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Am J Physiol Endocrinol Metab 293: E576-E586, 2007. First published May 22, 2007; doi:10.1152/ajpendo.00523.2006
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Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

Wen Guo,1 Siu Wong,1 Weisheng Xie,1 Tianluo Lei,1 and Zhijun Luo1,2

1Department of Medicine and 2Department of Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts

Submitted 26 September 2006 ; accepted in final form 9 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although fatty acids enhance preadipocyte differentiation in the presence of adequate hormone cocktails, little is known regarding their effects in the absence of these hormones. We have now shown that palmitate, a common long-chain saturated fatty acid, induced apoptosis in both mouse 3T3-L1 and rat primary preadipocytes grown in a normal serum-containing medium. Treatment of preadipocytes with palmitate induced multiple endoplasmic reticulum (ER) stress responses, evidenced by increased protein content of CHOP and GRP78 and splicing of XBP-1 mRNA, as well as altered phosphorylation of eIF2{alpha} and increased phosphorylation of JNK and Erk1/2. Intriguingly, palmitate induced an early activation of Akt but diminished both Akt activation and its protein mass after prolonged incubation (>6 h). In association with these changes, palmitate reduced expression of beta-catenin and its downstream target, c-Myc and cyclin D1, two key prosurvival proteins. Overexpression of constitutively active Akt did not block the apoptotic effect of palmitate. Cotreatment with unsaturated fatty acids (oleate, linoleate) or with LiCl (a glycogen synthase kinase-3beta inhibitor) attenuated the palmitate-induced apoptosis. Subsequent analysis suggested that the unsaturated fatty acids probably counteracted palmitate by reducing, not eliminating, ER stress, whereas LiCl probably improved viability by activating the Wnt signaling pathway. Cotreatment of palmitate with a standard adipogenic hormone cocktail also abolished the apoptotic effect and promoted adipocyte differentiation. Collectively, our results suggest that palmitate causes multiple cellular stresses that may lead to apoptosis in preadipocytes in the absence of adipogenic stimuli, highlighting the importance of exogenous hormones in directing cell fate in response to increased fatty acid influx.

insulin-like growth factor-1 signaling; Wnt signaling

ADIPOSE TISSUE MAY BE the most flexible tissue in the body in that it accommodates energy fluctuation by changing adipocyte cell size and number. When energy intake exceeds expenditure, the extra is deposited as fat. Obesity develops as a result of fat cell hypertrophy and hyperplasia. Eventually, the increase of fat cell size ceases and new fat cell recruitment slows down. This can lead to increased plasma free fatty acids that can damage proper function of nonadipose organs; now commonly termed "lipotoxicity" (11, 30).

Adipocytes are naturally immune from lipotoxicity because of their high capacity for fatty acid detoxification (27, 29). However, it should be noted that 15–50% of the cells in adipose tissue are fibroblast-like preadipocytes. These cells are able to divide or differentiate in response to extracellular cues (25). However, these cells have very limited capacity to synthesize and store neutral lipids (9, 18, 32). Previous studies on preadipocytes mostly focus on the mechanisms of adipogenesis induced by hormonal cocktails (3, 19, 28). Under these conditions, fatty acids have been shown to stimulate preadipocyte differentiation (2, 19, 41). Little is known about how preadipocytes respond to fatty acids in the absence of adipogenic hormones, except for one study showing that fatty acids do not activate adipogenesis without a high dose of insulin (12).

Although the "adipogenic" cocktails may mimic the enriched nutritional/hormonal conditions at an initial stage of fat tissue expansion (14), they may not reflect the conditions within overgrown fat tissues. Because of dampened blood flow in enlarged fat pads, preadipocytes may become relatively deprived of nonadipose endocrine factors such as insulin (10). Meanwhile, adipose-derived cytokines and other bioactive molecules, including fatty acids, may become locally enriched. Among these factors, tumor necrosis factor-{alpha} (TNF-{alpha}) and leptin inhibit (1, 42), whereas adiponectin promotes, adipogenesis (15); the roles of many others remain to be clearly established. Among other changes, obesity decreases adiponectin but increase TNF-{alpha} and leptin production, implying that the interior environment of enlarged fat tissue may become unfavorable for adipogenesis. Meanwhile, such changes are expected to increase fatty acid release further, because many of the fat tissue-derived cytokines are known to stimulate lipolysis. Learning how these conditions affect preadipocytes is important for understanding the mechanism of adipose dysfunction in obesity and type 2 diabetes. As the first step toward this goal, we tested the hypothesis that fatty acids can induce cellular "stress" in preadipocytes, as they do in several other cell types (5, 24, 30, 39). We also tested how different fatty acids act on preadipocytes. Our results demonstrate that the saturated fatty acid palmitate induced apoptosis in both 3T3-L1 cells and primary rat preadipocytes. In association with the proapoptotic effect, palmitate caused endoplasmic reticulum (ER) stress as evidenced by increased X-box-binding protein-1 (XBP-1) mRNA splicing, altered phosphorylation of eukaryotic initiation factor-2{alpha} (eiF2{alpha}), and increased expression of CEBP homology protein (CHOP) and glucose-regulated protein 78 (GRP78). Palmitate also induced hyperphosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-Jun NH2-terminal kinase (JNK) MAP kinases. Intriguingly, palmitate induced early activation (sustained for the first 6 h) of Akt but diminished basal and insulin-like growth factor-1 (IGF-1)-stimulated Akt activation after longer incubation. In association with these changes, palmitate reduced the protein content of beta-catenin, c-Myc, and cyclin D1, suggesting an impairment of the prosurvival Wnt pathway. Some of these effects were reversed or partially reversed by cotreatment with unsaturated fatty acids or with a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, LiCl, which also blunted the proapoptotic effects of palmitate. Collectively, our results demonstrate that palmitate can induce multiple cellular stresses in preadipocytes. This, if extrapolated to the in vivo conditions, may contribute to adipose tissue dysfunction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. 3T3-L1 preadipocytes were obtained from American Type Culture Collection (Manassas, VA). Cells were grown in DMEM containing 10% calf serum (CS) and antibiotics in a 5% CO2 incubator. Rat preadipocytes were isolated from the epididymal fat pads of Sprague-Dawley rats (350–400 g) and grown in {alpha}MEM containing 10% fetal bovine serum (FBS) and antibiotics as described previously (25). Cell culture supplies were obtained from Fisher Scientific (Agawam, MA).

Treatment with fatty acids. Sodium salts of palmitic acid, oleic acid, and linoleic acid, as well as fatty acid-free bovine serum albumin (BSA), were purchased from Sigma (St. Louis, MO). Fatty acid stock solution (10 mM) was prepared by dissolving the sodium salt in BSA (3.3 mM) solution. Cells were treated with fatty acids in the growth medium containing 10% serum. For selected experiments, 3T3-L1 cells were treated with palmitate for 24 h in DMEM with 10% FBS plus insulin (170 nM), dexamethasone (1 µM), and IBMX (0.5 mM). Pharmaceutical inhibitors for MEK1/2 (U0126, PD98059) and JNK (SP600125 and Dicoumarol), as well as LiCl, were purchased from Sigma or Calbiochem (San Diego, CA) and used as indicated.

Treatment with IGF-1. After preincubation with the corresponding fatty acids for 15–16 h, cells were washed twice with phosphate-buffered saline (PBS) and once with serum-free medium. Cells were then incubated in serum-free medium for 90 min before IGF-1 was added. After 15 min, cells were washed once with ice-cold PBS, snap frozen in liquid nitrogen, and stored at –80°C until use.

Flow cytometry. DNA fragmentation was analyzed using flow cytometry (fluorescence-activated cell sorting, FACS). Following treatment with palmitate, both nonadherent and trypsinized adherent cells were collected and fixed with ethanol (35%). After the removal of ethanol, cells were resuspended in PBS containing propidium iodide (PI; 20 µg/ml; Sigma) and RNase A (5 U/µl; Qiagen, Valencia, CA) and incubated at 37°C for 30 min. Cells were then analyzed using a Becton Dickinson fluorescent cytometer (BD Biosciences, San Jose, CA). Fluorescence of 25,000 cells was measured for each sample. The hypodiploid population with weaker PI staining than Go cells were considered apoptotic cells (30). The cell distribution among different sub-Go, Go/G1, G2/M, and S phases was analyzed using Cychred software.

Hoechst 33342 and PI staining and fluorescent microscopy. Cells were incubated under different conditions as indicated. At the end of the incubation period, Hoechst 33342 and PI were added together to the culture (10 µg/ml each) and incubated for an additional 10 min. Cells were then washed three times with Krebs-Ringer buffer (pH 7.4) and imaged immediately using an inverted phase-contrast/fluorescent microscope (Nikon, Melville, NY). Hoechst 33342 staining (blue) was examined with excitation wavelength at 330–380 nm and receiver (emission) band pass at 435–485 nm, and PI staining (pink) we examined with excitation wavelength at 426–446 nm and receiver band pass at 460–500 nm. Apoptotic cells were distinguished as those with characteristic chromatin condensation that stained more intensely than viable cells (4). Necrotic cells were distinguished from (early) apoptotic cells by PI staining, since this dye does not enter cells with intact plasma membranes.

MTT assay. Cells were grown in 96-well plates and treated with indicated conditions. Cell viability was measured using the MTT kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN) (31).

Western analysis. Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Sigma), 2 mM Na3VO4, and 1 mM of each of the following: NaF, imidazole, Na3MoO4, Na3P2O3, beta-glycerophosphate, and Na-tartarate. Protein concentration was quantified with the Bradford reagent (Sigma), and normalized protein (20 µg) was separated by electrophoresis using an 8% (for insulin receptor substrate-1, IRS-1) or 12% (for all other targets) Tris·HCl acrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane and used for Western analysis following the standard protocol. Primary antibodies for phospho-Akt (Ser473), phospho-Erk1/2, phospho-JNK, phospho-p38, phospho-IRS-1 (Ser307), and phospho-eiF2{alpha} (Ser 51), as well as antibodies for the total protein of each were obtained from Cell Signaling (Danvers, MA). Antibodies for GRP78 and CHOP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies were obtained from Santa Cruz. Proteins were visualized using chemiluminescence reagents (Pierce, Rockford, IL) and quantified by densitometry.

PCR analysis. Total RNA was isolated using the Trizol method (Invitrogen, Carlsbad, CA). Reverse transcription from mRNA to cDNA was done using the SuperScript cDNA synthesis kit from Invitrogen. SYBG-based real-time PCR was conducted using a Rotorgene 3000A system as described previously (41). For measurement of XBP-1 splicing, mouse XBP-1 (GenBank accession no. NM_013842) primer spanning the 26-bp intron splicing site was designed as forward: 5'-tgagaaccaggagttaagaacacgc-3' and reverse: 5'-ttctgggtagacctctgggagttcc-3'. This gave a PCR product of 326 bp for unspliced and 300 bp for spliced XBP-1. The PCR reaction was performed using Sigma JumpStart enzyme master mix as described previously (20). The products were separated by electrophoresis with a 4% agarose gel and visualized by ethidium bromide staining.

Immunoprecipitation of IRS-1 and phosphatidylinositol 3-kinase assay. Cells were lysed in a solution containing 0.5% Triton X-100, 120 mM NaCl, 50 mM Tris·HCl (pH 7.5), 5 mM NaF, 10 mM beta-glycerophosphate, 5 mM Na3P2O3, 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 2 mM imidazole, 1 mM Na3MoO4, 10% glycerol, and a protease inhibitor cocktail (Sigma). Crude protein extracts (500 µg/sample) were immunoprecipitated with an antibody against IRS-1 (Cell Signaling) at 4°C overnight with gentle rotation, and the immunocomplex was precipitated by addition of trisacryl protein A beads (Pierce) and incubation at 4°C for another 4 h. The beads were then separated into two equal fractions for phosphatidylinositol 3-kinase (PI3K) activity and immunoblotting analysis, respectively. PI3K was assayed as described previously (40).

Adenovirus-mediated overexpression of Akt and green fluorescent protein. Adenovirus encoding a constitutively active mutant of Akt (MyrAkt; Ref. 16) was a gift from Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA). Adenovirus encoding green fluorescent protein (GFP) was obtained from the Genecore of the University of Iowa (Ames, Iowa). For gene transfection, 3T3-L1 preadipocytes were grown to 90% confluence, detached from culture dish using an enzyme-free cell dissociation solution (Specialty Media, Phillipsburg, NJ). Each sample of 10 x 106 cells (in 0.7 ml of the cell dissociation solution) was mixed with 2 µg of viral DNA and subjected to three pulses of electroporation (160 V, 1,180 µF). Cells were then mixed with DMEM containing 10% CS and plated onto 60-mm dishes (3 x 106 cells/dish). The expression of GFP was examined under fluorescent microscope (typically 30–50% of the cells express green fluorescence), and the expression of Akt was measured using real-time PCR and Western analysis.

Statistical analysis. Quantitative results were analyzed for statistical significance by using either one-way or two-way ANOVA or Student's t-test. Multiple comparisons were analyzed using Duncan's test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Palmitate induces apoptosis in 3T3-L1 preadipcoytes in the absence of, but induces differentiation in the presence of, the adipogenic cocktail. Palmitate has been shown to induce apoptosis in several cell types (4, 5, 30) but to enhance adipogenesis in preadipocytes maintained in a differentiation cocktail (18). To test its effects on preadipocytes in the absence of adipogenic inducers, we treated 3T3-L1 preadipocytes with palmitate in normal growth medium. After 24 h, the density of adherent cells was reduced in proportion to the concentration of palmitate (Fig. 1A, top), concurrent with increases in detached cells (not shown). The adherent cells were then costained with Hoechst 33342 and PI. In all cultures examined, PI staining was minimal with no detectable difference between cells treated with and without palmitate (not shown), indicating that necrosis was not predominant if any. In contrast, palmitate induced a progressive increase in the number of cells that were intensely stained by Hoechst 33343 (Fig. 1A, bottom), reflecting increased apoptosis (4). A time course analysis showed that apoptosis became apparent between 6 and 12 h after exposure of cells to palmitate (Fig. 1B), temporally in agreement with the findings in other cell types (30). Using FACS analysis, we showed that palmitate caused a large increase in hypodiploid DNA (sub-Go phase, Fig. 1C), confirming the presence of DNA fragmentation (30).


Figure 1
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  		Fig. 1. Palmitate induces apoptosis of 3T3-L1 preadipocytes. A: cells were grown to 90% confluence before incubation in DMEM containing 10% calf serum (CS) added with palmitate [250 and 500 µM, fatty acid-BSA 3:1 molar ratio, with 85 µM BSA used in the control (con); the same conditions were used for the following experiments unless otherwise noted]. After 24 h, cells were stained with Hoechst 33342 and imaged with phase-contrast (top) and fluorescent microscopy (bottom). B: cells were treated with 500 µM palmitate, stained as described in A, and visualized with fluorescent microscopy at different time points. The fluorescently bright particles represent apoptotic cells. C: cells were treated with 500 µM palmitate for 48 h, fixed in ethanol, stained with propidium iodine, and examined with fluorescence-activated cell sorting (FACS). The sub-Go fraction represents apoptotic cells. Results are representative of 3 independent experiments. D, top: fluorescence photomicrograph of cells treated with 500 µM palmitate with or without the adipogenic hormone cocktail (MDI) for 20 h and stained with Neil red. D, bottom: mRNA expression of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), acyl-CoA synthetase (ACS-1), and C/EBP{alpha} in cells treated with palmitate for 20 h (dark shaded bars, MDI; light shaded bars, no MDI). Results are means ± SE (n = 4). P < 0.05, a < b < c < d.

 
Figure 1D shows the results of lipid staining (Neil red) of cells treated with or without palmitate. Compared with control cells (Fig. 1D, top left), incubation with palmitate for 20 h slightly increased the accumulation of lipid, but the staining was diffusive without well-defined lipid droplets (Fig. 1D, top right). Treatment with the adipogenic hormone cocktail (MDI) for 20 h induced a similar slight and diffusive increase of lipid staining (Fig. 1D, middle left), in agreement with previous reports that lipid accumulation is minimal within the first 2 days of adipogenic induction (41). In contrast, adding palmitate together with MDI caused a large increase of distinctive perinuclear lipid droplets (Fig. 1D, middle right), characteristic of a more advanced stage of differentiation. In agreement with previous studies (41), we showed that MDI induced expression of key adipogenic transcription factor peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and C/EBP{alpha} as well as the differentiation-dependent acyl-CoA synthetase (ACS-1) (Fig. 1D, bottom). This induction was greatly enhanced by palmitate, consistent with the morphological changes (Fig. 1D, middle right). No apoptosis was detected in the cells cotreated with MDI and palmitate (data not shown). We noticed that within the first 20 h, palmitate alone also increased mRNA of PPAR{gamma}, C/EBP{alpha}, and ACS-1 to an even greater extent than MDI alone (Fig. 1D, bottom). However, this initial induction of the adipogenic genes was not sufficient to counteract the simultaneous apoptotic effect.

Effects of IGF-1, oleate, and linoleate on the proapoptotic effect of palmitate. Using the quantitative MTT assay, we showed that palmitate induced a dose-dependent decrease in cell viability after 24 h of incubation (Fig. 2A, columns 1–3), in agreement with the morphological changes (Fig. 1A). This effect was accelerated at lower serum conditions (data not shown). Hence, we tested whether enrichment of a serum growth factor IGF-1 might confer protection. As expected, treatment of cells with IGF-1 for 24 h caused a large increase in MTT (Fig. 2A, column 4), indicating stimulation of cell proliferation. Surprisingly, IGF-1 did not prevent the palmitate-induced cell loss (Fig. 2A, column 5). In contrast, treatment with the unsaturated fatty acids oleate or linoleate did not cause apoptosis (Fig. 2A, columns 6 and 8, respectively), and these unsaturated fatty acids also reversed the palmitate-induced apoptosis (Fig. 2A, columns 7 and 9, respectively), consistent with previous reports in other cell types (4, 7, 30). However, when these cells were subsequently exposed to a low-serum (1%) medium, we found a greater cell loss in cultures pretreated with the fatty acid mixture than in nonpretreated cultures (Fig. 2B). This suggests that the protection role of the unsaturated fatty acids against palmitate requires the presence of serum. Even so, cells thus treated were still less resistant to the stress incurred by serum deprivation (see below), implying that preexposure to a palmitate-rich fatty acid mixture might weaken the cellular self-defense system to a sublethal level.


Figure 2
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  		Fig. 2. Effects of IGF-1, unsaturated fatty acids, and BSA on palmitate-induced apoptosis in 3T3-L1 preadipocytes as measured using the MTT assay. A: cells at 90% confluence were incubated for 20 h in DMEM containing 10% CS (1) with 250 µM palmitate (2) and 500 µM palmitate (3), 10 ng/ml IGF-1 (4), IGF-1 plus 500 µM palmitate (5), 500 µM oleate (6), 50 µM oleate plus 500 µM palmitate (7), 500 µM linoleate (8), and 50 µM linoleate plus 500 µM palmitate (9). B: cells were preincubated with DMEM plus 10% CS (A) containing 50 µM oleate and 500 µM palmitate (B), 500 µM oleate (C), 50 µM linoleate and 500 µM palmitate (D), and 500 µM linoleate (E). C: cells were treated with palmitate (pal) with incremental dosages of BSA for 20 h. Results are means ± SE (n = 8–16). P < 0.05, a > b > c> d.

 
Because fatty acids have low water solubility, BSA is commonly used as a delivery vehicle in cell culture, as we did in this study. Recently it was reported that BSA itself might have profound effects to the cells (34), raising the issue whether BSA might contribute to the apoptotic effects found in the palmitate-treated cells. We consider this unlikely, because for cells exposed to the same concentration of BSA, oleate or linoleate did not induce apoptosis (Fig. 2A). Nevertheless, we tested whether a variation of palmitate-to-BSA ratio might affect the apoptotic effect. As shown in Fig. 2C, BSA alone at concentrations range from 0 to 150 µM had no effect on cell viability. For cells treated with palmitate at 250 µM, apoptosis was significant when palmitate was delivered in the presence of 75 µM BSA but became undetectable as BSA was increased to 150 and 225 µM, which corresponds to a decrease of monomeric palmitate from 0.5 to 0.25 and 0.10 µM estimated based on the binding coefficient between palmitate and BSA (35). Similar effect of BSA was found for palmitate added at 500 µM. These results suggest that apoptosis was linked to the monomeric palmitate and was ameliorated by increasing BSA concentration, possibly by holding the fatty acids in the extracellular space. Under in vivo conditions, the plasma free fatty acid-to-serum albumin ratio is near 1:1. However, the ratio may be substantially higher at local areas when large amounts of fatty acids are acutely released by lipolysis. For this reason, most in vitro studies have used a fatty acid-to-BSA ratio range from 3:1 to 8:1 (43). A 3:1 molar ratio was used for the following experiments.

Palmitate alters IGF-1 signaling in preadipocytes. It has been shown that activation of the PI3K/Akt pathway, a major prosurvival mechanism downstream of IGF-1, protects palmitate-induced apoptosis in selected cell types (4). Since IGF-1 failed to protect the cells under our experimental conditions, we assessed whether the induction of Akt is impaired by palmitate. As shown in Fig. 3A, overnight incubation with palmitate caused a dose-dependent reduction of Akt phosphorylation (Ser473) in response to IGF-1. The inhibition was largely reversed by cotreatment with oleate or linoleate (Fig. 3A) in association with the protection of either against palmitate-induced apoptosis (Fig. 2A), in agreement with previous reports in other settings (4). Notably, overnight treatment with palmitate also caused a reduction of Akt mass, an effect that was reversed by cotreatment with oleate or linoleate as well (Fig. 3B). Figure 3C shows that palmitate induced a potent phosphorylation of Erk1/2, which was modestly increased further in response to IGF-1. Oleate or linoleate alone did not affect Erk1/2 phosphorylation (Fig. 3C). However, they did not reverse the hyperphosphorylation of Erk1/2 induced by palmitate either (Fig. 3C). None of these fatty acids affected Erk1/2 mass (Fig. 3D). Figure 3E shows that phospho-JNK (Thr183/Tyr185) was minimal in the control cells and was not affected by IGF-1, but it evidently was induced by palmitate. Oleate and linoleate each alone had no effect on phospho-JNK. Linoleate, but not oleate, blocked the induction of phospho-JNK by palmitate. These results indicate that the unsaturated fatty acids restored some (e.g., Akt) but not other (e.g., Erk1/2, JNK) signaling effects of palmitate. This supports our earlier prediction that cells treated with a palmitate-oleate mixture still endured significant cellular stress, albeit at a sublethal level. On the other hand, cotreatment with U0126 (5 µM), a specific inhibitor for MEK1/2 that prevented palmitate-induced Erk1/2 phosphorylation (data not shown), did not prevent or aggravate cell death induced by palmitate (Fig. 3, right). Cotreatment of palmitate with two different JNK inhibitors (SP600125 and Dicoumarol) each modestly increased cell viability, but the difference was very small (<15%, Fig. 3, right), suggesting that JNK might not be a dominant factor in this event.


Figure 3
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  		Fig. 3. Effect of fatty acids on IGF-1 signaling and effects of selected MAP kinase inhibitors on palmitate-induced apoptosis in 3T3-L1 preadipocytes. Left: cells were preincubated in DMEM containing 10% CS for 16 h with the indicated fatty acids (pal, palmitate; ole, oleate; lin, linoleate). After washing, cells were incubated with serum-free medium for an additional 90 min and then treated with IGF-1 (10 ng/ml) for 15 min. Cell lysates were immunoblotted with antibodies against phospho-Akt (Ser473) (A), total Akt (B), phospho-Erk1/2 (C), total Erk1/2 (D), phospho-JNK (E), total JNK (F), and beta-tubulin (G). Results are representative of 4 independent experiments. The degree of change (fold) was calculated as the optical density ratio of the target protein to beta-tubulin, normalized to the control cells. Right: cells were treated with 250 and 500 µM palmitate for 20 h, with or without the indicated inhibitors. Results are means ± SE (n = 8–16). *P < 0.05 compared with no inhibitor.

 
To assess the physiological relevance of our findings obtained with a clonal cell line, we tested the effects of palmitate in primary cultured rat preadipocytes. A significant decrease in MTT was found after treatment with palmitate for 24 h (≥250 µM; Fig. 4, left), which was consistent with the appearance of apoptotic cell morphology (not shown). Palmitate also inhibited IGF-I induced Akt phosphorylation, decreased Akt mass, and caused hyperphosphorylation of Erk1/2 and JNK without affecting the protein mass (Fig. 4, right). Hence, both the signaling and apoptotic effects of palmitate found in 3T3-L1 preadipocytes were reproduced in rat preadipocytes, suggesting similar mechanisms underlying palmitate actions in these cells of different donor origins.


Figure 4
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  		Fig. 4. Effects of palmitate on cultured primary rat preadipocytes. Rat preadipocytes were isolated from epididymal fat pads and cultured in {alpha}MEM supplemented with 10% fetal bovine serum. Cells were treated with palmitate at indicated concentrations for 20 h for MTT assay (left). Values are means ± SE. *P < 0.05 compared with control. In parallel, cells were incubated with palmitate for 16 h followed by treatment with IGF-1 (10 ng/ml) as described in Fig. 3, and cell extracts were then used for Western analysis with antibodies as indicated (right).

 
We then tested how palmitate affects the IRS-1 pathway, since this is an important mediator that transmits the mitogenic signal of IGF-1 (37). As shown in Fig. 5A, all the tested fatty acids moderately decreased IRS-1 expression. However, this could not explain their differential effects on Akt phosphorylation (Fig. 3A). Since it has been shown that serine phosphorylation of IRS-1 enhances its degradation (13), we tested the effects of these fatty acids on IRS-1 phosphorylation at Ser307 and Ser636/639, the two sites known to be induced by fatty acids (17) or other nutrients (through the mammalian target of rapamycin, mTOR) (38). Surprisingly, our results showed that all fatty acids tested caused a large decrease in phospho-IRS-1 (Ser307), and all except for palmitate decreased phospho-IRS-1 (Ser636/639). Hence, the decrease of IRS-1 protein could not be explained by serine phosphorylation at 307 or 636/639 residues. Whether serine phosphorylation at other residues is involved in this process remains to be determined.


Figure 5
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  		Fig. 5. Inhibition of insulin receptor substrate-1 (IRS-1)-mediated signaling by palmitate in 3T3-L1 preadipocytes. Cells were incubated with the indicated fatty acids for 16 h and used for Western analysis directly (A) or serum-starved for 90 min followed by IGF-1 (10 ng/ml) stimulation for 15 min (B). For the latter, cell lysates were prepared and immunoprecipitated with an IRS-1 antibody. The IRS-1 immunoprecipitates (IP) were then split into 2 fractions, one for assay of phosphatidylinositol 3-kinase (PI3K) activity and the other for Western analysis with a phospho-tyrosine antibody (Py). Values in B are means ± SE (n = 4). *P < 0.05 compared with control.

 
We next examined tyrosine phosphorylation of IRS-1 and its associated PI3K activity by immunoprecipitation of IRS-1. Figure 5B shows that treatment with palmitate overnight inhibited IGF-1-induced PI3K activity, an event that was prevented by cotreatment with oleate. Such changes were consistent with the changes in Akt phosphorylation (Fig. 3A). Interestingly, Western analysis of the immunoprecipitates revealed that IRS-1 tyrosine phosphorylation was reduced by treatment with the fatty acids (Fig. 5B), despite their differential effects on IRS-1-associated PI3K activity. The decrease of IRS-1 tyrosine phosphorylation is possibly a result of decreased IRS-1 protein mass (Fig. 5A). The reasons for the discordant effects of fatty acids on expression/phosphorylation of IRS-1 and associated PI3K activity are not clear and are worthy of further investigation.

Palmitate induces ER stress. Recent studies demonstrated that exogenous fatty acids, especially palmitate, cause ER stress, associated with increased expression of the proapoptotic transcription factor CHOP and inactivation of Akt (5, 23, 24, 39). As a self-protection mechanism, molecular chaperone GRP78 is upregulated, which is also often considered a molecular marker for ER stress. Using the same membrane blots from Figs. 3 and 4, we reprobed for CHOP and GRP78. As shown in Fig. 6A, palmitate induced expression of CHOP in 3T3-L1 and rat preadipocytes, whereas GRP78 was increased only in 3T3-L1 cells. Oleate or linoleate alone had no effect but did not prevent (oleate) or only partially prevented (linoleate) the induction of CHOP and GRP78 by palmitate. Real-time PCR analysis revealed that mRNA for these two proteins was not, or only moderately, affected by palmitate (Fig. 6B). Figure 6C shows that treatment with palmitate alone or mixed with oleate/linoleate induced partial but significant XBP-1 mRNA splicing, further evidence of increased ER stress (22). As a positive control, we showed that thapsigargin, a chemical inducer for ER stress, induced a greater increase of CHOP and GRP78 mRNA (Fig. 6B), as well as XBP-1 mRNA splicing (Fig. 6C), than palmitate.


Figure 6
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  		Fig. 6. Palmitate induced endoplasmic reticulum (ER) stress. A: protein expression of CEBP homology protein (CHOP) and glucose-regulated protein 78 (GRP78) in preadipocytes, under the same conditions as those shown in Fig. 3 (3T3-L1) and Fig. 4 (rat preadipocytes). The same membranes used for experiments in Figs. 3 and 4 were reprobed with the new antibodies. B: 3T3-L1 cells were incubated with the indicated fatty acids and thapsigargin (Tha) for 16 h, and RNA was harvested for qualitative RT-PCR analysis of CHOP and GRP78. Values are means ± SE (n = 4). C: the same RNA samples were used for conventional PCR with primers that flanked the cleavage site in XBP-1 mRNA. The products were separated by electrophoresis using 4% agarose gel. Results are representative of 4 independent experiments.

 
We next compared the time course of changes in apoptosis and the expression of CHOP and Akt. As shown in Fig. 7A, an increase in CHOP expression occurred within the first 6 h and continued throughout 24 h. This occurred before significant changes in Akt or phospho-Akt were detected (Fig. 7, B and C). Unexpectedly, an increase of phospho-Akt was detected in cells treated with palmitate at the 6-h time point, which was returned to control level after 12 h and followed with a substantial decrease after 24 h. This suggests that ER stress, marked by increased CHOP protein expression, was induced before the inactivation of Akt. In other words, impaired Akt activation detected at later time points might be the result, rather than the cause, of lost cell viability. To test this possibility further, we infected 3T3-L1 cells with MyrAkt adenovirus expressing a constitutively active form of Akt (16). The MyrAkt-transfected cells exhibited a substantial increase in Akt protein compared with the GFP-transfected control cells (Fig. 7D). Phosphorylation of FKHR (Ser256), a downstream target of Akt, was increased in MyrAkt- but not GFP-expressing cells (Fig. 7D). This confirms the functional activity of MyrAkt used for this experiment. Unexpectedly, palmitate further induced phosphorylation of FKHR in both GFP- and MyrAkt-transfected cells. This effect may be a result of the early activation of Akt (Fig. 7C). It is not known whether additional pathways are present that can increase phospho-FKHR in response to palmitate. MyrAkt blocked palmitate-induced hyperphosphorylation of Erk1/2 and JNK (Fig. 7D). However, MyrAkt did not prevent the palmitate induced CHOP expression (Fig. 7D). Consequently, we found that MyrAkt did not prevent cell loss when treated with palmitate at 500 µM (Fig. 7E).


Figure 7
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  		Fig. 7. Time-dependent effects of palmitate on CHOP and Akt and effects of expression of constitutively active Akt in 3T3 L1 preadipocytes. Cells were incubated in DMEM with 10% CS plus palmitate and harvested at different time points for Western analysis of CHOP (A), Akt (B), phospho-Akt (C), and beta-tubulin (not shown). Bars represent the optical density ratio of target protein to beta-tubulin after normalization to the control of each time point. Results are means ± SE (n = 3). P < 0.05, a > b > c. D: Western analysis of Akt and other indicated targets in cells overexpressing green fluorescent protein (GFP; g) or MyrAkt (a). E: MTT analysis of cell viability after a 20-h treatment with palmitate in cells transfected with GFP (open bars) or MyrAkt (filled bars). The transfection rate, evaluated by visual detection of GFP, was ~70% in D and E. Results in E are means ± SE (n = 16). P < 0.05, a > b > c.

 
LiCl attenuates palmitate-induced apoptosis. The above results suggest that palmitate induces cellular stress through multiple pathways, which might exceed the protection capacity of IGF-1/Akt. We then asked whether a global suppression of cellular stress might be beneficial for cell survival. To test this possibility, we treated 3T3-L1 cells for 24 h with palmitate together with LiCl, which is known to improve cell viability under diverse stress conditions (6). Indeed, LiCl (10 mM) largely blocked the palmitate-induced cell loss even at 500 µM, as evidenced by MTT assay (Fig. 8, left) and cell morphology (not shown). FACS analysis confirmed that LiCl reduced the number of cells in the sub-Go phase after 24 h of incubation with palmitate (Fig. 8, right).


Figure 8
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  		Fig. 8. LiCl suppresses palmitate-induced apoptosis in 3T3L1 preadipocytes. LiCl was added to the cells 30 min before palmitate, and incubation was continued for 20 h. Left: MTT assay for cell viability in cultures with 10 mM NaCl (light shaded bars), 10 mM LiCl (dark shaded bars), and 20 mM LiCl (open bars). Values are means ± SE (n = 24). P < 0.05, a > b > c. Top right: histogram of FACS analysis of cells treated for 24 h with palmitate (500 µM) and LiCl (10 mM). Bottom right: cell cycle distribution calculated from the FACS results. Values are means ± SE (n = 3). *P < 0.05; **P < 0.05, a > b > c. ***P < 0.05, a > b.

 
Since LiCl is a known GSK-3beta inhibitor, we analyzed how it interacts with palmitate in regulation of beta-catenin, a direct target of GSK-3beta and also a key protein in the canonical Wnt signaling pathway. As shown in Fig. 9A, palmitate caused a dose-dependent decrease of nonphosphorylated (active form) beta-catenin, an effect that was largely restored by LiCl. In parallel, palmitate decreased the expression of c-Myc and cyclin D1, two of the downstream targets of Wnt/beta-catenin pathway. This effect was also blocked by LiCl. In contrast, cotreatment of palmitate with oleate or linoleate did not prevent the loss of the Wnt signaling proteins (Fig. 9). These results suggest that although both inhibit palmitate-induced apoptosis, LiCl and the unsaturated fatty acids may act through different mechanisms. This became more apparent when we examined the effects of palmitate on eiF2{alpha} (see below).


Figure 9
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  		Fig. 9. Effects of palmitate on the expression of beta-catenin (beta-cat), c-Myc, cyclin D1, and eukaryotic initiation factor-2{alpha} (eiF2{alpha}) after 16 h of incubation, the early changes in eIF2{alpha}, and the cellular uptake of palmitate. A: cells were incubated for 16 h in DMEM containing 10% CS with indicated fatty acids and other reagents and used for Western analysis for the indicated targets. beta-tub, beta-Tubulin. B: cells were incubated with palmitate for 1, 3, and 7 h and used for analysis of eiF2{alpha}. Results are representative of 3 or more independent experiments. C: cells were incubated in a 24-well plate containing 2 ml of DMEM containing 10% CS added with [1-14C]palmitate (0.5 µCi/ml) at 250 or 500 µM, with or without LiCl (10 mM) and oleate (50 µM). At each time point, 100 µl of the medium was taken for scintillation counting. Results are means ± SE (n = 4).

 
A common cellular response to ER stress is to suppress mRNA translation and reduce new protein influx into ER, thereby reducing the buildup of misfolded proteins. Phosphorylation of eIF2{alpha} (Ser51) by RNA-dependent protein kinase-like ER eIF2{alpha} kinase (PERK) is the major contributor to this mechanism (21). However, we found that after 16 h of incubation, phospho-eIF2{alpha} was decreased, instead of being increased, by palmitate. At 500 µM, palmitate also diminished eIF2{alpha} protein mass. Cells cotreated with oleate and linoleate each showed little phospho-eIF2{alpha}, whereas cotreatment with LiCl induced a large increase in phospho-eIF2{alpha}. Both the unsaturated fatty acids and LiCl blocked the loss of total eIF2{alpha} protein induced by palmitate. To test whether palmitate might activate PERK at an earlier time point, we performed a time course study. As shown in Fig. 9B, at 1 h, palmitate did cause an increase in phospho-eIF2{alpha} to a greater extent at 250 µM than at 500 µM. A similar trend was also found after 3 and 7 h of incubation. Within the first 7 h, no decay of total eIF2{alpha} protein was detected. These results suggest that palmitate not only caused early onset of ER stress but also impaired cellular self-defense by inhibiting the activation PERK/eiF2{alpha} pathway.

Finally, we determined whether the antiapoptotic effect of oleate and LiCl might be related to altered cellular uptake of palmitate. Figure 9C shows that within the first 6 h, the uptake of palmitate was linearly correlated with the incubation time and was not affected by oleate. LiCl, on the other hand, increased the uptake in the first 2 h but blocked the continuous uptake thereafter. This again suggests that LiCl and oleate affected the cellular response to palmitate through different mechanisms.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Palmitate, oleate, and linoleate are the most common fatty acids in human diets as well as in animal and human fat tissue. Palmitate is also the end product of de novo fatty acid synthesis and is generated in large quantity in subjects on a low-fat diet. Most of the processed food products in the market contain high concentration of saturated fatty acid, which is dominated by palmitate. Negative impacts of palmitate on cell viability have been reported in several cell types (4, 26, 30). In this work, we present new evidence on how preadipocytes respond to exogenous palmitate and other fatty acids under both adipogenic and nonadipogenic conditions.

First, we showed that palmitate induced apoptosis in preadipocytes, in association with early increase of the proapoptotic transcription factor CHOP (Fig. 9). Meanwhile, we also detected simultaneous activation of several prosurvival pathways. These include 1) induction of adipogenic genes (Fig. 1D), 2) early increase of Akt phosphorylation (Fig. 7C), and 3) sustained phosphorylation of FKHR (Fig. 7D). These changes are normally found during adipogenic differentiation (44) and hence may be preadipocyte-specific responses to exogenous palmitate. However, in the absence of adipogenic hormones, these initial adaptive responses were not sufficient to induce full differentiation or to counteract the proapoptotic stress. In contrast, when mixed with the adipogenic stimuli, palmitate caused a much stronger induction of adipogenic genes (Fig. 1D), and cells progressed toward full differentiation without apoptosis (2). These results indicate that although preadipocytes have some innate ability to adapt to exogenous fatty acids, the cell fate between differentiation and apoptosis is still largely dependent on the coexisting hormonal conditions.

In the absence of adipogenic stimuli, palmitate induced multiple stress response in the preadipocytes. In particular, we detected the early induction of ER stress, evidenced by increased CHOP and phospho-eiF2{alpha} (Figs. 7A and 9B). At least two of the three major unfolded protein response (UPR) signaling pathways, IRE1 (inositol-requiring ER-to-nucleus signaling protein-1{alpha})/XBP-1 and PERK/eiF2{alpha}, were altered by palmitate. Intriguingly, we noticed that thapsigargin, a chemical ER stress inducer, caused more robust UPR than palmitate. This is evidenced by complete XBP-1 mRNA splicing (Fig. 6C), as well as strong and sustained increase of phospho-eiF2{alpha} (Fig. 9A), whereas palmitate only induced partial XBP-1 mRNA splicing (Fig. 6C) and early induction but late reduction of phospho-eiF2{alpha} (Fig. 9B). Thapsigargin, but not palmitate, also induced a large increase in GRP78 and CHOP mRNA (Fig. 6B). In association with these differences, we noticed that palmitate induced apoptosis at an earlier time point than thapsigargin (data not shown), which might be related to the less efficient UPR protection in palmitate-treated cells. In addition, palmitate also induced other cellular stress responses as evidenced by the hyperphosphorylation of Erk1/2 and JNK (Fig. 3), whereas thapsigargin had no effect on these two kinases (data not shown). This also distinguishes palmitate as a potent inducer of multiple stresses compared with thapsigargin as a more specific regulator of ER stress through altered Ca2+ balance. This may contribute to accelerate apoptosis in palmitate-treated cells.

The mechanism by which palmitate induces cellular stress is still not clear. We speculate that palmitate may modify the ER membrane lipid environment, making it unfavorable for proper protein folding, as implied by studies in other cell types (36). The earliest ER stress marker we detected was within 1 h (Fig. 9B, phospho-eiF2{alpha}). Other stress pathways appear to be activated at later time points. For instance, loss of phospho-Akt also occurred after 6–12 h (Fig. 7C), accompanied by a simultaneous increase of phospho-Erk1/2 (data not shown). Hence, we suggest that these later signs of cellular stress may be secondary to the initial ER stress.

A second interesting finding is that although the unsaturated fatty acids and LiCl each blocked palmitate-induced apoptosis (Figs. 2A and 8), the mechanisms seem to be largely different. It appears that LiCl did not block ER stress or other cellular stress, such as increased expression of CHOP, hyperphosphorylation of Erk1/2 and JNK, and splicing of XBP-1 mRNA (data not shown), but might improve cell viability through activation of the prosurvival Wnt/beta-catenin pathway and the UPR/eiF2{alpha} (Fig. 9A). Because there is no known mechanism by which LiCl may directly interact with PERK, and indeed, LiCl alone had no effect on phospho-eiF2{alpha}, we consider that the observed increase of phospho-eiF2{alpha} after cotreatment with palmitate may also be secondary to the improved cell viability through the activation of Wnt/beta-catenin by LiCl. In contrast, oleate or linoleate, while blocking apoptosis, only slightly ameliorated the palmitate-mediated reduction of beta-catenin, c-Myc, and cyclin D1, resulting in an expression level still much lower than the control cells or even the cells treated with 250 µM palmitate (Fig. 9A). These results suggest that the limited preservation of Wnt/beta-catenin pathway by oleate or linoleate may not be causally related to their cytoprotective effect.

A second noticeable difference between the unsaturated fatty acids and LiCl is their opposite effects on phospho-eiF2{alpha} in cells treated with palmitate (Fig. 9A). Lack of phospho-eiF2{alpha} may be a result of low ER stress that is below the threshold to activate PERK or stress that is too high and damages the UPR/PERK response. We believed that the latter may be applicable to the cells after long-term treatment with palmitate alone, which was also accompanied by decreased eiF2{alpha} protein mass (Fig. 9A). On the other hand, cotreatment with oleate or linoleate did not increase phospho-eiF2{alpha} either, but it did prevent the loss of total phospho-eiF2{alpha} mass, suggesting a lowering of cell stress. Another distinguishable effect of oleate or linoleate was to restore the prosurvival IGF-1/Akt pathway in the presence of palmitate (Fig. 3), also an indicator of lower cellular stress, whereas LiCl did not have such an impact (data not shown). However, overexpression of a constitutively active Akt did not prevent palmitate-induced apoptosis, implying that Akt alone was not able to counteract multiple stress pathways induced by palmitate.

Finally, it is worth pointing out that when the unsaturated fatty acids abolished palmitate-induced apoptosis in a full-serum medium (Fig. 2A), the fatty acid mixtures did induce substantial stress in the cells, as evidenced by increased expression of CHOP, XBP-1 mRNA splicing, and hyperphosphorylation of Erk1/2 and JNK (Figs. 3 and 6). Such stress might weaken the cellular defense as shown by the accelerated apoptosis during subsequent serum deprivation (Fig. 2B). It also is worth pointing out that palmitate in vivo is always present in mixtures with some unsaturated fatty acids, mostly oleate. The ratio of palmitate to oleate, however, can change substantially with diet and disease (7, 8). Additional studies on how preadipocyte respond to different fatty acid mixtures will be useful for better understanding of the physiological regulation of adipose tissue development.

In summary, our results support the hypothesis that palmitate, as well as palmitate-enriched fatty acid mixtures, can negatively regulate adipose tissue development under conditions deficient of adipogenic hormones. This may in turn promote ectopic fat storage in nonadipose organs (11). A recent study showed that rats fed a high-saturated fat diet develop smaller epididymal and abdominal fat pads but larger fat deposits in the intrathoracic space (33). This in vivo evidence appears to be in agreement with our hypothesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-59261 (to W. Guo) and GM057959 (to Z. Luo).


   ACKNOWLEDGMENTS
 
We thank Dr. K. Walsh for providing the adenovirus for MyrAkt and for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Guo, Dept. of Medicine, Boston Univ. School of Medicine, 670 Albany St., #207, Boston, MA 02118 (e-mail: wguo{at}bu.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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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