated with TNF-{alpha} for 0, 15, 30, or 180 min. Cells were then lysed for immunoblot analysis using the indicated antibodies. The experiments presented in C, D, and E were repeated for 2 or more times. Tyr-p, phosphotyrosine.

 
Because the PPAR-{gamma}-deficient adipocytes displayed impaired insulin-stimulated glucose uptake, we assessed the early insulin-induced phosphorylation events in these cells. First, adipocytes were infected with either control (shLUC) or shPPAR-{gamma} lentivirus, and insulin-stimulated phosphorylation steps were assayed by Western blot analysis 6 days later. Our data show that insulin-stimulated phosphorylation of IRbeta, IRS1, Akt, and ERK1/2 was not affected in shPPAR-{gamma} lentivirus-infected adipocytes (Fig. 5C, lanes 4–6 vs. lanes 1–3). Similarly, adipocytes were electroporated with either control (siLUC) or siPPAR-{gamma}, and the same measurements were examined. Again, siPPAR-{gamma} did not cause impaired insulin-stimulated phosphorylation of IRbeta, IRS1, Akt, and ERK1/2 (Fig. 5D, lanes 4 and 5 vs. lanes 1 and 2). However, it should be noted that depletion of PPAR-{gamma} was not complete, and it is possible that the small amount of residual PPAR-{gamma} can sustain early insulin signaling, even though glucose transport was inhibited.

Chronic insulin treatment causes desensitization of insulin signaling pathways, and we assessed the possible role of PPAR-{gamma} in this process. As shown in Fig. 5D, pretreatment of adipocytes with insulin for 20 h blunted the response to subsequent acute insulin stimulation in both control (Fig. 5D, lane 3 vs. lane 2) and PPAR-{gamma}-deficient cells (Fig. 5D, lane 6 vs. lane 5) with respect to IRbeta, IRS1, Akt, and ERK1/2 phosphorylation, and PPAR-{gamma} deficiency did not alter this effect (Fig. 5C, lanes 4–6 vs. lanes 1–3).

PPAR-{gamma} knockdown enhances TNF-{alpha}-induced activation of ERK and JNK. Activation of inflammatory pathway signaling can lead to insulin resistance, and it has been suggested that PPAR-{gamma} improves insulin sensitivity (1, 4, 35) by inhibiting inflammatory responses in macrophages and adipocytes (19, 33, 36, 38, 49). To assess this idea in our system, we determined whether PPAR-{gamma} deficiency influenced the effect of TNF-{alpha} to activate ERK and JNK in 3T3-L1 adipocytes. Control and PPAR-{gamma}-deficient adipocytes were incubated in the absence or presence of TNF-{alpha} for various periods (0, 15, 30, 180 min). TNF-{alpha}-induced phosphorylation of ERK and JNK was clearly enhanced in PPAR-{gamma}-deficient adipocytes compared with control cells, whereas PPAR-{gamma} deficiency had little effect on TNF-{alpha}-induced IKK phosphorylation (Fig. 5E) or on TNF receptor expression per se (not shown).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PPAR-{gamma} is essential for normal adipocyte differentiation (15, 44) and plays an important role in regulating insulin sensitivity and glucose homeostasis (1, 4, 35). Although a number of animal experiments and clinical studies have demonstrated the important role of PPAR-{gamma} in regulating insulin and glucose homeostasis, the precise molecular mechanisms remain unclear. In the present study, we demonstrated that PPAR-{gamma} knockdown prevented differentiation of 3T3-L1 preadipocytes into adipocytes but was not required to maintain the adipocyte differentiation state after the cells had undergone adipogenesis. Clearly, PPAR-{gamma} plays an essential role in the adipogenic differentiation program (15, 44), and this is fully supported by our PPAR-{gamma} knockdown studies in two independent differentiation models of 3T3-L1 cells. We showed that PPAR-{gamma} knockdown largely prevented adipogenic differentiation of 3T3-L1 cells induced by standard differentiation mix and completely abolished adipogenic differentiation of 3T3-L1 cells induced by the PPAR-{gamma} ligand rosiglitazone. In contrast, when PPAR-{gamma} was knocked down after full differentiation of the adipocytes, the cell retained their adipocyte phenotype, as assessed by morphology measurement of lipid content, and continued normal expression of proteins associated with the adipocyte phenotype. We noted that shPPAR-{gamma} lentivirus-mediated gene knockdown in differentiated adipocytes was somewhat less efficient than in the preadipocytes under our experimental conditions. One could argue that the residual PPAR-{gamma} is sufficient to retain the adipocytic characteristics of the cells. However, under fluorescent microscope, we observed that adipocytes with high expression of shPPAR-{gamma} (the bright green cells) retained full adipose cell phenotype even 20 days after transduction of shPPAR-{gamma} lentivirus (data not shown). These findings are further supported by studies in adipose tissue-specific PPAR-{gamma} knockout mice, which maintained some adipocyte islets that were even hypertrophic (8). Thus, in fully differentiated adipocytes, PPAR-{gamma} may not be essential to maintaining the adipocyte phenotype. These findings also suggest that PPAR-{gamma}'s effects on insulin sensitivity are not fully explained by the states of the adipose tissue and that effects on other tissues are also important.

One of the most important functions of PPAR-{gamma} is to maintain an appropriate expression level of key insulin signaling molecules for stimulation of glucose transport. Our present study demonstrated that PPAR-{gamma} knockdown attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes, providing direct evidence that PPAR-{gamma} is an important modulator of insulin-stimulated glucose uptake in these cells.

We recently demonstrated that both GLUT1 and GLUT4 contribute to glucose uptake in insulin-stimulated 3T3-L1 adipocytes (22). In the present study, we find that PPAR-{gamma} deficiency leads to impaired glucose uptake in insulin-stimulated adipocytes involving both GLUT1 and GLUT4 in these cells. Interestingly, we found that PPAR-{gamma} knockdown in the differentiated adipocytes did not affect the initial insulin-induced phosphorylation steps involving IRbeta, IRS1, Akt, or ERK. These results indicate that the decrease in insulin-stimulated glucose uptake in PPAR-{gamma}-depleted cells is related to some aspect of the insulin signaling/glucose transport system, downstream of Akt activation. In addition, expression levels of GLUT4 but not GLUT1 were reduced in PPAR-{gamma}-depleted cells, contributing to the reduction of insulin-stimulated glucose uptake.

It has been suggested that one mechanism whereby PPAR-{gamma} regulates insulin sensitivity is by inhibiting inflammatory pathway responses in macrophages and adipocytes. In the present study, we found that PPAR-{gamma} deficiency led to an enhanced ability of TNF-{alpha} to activate JNK and ERK, raising the possibility that PPAR-{gamma} normally restrains ERK and JNK signaling in 3T3-L1 adipocytes. Interestingly, both ERK and JNK have been shown to induce serine phosphorylation of IRS1, which can lead to decreased insulin-stimulated IRS1 tyrosine phosphorylation. However, in the present study, PPAR-{gamma} depletion did not appear to affect the insulin signaling steps, including IRS1 tyrosine phosphorylation. However, by modulating transcription of the target gene, it is possible that increased JNK and/or ERK activity leads to gene expression changes that result in decreased insulin sensitivity.

In summary, the present study shows that depletion of PPAR-{gamma} prevents differentiation of preadipocytes to adipocytes but does not induce dedifferentiation of cells once full differentiation has taken place. We further show that loss of PPAR-{gamma} results in a decrease in insulin-stimulated glucose transport into 3T3-L1 adipocytes and that this is due to a decrease in GLUT1 and GLUT4 function. Finally, it has been previously shown that PPAR-{gamma} activation can repress inflammatory pathway activity, and, consistent with this, we find that PPAR-{gamma}-deficient adipocytes display enhanced responses to TNF-{alpha}-induced activation of JNK and ERK.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by an American Heart Association National Scientist Development Grant (0235286N to W. Liao), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33651 (J. M. Olefsky), and a University of California Discovery Project Grant with matching fund from Pfizer Incorporated (Bio03-10383, BioStar, to J. M. Olefsky). J. M. Olefsky is a Consultant for Pfizer Incorporated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Olefsky, Dept. of Medicine, Division of Endocrinology and Metabolism, Univ. of California, San Diego, La Jolla, CA 92093 (e-mail: jolefsky{at}ucsd.edu)

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.


   REFERENCES
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 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Auwerx J. PPAR{gamma}, the ultimate thrifty gene. Diabetologia 42: 1033–1049, 1999.[CrossRef][ISI][Medline]
  2. Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med 53: 409–435, 2002.[CrossRef][ISI][Medline]
  3. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553, 2002.[Abstract/Free Full Text]
  4. Debril MB, Renaud JP, Fajas L, Auwerx J. The pleiotropic functions of peroxisome proliferator-activated receptor gamma. J Mol Med 79: 30–47, 2001.[CrossRef][ISI][Medline]
  5. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. A third-generation lentivirus vector with a conditional packaging system. J Virol 72: 8463–8471, 1998.[Abstract/Free Full Text]
  6. Evans RM, Barish GD, Wang YX. PPARs and the complex journey to obesity. Nat Med 10: 355–361, 2004.[CrossRef][ISI][Medline]
  7. Hansen JB, Petersen RK, Larsen BM, Bartkova J, Alsner J, Kristiansen K. Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation. J Biol Chem 274: 2386–2393, 1999.[Abstract/Free Full Text]
  8. He W, Barak Y, Hevener A, Olson P, Liao D, Le J, Nelson M, Ong E, Olefsky JM, Evans RM. Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle. Proc Natl Acad Sci USA 100: 15712–15717, 2003.[Abstract/Free Full Text]
  9. Hevener AL, He W, Barak Y, Le J, Bandyopadhyay G, Olson P, Wilkes J, Evans RM, Olefsky J. Muscle-specific PPAR-{gamma} deletion causes insulin resistance. Nat Med 9: 1491–1497, 2003.[CrossRef][ISI][Medline]
  10. Hotamisligil GS. Inflammatory pathways and insulin action. Int J Obes Relat Metab Disord 27, Suppl 3: S53–S55, 2003.[CrossRef][ISI]
  11. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased adipose tissue expression of tumor necrosis factor-{alpha} in human obesity and insulin resistance. J Clin Invest 95: 2409–2415, 1995.[ISI][Medline]
  12. Hotamisligil GS, Budavari A, Murray D, Spiegelman BM. Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-{alpha}. J Clin Invest 94: 1543–1549, 1994.[ISI][Medline]
  13. Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM. Tumor necrosis factor {alpha} inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 91: 4854–4858, 1994.[Abstract/Free Full Text]
  14. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-{alpha}: direct role in obesity-linked insulin resistance. Science 259: 87–91, 1993.[Abstract/Free Full Text]
  15. Hu E, Tontonoz P, Spiegelman BM. Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR {gamma} and C/EBP {alpha}. Proc Natl Acad Sci USA 92: 9856–9860, 1995.[Abstract/Free Full Text]
  16. Imamura T, Huang J, Dalle S, Ugi S, Usui I, Luttrell LM, Miller WE, Lefkowitz RJ, Olefsky JM. beta-Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport. J Biol Chem 276: 43663–43667, 2001.[Abstract/Free Full Text]
  17. Imamura T, Vollenweider P, Egawa K, Clodi M, Ishibashi K, Nakashima N, Ugi S, Adams JW, Brown JH, Olefsky JM. G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes. Mol Cell Biol 19: 6765–6774, 1999.[Abstract/Free Full Text]
  18. Ishibashi KI, Imamura T, Sharma PM, Huang J, Ugi S, Olefsky JM. Chronic endothelin-1 treatment leads to heterologous desensitization of insulin signaling in 3T3-L1 adipocytes. J Clin Invest 107: 1193–1202, 2001.[ISI][Medline]
  19. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 391: 82–86, 1998.[CrossRef][Medline]
  20. Lee MK, Miles PD, Khoursheed M, Gao KM, Moossa AR, Olefsky JM. Metabolic effects of troglitazone on fructose-induced insulin resistance in the rat. Diabetes 43: 1435–1439, 1994.[Abstract]
  21. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 270: 12953–12956, 1995.[Abstract/Free Full Text]
  22. Liao W, Nguyen MT, Imamura T, Singer O, Verma IM, Olefsky JM. Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes. Endocrinology 147: 2245–2252, 2006.[Abstract/Free Full Text]
  23. Martin G, Schoonjans K, Lefebvre AM, Staels B, Auwerx J. Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPAR{alpha} and PPAR{gamma} activators. J Biol Chem 272: 28210–28217, 1997.[Abstract/Free Full Text]
  24. Miles PD, Higo K, Romeo OM, Lee MK, Rafaat K, Olefsky JM. Troglitazone prevents hyperglycemia-induced but not glucosamine-induced insulin resistance. Diabetes 47: 395–400, 1998.[Abstract]
  25. Miles PD, Romeo OM, Higo K, Cohen A, Rafaat K, Olefsky JM. TNF-{alpha}-induced insulin resistance in vivo and its prevention by troglitazone. Diabetes 46: 1678–1683, 1997.[Abstract]
  26. Miyagishi M, Sumimoto H, Miyoshi H, Kawakami Y, Taira K. Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J Gene Med 6: 715–723, 2004.[CrossRef][ISI][Medline]
  27. Mukherjee R, Hoener PA, Jow L, Bilakovics J, Klausing K, Mais DE, Faulkner A, Croston GE, Paterniti JR Jr. A selective peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) modulator blocks adipocyte differentiation but stimulates glucose uptake in 3T3-L1 adipocytes. Mol Endocrinol 14: 1425–1433, 2000.[Abstract/Free Full Text]
  28. Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 93: 11382–11388, 1996.[Abstract/Free Full Text]
  29. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267, 1996.[Abstract]
  30. Norris AW, Chen L, Fisher SJ, Szanto I, Ristow M, Jozsi AC, Hirshman MF, Rosen ED, Goodyear LJ, Gonzalez FJ, Spiegelman BM, Kahn CR. Muscle-specific PPAR{gamma}-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones. J Clin Invest 112: 608–618, 2003.[CrossRef][ISI][Medline]
  31. Nugent C, Prins JB, Whitehead JP, Savage D, Wentworth JM, Chatterjee VK, O'Rahilly S. Potentiation of glucose uptake in 3T3-L1 adipocytes by PPAR-{gamma} agonists is maintained in cells expressing a PPAR-{gamma} dominant-negative mutant: evidence for selectivity in the downstream responses to PPAR-{gamma} activation. Mol Endocrinol 15: 1729–1738, 2001.[Abstract/Free Full Text]
  32. Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose DW, Willson TM, Rosenfeld MG, Glass CK. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-{gamma}. Nature 437: 759–763, 2005.[CrossRef][Medline]
  33. Peraldi P, Xu M, Spiegelman BM. Thiazolidinediones block tumor necrosis factor-{alpha}-induced inhibition of insulin signaling. J Clin Invest 100: 1863–1869, 1997.[ISI][Medline]
  34. Pfeifer A, Kessler T, Silletti S, Cheresh DA, Verma IM. Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2. Proc Natl Acad Sci USA 97: 12227–12232, 2000.[Abstract/Free Full Text]
  35. Picard F, Auwerx J. PPAR(gamma) and glucose homeostasis. Annu Rev Nutr 22: 167–197, 2002.[CrossRef][ISI][Medline]
  36. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-{gamma} is a negative regulator of macrophage activation. Nature 391: 79–82, 1998.[CrossRef][Medline]
  37. Ruan H, Lodish HF. Insulin resistance in adipose tissue: direct and indirect effects of tumor necrosis factor-{alpha}. Cytokine Growth Factor Rev 14: 447–455, 2003.[CrossRef][ISI][Medline]
  38. Ruan H, Pownall HJ, Lodish HF. Troglitazone antagonizes tumor necrosis factor-{alpha}-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-{kappa}B. J Biol Chem 278: 28181–28192, 2003.[Abstract/Free Full Text]
  39. Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Ihrig MM, McManus MT, Gertler FB, Scott ML, Van Parijs L. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33: 401–406, 2003.[CrossRef][ISI][Medline]
  40. Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA, Briggs M, Deeb S, Staels B, Auwerx J. PPAR{alpha} and PPAR{gamma} activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 15: 5336–5348, 1996.[ISI][Medline]
  41. Sethi JK, Hotamisligil GS. The role of TNF-{alpha} in adipocyte metabolism. Semin Cell Dev Biol 10: 19–29, 1999.[CrossRef][ISI][Medline]
  42. Subtil A, Lampson MA, Keller SR, McGraw TE. Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule. J Biol Chem 275: 4787–4795, 2000.[Abstract/Free Full Text]
  43. Tontonoz P, Hu E, Devine J, Beale EG, Spiegelman BM. PPAR {gamma} 2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene. Mol Cell Biol 15: 351–357, 1995.[Abstract]
  44. Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 79: 1147–1156, 1994.[CrossRef][ISI][Medline]
  45. Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM. PPAR{gamma} promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 93: 241–252, 1998.[CrossRef][ISI][Medline]
  46. Uysal KT, Wiesbrock SM, Hotamisligil GS. Functional analysis of tumor necrosis factor (TNF) receptors in TNF-{alpha}-mediated insulin resistance in genetic obesity. Endocrinology 139: 4832–4838, 1998.[Abstract/Free Full Text]
  47. Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS. Protection from obesity-induced insulin resistance in mice lacking TNF-{alpha} function. Nature 389: 610–614, 1997.[CrossRef][Medline]
  48. Way JM, Harrington WW, Brown KK, Gottschalk WK, Sundseth SS, Mansfield TA, Ramachandran RK, Willson TM, Kliewer SA. Comprehensive messenger ribonucleic acid profiling reveals that peroxisome proliferator-activated receptor gamma activation has coordinate effects on gene expression in multiple insulin-sensitive tissues. Endocrinology 142: 1269–1277, 2001.[Abstract/Free Full Text]
  49. Welch JS, Ricote M, Akiyama TE, Gonzalez FJ, Glass CK. PPAR{gamma} and PPAR{delta} negatively regulate specific subsets of lipopolysaccharide and IFN-{gamma} target genes in macrophages. Proc Natl Acad Sci USA 100: 6712–6717, 2003.[Abstract/Free Full Text]
  50. Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest 115: 1111–1119, 2005.[CrossRef][ISI][Medline]




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