Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes.

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor κB (NFκB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated 10,12 CLA–mediated production of reactive oxygen species (ROS), activation of ERK1/2 and cJun-NH2-terminal kinase (JNK), and induction of inflammatory genes. 10,12 CLA–mediated binding of NFκB to the promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 and induction of calcium-calmodulin kinase II (CaMKII) β were attenuated by TMB-8. KN-62, a CaMKII inhibitor, also suppressed 10,12 CLA–mediated ROS production and ERK1/2 and JNK activation. Additionally, KN-62 attenuated 10,12 CLA induction of inflammatory and integrated stress response genes, increase in prostaglandin F2α, and suppression of peroxisome proliferator activated receptor γ protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca2+]i levels, particularly calcium from the ER.


Culturing of human primary adipocytes
Abdominal white adipose tissue (WAT) was obtained, with consent from the institutional review boards at the University of North Carolina at Greensboro and Moses Cone Memorial Hospital, during abdominoplasty of nondiabetic Caucasian and African American females between the ages of 20 and 50 years with a body mass index р 32.0. Tissue was digested using collagenase; stromal vascular cells were isolated as previously described ( 5 ). Cultures containing newly differentiated human adipocytes were treated on day 6-12 of the differentiation program. Each independent experiment was repeated at least twice using a mixture of cells from two or three subjects, unless otherwise indicated.

Culturing of human Simpson-Golabi-Behmel Syndrome cells
Simpson-Golabi-Behmel Syndrome (SGBS) cells were generously provided by Dr. Martin Wabitsch at the University of Ulm, Ulm, Germany. They were grown to confl uence in DMEM/Nutrient Mixture F-12 Ham's supplemented with 10% bovine serum, 33 M biotin, 17 M pantothenate, 100 g/ml streptomycin and 62.5 g/ml penicillin. To induce differentiation, SGBS cells were washed repeatedly with PBS buffer and then cultured in serumfree medium supplemented with 10 nM insulin, 200 pM triiodothyronine, 1 M cortisol, 2 M BRL 49653, 0.115 mg/ml 1-methyl-3-isobutylxanthine (IBMX), 0.25 mmol/l dexamethasone (DEX), and 0.01 mg/ml human transferrin for 4 d. After 4 d, the medium was replaced with the differentiation medium lacking BRL 49653, IBMX, and DEX. These cells were used for the chromatin immunoprecipitation (ChIP) experiments on day 6 of differentiation.

Measuring ROS
For the DCF assay (Molecular Probes), primary human adipocytes were seeded in 96-well plates and differentiated for 6 d. On day 6, medium was changed to serum-and phenol red-free medium for 24 h. After 24 h, cells were preloaded with 5 M DCF at 37°C for 1 h and then treated with various treatments for 3 h. Cells were then washed once with HBSS and fl uorescence was immediately measured in a plate reader with an excitation/ emission wavelength of 485-528 nm. DCF values were calculated after normalizing background fl uorescence levels of DCF.

2+ ] i levels
We determined [Ca 2+ ] i levels using Fluo-3 AM. Briefl y, cells were preloaded with 5 M Fluo-3 AM and an anionic detergent, 10% Pluronic F-127, at 37°C for 30 min in the dark. Cells were then washed with a buffer consisting of HBSS, CaCl 2 , and probenecid, which prevents Fluo-3 AM leakage from cells. Baseline fl uorescence was measured using a Synergy Multidetection Microplate Reader (BioTek Inc., Winooski, VT). Cells were then Our research group has reported that 10,12 CLA, but not 9,11 CLA, inhibited human preadipocyte differentiation ( 6 ) and caused delipidation of newly differentiated human adipocytes ( 5 ). Isomer-specifi c delipidation of adipocytes by CLA was due largely to a decrease in adipogenic/lipogenic gene expression, uptake of glucose and fatty acids, and triglyceride (TG) synthesis as opposed to an increase in oxidation ( 5,7 ). Interestingly, 10,12 CLA suppression of glucose and fatty acid uptake was dependent on activation of mitogen-activated protein kinase/ extracellular kinase signal-regulated kinase (MEK/ERK) and nuclear factor B (NF B) signaling, as well as robust secretion or expression of the proinfl ammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF) ␣ ( 5,7,9 ). Consistent with these in vitro data, CLA supplementation in humans is associated with hyperglycemia, dyslipidemia, insulin resistance, and elevated levels of infl ammatory prostaglandins (PG) and cytokines (10)(11)(12)(13). However, the upstream mechanism(s) by which 10,12 CLA induces infl ammation, insulin resistance, and adipocyte delipidation remain unclear.
One possible upstream mediator of CLA-induced infl ammation and insulin resistance is intracellular calcium ([Ca 2+ ] i ), which is a vital second messenger for the activation of proteins involved in adipocyte proliferation, differentiation, and metabolism ( 14 ). Elevated levels of [Ca 2+ ] i have been reported to activate NF B (15)(16)(17), ERK1/2 ( 15,16,18 ), and phospholipase A2 (PLA 2 ) ( 19,20 ), leading to cytokine or PG production. Calmodulin (CaM), a major calcium-dependent protein, plays an important role in infl ammation and metabolism. CaM activates a number of protein kinases, such as CaMKI, II, and IV ( 21,22 ). Relevant to this study, CaMKII has been shown to inhibit adipocyte differentiation in response to infl ammatory PGs such as PGF 2 ␣ ( 23 ).
On the basis of the critical role of [Ca 2+ ] i in cell signaling, enzyme activation, and adipocyte differentiation, we hypothesized that [Ca 2+ ] i is an upstream mediator of 10,12 CLA-induced infl ammation and insulin resistance in human adipocytes. In this study, we demonstrated for the fi rst time that CLA increases [Ca 2+ ] i levels, leading to the production of reactive oxygen species (ROS), NF B, cJun-NH 2 -terminal kinase (JNK), ERK1/2, and ultimately, the induction of infl ammatory genes and PGs, and insulin resistance in human adipocytes.

Materials
All cell culture ware were purchased from Fisher Scientifi c (Norcross, GA). Western lightning Chemiluminescence Substrate was purchased from Perkin Elmer Life Science (Boston, MA). Immunoblotting buffers and precast gels were purchased from Invitrogen (Carlsbad, CA). The polyclonal antibody for anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc20357) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphospho (P) (Thr183/185) SAPK/JNK and anti-P (Thr-202/204) and total ERK1/2 antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Hyclone fetal bovine serum (FBS) was transcript according to Applied Biosystem's "Guide to Performing Relative Quantifi cation of Gene Expression Using Real-Time Quantitative PCR." Measurement of PGF 2 ␣ levels PGF 2 ␣ levels were measured according to a standard protocol provided by Cayman Chemicals for their PGF 2 ␣ enzyme immunoassay (EIA) kit. Medium was collected from cultures and assayed in duplicate at multiple dilutions.

Statistical analyses
One-way ANOVA was used to compare data unless otherwise indicated. Student's-t -test was used to compute individual pairwise comparisons of least square means ( P < 0.05). Data are expressed as mean ± SEM unless otherwise stated. All analyses were performed using JMP IN version 4.04 software (SAS Institute, Cary, NC).

10,12 CLA increases [Ca 2+ ] i
To examine the effect of CLA isomers on [Ca 2+ ] i levels, we utilized the calcium-sensitive fl uorescent dye Fluo-3 AM. Thapsigargin, a Ca 2+ -ATPase inhibitor that depletes ER calcium stores ( 24 ), was used as a positive control. Thapsigargin increased intracellular calcium levels in a dose-dependent manner ( Fig. 1A ). Consistent with our hypothesis, 50-150 M 10,12 CLA increased [Ca 2+ ] i in a dose-dependent manner ( Fig. 1B ). In contrast, 9,11 CLA modestly increased [Ca 2+ ] i levels compared to the vehicle control. Next, TMB-8 was used to determine the degree to which 10,12 CLA mobilized calcium specifi cally from the ER. As shown in Fig. 1C , TMB-8 attenuated the immediate increase in [Ca 2+ ] i mediated by thapsigargin and 10,12 CLA, suggesting that 10,12 CLA also stimulates calcium release from the ER. Whereas preincubation of Fluo-3loaded adipocytes with the [Ca 2+ ] i chelator BAPTA completely blocked thapsigargin-mediated increase in [Ca 2+ ] i ( Fig. 1D ), BAPTA attenuated the immediate, but not the sustained, effects of 10,12 CLA on [Ca 2+ ] i ( Fig. 1D ). In contrast, EGTA attenuated the sustained, but not the immediate 10,12 CLA-mediated increase of [Ca 2+ ] i, suggesting that extracellular calcium may play a role in the sustained regulation of [Ca 2+ ] i by 10,12 CLA ( Fig. 1E ). Lastly, to determine the extent to which CLA causes calcium release only from the ER, cultures were pretreated with thapsigargin followed by a second treatment with thapsigargin or 10,12 CLA, and vice-versa. As expected, thapsigargin pretreatment completely blocked thapsigargin from increasing [Ca 2+ ] i , demonstrating that thapsigargin depletes ER calcium in human adipocytes ( Fig. 1F ). Furthermore, thapsigargin pretreatment attenuated 10,12 treated with thapsigargin (positive control), CLA isomers, TMB-8, BAPTA, or EGTA and fl uorescence was monitored at 10-20-s intervals for 8 min. Excitation wavelength was 485 nm, and fl uorescence was collected at 528 nm. Changes in the ratio of calciumdependent fl uorescence to prestimulus background fl uorescence (F/F 0 ) were plotted over time. For simplicity, single representative experiments are shown.

Chromatin immunoprecipitation (ChIP) assay
ChIP experiments were performed as previously described with minor modifi cations ( 8 ). Briefl y, SGBS cells were grown and differentiated in 10-cm NUNC dishes and treated with BSA, CLA, or TMB-8. After treatment, cells were crosslinked, harvested in lysis buffer (i.e., 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 20 mM Tris [pH 8.0], and 1× Complete proteinase inhibitor cocktail), and sonicated. DNA concentration was determined by measuring absorbance at 260 nm (A 260 ). Samples were diluted to equal concentrations and preincubated for 3 h with 2 µg NF Β p65 antibody (sc-372 Santa Cruz) and 40 µg BSA in a total volume of 400 µl lysis buffer. Protein A beads (20 µl) were washed three times in lysis buffer, diluted in lysis buffer to a total volume of 100 µl, and incubated for 2 h with 10 µg BSA. Following preincubation, prepared beads were added to the chromatin samples and incubated overnight. Beads were washed as previously described, and DNA was purifi ed by phenol-chloroform extraction. Immunoprecipitated DNA and 5% input DNA were analyzed using real-time PCR.

Mitochondrial membrane integrity assays
Mitochondrial membrane potential and cytochrome C release were measured as indicators of potential mitochondrial stress caused by 10,12 CLA treatment. Chlorophenylhydrazone (CCCP) and antimycin A were used as positive controls for the membrane potential assay, and thapsigargin for cytochrome C release. The selective fl uorescent probe JC-1 (Molecular Probes) was used to measure the membrane potential according to the manufacturer's instructions. Briefl y, 5 g/ml JC-1 was added to the cultures during the last 15 min of a 12-h treatment. Cultures were then washed with PBS, and the fl uorescence (i.e., excitation 530 nm, emission 590 nm) was quantifi ed in a Synergy Multidetection Microplate Reader. For determining the abundance of cytochrome C in the cytosol, cultures were harvested after 24 h of treatment and then subjected to subcellular fractionation to separate the cytosol from the mitochondria fraction as previously described ( 7 ). Subsequently, cytosolic cytochrome C levels were determined by immunoblotting.

RNA isolation and PCR
Total RNA was isolated from the cultures using Tri Reagent purchased from Molecular Research Center (Cincinnati, OH), according to manufacturer's protocol. For real-time PCR, 2.0 g total RNA was converted into fi rst strand cDNA using Applied Biosystems High-Capacity cDNA Archive Kit (Foster City, CA). Real-time PCR was performed in an Applied Biosystems 7500 FAST Real-Time PCR System using Taqman Gene Expression Assays. To account for possible variation in cDNA input or the presence of PCR inhibitors, the endogenous reference gene GAPDH was simultaneously quantifi ed for each sample, and these data normalized accordingly. The Relative Standard Curve Method using seven, 2-fold dilutions ranging from 100 to 1.56 ng RNA was used to check primer effi ciency and linearity of each dependent on [Ca 2+ ] i , we investigated the effects of calcium chelators and inhibitors on ROS, ERK1/2, JNK, ATF3, and NF B in CLA-treated cultures. ROS production was increased by 50 M 10,12 CLA within 3 h in an isomerspecifi c manner ( Fig. 2A ; 9,11 CLA data not shown), which was blocked by BAPTA, TMB-8, and KN-62, a CaMKII inhibitor.
To investigate the role of calcium in mediating 10,12 CLA activation of NF B, cultures of human SGBS adipocytes were pretreated with 100 M TMB-8 for 1 h and then treated over time (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) h) with 30 M 10,12 CLA. SGBS cells were used because of the large number of cells needed to the perform ChIP assay. NF B binding to infl ammatory genes was subsequently analyzed using ChIP. Ten hour treatment with 10,12 CLA increased NF B binding to the IL-8 and COX-2 promoters, which was blocked by TMB-8 ( Fig. 2G ). These data suggest that 10,12 CLA induction of NF B binding to infl ammatory genes is mediated, in part, by calcium release from the ER.
Based on KN-62 attenuating CLA induction of ROS and MAPK activation, the impact of 10,12 CLA on CaMKII gest that PLC activity plays a role in 10,12 CLA mobilization of calcium, increase of ROS, and ISR and infl ammatory gene expression.

10,12 CLA activates the infl ammatory PG pathway independently of [Ca 2+ ] i
PGF 2 ␣ has been shown to suppress adipogenesis ( 23 ) and is associated with infl ammation and insulin resistance in humans consuming CLA supplements (10)(11)(12). Consistent with these fi ndings, 10,12 CLA increased COX-2 gene expression ( Fig. 4B ), which was decreased by BAPTA, TMB-8, and KN-62. For these reasons, we investigated the role of [Ca 2+ ] i in 10,12 CLA induction of the infl ammatory PG pathway. Activation of PLA 2 occurred within 12 h of 10,12 CLA treatment ( Fig. 6A ) and was isomer-specifi c ( Fig. 6B ). However, neither BAPTA nor TMB-8 suppressed 10,12 CLA-mediated phosphorylation of PLA 2 ( Fig. 6C ) nor increased levels of PGF 2 ␣ after 24 h of treatment ( Fig.  6D ), while KN-62 had only a modest effect. Collectively, these data show that chronic 10,12 CLA treatment robustly increases infl ammatory PGF 2 ␣ production in human adipocytes in vitro as it does in humans in vivo and that this effect appears to be relatively independent of [Ca 2+ ] i levels or CaMKII activity.

10,12 CLA-mediated insulin resistance is prevented by KN-62
KN-62 blocked 10,12 CLA suppression of PPAR ␥ protein levels after 48 h of treatment ( Fig. 7A ), while TMB-8 had no effect. Similarly, KN-62 blocked 10,12 CLA induction of SOCS-3 gene expression ( Fig. 7B ) and attenuation of insulin-stimulated glucose uptake after 48 h of treatment ( Fig. 7C ), but TMB-8 had no effect (data not shown). These data demonstrate that chronic treatment with 10,12 CLA causes insulin resistance in human adipocytes that appears to depend on CaMKII activation but not directly on calcium release from the ER.

DISCUSSION
The purpose of this study was to determine the role of [Ca 2+ ] i in mediating 10,12 CLA-induced ROS production, infl ammatory mRNA and protein levels, and insulin resistance in human adipocytes. Collectively these data provide support for our working model shown in Fig. 8 , suggesting that 10,12 CLA's impact on calcium signaling and markers of infl ammation and insulin resistance occurs in two response phases. The initial response to CLA begins with an immediate and sustained release of calcium from the ER, and possibly other sources like the extracellular compartment, which leads to ROS production, MAPK and NF B activation, and ISR and infl ammatory gene expression. The second and more chronic response begins with PLA 2 activation and PGF 2 ␣ production, which appears to be independent of calcium. This causes another phase of calcium signaling that further activates CaMKII, leading to the suppression of PPAR ␥ and the development of insulin resistance. Consistent with this second phase of calcium signaling involving CaMKII, PGF 2 ␣ mRNA levels was examined. We found that 10,12 CLA treatment for 12 h induced CaMKII ␤ mRNA by approximately 200% ( Fig. 2H ), which was modestly attenuated by TMB-8. However, 10,12 CLA did not increase the mRNA levels of the more abundant isoforms of CaMKII (i.e., ␣ , ␥ , and ␦ ; data not shown).

10,12 CLA-mediated increase of [Ca 2+ ] i is linked to infl ammatory gene expression but not to mitochondrial stress
The integrated stress response (ISR) has been recently shown to play a role in 10,12 CLA induction of infl ammation ( 25 ). To determine the effect of CLA on ISR and infl ammatory gene expression, cultures were treated with 9,11 CLA or 10,12 CLA for 6, 12, 24, or 48 h. Our results show that 30 M and 100 M 10,12 CLA (data not shown) increased the expression of ISR genes (i.e., ATF3, CHOP, GADD34) within 6-12 h ( Fig. 3A ), and infl ammatory genes (i.e., IL-6, IL-8, IL-1 ␤ , COX-2) by 12 h ( Fig. 3A ) in an isomer-specifi c manner. Because metabolic stress can cause calcium release from mitochondria, we analyzed two indicators of mitochondrial membrane integrity, mitochondrial JC-1 staining and cytochrome C release. However, neither JC-1 staining nor cytosolic cytochrome C abundance were affected by 10,12 CLA ( Fig. 3B ), suggesting that 10,12 CLA does not adversely affect mitochondrial membrane potential or integrity.
Next, we determined the ability of calcium release from the ER to induce infl ammation. Thapsigargin induced IL-8 and IL-6 gene expression, which was attenuated by TMB-8 ( Fig. 4A ). Subsequently, we investigated the role of [Ca 2+ ] i in mediating CLA induction of ISR and infl ammatory genes by pretreating cultures with BAPTA or TMB-8, and then treating with 30 M 10,12 CLA for 12 h. BAPTA completely blocked 10,12 CLA induction of IL-8, IL-6, CHOP, and GADD34 ( Fig. 4B ). In addition, BAPTA reduced COX-2 gene expression by 70%, while decreasing ATF3 gene expression by 44% ( Fig. 4B ). Treatment with TMB-8 suppressed the 10,12 CLA-induced expression of IL-8, IL-6, COX-2, CHOP, and GADD34 ( Fig. 4B ), while only modestly reducing ATF3 mRNA levels. To determine the role of CaMKII in mediating 10,12 CLA induction of ISR and infl ammatory genes, cultures were pretreated with KN-62. KN-62 attenuated or blocked induction of IL-8, IL-6, COX-2, CHOP, GADD34, and ATF3 gene expression ( Fig. 4B ). These data suggest that 10,12 CLA induction of ISR and infl ammatory genes is linked to CaMKII activation and calcium release from the ER. However, direct analysis of CaMKII activity is needed to verify this suggestion.

Phospholipase C activity plays a role in 10,12 CLA-mediated infl ammation
We investigated the role of phospholipase C (PLC), a membrane-bound, signal transduction protein involved in the release of calcium from intracellular stores ( 26 ), using the PLC inhibitor D609. D609 attenuated 10,12 CLA increase of [Ca 2+ ] i ( Fig. 5A ), ROS ( Fig. 5B ), and ISR and infl ammatory gene expression ( Fig. 5C ). These data sug- For the JC-1 assay, fl uorescence was measured at 590 and 530 nm. Means (± SE; n = 6-12) that do not share a common lowercase letter differ ( P < 0.05). Data are representative of two independent experiments. One-way ANOVA was used to compare data. For the cytochrome C release assay, cells were harvested and subcellular fractionation was performed to collect mitochondrial and cytosolic proteins. The cytosolic fraction was immunoblotted for cytochrome C and GAPDH. Data are representative of one independent experiment.
[Ca 2+ ] i levels from sources other than the ER. Although KN-62 was initially identifi ed as a calcium-CaMKII inhibitor, this inhibitor has also been reported to block voltagesensitive channels (28)(29)(30)(31). Thus, it seemed possible that 10,12 CLA increases Ca 2+ infl ux across the plasma membrane. Although, KN-62 (data not shown) and the extracellular calcium chelator EGTA did not dramatically affect 10,12 CLA's immediate increase of [Ca 2+ ] i ( Fig. 1E ), EGTA attenuated the sustained effects of 10,12 CLA on [Ca 2+ ] i , suggesting that extracellular calcium infl ux may be an important for sustaining [Ca 2+ ] i , at least for the short term. In agreement with other studies, the phosphatidylcholine-specifi c (PC)-PLC inhibitor D609 ( Fig. 5A ) attenuated 10,12 CLA's immediate increase in [Ca 2+ ] i levels. Activation of PC-PLC has been reported to increase [Ca 2+ ] i levels via conversion of diacylglycerol (DAG) by DAG kinase to phosphatidic acid, which mobilizes calcium from inositiol-3-phosphate-independent calcium pools ( 26,(31)(32)(33). Therefore, it appears that 10,12 CLA initially stimulates an effl ux of calcium from intracellular stores like the ER, followed by calcium has been reported to increase [Ca 2+ ] i levels and activate CaMKII in murine adipocytes, leading to decreased adipocyte differentiation ( 23 ).
In support of our fi ndings, calcium and ROS have been implicated in the activation of NF B and infl ammation ( 27 ). Likewise, we found that blocking calcium signaling with BAPTA, TMB, or KN-62 prevented CLAmediated ROS production ( Fig. 2A ), suggesting that increased [Ca 2+ ] i levels precede ROS production in human adipocytes. Furthermore, these compounds attenuated 10,12 CLA-mediated activation of MAPKs ( Fig. 2B-F ) and NF B ( Fig. 2G ), and the expression of ISR and infl ammatory genes ( Fig. 4 ). Consistent with these data, the PLC inhibitor D609 attenuated 10,12 CLA increase in [Ca 2+ ] i levels, ROS, and infl ammatory gene expression ( Fig. 5 ). Collectively, these data demonstrate the important role of [Ca 2+ ] i in mediating ROS and infl ammatory responses to 10,12 CLA in cultures of human adipocytes.
The prevention of CLA-mediated insulin resistance by KN-62, but not TMB-8, suggests that 10,12 CLA increased mitochondrial respiration increases [Ca 2+ ] i levels and activates CaMKIV ( 36 ). To this end, we investigated whether 10,12 CLA could affect mitochondria function by examining two indicators of altered mitochondrial membrane potential or integrity (i.e., the JC-1 assay and cytochrome C release from mitochondria, respectively). Neither marker of mitochondrial dysfunction was affected by 10,12 CLA ( Fig. 3B ), suggesting that acute 10,12 CLA treatment does not directly cause mitochondrial dysfunction.
infl ux from extracellular sources, possibly to replenish ER calcium.
The mitochondria can also store and release calcium, potentially providing another source of [Ca 2+ ] i for signaling ( 34 ). Mitochondrial dysfunction or stress, similar to ER stress, can adversely affect the mitochondria's capacity for calcium storage or regulated release, thereby increasing [Ca 2+ ] i levels. For example, treating cells with a mitochondrial uncoupler increases [Ca 2+ ] i levels, thereby activating ERK1/2 ( 35 ). Similarly, impairing . Emitted fl uorescence intensities were measured using a multidetection microplate reader. Excitation wavelength was 485 nm, and fl uorescence was collected at 528 nm. Means (± SEM; n = 3-12) are representative of three independent experiments. C: Cultures were pretreated for 30 min with 50 M D609 (D) followed by 12-h treatment with BSA vehicle (V) or 50 M 10,12 CLA (10). RNA was subsequently isolated and mRNA levels of IL-8, COX-2, ATF-3, GADD34, and GAPDH were measured by real-time PCR. Data are normalized to the vehicle controls. Means (± SEM; n = 2) that do not share a common lower case letter differ ( P < 0.05). Data are representative of three independent experiments. One-way ANOVA was used to compare data. resistance in insulin-sensitive cells (43)(44)(45), including rat adipocytes ( 46 ). In vivo, BAPTA was shown to reverse insulin resistance in rats fed a high-fat diet ( 47 ). Unfortunately, we could not use BAPTA for our glucose uptake studies due to its toxicity when used chronically (48 h). Furthermore, we were unable to consistently demonstrate that CLA increased phosphorylation or total CaMKII protein levels (data not shown). Therefore, although 10,12 CLA increased CaMKII ␤ mRNA by 200%, we could not determine whether it increases CaMKII ␤ protein abundance or activity. Future studies directly measuring CaMKII ␤ activity and employing gene silencing techniques are needed to defi ne the specifi c role of CaMKII ␤ signaling on infl ammation and insulin sensitivity in CLA-treated cultures of human adipocytes. G protein-coupled receptor (GPCR) agonists, depolarizing stimuli, or ionophores have been reported to increase free [Ca 2+ ] i levels, thereby activating CaMKII and its downstream targets such as ERK1/2 (37)(38)(39)(40)(41). Similarly, we previously demonstrated that the GPCR antagonist pertussis toxin blocked 10,12 CLA-mediated activation of ERK1/2 ( 4 ). In the present study, we demonstrated the KN-62 also attenuates 10,12 CLA phosphorylation of ERK1/2 and JNK ( Fig. 2C, F ). KN-93, another CaMK inhibitor, has also been shown to attenuate lipopolysaccharide activation of ERK1/2, JNK, NF B, and AP-1, a target of JNK ( 42 ). Collectively, these data support our hypothesis that CLA's activation of ERK1/2 and JNK are closely linked to [Ca 2+ ] i and CaMK activity. We provide data demonstrating that the CaMKII inhibitor KN-62 prevents 10,12 CLA-mediated insulin resistance ( Fig. 7 ). Elevated [Ca 2+ ] i levels have been linked to insulin . Conditioned media were subsequently collected and PGF 2 ␣ levels were measured using a commercially available EIA kit. Means (± SEM; n = 2) not sharing a common lower case letter differ ( P < 0.05). Data in all panels are representative of three independent experiments. One-way ANOVA was used to compare data. 3 H]2deoxy-glucose was subsequently measured in cultures treated without ( Ϫ ) or with (+) insulin. Means (± SEM; n = 3) that do not share a common lower case letter differ ( P < 0.05). Data in all panels are representative of three independent experiments. One-way ANOVA was used to compare data. Delipidation of murine adipocytes by 10,12 CLA in vivo and in vitro has recently been linked to the ISR pathway ( 25 ). These authors demonstrated that 10,12 CLA, but not 9,11 CLA, induced markers of the ISR pathway in mouse and 3T3-L1 adipocytes. They also suggest that the ISR precedes the later induction of infl ammatory gene expression and contributes to delipidation by 10,12 CLA. Our data in cultures of human adipocytes are similar to these published data in murine adipocytes. In addition, 10,12 CLA has been shown to cause atypical ER stress in a murine mammary carcinogenesis model ( 48 ). Similarly, the rapid (1 min) and chronic increase in [Ca 2+ ] i levels by 10,12 CLA appears to increase ROS production (3 h) and activate specifi c MAPKs (6 h) and ISR stress response genes (6-12 h) relatively early, which in turn, may lead to chronic (48 h) activation of CaMKII and subsequent insulin resistance in cultures of human adipocytes. However, the specifi c mechanism by which 10,12 CLA increases [Ca 2+ ] i , and its direct effect on CaMKII activity require further investigation. ] i levels, which are dependent on PLC activity and mobilization of calcium from the ER initially, which is sustained by an infl ux of calcium from extracellular sources. This increase activates ROS and CaMKII, which in turn activate MAPK and NF B that trigger ISR and infl ammatory gene expression. The second and more chronic response to 10,12 CLA begins with PLA 2 activation and PGF 2 ␣ production, which appears to be independent of calcium. This causes another phase of calcium signaling that further activates CaMKII, leading to the suppression of PPAR ␥ and the development of insulin resistance.