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. 2013 Mar 6;143(5):584–590. doi: 10.3945/jn.112.173161

The Phospholipase C Inhibitor U73122 Attenuates trans-10, cis-12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes1,2

Wan Shen 1, Kristina Martinez 1, Chia Chi Chuang 1, Michael McIntosh 1,*
PMCID: PMC3738231  PMID: 23468551

Abstract

We have demonstrated that trans-10, cis-12 conjugated linoleic acid (18:2t10,c12)-mediated delipidation of human adipocytes was dependent on increased intracellular calcium and activation of inflammatory signaling in human primary adipocytes. These data are consistent with the actions of diacylglycerol and inositol triphosphate derived from phospholipase C (PLC)-dependent cell signaling. To test the hypothesis that PLC was an upstream activator of these cellular responses to 18:2t10,c12, primary cultures of human adipocytes were pretreated with 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), a universal PLC inhibitor, followed by 18:2t10,c12 treatment. U73122 attenuated 18:2t10,c12-mediated insulin resistance within 48 h and suppression of the mRNA levels of peroxisome proliferator-activated receptor (PPAR)γ, insulin-stimulated glucose transporter-4, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase-1, and the protein levels of PPARγ within 18–24 h. U73122 inhibited 18:2t10,c12-mediated induction of the inflammatory-related genes calcium/calmodulin-dependent protein kinase-β, cyclooxygenase-2, monocyte chemoattractant protein-1, interleukin (IL)-6, and IL-8, secretion of IL-6 and IL-8, and the activation of extracellular signal-related kinase, c-Jun N-terminal kinase, and c-Jun within 18–24 h. Moreover, 18:2t10,c12 increased the mRNA levels of heat shock proteins within 6–24 h and intracellular calcium concentrations within 3 min, which were inhibited by U73122. Lastly, 18:2t10,c12 increased the abundance of PLCγ1 in the plasma membrane within 3 min. Taken together, these data suggest that PLC plays an important role in 18:2t10,c12-mediated activation of intracellular calcium accumulation, inflammatory signaling, delipidation, and insulin resistance in human primary adipocytes.

Introduction

Obesity is currently one of the most prevalent nutrition-related diseases in the US. The CDC estimated that 35.7% of U.S. adults were obese in 2010 (1). Consuming conjugated linoleic acid (CLA)3-containing supplements and fortified foods, which were given Generally Recognized as Safe status by the FDA in 2008, has become a popular method for weight management. Within the last decade, at least 15 clinical studies (2, 3) and 30 animal (for review, see 4) studies showed that consuming an equal mixture of trans-10, cis-12 (18:2t10,c12) and cis-9, trans-11 (18:2c9,t11) CLA or 18:2t10,c12 alone reduced body fat or increased fat-free mass. 18:2c9,t11 and 18:2t10,c12 are the 2 major CLA isomers with known biological functions (4, 5). 18:2c9,t11 is the predominant CLA isomer in dairy products, whereas an equal mixture of 18:2c9,t11 and 18:2t10,c12 is found in most CLA-containing weight loss supplements and fortified foods. However, it is only the 18:2t10,c12 isomer that causes adipocyte delipidation (6, 7) and loss of body fat (8, 9).

The proposed antiobesity mechanisms of 18:2t10,c12 include increasing energy expenditure, inhibiting adipogenesis and lipogenesis, stimulating lipolysis, and promoting adipocyte apoptosis (4). We previously demonstrated that 18:2t10,c12-mediated suppression of adipogenesis and lipogenesis and adipocyte delipidation were dependent on inflammatory signaling (7, 10, 11) and linked to endoplasmic reticulum (ER) release of calcium and increased intracellular calcium accumulation (12). Upregulation of inflammatory pathways by 18:2t10,c12 increased the activity of nuclear factor kappa B (NFκB), activator protein (AP)-1, and mitogen-activated protein kinases (MAPK) (7, 10, 11), which lead to inhibition of PPARγ abundance and activity (13–15), reduction of glucose and fatty acid uptake, and de novo lipogenesis (6, 7, 14). However, the upstream signals activated by 18:2t10,c12 that initiate this inflammatory signaling cascade are unclear (11).

Heat shock proteins (HSPs) exist ubiquitously in cellular organelles, including the cytoplasm, mitochondria, golgi, and nucleus (16). They respond to various stressors, including heat, pH changes, inflammation, oxidative injury, and viral agents. They have strong cytoprotective effects and function as molecular chaperones in protein folding, transport, and degradation (17). Relevant to this study, several HSPs (e.g., the HSPA family of HSP70s) are linked to intracellular calcium accumulation (18) and inflammatory signaling pathways, including NFκB, extracellular signal-related kinases (ERKs), and phospholipase C (PLC) (19–21). However, the role of HSPs in 18:2t10,c12-mediated inflammatory signaling is unknown.

We recently discovered that blocking diacyglycerol kinase (DGK) activity using the chemical compound R59022 or RNA interference targeting DGK attenuated 18:2t10,c12-induced intracellular calcium accumulation, inflammatory signaling, and suppression of lipogenesis (22), suggesting a role for DGK in 18:2t10,c12-regulated cell signaling. Based on the well-known role of PLC as an upstream enzyme that produces diacylglycerol (DAG), a substrate for DGK, and inositol-3-phosphate (IP3), an inducer of calcium release from the ER, we hypothesized that PLC is an upstream activator of these cellular responses to 18:2t10,c12. To test this hypothesis, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), a universal PLC inhibitor, was used in primary cultures of human adipocytes treated with 18:2t10,c12. Data from this study suggest that PLC plays an important role in 18:2t10,c12-mediated activation of inflammatory signaling, insulin resistance, and delipidation.

Materials and Methods

Materials.

All cell culture ware and Hyclone FBS were purchased from Fisher Scientific. Taqman assays were purchased from Applied Biosytems Inc, Foster City, CA. Lightning Chemiluminescence Substrate was purchased from Perkin Elmer Life Science. Immunoblotting buffers and precast gels were purchased from Invitrogen Life Technologies. Polyclonal antibodies for GAPDH, α-caveolin-1, and PLCδ4 and a monoclonal antibody for anti-PPARγ were obtained from Santa Cruz Biotechnology. Anti-total and anti-phospho (P) ERK, c-Jun-NH2-terminal kinase (JNK), cJun, and PLCγ1 antibodies were purchased from Cell Signaling Technologies. 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya. Fluo-3 acetyloxymethyl ester, pluronic F-127, and probenecid were purchased from Invitrogen by Life Technologies. Thapsigargin and ionomycin were purchased from Calbiochem-EMD Biosciences. The PLC inhibitor, U73122, was purchased from Sigma Aldrich. Adipocyte media were purchased from Zen Bio. All other reagents and chemicals were purchased from Sigma Aldrich unless otherwise stated.

Culturing of human primary adipocytes.

Abdominal white adipose tissue was obtained from Caucasian and African American females aged between 30 and 50 y with a BMI ≤32.0 during elective abdominoplasty with consent approved from the Institutional Review Board at University of North Carolina at Greensboro and the Moses Cone Memorial Hospital. Tissue was digested, stromal vascular cells were isolated, and cells were differentiated for 7–14 d as described (6, 7). Cells were pretreated with or without 5, 10, or 15 μmol/L of the PLC inhibitor U73122, followed by administration of BSA vehicle control, 18:2c9,t11, or 18:2t10,c12 for 10 min to 48 h, depending on the outcome measured. U73122 inhibits PLC by decreasing the activity of PLC’s substrate phosphatidylinositol 4,5-bisphosphate (PIP2) (23). Each treatment was normalized to the same amount of BSA or ethanol vehicle control. Each independent experiment was repeated at least twice using a mixture of cells from 3 participants unless otherwise indicated.

Fatty acid preparation.

18:2c9,t11 and 18:2t10,c12 were complexed to 7.5% fatty acid-free BSA (Sigma A7030, lot no. 040M1649) at a 4:1 molar ratio to make a 4-mmol/L stock concentration as previously described (7). 18:2c9,t11 and 18:2t10,c12 were given at physiological levels (50 μmol/L) unless otherwise indicated (24, 25).

RNA isolation and real-time qPCR.

Total RNA was extracted, single-strand RNA was reverse- transcribed into complementary DNA, and real-time qPCR was performed as previously described (12).

3H-2-deoxy-d-glucose uptake.

On d 10 of differentiation, serum-starved cultures were pretreated with or without U73122 for 30 min, followed by BSA vehicle control or 18:2t10,c12 for 48 h. Subsequently, 3H-2-deoxy-d-glucose uptake was measured as previously described (26).

Immunoblotting.

Immunoblotting was conducted as previously described (7) using primary antibodies for P-ERK, total ERK, P-JNK, total JNK, P-cJun, total cJun, PLCδ4, PLCγ1, and caveolin-1 at 1:1000 dilutions followed by 1 h of exposure at room temperature to HRP-conjugated secondary antibodies at 1:5000 dilutions unless otherwise indicated. Primary and secondary antibodies targeting PPARγ were used at dilutions of 1:200 and 1:2000, respectively. Blots were exposed to chemiluminescence reagent for 1 min and X-ray films were developed using a SRX-101A Konica Minolta film developer.

Adipokine secretion in the media.

Media were collected from the same set of cells that were used for immunoblotting. The supernatant from these cells was centrifuging at 13,200 × g for 10 min at 4°C to remove cell debris and kept at −80°C until analysis. The assay kits were obtained from Bio-Rad and the concentrations of adipocytokines were measured in the media following the manufacturer’s protocol using a BioPlex Suspension Array System (Bio-Rad).

Intracellular calcium concentrations.

Intracellular calcium concentrations were measured using a calcium-sensitive fluorescent dye fluo-3 as previously described (12).

Plasma membrane fractionation.

PLC isoforms acutely translocate to the cell membrane following activation and thus measuring their appearance in the membrane provides an indicator of activity. To determine if 18:2t10,c12 increased the activity of specific PLC isoforms, cells were treated with controls or 18:2t10,c12 and then the plasma membrane fractions were isolated and processed for immunoblotting using isoform-specific PLC antibodies. Briefly, cultures were changed to serum-free media 1 d prior to the experiment. On the day of the assay, cells were treated with BSA vehicle control, 18:2t10,c12, or thapsigargin, a positive control that induces intracellular calcium release, for 1, 3, or 6 min and immediately put on ice for processing. After washing with ice-cold HBSS twice, cells were gently scraped in 300 μL of Tris-buffered saline (pH 7.4) with 1% protease inhibitor containing 20 mmol/L Tris-HCl, 225 mmol/L sucrose, and 1 mmol/L EDTA and homogenized using a prechilled potter-elvahjem homogenizer system. Lysate was centrifuged at 16,000 × g for 20 min, supernatant was removed, and the pellet was resuspended in 500 μL Tris-buffered saline, rehomogenized, and subsequently layered onto a 1.2-mol/L sucrose cushion in a ultracentrifuge tube and centrifuged at 100,000 × g for 20 min as previously described (10). The suspended plasma membrane interphase was collected using a syringe and pelleted after centrifuging at 100,000 × g for 30 min. The plasma membrane pellets were then resuspended in RIPA buffer containing protease inhibitor and kept on ice prior to determining the protein concentration for immunoblotting to detect PLC specific proteins and caviolin-1, a loading control.

Statistical analyses.

Data for the dose-response studies were analyzed using 1-way ANOVA and Student’s t test to compute individual pairwise comparisons of means (P < 0.05). Data for the time course x fatty acid treatment studies were analyzed by 2-way ANOVA testing the main effects of time (3, 6, 12, and 24 h) and fatty acid type (BSA, 18:2c9,t11, 18:2t10,c12) and their full-factorial interaction (time × fatty acid type). Tukey’s multi-comparison test was conducted to detect treatment differences among the interactions (P < 0.05). All analyses were conducted on the JMP version 10.0 program (SAS). Data are expressed as means ± SEMs.

Results

The PLC inhibitor U73122 attenuates 18:2t10,c12-mediated insulin resistance and suppression of lipogenic protein or gene expression.

We hypothesized that PLC was involved in 18:2t10,c12-mediated activation of inflammatory signaling and suppression of PPARγ activity and lipogenesis (7, 14) based on our published data showing that the PLC-phosphatidylcholine (PC)-specific inhibitor tricyclodecan-9-yl potassium xanthate (D609) attenuated markers of inflammation in 18:2t10,c12-treated adipocytes (12). Indeed, 50 μmol/L 18:2t10,c12 impaired insulin-stimulated glucose uptake within 48 h (Fig. 1A), PPARγ protein abundance within 24 h (Fig. 1B), and the mRNA levels of PPARγ and several of its target genes [e.g., insulin-dependent glucose transporter (GLUT)-4, acetyl CoA carboxylase (ACC)-1, and stearoyl-coenzyme A desaturase (SCD)-1] within 18 h of treatment (Fig. 1C). Consistent with our hypothesis, pretreatment with low amounts (i.e., 5–15 μmol/L) of the PLC inhibitor U73122 blocked or attenuated these antilipogenic effects of 18:2t10,c12.

FIGURE 1.

FIGURE 1

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The PLC inhibitor U73122 attenuates 18:2t10,c12-mediated insulin resistance and suppression of lipogenic protein or gene expression in human adipocytes. (A) Human primary adipocytes were incubated in serum-free, low-glucose DMEM media for 24 h and pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control (B) for 48 h. Insulin-stimulated glucose uptake was measured by scintillation radioactivity counting on the day of assay (n = 4/treatment). (B) Cultures were pretreated as in A, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control treatment for 24 h. The protein abundance of PPARγ was measured by immunoblotting (n = 3/treatment). (C) Cultures were pretreated as in A, followed by treatment of 50 μmol/L 18:2t10,c12 or BSA vehicle control for 18 h. The expression of PPARγ, GLUT4, ACC-1, and SCD-1 was measured by qPCR (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A–C are representative of at least 3 independent experiments. ACC, acetyl-CoA carboxylase; GLUT4, insulin-dependent glucose transporter 4; PLC, phospholipase C; SCD, stearoyl-CoA desaturase; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

U73122 inhibits 18:2t10,c12-mediated inflammatory signaling.

To determine the extent to which 18:2t10,c12-mediated inflammatory signaling was dependent on PLC activation, primary human adipocytes were pretreated with U73122 for 30 min and then treated with 50 μmol/L 18:2t10,c12 for 18–24 h, and inflammatory gene and protein expression were measured. 18:2t10,c12-mediated induction of inflammatory genes [Fig. 2A; Ca2+/calmodulin-dependent protein kinase (CaMK2)-β, cyclooxygenase (COX)-2, monocyte chemoattractant protein (MCP)-1, IL-6, and IL-8], secretion of inflammatory adipokines (Fig. 2B; IL-6 and IL-8), and phosphorylation of ERK, JNK, and cJun (Fig. 2C) were attenuated by pretreatment with micromolar amounts of U73122. Thus, U73122 inhibited 18:2t10,c12-mediated inflammatory signaling.

FIGURE 2.

FIGURE 2

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U73122 inhibits 18:2t10,c12-mediated inflammatory signaling in human adipocytes. (A) Human primary adipocytes were pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control (B) for 18 h. Gene expression of CaMK2-β, COX-2, MCP-1, IL-6, and IL-8 was measured by qPCR (n = 3–4/treatment). (B) Cultures were pretreated as in A, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control treatment for 24 h. Media was collect to measure secreted proinflammatory markers (n = 3–4/treatment). (C) Cultures were used from B. Activation of inflammatory mediators, including ERK, JNK, c-Jun, and GAPDH, were measured by immunoblotting (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A–C are representative of at least 3 independent experiments. CaMK, Ca2+/calmodulin-dependent protein kinase; COX, cyclooxygenase; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; MCP, monocyte chemoattractant protein; P, phosphorylated; T, total; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

U73122 attenuates 18:2t10,c12-mediated induction of HSPs.

18:2t10,c12 causes cell stress, including the integrated cell stress response (27) or ER stress (12, 28). Therefore, we wanted to determine the extent to which 18:2t10,c12 induced HSPs and if PLC was involved in this induction. Indeed, 50 μmol/L 18:2t10,c12 increased the mRNA levels of several HSPs, including HSPA1A, HSPA6, and HSPH1 as early as 6 h, with the greatest induction at 12 h of treatment (Fig. 3A). Pretreatment with U73122 attenuated the induction of HSPs by 18:2t10,c12 (Fig. 3B), suggesting that PLC is involved in 18:2t10,c12-mediated induction of several isoforms of HSPs associated with inflammatory signaling.

FIGURE 3.

FIGURE 3

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U73122 attenuates 18:2t10,c12-mediated induction of HSPs in human adipocytes. (A) Human primary adipocytes were treated with 50 μmol/L 18:2c9,t11, 18:2t10,c12, or BSA vehicle control (B) for 3, 6, 12, or 24 h. Gene expression of HSPA1A, HSPA6, and HSPH1 was measured by qPCR (n = 3–4/treatment). (B) Cultures were pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by treatment with 50 μmol/L 18:2t10,c12 or BSA vehicle control (B) for 12 h. Gene expression of HSPA1A, HSPA6, and HSPH1 were measure by qPCR (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A,B are representative of 2–3 independent experiments. 18:2c9,t11, cis-9, trans-11; HSP, heat shock protein; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

U73122 attenuates 18:2t10,c12-mediated intracellular calcium accumulation.

The rapid increase in intracellular calcium accumulation caused by 18:2t10,c12 may serve as an initial inflammatory signal (12), because blocking calcium release from the ER prevented 18:2t10,c12-mediated inflammatory signaling and insulin resistance (5, 12). To determine the extent to which 18:2t10,c12-mediated increase in intracellular calcium was dependent on PLC signaling, adipocytes were pretreated with U73122 for 30 min and then treated with 18:2t10,c12 for up to 10 min and intracellular calcium concentrations were measured. As expected, 18:2t10,c12 increased the accumulation of intracellular calcium within several minutes, reaching a peak after 3 min (Fig. 4). Notably, this increase of intracellular calcium was attenuated by pretreating the cells with low amounts of U73122 (Fig. 4), suggesting that PLC is involved in 18:2t10,c12-mediated intracellular calcium accumulation.

FIGURE 4.

FIGURE 4

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U73122 attenuates 18:2t10,c12-mediated intracellular calcium accumulation in human adipocytes. Human primary adipocytes were incubated in 5 μmol/L Fluo-3 for 30 min, then treated with 5 or 10 μmol/L U73122 for 10 min and followed by injection of 150 μmol/L 18:2t10,c12, 5 μmol/L thapsigargin (Tg), a positive control for stimulating calcium release from the ER, or vehicle (NT). A kinetic intracellular calcium curve was generated by measuring the change in intensity of fluorescence over time. Data (n = 4–6/treatment) are representative of 3 independent experiments. ER, endoplasmic reticulum; F/Fo, changes in the ratio of calcium-dependent fluorescence to pre-stimulus background fluorescence. 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

18:2t10,c12 increases mRNA levels of PLCδ4 and the translocation of PLCγ1 protein.

Fifty micromolars of 18:2t10,c12, but not 18:2c9,t11, induced the mRNA levels of PLCδ4 after 12 h of treatment (Fig. 5A) and this induction was inhibited in a dose-dependent manner by U73122 (Fig. 5B). However, 18:2t10,c12 did not acutely increase the translocation of PLCδ4 to the plasma membrane (Fig. 5C). In contrast, 18:2t10,c12 increased the translocation of PLCγ1 protein to the plasma membrane within 3 min (Fig. 5C) but did not induce the mRNA levels of PLCγ1 (Fig. 5A,B). Collectively, these data suggest that 18:2t10,c12 increases the activity of 1 (i.e., PLCγ) of the 3 (i.e., PLCγ, PLCδ, PLCβ) major PLC isoforms found in human adipose tissue. However, gene silencing studies targeting these candidate isoforms of PLC are needed to confirm these findings, given the lack of specificity of many chemical inhibitors.

FIGURE 5.

FIGURE 5

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18:2t10,c12 increases mRNA levels of PLCδ4 and the translocation of PLCγ1 protein in human adipocytes. (A) Human primary adipocytes were treated with 50 μmol/L 18:2c9,t11, 18:2t10,c12, or BSA vehicle control (B) for 3, 6, 12, or 24 h. Gene expression of PLCδ4 and PLCγ1 was measured by qPCR (n = 3/treatment). (B) Another set of cells was pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control (B). Gene expression of PLCδ4 and PLCγ1 were measured by qPCR (n = 3/treatment) (C). For measuring protein abundance, cultures were treated with vehicle control (B), 50 μmol/L 18:2t10,c12 (C), or 5 μmol/L thapsigargin (T) for 1, 3, or 6 min. Plasma membranes were then isolated and candidate PLC isomers were measured by immunoblotting (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A,B are representative of at least 2 independent experiments. Cav-1, caveolin-1; 18:2c9,t11, cis-9, trans-11; PLC, phospholipase C; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

Discussion

Based on data presented in this article along with our previously published work on 18:2t10,c12, we proposed the following working model (Fig. 6). We postulate that 18:2t10,c12 activates within seconds the specific cell surface receptors, including G protein coupled receptor (GPCR), G protein receptor (GPR), or protein tyrosine kinases. Activation of these receptors stimulates the translocation of specific isoforms of PLC (i.e., PLCγ1) to the plasma membrane within 3 min, thereby generating DAG and IP3 from PIP2. DAG is rapidly converted to phosphatidic acid and, along with IP3, triggers calcium release from ER within 3 min. The 18:2t10,c12-mediated increase in intracellular calcium in turn: 1) increases the transcription of HSPs within 6–12 h; 2) upregulates the transcription of calcium-specific isoforms of PLC (e.g., PLCδ4) within 12 h; and 3) activates inflammatory signaling within 24 h. Inflammatory signaling subsequently antagonizes PPARγ abundance and activity within 24 h, thereby suppressing insulin-stimulated glucose uptake and lipogenesis within 48 h. These 18:2t10,c12-mediated events cause adipocyte delipidation. Taken together, these data suggest an important role of PLC in mediating inflammatory signaling and delipidation of adipocytes by 18:2t10,c12. However, isoform-specific, PLC knockdown studies are needed to validate this hypothesis.

FIGURE 6.

FIGURE 6

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Working model in human adipocytes. 18:2t10,c12 activates within seconds the specific cell surface receptors, including GPCRs, GPRs, or protein tyrosine kinases. Activation of these receptors stimulates the translocation of specific isoforms of PLC (i.e., PLCγ1) to the plasma membrane within 3 min, thereby generating DAG and IP3 from PIP2. DAG is rapidly converted to PA, and along with IP3, triggers calcium release from ER within 3 min. The 18:2t10,c12-mediated increase in intracellular calcium, in turn: 1) increases the transcription of HSPs within 6–12 h; 2) upregulates the transcription of calcium-specific isoforms of PLC (e.g., PLCδ4) within 12 h; and 3) activates inflammatory signaling within 24 h. Inflammatory signaling subsequently antagonizes PPARγ abundance and activity within 24 h, thereby suppressing insulin-stimulated glucose uptake and lipogenesis within 48 h. Together, these 18:2t10,c12-mediated events cause adipocyte delipidation. DAG, diacylglycerol; ER, endoplasmic reticulum; GPCR, G protein coupled receptor; GPR, G protein receptor; IP3, inositol-3-phosphate; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

HSPs and 18:2t10,c1.

HSPs are molecular chaperones or stress-related proteins that help prevent or reverse protein misfolding, aggregation, and misassembly in response to cellular stressors (for review, see 29, 30). The main HSPs activated by stress include HSPA (i.e., HSP70s), HSPB (i.e., small HSPs), HSPC (i.e., HSP90s), and HSPH (i.e., HSP105/110) (for review, see 31). Relevant to the current study, the protein levels of HSP70 were decreased by U73122 in A431 epidermoid carcinoma cells, demonstrating their dependence on PLC activation (21). Furthermore, HSP70 has been shown to be activated by increased cytosolic Ca2+ concentrations (18). However, HSP70 has also been reported to have antiinflammatory actions, as demonstrated by inhibiting MAPK and NFκB pathways (19, 20; for review, see 32). HSPHs and HSPAs have been reported to suppress heat-induced stress as well (for review, see 33, 34). Consistent with these findings, our data suggest that 18:2t10,c12 induces cell stress as evidenced by increased concentrations of calcium within 3 min, several HSP family members within 6 h, inflammatory genes within 18 h, and insulin resistance within 48 h. 18:2t10,c12-mediated induction of HSPA1A, HSPA6, and HSPH1 were inhibited by U73122, suggesting that their induction was dependent on PLC.

PLC and 18:2t10,c12.

PLC can be classified as PC or phosphoinositide (PI) specific, depending on its phospholipid substrate. PLC-PC is involved in cell proliferation, differentiation, apoptosis, inflammation, generation of reactive oxygen species, and hypoxia (for review, see, 35). Its specific inhibitor, D609, has been reported to decrease PC-PLC activity, possibly by chelation of Zn2+ on the active sites of the enzyme (35; for review, see 36). Consistent with these data, we previously reported that D609 blocked a 18:2t10,c12-mediated increase in the concentrations of intracellular calcium and reactive oxygen species and the expression of IL-8, COX-2, activating transcription factor 3, and GADD34 (12). However, D609 did not prevent 18:2t10,c12-mediated suppression of adipogenic and lipogenic genes, e.g., PPARγ, GLUT4, and adipocyte fatty acid binding protein 4 (data not shown). Collectively, these data suggest that some, but not all of 18:2t10,c12’s effects in adipocytes are dependent on PC-specific PLC.

Currently, 6 families and 13 isoforms of PI-specific PLC have been identified (for review, see 37). Most PLC isozymes are activated by GPCRs, protein tyrosine kinase, or both (38, 39; for review, see 40). Consistent with these data, we recently reported that 18:2t10,c12 increased the expression of GPR56 and GPCR5A, 2 tumor suppressor proteins found in inflamed tissues, and decreased the expression of GPR120, an antiinflammatory protein (41). Furthermore, 18:2t10,c12’s induction of inflammatory genes was blocked by the GPR40/120 agonist GW9508 (41). In the current study, we examined 2 PLC isoforms based on the human UniGene database showing that PLCγ and PLCδ are 2 of the 3 adipose tissue-specific PI-PLC isozymes, PLCβ being the third (for review, see 42), and our microarray data suggesting that 18:2t10,c12 increased PLCδ4 expression (data not shown). PLCγ can be directly activated by protein tyrosine kinases or by several lipid-derived second messengers, including phosphatidic acid and arachidonic acid, in the absence of protein tyrosine kinase activation (40). In contrast, PLCδ can be activated by increased intracellular calcium concentrations (40) due to its calcium-binding C2 domain (43).

In the present study, 18:2t10,c12 increased intracellular calcium concentrations within 3 min of treatment, whereas it increased the expression of PLCδ4 after 12 h of treatment but had no effect on PLCδ4 translocation. In contrast, 18:2t10,c12 had no effect on the expression of PLCγ1 but increased PLCγ1 translocation to the plasma membrane within 3 min. The time frame of this acute translocation of PLCγ1 to plasma membrane is consistent with work by Matsuda et al. (44). Notably, these 2 PLC isoforms have been linked to downstream inflammation. For example, overexpression of PLCδ4 has been reported to upregulate inflammatory ERK signaling in MCF-7 cells (45). Similarly, generation of DAG from PLCγ activates NFκB and MAPK signaling pathways (for review, see 46).

Interestingly, SFAs like palmitic and stearic acids also induce inflammatory signaling and insulin resistance in adipose tissue (for review, see 47). Consistent with these data, we previously demonstrated that stearic acid increased the mRNA levels of several proinflammatory genes in human adipocytes, which was further enhance by co-supplementation with 18:2t10,c12 (41). Although we showed that 18:2t10,c12 activates several inflammatory pathways shared by SFAs (for review, see 4, 47), including ERK, JNK, cJun, and NFκB (7, 10, 12, 22, 26, 41), we have not observed increases in the expression levels of toll-like receptor (TLR)4 or TLR2 in adipocytes treated with 18:2t10,c12 (data not shown). However, we have not chemically inhibited or silenced TLR4 or TRL2 to determine their role in 18:2t10,c12-mediated inflammatory signaling and insulin resistance. Currently, we speculate that 18:2t10,c12 activates inflammatory signaling via GPCR, GPR, or protein tyrosine kinase as shown in Figure 6.

Taken together, these data suggest that the rapid 18:2t10,c12-mediated increase in PLCγ1 is associated with the rapid increase of intracellular calcium, which triggers downstream inflammatory pathways that promote adipocyte delipidation. However, loss-of-function studies for PLCγ1 are needed to confirm this hypothesis. Alternatively, PLCβ, another adipose tissue-specific PI-PLC isozyme, can also be activated by cell surface GPCR and warrants investigation.

Acknowledgments

W.S. conducted all of the assays with assistance from K.M. for culturing cells and conducting the glucose uptake, calcium, bioplex, and cell fractionation assays and with assistance from C.C.C. for culturing cells and conducting the glucose uptake assays; M.M. and W.S. designed the studies; W.S. analyzed the data; and M.M. and W.S. wrote the manuscript. All authors read and approved the final manuscript.

Footnotes

3

Abbreviations used: ACC, acetyl-CoA carboxylase; AP-1, activator protein-1; CaMK, Ca2+/calmodulin-dependent protein kinase; CLA, conjugated linoleic acid; 18:2c9,t11, cis-9, trans-11; 18:2t10,c12, trans-10, cis-12; COX, cyclooxygenase; DAG, diacylglycerol; DGK, diacylglycerol kinase; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; GLUT4, insulin-dependent glucose transporter 4; GPCR, G protein coupled receptor; GPR, G protein receptor; HSP, heat shock protein; IP3, inositol-3-phosphate; JNK, c-Jun-NH2-terminal kinase; MAPK, mitogen-activated protein kinases; MCP, monocyte chemoattractant protein; NFκB, nuclear factor kappa B; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PC, phosphatidylcholine; PI, phosphoinositide; SCD, stearoyl-CoA desaturase; TLR, toll-like receptor; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione

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