Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes
2012; Elsevier BV; Volume: 54; Issue: 3 Linguagem: Inglês
10.1194/jlr.m031211
ISSN1539-7262
AutoresKristina Martinez, Shruthi Shyamasundar, Arion Kennedy, Chia‐Chi Chuang, Angel Marsh, Jennifer Kincaid, Tanya Reid, Michael McIntosh,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoDiacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes. Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes. acetyl-CoA carboxylase adipocyte media activator protein adipocyte fatty acid binding protein adiponectin intracellular calcium calmodulin calmodulin kinase conjugated linoleic acid t11 CLA, cis-9, trans-11 CLA c12 CLA, trans-10, cis-12 CLA cyclooxygenase diacylglycerol diacylglycerol kinase endoplasmic reticulum extracellular signal-regulated kinase free fatty acid receptor 1 insulin-dependent glucose transporter 4 G-protein coupled receptor 1-methyl-3-isobutylxanthine interleukin inositol phosphate insulin receptor substrate c-Jun-NH2-terminal kinase mitogen-activated protein kinase monocyte chemoattractant protein mitogen-activated protein kinase kinase mammalian target of rapamycin nuclear factor kappa B phosphatidic acid phosphatidyl choline prostaglandin phosphatidyl inositol phosphatidylinositol-bisphosphate protein kinase C phospholipase C peroxisome proliferator activated receptor reactive oxygen species S6 kinase stearoyl-CoA desaturase small interfering RNA triglyceride tumor necrosis factor Overweight and obesity are global health issues affecting 1.6 billion individuals worldwide (1World Health Organization. Obesity and overweight. Accessed March 7, 2011, at www.who.int/mediacentre/factsheets/fs311/en/index.html.Google Scholar). One potential strategy for reducing adiposity is consumption of conjugated linoleic acid (CLA), a group of conjugated octadecadienoic acid isomers derived from linoleic acid, a fatty acid (FA) that contains 18 carbons and 2 double bonds in the cis configuration at the 9th and 12th carbons (i.e., cis-9, cis-12 octadecadienoic acid) (2Martinez, K., Kennedy, A., McIntosh, M., . 2010. Conjugated linoleic acid. In Encyclopedia of Dietary Supplements. 2nd edition. Coates, P. M., Betz, J. M., Blackman, M. R., ., editors. Informa Healthcare, London. 166–174.Google Scholar). CLA is found in ruminant meats and dairy products, as microbes in the gastrointestinal tract of ruminant animals convert linoleic acid into different isoforms of CLA through biohydrogenation (2Martinez, K., Kennedy, A., McIntosh, M., . 2010. Conjugated linoleic acid. In Encyclopedia of Dietary Supplements. 2nd edition. Coates, P. M., Betz, J. M., Blackman, M. R., ., editors. Informa Healthcare, London. 166–174.Google Scholar). This process changes the position and configuration of the double bonds, resulting in a single bond between the two double bonds. The major isomers produced include cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA. Food sources of CLA contain ∼80% c9,t11 CLA and 10% t10,c12 CLA, and the remaining 10% is composed of other isomers (2Martinez, K., Kennedy, A., McIntosh, M., . 2010. Conjugated linoleic acid. In Encyclopedia of Dietary Supplements. 2nd edition. Coates, P. M., Betz, J. M., Blackman, M. R., ., editors. Informa Healthcare, London. 166–174.Google Scholar). CLA is also produced chemically from linoleic acid for inclusion in supplements and fortified foods, yielding a composition containing ∼40% c9,t11 CLA, ∼40% t10,c12 CLA isomers, and the remaining 20% other isomers (2Martinez, K., Kennedy, A., McIntosh, M., . 2010. Conjugated linoleic acid. In Encyclopedia of Dietary Supplements. 2nd edition. Coates, P. M., Betz, J. M., Blackman, M. R., ., editors. Informa Healthcare, London. 166–174.Google Scholar). Consuming a mixture of c9,t11 and t10,c12 CLA isomers or consuming t10,c12 CLA alone reduces body fat mass in rodents, particularly mice, and in some humans (3Kennedy A. Martinez K. Schmidt S. Mandrup S. Lapoint K. McIntosh M. Anti-obesity mechanisms of action of conjugated linoleic acid.J. Nutr. Biochem. 2010; 21: 171-179Crossref PubMed Scopus (194) Google Scholar). However, the isomer-specific mechanism by which CLA reduces adiposity is unclear. Proposed antiobesity mechanisms of t10, c12 CLA include regulation of i) energy metabolism, ii) adipogenesis, iii) lipid metabolism, iv) inflammation, and v) adipocyte apoptosis (reviewed in Ref. 3Kennedy A. Martinez K. Schmidt S. Mandrup S. Lapoint K. McIntosh M. Anti-obesity mechanisms of action of conjugated linoleic acid.J. Nutr. Biochem. 2010; 21: 171-179Crossref PubMed Scopus (194) Google Scholar). However, direct linkage of these potential mechanisms to body fat loss, especially inflammation, is unclear. We have demonstrated that activation of extracellular signal-regulated kinase (ERK) (4Brown J.M. Boysen M. Chung S. Fabiyi O. Morrison R. Mandrup S. McIntosh M. Conjugated linoleic acid (CLA) induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines.J. Biol. Chem. 2004; 279: 26735-26747Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and nuclear factor kappa B (NFκB) play a role in t10,c12 CLA-mediated delipidation and insulin resistance (5Chung S. Brown J.M. Provo J.N. Hopkins R. McIntosh M. Conjugated linoleic acid promotes human adipocyte insulin resistance through NFkappaB-dependent cytokine production.J. Biol. Chem. 2005; 280: 38445-38456Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). We have also shown that t10,c12 CLA-mediated activation of ERK, cJun N-terminal kinase (JNK), NFκB, and production of reactive oxygen species (ROS) was dependent on accumulation of intracellular calcium levels (6Kennedy A. Martinez K. Chung S. LaPoint K. West T. Hopkins R. Schmidt S. Andersen K. Mandrup S. McIntosh M. Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid are dependent on intracellular calcium levels in primary cultures of human adipocytes.J. Lipid Res. 2010; 51: 1906-1917Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Additionally, we demonstrated that TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), prevented t10,c12 CLA-mediated NFκB binding to promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 (6Kennedy A. Martinez K. Chung S. LaPoint K. West T. Hopkins R. Schmidt S. Andersen K. Mandrup S. McIntosh M. Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid are dependent on intracellular calcium levels in primary cultures of human adipocytes.J. Lipid Res. 2010; 51: 1906-1917Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Moreover, activated NFκB (7Poirier H. Shapiro H. Kim R. Lazar M. Nutritional supplementation with trans-10, cis-12 conjugated linoleic acid induces inflammation of white adipose tissue.Diabetes. 2006; 55: 1634-1641Crossref PubMed Scopus (163) Google Scholar–9Ruan H. Pownall H. Lodish H. Troglitazone antagonizes tumor necrosis factor-a induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NFkB.J. Biol. Chem. 2003; 278: 28181-28192Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and ERK (10Suzawa M. Takada I. Yanagisawa J. Ohtake F. Ogawa S. Yamauchi T. Kadowaki T. Takeuchi Y. Shibuya H. Gotoh Y. et al.Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade.Nat. Cell Biol. 2003; 5: 224-230Crossref PubMed Scopus (257) Google Scholar–12Camp H.S. Tafuri S.R. Regulation of peroxisome proliferators-activated receptor γ activity by mitogen activated protein kinase.J. Biol. Chem. 1997; 272: 10811-10816Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar) induce markers of inflammation and antagonize peroxisome proliferator activated receptor (PPAR)γ activity, thereby causing insulin resistance. These data suggest that t10,c12 CLA mediates inflammatory signaling that antagonizes adipogenic processes in adipocytes. However, the upstream signals responsible for t10,c12 CLA-mediated increases in intracellular calcium levels, inflammatory signaling, insulin resistance, and reduced triglyceride (TG) content in human adipocytes are unknown. Diacylglycerol kinases (DGK) are a family of kinases that phosphorylate diacylglycerol (DAG), resulting in the conversion of DAG into phosphatidic acid (PA). DAG and PA act as second messengers that activate an array of target proteins, resulting in significant changes in cellular signaling (reviewed in Ref. 13Sakane F. Imai S.I. Kai M. Yasuda S. Kanoh H. Diacylglycerol kinases: why so many of them?.Biochim. Biophys. Acta. 2007; 1771: 793-806Crossref PubMed Scopus (254) Google Scholar). For example, DAG activates conventional protein kinase C (cPKC), Unc-13, and protein kinase D, whereas PA activates atypical PKC, phosphatidylinositol (PI)-4-phosphate 5-kinase, and mammalian target of rapamycin (mTOR), RasGAP, and Raf-1 kinase (reviewed in Ref. 13Sakane F. Imai S.I. Kai M. Yasuda S. Kanoh H. Diacylglycerol kinases: why so many of them?.Biochim. Biophys. Acta. 2007; 1771: 793-806Crossref PubMed Scopus (254) Google Scholar). Therefore, DGKs are critical in terminating DAG signaling and initiating PA signaling. In addition to this well-characterized function, DGKs act as scaffolding proteins and regulate subcellular signaling via endosomal and nuclear transport. To date, 10 different DGK isozymes have been identified. Each of the DGKs has up to three PKC-like C1 domains and a catalytic region. DGKs are grouped into five different types, based on their structural and functional features. For example, type 1 DGKs, which include DGK α, β, and γ, contain recoverin homology domains and EF-hand motifs that serve as calcium-binding domains (reviewed in Ref. 13Sakane F. Imai S.I. Kai M. Yasuda S. Kanoh H. Diacylglycerol kinases: why so many of them?.Biochim. Biophys. Acta. 2007; 1771: 793-806Crossref PubMed Scopus (254) Google Scholar). Thus, these DGKs are activated in part by calcium binding. Type II DGKs, including DGK δ, η, and κ, contain pleckstrin homology domains, sterile α motif domain, and a separated catalytic region. Type III DGKs, including DGK ∊, contain no additional functional domains different than other DGK isoforms. Type IV DGKs, including DGK ζ and ι, contain a nuclear localization signal, a myristolated alanine-rich C kinase substrate phosphorylation domain, and four ankyrin repeats. Type V DGKs, including DGK θ, contain three C1 domains, a Gly/Pro-rich domain, and a PH-domain-like region (reviewed in Ref. 13Sakane F. Imai S.I. Kai M. Yasuda S. Kanoh H. Diacylglycerol kinases: why so many of them?.Biochim. Biophys. Acta. 2007; 1771: 793-806Crossref PubMed Scopus (254) Google Scholar). DGKs also display tissue-specific expression. DGKs are highly expressed in the brain, thymus, and muscle (13Sakane F. Imai S.I. Kai M. Yasuda S. Kanoh H. Diacylglycerol kinases: why so many of them?.Biochim. Biophys. Acta. 2007; 1771: 793-806Crossref PubMed Scopus (254) Google Scholar). However, DGK expression in adipose tissue or primary human adipocytes is poorly defined. Thus, DGKs are a complex family of kinases, and little is known regarding their potential function in adipose tissue. Several lines of evidence support the involvement of DGKs in t10,c12 CLA-mediated inflammation and insulin resistance. First, DGK-generated PA levels activate mTOR and S6 kinase (S6K) in HEK 293 cells (14Avila-Flores A. Santos T. Rincon E. Merida I. Modulation of the mammalian target of rapamycin pathway by diacylglycerol kinase-produced phosphatidic acid.J. Biol. Chem. 2005; 280: 10091-10099Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar); and we reported that t10,c12 CLA activated these two proteins in primary human adipocytes (15Chung S. Brown J.M. Sandberg M.B. McIntosh M. Trans-10, cis-12 CLA increases adipocyte lipolysis and alters lipid droplet-associated proteins: role of mTOR and ERK signaling.J. Lipid Res. 2005; 46: 885-895Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Second, mTOR and S6K activation have been implicated in the development of insulin resistance, a side effect of CLA supplementation (16Zhang J. Gao Z. Yin J. Quon M.J. Ye J. S6K directly phophorylates IRS-1 on Ser-270 to promote insulin resistnace in reponse to TNF-α signaling through IKK2.J. Biol. Chem. 2008; 283: 35375-35382Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 17Khamzina L. Veilleux A. Bergeron S. Marette A. Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance.Endocrinology. 2005; 146: 1473-1481Crossref PubMed Scopus (444) Google Scholar). Third, DGK-mediated PA production has also been shown to increase calcium release from the ER (18Camiña J. Casabiell X. Casanueva F.F. Inositol 1,4,5-trisphophate-independent Ca2-mobilization triggered by a lipid factor isolated from vitreous body.J. Biol. Chem. 1999; 274: 28134-28141Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). This finding could provide a mechanism by which t10,c12 CLA increases intracellular calcium levels in newly differentiated primary human adipocytes (6Kennedy A. Martinez K. Chung S. LaPoint K. West T. Hopkins R. Schmidt S. Andersen K. Mandrup S. McIntosh M. Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid are dependent on intracellular calcium levels in primary cultures of human adipocytes.J. Lipid Res. 2010; 51: 1906-1917Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Moreover, DGKη has been reported to regulate ERK activation, which we have found to be necessary, in part, for t10,c12 CLA-mediated insulin resistance (4Brown J.M. Boysen M. Chung S. Fabiyi O. Morrison R. Mandrup S. McIntosh M. Conjugated linoleic acid (CLA) induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines.J. Biol. Chem. 2004; 279: 26735-26747Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 19Yasuda S. Kai M. Imai S. Takeishi K. Taketomi A. Toyota M. Kanoh H. Sakane F. Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.J. Biol. Chem. 2009; 284: 29559-29570Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Indeed, Yasuda et al. (19Yasuda S. Kai M. Imai S. Takeishi K. Taketomi A. Toyota M. Kanoh H. Sakane F. Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.J. Biol. Chem. 2009; 284: 29559-29570Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) found that DGKη facilitated the transport of c-Raf to the plasma membrane, upstream of MEK/ERK activation in response to epidermal growth factor treatment in HeLa cells. Therefore, it is tempting to speculate that t10,c12 CLA-mediated activation of MEK/ERK may involve similar signaling mechanisms. Additionally, DGKα has been shown to regulate tumor necrosis factor (TNF)α-mediated NFκB activation (20Kai M. Yasuda S. Imai S. Toyota M. Kanoh H. Sakane F. Diacylglycerol kinase α enhances protein kinase cζ-dependent phosphorylation at Ser311 of p65/relA subunit of nuclear factor-κB.FEBS Lett. 2009; 583: 3265-3268Crossref PubMed Scopus (28) Google Scholar), which we have reported to be activated by t10,c12 CLA treatment in adipocytes (5Chung S. Brown J.M. Provo J.N. Hopkins R. McIntosh M. Conjugated linoleic acid promotes human adipocyte insulin resistance through NFkappaB-dependent cytokine production.J. Biol. Chem. 2005; 280: 38445-38456Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Finally, a mixture of CLA isomers has been shown to increase expression of DGKζ and increase PA levels in cardiomyocytes (21Alibin C.P. Kopilas M.A. Anderson H.D.I. Suppression of cardiac myocyte hypertrophy by conjugated linoleic acid: role of peroxisome proliferator-activated receptors α and γ.J. Biol. Chem. 2008; 283: 10707-10715Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Taken together, there are several interesting findings in the literature that suggest a potential role for DGKs in t10,c12 CLA-mediated signaling in primary human adipocytes. Based on the close similarity between pathways activated by DGK and t10,c12 CLA, we hypothesized that DGKs play an important role in t10,c12 CLA-mediated inflammation, insulin resistance, and delipidation. To test this hypothesis, we employed the chemical DGK inhibitor R59022 and siRNA targeting DGKη. In this study, we demonstrated that DGKs, particularly DGKη, may be involved in the regulation of t10,c12 CLA-mediated inflammatory signaling, insulin resistance, and delipidation in primary human adipocytes. All cell cultureware was purchased from Fisher Scientific (Norcross, GA). Lightning chemiluminescence substrate was purchased from Perkin Elmer Life Science (Boston, MA). Immunoblotting buffers and precast gels were purchased from Invitrogen by Life Technologies (Carlsbad, CA). Polyclonal antibodies for anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Beta (β)-actin, and monoclonal antibody for anti-PPARγ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody for DGKη was purchased from Abcam (Cambridge, MA). Anti-total and anti-phospho (P) ERK, JNK, and P-cJun antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Hyclone fetal bovine serum was purchased from Fisher Scientific. Isomers of CLA (+98% pure) were purchased from Matreya (Pleasant Gap, PA). Fluo-3 acetyloxymethyl ester (Fluo-3 AM), pluronic F-127, and probenecid were purchased from Invitrogen by Life Technologies. Thapsigargin and ionomycin were purchased from Calbiochem-EMD Biosciences, Inc. (La Jolla, CA). The cell permeable DGK inhibitor R59022 (6-{2-{4-[(4-fluorophenyl)phenylmethylene]1-piperidinyl}ethyl}-7-methyl-5H-thiazolo-(3,2-a)pyrimidin-5-one) was purchased from EMD Chemicals (Gibbstown, NJ). This inhibitor functions by inhibiting DAG phosphorylation, which is more specific to the type I, calcium-sensitive DGKs (22Jiang Y. Sakane F. Kanoh H. Walsh J. Selectivity of the diacylglycerol kinase inhibitor 3-{2-(4-[bis-(4-Fluorophenyl)methylene]-1-piperidinyl)ethyl}-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) among diacylglycerol kinase subtypes.Biochem. Pharmacol. 2000; 59: 763-772Crossref PubMed Scopus (112) Google Scholar). Dharmacon DGKη, GAPDH, and nontargeting ON-TARGETplus siRNA, and Dharmafect 1 transfection reagent were purchased from ThermoScientific (Lafayette, CO). All other reagents and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Abdominal white adipose tissue was obtained with consent from the Institutional Review Boards at the University of North Carolina at Greensboro and the Moses Cone Memorial Hospital during elective abdominoplasty of nondiabetic Caucasian and African American females between the ages of 20 and 50 years with a body mass index ≤ 32.0. These selection criteria allowed for reduced variation in gender, age, and obesity status. Tissue was digested using collagenase; stromal vascular cells were isolated as previously described (4Brown J.M. Boysen M. Chung S. Fabiyi O. Morrison R. Mandrup S. McIntosh M. Conjugated linoleic acid (CLA) induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines.J. Biol. Chem. 2004; 279: 26735-26747Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Stromal vascular cells were differentiated with adipocyte media (AM-1) containing 1 μM rosiglitazone and 250 μM 1-methyl-3-isobutylxanthine for three days, which yielded cultures containing ∼30–50% adipocytes. On days 7–14, cells were pretreated with 0.1, 1, 3, 10, or 30 μM R59022 dissolved in DMSO for 30 min and subsequently treated with 50–150 μM t10,c12 CLA or BSA (BSA) vehicle control for 5 min to 48 h depending on the experimental outcome measured. All cultures were normalized to contain the same amount of BSA and DMSO vehicles. Each independent experiment was repeated at least twice using a mixture of cells from three subjects, unless otherwise indicated. t10,c12 and c9,t11 CLA were delivered as free FAs complexed to 7.5% FA-free BSA (Sigma A7030, lot #040M1649) at a 4:1 molar ratio as previously described (4Brown J.M. Boysen M. Chung S. Fabiyi O. Morrison R. Mandrup S. McIntosh M. Conjugated linoleic acid (CLA) induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines.J. Biol. Chem. 2004; 279: 26735-26747Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This BSA was chosen based on its decreased capacity to induce inflammatory gene expression compared with other BSA samples tested (unpublished data). For measuring [Ca2+]i levels, t10,c12 CLA dissolved in a 0.1M KOH solution was used. TG levels were determined with a modified commercially available TG assay as previously described (23Brown J.M. Sandberg-Boysen M. Skov S. Morrison R. Storkson J. Lea-Currie R. Pariza M. Mandrup S. McIntosh M. Isomer-specific regulation of metabolism and PPARγ by conjugated linoleic acid (CLA) in human preadipocytes.J. Lipid Res. 2003; 44: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Cultures of human adipocytes were supplemented with low-glucose DMEM on day 10. The following day, cultures were pretreated with 0.1, 1, or 10 μM R59022 for 30 min and subsequently treated with BSA vehicle or 50 µM t10,c12 CLA for 48 h. Cultures were stimulated for 10 min with 100 nM insulin and treated with 4 nmol 3H-2-deoxyglucose for 90 min. The amount of 3H-2-deoxyglucose was measured via scintillation counting as described (23Brown J.M. Sandberg-Boysen M. Skov S. Morrison R. Storkson J. Lea-Currie R. Pariza M. Mandrup S. McIntosh M. Isomer-specific regulation of metabolism and PPARγ by conjugated linoleic acid (CLA) in human preadipocytes.J. Lipid Res. 2003; 44: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Cultures of human primary adipocytes were supplemented with low-glucose DMEM on day 10. The following day, cultures were pretreated with 0.1, 1, or 10 μM R59022 for 30 min and subsequently treated with BSA vehicle or 50 µM t10,c12 CLA for 48 h. Cultures were treated with 12.5 nM 14C-oleic acid (0.2 µCi; specific activity = 40–60 mCi/mmol) for 120 min. The amount of 14C-oleic acid oleic acid was measured via scintillation counting as described previously (23Brown J.M. Sandberg-Boysen M. Skov S. Morrison R. Storkson J. Lea-Currie R. Pariza M. Mandrup S. McIntosh M. Isomer-specific regulation of metabolism and PPARγ by conjugated linoleic acid (CLA) in human preadipocytes.J. Lipid Res. 2003; 44: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). After experimental treatments, cultures were washed once with ice-cold HBSS. The cells were then solubilized by the direct addition of a lysis buffer containing PBS (pH 7.5), 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM Na3VO4, 20 mM β-glycerophosphate, 10 mM NaF, and a protease inhibitor mixture (Calbiochem) including 500 μM AEBSF, 1 μg/ml aprotinin, 1 μM E-64, 500 μM EDTA, and 1 μM leupeptin. Monolayers were scraped and transferred to prechilled microfuge tubes. Cell lysates were then sonicated three times for 5 s and stored on ice for an additional 20 min. Cell debris was pelleted by centrifugation at 14,000 rpm at 4°C, and the resulting supernatant was collected for analysis. The protein concentration of each sample was determined using a bicinchoninic acid assay (Pierce). Subsequently, 20 μg of protein from each sample was prepared with Nu-Page LDS sample buffer (Life Technologies) for denaturing gel electrophoresis using 4–12% NuPage precasted gels. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes that were next blocked with 5% milk in TBST for 1 h and washed three times in TBST for 5 min. Blots were incubated overnight at 4°C with primary antibodies targeting DGKη, P-ERK, P-JNK, P-cJun, and total cJun at a dilution of 1:1,000, and then subsequently incubated in the respective horseradish peroxidase-conjugated secondary antibody at a dilution of 1:5,000 at room temperature for 1 h. Primary and secondary antibodies targeting GAPDH were used at a 1:5,000 dilution. Primary and secondary antibodies targeting PPARγ were used at dilutions 1:200 and 1:2,000, respectively. After washing, blots were treated with chemiluminescence reagent for 1 min, and film was exposed using a SRX-101A Konica Minolta film developer. To quantify treatment differences, densitometry was performed using a Kodak Image Station 440. Data were normalized to β-actin loading control. Total RNA was isolated from the cultures using Tri Reagent purchased from Molecular Research Center (Cincinnati, OH), according to manufacturer's protocol. For quantitative real-time PCR, 1.0 μg total RNA was converted into first-strand cDNA using high-capacity cDNA archive kit (Applied Biosystems, 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 quantified for each sample, and these data were normalized accordingly. Due to RNA interference of GAPDH in Fig. 6, TATA box binding protein (TBP) was used as the endogenous reference gene. The relative standard curve method using seven 2-fold dilutions ranging from 100 to 1.56 ng RNA was used to check primer efficiency and linearity of each transcript according to Applied Biosystems's Guide to Performing Relative Quantification of Gene Expression Using Real-Time Quantitative PCR. The concentrations of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 were determined using the Bio-Plex suspension array system from Bio-Rad (Hercules, CA) following the manufacturer's protocol. Briefly, media was collected from cultures that were pretreated with 30 μM R59022 for 30 min, and subsequently treated with 50 μM t10,c12 CLA or BSA for 24 h. This time point was based on previous time course studies showing that the maximum level of cytokine secretion occurred after 24 h of t10,c12 CLA treatment (5Chung S. Brown J.M. Provo J.N. Hopkins R. McIntosh M. Conjugated linoleic acid promotes human adipocyte insulin resistance through NFkappaB-dependent cytokine production.J. Biol. Chem. 2005; 280: 38445-38456Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The media was centrifuged at 13,200 g for 10 min at 4°C to clear the samples of cellular debris. Samples and standards were run in duplicate. Based on the manufacturer's report Bio-Plex Pro Human Cytokine, Chemokine, and Growth Factor Assays - Bulletin 5828, the intra-assay percentage coefficient of variation for IL-6, IL-8, and MCP-1 are 7, 9, and 9%, respectively. The interassay percentage CVs a
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