Distinct gene expression profiles characterize cellular responses to palmitate and oleate.

Obese individuals are both insulin resistant and have high levels of circulating free fatty acids (FFAs). In cell culture, saturated but not unsaturated fatty acids induce endoplasmic reticulum (ER) stress. We hypothesized that chronic exposure to low dose fatty acids would significantly attenuate the acute stress response to a saturated fatty acid challenge and that unsaturated fatty acids (oleate) would be more protective than saturated fatty acids (palmitate). The ER stress response to palmitate was reduced after low dose fatty acid exposure in human hepatoma cells. Palmitate and oleate gave distinctive transcript responses, both acutely and after chronic low dose exposure. Differentially regulated pathways included lipid, cholesterol, fatty acid, and triglyceride metabolism, and IκB kinase and nuclear factor κB kinase inflammatory cascades. Oleate reduced palmitate-induced changes significantly more than low dose palmitate and completely blocked palmitate-induced phosphoinositide 3 kinase inhibitor (PIK3IP1) as well as induction of GADD45A and B. These changes are predicted to alter the PI3 kinase pathway and the pro-apoptotic p38 MAPK pathway. We recapitulated the oleate response by small interfering RNA-mediated block of PIK3IP1 stimulation with palmitate and significantly protected cells from palmitate-mediated ER stress. We show that transcriptional responses to oleate and palmitate are distinct, broad, and often discordant. We identified several potential candidates that may direct the transcriptional networks and demonstrate that PIK3IP1 partially accounts for the protective effects of oleate.

prior studies in our laboratory, which were within the observed physiologic range of total FFA in obese humans. We selected 0.25 mM FFA as an adaptive dose, because it does not induce ER stress in HepG2 cells at this time point. In a separate experiment, we treated HepG2 cells with palmitate, oleate, BSA control, or a mixture of palmitate and oleate for 12 h.

Inhibitor treatment
The PI3 kinase inhibitor LY294002 was dissolved in DMSO and used in a fi nal concentration of 25-50 M. The LY294002 dose was based on published literature and vendor recommendations. Cells were pretreated with LY294002 or DMSO (vehicle control) for 1 h and then treated with palmitate (0.5 mM), a mixture of 0.5 mM palmitate and 0.25 mM oleate, or BSA (control) for 12 h in serum free condition.

siRNA targeting of PIK3IP1
HepG2 cells were transfected with 100 nM PIK3IP1 siRNA or a nontargeting siRNA pool (Dharmacon, Chicago, IL) using DharmaFECT 4 transfection reagent (Dharmacon) according to an optimized protocol modifi ed based on the manufacturer's instructions. Further details of our transfection protocol are described in supplementary Methods. After 24 h, siRNAtransfected cells were treated with either palmitate (0.5 mM) or BSA (control) for 12 h under serum and antibiotic-free conditions. The cells were treated as indicated in the fi gure legends and processed for RNA isolation and quantitative realtime PCR to analyze transcript level expression of ER stress pathway genes.

RNA isolation
RNA was isolated as described previously ( 10 ) (supplementary Data and Methods).

Quantitative real-time PCR to analyze transcript level
Measurements were conducted in at least two independent experiments for each condition, each in turn with two biological replicates yielding between four and six measures for each condition. Real-time quantitative PCR was performed as described ( 10 ) (supplementary Methods).

Immunoblot analysis
Phosphorylation of eIF2 ␣ , JNK1, and C-Jun were analyzed by immunoblot ( 10,21 ) normalized to ␤ -actin (supplementary Methods) for at least three independent experiments, each with two technical replicates. vated fasting FFA punctuated by acute postprandial and nocturnal increases, published cell culture experiments were based on acute FFA exposure. Recent studies demonstrated that chronic exposure of cells to low levels of the classic ER stress inducers tunicamycin and thapsigargin were protective of ER stress response and apoptosis induced by subsequent acute challenge ( 20 ). A similar model of long-term FFA exposure followed by an acute challenge would more closely recapitulate the physiologic situation, but whether chronic FFA elevations are toxic or protective is, to our knowledge, unknown and untested.
Based on these considerations, we hypothesized that chronic exposure to low levels of FFA would similarly attenuate the acute response to FFA levels comparable to those observed physiologically in the postprandial state. Furthermore, we hypothesized that low levels of unsaturated FFA would be more protective than saturated FFA. Finally, we sought to defi ne the molecular mechanisms behind the differential response to saturated and unsaturated FFA. To test these hypotheses, we developed an in vitro system based on the human HepG2 hepatoma cell line in which cells were adapted in the presence of low doses of either palmitate (C16:0) or oleate (C18:1). We then challenged cells acutely with high doses of either FFA. After demonstrating the differential induction of ER stress, we explored the molecular basis of the protection using genome-wide transcript profi ling. We further used genome wide transcript profi les to determine whether simultaneous exposure to oleate in the presence of palmitate induced the same protective response observed in the adaptation experiments. Finally, we demonstrated that the protective response of oleate is mediated in part by downregulation of the phosphoinositide 3 kinase inhibitor (PIK3IP1) by recapitulating the oleate effect with small interfering RNA (siRNA)-mediated knock-down of the PIK3IP1 gene.

Experimental reagents
Palmitic and oleic acids were conjugated to fatty acid-free BSA as described ( 10,18 ) (supplementary Data and Methods).

Cell culture
Human hepatoma (HepG2; American Type Culture Collection, Manassas, VA) cells were cultured in 5.6 mM glucose under standard conditions. For adaptation experiments, cells were grown for two passages in the presence of 0.25 mM oleate, 0.25 mM palmitate, or BSA (control) with exchange of culture medium daily to maintain the FFA concentration. Because palmitate-treated cells grew more slowly, more cells were seeded to achieve similar confl uence. At 80-90% confl uence in the third passage (treatment day 14), cells were washed and transferred to serum-free medium containing either 0.25 mM or 1 mM FFA (BSA-conjugated oleate or palmitate) or BSA control ( Fig. 1 ). Cells were harvested after 6 h. Our fatty acid adaptive stress model was based on the adaptive stress model in MEF cells using chemically induced ER stress (tunicamycin and thapsigargin) ( 20 ). We selected an acute FFA dose that signifi cantly induced ER stress and resulted in JNK1 and c-JUN phosphorylation by 6 h based on

Short-term oleate and palmitate induce unique gene expression profi les
We showed previously that palmitate but not oleate at comparable levels induced genes of the ER stress pathway ( 10 ). In confi rmation of our previous studies ( 10 ), treatment of HepG2 cells with 1 mM palmitate for 6 h elevated the transcript levels of genes downstream of the PERK (EIF2AK3)-eIF2 ␣ ER stress pathway, including ATF3 ( P = 0.009), DDIT3 (CHOP, P = 0.006), and PPP1R15A

Genome wide expression analysis
Genome wide transcriptome analysis and initial array processing was performed by GenUs Biosystems (Northbrook, IL) using Human Whole Genome 4 × 44k arrays (Agilent Technologies, Santa Clara, CA). We included genes in subsequent analyses if they were both above background and differed by <40% between biological replicates. Fold change was compared with BSA control. Additional details are provided in supplementary Methods.

Statistical and bioinformatic analysis
Differences between responses for quantitative real-time PCR and immunoblot were evaluated by Student's t -test, with a P < 0.05 considered signifi cant. We used PermutMatrix version 1.9.3EN software ( 22 ) for unsupervised hierarchical clustering. Differentially expressed genes were categorized and annotated using singular and modular enrichment analysis implemented in the DAVID package ( 23, 24 ) (http://david.abcc.ncifcrf.gov/), Gene Set Enrichment Analysis (GSEA) using GeneTrail software Data are for HepG2 cells treated with 1 mM palmitate for 6 h and compared with controls (BSA). We show selected DAVID functional annotation cluster analysis groups with a geometric mean P -value р 0.05. a Median P -value of all categories in a functional group. b Geometric mean P -value of all categories in a functional group. Selected enriched categories common between acute 1 mM palmitate and 1 mM oleate treatment for 6 h in HepG2 cells compared with controls.
a Benjamini and Hochberg corrected P -value. dose tunicamycin and thapsigargin. We asked whether HepG2 cells adapt similarly to low levels of palmitate or oleate over several passages. Thus, cells were adapted to 0.25 mM oleate or palmitate followed by a 1 mM acute challenge ( Fig. 1 ). Indeed, adaptation to either low dose palmitate or low dose oleate blunted the expected increase in ER stress transcripts and phosphorylation response (eIF2 ␣ , JNK1, c-Jun) in response to 1 mM palmitate ( Fig.  2 ), with oleate providing more protection.  Table VII). Surprisingly, these adaptive responses shared only 417 upregulated genes and 452 downregulated genes. Among the shared upregulated pathways were mitochondrial activity, electron transport, oxidative phosphorylation, oxidoreductase activity, and cell cycle regulation. Shared downregulated pathways included phospholipid binding, glycerol metabolism, cytokine binding and cytokine-cytokine receptor interaction, immune response, ER membrane genes, insulin receptor signaling pathway, and cell adhesion ( Table 3 ). Among the discordantly regulated pathways were lipid metabolism (endothelial lipase, hydroxysteroid dehydrogenase like 2, steroidogenic acute regulatory protein, phospholipase A2 group IIA, apolipoprotein L3), the I-Β kinase/NF-Β cascades, fatty acid metabolism and long-chain fatty acid-CoA ligase activity, fatty acid biosynthesis, acute infl ammatory response, and hypoxia response genes. As with the acute responses, the adaptive responses to 2 week exposure to 0.25 mM palmitate and oleate were distinct when the 5,953 differentially expressed probes were examined by hierarchical clustering (supplementary Data and Fig.  II ), despite the shared pathways.

Responses to 1 mM palmitate challenge after oleate and palmitate adaptation are distinct
We selected 203 probes that showed at least a 2-fold change with 6 h, 1 mM palmitate exposure in unadpated cells (189 increased, 14 decreased). Despite the similar effects on the narrower class of ER stress response transcripts, hierarchical clustering again showed distinct profi les after palmitate or oleate adaptation. Whereas palmitate-induced changes were reduced by at least 50% in 82 probes after oleate adaptation, palmitate adaptation similarly reduced the response by 50% or more in only 62 probes. This difference was readily apparent by hierarchical cluster analysis ( Fig. 3A ). Among the individual genes showing striking differences was PIK3IP1, which was in-(GADD34, P = 0.014). Activation of the IRE1 ␣ (ERN1)-XBP1 ER stress pathway was also observed, with induction of ERN1 ( P = 0.027) and splicing of XBP1. Palmitate also induced expression of STC2 ( P = 0.037) and NRF2 (NFE2L2, P = 0.012) but not the major ER stress chaperone, HSPA5 (BiP/GRP78). Phosphorylation of eIF2 ␣ , JNK1, and c-JUN was induced by palmitate. In contrast, oleate did not induce the ER stress genes.
To expand this work, we compared the global gene expression response in HepG2 cells to palmitate (1 mM), oleate (1 mM), or BSA control. Palmitate induced 513 genes at least a 1.5-fold and reduced expression of 263 genes by at least 1.5-fold (supplementary Data and Table  I ). The most strongly upregulated gene was ATF3 (10.3fold increased), whereas oxysterol binding protein 2 was 2.5-fold decreased. In contrast, 1 mM oleate upregulated only 51 transcripts but downregulated 306 unique genes (supplementary Data and Table II ). Of 19 genes with у 1.5fold change by both palmitate and oleate, 4 were concordantly upregulated, 10 were concordantly downregulated, and 5 were discordant including the key ER stress response gene , stanniocalcin (supplementary Data and Table III ).
We considered all probes showing a 1.5-fold or greater change with either palmitate or oleate (1531 probes). Hierarchical clustering showed distinctive profi les (supplementary Data; Fig. 1A ). Modular enrichment analysis ( 23,24 ) showed that palmitate altered pathways of apoptosis, infl ammatory and stress response, oxidative stress, NF ␤ activation, JAK-STAT signaling, and MAPK signaling ( Table 1 ), whereas these pathways were not involved in the oleate response (supplementary Data and Table IV ). In contrast, oleate primarily altered pathways of rhodopsin-like receptor (G-protein coupled receptor) activity, oxidative phosphorylation, and sterol and lipid biosynthesis.
GSEA considers all expressed genes by rank without a fold-change threshold ( 25 ) and thus may identify altered pathways in which no individual members are altered by 1.5-fold. Indeed, GSEA identifi ed additional palmitateinduced changes in pathways of oxidative phosphorylation, fatty acid and lipid synthesis, lipid binding, and lipid transport ( Table 2 ; supplementary Data and Table V). Again, oleate and palmitate acted on distinct pathways, with only 4 of 25 gene ontology (GO) categories showing concordant responses ( Table 2 ). Whereas GO categories involving oxidative phosphorylation, mitochondrial electron transport, and lipid biosynthesis were all downregulated by palmitate, they were upregulated by oleate ( Table  2 ). Notably, oleate and palmitate had strikingly different profi les for genes involved in oxidative phosphorylation (supplementary Data and Fig. IB ).
Low dose palmitate and oleate attenuate the ER stress response to high dose palmitate Rutkowski et al. ( 20 ) demonstrated protective adaptation and reduced ER stress response after exposure to low arbitrary unit of protein expression. XBP1 transcripts amplifi ed from cDNA by PCR (197 bp XBP1-unspliced and 171 bp XBP1-spliced) were examined by agarose gel electrophoresis. duced by 2.6-fold by palmitate in unadapted cells. This response was reduced by 32% after palmitate adaptation but completely abolished by oleate adaptation. Table 4 lists other genes showing marked differences in response following oleate or palmitate adaptation. We used GSEA to identify the pathways that characterized these responses. Oleate adaptation resulted in an attenuated palmitate response in pathways of cholesterol and triglyceride metabolism (GO categories GO 0008203 and GO 0006641 with 61 and 17 genes, respectively; corrected P < 2 × 10 Ϫ 5 for both). Oleate but not palmitate adaptation completely blocked the palmitate induction of 3-hydroxy-3-methylglutaryl-CoA synthase, mevalonate kinase, and phosphoenolpyruvate carboxykinase 1. A somewhat different picture emerged when we instead selected 23 genes from the interaction network that was most signifi cantly upregulated by 1 mM palmitate (Ingenuity; https://analysis.ingenuity.com). This network included ATF3, CEBPB, DUSP1/8, GADD45A/B, JUN/JUNB/ JUND, KLF5, LDLR, and LIPG (signifi cance score 34; supplementary Data and Fig. III A). Oleate adaptation reduced the palmitate response for the 31 probes covering this network by an average 54% ( P = 0.00003), with the most marked effect on genes upstream of the JNK and p38 pathways (DUSP1 and DUSP8 at 72% and 97% reduced response, respectively; supplementary Data and Fig. III B). In marked contrast, palmitate adaptation had no signifi cant effect on the genes in this interactive network ( P = 0.22).

Oleate protects against the saturated fatty acid response when mixed with palmitate
In contrast to the adaptation model, physiologic exposure to FFA involves a mixture of saturated and unsaturated fatty acids. Hence, we asked whether the preadaptation response was unique or whether unsaturated fatty acids could protect also against the saturated fatty acid stress response comparing 0.25 mM to 0.5 mM oleate was 54% versus 90% for upregulated and 29% versus 73% for downregulated transcripts (supplementary Data and Table VIII). Thus, oleate when mixed with palmitate provided similar protection to that observed when cells were adapted to the same oleate concentration for 14 days.

PIK3IP1 mediates the palmitate-induced ER stress response
PIK3IP1 was induced by palmitate in unadapted cells. This induction was reduced by 32% after palmitate adaptation and completely abolished by oleate. PIK3IP1 was identifi ed recently as a negative regulator of the p110 catalytic when mixed in an acute challenge. The ER stress response to 0.5 mM palmitate was nearly completely abolished by the addition of 0.25 mM oleate ( Fig. 4 ). Additionally, oleate when added to palmitate attenuated the induction of GADD45A, GADD45B, and PI3KIP1. We considered the 405 transcripts that differed by at least 2-fold in response to 0.5 mM oleate and 0.5 mM palmitate ( Fig. 3B ). Oleate acted in a dose-dependent manner to attenuate the palmitate response, with 0.25 mM oleate reducing the response in 232/261 upregulated and 140/144 downregulated probes, and 0.5 mM oleate attenuating 260/261 upregulated and 143/144 downregulated transcript responses. The mean reduction in palmitate response when Fig. 3. Hierarchical clustering and heat maps of transcript profi le in responses to oleate and palmitate. A: Hierarchical clustering with heat map of 203 probes that showed at least 2-fold change with 1 mM palmitate challenge with and without palmitate or oleate preexposure. B: Hierarchical clustering with heat map of 405 probes that showed at least a 2-fold change in expression after 12 h palmitate exposure (0.5 mM) when exposed to variable palmitate and oleate ratios. Only genes with at least 2-fold upregulation by 1 mM palmitate exposure (6 h) in HepG2 cells and discordance between oleate and palmitate adaptation are shown.
a Fold change is taken from the acute 1 mM response to palmitate compared with control (BSA).
PIK3IP1 is an important mediator of the palmitateinduced ER stress response and part of the protective mechanism of oleate.

DISCUSSION
The principal aims of our study were to understand the differences in cellular response to saturated and unsaturated fatty acids and to determine whether the protective adaptation seen with preexposure to low levels of tunicamycin and thapsigargin could be replicated with FFAs. While the motivation for our studies was to mimic the exposure of liver to fatty acids in human obesity, we were necessarily limited to performing these experiments in human-derived cell lines and using nonphysiologic preparations of palmitate and oleate. Nonetheless, our results suggest important differences in oleate and palmitate responses. We have identifi ed important pathways that are regulated in cells undergoing the adaptive response, and these pathways may refl ect human physiology.
Previous studies have examined the cellular response to saturated and unsaturated fatty acids in HepG2 cells but at lower exposure levels. Vock et al. ( 27,28 ) examined 0.2 mM oleate for 24 h and 50 M palmitate. They identifi ed subunit of PI3 kinase ( 26 ), suggesting that downregulation of PIK3IP1 might mediate the protective effects of oleate. We fi rst tested the effects of the pharmacologic PI3 kinase inhibitor LY294002 in cells treated with palmitate (0.5 mM) or palmitate and oleate (0.5 mM and 0.25 mM, respectively). As expected, LY294002 recapitulated the palmitate-induced ER stress and signifi cantly reduced the oleate-mediated protection (supplementary Data and Fig. IV ). In the presence of LY294002, proapoptotic factors CHOP ( P = 0.004), ATF3 ( P = 0.04), ATF4 ( P = 0.02), and GADD34 ( P = 0.04) were all increased in cells treated with both palmitate and oleate.
Based on evidence that PIK3IP1 may mediate the oleate response, we sought to recapitulate the oleate effect by siRNA-mediated silencing of the PIK3IP1 transcriptional response to palmitate. Palmitate-induced PIK3IP1 was signifi cantly ( P = 0.002) attenuated in HepG2 cells treated with PIK3IP1-siRNA compared with similar nontarget siRNA ( Fig. 5A and supplementary Fig. VI ). Furthermore, siRNA-mediated reductions in PIK3IP1 signifi cantly protected cells from palmitate-induced ER stress, with significant reductions in CHOP ( P = 0.01), ATF3 ( P = 0.01), and ATF4 ( P = 0.04) ( Fig. 5B ). The palmitate-mediated XBP1 splicing was signifi cantly attenuated ( P = 0.001) by reduced PIK3IP1 ( Fig. 5C ). These results suggest that guished in steady-state measures of transcript levels and may both infl uence our observations. In the current manuscript, we have not attempted to distinguish the mechanism for differential transcript levels. Interestingly, GSEA analysis, which uses information from all expressed genes, showed differential expression of the same gene sets by both oleate and palmitate, but in distinctly opposite directions. Genes in electron transport chain and oxidative phosphorylation were upregulated by acute oleate exposure, whereas acute palmitate exposure downregulated these same processes. Given that protein folding in the ER is highly energy dependent, the downregulation of electron transport chain and oxidative phosphorylation by palmitate would be expected to reduce cellular ATP production and induce ER stress. Indeed, this model is supported by recent studies showing induction of ER stress response by chemicals that impair mitochondrial function by activating the JNK pathway ( 32 ); JNK inhibitors both ameliorated ER stress and restored mitochondrial function ( 32 ).
Palmitate exposure in HepG2 cells induced the PERK-eIF2 ␣ -ATF4 and IRE1-XBP1 arms of the ER stress pathways. Genes downstream of these arms were also upregulated, as were genes in other pathways ( Table 1 ; supplementary  Table V). Because of signifi cant cross-talk between pathways, many differentially expressed genes overlap between pathways. This complex network of palmi tate response is evident in our Ingenuity Pathway Analysis, which showed signifi cant enrichment of 41 different canonical pathways, each in turn with 5-21 gene members (supplementary Fig. 5. Silencing of the PIK3IP1 transcriptional response to palmitate protects cells against ER stress. A: Signifi cant attenuation of palmitate-mediated induction of PIK3IP1 expression was achieved in HepG2 cells treated with PIK3IP1-siRNA compared with similar nontarget siRNA-treated cells. SiRNA-mediated knock-down of PIK3IP1 signifi cantly attenuated palmitate-mediated ER stress response (B), including XBP1 splicing (C). XBP1 transcripts amplifi ed from cDNA by PCR (197 bp XBP1-unspliced and 171 bp XBP1-spliced) were examined by agarose gel electrophoresis. Data are shown as mean ± SD for three biological replicates. NT-siRNA, nontargeting siRNA pool. only 14 genes altered by oleate and 11 genes altered by palmitate. Swagell et al. ( 29 ) found over 1.6-fold altered expression in 162 genes in the huh-7 human hepatocyte line after 48 h exposure to 150 M palmitate. These experiments were similar in palmitate concentration and duration to the adaptive experiments performed in the present study, and in our hands, these concentrations did not induce markers of ER stress in HepG2 cells. In contrast, Li et al. ( 30) and Srivastava et al . (31 ) tested in HepG2 cells a 24 h exposure of 700 µM palmitate, oleate, and linoleate, a level that would induce ER stress and cytotoxicity with palmitate. Although these studies focused on the analytical approach, they reported fatty acid oxidation and electron transport as key markers of cytotoxicity and NADH dehydrogenase, mitogen activated protein kinases, extracellular signal regulated kinase, and JNK as potential regulators of cytotoxicity and lipid accumulation. Previous studies have not, however, evaluated the differences between chronic and acute exposure to saturated and unsaturated fatty acid or the adaptive response to prolonged, low level saturated or unsaturated fatty acid exposure and the effect on the response to a subsequent challenge with cytotoxic levels of saturated fatty acids.
We observed almost no overlap in the genes showing a 1.5-fold or greater response to palmitate when compared with oleate in HepG2 cells, thus suggesting distinct transcriptional responses. Notably, the transcript profi les we observed could be the result of altered gene transcription or altered mRNA stability; these mechanisms cannot be distin-HepG2 cells from palmitate-mediated ER stress. With our data, these studies strongly suggest that PIK3IP1 is one of the mediators of the palmitate-induced stress response and that adaptation acts in part to suppress this activation.
We also found evidence that a highly upregulated interaction network comprising 23 genes was involved in the adaptive response. This network includes GADD45A and GADD45B, which in turn bind to and activate MTK1 kinase, which is upstream of the JNK and p38 MAPK pathway. Thus, palmitate may act through GADD45A and GADD45B to induce apoptosis ( 35 ). Like PIK3IP1, palmitate-induced GADD45A and GADD45B transcription was reduced in HepG2 cells adapted fi rst to oleate, thus providing another mechanism by which oleate protected against cellular stress responses and apoptosis. We made similar observations when palmitate and oleate were present simultaneously as a mixture.
Recently, palmitate conversion to lysophosphatidylcholine (LPC) but not ceramide was shown to cause apoptosis in hepatocytes ( 36 ). These authors suggested that oleate was protective by diverting palmitate from LPC to triglyceride formation. Other studies have shown that fatty acids that cannot be metabolized do not induce ER stress ( 37,38 ). In the current study, palmitate upregulated genes involved in cholesterol and triglyceride metabolism, including HMGSC1, MVK, and PCK1. This upregulation was abolished by oleate pretreatment. Furthermore, the LPC acyltransferase 1 (AYTL2) gene was downregulated by palmitate (supplementary Data and Fig. V ). Putting these observations together with the evidence that LPC may induce cellular stress, we propose another mechanism by which oleate may protect against palmitate-induced cellular stress. Oleate appears to alter saturated triglyceride metabolism, cholesterol metabolism, and LPC synthesis, which in turn may reduce the remodeling of the ER and mitochondrial membranes.
We have identifi ed multiple important genes and pathways that are altered in cells undergoing an adaptive response to unsaturated fatty acids. These pathways may refl ect the physiological response in humans and may provide targets to prevent lipotoxicity in the liver and ␤ -cell. Our in vitro study validates the role of PIK3IP1 and the PI3 kinase pathway as mediators that partially account for the protective effects of oleate against palmitate-induced ER stress. Functional analyses of other novel pathways that have emerged from our study are required to further test the hypotheses generated by the transcriptional profi ling reported here.