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Physiol. Genomics 31: 544-553, 2007. First published September 18, 2007; doi:10.1152/physiolgenomics.00156.2007
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Received 13 July 2007; accepted in final form 11 September 2007.
Physiological Genomics 31:544-553 (2007)
1094-8341/06 $8.00 © 2007 American Physiological Society

Trans-10, cis-12 conjugated linoleic acid activates the integrated stress response pathway in adipocytes

P. Christopher LaRosa 1, Jean-Jack M. Riethoven 1, Han Chen 1, Yuannan Xia 1, You Zhou 1, Mei Chen 1, Jess Miner 2 and Michael E. Fromm 1

1 Center for Biotechnology
2 Department of Animal Science, University of Nebraska, Lincoln, Nebraska


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse white adipose tissue (WAT) and adipocytes in culture. The early transcriptome changes in treated WAT and 3T3-L1 adipocytes were analyzed using high-density microarrays to better characterize the signaling pathways responding to t10c12 CLA. Gene expression responses between 4 and 24 h after treatment showed a common set of early gene expression changes indicative of an integrated stress response (ISR). The responses of 3T3-L1 preadipocytes treated with t10c12 CLA or adipocytes treated with the cis-9, trans-11 isomer of CLA did not show the ISR, indicating the effect is specific to adipocytes responding to t10c12 CLA. Western blot analysis found increased phosphorylation of eIF2{alpha} and increased production of ATF4 confirming at least part of the response to t10c12 CLA is mediated through the ISR pathway. Immunofluorescence microscopy found that the cell type expressing ATF3, an indicator of the ISR, was early stage adipocytes containing oil droplets but lacking the abundant levels of fatty acid binding protein-4 (FABP4) (AP2) found in mature adipocytes. Our data suggests that the ISR precedes and is possibly the cause of the later induction of proinflammatory cytokines observed in t10c12 CLA treated adipocytes. The release of proinflammatory cytokines may explain how the ISR in early stage adipocytes causes lipid loss in mature adipocytes.

activating transcription factor 4; activating transcription factor 3; microarray; Akt


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DIETARY conjugated linoleic acid (CLA), either as mixed isomers or the trans-10, cis-12 isomer (t10c12 CLA), dramatically reduces fat content in white adipose tissues (WAT) in mice (10, 40). At the cellular level, evidence supports both apoptotic loss of cells (23, 32, 47) and reduced triglyceride content in adipocytes (13, 32, 47). Inflammation appears to play a key role (42) as proinflammatory cytokines and chemokine levels, including those of TNF-{alpha}, IL-6, and IL-8, are increased (3, 47), as are chemokines in the CC and CXC families (32). NF{kappa}B activation is necessary for the reduction of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and glucose transporter 4 (GLUT4) protein or activity levels and for induction of IL-6 (6). Despite this progress, additional signaling pathways participating in the response to t10c12 CLA need to be determined.

Phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}) is a translation control response to stress that is mediated by at least four eIF2 kinases. Many different stresses result in activation of one or more of these eIF2 kinases, and the participation of eIF2{alpha} phosphorylation in these stress responses has been called the integrated stress response (ISR; Refs. 35, 48). Two of the eIF2 kinases, general control non-derepressible-2 (GCN2) and PKR-like eukaryotic initiation factor 2{alpha} kinase (PERK), are predominantly activated by amino acid deprivation and endoplasmic reticulum (ER) stress, respectively. ER stress can also initiate a more extensive response, which includes the ISR, called the unfolded protein response (UPR).

Phosphorylated eIF2{alpha} reduces the rate of translation initiation to reduce the amount of protein being processed by the ER and to allow for the selective translation of specific stress-related mRNAs (48). The translation of stress-related activating transcription factor 4 (ATF4) mRNA increases under these conditions, and increased amounts of ATF4 induce the transcription of many of the ISR genes. In particular, ATF4 induces the expression of activating transcription factor 3 (ATF3), which then induces the expression of CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), pseudokinase Tribbles 3/SKIP 3 (TRIB3), and the phosphatase regulatory subunit, growth arrest and DNA damage inducible protein (GADD34; Ref. 29).

ER stress conditions are perceived by one or more of the three ER transmembrane receptors: PERK, activating transcription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE1; Ref. 48). When activated by ER stress, transcription factor ATF6 is proteolytically released from the ER membrane, and the sequence-specific RNase domain of IRE1 specifically splices the X-box binding protein (XBP1) mRNA to create the coding region for the more stable XBP1s transcription factor, both of which transcriptionally regulate UPR-related genes (1). If the cell's UPR fails to remove the unfolded proteins, proapoptotic pathways are then activated and can result in programmed cell death (45).

In the present study, we analyzed the response of mouse WAT and 3T3-L1 adipocytes 4 to 24 h after exposure to t10c12 CLA. A combined analysis using microarray profiling, Western blots, and immunofluorescence microscopy indicates the ISR pathway is activated early after t10c12 CLA treatment, and this occurs before the induction of proinflammatory cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice, cells, tissue culture, and fatty acid treatments.
All animal experiments were carried out with male C57BL/6J mice (Charles River Laboratories) as described previously (32) following a protocol (no. 0306-021) approved by the Institutional Animal Care and Use Committee of University of Nebraska-Lincoln and under the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (Publication No. 80-23; revised in 1996). Mice were acclimated to fasting overnight for 1 wk before experiments. For experiments, mice were fasted overnight and gavaged (t = 0) with 15 µl of control soy oil or t10c12 CLA oil and then offered modified AIN-93G pellets (soy protein replaces casein; Dyets) containing 7% soy oil or 6.5% soy oil and 0.5% t10c12 CLA (Nu-Check Prep, Elysian, MN). Low-passage 3T3-L1 preadipocytes (18) were cultured (33) and treated with 100 µM fatty acids prepared as reported (6).

Western blots.
Cytoplasmic and nuclear extracts were prepared following the manufacturer's protocol (Active Motif), with the addition of phosphatase and protease inhibitors to the hypotonic buffer. Antibodies to ATF3, ATF4, nucleoporin (Santa Cruz Biotechnology), phospho-eIF2{alpha} (p-eIF2{alpha}), and eIF2{alpha} (Invitrogen) were used at the manufacturer's suggested concentrations. All Western blot analyses were repeated at least three times on different biological samples.

Immunofluorescence confocal microscopy and quantitative image analysis.
3T3-L1 adipocytes in eight-well chamber slides were fixed and stained using standard methods (32). Primary antibodies were to either ATF3, Akt, phospho-Akt (p-Akt), or adipocyte marker fatty acid binding protein-4 (FABP4; Santa Cruz Biotechnology). Secondary antibodies were Alexa Fluor 488 conjugated donkey anti-rabbit or Alexa Fluor 647 conjugated donkey anti-goat IgG (Invitrogen).

For quantitative image analysis of ATF3 expression in response to t10c12 CLA or linoleic acid (LA) treatment, optical images were collected using a x10 lens and a 512 x 512 frame size, under the same confocal settings (e.g., laser power, gain/offset levels). At least four images from each well for each treatment were collected randomly from each slide. The quantitative data for ATF3 expression were obtained by automated image analysis using Olympus Soft Imaging AnalySIS (v3.2) software. Particle counts and analyses were performed under a preset threshold for 1) upregulated ATF3 expression, 2) low-level ATF3 expression (for total number of cells), and 3) FABP4-positive cells (adipocyte markers), respectively.

RNA isolation and labeling.
RNA from mouse WAT or 3T3-L1 cells was isolated and analyzed by Affymetrix GeneChip Mouse Genome 430 2.0 arrays as previously reported (32). The number of replica samples for each cell type and treatment are shown in Table 1.


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  		Table 1. Number of gene expression changes in treated cells and their percentage overlap with changes from t10c12 CLA-treated WAT or tunicamycin-treated 3T3-L1 adipocytes

 
Affymetrix microarray data analysis.
Affymetrix GeneChip Operating Software (GCOS) generated the image and probe set signal values as CEL files. The CEL files were screened for outlying arrays by means of a combination of box plots, density plots, and M versus A (MvA) plots on both raw and normalized signal values, where normalized signal values were acquired by using the robust multiarray analysis algorithm [RMA (27)] via the R/Bioconductor affy package (17). MvA plots show M = (X – Y) on the x-axis and A = (X + Y)/2 on the y-axis, where X is the raw or normalized log2 signal value of an array, and Y is the same for another array or a synthesized array created by taking the median of all similar arrays (i.e., control, CLA cells, tunicamycin cells, etc); both pairwise and array-median array comparisons where done. CEL files with acceptable MvA plots were loaded into the Rosetta Resolver (version 6.0, build 6.0.0.311; Rosetta Inpharmatics LLC, Seattle, WA). For each treatment-time combination, the replicate arrays were combined with their control replicates into ratio experiments in Rosetta Resolver and analyzed using the software's proprietary ratio error model (50). From the ratio experiments, lists were generated of Affymetrix probe set identifiers that were expressed at P ≤ 0.05 and where the arithmetic mean intensity for the control or the treatment were above the 50% percentile, and the expression ratio was at least 2. The series records of the data submitted to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus are GSE8679, GSE8681, GSE8682, GSE8683, and GSE8684.

Quantitative RT-PCR.
Quantitative RT-PCR was performed as described previously (32).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Microarray analysis.
Differentiated 3T3-L1 adipocytes treated with LA or t10c12 CLA for 2 days showed a reduction in the amount of oil-red staining lipids in the t10c12 CLA-treated adipocytes relative to the LA-treated adipocytes (Fig. 1). The gene expression changes causing this were measured by microarray analysis of 3T3-L1 adipocytes treated with LA or t10c12 CLA for 4, 8, 12, or 24 h (Table 1). Microarray analysis of the mRNA from the WAT of mice fed diet containing control or 1% t10c12 CLA fatty acids for 4, 12, or 24 h was also performed to find common gene expression changes (Table 1). Differentiated 3T3-L1 adipocyte cultures were observed to contain ~30% undifferentiated preadipocytes. Therefore, the parental line of undifferentiated 3T3-L1 preadipocytes was also exposed to LA or t10c12 CLA for similar times, and the mRNA was analyzed by microarray hybridization to define their possible contribution to gene expression changes in the 3T3-L1 adipocyte cultures (Table 1).


Figure 1
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  		Fig. 1. Oil red staining of treated adipocytes. 3T3-L1 adipocyte cultures were treated for 2 days with 100 µM linoleic acid (LA) or trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) and then stained with oil red to visualize the amount of lipid in the adipocytes.

 
As described in more detail below, initial microarray analysis indicated an ER stress response in WAT and 3T3-L1 adipocytes exposed to t10c12 CLA. Therefore, 3T3-L1 adipocytes were exposed to tunicamycin, a known inducer of ER stress, for comparison to the above microarray experiments (Table 1). The number of genes that increased or decreased expression by a ratio of two or more in these various treatments is summarized in Table 1 (Individual gene values are shown in Supplemental Table 11 ). Also shown in Table 1 are the number of genes with expression changes in 3T3-L1 adipocytes or preadipocytes that are in common with either the mouse WAT treatments or the tunicamycin-treated 3T3-L1 adipocytes.

The experiments in this report were performed using LA as the control fatty acid. As a further comparison of the specificity of the gene expression response to t10c12 CLA, a microarray analysis of the gene expression changes of cis-9, trans-11 (c9t11) CLA or t10c12 CLA treated 3T3-L1 adipocytes was performed for 8 or 12 h. When analyzed by either combining data from the 8- and 12-h samples or compared individually, the c9t11 CLA gene expression changes showed little in common with the t10c12 CLA responses (data not shown). This indicates the pattern of t10c12 CLA-induced gene expression changes distinguish it from the changes caused by the closely related LA or c9t11 CLA fatty acid isomers.

The reliability of the microarray data was analyzed by performing quantitative real-time PCR (qRT-PCR). A set of seven genes that showed different amounts of gene expression changes in the microarray analysis 12 h after feeding mice t10c12 CLA was further analyzed by qRT-PCR of cDNA made from WAT mRNA. The signals from ATF3, DNA damage-inducible transcript 3 (DDIT3; CHOP), TRIB3, Serpine1, and IER3 all showed increases, XBP1 showed no significant change, and THRSP (spot 14) showed a modest decrease, in good agreement with the microarray results (Table 2).


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  		Table 2. Comparison of real-time PCR and microarray values for selected genes from WAT after 12 h of t10c12 CLA treatment

 
A more detailed analysis of the early response was done to find the gene expression changes after 4 or 8 h of t10c12 CLA treatment of 3T3-L1 adipocytes. These gene expression changes were used to find those in common in mouse WAT after t10c12 CLA treatment (Table 3; ratio values are given in Supplemental Table 2). The overlap approach focuses on gene changes in common between the two systems and increases the number of microarrays used in the combined analysis to reduce the false signals in the experiments.


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  		Table 3. Gene expression changes at 4 and 8 h after the indicated treatments that are in common with those in WAT from t10c12 CLA-fed mice

 
The overlap between the gene-specific responses of the t10c12 CLA-treated mouse WAT and tunicamycin-treated 3T3-L1 adipocytes was examined to compare the t10c12 CLA response to that from a known UPR and to compare the t10c12 CLA response of undifferentiated 3T3-L1 preadipocytes to the non-adipocyte cell-type response (Table 3). The genes in Table 3 are further organized into either ISR/UPR, inflammatory, lipid biosynthesis (32), or grouped as "other" categories (Table 3). The ISR/UPR grouping used a list of ISR/UPR-responsive genes from reported experiments (22, 34, 44), and this list is provided (Supplemental Table 3); the GO classifications of the "other" genes is in Supplemental Table 4. Twenty six percent of the genes induced by t10c12 CLA treatment in both WAT and 3T3-L1 adipocytes after 4 h are ISR/UPR related genes (Table 3). Twenty percent of the genes induced in common in the t10c12 CLA-treated mouse WAT and in 3T3-L1 adipocytes 4 h after tunicamycin treatment are ISR/UPR genes.

Western blot analysis of the ISR/UPR signaling pathway.
The three signaling pathways known to participate in initiating a UPR, including the ISR pathway, were analyzed to determine if they were activated by t10c12 CLA treatments. Western blots of phosphorylated and total eIF2{alpha} protein indicated at least a 100% increase in phosphorylated eIF2{alpha} in tunicamycin-treated 3T3-L1 adipocytes (data not shown) and a 20–40% increase at 2, 4, and 8 h after treating 3T3-L1 adipocytes with t10c12 CLA (P ≤ 0.002; Fig. 2A). t10c12 CLA treatment of 3T3-L1 adipocytes induced ATF4 levels after 1, 2, 4, and 8 h and ATF3 levels after 4 and 8 h (Fig. 2B). ATF4 mRNA levels in the microarray data indicated changes of less than a 100% increase in t10c12 CLA-treated 3T3-L1 adipocytes, suggesting the up to 500% increase in ATF4 protein is in part due to translation increases.


Figure 2
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  		Fig. 2. Protein and RNA analysis of integrated stress response (ISR)/unfolded protein response (UPR) signaling. Western blot or RNA analyses of 3T3-L1 adipocytes treated with LA or t10c12 CLA are shown. Each experiment was repeated at least 3 times, and a representative example is shown. A: amount of phosphorylated (p-EIF2a) and total eukaryotic initiation factor 2{alpha} (eIF2a) protein levels at various times in cytoplasmic extracts. Ratio of the amount of phosphorylated to total eIF2{alpha} was determined for the t10c12 CLA and LA (–) treatments at each time point, and the relative CLA/LA ratio is shown below for 3 independent experiments. The SD (n = 3, except where noted) of the ratio for the average of 3 are shown. The abundance of p-eIF2{alpha} over the whole time series for the CLA-treated sample is statistically higher than for the LA-treated sample as measured on the raw pixel intensity data (Wilcoxon signed rank test; P ≤ 0.00013; H0: {theta} = 0; HA: {theta} > 0). For the 2- to 8-h period only, the same test is significant at P ≤ 0.002. B, inset: tunicamycin (Tm) does not induce activating transcription factor 3 (ATF3) until 4 h. B and C: antibody detection of ATF3 and ATF4 (B) or ATF6 and ATF6n (B) (50 kDa) in nuclear extracts at the indicated times. The amount of the nucleoporin protein (NP) present in each lane is shown. D: amount of inositol-requiring enzyme 1 (IRE1)-dependent X-box binding protein-1 (XBP1) mRNA splicing was measured by RT-PCR of cDNA from 3T3-L1 adipocytes or white adipose tissue (WAT) from mice 12 h after treatment with LA (–), t10c12 CLA (CLA+), or Tm. A 200-bp DNA marker is shown in lane M.

 
ATF6{alpha} is reported to undergo proteolytic processing in the ER membrane on activation and then be released to the nucleus as a 50-kDa form (39). Comparison of LA- and t10c12 CLA-treated adipocytes showed no change in the amount of the 50-kDa form of ATF6{alpha}, suggesting this pathway is not activated by t10c12 CLA treatment (Fig. 2C).

XBP1 mRNA requires an IRE1-dependent splicing reaction to remove an intron to produce the spliced form with the correct reading frame for the XBP1s protein. 3T3-L1 adipocytes show an increase from ~5% in control samples to over 50% spliced XBP1s mRNA in tunicamycin-treated cells when measured by RT-PCR (Fig. 2D). t10c12 CLA-treated mouse WAT or 3T3-L1 adipocytes show no change in the amount of spliced XBP1s mRNA compared with the LA-treated samples (Fig. 2D). Taken together, these results indicate the phosphorylation of eIF2{alpha} and increased translation of ATF4 is likely mediating the induction of the ISR genes observed in the microarray response (Table 3).

Immunofluorescence microscopy analysis of ATF3.
The type of cell showing an ISR after t10c12 CLA treatment was examined by immunofluorescence of ATF3 in 3T3-L1 adipocyte cultures. These cultures are ~70% adipocytes, where the adipocytes are defined as cells containing oil droplets. These adipocytes have varying amounts of the mature adipocyte marker protein FABP4 (AP2), as measured by immunofluorescence. One class of adipocytes has oil droplets but little detectable FABP4, and these are considered early-stage adipocytes.

A background level of ~10% of the cells in LA- or t10c12 CLA-treated cells show bright ATF3 immunofluorescence at early time points (Fig. 3 and Table 4). The background level of ATF3-positive cells is reduced over time in LA-treated cells but increases to 38 percent of the t10c12 CLA-treated cells at 6 h after treatment (Table 4). A higher-power magnification of the cells indicates that early adipocytes, as indicated by the presence of oil droplets and little or no FABP4 immunofluorescence, are the cells that show ATF3 induction (Fig. 3, bottom). Mature adipocytes with high levels of FABP4 do not show the bright ATF3-containing nuclei that the early stage adipocytes show (Fig. 3, bottom). The oil red staining (Fig. 1) examined at higher-power magnification indicates that t10c12 CLA treatment prevents oil accumulation and/or mediates oil reduction in all adipocytes (data not shown).


Figure 3
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  		Fig. 3. Immunofluorescence of ATF3 and fatty acid binding protein-4 (FABP4). Immunofluorescence images from binding of antibodies to either ATF3 (green) or FABP4 (red) or the merged view are shown for 3T3-L1 adipocyte cultures treated with either LA or CLA for the indicated times. Bars: 200 µm (top 2 rows) and 50 µm (bottom row).

 

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  		Table 4. Percentage of cells highly expressing ATF3 in response to t10c12 CLA or LA treatment

 
Immunofluorescence microscopy analysis of p-AKT.
TRIB3 mRNA, and presumably TRIB3 protein, is induced after 4 h by tunicamycin or t10c12 CLA treatment of 3T3-L1 adipocytes or WAT (Table 3). TRIB3 protein has been reported to interact with protein kinase Akt family members (12). We examined the intracellular distribution of Akt family members and their phosphorylated form (p-Akt phosphorylated at Thr308) in adipocytes treated with t10c12 CLA. Immunofluorescence microscopic analysis of 3T3-L1 adipocytes treated with LA or t10c12 CLA did not find a difference in Akt subcellular distribution (data not shown). However, t10c12 CLA treatment caused the phosphorylated forms of p-Akt (pThr308) to become mostly excluded from the nucleus 24 h after t10c12 CLA addition (Fig. 4). No observable difference was detected at 6 h (Fig. 4).


Figure 4
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  		Fig. 4. Immunofluorescence of phospho-Akt (p-Akt). Immunofluorescence due to binding of antibody to p-Akt is shown for 3T3-L1 adipocyte cultures treated with either LA or CLA for 6 or 24 h. Bar = 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
T10c12 CLA responses in WAT and 3T3-L1 adipocytes.
Both mouse WAT and 3T3-L1 adipocyte cultures showed inhibition of oil accumulation in adipocytes when exposed to t10c12 CLA (13, 42), indicating similar responses in the adipocytes. Comparison of the gene expression changes that are in common reduced the complexity of the transcriptome responses due to the different cellular environments and the additional cell types present in either mouse WAT or 3T3-L1 adipocyte cultures.

At the earliest 4-h time point following t10c12 CLA treatment, a limited number of genes were induced in both WAT and 3T3-L1 adipocyte cultures. These genes included ATF3, DDIT3 (CHOP), eIF2 protein phosphatase-targeting subunit GADD34, and pseudokinase TRIB3, which are indicative of an ISR (21, 29, 34, 51). This indicated that t10c12 CLA might be acting through a stress pathway that activates ISR through eIF2{alpha} phosphorylation. This hypothesis was confirmed by finding increases in the amounts of ATF4 and phosphorylated eIF2{alpha} proteins. The ATF4 transcript is translationally controlled, and ATF4 protein levels increase under the reduced translation conditions present when eIF2{alpha} is phosphorylated (48). The 20–40% increase in the amount of eIF2{alpha} phosphorylation in t10c12 CLA-treated adipocytes is small compared with the >100% increase we found when the cells were treated with tunicamycin. The smaller-magnitude response is likely due to the participation of a limited part of the cell population, 30% or less, that ATF3 immunofluorescence found to be responding to t10c12 CLA. An early UPR did not seem to be occurring as neither the ATF6 nor IRE-1/XBP1 pathways appeared to be activated.

Insight into the extent of the t10c12 CLA response that is possibly mediated by the ISR is gained through comparison of the gene expression responses to those from tunicamycin treatments of 3T3-L1 adipocytes. Tunicamycin inhibits protein glycosylation and is a known inducer of the ISR/UPR that increases eIF2{alpha} phosphorylation (48). The tunicamycin-induced gene expression changes in 3T3-L1 adipocytes include a later inflammatory response and downregulation of many of the genes in the lipid biosynthetic pathway that are characteristic of the t10c12 CLA response in WAT and adipocytes (25, 32). This suggests that a sustained ISR/UPR is sufficient to cause many of the subsequent transcriptional changes observed with t10c12 CLA treatments. Differences in the sets of induced genes indicate the t10c12 CLA and tunicamycin responses are not identical and may affect other pathways differently. Tunicamycin, but not t10c12 CLA, activated a UPR through ATF6 cleavage and IRE1-mediated splicing of XBP1 (1) in these 3T3-L1 adipocyte cultures.

In 3T3-L1 adipocyte cultures the cytokine-related genes expressed at the most time points are cardiotrophin-like cytokine factor 1 (CLCF1) in the IL-6 family, suppressor of cytokine signaling 3 (SOCS3), and CXCL2 (an IL-8-related proinflammatory chemokine). Only cytokine CLCF1 was induced at the earliest 4-h time point in 3T3-L1 adipocytes. CLCF1 is related to cardiotrophin 1 (CTF1), which has been shown to induce SOCS3 and increase insulin resistance (52). CLCF1 may be partially substituting for IL-6 in 3T3-L1 adipocyte cultures and initiating an IL-6-dependent fat loss (19). SOCS3 can be induced by IL-6 and CTF1, suppress cytokine signaling, inhibit insulin signaling (28), and reduce lipid biosynthesis. CXCL2 (MIP2) is a proinflammatory cytokine that induces other cytokines, including IL-6, possibly helping to maintain the inflammatory response. Cells under ER stress also induce cyclooxygenase-2 [COX-2 (PTGS2)] through NF{kappa}B (26), and we found COX-2 mRNA to be highly induced by t10c12 CLA treatment in 3T3-L1 adipocytes and to a lesser extent in WAT. t10c12 CLA-induced COX-2 would be expected to increase prostaglandin levels and maintain the inflammatory state. Proinflammatory cytokines increase triglyceride breakdown levels (30).

Additional support for the hypothesis that phosphorylated eIF2{alpha} may be the key pathway causing the inflammation and reduced lipogenesis found in t10c12 CLA-treated WAT and 3T3-L1 adipocytes is found in examples where other chemical inducers of ISR/UPR cause similar phenotypes. For example, human immunodeficiency virus (HIV) protease inhibitors have been found to cause a lipodystrophy in patients that result in a loss of peripheral body fat and hepatic steatosis (4). These HIV protease inhibitors were shown to induce a UPR in 3T3-L1 adipocytes, apparently by inhibition of the proteasome and protein processing (41). Oxidized fatty acids have been found to induce a UPR in human aortic endothelial cells with a subsequent inflammation response (15, 16). This response was found to be mediated by the UPR transcription factors ATF4 and XBP1 (15). Leucine deprivation in the diets of mice also activates an ISR through GCN2-mediated phosphorylation of eIF2{alpha} and results in dramatic fat loss in mouse WAT (20). The involvement of the ISR pathway can explain some of the observed gene expression changes in individual genes in their response to t10c12 CLA (Fig. 5).


Figure 5
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  		Fig. 5. Hypothetical model of t10c12 CLA signaling through the ISR pathway. t10c12 CLA (or the other inducers) activates an eIF2{alpha} kinase through an unknown pathway, resulting in increased eIF2{alpha} phosphorylation. The reduced rate of translation increases ATF4 protein production but generally reduces protein production, including that for the unstable I{kappa}B{alpha}. ATF4 induces the ISR genes while reduced amounts of I{kappa}B{alpha} release NF{kappa}B to the nucleus. Stress signaling, possibly via ATF4, induces the immediate early (IE) proteins and activates ERK, which can phosphorylate and activate NF{kappa}B. Active NF{kappa}B induces the expression of a number of inflammatory genes, including cytokines, PTGS2 for prostaglandin synthesis, and suppressor of cytokine signaling 3 (SOCS3), which can inhibit insulin signaling. In early adipocytes, Tribbles 3/SKIP 3 (TRIB3) can inhibit Akt in the insulin-signaling pathway, including glucose transporter GLUT4, and acetyl-coenzyme A carboxylase (ACCase) in the lipid biosynthesis pathway, while DNA damage-inducible transcript 3 (DDIT3) can inhibit CCAAT/enhancer binding protein (C/EBP)-ß and downstream lipid regulatory proteins peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and C/EBP{alpha}. In mature adipocytes, inflammatory cytokines and prostaglandin signaling reduce lipid regulatory proteins. Reduced amounts of active lipid regulatory proteins, including sterol-regulating element binding protein 1c (SREBP1c), PPAR{gamma}, and C/EBP{alpha}, reduce the amount of key lipid biosynthetic (biosyn) proteins, including fatty acid synthase (FASN), LIPN1, and steroyl-CoA desaturase (SCD). GSK3ß, glycogen synthase kinase-ß; CLCF1, cardiotrophin-like cytokine factor 1; COX-2, cyclooxygenase-2; GADD34, growth arrest and DNA damage-inducible protein; LIPE, hormone-sensitive lipase; CXCL2 and CXCL3, CXC chemokine ligand-2 and -3, respectively.

 
Signaling mechanisms downstream of eIF2{alpha} translation control: Immediate early genes Egr-1, c-myc, BTG2, ZF36, fra-1, c-jun.
A number of the immediate early (IE) stress genes are induced by t10c12 CLA treatment, particularly in 3T3-L1 adipocytes after 4–8 h of treatment. IE genes are known to be induced by many stresses, including ER stress. Depending on the specific type of ER stress-inducing chemical, the induction of the IE genes Egr-1, c-myc, BTG2, ZF36, fra-1 (fosl-1), and c-jun that are induced by t10c12 CLA can be either partially dependent or dependent on PERK phosphorylation of eIF2{alpha} (34). The signaling pathways activating these IE genes may also be involved in activating or interacting with the NF{kappa}B pathway (Fig. 5) as NF{kappa}B activation was found to require an active ERK pathway in the t10c12 CLA response in human adipocytes (6).

Inflammation.
A possible mechanism for the induction of proinflammatory cytokines subsequent to the ISR is through activation of NF{kappa}B through reduction of the protein levels of its cytoplasmic inhibitor I{kappa}B (Fig. 5). Phosphorylated eIF2{alpha} reduces the translation of most mRNAs, including the mRNA for the I{kappa}B{alpha} subunit of I{kappa}B, leading to a decrease in the amount of the unstable I{kappa}B{alpha} protein (11). I{kappa}B{alpha} protein levels were reported to be reduced in response to t10c12 CLA treatment in human adipocytes (6). The requirement of ATF4 and XBP1s for an inflammatory response in endothelial cells responding to oxidized phospholipids (15) indicates the interactions between the ISR and NF{kappa}B pathways are more complex than just a requirement for eIF2{alpha} phosphorylation to reduce I{kappa}B{alpha} levels.

p-Akt and TRIB3.
Protein kinase Akt has three family members [which are also known as protein kinase B (PKB)] and is a key component of many signaling pathways, including insulin signaling (2) (Fig. 5). The insulin-signaling pathway activates Akt by increased phosphorylation, and p-Akt regulates glucose, glycogen, and lipid metabolism. The phosphorylation of the synip protein (Syntaxin4 interacting protein) by p-Akt allows GLUT4, the key glucose transporter of adipocytes, to translocate to the plasma membrane where it is active. The active p-Akt also phosphorylates the constitutively active glycogen synthase kinase-ß (GSK3ß), converting GSK3ß into its inactive form (9). The phosphorylated, inactive GSK3ß is prevented from phosphorylating, and thereby reducing the activities, of glycogen synthase and sterol-regulatory element binding protein 1 (SREBP1) (31). Any inhibition of Akt protein kinase activity would be expected to reverse these signals, increase insulin resistance, and reduce glucose uptake, glycogen synthesis, and lipid biosynthesis (Fig. 5).

A t10c12 CLA-induced gene encoding a candidate protein reported to bind to and inhibit Akt is the pseudokinase TRIB3 (12, 24). TRIB3 is a metabolic regulatory protein induced by either low glucose (51), fasting (12), or ER stress (38, 51), or after t10c12 CLA treatment (25, 32). Mice overexpressing TRIB3 have higher energy expenditures and a lean phenotype (43). TRIB3 has been reported to also initiate a ubiquitin-mediated degradation of acetyl-coenzyme A carboxylase (ACCase), the rate-limiting enzyme in fatty acid biosynthesis (43). The t10c12 CLA-induced TRIB3 could affect Akt activity and ACCase degradation to contribute to the reduced lipid biosynthesis and increased fatty acid oxidation that occurs in mice fed t10c12 CLA (46) or overexpressing TRIB3 (43) (Fig. 5).

TRIB3 binding to p-Akt might not explain the exclusion of p-Akt from the nucleus that we observed as TRIB3 mRNA appears early and the p-Akt exclusion from the nucleus occurs only after 24 h. Alternative interactions are likely because TRIB3 has been reported to bind poorly to a phosphorylation mimic of p-Akt2 [Thr308 to Asp308 (12)]. TRIB3 would be expected to bind preferentially to Akt while we see nuclear exclusion of p-Akt but not of Akt. Nuclear exclusion of the active phosphorylated form of Akt is likely to change its ability to function in the insulin-signaling pathway, as the majority of p-Akt was observed to be in the nucleus of control adipocytes. Reductions in insulin signaling through sequestration of p-Akt could explain some of the observed reductions in mRNAs encoding lipid biosynthetic enzymes and regulatory proteins occurring at later times after t10c12 CLA treatment (Fig. 5).

Additional adipocyte regulatory proteins.
Some of the key transcription factors required for adipocyte metabolism (5, 14, 30) are PPAR{gamma}, PPAR{gamma} coactivator 1, MLXIPL (CHREBP), C/EBP{alpha}, Krupple-like zinc finger transcription factor 15 [KLF15 (37)], FoxO1 (36) together with SREBP1c. These adipocyte regulatory transcripts are all downregulated by expression ratios of two to four after 24 h of t10c12 CLA-treatment in WAT (32) and generally to a similar extent in 3T3-L1 adipocytes. The presumed reductions in the corresponding proteins of these key transcription factors may account for the global reduction in many transcripts required for lipid metabolism at 24 h or later in mice (25, 32). The alterations in p-Akt insulin signaling together with the previously reported increases in NF{kappa}B activity and proinflammatory cytokines (7, 32), and inhibition of transcription factor C/EBPß by heterodimerization with DDIT3 (CHOP), which reduces the C/EBPß-dependent expression of C/EBP{alpha} and PPAR{gamma} (33), are possible mechanisms (Fig. 5) for causing these later reductions in lipid biosynthesis-related transcripts.

Summary.
Recent reports suggest that stromal vascular adipocyte precursors express the majority of the initial proinflammatory cytokines in adipocyte cultures (6, 8). Our observation that early-stage adipocytes show an ISR after t10c12 CLA exposure and presumably are the source of the induced proinflammatory cytokines seems similar to this model as mature adipocytes do not show the ISR. Nonetheless, these mature adipocytes respond to t10c12 CLA by fat loss in WAT (32) and 3T3-L1 cultures. Induction of secreted proinflammatory cytokines (25, 32) is a plausible model (6) for the nonautonomous effects of ISR in early-stage adipocytes (Fig. 5). The initial ISR appears to progress into a UPR in WAT as judged by the later induction of GRP78 (HSPA5), a diagnostic marker for UPR. Persistent UPR and the ISR-induced DDIT3 (CHOP) can induce proapoptotic pathways, and this may explain the frequent reports of t10c12 CLA-mediated induction of apoptosis in WAT (23, 32, 47). Additional research is required to further delineate the early signaling pathways that initiate the ISR in early-stage adipocytes, to functionally demonstrate that early ISR leads to activation of the NF{kappa}B pathway and induction of inflammatory cytokines , and to understand the mechanisms that prevent lipid loss from occurring during the similar inflammatory and ER stress conditions present in obese WAT (19, 49).


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Science Foundation Grant EPS-0346476.


   ACKNOWLEDGMENTS
 
We thank Steve Kachman for advice on the statistical analysis of microarrays and Lucas Knoflicek for help with making the figures.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: M. E. Fromm, E205 Beadle Center, Univ. of Nebraska, 1901 Vine St., Lincoln, NE 68588-0665 (e-mail: mfromm@unlnotes.unl.edu).

1 Supplemental material is available with the online version of this article. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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